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4c:T1cc6,Role and Mechanism of MDK in Promoting Vascular Mimicry in Breast Cancer 1. Overview Scientific rationale and importance: Vascular mimicry (VM) enables aggressive tumors to form vessel-like structures independent of endothelial cells, contributing to therapy resistance and poor prognosis. My preliminary data indicate that Midkine (MDK), a secreted growth factor stabilized by USP12, promotes VM in triple-negative breast cancer (TNBC) cells. Proteomic analysis identified Vitronectin (VTN) as a potential downstream target of MDK, and high MDK expression correlates with increased VE-cadherin in patient tumors. This project aims to validate VTN as a bona fide mediator of MDK-induced VM, thereby providing a novel therapeutic target. Funding/facilities/ethics approvals: The proposed work will be conducted at Northern Jiangsu People's Hospital, with full access to molecular biology, histology, and imaging facilities. Institutional ethics approval for human tissue use and animal experiments will be secured before study initiation. I obtained my PhD in Cell Biology from Peking Union Medical College in 2021 and subsequently began working as a physician at Northern Jiangsu People's Hospital, one of the largest and most historic hospitals in Jiangsu Province. Our hospital has well-established research facilities and adequate clinical samples to support both basic and translational research. My research background in cancer biology, supported by a strong publication track record, demonstrates my ability to serve as the PI of this proposal. This mini grant will enable me to generate preliminary data to secure a full-scale research grant, establish my own research laboratory, and continue investigating the molecular mechanisms and novel therapeutic targets in breast cancer. To facilitate fund transfer, I have obtained approval from our collaborating organization, One Patient One Cure, a US-based nonprofit, to receive the funding. Estimated timeline: 12 months (September 1, 2025 – August 31, 2026). Data sharing commitment: All raw and processed data, including imaging and proteomics datasets, will be deposited in public repositories (e.g., GEO, PRIDE) within six months of publication. 2. Introduction Background: VM is a key alternative angiogenesis mechanism in breast cancer, particularly under hypoxic stress, driven by EMT and signaling pathways such as Wnt/β-catenin and TGF-β. Current anti-VEGF therapies fail to suppress VM, underscoring the need for alternative targets. MDK is overexpressed in various malignancies and promotes proliferation, EMT, angiogenesis, and therapy resistance. In TNBC, MDK overexpression enhances tube formation and VM marker expression. Our proteomics and immunoprecipitation data suggest that MDK may act through VTN, an extracellular matrix glycoprotein implicated in cell adhesion, migration, and angiogenesis. Research question: Does VTN mediate MDK-driven VM in breast cancer? Hypotheses: H1: MDK promotes VM in breast cancer through upregulation of VTN. H2: Disruption of VTN abrogates MDK-induced VM in vitro and in vivo. Impact statement: Confirmation of the MDK–VTN axis as a driver of VM will open new therapeutic avenues, particularly for TNBC, by targeting VM to improve anti-angiogenic therapy outcomes. 3. Methods Participants/Sample Cell lines: TNBC cell lines such as MDA-MB-231 and BT549 with MDK overexpression or knockdown. Animal models: Orthotopic TNBC xenografts in immunodeficient mice. Clinical samples: Preserved breast cancer tissues. Materials and Procedures Aim 1 – Verify VTN regulation by MDK Western blot and qRT-PCR to measure VTN levels in MDK-overexpressing and knockdown cells compared to controls. Immunoprecipitation to assess MDK–VTN interaction. Aim 2 – Assess functional role of VTN in MDK-induced VM In vitro tube formation assays: VTN overexpression in MDK-knockdown cells to assess restoration of the VM phenotype (rescue experiment). In vivo assays: Orthotopic xenografts with manipulated MDK/VTN expression; assess VM structures via PAS staining and VM marker IHC. Aim 3 – Clinical correlation Immunohistochemistry for MDK, VTN, and VE-cadherin in TNBC patient tissues. Statistical correlation of MDK and VTN expression with VM prevalence and patient outcomes. Planned Analyses Statistical tests: Two-tailed t-tests or ANOVA for group comparisons; Pearson/Spearman for correlation; Kaplan–Meier survival curves for prognostic value. Alternative strategy: If VTN overexpression only partially affects MDK-induced VM in vitro, we will screen for cofactors via proteomics. Ethics and Data Management Ethics: Institutional Review Board and Animal Care Committee approval will be obtained before patient sample analysis and animal experiments. Confidentiality: All human data will be anonymized before analysis. Data sharing: Raw data will be deposited in GEO/PRIDE; protocols will be made publicly available (e.g., protocols.io). 4. Pilot Data (Optional) MDK overexpression in MDA-MB-231 cells increases tubular structure formation. Proteomics identified VTN as significantly upregulated in MDK-overexpressing cells. High-MDK tumors in clinical samples show elevated VE-cadherin 5. Budget Consumable reagents and assay kits: $2,000 Animal studies: $2,300 Imaging and histology: $500 Data analysis software: $200 Total: $5,000 6. References 1 Cerezo-Wallis, D. et al. Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state. Nat Med 26, 1865-1877 (2020). https://doi.org/10.1038/s41591-020-1073-3 2 Christou, C., Stylianou, A. & Gkretsi, V. Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker. Cells 13 (2024). https://doi.org/10.3390/cells13020136 3 Han, B. et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent 4, 47-53 (2024). https://doi.org/10.1016/j.jncc.2024.01.006 4 Kadomatsu, K., Tomomura, M. & Muramatsu, T. cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun 151, 1312-1318 (1988). https://doi.org/10.1016/s0006-291x(88)80505-9 5 Lapkina, E. Z., Esimbekova, A. R. & Ruksha, T. G. [Vasculogenic mimicry]. Arkh Patol 85, 62-69 (2023). https://doi.org/10.17116/patol20238506162 6 Maeda, N. et al. A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta. J Biol Chem 274, 12474-12479 (1999). https://doi.org/10.1074/jbc.274.18.12474 7 Muramatsu, T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proc Jpn Acad Ser B Phys Biol Sci 86, 410-425 (2010). https://doi.org/10.2183/pjab.86.410 8 Sheng, B., Wei, Z., Wu, X., Li, Y. & Liu, Z. USP12 promotes breast cancer angiogenesis by maintaining midkine stability. Cell Death Dis 12, 1074 (2021). https://doi.org/10.1038/s41419-021-04102-y 9 Shin, D. H. et al. Midkine Is a Potential Therapeutic Target of Tumorigenesis, Angiogenesis, and Metastasis in Non-Small Cell Lung Cancer. Cancers (Basel) 12 (2020). https://doi.org/10.3390/cancers12092402 10 Wei, X. et al. Mechanisms of vasculogenic mimicry in hypoxic tumor microenvironments. Mol Cancer20, 7 (2021). https://doi.org/10.1186/s12943-020-01288-14d:T1db7,Proposal Template 1. Overview Scientific rationale and importance: Vascular mimicry (VM) enables aggressive tumors to form vessel-like structures independent of endothelial cells, contributing to therapy resistance and poor prognosis. My preliminary data indicate that Midkine (MDK), a secreted growth factor stabilized by USP12, promotes VM in triple-negative breast cancer (TNBC) cells. Proteomic analysis identified Vitronectin (VTN) as a potential downstream target of MDK, and high MDK expression correlates with increased VE-cadherin in patient tumors. This project aims to validate VTN as a bona fide mediator of MDK-induced VM, thereby providing a novel therapeutic target. Funding/facilities/ethics approvals: The proposed work will be conducted at Northern Jiangsu People's Hospital, with full access to molecular biology, histology, and imaging facilities. Institutional ethics approval for human tissue use and animal experiments will be secured before study initiation. I obtained my PhD in Cell Biology from Peking Union Medical College in 2021 and subsequently began working as a physician at Northern Jiangsu People's Hospital, one of the largest and most historic hospitals in Jiangsu Province. Our hospital has well-established research facilities and adequate clinical samples to support both basic and translational research. My research background in cancer biology, supported by a strong publication track record, demonstrates my ability to serve as the PI of this proposal. This mini grant will enable me to generate preliminary data to secure a full-scale research grant, establish my own research laboratory, and continue investigating the molecular mechanisms and novel therapeutic targets in breast cancer. To facilitate fund transfer, I have obtained approval from our collaborating organization, One Patient One Cure, a US-based nonprofit, to receive the funding. Estimated timeline: 12 months (September 1, 2025 – August 31, 2026). Data sharing commitment: All raw and processed data, including imaging and proteomics datasets, will be deposited in public repositories (e.g., GEO, PRIDE) within six months of publication. 2. Introduction Background: VM is a key alternative angiogenesis mechanism in breast cancer, particularly under hypoxic stress, driven by EMT and signaling pathways such as Wnt/β-catenin and TGF-β. Current anti-VEGF therapies fail to suppress VM, underscoring the need for alternative targets. MDK is overexpressed in various malignancies and promotes proliferation, EMT, angiogenesis, and therapy resistance. In TNBC, MDK overexpression enhances tube formation and VM marker expression. Our proteomics and immunoprecipitation data suggest that MDK may act through VTN, an extracellular matrix glycoprotein implicated in cell adhesion, migration, and angiogenesis. Research question: Does VTN mediate MDK-driven VM in breast cancer? Hypotheses: H1: MDK promotes VM in breast cancer through upregulation of VTN. H2: Disruption of VTN abrogates MDK-induced VM in vitro and in vivo. Impact statement: Confirmation of the MDK–VTN axis as a driver of VM will open new therapeutic avenues, particularly for TNBC, by targeting VM to improve anti-angiogenic therapy outcomes. 3. Methods Participants/Sample Cell lines: TNBC cell lines such as MDA-MB-231 and BT549 with MDK overexpression or knockdown. Animal models: Orthotopic TNBC xenografts in immunodeficient mice. Clinical samples: Preserved breast cancer tissues. Materials and Procedures Aim 1 – Verify VTN regulation by MDK Western blot and qRT-PCR to measure VTN levels in MDK-overexpressing and knockdown cells compared to controls. Immunoprecipitation to assess MDK–VTN interaction. Aim 2 – Assess functional role of VTN in MDK-induced VM In vitro tube formation assays: VTN overexpression in MDK-knockdown cells to assess restoration of the VM phenotype (rescue experiment). In vivo assays: Orthotopic xenografts with manipulated MDK/VTN expression; assess VM structures via PAS staining and VM marker IHC. Aim 3 – Clinical correlation Immunohistochemistry for MDK, VTN, and VE-cadherin in TNBC patient tissues. Statistical correlation of MDK and VTN expression with VM prevalence and patient outcomes. Planned Analyses Statistical tests: Two-tailed t-tests or ANOVA for group comparisons; Pearson/Spearman for correlation; Kaplan–Meier survival curves for prognostic value. Alternative strategy: If VTN overexpression only partially affects MDK-induced VM in vitro, we will screen for cofactors via proteomics. Ethics and Data Management Ethics: Institutional Review Board and Animal Care Committee approval will be obtained before patient sample analysis and animal experiments. Confidentiality: All human data will be anonymized before analysis. Data sharing: Raw data will be deposited in GEO/PRIDE; protocols will be made publicly available (e.g., protocols.io). 4. Pilot Data (Optional) MDK overexpression in MDA-MB-231 cells increases tubular structure formation. Proteomics identified VTN as significantly upregulated in MDK-overexpressing cells. High-MDK tumors in clinical samples show elevated VE-cadherin 5. Budget Consumable reagents and assay kits: $2,000 Animal studies: $2,300 Imaging and histology: $500 Data analysis software: $200 Total: $5,000 6. References 1 Cerezo-Wallis, D. et al. Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state. Nat Med 26, 1865-1877 (2020). https://doi.org/10.1038/s41591-020-1073-3 2 Christou, C., Stylianou, A. & Gkretsi, V. Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker. Cells 13 (2024). https://doi.org/10.3390/cells13020136 3 Han, B. et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent 4, 47-53 (2024). https://doi.org/10.1016/j.jncc.2024.01.006 4 Kadomatsu, K., Tomomura, M. & Muramatsu, T. cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun 151, 1312-1318 (1988). https://doi.org/10.1016/s0006-291x(88)80505-9 5 Lapkina, E. Z., Esimbekova, A. R. & Ruksha, T. G. [Vasculogenic mimicry]. Arkh Patol 85, 62-69 (2023). https://doi.org/10.17116/patol20238506162 6 Maeda, N. et al. A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta. J Biol Chem 274, 12474-12479 (1999). https://doi.org/10.1074/jbc.274.18.12474 7 Muramatsu, T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proc Jpn Acad Ser B Phys Biol Sci 86, 410-425 (2010). https://doi.org/10.2183/pjab.86.410 8 Sheng, B., Wei, Z., Wu, X., Li, Y. & Liu, Z. USP12 promotes breast cancer angiogenesis by maintaining midkine stability. Cell Death Dis 12, 1074 (2021). https://doi.org/10.1038/s41419-021-04102-y 9 Shin, D. H. et al. Midkine Is a Potential Therapeutic Target of Tumorigenesis, Angiogenesis, and Metastasis in Non-Small Cell Lung Cancer. Cancers (Basel) 12 (2020). https://doi.org/10.3390/cancers12092402 10 Wei, X. et al. Mechanisms of vasculogenic mimicry in hypoxic tumor microenvironments. Mol Cancer20, 7 (2021). https://doi.org/10.1186/s12943-020-01288-14e:T8612,{"type":"doc","content":[{"type":"heading","attrs":{"id":"21bd51b8-6618-454c-81fd-a25b3218d109","data-toc-id":"21bd51b8-6618-454c-81fd-a25b3218d109","textAlign":"left","level":1},"content":[{"type":"text","text":"Proposal Template"}]},{"type":"heading","attrs":{"id":"dc31eeb1-4c24-40ea-b9ac-e3b63b3fb2cd","data-toc-id":"dc31eeb1-4c24-40ea-b9ac-e3b63b3fb2cd","textAlign":"left","level":2},"content":[{"type":"text","text":"1. Overview"}]},{"type":"paragraph","attrs":{"id":"407c90b7-84fd-436b-a888-2a8f5fe75d70","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Scientific rationale and importance:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Vascular mimicry (VM) enables aggressive tumors to form vessel-like structures independent of endothelial cells, contributing to therapy resistance and poor prognosis. My preliminary data indicate that Midkine (MDK), a secreted growth factor stabilized by USP12, promotes VM in triple-negative breast cancer (TNBC) cells. Proteomic analysis identified Vitronectin (VTN) as a potential downstream target of MDK, and high MDK expression correlates with increased VE-cadherin in patient tumors. This project aims to validate VTN as a bona fide mediator of MDK-induced VM, thereby providing a novel therapeutic target."}]},{"type":"paragraph","attrs":{"id":"ff7c073f-efc4-44e6-8bf4-8b5430829a23","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Funding/facilities/ethics approvals:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"The proposed work will be conducted at Northern Jiangsu People's Hospital, with full access to molecular biology, histology, and imaging facilities. Institutional ethics approval for human tissue use and animal experiments will be secured before study initiation."}]},{"type":"paragraph","attrs":{"id":"6765bbaa-aa6b-43e1-9ba7-2c9656dfa6e1","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"I obtained my PhD in Cell Biology from Peking Union Medical College in 2021 and subsequently began working as a physician at Northern Jiangsu People's Hospital, one of the largest and most historic hospitals in Jiangsu Province. Our hospital has well-established research facilities and adequate clinical samples to support both basic and translational research. My research background in cancer biology, supported by a strong publication track record, demonstrates my ability to serve as the PI of this proposal."}]},{"type":"paragraph","attrs":{"id":"22646ac7-6fdf-4f13-8b9e-53c8e947f8c7","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"This mini grant will enable me to generate preliminary data to secure a full-scale research grant, establish my own research laboratory, and continue investigating the molecular mechanisms and novel therapeutic targets in breast cancer. To facilitate fund transfer, I have obtained approval from our collaborating organization, One Patient One Cure, a US-based nonprofit, to receive the funding."}]},{"type":"paragraph","attrs":{"id":"c03e88ff-0d47-4d12-8d26-fb6ea29dc3b5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Estimated timeline:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"12 months (September 1, 2025 – August 31, 2026)."}]},{"type":"paragraph","attrs":{"id":"d8eb9d94-fabb-4dd5-8aa2-268a48123b0c","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Data sharing commitment:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"All raw and processed data, including imaging and proteomics datasets, will be deposited in public repositories (e.g., GEO, PRIDE) within six months of publication."}]},{"type":"heading","attrs":{"id":"25605e66-40a8-4d28-8e0c-f54f40b938bf","data-toc-id":"25605e66-40a8-4d28-8e0c-f54f40b938bf","textAlign":"left","level":2},"content":[{"type":"text","text":"2. Introduction"}]},{"type":"paragraph","attrs":{"id":"6aa560f7-3804-43f0-8ba9-f1ffa04c9119","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Background:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"VM is a key alternative angiogenesis mechanism in breast cancer, particularly under hypoxic stress, driven by EMT and signaling pathways such as Wnt/β-catenin and TGF-β. Current anti-VEGF therapies fail to suppress VM, underscoring the need for alternative targets."