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Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.

BackgroundTo understand fundamental mechanisms of immune escape, we leveraged our functional ex vivo platform of murine derived organotypic tumor spheroids (DOTS)1 to determine if drug-tolerant persister cells analogous to oncogene targeted therapies limit efficacy of programmed death (PD)-1 blockade, and to identify therapeutic vulnerabilities to overcome anti-PD-1 (αPD-1) resistance.MethodsMurine syngeneic cancer models with well-characterized response to αPD-1 therapy were chosen: MC38 (sensitive) and CT26 (partially resistant). Bulk and single-cell (sc) RNA-sequencing (RNA-seq) were performed on αPD-1 treated DOTS. In vitro culture studies were conducted with or without cytokines (100 ng/ml) or drugs (500 nM). In vivo studies in mice bearing MC38 or CT26 tumors evaluated the combinatorial strategy with PD-1 blockade. We further evaluated our findings in scRNA-seq of an αPD-1 refractory colorectal cancer (CRC) patient tumor.2ResultsBulk RNA-seq of αPD-1 treated DOTS revealed a mesenchymal resistant phenotype with upregulated TNF-α/NFκB signaling (figure 1). scRNA-seq further identified a discrete sub-population of immunotherapy persister cells (IPCs). These cells expressed a stem-like phenotype including downregulation of E2F targets indicative of quiescence, suppression of interferon-γ response genes, induction of hybrid epithelial-to-mesenchymal state, and active IL-6 signaling (figure 1). Ly6a/stem cell antigen-1 (Sca-1) and Snai1 were found to be differentially upregulated in IPCs resistant to PD-1 blockade (not shown). Sca-1 positivity was confirmed in pre-existing tumor populations in vitro (figure 2). When enriched via sorting, these cells remained more persistently Sca-1+ at 96 hours in culture of CT26 compared to MC38 cells, related to increased autocrine IL-6 production by CT26 Sca-1+ cells. Indeed, IL-6 supplementation was capable of expanding Sca-1+ cells in culture (figure 2). Sca-1+ cells expressing ovalbumin peptide were refractory to OT-1 T cell mediated killing and failed to upregulate MHC class-1 antigen presentation (H-2Kb) in response to IL-6, in contrast to interferon-γ (not shown). Analysis of RNA-seq data further identified Birc2/3 as potential targets limiting TNF-mediated apoptosis of these cells (not shown). Notably, Birc2/3 antagonism depleted Sca-1+ IPCs in vitro and significantly potentiated the impact of PD-1 blockade in vivo in MC38, and less robustly in CT26 (figure 3). Evaluation in a microsatellite-instability high CRC patient identified a pre-existent IPC subpopulation within the αPD-1 refractory pre-treatment tumor, with high SNAI1 expression compared to CRC samples in TCGA (figure 4).Abstract 248 Figure 1Bulk and single-cell (sc) RNA-sequencing (RNA-seq) of MDOTS identifies an anti-PD-1 (αPD-1) resistant subpopulation of persister cells. IgG= isotype control[Figure omitted. See PDF]Abstract 248 Figure 2Pre-existent population of stem cell antigen-1 (Sca-1)+ cells expands in response to interleukin-6 (IL-6), as characterized by flow cytometry evaluation in murine syngeneic cancer models at baseline and after purification by fluorescence-activated cell sorting (FACS). H = hours[Figure omitted. See PDF]Abstract 248 Figure 3Combination of anti-PD-1 therapy with Birc2/3 antagonism increases tumor responses and improves survival. CR = complete response[Figure omitted. See PDF]Abstract 248 Figure 4Single-cell RNA-sequencing (scRNA-seq) of a pre-treatment microsatellite-instability (MSI-H) colorectal cancer (CRC) patient tumor, refractory to anti-PD-1 (αPD-1) therapy, reveals presence of SNAI1-high immunotherapy persister cells[Figure omitted. See PDF]ConclusionsHigh-resolution functional ex vivo profiling identified Sca-1+/Snai1high stem-like ‘immunotherapy persister cells‘ and uncovered their anti-apoptotic dependencies targetable with Birc2/3 antagonism to augment αPD-1 efficacy.Ethics ApprovalThis study was approved by the Dana-Farber Animal Care and Use Committee and Novartis Institutional Animal Care and Use Committee. Informed written consent to participate in Dana-Farber/Harvard Cancer Center institutional review board (IRB)-approved research protocols was obtained from the human subject. A copy of the written consent is available for review by the Editor of this journal. The study was conducted per the WMA Declaration of Helsinki and IRB-approved protocols.ReferencesJenkins RW, Aref AR, Lizotte PH, Ivanova E, Stinson S, Zhou CW, et al. Ex Vivo Profiling of PD-1 Blockade using organotypic tumor spheroids. Cancer Discov. 2018;8(2):196–668 215.Gurjao C, Liu D, Hofree M, AlDubayan SH, Wakiro I, Su MJ, et al. intrinsic resistance to immune checkpoint blockade in a mismatch repair-deficient colorectal cancer. Cancer Immunol Res 2019;7(8):1230–6.