Abstract
Simple SummaryClear cell renal cell carcinoma (ccRCC) is the most common kidney cancer. Cell culture studies have the potential to explain how interactions between cancer cells and their support cells (stroma) determine growth and malignancy of ccRCC. The ability to grow tumor cells together with stroma from patient tumors is critical for studies of these interactions, but conventional culture methods do not provide representation of both cell types. We hypothesized that mimicking the extracellular environment of the tumor would promote growth of both tumor and stromal cells. We characterized the extracellular protein composition of patient ccRCCs and defined a nine-component protein blend to mimic the tumor microenvironment. Adherence of tumor cells, supporting stromal cells, and immune cells was demonstrated. Furthermore, we found that cells from patient tumors grown in our protein blend maintained representation of both tumor cells and cancer-associated fibroblasts (CAFs), a stromal cell type that plays a defining but poorly understood role in establishing the tumor microenvironment. This study demonstrates the dependence of CAFs on the extracellular protein composition and provides a technology to investigate interactions between tumor cells and CAFs isolated from patient ccRCCs.Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and is often caused by mutations in the oxygen-sensing machinery of kidney epithelial cells. Due to its pseudo-hypoxic state, ccRCC recruits extensive vasculature and other stromal components. Conventional cell culture methods provide poor representation of stromal cell types in primary cultures of ccRCC, and we hypothesized that mimicking the extracellular environment of the tumor would promote growth of both tumor and stromal cells. We employed proteomics to identify the components of ccRCC extracellular matrix (ECM) and found that in contrast to healthy kidney cortex, laminin, collagen IV, and entactin/nidogen are minor contributors. Instead, the ccRCC ECM is composed largely of collagen VI, fibronectin, and tenascin C. Analysis of single cell expression data indicates that cancer-associated fibroblasts are a major source of tumor ECM production. Tumor cells as well as stromal cells bind efficiently to a nine-component ECM blend characteristic of ccRCC. Primary patient-derived tumor cells bind the nine-component blend efficiently, allowing to us to establish mixed primary cultures of tumor cells and stromal cells. These miniature patient-specific replicas are conducive to microscopy and can be used to analyze interactions between cells in a model tumor microenvironment.
Highlights
Renal cell carcinoma (RCC) is among the ten most common cancers [1], and the most common histological subtype is clear cell renal cell carcinoma [2]
We found that all cell types adhered more efficiently to our Clear cell renal cell carcinoma (ccRCC) extracellular matrix (ECM) blend compared to Matrigel with the exception of endothelial cells (Figure 4E)
This suggests that the ECM profile of ccRCCs defined in our analysis reduces tissue rigidity in comparison with the healthy surrounding tissue, perhaps contributing to the indolent progression of ccRCC tumors, which genetic analysis has revealed generally develop over decades [85]
Summary
Renal cell carcinoma (RCC) is among the ten most common cancers [1], and the most common histological subtype is clear cell renal cell carcinoma (ccRCC) [2]. Loss of the tumor suppressor VHL through genetic or epigenetic mechanisms is seen in over 90% of ccRCCs [3]. VHL is an essential component of the cellular oxygen sensor [4], and its loss induces a persistent pseudo-hypoxic state that results in a strong angiogenic profile of tumors [5]. Epithelial cells within the kidney cortex are thought to be the cells of origin for ccRCC, most likely proximal tubule epithelial cells [6]. Within the tumor, transformed epithelial cells are interspersed with a network of blood vessels, and the ccRCC stroma contains interstitial fibroblasts and inflammatory and immune cells [7]. Cellular components of the tumor are embedded in the extracellular matrix (ECM)
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