}]},{"type":"paragraph","attrs":{"id":"d1fb735b-96e6-4213-9853-dabe6ce0cb41","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"MDK is overexpressed in various malignancies and promotes proliferation, EMT, angiogenesis, and therapy resistance. In TNBC, MDK overexpression enhances tube formation and VM marker expression. Our proteomics and immunoprecipitation data suggest that MDK may act through VTN, an extracellular matrix glycoprotein implicated in cell adhesion, migration, and angiogenesis."}]},{"type":"paragraph","attrs":{"id":"ec60dcad-9897-4314-b88d-55b7c9f62135","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Research question:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Does VTN mediate MDK-driven VM in breast cancer?"}]},{"type":"paragraph","attrs":{"id":"78dbeb6a-19d2-4380-bf78-4da30dda99a1","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Hypotheses:"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"628a41ae-d348-4c1d-bc67-2c753dd9cf1c","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"H1: MDK promotes VM in breast cancer through upregulation of VTN."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"88c6bc49-ef44-4c5b-aa7e-6c933cce5fff","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"H2: Disruption of VTN abrogates MDK-induced VM in vitro and in vivo."}]}]}]},{"type":"paragraph","attrs":{"id":"8a87fc79-c32a-45fb-b115-c6d0fc0b1f2b","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Impact statement:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Confirmation of the MDK–VTN axis as a driver of VM will open new therapeutic avenues, particularly for TNBC, by targeting VM to improve anti-angiogenic therapy outcomes."}]},{"type":"heading","attrs":{"id":"ca433291-7526-4f84-b27a-b0b46eee9d4e","data-toc-id":"ca433291-7526-4f84-b27a-b0b46eee9d4e","textAlign":"left","level":2},"content":[{"type":"text","text":"3. Methods"}]},{"type":"heading","attrs":{"id":"ed97f88e-b5c2-4a35-b550-65dcfafed1df","data-toc-id":"ed97f88e-b5c2-4a35-b550-65dcfafed1df","textAlign":"left","level":3},"content":[{"type":"text","text":"Participants/Sample"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"a5c0bbf5-b780-4ada-a5b4-8650d83a82b6","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Cell lines: TNBC cell lines such as MDA-MB-231 and BT549 with MDK overexpression or knockdown."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"dd3b5082-329c-4389-a454-0bdcaa5afd31","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Animal models: Orthotopic TNBC xenografts in immunodeficient mice."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"8b051e0f-0900-4fa2-b92e-29968548258f","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Clinical samples: Preserved breast cancer tissues."}]}]}]},{"type":"heading","attrs":{"id":"8817c620-3f60-4956-bda5-beea4a777f97","data-toc-id":"8817c620-3f60-4956-bda5-beea4a777f97","textAlign":"left","level":3},"content":[{"type":"text","text":"Materials and Procedures"}]},{"type":"paragraph","attrs":{"id":"01775a43-8a1e-43ff-8bef-e519d266ffbd","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Aim 1 – Verify VTN regulation by MDK"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"bfa9350b-86ec-4507-b43a-92ab14f739ac","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Western blot and qRT-PCR to measure VTN levels in MDK-overexpressing and knockdown cells compared to controls."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"eb550025-f215-48fe-b1e5-13f708f759b0","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Immunoprecipitation to assess MDK–VTN interaction."}]}]}]},{"type":"paragraph","attrs":{"id":"c2f61a8a-a433-478c-a5bd-39730af170dc","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Aim 2 – Assess functional role of VTN in MDK-induced VM"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"bc29722b-0939-4234-8b77-850fbed72285","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"In vitro tube formation assays:"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" VTN overexpression in MDK-knockdown cells to assess restoration of the VM phenotype (rescue experiment)."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"ec76c5f7-a760-455c-989e-b8e0b05c6c98","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"In vivo assays:"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" Orthotopic xenografts with manipulated MDK/VTN expression; assess VM structures via PAS staining and VM marker IHC."}]}]}]},{"type":"paragraph","attrs":{"id":"444174c7-e5ee-4b27-a81b-a01eb8f8e0a5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Aim 3 – Clinical correlation"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"5700b1fc-57ea-4850-a04e-0e9c345a74f4","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Immunohistochemistry for MDK, VTN, and VE-cadherin in TNBC patient tissues."}]}]}]},{"type":"paragraph","attrs":{"id":"3b82e61e-436e-4d4a-a4cc-8ff7eb82c73d","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"rgb(0, 0, 0)"}}],"text":"Statistical correlation of MDK and VTN expression with VM prevalence and patient outcomes."}]},{"type":"heading","attrs":{"id":"49dee47f-4a5d-4479-8380-1c769df45c26","data-toc-id":"49dee47f-4a5d-4479-8380-1c769df45c26","textAlign":"left","level":3},"content":[{"type":"text","text":"Planned Analyses"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"dca64fa2-a7f2-454a-907d-34745b36e07a","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Statistical tests: Two-tailed t-tests or ANOVA for group comparisons; Pearson/Spearman for correlation; Kaplan–Meier survival curves for prognostic value."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"98bf208a-fca8-4d7c-a433-d847efa13a6c","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Alternative strategy: If VTN overexpression only partially affects MDK-induced VM in vitro, we will screen for cofactors via proteomics."}]}]}]},{"type":"heading","attrs":{"id":"ed994363-1076-4c56-bb45-46b55a5130e2","data-toc-id":"ed994363-1076-4c56-bb45-46b55a5130e2","textAlign":"left","level":3},"content":[{"type":"text","text":"Ethics and Data Management"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"280c3b63-119b-4e73-b685-f536ee1bf4d6","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Ethics: Institutional Review Board and Animal Care Committee approval will be obtained before patient sample analysis and animal experiments."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"5189580c-e0fc-4cc9-98ec-a423692620d5","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Confidentiality: All human data will be anonymized before analysis."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"d8781ea1-6d95-4194-9eb2-d84801f2c4b9","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Data sharing: Raw data will be deposited in GEO/PRIDE; protocols will be made publicly available (e.g., "},{"type":"text","marks":[{"type":"link","attrs":{"href":"http://protocols.io","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"protocols.io"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":")."}]}]}]},{"type":"heading","attrs":{"id":"05f07119-09c5-41c7-8c08-45ee134a0e9c","data-toc-id":"05f07119-09c5-41c7-8c08-45ee134a0e9c","textAlign":"left","level":2},"content":[{"type":"text","text":"4. Pilot Data (Optional)"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"6f8ed375-1901-4a53-a8ec-3b77d93e36e5","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"MDK overexpression in MDA-MB-231 cells increases tubular structure formation."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"7bdec09e-b999-47e7-935a-e578a81510f1","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Proteomics identified VTN as significantly upregulated in MDK-overexpressing cells."}]}]}]},{"type":"paragraph","attrs":{"id":"da71c5dd-7cbd-4a78-88e8-9a629a5798c5","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"rgb(0, 0, 0)"}}],"text":"High-MDK tumors in clinical samples show elevated VE-cadherin"}]},{"type":"heading","attrs":{"id":"65b27884-304d-45a1-a093-8dff762f2535","data-toc-id":"65b27884-304d-45a1-a093-8dff762f2535","textAlign":"left","level":2},"content":[{"type":"text","text":"5. Budget"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"336a1b0b-a162-48e7-b849-6590207496e9","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Consumable reagents and assay kits: $2,000"}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"8db4a0af-2311-4d0d-a359-31c0d43a55dd","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Animal studies: $2,300"}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"71c8dd25-7bea-4ae3-80fc-50a706615b05","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Imaging and histology: $500"}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"77ee3535-71da-49b1-ad84-0bad9af6786e","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Data analysis software: $200"}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"6115875c-5349-408f-bf1e-c0e2d579837f","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Total: $5,000"}]}]}]},{"type":"heading","attrs":{"id":"1b54423e-33ea-4947-922c-40e17bb3e9b8","data-toc-id":"1b54423e-33ea-4947-922c-40e17bb3e9b8","textAlign":"left","level":2},"content":[{"type":"text","text":"6. 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"},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.1016/s0006-291x(88)80505-9","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.1016/s0006-291x(88)80505-9"}]},{"type":"paragraph","attrs":{"id":"71dbec2d-f902-4ea5-b519-d95a88dc3843","class":"EndNoteBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"5 Lapkina, E. Z., Esimbekova, A. R. & Ruksha, T. G. [Vasculogenic mimicry]. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"Arkh Patol"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"bold"}],"text":"85"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":", 62-69 (2023). "},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.17116/patol20238506162","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.17116/patol20238506162"}]},{"type":"paragraph","attrs":{"id":"8cc627b0-5c9d-4e1f-b216-7241091292d9","class":"EndNoteBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"6 Maeda, N."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":" et al."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"J Biol Chem"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"bold"}],"text":"274"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":", 12474-12479 (1999). "},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.1074/jbc.274.18.12474","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.1074/jbc.274.18.12474"}]},{"type":"paragraph","attrs":{"id":"2f07e13e-4b96-4e15-8a51-f3093d1df30e","class":"EndNoteBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"7 Muramatsu, T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"Proc Jpn Acad Ser B Phys Biol Sci"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"bold"}],"text":"86"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":", 410-425 (2010). "},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.2183/pjab.86.410","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.2183/pjab.86.410"}]},{"type":"paragraph","attrs":{"id":"ca36e920-abe7-4aae-a42f-fd44e3190c53","class":"EndNoteBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"8 Sheng, B., Wei, Z., Wu, X., Li, Y. & Liu, Z. USP12 promotes breast cancer angiogenesis by maintaining midkine stability. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"Cell Death Dis"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"bold"}],"text":"12"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":", 1074 (2021). "},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.1038/s41419-021-04102-y","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.1038/s41419-021-04102-y"}]},{"type":"paragraph","attrs":{"id":"3f53f9b5-59ea-4d30-a534-a8617305ac44","class":"EndNoteBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"9 Shin, D. H."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":" et al."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" Midkine Is a Potential Therapeutic Target of Tumorigenesis, Angiogenesis, and Metastasis in Non-Small Cell Lung Cancer. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"Cancers (Basel)"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"bold"}],"text":"12"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" (2020). "},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.3390/cancers12092402","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.3390/cancers12092402"}]},{"type":"paragraph","attrs":{"id":"aa645d87-8820-4993-b89e-f23b21ce5f83","class":"EndNoteBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"10 Wei, X."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":" et al."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" Mechanisms of vasculogenic mimicry in hypoxic tumor microenvironments. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"Mol Cancer"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"bold"}],"text":"20"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":", 7 (2021). "},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.1186/s12943-020-01288-1","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.1186/s12943-020-01288-1"}]}]}4f:T1db7,Proposal Template 1. Overview Scientific rationale and importance: Vascular mimicry (VM) enables aggressive tumors to form vessel-like structures independent of endothelial cells, contributing to therapy resistance and poor prognosis. My preliminary data indicate that Midkine (MDK), a secreted growth factor stabilized by USP12, promotes VM in triple-negative breast cancer (TNBC) cells. Proteomic analysis identified Vitronectin (VTN) as a potential downstream target of MDK, and high MDK expression correlates with increased VE-cadherin in patient tumors. This project aims to validate VTN as a bona fide mediator of MDK-induced VM, thereby providing a novel therapeutic target. Funding/facilities/ethics approvals: The proposed work will be conducted at Northern Jiangsu People's Hospital, with full access to molecular biology, histology, and imaging facilities. Institutional ethics approval for human tissue use and animal experiments will be secured before study initiation. I obtained my PhD in Cell Biology from Peking Union Medical College in 2021 and subsequently began working as a physician at Northern Jiangsu People's Hospital, one of the largest and most historic hospitals in Jiangsu Province. Our hospital has well-established research facilities and adequate clinical samples to support both basic and translational research. My research background in cancer biology, supported by a strong publication track record, demonstrates my ability to serve as the PI of this proposal. This mini grant will enable me to generate preliminary data to secure a full-scale research grant, establish my own research laboratory, and continue investigating the molecular mechanisms and novel therapeutic targets in breast cancer. To facilitate fund transfer, I have obtained approval from our collaborating organization, One Patient One Cure, a US-based nonprofit, to receive the funding. Estimated timeline: 12 months (September 1, 2025 – August 31, 2026). Data sharing commitment: All raw and processed data, including imaging and proteomics datasets, will be deposited in public repositories (e.g., GEO, PRIDE) within six months of publication. 2. Introduction Background: VM is a key alternative angiogenesis mechanism in breast cancer, particularly under hypoxic stress, driven by EMT and signaling pathways such as Wnt/β-catenin and TGF-β. Current anti-VEGF therapies fail to suppress VM, underscoring the need for alternative targets. MDK is overexpressed in various malignancies and promotes proliferation, EMT, angiogenesis, and therapy resistance. In TNBC, MDK overexpression enhances tube formation and VM marker expression. Our proteomics and immunoprecipitation data suggest that MDK may act through VTN, an extracellular matrix glycoprotein implicated in cell adhesion, migration, and angiogenesis. Research question: Does VTN mediate MDK-driven VM in breast cancer? Hypotheses: H1: MDK promotes VM in breast cancer through upregulation of VTN. H2: Disruption of VTN abrogates MDK-induced VM in vitro and in vivo. Impact statement: Confirmation of the MDK–VTN axis as a driver of VM will open new therapeutic avenues, particularly for TNBC, by targeting VM to improve anti-angiogenic therapy outcomes. 3. Methods Participants/Sample Cell lines: TNBC cell lines such as MDA-MB-231 and BT549 with MDK overexpression or knockdown. Animal models: Orthotopic TNBC xenografts in immunodeficient mice. Clinical samples: Preserved breast cancer tissues. Materials and Procedures Aim 1 – Verify VTN regulation by MDK Western blot and qRT-PCR to measure VTN levels in MDK-overexpressing and knockdown cells compared to controls. Immunoprecipitation to assess MDK–VTN interaction. Aim 2 – Assess functional role of VTN in MDK-induced VM In vitro tube formation assays: VTN overexpression in MDK-knockdown cells to assess restoration of the VM phenotype (rescue experiment). In vivo assays: Orthotopic xenografts with manipulated MDK/VTN expression; assess VM structures via PAS staining and VM marker IHC. Aim 3 – Clinical correlation Immunohistochemistry for MDK, VTN, and VE-cadherin in TNBC patient tissues. Statistical correlation of MDK and VTN expression with VM prevalence and patient outcomes. Planned Analyses Statistical tests: Two-tailed t-tests or ANOVA for group comparisons; Pearson/Spearman for correlation; Kaplan–Meier survival curves for prognostic value. Alternative strategy: If VTN overexpression only partially affects MDK-induced VM in vitro, we will screen for cofactors via proteomics. Ethics and Data Management Ethics: Institutional Review Board and Animal Care Committee approval will be obtained before patient sample analysis and animal experiments. Confidentiality: All human data will be anonymized before analysis. Data sharing: Raw data will be deposited in GEO/PRIDE; protocols will be made publicly available (e.g., protocols.io). 