P2.14-40 Tumor-Stromal Microenvironment Interactions in a PDX Model of EGFR TKI Drug Tolerance

Abstract

Sixty to eighty percent of advanced stage lung adenocarcinoma patients with sensitizing epidermal growth factor receptor (EGFR) mutated tumors respond to EGFR tyrosine kinase inhibitors (TKIs). However, the vast majority of patients eventually progress due to acquired resistance. In vitro evidence suggests that minor populations of drug tolerant cells (DTCs) may be important for tumors surviving TKI. These studies cannot investigate changes in non-cancerous cell populations found within tumors. Yet, stromal cells have been implicated in protecting cancer cells from treatment-induced death and early stage lung adenocarcinomas responding to neoadjuvant EGFR TKI exhibited DTCs within large areas of fibrosis (NCT00188617). We hypothesize that molecularly characterizing DTCs in vivo in comparison to an untreated tumor in a patient-derived xenograft (PDX) model may delineate stromal changes that sustain DTCs, and potentially mimic clinical events. DTCs were harvested after one month of chronic erlotinib exposure in a lung adenocarcinoma PDX model harboring an exon 19 deletion; an untreated baseline (BL) tumor was also harvested. Histological characterization and single-cell RNA-sequencing (scRNA-seq) of DTCs and BL tumors were compared. ScRNAseq cell-types were assigned using reference component analysis. RNA expression levels of receptor/ligands were explored in cell populations. Post-erlotinib treatment, cell-type proportions within the tumor shifted dramatically, with substantially fewer cancer cells and more fibroblasts, mesenchymal stem cells (MSCs), and natural killer cells (NKCs). Two antigen presenting cell transcriptomic states (APC1 and APC2) were identified in both DTC and BL tumors: APC1s exhibited translation-related gene expression profiles while APC2s exhibited immune-response profiles. BL tumors contained mostly APC1s, whereas DTC tumors exhibited more equal proportions of both APC types. Expression profiles for some cell-types also shifted after treatment. Fibroblasts and NKCs exhibited shifts toward more inflammatory and immune-responsive expression profiles post-treatment. Fibroblasts and endothelial cells demonstrated gene expression shifts towards decreased angiogenesis and vasculature development. Paired ligand-receptor interactions between cancer-stromal cells were increased or decreased congruently post-treatment. Specifically, fibroblasts exhibited a shift from alpha-SMA+ myofibroblastic to more IL6+ inflammatory phenotypes, by mRNA and equivalent immunohistochemistry, post-treatment. Cancer cells exhibited a reciprocal increase in IL6R receptor expression post-treatment. Using an EGFR mutant PDX model sensitive to EGFR TKIs, we see substantial post-treatment changes after chronic TKI exposure in non-cancerous (stromal) cell population composition involving their proportions, expression profiles, and their inferred communication with cancer cells. Understanding these potentially protective shifts in non-cancerous cell populations post-TKI-treatment may help identify clinically-relevant mechanisms of drug tolerance.