Dynamic single-cell RNA sequencing identifies immunotherapy persister cells following PD-1 blockade
Kartik Sehgal, Andrew Portell, Elena V. Ivanova, Patrick H. Lizotte, Navin R. Mahadevan, Jonathan R. Greene, Amir Vajdi, Carino Gurjao, Tyler Teceno, Luke J. Taus, Tran C. Thai, Shunsuke Kitajima, Derek Liu, Tetsuo Tani, Moataz Noureddine, Christie J. Lau, Paul T. Kirschmeier, David Liu, Marios Giannakis, Russell W. Jenkins, Prafulla C. Gokhale, Silvia Goldoni, Maria Pinzon-Ortiz, William D. Hastings, Peter S. Hammerman, Juan J. Miret, Cloud P. Paweletz, David A. Barbie
Kartik Sehgal, Andrew Portell, Elena V. Ivanova, Patrick H. Lizotte, Navin R. Mahadevan, Jonathan R. Greene, Amir Vajdi, Carino Gurjao, Tyler Teceno, Luke J. Taus, Tran C. Thai, Shunsuke Kitajima, Derek Liu, Tetsuo Tani, Moataz Noureddine, Christie J. Lau, Paul T. Kirschmeier, David Liu, Marios Giannakis, Russell W. Jenkins, Prafulla C. Gokhale, Silvia Goldoni, Maria Pinzon-Ortiz, William D. Hastings, Peter S. Hammerman, Juan J. Miret, Cloud P. Paweletz, David A. Barbie
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Research Article Immunology Oncology

Dynamic single-cell RNA sequencing identifies immunotherapy persister cells following PD-1 blockade

Abstract

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.

Authors

Kartik Sehgal, Andrew Portell, Elena V. Ivanova, Patrick H. Lizotte, Navin R. Mahadevan, Jonathan R. Greene, Amir Vajdi, Carino Gurjao, Tyler Teceno, Luke J. Taus, Tran C. Thai, Shunsuke Kitajima, Derek Liu, Tetsuo Tani, Moataz Noureddine, Christie J. Lau, Paul T. Kirschmeier, David Liu, Marios Giannakis, Russell W. Jenkins, Prafulla C. Gokhale, Silvia Goldoni, Maria Pinzon-Ortiz, William D. Hastings, Peter S. Hammerman, Juan J. Miret, Cloud P. Paweletz, David A. Barbie

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Figure 5

Sca-1+ IPCs expand in response to IL-6 and show differential thresholds to TNF cytotoxicity.

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Sca-1+ IPCs expand in response to IL-6 and show differential thresholds ...
(A) Summary of experiments showing proportion of Sca-1+ cells in 96-hour cultures of Sca-1+ purified MC38 cells supplemented with growth factors (100 ng/mL) versus media (negative control) and IFN-γ (positive control) (n = 3). (B) Left, representative histogram of MFI of H-2Kb of MC38 Sca-1+ cells in 96-hour culture of Sca-1+ purified fractions (media alone vs. IFN-γ vs. IL-6 stimulation [100 ng/mL]). Right, summary of experiments [n = 3]. (C) Representative immunofluorescence images and summary of experiments showing Sca-1 expression in MC38 MDOTS after treatment with IL-6 versus control (IgG) (scale bar: 200 μm). Bottom, data are median (solid line) with first and third quartiles (dashed lines) and were analyzed by Wilcoxon rank sum test. (D) Representative flow cytometry plots showing effect of stimulation by IL-6 on expansion of Sca-1+ cells in 96-hour cultures of Sca-1+ purified fractions. Right, summary of experiments (n = 4 for MC38/ CT26, n = 3 for LLC/CMT167). (E) Summary of experiments showing effect of stimulation by IL-6, TNF-α, or IL-6 + TNF-α (100 ng/mL) on fold change of proportion of Sca-1+ cells in 96-hour cultures of Sca-1+ purified fractions of MC38 and CT26 cells versus media alone (negative control) (n = 3). (F) Heatmap showing log2 fold change (log2F) of expression of genes involved in apoptosis pathway in MC38 Sca-1+ cells stimulated with TNF-α (100 ng/mL) for 2 and 6 hours versus media alone. Asterisks denote genes upregulated on RNA-Seq analysis of MC38 MDOTS after anti–PD-1 therapy. (A, B, D, and E) Data are mean ± SEM and were analyzed by multiple t tests with Bonferroni’s correction (A and E), 1-way ANOVA with Bonferroni’s correction (B), or 2-tailed Student’s t test (D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.