Lung CAFs Show T Cells the Velvet Rope

Immune-checkpoint blockade is an effective therapeutic approach in several cancer types, including non–small cell lung cancer (NSCLC). However, only a subset of NSCLC patients exhibit durable responses to immunotherapy, motivating efforts to understand tumor features and underlying mechanisms associated with resistance to these interventions. Heterogeneity in response to immunotherapy is explained in part by differential T-cell abundance in tumors across the patient population and specifically the variable frequencies of T cells in proximity to tumor cells (1). Previous efforts to understand mechanisms of T-cell exclusion from tumor nests implicated extracellular matrix (ECM) fibers in the restriction of T-cell motility (2). Real-time imaging of viable human lung cancer specimens showed chemokine-driven T-cell movement through tumor regions harboring loose fibronectin and collagen, but poor T-cell migration into and through regions of dense ECM featuring aligned collagen fibers. These prior findings motivate efforts to understand the cellular orchestrators of tumor architecture that restrict T-cell migration into tumor nests, as well as the specific molecular mediators of T-cell exclusion. Grout and colleagues set out to address these questions and identified two novel cancer-associated fibroblast (CAF) populations with immune-regulatory potential in human lung cancer in the process.

To identify cell populations mediating T-cell exclusion from tumor nests, the authors focused on CAFs in human lung cancer (3). Like fibroblasts in secondary lymphoid organs, CAFs regulate immune responses in diverse tumor types, regulating the abundance and phenotypes of various lymphoid and myeloid cell populations including intratumoral T cells (4). Using single-cell RNA sequencing (scRNA-seq), the authors identified diverse CAF populations in human lung cancer specimens (Fig. 1), which were validated by mass cytometry by time of flight (CyTOF) and multiplex IHC and which indicated intra- and intertumor heterogeneity. ADH1B marked a CAF population also characterized by low or absent expression of conventional CAF markers including FAP. The ADH1B⁺ CAF population had a notably similar expression profile to alveolar fibroblasts in benign adjacent lung tissue, suggesting a potential tissue-resident cell of origin. The common CAF marker FAP marked three distinct clusters, which harbored a variable expression of an additional CAF marker, αSMA, and included prominent FAP⁺αSMA⁺ CAFs. Analysis of genes associated with ADH1B⁺ versus FAP⁺ CAFs in publicly available datasets showed a significant separation of the two gene groups in pancreatic, breast, and colon cancers, highlighting the relevance of these findings in diverse solid tumor types. An additional, clearly distinct CAF cluster lacked FAP expression but had high expression of αSMA as well as MYH11, a marker of diverse mesenchymal and smooth muscle cell types. As multiplex IHC captures these heterogeneous CAF populations as well as their spatial relationships within the tumor microenvironment, this analysis demonstrated that the MYH11⁺αSMA⁺ CAFs form a single layer surrounding tumor nests, similar to a subset of αSMA^hi myofibroblastic CAFs documented in pancreatic cancer (5). In contrast, the ALDH1B⁺ CAFs and FAP⁺ CAFs generally localized to the stroma. The authors further analyzed these CAF populations with respect to tumor stage and found that low-stage lung cancer samples were dominated by ADH1B⁺ CAFs and sometimes had or sometimes lacked MYH11⁺αSMA⁺ CAFs, whereas higher-stage lung cancer was dominated by FAP⁺ CAFs including FAP⁺αSMA⁺ CAFs. These changes to CAF composition over the course of lung cancer progression may reflect the accumulation of CAFs of distinct cellular origins over time or may reflect similar mesenchymal cell types responding to a different milieu of secreted factors at distinct stages of lung tumorigenesis. Heterogeneity among tumor stages was accompanied by heterogeneity among patients, with some patient samples rich in FAP⁺ CAFs but lacking MYH11⁺αSMA⁺ CAFs, whereas others—about half of the tumors rich in ALDH1B⁺ CAFs—harbored prominent populations of MYH11⁺αSMA⁺ CAFs. The significance of tumor genotype, etiology, or subtype as a determinant of CAF composition remains to be determined for these populations but may hold relevance to disease biology and response to therapy.

