Spatial single-cell proteotyping reveals immunotherapy-resistant features within the complex tumor microenvironment of metastatic NSCLC
Kohsuke Isomoto, Koji Haratani, Takahiro Tsujikawa, Shuta Tomida, Yusuke Makutani, Masayuki Takeda, Kimio Yonesaka, Kaoru Tanaka, Tsutomu Iwasa, Kazuko Sakai, Kazuto Nishio, Akihiko Ito, Kazuhiko Nakagawa, Hidetoshi Hayashi
Kohsuke Isomoto, Koji Haratani, Takahiro Tsujikawa, Shuta Tomida, Yusuke Makutani, Masayuki Takeda, Kimio Yonesaka, Kaoru Tanaka, Tsutomu Iwasa, Kazuko Sakai, Kazuto Nishio, Akihiko Ito, Kazuhiko Nakagawa, Hidetoshi Hayashi
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Clinical Research and Public Health Clinical Research Immunology Oncology

Spatial single-cell proteotyping reveals immunotherapy-resistant features within the complex tumor microenvironment of metastatic NSCLC

Abstract

BACKGROUND Immune checkpoint inhibitors (ICIs) targeting the programmed cell death 1 axis have revolutionized metastatic non–small cell lung cancer (mNSCLC) treatment. However, disease progression remains a concern, and the role of the complex tumor microenvironment (TME) in treatment failure is not fully understood.METHODS In this biomarker study involving 103 patients with mNSCLC, including 81 patients who received ICI treatment, we evaluated the association between heterogeneous immune cell subsets and ICI efficacy through single-cell spatial profiling of pretreatment tumor tissue, using a 29-marker multiplex IHC platform built for in-depth dissection of the TME.RESULTS Among various types of intratumoral lymphocytes, including Th1, Treg, and NK cells, only CD8+ T cells (tumor-infiltrating lymphocytes [TILs]) were associated with ICI efficacy. Computational tissue segmentation underscored the importance of direct physical interactions between CD8+ TILs and cancer cells for ICI efficacy. TIL phenotyping identified CD39/CD103/Ki-67 positivity as a hallmark of exhausted yet functional tumor-reactive CD8+ TILs. Immunosuppressive tumor-associated macrophages (TAMs) and cancer-associated fibroblasts were independent unfavorable adversaries. High CD73 expression on cancer cells was suggested to confer tolerance to ICI in EGFR/ALK-oncogene+ NSCLC, potentially through M2-TAM accumulation and aberrant angiogenesis.CONCLUSION Our study delineates the clinical relevance of heterogeneous immune cell subsets in ICI-treated mNSCLC, aiding the development of targeted therapeutic strategies.FUNDING Osaka Cancer Society, KANAE Foundation for the Promotion of Medical Science, SGH Foundation, and YOKOYAMA Foundation for Clinical Pharmacology.

Authors

Kohsuke Isomoto, Koji Haratani, Takahiro Tsujikawa, Shuta Tomida, Yusuke Makutani, Masayuki Takeda, Kimio Yonesaka, Kaoru Tanaka, Tsutomu Iwasa, Kazuko Sakai, Kazuto Nishio, Akihiko Ito, Kazuhiko Nakagawa, Hidetoshi Hayashi

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Figure 10

EGFR-mutation–associated gene expression and spatial immune features.

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EGFR-mutation–associated gene expression and spatial immune features. (...
(A) Volcano plot showing −log10(P value) and log2(fold change [FC]) of gene expression levels (EGFR-mutant [N = 9] vs. EGFR-wt [N = 18]) for 726 genes (left). The top 30 upregulated genes in EGFR-mutation+ tumors are listed with q values (right), ranked by the log2(FC). (B) Gene expression levels of CD8A, CD8B, and FOXP3 compared between EGFR-mutation– NSCLC (N = 18) and EGFR-mutation+ NSCLC (N = 9). (C and D) Gene expression signature scores (antigen presentation, angiogenesis, and TGF-β signaling) compared between EGFR-mutation– NSCLC (N = 18) and EGFR-mutation+ NSCLC (N = 9). (E) Gene expression levels of NT5E compared between EGFR-mutation– NSCLC (N = 18) and EGFR-mutation+ NSCLC (N = 9). (F) CD73 protein positivity in cancer cells compared between EGFR/ALK-oncogene– tumors (N = 71) and EGFR/ALK-oncogene+ tumors (N = 32). (G) Autophagy signature scores compared between EGFR-mutation– NSCLC (N = 18) and EGFR-mutation+ NSCLC (N = 9). (H) Representative mIHC images of M2-TAM (CD68+CD206+), CD73-expressing cancer cells, Trm-like (CD39+CD103+) CD8+ T cells, and Tregs (FOXP3+) are shown for EGFR-wt NSCLC (left) and EGFR-oncogene+ NSCLC (right). The EGFR-oncogene+ NSCLC presented a higher intratumoral M2-TAM infiltration and CD73 expression on cancer cells compared with the WT tumors. Substantial Trm-like CD8+ T cell infiltration was verified in the EGFR-oncogene+ tumor as equivalent to the WT tumor. Treg densities were comparable between the 2 distinctive tumor types. Arrows represent overlaid color markers. Scale bars: 100 μm. Data are presented in violin plots in F, with each dot representing 1 patient; tumors with EGFR-mutations are colored red, and those with ALK-fusions are colored green. Data are presented as mean ± SEM in dot plots in B–E and G. P values were determined by unpaired 2-tailed t tests in A and Mann-Whitney U test in B–G.