4. Pilot Data (Optional) MDK overexpression in MDA-MB-231 cells increases tubular structure formation. Proteomics identified VTN as significantly upregulated in MDK-overexpressing cells. High-MDK tumors in clinical samples show elevated VE-cadherin 5. Budget Consumable reagents and assay kits: $2,000 Animal studies: $2,300 Imaging and histology: $500 Data analysis software: $200 Total: $5,000 6. References 1 Cerezo-Wallis, D. et al. Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state. Nat Med 26, 1865-1877 (2020). https://doi.org/10.1038/s41591-020-1073-3 2 Christou, C., Stylianou, A. & Gkretsi, V. Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker. Cells 13 (2024). https://doi.org/10.3390/cells13020136 3 Han, B. et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent 4, 47-53 (2024). https://doi.org/10.1016/j.jncc.2024.01.006 4 Kadomatsu, K., Tomomura, M. & Muramatsu, T. cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun 151, 1312-1318 (1988). https://doi.org/10.1016/s0006-291x(88)80505-9 5 Lapkina, E. Z., Esimbekova, A. R. & Ruksha, T. G. [Vasculogenic mimicry]. Arkh Patol 85, 62-69 (2023). https://doi.org/10.17116/patol20238506162 6 Maeda, N. et al. A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta. J Biol Chem 274, 12474-12479 (1999). https://doi.org/10.1074/jbc.274.18.12474 7 Muramatsu, T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proc Jpn Acad Ser B Phys Biol Sci 86, 410-425 (2010). https://doi.org/10.2183/pjab.86.410 8 Sheng, B., Wei, Z., Wu, X., Li, Y. & Liu, Z. USP12 promotes breast cancer angiogenesis by maintaining midkine stability. Cell Death Dis 12, 1074 (2021). https://doi.org/10.1038/s41419-021-04102-y 9 Shin, D. H. et al. Midkine Is a Potential Therapeutic Target of Tumorigenesis, Angiogenesis, and Metastasis in Non-Small Cell Lung Cancer. Cancers (Basel) 12 (2020). https://doi.org/10.3390/cancers12092402 10 Wei, X. et al. Mechanisms of vasculogenic mimicry in hypoxic tumor microenvironments. Mol Cancer20, 7 (2021). https://doi.org/10.1186/s12943-020-01288-150:T8612,{"type":"doc","content":[{"type":"heading","attrs":{"id":"21bd51b8-6618-454c-81fd-a25b3218d109","data-toc-id":"21bd51b8-6618-454c-81fd-a25b3218d109","textAlign":"left","level":1},"content":[{"type":"text","text":"Proposal Template"}]},{"type":"heading","attrs":{"id":"dc31eeb1-4c24-40ea-b9ac-e3b63b3fb2cd","data-toc-id":"dc31eeb1-4c24-40ea-b9ac-e3b63b3fb2cd","textAlign":"left","level":2},"content":[{"type":"text","text":"1. Overview"}]},{"type":"paragraph","attrs":{"id":"407c90b7-84fd-436b-a888-2a8f5fe75d70","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Scientific rationale and importance:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Vascular mimicry (VM) enables aggressive tumors to form vessel-like structures independent of endothelial cells, contributing to therapy resistance and poor prognosis. My preliminary data indicate that Midkine (MDK), a secreted growth factor stabilized by USP12, promotes VM in triple-negative breast cancer (TNBC) cells. Proteomic analysis identified Vitronectin (VTN) as a potential downstream target of MDK, and high MDK expression correlates with increased VE-cadherin in patient tumors. This project aims to validate VTN as a bona fide mediator of MDK-induced VM, thereby providing a novel therapeutic target."}]},{"type":"paragraph","attrs":{"id":"ff7c073f-efc4-44e6-8bf4-8b5430829a23","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Funding/facilities/ethics approvals:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"The proposed work will be conducted at Northern Jiangsu People's Hospital, with full access to molecular biology, histology, and imaging facilities. Institutional ethics approval for human tissue use and animal experiments will be secured before study initiation."}]},{"type":"paragraph","attrs":{"id":"6765bbaa-aa6b-43e1-9ba7-2c9656dfa6e1","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"I obtained my PhD in Cell Biology from Peking Union Medical College in 2021 and subsequently began working as a physician at Northern Jiangsu People's Hospital, one of the largest and most historic hospitals in Jiangsu Province. Our hospital has well-established research facilities and adequate clinical samples to support both basic and translational research. My research background in cancer biology, supported by a strong publication track record, demonstrates my ability to serve as the PI of this proposal."}]},{"type":"paragraph","attrs":{"id":"22646ac7-6fdf-4f13-8b9e-53c8e947f8c7","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"This mini grant will enable me to generate preliminary data to secure a full-scale research grant, establish my own research laboratory, and continue investigating the molecular mechanisms and novel therapeutic targets in breast cancer. To facilitate fund transfer, I have obtained approval from our collaborating organization, One Patient One Cure, a US-based nonprofit, to receive the funding."}]},{"type":"paragraph","attrs":{"id":"c03e88ff-0d47-4d12-8d26-fb6ea29dc3b5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Estimated timeline:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"12 months (September 1, 2025 – August 31, 2026)."}]},{"type":"paragraph","attrs":{"id":"d8eb9d94-fabb-4dd5-8aa2-268a48123b0c","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Data sharing commitment:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"All raw and processed data, including imaging and proteomics datasets, will be deposited in public repositories (e.g., GEO, PRIDE) within six months of publication."}]},{"type":"heading","attrs":{"id":"25605e66-40a8-4d28-8e0c-f54f40b938bf","data-toc-id":"25605e66-40a8-4d28-8e0c-f54f40b938bf","textAlign":"left","level":2},"content":[{"type":"text","text":"2. Introduction"}]},{"type":"paragraph","attrs":{"id":"6aa560f7-3804-43f0-8ba9-f1ffa04c9119","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Background:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"VM is a key alternative angiogenesis mechanism in breast cancer, particularly under hypoxic stress, driven by EMT and signaling pathways such as Wnt/β-catenin and TGF-β. Current anti-VEGF therapies fail to suppress VM, underscoring the need for alternative targets."}]},{"type":"paragraph","attrs":{"id":"d1fb735b-96e6-4213-9853-dabe6ce0cb41","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"MDK is overexpressed in various malignancies and promotes proliferation, EMT, angiogenesis, and therapy resistance. In TNBC, MDK overexpression enhances tube formation and VM marker expression. Our proteomics and immunoprecipitation data suggest that MDK may act through VTN, an extracellular matrix glycoprotein implicated in cell adhesion, migration, and angiogenesis."}]},{"type":"paragraph","attrs":{"id":"ec60dcad-9897-4314-b88d-55b7c9f62135","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Research question:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Does VTN mediate MDK-driven VM in breast cancer?"}]},{"type":"paragraph","attrs":{"id":"78dbeb6a-19d2-4380-bf78-4da30dda99a1","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Hypotheses:"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"628a41ae-d348-4c1d-bc67-2c753dd9cf1c","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"H1: MDK promotes VM in breast cancer through upregulation of VTN."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"88c6bc49-ef44-4c5b-aa7e-6c933cce5fff","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"H2: Disruption of VTN abrogates MDK-induced VM in vitro and in vivo."}]}]}]},{"type":"paragraph","attrs":{"id":"8a87fc79-c32a-45fb-b115-c6d0fc0b1f2b","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Impact statement:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Confirmation of the MDK–VTN axis as a driver of VM will open new therapeutic avenues, particularly for TNBC, by targeting VM to improve anti-angiogenic therapy outcomes."}]},{"type":"heading","attrs":{"id":"ca433291-7526-4f84-b27a-b0b46eee9d4e","data-toc-id":"ca433291-7526-4f84-b27a-b0b46eee9d4e","textAlign":"left","level":2},"content":[{"type":"text","text":"3. Methods"}]},{"type":"heading","attrs":{"id":"ed97f88e-b5c2-4a35-b550-65dcfafed1df","data-toc-id":"ed97f88e-b5c2-4a35-b550-65dcfafed1df","textAlign":"left","level":3},"content":[{"type":"text","text":"Participants/Sample"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"a5c0bbf5-b780-4ada-a5b4-8650d83a82b6","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Cell lines: TNBC cell lines such as MDA-MB-231 and BT549 with MDK overexpression or knockdown."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"dd3b5082-329c-4389-a454-0bdcaa5afd31","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Animal models: Orthotopic TNBC xenografts in immunodeficient mice."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"8b051e0f-0900-4fa2-b92e-29968548258f","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Clinical samples: Preserved breast cancer tissues."}]}]}]},{"type":"heading","attrs":{"id":"8817c620-3f60-4956-bda5-beea4a777f97","data-toc-id":"8817c620-3f60-4956-bda5-beea4a777f97","textAlign":"left","level":3},"content":[{"type":"text","text":"Materials and Procedures"}]},{"type":"paragraph","attrs":{"id":"01775a43-8a1e-43ff-8bef-e519d266ffbd","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Aim 1 – Verify VTN regulation by MDK"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"bfa9350b-86ec-4507-b43a-92ab14f739ac","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Western blot and qRT-PCR to measure VTN levels in MDK-overexpressing and knockdown cells compared to controls."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"eb550025-f215-48fe-b1e5-13f708f759b0","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Immunoprecipitation to assess MDK–VTN interaction."}]}]}]},{"type":"paragraph","attrs":{"id":"c2f61a8a-a433-478c-a5bd-39730af170dc","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Aim 2 – Assess functional role of VTN in MDK-induced VM"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"bc29722b-0939-4234-8b77-850fbed72285","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"In vitro tube formation assays:"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" VTN overexpression in MDK-knockdown cells to assess restoration of the VM phenotype (rescue experiment)."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"ec76c5f7-a760-455c-989e-b8e0b05c6c98","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"In vivo assays:"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" Orthotopic xenografts with manipulated MDK/VTN expression; assess VM structures via PAS staining and VM marker IHC."}]}]}]},{"type":"paragraph","attrs":{"id":"444174c7-e5ee-4b27-a81b-a01eb8f8e0a5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Aim 3 – Clinical correlation"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"5700b1fc-57ea-4850-a04e-0e9c345a74f4","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Immunohistochemistry for MDK, VTN, and VE-cadherin in TNBC patient tissues."}]}]}]},{"type":"paragraph","attrs":{"id":"3b82e61e-436e-4d4a-a4cc-8ff7eb82c73d","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"rgb(0, 0, 0)"}}],"text":"Statistical correlation of MDK and VTN expression with VM prevalence and patient outcomes."}]},{"type":"heading","attrs":{"id":"49dee47f-4a5d-4479-8380-1c769df45c26","data-toc-id":"49dee47f-4a5d-4479-8380-1c769df45c26","textAlign":"left","level":3},"content":[{"type":"text","text":"Planned Analyses"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"dca64fa2-a7f2-454a-907d-34745b36e07a","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Statistical tests: Two-tailed t-tests or ANOVA for group comparisons; Pearson/Spearman for correlation; Kaplan–Meier survival curves for prognostic value."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"98bf208a-fca8-4d7c-a433-d847efa13a6c","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Alternative strategy: If VTN overexpression only partially affects MDK-induced VM in vitro, we will screen for cofactors via proteomics."}]}]}]},{"type":"heading","attrs":{"id":"ed994363-1076-4c56-bb45-46b55a5130e2","data-toc-id":"ed994363-1076-4c56-bb45-46b55a5130e2","textAlign":"left","level":3},"content":[{"type":"text","text":"Ethics and Data Management"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"280c3b63-119b-4e73-b685-f536ee1bf4d6","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Ethics: Institutional Review Board and Animal Care Committee approval will be obtained before patient sample analysis and animal experiments."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"5189580c-e0fc-4cc9-98ec-a423692620d5","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Confidentiality: All human data will be anonymized before analysis."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"d8781ea1-6d95-4194-9eb2-d84801f2c4b9","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Data sharing: Raw data will be deposited in GEO/PRIDE; protocols will be made publicly available (e.g., "},{"type":"text","marks":[{"type":"link","attrs":{"href":"http://protocols.io","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"protocols.io"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":")."}]}]}]},{"type":"heading","attrs":{"id":"05f07119-09c5-41c7-8c08-45ee134a0e9c","data-toc-id":"05f07119-09c5-41c7-8c08-45ee134a0e9c","textAlign":"left","level":2},"content":[{"type":"text","text":"4. Pilot Data (Optional)"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"6f8ed375-1901-4a53-a8ec-3b77d93e36e5","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"MDK overexpression in MDA-MB-231 cells increases tubular structure formation."}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"7bdec09e-b999-47e7-935a-e578a81510f1","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Proteomics identified VTN as significantly upregulated in MDK-overexpressing cells."}]}]}]},{"type":"paragraph","attrs":{"id":"da71c5dd-7cbd-4a78-88e8-9a629a5798c5","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"rgb(0, 0, 0)"}}],"text":"High-MDK tumors in clinical samples show elevated VE-cadherin"}]},{"type":"heading","attrs":{"id":"65b27884-304d-45a1-a093-8dff762f2535","data-toc-id":"65b27884-304d-45a1-a093-8dff762f2535","textAlign":"left","level":2},"content":[{"type":"text","text":"5. Budget"}]},{"type":"bulletList","content":[{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"336a1b0b-a162-48e7-b849-6590207496e9","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Consumable reagents and assay kits: $2,000"}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"8db4a0af-2311-4d0d-a359-31c0d43a55dd","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Animal studies: $2,300"}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"71c8dd25-7bea-4ae3-80fc-50a706615b05","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Imaging and histology: $500"}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"77ee3535-71da-49b1-ad84-0bad9af6786e","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Data analysis software: $200"}]}]},{"type":"listItem","content":[{"type":"paragraph","attrs":{"id":"6115875c-5349-408f-bf1e-c0e2d579837f","class":null,"textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"Total: $5,000"}]}]}]},{"type":"heading","attrs":{"id":"1b54423e-33ea-4947-922c-40e17bb3e9b8","data-toc-id":"1b54423e-33ea-4947-922c-40e17bb3e9b8","textAlign":"left","level":2},"content":[{"type":"text","text":"6. 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"},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.2183/pjab.86.410","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.2183/pjab.86.410"}]},{"type":"paragraph","attrs":{"id":"ca36e920-abe7-4aae-a42f-fd44e3190c53","class":"EndNoteBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"8 Sheng, B., Wei, Z., Wu, X., Li, Y. & Liu, Z. USP12 promotes breast cancer angiogenesis by maintaining midkine stability. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"Cell Death Dis"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"bold"}],"text":"12"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":", 1074 (2021). "},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.1038/s41419-021-04102-y","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.1038/s41419-021-04102-y"}]},{"type":"paragraph","attrs":{"id":"3f53f9b5-59ea-4d30-a534-a8617305ac44","class":"EndNoteBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"9 Shin, D. H."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":" et al."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" Midkine Is a Potential Therapeutic Target of Tumorigenesis, Angiogenesis, and Metastasis in Non-Small Cell Lung Cancer. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"Cancers (Basel)"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"bold"}],"text":"12"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" (2020). "},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.3390/cancers12092402","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.3390/cancers12092402"}]},{"type":"paragraph","attrs":{"id":"aa645d87-8820-4993-b89e-f23b21ce5f83","class":"EndNoteBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":"10 Wei, X."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":" et al."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":" Mechanisms of vasculogenic mimicry in hypoxic tumor microenvironments. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"italic"}],"text":"Mol Cancer"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"bold"}],"text":"20"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}}],"text":", 7 (2021). "},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://doi.org/10.1186/s12943-020-01288-1","target":"_blank","rel":"noopener noreferrer nofollow","class":"link"}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":""}},{"type":"underline"}],"text":"https://doi.org/10.1186/s12943-020-01288-1"}]}]}51:T49c1,

