Signatures of plasticity and immunosuppression in a single-cell atlas of human small cell lung cancer.
Authors: Chan Joseph Minhow , Quintanal-Villalonga Alvaro , Gao Vianne , Allaj Viola , Masilionis Ignas , Chaudhary Ojasvi , Egger Jacklynn V. , Chow Andrew , Walle Thomas , Mattar Marissa , Offin Michael , Lai W. Victoria Victoria , Bott Matthew , Hollman Travis , Nawy Tal , Mazutis Linas , Sen Triparna , Pe'er Dana , Rudin Charles M. 
Published: June 2021

8509

Background: Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively), which are associated with distinct therapeutic vulnerabilities. The emerging consensus on SCLC subtypes has led to new questions, such as whether subtypes are associated with different disease stages, metastatic potential, or immune microenvironments; whether there is plasticity between subtypes; and whether novel SCLC phenotypes exist. Single cell RNA sequencing (scRNA-seq) offers a unique opportunity to address these questions by dissecting intratumoral transcriptional heterogeneity and the surrounding tumor microenvironment (TME). However, efforts to apply this technology to human SCLC tumors have been limited, as these tumors are infrequently resected. Methods: We have optimized protocols to process both surgical resections and biopsies to construct the first single-cell atlas of SCLC patient tumors (N = 21), with comparative lung adenocarcinoma (LUAD) and normal lung data. We leverage computational methods including diffusion maps and non-negative matrix factorization to perform a deep annotation of SCLC phenotypes and the surrounding immune TME. We perform validation experiments using flow cytometry, Vectra, and immunohistochemistry in independent SCLC cohorts, as well as genetic manipulation in preclinical SCLC models. Results: Our data reveals substantial transcriptional heterogeneity in SCLC both within and across tumors and confirms a pro-metastatic gene program in SCLC-N subtype characterized by epithelial-mesenchymal transformation and axonogenesis. Beyond known subtypes, we discover a PLCG2-high tumor cell population with stem-like, pro-metastatic features that recurs across subtypes and predicts significantly worse overall survival. Manipulation of PLCG2 expression in cells confirms correlation with key metastatic markers. Treatment and subtype are associated with substantial phenotypic changes in the SCLC immune microenvironment, with greater T-cell dysfunction in SCLC-N than SCLC-A. Moreover, the recurrent, PLCG2-high subclone is associated with exhausted CD8+ T-cells and a pro-fibrotic, immunosuppressive monocyte/macrophage population, suggesting possible tumor-immune coordination to promote metastasis. Conclusions: This atlas of SCLC illustrates how canonical subtypes and a novel PLCG2-high recurrent tumor subclone enlist diverse gene programs to create tumor heterogeneity and facilitate metastasis in a profoundly immunosuppressed TME. Our dataset provides further insight into tumor and immune biology in SCLC at single-cell resolution, with potential implications for design of novel targeted therapies and immunotherapeutic approaches.