Defining cellular determinants of resistance to PD-1 pathway blockade in non-small-cell lung cancer

Despite sustained clinical responses to immune-checkpoint blockade (ICB) therapies in non-small-cell lung cancer (NSCLC), the majority of patients derive no clinical benefits, and the cellular and molecular underpinnings of such resistance remain incompletely understood. To identify cell types that may influence immunotherapy responses, we first integrated newly generated and previously published single-cell RNA sequencing data from 110 treatment-naive patients with NSCLC. Among tumor-resident cell types, we identified MMP1+ cancer-associated fibroblasts (CAFs), which were inversely correlated with the level of tumor-reactive T cells--a key determinant of response to ICB. Further single-cell analysis for newly collected 21 tumor samples from NSCLC patients treated with anti-PD-1/PD-L1 agents revealed that MMP1+ fibroblasts were indeed enriched in treatment-refractory patients, and this observation was also validated in an independent dataset of bulk RNA sequencing from 344 NSCLC patients treated with PD-L1 agents. Examination of the spatial architecture showed that MMP1+ fibroblasts were located at the tumor-stroma boundary, forming a single-cell layer that encircled the cancer cell aggregates, and we hence defined MMP1+ fibroblasts as tumor-stroma boundary (tsb)CAFs. Such tsbCAFs likely promote resistance to ICB by functioning as a physical barrier that prevents tumor-reactive T cells from recognizing and killing cancer cells. Our study provides a new framework to identify cellular underpinnings of resistance to ICB and suggests new strategies to overcome ICB resistance. Highlights{lozenge} Identification and characterization of MMP1+ fibroblasts in lung cancer. {lozenge}Single-cell meta-analysis reveals cell populations impeding the accumulation of tumor-reactive T cells. {lozenge}MMP1+ fibroblasts correlate with the low infiltration of tumor-reactive T cells and the resistance to anti-PD-1/PD-L1 treatment. {lozenge}MMP1+ fibroblasts appear to form a space barrier between malignant and T cells.