Background Persister tumor cells are a discrete and typically undetectable sub-population of cells that survive anti-cancer therapy and are thought to be a major cause of tumor recurrence. Persisters have been identified following both drug- and immunotherapy. The focus of this study is to examine any similarities or differences between immunotherapy persister cells (IPCs) and drug tolerant persister cells (DTPs). Pharmacological agents and immune cells often kill via apoptosis. Therefore, we investigated if reduced apoptotic sensitivity is a shared mechanism common to IPCs and DTPs and whether the use of BH3 mimetics to enhance apoptotic sensitivity could sensitize IPCs and DTPs. We also investigated if IPCs have an altered sensitivity profile to conventional chemotherapy, radiotherapy and epigenetic modulators. Similarly, we assessed the sensitivity of DTPs to T cell attack and radiotherapy.
Methods IPCs were generated by chronically co-culturing tumor cell lines (murine B16-OVA melanoma cells or human HeLa-EGFR cervical tumor cells) with T cells (OVA-T-1 murine T cells or human EGFR-CAR T cells) for 7 days. Six different DTP cell lines were generated by chronically treating tumor cells for 7 days with a high dose of drug that killed approximately 90% of the cells after 3 days (5-FU/oxaliplatin/docetaxel/etoposide/dinaciclib/panobinostat). Using a CellTiter-glo assay, we determined the sensitivity of IPCs and DTPs to drugs, radiation and T cell attack. Microscopy-based BH3 profiling was employed to measure the mitochondrial apoptotic sensitivity of persisters and to determine if there exists any anti-apoptotic dependencies that persisters might rely on to enhance their survival.
Results IPCs acquired a reduced sensitivity to multiple drug classes and radiotherapy. Likewise, DTPs also developed a reduced sensitivity to multiple drug classes and radiotherapy but also acquired a reduced sensitivity to T cell killing. Depending on the cell line and therapy, some persisters developed either an increased or decreased sensitivity to apoptosis. Inhibition of specific anti-apoptotic dependencies using BH3 mimetics increased apoptotic sensitivity of persister cells.
Conclusions We identified that reduced apoptotic sensitivity is a shared mechanism of survival common to many IPCs and DTPs. IPCs and DTPs have distinct anti-apoptotic dependencies compared to their parental cell lines wherein pharmacological inhibition of such dependencies enhanced apoptotic sensitivity. IPCs and DTPs also displayed a reduced sensitivity to other therapies that they were never exposed to suggesting that the acquired mechanisms of resistance to T cell killing or drug therapy also conferred a reduced sensitivity to many drug classes, cellular immunotherapies and radiation.
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