Role and Mechanism of MDK in Promoting Vascular Mimicry in Breast Cancer

1. Overview

Scientific rationale and importance:
Vascular mimicry (VM) enables aggressive tumors to form vessel-like structures independent of endothelial cells, contributing to therapy resistance and poor prognosis. My preliminary data indicate that Midkine (MDK), a secreted growth factor stabilized by USP12, promotes VM in triple-negative breast cancer (TNBC) cells. Proteomic analysis identified Vitronectin (VTN) as a potential downstream target of MDK, and high MDK expression correlates with increased VE-cadherin in patient tumors. This project aims to validate VTN as a bona fide mediator of MDK-induced VM, thereby providing a novel therapeutic target.

Funding/facilities/ethics approvals:
The proposed work will be conducted at Northern Jiangsu People's Hospital, with full access to molecular biology, histology, and imaging facilities. Institutional ethics approval for human tissue use and animal experiments will be secured before study initiation.

I obtained my PhD in Cell Biology from Peking Union Medical College in 2021 and subsequently began working as a physician at Northern Jiangsu People's Hospital, one of the largest and most historic hospitals in Jiangsu Province. Our hospital has well-established research facilities and adequate clinical samples to support both basic and translational research. My research background in cancer biology, supported by a strong publication track record, demonstrates my ability to serve as the PI of this proposal.

This mini grant will enable me to generate preliminary data to secure a full-scale research grant, establish my own research laboratory, and continue investigating the molecular mechanisms and novel therapeutic targets in breast cancer. To facilitate fund transfer, I have obtained approval from our collaborating organization, One Patient One Cure, a US-based nonprofit, to receive the funding.

Estimated timeline:
12 months (September 1, 2025 – August 31, 2026).

Data sharing commitment:
All raw and processed data, including imaging and proteomics datasets, will be deposited in public repositories (e.g., GEO, PRIDE) within six months of publication.

2. Introduction

Background:
VM is a key alternative angiogenesis mechanism in breast cancer, particularly under hypoxic stress, driven by EMT and signaling pathways such as Wnt/β-catenin and TGF-β. Current anti-VEGF therapies fail to suppress VM, underscoring the need for alternative targets.

MDK is overexpressed in various malignancies and promotes proliferation, EMT, angiogenesis, and therapy resistance. In TNBC, MDK overexpression enhances tube formation and VM marker expression. Our proteomics and immunoprecipitation data suggest that MDK may act through VTN, an extracellular matrix glycoprotein implicated in cell adhesion, migration, and angiogenesis.

Research question:
Does VTN mediate MDK-driven VM in breast cancer?

Hypotheses:

H1: MDK promotes VM in breast cancer through upregulation of VTN.
H2: Disruption of VTN abrogates MDK-induced VM in vitro and in vivo.

Impact statement:
Confirmation of the MDK–VTN axis as a driver of VM will open new therapeutic avenues, particularly for TNBC, by targeting VM to improve anti-angiogenic therapy outcomes.

3. Methods

Participants/Sample

Cell lines: TNBC cell lines such as MDA-MB-231 and BT549 with MDK overexpression or knockdown.
Animal models: Orthotopic TNBC xenografts in immunodeficient mice.
Clinical samples: Preserved breast cancer tissues.

Materials and Procedures

Aim 1 – Verify VTN regulation by MDK

Western blot and qRT-PCR to measure VTN levels in MDK-overexpressing and knockdown cells compared to controls.
Immunoprecipitation to assess MDK–VTN interaction.

Aim 2 – Assess functional role of VTN in MDK-induced VM

In vitro tube formation assays: VTN overexpression in MDK-knockdown cells to assess restoration of the VM phenotype (rescue experiment).
In vivo assays: Orthotopic xenografts with manipulated MDK/VTN expression; assess VM structures via PAS staining and VM marker IHC.

Aim 3 – Clinical correlation

Immunohistochemistry for MDK, VTN, and VE-cadherin in TNBC patient tissues.

Statistical correlation of MDK and VTN expression with VM prevalence and patient outcomes.

Planned Analyses

Statistical tests: Two-tailed t-tests or ANOVA for group comparisons; Pearson/Spearman for correlation; Kaplan–Meier survival curves for prognostic value.
Alternative strategy: If VTN overexpression only partially affects MDK-induced VM in vitro, we will screen for cofactors via proteomics.

Ethics and Data Management

Ethics: Institutional Review Board and Animal Care Committee approval will be obtained before patient sample analysis and animal experiments.
Confidentiality: All human data will be anonymized before analysis.
Data sharing: Raw data will be deposited in GEO/PRIDE; protocols will be made publicly available (e.g., protocols.io).

4. Pilot Data (Optional)

MDK overexpression in MDA-MB-231 cells increases tubular structure formation.
Proteomics identified VTN as significantly upregulated in MDK-overexpressing cells.

High-MDK tumors in clinical samples show elevated VE-cadherin

5. Budget

Consumable reagents and assay kits: $2,000
Animal studies: $2,300
Imaging and histology: $500
Data analysis software: $200
Total: $5,000

6. References

1 Cerezo-Wallis, D. et al. Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state. Nat Med 26, 1865-1877 (2020). https://doi.org/10.1038/s41591-020-1073-3

2 Christou, C., Stylianou, A. & Gkretsi, V. Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker. Cells 13 (2024). https://doi.org/10.3390/cells13020136

3 Han, B. et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent 4, 47-53 (2024). https://doi.org/10.1016/j.jncc.2024.01.006

4 Kadomatsu, K., Tomomura, M. & Muramatsu, T. cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun 151, 1312-1318 (1988). https://doi.org/10.1016/s0006-291x(88)80505-9

5 Lapkina, E. Z., Esimbekova, A. R. & Ruksha, T. G. [Vasculogenic mimicry]. Arkh Patol 85, 62-69 (2023). https://doi.org/10.17116/patol20238506162

6 Maeda, N. et al. A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta. J Biol Chem 274, 12474-12479 (1999). https://doi.org/10.1074/jbc.274.18.12474

7 Muramatsu, T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proc Jpn Acad Ser B Phys Biol Sci 86, 410-425 (2010). https://doi.org/10.2183/pjab.86.410

8 Sheng, B., Wei, Z., Wu, X., Li, Y. & Liu, Z. USP12 promotes breast cancer angiogenesis by maintaining midkine stability. Cell Death Dis 12, 1074 (2021). https://doi.org/10.1038/s41419-021-04102-y

9 Shin, D. H. et al. Midkine Is a Potential Therapeutic Target of Tumorigenesis, Angiogenesis, and Metastasis in Non-Small Cell Lung Cancer. Cancers (Basel) 12 (2020). https://doi.org/10.3390/cancers12092402

10 Wei, X. et al. Mechanisms of vasculogenic mimicry in hypoxic tumor microenvironments. Mol Cancer20, 7 (2021). https://doi.org/10.1186/s12943-020-01288-1

52:T49c1,

Role and Mechanism of MDK in Promoting Vascular Mimicry in Breast Cancer

1. Overview

Estimated timeline:
12 months (September 1, 2025 – August 31, 2026).

Data sharing commitment:
All raw and processed data, including imaging and proteomics datasets, will be deposited in public repositories (e.g., GEO, PRIDE) within six months of publication.

2. Introduction

Research question:
Does VTN mediate MDK-driven VM in breast cancer?

Hypotheses:

H1: MDK promotes VM in breast cancer through upregulation of VTN.
H2: Disruption of VTN abrogates MDK-induced VM in vitro and in vivo.

Impact statement:
Confirmation of the MDK–VTN axis as a driver of VM will open new therapeutic avenues, particularly for TNBC, by targeting VM to improve anti-angiogenic therapy outcomes.

3. Methods

Participants/Sample

Cell lines: TNBC cell lines such as MDA-MB-231 and BT549 with MDK overexpression or knockdown.
Animal models: Orthotopic TNBC xenografts in immunodeficient mice.
Clinical samples: Preserved breast cancer tissues.

Materials and Procedures

Aim 1 – Verify VTN regulation by MDK

Western blot and qRT-PCR to measure VTN levels in MDK-overexpressing and knockdown cells compared to controls.
Immunoprecipitation to assess MDK–VTN interaction.

Aim 2 – Assess functional role of VTN in MDK-induced VM

In vitro tube formation assays: VTN overexpression in MDK-knockdown cells to assess restoration of the VM phenotype (rescue experiment).
In vivo assays: Orthotopic xenografts with manipulated MDK/VTN expression; assess VM structures via PAS staining and VM marker IHC.

Aim 3 – Clinical correlation

Immunohistochemistry for MDK, VTN, and VE-cadherin in TNBC patient tissues.

Statistical correlation of MDK and VTN expression with VM prevalence and patient outcomes.

Planned Analyses

Statistical tests: Two-tailed t-tests or ANOVA for group comparisons; Pearson/Spearman for correlation; Kaplan–Meier survival curves for prognostic value.
Alternative strategy: If VTN overexpression only partially affects MDK-induced VM in vitro, we will screen for cofactors via proteomics.

Ethics and Data Management

Ethics: Institutional Review Board and Animal Care Committee approval will be obtained before patient sample analysis and animal experiments.
Confidentiality: All human data will be anonymized before analysis.
Data sharing: Raw data will be deposited in GEO/PRIDE; protocols will be made publicly available (e.g., protocols.io).

4. Pilot Data (Optional)

MDK overexpression in MDA-MB-231 cells increases tubular structure formation.
Proteomics identified VTN as significantly upregulated in MDK-overexpressing cells.

