Dynamic single-cell RNA sequencing identifies immunotherapy persister cells following PD-1 blockade
Kartik Sehgal, … , Cloud P. Paweletz, David A. Barbie
Kartik Sehgal, … , Cloud P. Paweletz, David A. Barbie
Published November 5, 2020
Citation Information: J Clin Invest. 2021;131(2):e135038. https://doi.org/10.1172/JCI135038.
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Research Article Immunology Oncology Article has an altmetric score of 7

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 6

Birc2/3 degradation by LCL161 further sensitizes IPCs to TNF-α and promotes durable αPD-1 response.

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Birc2/3 degradation by LCL161 further sensitizes IPCs to TNF-α and prom...
(A) Representative flow cytometry plots showing effects on proportion of Sca-1+ cells in culture of Sca-1+ purified MC38 and CT26 cells at 96 hours with or without treatment with Birc2/3 antagonist LCL161 in the presence of TNF-α, IL-6, TNF-α + IL-6 (both at 100 ng/mL), or media alone (n = 3). (B) Representative immunofluorescence microscopic images and quantification of CD45 and DAPI-stained tumor specimens from MC38 tumor-containing mice 1 week after treatment with negative controls (isotype control IgG + vehicle), αPD-1 monotherapy, LCL161 monotherapy, or αPD-1 + LCL161 combination therapy (n = 6 mice per group, except 4 for IgG + vehicle) (scale bar: 100 μm). Data are box-and-whisker graphs, with box representing IQR, solid line representing median, and all points ranging from minimum to maximum, and were analyzed by Kruskal-Wallis test. (C and D) Summary of in vivo experiments with Kaplan-Meier curves showing tumor volumes (mm3) over time (days) in MC38 (C) or CT26 (D) tumor-containing mice treated weekly with negative controls (vehicle + IgG), αPD-1, LCL161, or αPD-1 + LCL161 combination therapy. Box with shaded region represents duration of treatment. Mice with complete response (CR)/total number of mice tested are presented for each group. (C) For MC38, n = 11 mice, except 10 in LCL161 monotherapy group. (D) For CT26, n = 8 mice, except 7 in LCL161 monotherapy group. Some mice were euthanized because of tumor necrosis before they reached threshold for progression (2 mice in vehicle + IgG, 5 in αPD-1, 6 in LCL161, and 2 in combination therapy groups); 1 mouse in combination therapy group that achieved CR had accidental death on day 15. *P < 0.05, **P < 0.01, ***P < 0.001.