Incorporation of Tumor Spheroids into an Ex‐vivo Tissue Culture Model for Investigating Cancer Cell–Microvascular Interactions

Abstract

A challenge in cancer research is the lack of biomimetic models that incorporate cancer cells, blood vessels, lymphatic vessels, and immune cells. This challenge highlights the gap between an in vivo system and common in vitro assays, which do not recapitulate the tumor microenvironment. To bridge this gap, our lab has introduced the rat mesentery culture model, which provides a complex and physiologically relevant culture system that enables the real‐time tracking of multiple cell types during angiogenesis and lymphangiogenesis. The objective of this study was to demonstrate the feasibility of culturing transplanted tumor spheroids on rat mesenteric tissues for investigating cancer cell‐angiogenesis interactions. Two lung carcinoma cell lines, A549 and H1299, were labeled with DiI and suspended in a hanging drop culture system consisting of MEM + 10% FBS for 4 days to form fluorescent spheroids. These spheroids were then seeded onto excised rat mesentery tissues (approximately 20 spheroids per tissue). Tissues with transplanted spheroids were cultured for 3 days. To identify blood vessels during various culture timepoints, tissues were labeled with FITC‐conjugated lectin and imaged on Day 2 and Day 3. On Day 3, tissues were labeled for PECAM, NG2, and CD11b to identify endothelial cells, pericytes, and macrophages respectively. Labeling a subset of tissues for Calcien AM enabled the detection of live cells. Observations confirmed that tumor spheroids remained viable and intact during culture. The apparent increase in individual cells along the periphery of the spheroids suggests radial cell migration into the tissue. Spheroids were observed in the same regions of angiogenic microvascular networks characterized by increased sprouting and vessel density. Angiogenesis was also confirmed by comparing time‐lapse lectin labeling of vessels. For some spheroids, directed sprouting towards spheroids was also observed. The presence of lymphatic vessels (identified by PECAM labeling morphology), NG2+ pericytes, and CD11b+ macrophages confirmed a multi‐cellular environment. These results support the feasibility of co‐culturing tumor spheroids and rat mesenteric tissues as well as a new approach for exploring how cancer cells interact with a surrounding microvasculature and their local environment.