Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient–derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8+ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF–MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

Cancer immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD); however, a significant proportion of patients do not respond. Recent transcriptomic studies to understand determinants of immunotherapy response have pinpointed stromal-mediated resistance mechanisms. To gain a better understanding of stromal biology at the cellular and molecular level in LUAD, we performed single-cell RNA sequencing of 256,379 cells, including 13,857 mesenchymal cells, from 9 treatment-naïve patients. Among the mesenchymal cell subsets, FAP+PDPN+ cancer-associated fibroblasts (CAF) and ACTA2+MCAM+ pericytes were enriched in tumors and differentiated from lung-resident fibroblasts. Imaging mass cytometry revealed that both subsets were topographically adjacent to the perivascular niche and had close spatial interactions with endothelial cells (EC). Modeling of ligand and receptor interactomes between mesenchymal and ECs identified that NOTCH signaling drives these cell-to-cell interactions in tumors, with pericytes and CAFs as the signal receivers and arterial and PLVAPhigh immature neovascular ECs as the signal senders. Either pharmacologically blocking NOTCH signaling or genetically depleting NOTCH3 levels in mesenchymal cells significantly reduced collagen production and suppressed cell invasion. Bulk RNA sequencing data demonstrated that NOTCH3 expression correlated with poor survival in stroma-rich patients and that a T cell–inflamed gene signature only predicted survival in patients with low NOTCH3. Collectively, this study provides valuable insights into the role of NOTCH3 in regulating tumor stroma biology, warranting further studies to elucidate the clinical implications of targeting NOTCH3 signaling. Significance: NOTCH3 signaling activates tumor-associated mesenchymal cells, increases collagen production, and augments cell invasion in lung adenocarcinoma, suggesting its critical role in remodeling tumor stroma.

Immunotherapy successfully complements traditional cancer treatment. However, primary and acquired resistance might limit efficacy. Reduced antigen presentation by MHC-I has been identified as potential resistance factor. Here we show that the epigenetic regulator ubiquitin-like with PHD and ring finger domains 1 (UHRF1), exhibits altered expression and aberrant cytosolic localization in cancerous tissues, where it promotes MHC-I ubiquitination and degradation. Cytoplasmic translocation of UHRF1 is induced by its phosphorylation on a specific serine in response to signals provided by factors present in the tumor microenvironment (TME), such as TGF-β, enabling UHRF1 to bind MHC-I. Downregulation of MHC-I results in suppression of the antigen presentation pathway to establish an immune hostile TME. UHRF1 inactivation by genetic deletion synergizes with immune checkpoint blockade (ICB) treatment and induces an anti-tumour memory response by evoking low-affinity T cells. Our study adds to the understanding of UHRF1 in cancer immune evasion and provides a potential target to synergize with immunotherapy and overcome immunotherapeutic resistance.

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by synovial inflammation and progressive joint destruction. There is no cure, and patient responses to current therapies vary, reflecting underlying pathogenic heterogeneity. Leveraging single-cell RNA sequencing (scRNA-seq) of RA synovium, we identified a PLAUR/uPAR-high myeloid subset that co-expresses pathogenic mediators, including IL1B and CXCL8. To target these cells, we developed anti-uPAR antibody–drug conjugates (ADCs) and evaluated various payloads in vitro and in vivo. ADCs bearing BCL-2 family inhibitors selectively induced apoptosis in proinflammatory human monocytes and macrophages with elevated uPAR, while sparing unstimulated monocytes with low basal uPAR in vitro. The treatment also reduced CXCL8 secretion. Given that murine myeloid cells exhibited lower uPAR expression and reduced sensitivity to BCL-2 family inhibitors, we used a monomethyl auristatin F (MMAF) payload to demonstrate in vivo proof-of-concept. In an air-pouch model, the anti-uPAR–MMAF conjugate reduced uPAR^highCD11b⁺F4/80⁺ macrophages by 39% compared with the isotype control. Together, our study underscores the potential of ADCs to eliminate disease-relevant cell types with inducible cell surface markers. This work opens new avenues for exploring cytotoxic ADCs as targeted therapies for autoimmune and inflammatory diseases.

The IMC antibody and nuclear staining list