What question did this study set out to answer?

To explore how ASCL2 drives the transition of tumor-associated macrophages into immunosuppressive cancer-associated fibroblasts in bone metastases.

May 20, 2026Journal of Experimental & Clinical Cancer Research0 citationsOpen Access

ASCL2-mediated macrophage-myofibroblast transition generates immunosuppressive CAF₇ in NSCLC bone metastases

JWJinfeng Wang JQJin Qian XYXiujia Yang

Key Points

To explore how ASCL2 drives the transition of tumor-associated macrophages into immunosuppressive cancer-associated fibroblasts in bone metastases.
Integrated single-cell RNA sequencing and regulon analyses of lung tumors and bone metastases.
In vitro assays assessed induction of CAF7-like characteristics in macrophages and their immunosuppressive functions.
In vivo studies involved macrophage manipulation and ASCL2 inhibition in a bone-metastasis mouse model.
Identified seven CAF subsets, with CAF7 linked to higher Treg infiltration and exhausted CD8+ T cells.
ASCL2 inhibition blocked macrophage transition to CAF7-like cells, reducing tumor growth and immune suppression.
Combining ASCL2 silencing with anti-PD-1 therapy improved tumor control and extended survival.

Abstract

Abstract Background NSCLC frequently metastasizes to bone, where the microenvironment becomes immunosuppressive, limiting immunotherapy efficacy. Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) contribute to immune evasion, but CAF subset origins and roles in bone metastases remain unclear. We investigated whether TAMs undergo macrophage-to-myofibroblast transition (MMT) to generate an immunosuppressive CAF subpopulation in bone lesions, driven by ASCL2. Methods We integrated single-cell RNA sequencing of primary lung tumors and bone metastases with pseudotime and regulon analyses to resolve CAF heterogeneity and nominate MMT regulators. Findings were validated by immunofluorescence and flow cytometry in patient tissues and a murine bone-metastasis model. In vitro, TGF-β1-induced MMT with ASCL2 knockdown/rescue and T-cell co-culture assays were used to assess CAF₇-like induction and immunosuppressive function. In vivo, macrophage depletion/reconstitution, donor and endogenous tracing, and macrophage-directed ASCL2 or Il6ra knockdown were performed in a mouse bone-metastasis model to assess CAF₇-like abundance, tumor burden, and T-cell states; ASCL2 inhibition was further evaluated with PD-1 blockade for tumor control and survival. Results Seven CAF subsets were identified, including a bone metastasis–enriched subset (CAF₇) that co-expressed macrophage, MHC-II, and stromal markers. Trajectory analyses, together with the observation that LLC-conditioned medium induced a CAF₇-like shift in bone marrow–derived macrophages in vitro and that both exogenous GFP-labeled BMDMs and endogenous macrophage-lineage-traced cells acquired CAF₇-like features in the bone metastatic microenvironment, supported TAM-to-CAF₇-like transition via MMT. CAF₇ accumulation correlated with increased Treg infiltration, CD8⁺ T cell exhaustion, and poorer survival. Regulon analysis highlighted ASCL2 as a CAF₇-associated regulator; ASCL2 increased in vitro during MMT, and in vivo ASCL2 protein and ASCL2⁺ CAF₇-like cells were enriched in bone-metastasis lesions versus sham marrow or subcutaneous tumors. ASCL2 knockdown in macrophages blocked MMT, reducing CAF₇-like cells and associated Treg and exhausted CD8⁺ T cells, and suppressing tumor growth. Mechanistically, ASCL2 directly transactivated Il6ra, and macrophage Il6ra knockdown phenocopied ASCL2 silencing in vivo. Notably, ASCL2 silencing synergized with anti–PD-1 therapy, improving tumor control and extending survival. Conclusions In NSCLC bone metastases, ASCL2-driven MMT converts TAMs into immunosuppressive CAF₇ that promotes immune escape. Targeting ASCL2 disrupts this transition, restores anti-tumor immunity, and may improve immunotherapy efficacy. Graphical Abstract

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