The ability of tumor cells to reestablish a niche and cause recurrence of the tumor, either within the primary tissue that the tumor initially formed or to a secondary site within the body, enhances tumor malignancy and causes patient morbidity and mortality. The propensity of tumor cells to migrate away from their primary site poses several challenges. First is the high variability in driver mutations between patients, as well as the variability in patient background genetics,
both of which can induce variability in tumor cell migratory and metastatic propensity. Second, is a lack of universal markers, either genetic or molecular, which can accurately predict tumor cell migratory and metastatic potential. Together these challenges prevent the stratification of different tumors and prevents the implementation of patient-specific interventions tailored to prevent tumor cell migration and metastasis.
An emerging field which is providing insight into the tumor metastatic process is biophysical analysis. However, the field is currently focused on analyzing the presence of circulating tumor cell through turbulent flow microfluidics as an indicator for metastasis, not predicting tumor metastatic potential from the primary site. Therefore, there is a need for a rapid and quantitative biophysical metric which can predict the migratory and metastatic potential of those primary tumors. This dissertation addresses the need to predict tumor migratory and metastatic potential by gaining an understanding of how those characteristics are linked to cell adhesion in glioblastoma and mammary tumors, respectively.
In order to determine the relationship between adhesion strength and cell migratory and metastatic potential first we characterized the adhesion strength of metastatic and non-metastatic mammary cancer cells. In order to evaluate cellular adhesion strength as well as understand the effects of the tumor microenvironment on cell biophysical characteristics, we built a spinning disk shear device. This device gave us the capability of interrogating cellular adhesion characteristics in a quantitative and high throughput manner, as well as being able to modulate extracellular divalent cation conditions Mg2+ and Ca2+ to mimic those found in patients. We found that in the absence of divalent cations those metastatic cells showed an overall decrease in the cellular adhesion strength as well as a broader range of adhesion characteristics
than those non-metastatic cell lines. Comparison of those metastatic cells to their non-metastatic counterparts demonstrated a decrease in the assembly state of focal adhesions in both number and size. Similarly, the exposure of those non-metastatic cells to cyclic RGD peptides also induced a decrease in focal adhesion assembly state as well as decreasing cellular adhesion strength and increasing cell migratory phenotype. Together, these data suggest that there is a correlation between decreased cellular adhesion strength, an increase in metastatic potential, and that this correlation is due to altered assembly state of focal adhesion structures.
Next, I wanted to understand if the correlation between decreased cellular adhesion strength and increased cell migratory phenotype extended beyond mammary tumors to glioblastoma (GBM). In order to investigate the effects of common GBM mutations on cellular adhesion strength, isogenic murine astrocytes were exposed to fluidic shear stress via spinning disk assay. Specifically, astrocytes with combinations of CDKN2A/B deletion (occurring in 61% of patients), Pten deletion (occurring in 41% of patients), or alteration of epidermal growth factor receptor (EGFR) (occurring in 57% of patients) were analyzed. This analysis showed that unlike mammary tumors, decreased astrocyte adhesion strength was dependent on the presence of divalent cations. Furthermore, I found that the decrease in adhesion strength was limited to those cells expressing EGFRvIII independent of other mutations, and correlated with increased migratory phenotype. Further analysis found that this change in EGFRvIII expressing cells biophysical phenotype is a result of a signaling-dependent decrease in integrin expression which results in alteration of focal adhesion assembly state. In order to investigate what EGFRvIII dependent signaling cascades modulate decreased adhesion strength, I utilized small-molecule inhibitors to target multiple pathway nodes such as: the EGFR receptor itself, MEK, SRC, and Stat3
in a systematic manner. I found that those cells treated with MEK inhibitor or EGFR inhibitor showed an increase in EGFRvIII cell adhesion strength, similar to non-EGFRvIII expressing cells. Lastly, it has been well documented that cell-cell communication allows those EGFRvIII cells to affect behavior of non-EGFRvIII cells within that tumor. In order to understand if EGFRvIII cells are capable of altering cell adhesion strength of other cells, I educated wtEGFR with EGFRvIII conditioned media prior to analyzing cellular adhesion strength. I found that those wtEGFR expressing cells altered their adhesion strength after education with vIII CM, and that this decrease in adhesion strength was dependent on the presence of the soluble factor TNF-α.
Throughout this dissertation I will demonstrate the value of utilizing fluidic shear as a methodology for analyzing cellular adhesion strength and its application for predicting tumor migratory and metastatic potential. Furthermore, I will also demonstrate how fluidic shear can be used to understand the genetic and molecular mechanisms that contribute to and enhance tumor cell malignancy.