Tumor metastasis is the leading cause of cancer-related mortality. During the metastatic process, disseminated tumor cells establish metastatic tumor microenvironments. Tumor cells can reshape specific immune microenvironments through intrinsic genetic alterations, thereby establishing immune barriers that limit therapeutic responses.

Conventional tumor immunological screening approaches typically rely on tissue dissociation, resulting in the loss of critical spatial information within the microenvironment. A systematic understanding of how tumor-intrinsic genetic alterations shape specific immune microenvironments and ultimately determine immunotherapy responses remains limited.

In a study published in Cell, a team led by Prof. WANG Guangchuan from the Center for Excellence in Molecular Cell Science (Shanghai Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences, along with collaborators from Shanghai Jiao Tong University and the Guangzhou National Laboratory, established causal links between tumor-intrinsic mutations, the spatial organization of lung metastatic microenvironments, and immunotherapy responses, and identified key targets that remodel metastatic niches to enhance T cell infiltration and therapeutic efficacy.

The researchers first developed a novel spatial perturbation screening platform termed CRISPR-laser-captured microdissection integration mapping of tumor immune microenvironment (CLIM-TIME). This platform integrates CRISPR-based genetic screening with laser capture microdissection (LCM) of metastatic lesions. Through transcriptomic profiling, deconvolution analysis, and immunofluorescence imaging, the platform enables systematic spatial dissection of metastatic tumor microenvironments.

By using the CLIM-TIME platform, the researchers analyzed lung metastatic lesions driven by the loss of 391 commonly mutated tumor suppressor genes, and profiled their transcriptomic features, spatial immune cell distribution, immune evasion capacity, and responses to T cell–based therapies. They classified metastatic tumor microenvironments induced by distinct genetic perturbations into seven major types, with each characterized by distinct transcriptional programs.

The researchers revealed that metastatic lesions resistant to T cell therapy predominantly exhibited two microenvironmental states: a myeloid-rich, T cell–excluded phenotype (T-IE/M-II) and an immune desert (ID) phenotype. Notably, the T-IE/M-II microenvironment was marked by significant enrichment of collagen-associated extracellular matrix (ECM) remodeling pathways and signaling pathways involving focal adhesion kinase (FAK).

Moreover, the researchers demonstrated that loss-of-function mutations in multiple tumor suppressor genes within the Hippo signaling pathway preferentially induced the formation of T-IE/M-II metastatic niches and conferred resistance to T cell-based therapies. Mechanistic studies using Nf2 loss as a representative example revealed markedly reduced T cell infiltration, increased myeloid cell accumulation, and enhanced collagen deposition within these metastatic lesions.

At the molecular level, Nf2-deficient tumor cells upregulated a suite of ECM-related genes, including the collagen crosslinking enzyme LOXL2, and simultaneously reprogrammed recruited myeloid cells. These changes cooperatively promoted T cell exclusion and functional exhaustion. Clinical data analyses showed that metastatic tumors with high LOXL2 expression exhibited significantly reduced T cell infiltration and poor responses to immunotherapy.

Genetic ablation of LOXL2 or mutation of its catalytic site led to decreased collagen deposition and myeloid cell infiltration, accompanied by increased T cell infiltration, effectively converting "T cell-resistant" metastatic lesions into therapy-responsive states. In mouse and human metastatic tumor models, both genetic targeting and small-molecule inhibition of LOXL2 greatly enhanced the efficacy of TCR-T and CAR-T cell therapies.

This study develops CLIM-TIME, a high-throughput perturbation-mapping platform that links tumor suppressor gene loss to metastatic tumor microenvironment remodeling and immunotherapy responses, and identifies key causal genes that determine immune states within metastatic tumors. It offers theoretical and experimental foundations for overcoming immunotherapy resistance in metastatic cancer and the development of novel therapeutic strategies.