One of the hallmarks of cancer development is

One of the hallmarks of cancer development is alterations in cellular metabolism (Hanahan and Weinberg, 2011). It has long been postulated that cancer cells upregulate aerobic glycolysis in order to provide the cancer cells with the building blocks necessary to rapidly proliferate (Vander Heiden et al., 2009; Warburg, 1956; Warburg et al., 1927). Recently, the role of the mitochondria as a biosynthetic “factory” for cancer cell proliferation has become more apparent (Ahn and Metallo, 2015), while alterations in metabolism have been found to change depending on tumor type and the environment around the tumor (Xie et al., 2014; Choi et al., 2013; Gordon et al., 2015). These studies have shown that cancer cell metabolism is not a stagnant, predetermined process but is altered based on the needs of the cell and the conditions within which the cell is growing. While the majority of studies on the metabolism of cancer in vitro have been completed in 2D monolayer cell cultures, a growing number of studies have shown the importance of the extracellular environment on tumor cell metabolism. A recent study showed that successful metastasis to various organ sites was dependent upon differential metabolic profiles of the same primary tumor cells (Dupuy et al., 2015). The flux of metabolites through glycolysis and the tricarboxylic bcrp inhibitor (TCA) cycle is decreased when breast cancer cells are grown in anchorage independent conditions (Grassian et al., 2011). Additionally, the metabolism of circulating tumor cells is different than that of primary tumor cells, with a predilection for increased oxidative phosphorylation in circulating tumor cells (Lebleu et al., 2014). Cellular metabolism is a key first responder to changes in the chemical and mechanical environment (Kamel et al., 2014). Despite this small but growing data, the direct effect of collagen density on cellular metabolism has not been well established. In this study we investigated whether the metabolism of cancer cells is altered in response to changes in collagen ECM density. We sought to determine the alterations in cellular metabolism in two mammary breast cancer cell lines in response to changes in collagen matrix density. The two cell lines chosen (4T1 and 4T07) arose from the same spontaneous mouse mammary tumor and were separated based on metastatic potential such that 4T1 cells traffic to and form metastatic lesions in the mouse lung, whereas 4T07 cells traffic to the mouse lung but fail to proliferate, arresting to quiescence (Heppner et al., 2000; Aslakson and Miller, 1992; Miller et al., 1987). Recent studies using this clonal cell line panel have shown that metabolic plasticity in response to the local microenvironment is greater in the metastatic cell lines than in the non-metastatic cell lines (Simões et al., 2015; Dupuy et al., 2015). Surprisingly, we found that the more metastatic 4T1 cells showed altered metabolism and associated changes in gene expression in response to changes in extracellular collagen matrix density, while 4T07 cell metabolism was more refractory toward these changes in response to changes in density. Our data demonstrate how breast carcinoma cells may use adaptable mechanisms to alter their metabolism in response to changes in the extracellular matrix composition and density.
Materials and Methods
Results
Discussion It has long been observed that cancer cells alter their metabolism to support progression and resiliency (Warburg, 1956; Warburg et al., 1927; Hanahan and Weinberg, 2011). However, the influence of the local extracellular matrix in the tumor microenvironment on cellular metabolism is not well understood. Here, we report that the density of the collagen microenvironment can induce metabolic shifts in cancer cells. We consistently observe that the cellular metabolism of two mammary carcinoma cell lines, 4T1 and 4T07, respond significantly to changes in the collagen density of their microenvironment. Both cell lines show decreased oxygen consumption and ATP production in response to changes in density. The functional metabolism findings in this study are not just a peculiarity of the 4T1 clonal cell lines. Indeed, we find changes in OCR and ECAR in response to changes in collagen density in both a normal mouse mammary gland cell line and a human breast carcinoma cell line (Supplemental Fig. 2a–d), suggesting that collagen matrix density alters functional metabolism broadly across mammary cells.