To assess the immune-modulatory potential of these defined CAF populations, Grout and colleagues performed multiplex imaging of formalin-fixed, paraffin-embedded lung cancer samples and analyzed spatial relationships among CAFs and immune cells. Immune-modulatory potential was also probed using scRNA-seq data to determine the expression of different ligands for immune cell types of interest by CAFs. A subset of ALDH1B⁺ CAFs expressed high levels of T-cell chemoattraction or retention factors such as CCL19, CCL21, and VCAM1, like fibroblastic reticular cells in lymph nodes. Interestingly, this ALDH1B⁺ CAF subset was found exclusively within tertiary lymphoid structures and expressed MHCII. Although MHCII expression by these CAFs was substantially lower than expression among type I dendritic cells, these CAFs certainly may participate in meaningful antigen presentation to T cells. This notion is consistent with a recent study documenting MHCII-expressing, antigen-presenting CAFs (apCAF) in human lung tumors (6). These lung apCAFs actively promoted CD4⁺ T cell–mediated antitumor immunity in part by activating the T-cell receptors of effector CD4⁺ T cells and in part by secreting C1q to rescue these T cells from apoptosis. In contrast, mesothelial cell–derived apCAFs in pancreatic cancer were recently shown to suppress antitumor immunity at least in part by inducing naïve CD4⁺ T cells into regulatory T cells in an antigen-specific manner (7). This context dependency with respect to interactions between immune and nonimmune stroma likely extends beyond apCAFs and may reflect conserved immune responses in the tissue of origin.

In contrast to these potentially immune-activating CAF populations, other CAF subtypes identified by Grout and colleagues appear to suppress antitumor immunity. Spatial distribution of CD3⁺ and CD8⁺ T cells was independent of total FAP⁺ and ALDH1B⁺ CAF frequencies, but in tumor lesions with MYH11⁺αSMA⁺ CAFs, the frequencies of CD3⁺ and CD8⁺ T cells infiltrating tumor nests was markedly reduced among stage I patients compared with patients lacking this CAF subset. The subset of FAP⁺ CAFs with high αSMA expression also associated with the poor infiltration of CD3⁺ and CD8⁺ T cells into tumor nests, prompting the authors to investigate factors produced by these two CAF populations potentially implicated in the spatial regulation of T cells. MYH11⁺αSMA⁺ CAFs produced high levels of TGFB1, WNT5A, and WNT11, all of which may suppress T-cell activation or function. ECM components that may also contribute to T-cell exclusion from regions adjacent to tumor cells included COL9A1, COL27A1, COL4A1, and COL4A2. Notably, collagen IV fibers lined tumor nests surrounded by MYH11⁺αSMA⁺ CAFs but not other CAF subtypes, implicating collagen IV derived from these CAFs as a potential barrier to T-cell infiltration. In contrast, FAP⁺αSMA⁺ CAFs produced high levels of COL11A1 and COL12A1, and collagen XI and XII abundance specifically correlated with αSMA. Consistent with these findings, FAP⁺ CAFs limit the efficacy of immune-checkpoint blockade and restrict T-cell trafficking into tumor cell–adjacent regions in mouse models of pancreatic cancer (8); whether these findings reflect the functions of a subset of FAP⁺ CAFs and a function of specific ECM component production remains to be determined. Lung CAFs were recently shown to suppress antitumor immunity in the context of breast cancer lung metastasis, albeit by a distinct mechanism (9). In this setting, a population of PTGS2 (COX-2)-expressing CAFs produced prostaglandin E2 and IL1β, together promoting dysfunctional dendritic cells and suppressive monocytes to support a premetastatic niche. Together, these findings demonstrate that host tissue as well as cancer tissue of origin dictates CAF composition and function.

This study by Grout and colleagues highlights several important areas for future investigation. The relative significance of CAF-derived ECM proteins versus soluble mediators for T-cell spatial regulation will be meaningful to parse to help identify combination therapeutic strategies that may potentially bolster the efficacy of immune-checkpoint blockade in patients with lung cancer harboring high levels of these T cell–restraining CAF populations. The relationships between tumor genotype or cancer cell–intrinsic signaling pathways and CAF composition will also be important to assess as a potential basis for patient stratification and to inform us about the heterogeneous biology of lung tumor–stroma interactions. The significance of ALDH1B⁺ CAFs within tertiary lymphoid structures may also inform us on determinants of antitumor immunity in lung cancer, with potential relevance in other tumor types. Overall, this study underscores the power of combined gene expression profiling and spatial analysis of stromal cell types to discover novel heterocellular interactions regulating tumor immunity.

No disclosures were reported.