Tumor nutrient stress gives rise to a drug tolerant cell state in pancreatic cancer

Cytotoxic chemotherapy remains the standard-of-care treatment for patients with pancreatic ductal adenocarcinoma (PDAC). However, chemotherapy only has modest effects at improving overall survival due to primary or rapidly acquired chemoresistance. The biological underpinnings of PDAC therapy resistance are incompletely defined, but the tumor microenvironment is known to be a major contributor to chemoresistance. We have found chemoresistance is imprinted on PDAC cells by the tumor microenvironment and persists for a period of days after PDAC cells are removed from tumors. However, PDAC chemoresistance is lost upon long term culture in standard laboratory conditions. Interestingly, culture of PDAC cells in Tumor Interstitial Fluid Medium (TIFM), a culture medium we developed to recapitulate the nutrient availability of the tumor microenvironment, maintains PDAC cells in a chemoresistant state even after long term culture ex vivo. These findings suggest that microenvironmental metabolic stress keeps PDAC cells in a physiologically relevant, therapy resistant cell state that standard culture models fail to maintain. Using TIFM culture, we sought to understand how PDAC cells in this state resist chemotherapeutic challenge. We found that chemotherapy drugs largely retain on-target activity within TIFM medium but fail to activate cell death, enabling a “chemotolerant” cell state, which is also observed in PDAC tumors. This chemotolerant state is driven by suppression of apoptotic priming and can be overcome by targeting the anti-apoptotic regulator BCL-xL. Taken together, these findings suggest that reprogramming of cell death mechanisms by the PDAC nutrient microenvironment is a key contributor to therapy resistance in this disease. Overall design: Murine pancreatic cancer cells (mPDAC3) were orthotopically implanted in C57BL/6 mice and allowed to establish tumors after 3 weeks. Just prior to euthanasia, mice were treated with a single I.V. injection of 20mg/kg hoechst 33342 dye to label the vascularized space within tumors. Tumors were then dissected, finely minced, and enzymatically dissociated into a single cell suspension. Dissociated tumor cells were stained for the PDAC cell surface marker, mesothelin (MSLN) and processed by FACS for isolation of well perfused (hoechst-high) and poorly perfused (hoechst-low) cancer cells. Sorting populations were gated by positive labeling for MSLN, and the highest/lowest 10% of labeling for hoechst. Cells were directly sorted into RLT extraction buffer. RNA was then isolated by column purification and sent for library prep and sequencing with the University of Chicago Genomics Core.