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Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy. Fibroblast heterogeneity is a prominent but poorly understood feature of solid tumours. Here three major fibroblast subpopulations in non-small cell lung cancer are identified and characterised through single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry.

Idiopathic pulmonary fibrosis (IPF), the prototypic progressive fibrotic interstitial lung disease, is thought to be a consequence of repetitive micro-injuries to an ageing, susceptible alveolar epithelium. Ageing is a risk factor for IPF and incidence has been demonstrated to increase with age. Decreased (macro)autophagy with age has been reported extensively in a variety of systems and diseases, including IPF. However, it is undetermined whether the role of faulty autophagy is causal or coincidental in the context of IPF. Here, we report that in alveolar epithelial cells inhibition of autophagy promotes epithelial–mesenchymal transition (EMT), a process implicated in embryonic development, wound healing, cancer metastasis and fibrosis. We further demonstrate that this is attained, at least in part, by increased p62/SQSTM1 expression that promotes p65/RELA mediated-transactivation of an EMT transcription factor, Snail2 ( SNAI2 ), which not only controls EMT but also regulates the production of locally acting profibrogenic mediators. Our data suggest that reduced autophagy induces EMT of alveolar epithelial cells and can contribute to fibrosis via aberrant epithelial–fibroblast crosstalk.

Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis. Idiopathic pulmonary fibrosis (IPF) is a devastating disease of the lung, which scars the tissue and gradually destroys the organ, ultimately leading to death. It is still unclear what exactly causes this scarring, but it is thought that increasing amounts of proteins in the space surrounding the cells of the lungs, the extracellular matrix, could play a role. These proteins, including collagen, normally form a ‘scaffold’ to stabilize cells, but if they accumulate uncontrollably, they can render tissues rigid. It has been assumed that these changes are a consequence of the disease. However, recent evidence suggests that the increased stiffness itself could stimulate cells to produce even more extracellular matrix, driving the progression of the disease. A better understanding of what exactly causes the tissue to become gradually stiffer may identify new ways to block the progression of IPF. Now, Jones et al. compared measurements of the tissue stiffness and the collagen structure taken from samples of patients with IPF. The results showed that the collagen fibres were faulty and had an abnormal shape. This suggests that these problems, rather than an increased amount of collagen, alter the flexibility of the lung tissue. Jones et al. also found that a specific family of proteins, which helps to connect the collagen fibres, was increased in the tissue of patients with IPF. When these proteins were blocked with a newly developed drug, the collagen structure returned to normal and the stiffness of the tissue decreased. As a consequence, the lung capacity improved. This suggests that treatment approaches that help to maintain a normal collagen structure, may in future prevent the stiffening of the lung tissue and so limit feed-forward mechanisms that drive progressive IPF. Moreover, it indicates that measurements of the structure of collagen rather than the its total concentration could serve as a more suitable indicator for the disease.

Background Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. Methods We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. Results We report that TAMs are FcγRIIb bright relative to healthy tissue counterparts and under hypoxic conditions , mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb + / + transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. Conclusion Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

Vijayanand and colleagues use genome-wide RNA sequencing for transcriptional profiling of CD8 + T cells from tumors and adjacent uninvolved lung tissue from patients with early-stage lung cancer. A tissue-resident memory signature is associated with enhanced cytotoxicity and improved survival. Therapies that boost the anti-tumor responses of cytotoxic T lymphocytes (CTLs) have shown promise; however, clinical responses to the immunotherapeutic agents currently available vary considerably, and the molecular basis of this is unclear. We performed transcriptomic profiling of tumor-infiltrating CTLs from treatment-naive patients with lung cancer to define the molecular features associated with the robustness of anti-tumor immune responses. We observed considerable heterogeneity in the expression of molecules associated with activation of the T cell antigen receptor (TCR) and of immunological-checkpoint molecules such as 4-1BB, PD-1 and TIM-3. Tumors with a high density of CTLs showed enrichment for transcripts linked to tissue-resident memory cells (T RM cells), such as CD103 , and CTLs from CD103 hi tumors displayed features of enhanced cytotoxicity. A greater density of T RM cells in tumors was predictive of a better survival outcome in lung cancer, and this effect was independent of that conferred by CTL density. Here we define the 'molecular fingerprint' of tumor-infiltrating CTLs and identify potentially new targets for immunotherapy.

