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Updated: Jun 28, 2026
Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection
Published on: July 6, 2022
Spatial proteomics profiling reveals oncogene-specific immune niches and prognostic markers in NSCLC
Rajender Nandigama1,2, Bassem Ben Cheikh3, Jochen Wilhelm1,4
1Institute for Lung Health (ILH), Justus Liebig University, Giessen, Germany.
Rationale:
Only subset of patients with Non-Small Cell Lung Cancer (NSCLC) benefit from immunotherapy and this is partly due to limited understanding of how oncogenic mutations shape the tumor microenvironment (TME).
Objectives:
To define how EGFR and KRAS mutations influence the spatial organization and immune composition of the NSCLC TME.
Methods:
We conducted 38-marker high-plex immunofluorescence to 197 NSCLC tumors (>2 million cells) stratified by EGFR and KRAS genotypes. Quantitative phenotyping and spatial analyses, including cellular neighborhoods (CN), nearest neighbors (NNs = 5-20), and spatial proximity profiling (25-100 μm), were performed to assess immune architecture and clinical correlations.
Measurements And Main Results:
EGFR- and KRAS-mutant tumors showed higher tumor cell density and reduced immune infiltration compared with wild-type tumors. Both mutation types were associated with depletion of cytotoxic T cells, dendritic cells, and granulocytes, while EGFR-mutant tumors showed enrichment of M2-like tumor-associated macrophages (TAMs). CN-analysis identified 14 spatial clusters, with reduced cytotoxic and helper T cell-rich neighborhoods in mutant tumors. NN-analysis revealed shorter distances between M2-like TAMs in EGFR-mutant tumors and greater immune exclusion in non-mutants. Spatial proximity revealed higher densities of T-regs, TAMs near tumor cells in KRAS-mutant tumors. All spatial metrics correlated significantly with prognosis in Cox proportional hazards models, highlighting immune cell positioning as an important predictor of outcome.
Conclusions:
EGFR- and KRAS-mutant tumors remodel the NSCLC immune landscape, creating distinct immunosuppressive and immune-excluded niches. Spatial proteomics reveals prognostic immune architectures that may guide mutation-directed immunotherapy strategies.
Insights
Oncogenic mutations in EGFR and KRAS significantly alter the Non-Small Cell Lung Cancer (NSCLC) tumor microenvironment, creating immune-suppressive niches. Understanding these spatial immune architectures is key for developing targeted immunotherapy strategies.
Area of Science:
- Oncology
- Immunology
- Cancer Biology
Background:
- Only a subset of Non-Small Cell Lung Cancer (NSCLC) patients benefit from immunotherapy.
- Limited understanding exists on how oncogenic mutations shape the tumor microenvironment (TME).
Purpose of the Study:
- To define how EGFR and KRAS mutations influence the spatial organization and immune composition of the NSCLC TME.
- To correlate spatial immune architecture with clinical outcomes.
Main Methods:
- Conducted 38-marker high-plex immunofluorescence on 197 NSCLC tumors.
- Performed quantitative phenotyping and spatial analyses (cellular neighborhoods, nearest neighbors, spatial proximity).
Main Results:
- EGFR- and KRAS-mutant tumors exhibited higher tumor cell density and reduced immune infiltration compared to wild-type.
- Mutant tumors showed depletion of cytotoxic T cells and dendritic cells; EGFR-mutant tumors enriched M2-like TAMs.
- Spatial analyses revealed distinct immunosuppressive and immune-excluded niches, with spatial metrics significantly correlating with prognosis.
Conclusions:
- EGFR and KRAS mutations remodel the NSCLC immune landscape into distinct immunosuppressive niches.
- Spatial proteomics identified prognostic immune architectures.
- Findings may guide mutation-directed immunotherapy strategies in NSCLC.
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