Abstract
Drug treatments have been designed to inhibit tumor angiogenesis in hope of stopping tumor growth. However, not all tumor types respond to this type of treatment. A screening method which identifies angiogenesis inducing cancer types would help predict the efficacy of angiogenesis-inhibiting drugs for the patients. Our goal is to develop (1) a cell assay to assess the angiogenic induction potential of patient-derived tumor cells, and (2) a protocol for culturing cancer cells on a vascular platform. We optimized the media composition and seeding density of cells (hASC, HUVEC, and cancer cells) to 48-, 96-, and even 384-well plate sizes to allow vascular formation and cancer cell proliferation and subsequent analysis with high throughput. The angiogenic induction potential of patient-derived cancer cells was investigated by quantifying the formation of tubular structures and the drug response of cancer cells grown on a vascular platform was evaluated using gene expression and cell viability (WST-1) assay. Immunocytochemistry was performed with von Willebrand factor, collagen IV, CD44, cytokeratin 19 and ALDH1A1. The angiogenic induction potential test was shown to be responsive to the induction of angiogenesis by cancer cells. The responses of cancer cells were different when grown on a vascular platform or on plastic, seen in gene expression level and viability results. These two protocols are promising novel tools for aiding the selection of efficient cancer drugs for personalized medicine and as an alternative cancer cell culture platform.
Highlights
Blood supply is essential for tumor growth and progression
The cell densities of human adipose stromal cell (hASC) and human umbilical cord vein endothelial cell (HUVEC) (20000 and 4100 cells/cm2, respectively) were based on the previously published angiogenesis model, which has been intra-laboratory validated according to OECD guidance [14]
These cell numbers allowed enough room for cells to proliferate during the culture period and there were enough cancer cells compared to the number of vasculature cells, i.e., hASC and HUVEC
Summary
Blood supply is essential for tumor growth and progression. It is needed to ensure the supply of nutrients and oxygen, and to remove waste products from the tumor. Due to the need of blood vessels, one effective means to prevent growth and expansion of tumors is the inhibition of angiogenesis. Patient-derived cancer cell cultures have been used to test the efficacy of the drugs. To date, this has been carried out by using cell culture methods based on the culture of cancer cells on cell culture plastic. This has been carried out by using cell culture methods based on the culture of cancer cells on cell culture plastic These plastic 2D cultures fail to recapitulate the environment of in vivo tumors as they lack the correct cell-to-cell interactions, extracellular matrix composition, and structure of in vivo tissue, among others [2,3].
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