UC San Diego

While metastasis of cells from the primary tumor is what leads to mortality in cancer patients, the likelihood of metastasis varies from patient-to-patient as well as for cells within a given tumor. However, there is no universal biological marker that can identify heterogeneity in aggressiveness. Cell-ECM dynamics play a profound role in the dissemination of tumor cells from the primary tumor and variations in cell-ECM dynamics in cancer cells can be exploited to distinguish more aggressive cancers from their non-aggressive counterparts. This dissertation aims to demonstrate the utility of studying cell-ECM dynamics—specifically cellular adhesion to the ECM—in order to identify the most aggressive cancer cells.

We first examined how adhesion strength of metastatic and non-metastatic cancer cells varies in the presence of tumoral and stromal cation conditions. We built a spinning-disk shear assay to quantify population adhesion strength in the presence or absence of Mg2+ and Ca2+. Metastatic cells displayed a decrease in cellular adhesion strength in low Mg2+ and Ca2+ conditions as well as heterogeneity in adhesion strength that was not present in the non-metastatic cells. These differences were correlated with differing rates of focal adhesion disassembly between metastatic and non-metastatic cells. When non-metastatic cells were exposed to RGD to decrease their adhesion, they recapitulated the decreased adhesion, increased focal adhesion disassembly, and increased migration associated with metastatic cells. These data suggest that decreased cellular adhesion is a marker of metastatic cell populations.

We next examined how heterogeneity in adhesion strength within a population could lead to intrapopulation heterogeneity in metastatic ability. We utilized a parallel plate flow chamber to isolate distinct fractions of cells from a heterogeneous population. Weakly adherent cells within a population displayed increased 2D and 3D migration compared to their strongly adherent counterparts. These differences were due to increased focal adhesion disassembly and contractility in the weakly adherent cells, which is consistent with previous findings comparing metastatic and non-metastatic cell lines. RNA sequencing revealed differences in transcriptomic expression of cytoskeletal proteins, particularly those corresponding to the microtubule network, between the weakly and strongly adherent cells. When we compared triple negative patient datasets to the expression profiles of weakly and strongly adherent cells, we found that patients with gene expression profiles that matched weakly adherent cells had significantly lower disease-free intervals than the patients that matched strongly-adherent cells.

Finally, we examined how adhesion strength can correlate with invasive and metastatic potential in vivo. We injected MDA-MB231 cells into the mammary fat pads of NOD/SCIDgamma mice, allowed the tumors to grow until localized invasion occurred, resected the fat pad and manually separated the tumor from stroma, and isolated the cancer cells from tumor and stroma respectively. We observed that cells that had invaded into the stroma had a decreased adhesion strength compared to cells within the tumor. This further suggests that the least adherent cells are the most invasive and have the greatest metastatic potential.

These studies demonstrate the universality of using cellular adhesion strength as a biophysical marker of metastatic potential. In addition, the shear separation technique can be used to study heterogeneous cell fractions in other diseases where adhesion plays a significant role.