High-MDK tumors in clinical samples show elevated VE-cadherin

5. Budget

Consumable reagents and assay kits: $2,000
Animal studies: $2,300
Imaging and histology: $500
Data analysis software: $200
Total: $5,000

6. References

1 Cerezo-Wallis, D. et al. Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state. Nat Med 26, 1865-1877 (2020). https://doi.org/10.1038/s41591-020-1073-3

2 Christou, C., Stylianou, A. & Gkretsi, V. Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker. Cells 13 (2024). https://doi.org/10.3390/cells13020136

3 Han, B. et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent 4, 47-53 (2024). https://doi.org/10.1016/j.jncc.2024.01.006

5 Lapkina, E. Z., Esimbekova, A. R. & Ruksha, T. G. [Vasculogenic mimicry]. Arkh Patol 85, 62-69 (2023). https://doi.org/10.17116/patol20238506162

8 Sheng, B., Wei, Z., Wu, X., Li, Y. & Liu, Z. USP12 promotes breast cancer angiogenesis by maintaining midkine stability. Cell Death Dis 12, 1074 (2021). https://doi.org/10.1038/s41419-021-04102-y

10 Wei, X. et al. Mechanisms of vasculogenic mimicry in hypoxic tumor microenvironments. Mol Cancer20, 7 (2021). https://doi.org/10.1186/s12943-020-01288-1

