Cancer cell seeding density as a mechanism of chemotherapy resistance: a novel cancer cell density index based on IC50-Seeding Density Slope (ISDS) to assess chemosensitivity

Although the 50% inhibitory concentration (IC50) is a commonly used measurement of chemosensitivity in cancer cells, it has been known to vary with the density of the treated cells (in that more densely seeded cells are more resistant to chemotherapeutic agents). Indeed, density-dependent chemoresistance may be a significant independent mechanism of therapy resistance. We examine the nature of cell density-dependent chemoresistance and explore possible underlying mechanisms. CellTiter-Glo assays and ethidium homodimer staining revealed that response to chemotherapy is density-dependent in all cancer cell lines tested. Our results prompted us to develop a novel cancer cell seeding density index of chemosensitivity, the ISDS (IC50-Seeding Density Slope), which we propose can serve as an improved method of analyzing how cancer cells respond to chemotherapeutic treatment compared to the widely-used IC50. Furthermore, western blot analysis suggests that levels of autophagy and apoptotic markers are modulated by cancer cell density. Cell viability experiments using the autophagy inhibitor chloroquine showed that chloroquine's efficacy was reduced at higher cell densities and that chloroquine and cisplatin exhibited synergy at both higher and lower cell densities in TOV-21G cells. We discuss alternative mechanisms of density-dependent chemoresistance and in vivo/clinical applications, including challenges of adjuvant chemotherapy and minimal residual disease. Taken together, our findings show that cell density is a significant contributor in shaping cancer chemosensitivity, that the ISDS (aka the Ujwal Punyamurtula/Wafik El-Deiry or Ujwal-WAF Index) can be used to effectively assess cell viability and that this phenomenon of density-dependent chemoresistance may be leveraged for a variety of biologic and cancer therapeutic applications.