Abstract
Tumor vasculature is the major extrinsic factor that shapes Intra-tumoral heterogeneity (ITH). Non-uniform exposure of microenvironmental cues greatly impacts cancer cell phenotypes leading to ITH, which exacerbates therapy resistance. This raises a need to study the influence of non-uniform perfusion patterns and the resulting heterogeneity that persists within the tumor microenvironment (TME). A method was developed to identify cancer cells based on their proximity to functional blood vessels (BVs) called perfusion-based fluorescent dye labeling of cells (PFDLC). PFDLC works on the principle of perfusion, where a freely diffusible nuclear binding fluorescent dye (Hoechst 33342) is injected intravenously (i.v.) through a tail vein into atumor-bearing mice. The tumors are retrieved post dye perfusion, dissociated into single cells, and sorted based on their dye uptake proportional to their distance from the nearest blood capillary. This method is amenable to multi-omics as well as functional assays.
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Acknowledgments
The author would like to thank Prof. Eli Keshet and his lab members for all the necessary help during the standardization of this protocol at the Hebrew University of Jerusalem, Israel. This work was supported by the Indian Institute of Technology, Delhi (Institutional New Faculty Grant: NFG 134) to S.K. The graphical figures were designed using Biorender webtool.
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Kumar, S. (2023). Perfusion-Based Fluorescent Dye Labeling to Sort Cancer Cells Based on Their Distance from Blood Vessels. In: Ribatti, D. (eds) Tumor Angiogenesis Assays. Methods in Molecular Biology, vol 2572. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2703-7_4
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DOI: https://doi.org/10.1007/978-1-0716-2703-7_4
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