53:T4db,This proposal investigates the role of Midkine (MDK) and Vitronectin (VTN) in promoting vascular mimicry (VM) in triple-negative breast cancer. The scientific rationale is sound and grounded in established VM biology; the MDK-VTN mechanistic hypothesis is novel and biologically plausible, supported by preliminary proteomics and immunoprecipitation data. The experimental design is methodologically appropriate, combining in vitro rescue experiments, in vivo orthotopic xenografts, and clinical tissue correlation. However, the proposal has significant limitations: the investigator's publication record is minimal (h-index 0/0 per ResearchHub profile), creating concerns about research independence and execution capacity; the statistical analysis plan lacks power calculations and detailed specifications; the 12-month timeline is ambitious for the scope of work (three aims spanning molecular, functional, and clinical validation); and the budget ($5,000) appears insufficient for the proposed animal studies and comprehensive analyses. The proposal is well-written and addresses an important therapeutic gap, but feasibility concerns and the junior investigator status warrant careful scrutiny of team support and institutional resources.54:T1dee,Role and Mechanism of MDK in Promoting Vascular Mimicry in Breast Cancer 1. Overview Scientific rationale and importance: Vascular mimicry (VM) enables aggressive tumors to form vessel-like structures independent of endothelial cells, contributing to therapy resistance and poor prognosis. My preliminary data indicate that Midkine (MDK), a secreted growth factor stabilized by USP12, promotes VM in triple-negative breast cancer (TNBC) cells. Proteomic analysis identified Vitronectin (VTN) as a potential downstream target of MDK, and high MDK expression correlates with increased VE-cadherin in patient tumors. This project aims to validate VTN as a bona fide mediator of MDK-induced VM, thereby providing a novel therapeutic target. Funding/facilities/ethics approvals: The proposed work will be conducted at Northern Jiangsu People's Hospital, with full access to molecular biology, histology, and imaging facilities. Institutional ethics approval for human tissue use and animal experiments will be secured before study initiation. I obtained my PhD in Cell Biology from Peking Union Medical College in 2021 and subsequently began working as a physician at Northern Jiangsu People's Hospital, one of the largest and most historic hospitals in Jiangsu Province. Our hospital has well-established research facilities and adequate clinical samples to support both basic and translational research. My research background in cancer biology, supported by a strong publication track record, demonstrates my ability to serve as the PI of this proposal. This mini grant will enable me to generate preliminary data to secure a full-scale research grant, establish my own research laboratory, and continue investigating the molecular mechanisms and novel therapeutic targets in breast cancer. To facilitate fund transfer, I have obtained approval from our collaborating organization, One Patient One Cure, a US-based nonprofit, to receive the funding. Estimated timeline: 12 months (September 1, 2025 – August 31, 2026). Data sharing commitment: All raw and processed data, including imaging and proteomics datasets, will be deposited in public repositories (e.g., GEO, PRIDE) within six months of publication. 2. Introduction Background: VM is a key alternative angiogenesis mechanism in breast cancer, particularly under hypoxic stress, driven by EMT and signaling pathways such as Wnt/β-catenin and TGF-β. Current anti-VEGF therapies fail to suppress VM, underscoring the need for alternative targets. MDK is overexpressed in various malignancies and promotes proliferation, EMT, angiogenesis, and therapy resistance. In TNBC, MDK overexpression enhances tube formation and VM marker expression. Our proteomics and immunoprecipitation data suggest that MDK may act through VTN, an extracellular matrix glycoprotein implicated in cell adhesion, migration, and angiogenesis. Research question: Does VTN mediate MDK-driven VM in breast cancer? Hypotheses: H1: MDK promotes VM in breast cancer through upregulation of VTN. H2: Disruption of VTN abrogates MDK-induced VM in vitro and in vivo. Impact statement: Confirmation of the MDK–VTN axis as a driver of VM will open new therapeutic avenues, particularly for TNBC, by targeting VM to improve anti-angiogenic therapy outcomes. 3. Methods Participants/Sample Cell lines: TNBC cell lines such as MDA-MB-231 and BT549 with MDK overexpression or knockdown. Animal models: Orthotopic TNBC xenografts in immunodeficient mice. Clinical samples: Preserved breast cancer tissues. Materials and Procedures Aim 1 – Verify VTN regulation by MDK Western blot and qRT-PCR to measure VTN levels in MDK-overexpressing and knockdown cells compared to controls. Immunoprecipitation to assess MDK–VTN interaction. Aim 2 – Assess functional role of VTN in MDK-induced VM In vitro tube formation assays: VTN overexpression in MDK-knockdown cells to assess restoration of the VM phenotype (rescue experiment). In vivo assays: Orthotopic xenografts with manipulated MDK/VTN expression; assess VM structures via PAS staining and VM marker IHC. Aim 3 – Clinical correlation Immunohistochemistry for MDK, VTN, and VE-cadherin in TNBC patient tissues. Statistical correlation of MDK and VTN expression with VM prevalence and patient outcomes. Planned Analyses Statistical tests: Two-tailed t-tests or ANOVA for group comparisons; Pearson/Spearman for correlation; Kaplan–Meier survival curves for prognostic value. Alternative strategy: If VTN overexpression only partially affects MDK-induced VM in vitro, we will screen for cofactors via proteomics. Ethics and Data Management Ethics: Institutional Review Board and Animal Care Committee approval will be obtained before patient sample analysis and animal experiments. Confidentiality: All human data will be anonymized before analysis. Data sharing: Raw data will be deposited in GEO/PRIDE; protocols will be made publicly available (e.g., protocols.io). 4. Pilot Data (Optional) MDK overexpression in MDA-MB-231 cells increases tubular structure formation. Proteomics identified VTN as significantly upregulated in MDK-overexpressing cells. High-MDK tumors in clinical samples show elevated VE-cadherin 5. Budget Consumable reagents and assay kits: $2,000 Animal studies: $2,300 Imaging and histology: $500 Data analysis software: $200 Total: $5,000 6. References 1 Cerezo-Wallis, D. et al. Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state. Nat Med 26, 1865-1877 (2020). https://doi.org/10.1038/s41591-020-1073-3 2 Christou, C., Stylianou, A. & Gkretsi, V. Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker. Cells 13 (2024). https://doi.org/10.3390/cells13020136 3 Han, B. et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent 4, 47-53 (2024). https://doi.org/10.1016/j.jncc.2024.01.006 4 Kadomatsu, K., Tomomura, M. & Muramatsu, T. cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun 151, 1312-1318 (1988). https://doi.org/10.1016/s0006-291x(88)80505-9 5 Lapkina, E. Z., Esimbekova, A. R. & Ruksha, T. G. [Vasculogenic mimicry]. Arkh Patol 85, 62-69 (2023). https://doi.org/10.17116/patol20238506162 6 Maeda, N. et al. A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta. J Biol Chem 274, 12474-12479 (1999). https://doi.org/10.1074/jbc.274.18.12474 7 Muramatsu, T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proc Jpn Acad Ser B Phys Biol Sci 86, 410-425 (2010). https://doi.org/10.2183/pjab.86.410 8 Sheng, B., Wei, Z., Wu, X., Li, Y. & Liu, Z. USP12 promotes breast cancer angiogenesis by maintaining midkine stability. Cell Death Dis 12, 1074 (2021). https://doi.org/10.1038/s41419-021-04102-y 9 Shin, D. H. et al. Midkine Is a Potential Therapeutic Target of Tumorigenesis, Angiogenesis, and Metastasis in Non-Small Cell Lung Cancer. Cancers (Basel) 12 (2020). https://doi.org/10.3390/cancers12092402 10 Wei, X. et al. Mechanisms of vasculogenic mimicry in hypoxic tumor microenvironments. 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My main research direction is the molecular mechanism of breast cancer metastasis.","h_index":0,"i10_index":0,"profile_image":null,"author_score":0,"university":null,"orcid_id":null,"openalex_ids":[],"education":[{"id":5202,"name":"Peking University","year":{"label":"2021","value":"2021"},"major":"","degree":{"label":"Doctor of Medicine","value":"MD"},"summary":"MD '21, Peking University","is_public":false,"university":{"id":5202,"city":"","name":"Peking University","state":"","country":"China","updated_date":"2020-11-24T21:44:27.612191Z"}}],"headline":"Researcher at Subei Hospital of Jiangsu Province","facebook":null,"twitter":"","linkedin":"","google_scholar":"","claimed":true,"country_code":null,"created_source":"RESEARCHHUB","last_full_fetch_from_openalex":null,"two_year_mean_citedness":0,"merged_with_author":null,"added_as_editor_date":null,"is_hub_editor_of":[],"is_hub_editor":false,"num_posts":1,"is_orcid_connected":false,"reputation":100,"suspended_status":{"probable_spammer":false,"is_suspended":false},"total_score":null,"wallet":null,"is_verified":true},"balance":null,"balances":null,"created_date":"2025-08-14T02:34:23.135564Z","has_seen_first_coin_modal":false,"has_seen_orcid_connect_modal":false,"is_funder":false,"is_staking_opted_in":true,"is_suspended":false,"probable_spammer":false,"is_verified":true,"moderator":false,"reputation":100,"updated_date":"2025-09-09T03:09:01.611695Z","upload_tutorial_complete":false,"hub_rep":null,"time_rep":null},"created_date":"2025-08-14T15:05:20.250741Z","discussion_count":2,"document_type":"PREREGISTRATION","doi":"10.55277/researchhub.t3knw0hm","editor_type":"CK_EDITOR","full_markdown":"$52","grants":[{"id":3,"short_title":"Cancer Biology","status":"OPEN","organization":"ResearchHub Foundation","amount":"5000.00","currency":"USD","post_id":4222,"image_url":"https://storage.prod.researchhub.com/uploads/posts/users/39602/4ef11956-d243-48c9-bda2-ae1a859aed8b/chatgpt-image-jul-21-2025-12_30_33-pm.png","title":"Request for Proposals - Cancer Biology Research Project","applicant_count":5,"application_visibility":"OPTIONAL","proposal":{"unified_document_id":7555781,"ai_peer_review":{"id":59,"unified_document_id":7555781,"grant_id":3,"created_by_id":39602,"status":"completed","overall_rating":"good","overall_rationale":"The proposal presents a scientifically sound and novel hypothesis addressing an unmet clinical need (VM-driven therapy resistance in TNBC). The experimental design is rigorous and aligned with current breast-cancer VM literature. However, critical gaps in statistical rigor (no power analysis, limited analysis detail), feasibility concerns (tight timeline, minimal budget, junior investigator with no prior publications), and incomplete specification of team support and institutional capacity prevent a rating of 'excellent'. The work is fundable as a pilot/exploratory grant for a junior researcher, but execution risk is moderate to high.","overall_score_numeric":2,"result_data":{"categories":{"methods_rigor":{"items":{"methods_detail":{"decision":"Partial","justification":"The proposal describes the general approach (Western blot, qRT-PCR, IP, tube formation, xenografts, IHC) but lacks critical details: no specification of transfection/transduction methods, no quantification thresholds for tube formation or VM structures, no details on PAS-staining scoring, and no description of imaging parameters or software used for analysis."},"controls_defined":{"decision":"Partial","justification":"The proposal mentions 'controls' (e.g., 'compared to controls' in Aim 1) but does not explicitly define them. For Aim 1, it is unclear whether controls are wild-type cells, empty-vector cells, or scrambled-shRNA cells. For Aim 2, the rescue experiment design is clear (VTN overexpression in MDK-knockdown cells), but negative controls (e.g., VTN overexpression alone) are not mentioned. For Aim 3, no control tissues are specified."},"parameters_specified":{"decision":"Partial","justification":"Key parameters are missing: cell-line passage numbers, MDK overexpression/knockdown efficiency targets, VTN rescue dosages, animal numbers per group, tumor-growth endpoints, and imaging resolution/settings are not specified. The proposal mentions 'orthotopic xenografts' but does not state injection site, cell number, or monitoring frequency."},"model_choice_justified":{"decision":"Yes","justification":"The choice of MDA-MB-231 and BT549 TNBC cell lines is well-justified (both are standard models for VM studies in breast cancer). The use of orthotopic xenografts in immunodeficient mice is appropriate for assessing in vivo VM. The proposal does not justify why immunodeficient (rather than immunocompetent) mice are used, but this is a reasonable choice for a pilot study focused on cell-intrinsic VM mechanisms."},"outcomes_linked_to_aims":{"decision":"Yes","justification":"Each aim has clearly linked outcomes: Aim 1 measures VTN upregulation and MDK-VTN interaction; Aim 2 assesses tube formation and VM structures; Aim 3 correlates MDK/VTN/VE-cadherin expression with VM and patient outcomes. All outcomes directly test the stated hypotheses."}},"score":"Medium","rationale":"The experimental design is methodologically sound and follows best practices in cancer biology (rescue experiments, in vivo validation, clinical correlation). However, critical gaps in specification reduce rigor: sample sizes are not stated, controls are mentioned but not fully detailed, and the alternative strategy (proteomics screening if VTN rescue is partial) is vague. The proposal does not specify how MDK overexpression/knockdown will be achieved (lentiviral, transient transfection, CRISPR), what constitutes a 'positive' tube-formation assay, or how VM structures will be quantified in vivo. These omissions prevent a High rating."},"scientific_impact":{"items":{"generalizability":{"decision":"Partial","justification":"The proposal focuses on TNBC, which is appropriate given the clinical importance of this subtype. However, it does not address whether the MDK-VTN-VM axis operates in other breast-cancer subtypes (luminal, HER2+) or other cancer types. The use of standard cell lines (MDA-MB-231, BT549) and orthotopic xenografts is appropriate for TNBC but limits generalizability. The clinical correlation (Aim 3) is restricted to TNBC tissues, so the findings may not apply broadly."},"opens_new_directions":{"decision":"Yes","justification":"If the MDK-VTN-VM axis is validated, it could open new therapeutic directions: targeting MDK (e.g., with neutralizing antibodies or small-molecule inhibitors), targeting VTN (e.g., with blocking antibodies or integrin antagonists), or combining MDK/VTN inhibition with anti-VEGF therapy to overcome VM-driven resistance. The proposal explicitly states this as an impact goal."},"advances_understanding":{"decision":"Yes","justification":"If validated, the MDK-VTN-VM axis would advance understanding of how secreted growth factors promote VM in breast cancer. This addresses a gap in the literature (VM is known to be driven by EMT and hypoxia, but the role of specific growth factors like MDK is less well characterized). The work would clarify a mechanistic link between MDK (a known pro-tumor factor) and VM (a therapy-resistance mechanism)."}},"score":"High","rationale":"The proposal addresses an important scientific question (the role of MDK-VTN in VM) that could advance understanding of therapy-resistant breast cancer. The MDK-VTN hypothesis is novel and, if validated, would open new therapeutic avenues. However, the scientific impact is limited by the exploratory nature of the work (pilot data only), the lack of mechanistic depth (the proposal does not investigate downstream signaling, transcriptional regulation, or how MDK-VTN-VM relates to other VM drivers like Wnt/beta-catenin or TGF-beta), and the narrow focus on TNBC (generalizability to other breast-cancer subtypes or cancer types is unclear). The proposal does not propose new methods or technologies; it applies standard techniques to a novel hypothesis."},"funding_opportunity_fit":{"items":{"fit_aims":{"decision":"Yes","justification":"The three aims (molecular validation, functional rescue, clinical correlation) directly test the stated hypothesis and are aligned with the goal of identifying novel therapeutic targets in breast cancer."},"fit_scope":{"decision":"Yes","justification":"The scope is appropriate for a $5,000 pilot grant with a 12-month timeline. The three aims are focused and feasible in principle, though execution risk is moderate."},"fit_modality":{"decision":"Yes","justification":"The proposal employs standard cancer-biology methods (cell culture, molecular biology, animal models, clinical tissue analysis) appropriate for a junior researcher pilot grant."},"fit_deliverables":{"decision":"Yes","justification":"The proposal commits to data sharing (GEO/PRIDE), protocol sharing (protocols.io), and publication, which are standard deliverables for a research grant."}},"score":"High","rationale":"The proposal aligns well with the ResearchHub Foundation's stated goal of funding 'a junior researcher in pursuing an innovative cancer biology project.' The MDK-VTN-VM hypothesis is innovative and addresses an important unmet need in breast cancer therapy. The scope is appropriate for a pilot/exploratory grant ($5,000 budget, 12-month timeline). The investigator is clearly junior (no prior publications), and the work is positioned as preliminary data for a larger grant, which matches the stated intent."},"feasibility_and_execution":{"items":{"team_environment":{"decision":"Partial","justification":"The proposal states that the work will be conducted at Northern Jiangsu People's Hospital with 'well-established research facilities,' but does not specify the composition of the research team. No co-investigators, mentors, biostatisticians, or animal-care staff are named. The PI is described as a 'medical laboratory worker' with no prior publications, raising concerns about independent execution. The role of 'One Patient One Cure' (a US-based nonprofit) in fund transfer is mentioned but not explained; it is unclear whether this organization provides scientific oversight or is merely a fiscal intermediary. The proposal does not address how the PI will gain expertise in animal surgery, imaging, or statistical analysis if these are new areas."},"ethics_data_quality":{"decision":"Partial","justification":"The proposal commits to obtaining IRB and Animal Care Committee approval 'before study initiation,' but approval has not yet been obtained. This creates execution risk and potential delays. The proposal commits to anonymizing human data and depositing raw data in public repositories, which are positive. However, no data-quality assurance plan is described (e.g., how will data integrity be verified, who will audit the analysis, how will outliers be handled)."},"procedures_feasible":{"decision":"Partial","justification":"Standard molecular biology (Western blot, qRT-PCR, IP) and cell-culture procedures are feasible. Tube-formation assays are routine. However, orthotopic xenografts require animal-care expertise, surgical skill, and tumor-monitoring infrastructure. The proposal does not specify who will perform surgeries, how tumors will be monitored (caliper, imaging), or whether the institution has experience with orthotopic breast-cancer models. The 12-month timeline must accommodate animal breeding, tumor growth (typically 3-4 weeks), and tissue processing, leaving limited time for data analysis and troubleshooting."},"timeline_milestones":{"decision":"Partial","justification":"The proposal states a 12-month timeline (September 2025 - August 2026) but does not provide a detailed milestone schedule. Aim 1 (molecular validation) is likely feasible in 2-3 months. Aim 2 (functional rescue in vitro and in vivo) requires 4-6 months (including animal acclimation, tumor growth, and tissue processing). Aim 3 (clinical correlation) requires tissue collection, IHC, and statistical analysis (2-3 months). The timeline is tight and does not account for ethics approval delays, reagent procurement, or troubleshooting. No contingency time is specified. This is a critical-fail item per the rubric."},"recruitment_feasible":{"decision":"Yes","justification":"Northern Jiangsu People's Hospital is described as 'one of the largest and most historic hospitals in Jiangsu Province' with 'adequate clinical samples.' TNBC patient tissues are likely available. The proposal does not specify sample-size targets, but recruitment of preserved tissues for retrospective analysis is generally feasible."}},"score":"Medium","rationale":"The proposal is feasible in principle but faces significant execution risks. Recruitment of TNBC patient samples is likely feasible at a large hospital, and the cell-line and animal-model work is standard. However, the 12-month timeline is tight for three aims, the $5,000 budget is minimal, and the investigator's lack of prior publications raises concerns about independent execution. The proposal does not clearly specify team support, mentorship, or institutional oversight. Ethics approval is stated as 'to be obtained,' creating a potential delay. The proposal does not address potential challenges (e.g., low VM prevalence in some tumors, variable MDK expression in patient samples) or contingency plans."},"statistical_analysis_plan":{"items":{"power_analysis":{"decision":"No","justification":"No power analysis is provided for any aim. No sample sizes are justified. For Aim 1, the number of cell-line replicates is not stated. For Aim 2, the number of animals per group is not specified. For Aim 3, the number of patient samples required to detect a significant correlation is not calculated. This is a critical-fail item."},"metrics_defined":{"decision":"Partial","justification":"Primary metrics are identified (VTN protein/mRNA levels, tube-formation area, VM structures via PAS staining, VE-cadherin expression, survival curves) but not formally defined. No quantification thresholds, no specification of how 'tube formation' will be measured (total length, number of nodes, area covered), and no definition of what constitutes a 'VM structure' in histology."},"analysis_present":{"decision":"Partial","justification":"The proposal lists statistical tests (t-tests, ANOVA, correlations, survival curves) but does not provide a formal statistical analysis plan. No specification of primary vs. secondary outcomes, no pre-specified hypotheses for each test, and no description of how data will be prepared or checked for assumptions (normality, homogeneity of variance)."},"multiple_comparisons":{"decision":"No","justification":"The proposal does not address multiple-comparison correction. Aim 1 will likely involve multiple Western blots and qRT-PCR measurements; Aim 2 will compare multiple groups (MDK-overexpressing, MDK-knockdown, VTN-overexpressing, rescue, controls); Aim 3 will test multiple correlations. No Bonferroni, FDR, or other correction is mentioned."},"analysis_matches_design":{"decision":"Partial","justification":"The proposed tests (t-tests for group comparisons, correlations for clinical data, survival curves for prognostic value) are generally appropriate for the experimental design. However, the design includes both within-group (e.g., rescue experiments) and between-group comparisons, and it is unclear whether paired or unpaired tests will be used. The clinical correlation (Aim 3) may involve confounders (age, stage, treatment) that are not addressed in the analysis plan."}},"score":"Low","rationale":"The proposal mentions statistical tests (t-tests, ANOVA, Pearson/Spearman, Kaplan-Meier) but provides no power analysis, sample-size justification, or detailed specifications. No effect sizes, alpha levels, or multiple-comparison corrections are stated. The proposal does not specify how many replicates (biological or technical) will be used for cell-line experiments, how many animals per group, or how many patient samples will be analyzed. The alternative strategy (proteomics screening if VTN rescue is partial) is mentioned but not formally specified. These critical omissions result in a Low score and trigger the critical-fail cap on this category."},"societal_and_broader_impact":{"items":{"societal_challenge":{"decision":"Yes","justification":"Breast cancer is a major public-health challenge, and TNBC is particularly aggressive with poor prognosis. The proposal addresses a real clinical need (overcoming therapy resistance), which has clear societal relevance. Improving outcomes in TNBC would have significant public-health impact."},"commercial_potential":{"decision":"Partial","justification":"If MDK or VTN inhibitors are developed, there is clear commercial potential (new cancer therapeutics have significant market value). However, the proposal does not discuss intellectual property, patent strategy, or commercialization plans. The involvement of 'One Patient One Cure' (a nonprofit) suggests a non-profit orientation, but the commercial potential is not explicitly addressed."},"public_communication":{"decision":"Partial","justification":"The proposal commits to data sharing and protocol sharing, which supports scientific transparency and reproducibility. However, it does not explicitly plan for public communication, patient engagement, or dissemination to non-scientific audiences. No mention of press releases, patient-advocacy partnerships, or educational outreach."}},"score":"Medium","rationale":"The proposal addresses a significant societal challenge (breast cancer, particularly TNBC, which has poor prognosis and disproportionately affects certain populations). The work could have broad impact if it leads to new therapies. However, the proposal does not explicitly discuss societal or public-communication aspects. It does not address health disparities, patient engagement, or plans to communicate findings to the public or patient communities. The data-sharing commitment (GEO/PRIDE, protocols.io) is positive for scientific transparency but is not framed as a broader-impact activity."},"clinical_or_translational_impact":{"items":{"unmet_need":{"decision":"Yes","justification":"The proposal clearly articulates an unmet clinical need: current anti-VEGF therapies fail to suppress VM, and VM is a driver of therapy resistance and poor prognosis in TNBC. This is well-established in the literature and is a genuine clinical problem. The proposal positions MDK-VTN as a potential solution to this unmet need."},"clinical_pathway":{"decision":"Partial","justification":"The proposal identifies MDK-VTN as a potential therapeutic target but does not outline a clear path to clinical translation. It does not discuss existing MDK or VTN inhibitors, preclinical development requirements, or regulatory pathways. The statement that 'confirmation of the MDK-VTN axis will open new therapeutic avenues' is aspirational but not concrete. A stronger proposal would specify potential drug candidates, preclinical models for efficacy testing, or partnerships with pharmaceutical companies."},"milestones_defined":{"decision":"No","justification":"The proposal does not define milestones for clinical translation. It does not specify what evidence would be required to move from this pilot study to preclinical drug development, what the timeline would be, or what partnerships or funding would be needed. The proposal is framed as preliminary data for a larger grant, but no specific next steps or milestones are outlined."}},"score":"Medium","rationale":"The proposal has a clear translational goal: identifying MDK-VTN as a novel therapeutic target to overcome VM-driven therapy resistance in TNBC. This addresses an unmet clinical need (current anti-VEGF therapies fail to suppress VM). However, the translational pathway is not fully developed. The proposal does not discuss how MDK or VTN could be targeted clinically (e.g., are there existing inhibitors, what would be the development timeline, what are the potential toxicities). The clinical correlation (Aim 3) is correlative, not predictive; it does not establish that MDK-VTN-VM status predicts treatment response or prognosis. No milestones for moving toward clinical translation are specified."}},"fatal_flaws":["No power analysis or sample-size justification: The statistical analysis plan does not include power calculations for any aim. This violates standard NIH grant expectations and is a critical-fail item per the rubric, capping the statistical_analysis_plan category at Medium.","Insufficient detail on statistical methods: The proposal does not specify how multiple comparisons will be controlled, what effect sizes are expected, or how confounders will be handled in clinical correlations. This is a critical-fail item (analysis_present is only partially specified)."],"overall_rating":"good","major_strengths":["Novel and biologically plausible hypothesis: The MDK-VTN-VM axis is well-motivated by preliminary proteomics and immunoprecipitation data, and addresses a genuine therapeutic gap (VM resistance to anti-VEGF therapy). The hypothesis is sufficiently original to warrant investigation.","Methodologically sound experimental design: The three-aim structure (molecular validation, functional rescue, clinical correlation) follows best practices in cancer biology. In vitro rescue experiments combined with in vivo orthotopic xenografts and human tissue analysis provide complementary evidence levels.","Appropriate choice of readouts and models: Use of VE-cadherin, PAS staining, and tube-formation assays aligns with established VM literature. MDA-MB-231 and BT549 cell lines are standard for TNBC VM studies, and orthotopic xenografts are appropriate for in vivo validation.","Strong commitment to data sharing and reproducibility: Explicit commitment to deposit raw data in GEO/PRIDE within six months of publication and to share protocols on protocols.io demonstrates transparency and reproducibility standards.","Clear research question and hypotheses: The proposal articulates a specific, testable hypothesis with two clearly stated predictions (H1 and H2) that directly address the research question."],"overall_summary":"$53","major_weaknesses":["Investigator track record and research independence: The ResearchHub profile shows h-index 0/0 and describes the PI as a 'medical laboratory worker' with no documented prior publications. This raises serious concerns about the PI's ability to independently execute a multi-aim research project, manage animal studies, and lead data analysis. The proposal does not clearly specify mentorship, co-investigator support, or institutional oversight structures.","Statistical analysis plan lacks rigor: No power analysis is provided for any aim. Sample sizes for cell-line experiments, animal studies, and clinical correlations are not specified. The proposal mentions 'two-tailed t-tests or ANOVA' and 'Pearson/Spearman' correlations but does not specify effect sizes, alpha levels, or multiple-comparison corrections. This is a critical-fail item per the rubric.","Timeline and budget appear insufficient: The 12-month timeline must accommodate three distinct aims (molecular, functional, in vivo, and clinical), including animal breeding/acclimation, tumor growth, and tissue processing. The $5,000 budget is extremely modest for orthotopic xenografts (typically $2,300 stated here), imaging, histology, and reagents. No personnel costs, travel, or contingency are included.","Incomplete specification of team and institutional capacity: While the proposal mentions 'well-established research facilities' at Northern Jiangsu People's Hospital, it does not detail the availability of animal-care staff, biostatisticians, or senior mentors. The role of 'One Patient One Cure' (a US-based nonprofit) in fund transfer is mentioned but not explained; this raises questions about institutional oversight and compliance.","Limited detail on clinical sample availability and ethics: The proposal states 'preserved breast cancer tissues' will be used but does not specify sample size, inclusion/exclusion criteria, or the status of IRB approval. The statement that 'ethics approval will be secured before study initiation' suggests it has not yet been obtained, creating execution risk."],"overall_rationale":"The proposal presents a scientifically sound and novel hypothesis addressing an unmet clinical need (VM-driven therapy resistance in TNBC). The experimental design is rigorous and aligned with current breast-cancer VM literature. However, critical gaps in statistical rigor (no power analysis, limited analysis detail), feasibility concerns (tight timeline, minimal budget, junior investigator with no prior publications), and incomplete specification of team support and institutional capacity prevent a rating of 'excellent'. The work is fundable as a pilot/exploratory grant for a junior researcher, but execution risk is moderate to 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59:T375e,

1. Overview

Estimated timeline:
12 months (September 1, 2025 – August 31, 2026).

Data sharing commitment:
All raw and processed data, including imaging and proteomics datasets, will be deposited in public repositories (e.g., GEO, PRIDE) within six months of publication.

2. Introduction

Research question:
Does VTN mediate MDK-driven VM in breast cancer?

Hypotheses:

H1: MDK promotes VM in breast cancer through upregulation of VTN.
H2: Disruption of VTN abrogates MDK-induced VM in vitro and in vivo.

Impact statement:
Confirmation of the MDK–VTN axis as a driver of VM will open new therapeutic avenues, particularly for TNBC, by targeting VM to improve anti-angiogenic therapy outcomes.

3. Methods

Participants/Sample

Cell lines: TNBC cell lines such as MDA-MB-231 and BT549 with MDK overexpression or knockdown.
Animal models: Orthotopic TNBC xenografts in immunodeficient mice.
Clinical samples: Preserved breast cancer tissues.

Materials and Procedures

Aim 1 – Verify VTN regulation by MDK

Western blot and qRT-PCR to measure VTN levels in MDK-overexpressing and knockdown cells compared to controls.
Immunoprecipitation to assess MDK–VTN interaction.

Aim 2 – Assess functional role of VTN in MDK-induced VM

In vitro tube formation assays: VTN overexpression in MDK-knockdown cells to assess restoration of the VM phenotype (rescue experiment).
In vivo assays: Orthotopic xenografts with manipulated MDK/VTN expression; assess VM structures via PAS staining and VM marker IHC.

Aim 3 – Clinical correlation

Immunohistochemistry for MDK, VTN, and VE-cadherin in TNBC patient tissues.

Statistical correlation of MDK and VTN expression with VM prevalence and patient outcomes.

Planned Analyses

Statistical tests: Two-tailed t-tests or ANOVA for group comparisons; Pearson/Spearman for correlation; Kaplan–Meier survival curves for prognostic value.
Alternative strategy: If VTN overexpression only partially affects MDK-induced VM in vitro, we will screen for cofactors via proteomics.

Ethics and Data Management

Ethics: Institutional Review Board and Animal Care Committee approval will be obtained before patient sample analysis and animal experiments.
Confidentiality: All human data will be anonymized before analysis.
Data sharing: Raw data will be deposited in GEO/PRIDE; protocols will be made publicly available (e.g., protocols.io).

4. Pilot Data (Optional)

MDK overexpression in MDA-MB-231 cells increases tubular structure formation.
Proteomics identified VTN as significantly upregulated in MDK-overexpressing cells.

High-MDK tumors in clinical samples show elevated VE-cadherin

5. Budget

Consumable reagents and assay kits: $2,000
Animal studies: $2,300
Imaging and histology: $500
Data analysis software: $200
Total: $5,000

6. References

1 Cerezo-Wallis, D. et al. Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state. Nat Med 26, 1865-1877 (2020). https://doi.org/10.1038/s41591-020-1073-3

2 Christou, C., Stylianou, A. & Gkretsi, V. Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker. Cells 13 (2024). https://doi.org/10.3390/cells13020136

3 Han, B. et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent 4, 47-53 (2024). https://doi.org/10.1016/j.jncc.2024.01.006

5 Lapkina, E. Z., Esimbekova, A. R. & Ruksha, T. G. [Vasculogenic mimicry]. Arkh Patol 85, 62-69 (2023). https://doi.org/10.17116/patol20238506162

8 Sheng, B., Wei, Z., Wu, X., Li, Y. & Liu, Z. USP12 promotes breast cancer angiogenesis by maintaining midkine stability. Cell Death Dis 12, 1074 (2021). https://doi.org/10.1038/s41419-021-04102-y

10 Wei, X. et al. Mechanisms of vasculogenic mimicry in hypoxic tumor microenvironments. Mol Cancer20, 7 (2021). https://doi.org/10.1186/s12943-020-01288-1

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Role and Mechanism of MDK in Promoting Vascular Mimicry in Breast Cancer

Role and Mechanism of MDK in Promoting Vascular Mimicry in Breast Cancer

Funding Progress

Funders

Peer Reviews

Related Funding

Request for Proposals - Cancer Biology Research Project

Nonprofit Recipient

DOI