Immune-checkpoint blockade (ICB) has shown remarkable clinical success in boosting antitumor immunity. However, the breadth of its cellular targets and specific mode of action remain elusive. We find that tumor-infiltrating follicular regulatory T (T FR ) cells are prevalent in tumor tissues of several cancer types. They are primarily located within tertiary lymphoid structures and exhibit superior suppressive capacity and in vivo persistence as compared with regulatory T cells, with which they share a clonal and developmental relationship. In syngeneic tumor models, anti-PD-1 treatment increases the number of tumor-infiltrating T FR cells. Both T FR cell deficiency and the depletion of T FR cells with anti-CTLA-4 before anti-PD-1 treatment improve tumor control in mice. Notably, in a cohort of 271 patients with melanoma, treatment with anti-CTLA-4 followed by anti-PD-1 at progression was associated with better a survival outcome than monotherapy with anti-PD-1 or anti-CTLA-4, anti-PD-1 followed by anti-CTLA-4 at progression or concomitant combination therapy. Vijayanand and colleagues show highly suppressive CD4 + CTLA-4 + PD-1 + follicular regulatory T (T FR ) cells reside within tumor microenvironments. Depleting T FR cells or blocking their activity with CTLA-4-depleting antibodies before anti-PD-1 checkpoint blockade therapy improved the efficacy of anti-PD-1 treatment in mouse tumor models and was also associated with better survival outcomes in a large cohort of patients with melanoma.

The contribution of epithelial–mesenchymal transition (EMT) to human lung fibrogenesis is controversial. Here we provide evidence that ZEB1-mediated EMT in human alveolar epithelial type II (ATII) cells contributes to the development of lung fibrosis by paracrine signalling to underlying fibroblasts. Activation of EGFR–RAS–ERK signalling in ATII cells induced EMT via ZEB1. ATII cells had extremely low extracellular matrix gene expression even after induction of EMT, however conditioned media from ATII cells undergoing RAS-induced EMT augmented TGFβ-induced profibrogenic responses in lung fibroblasts. This epithelial–mesenchymal crosstalk was controlled by ZEB1 via the expression of tissue plasminogen activator (tPA). In human fibrotic lung tissue, nuclear ZEB1 expression was detected in alveolar epithelium adjacent to sites of extracellular matrix (ECM) deposition, suggesting that ZEB1-mediated paracrine signalling has the potential to contribute to early fibrotic changes in the lung interstitium. Targeting this novel ZEB1 regulatory axis may be a viable strategy for the treatment of pulmonary fibrosis.

The role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor's anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer. Macrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8 tissue-resident memory T cells (T ) in tumors and survival data from an independent cohort of 393 patients with lung cancer. TAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1 ). Importantly, there was a strong association between the density of M1 TAMs and T cells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1 TAMs may recruit T cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which T depend. We showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1 TAMs was associated with a strong T tumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1 phenotype are likely to augment the adaptive antitumor responses.

Background: We systematically assessed the prognostic and predictive value of infiltrating adaptive and innate immune cells in a large cohort of patients with advanced mesothelioma. Methods: A tissue microarray from 302 samples was constructed. Markers of adaptive immune response in T-cells (CD8 + , FOXP3 + , CD4 + , CD45RO + , CD3 + ) and B-cells (CD20 + ), and of innate immune response; neutrophils (NP57 + ), natural killer cells (CD56 + ) and macrophages (CD68 + ) were evaluated. Results: We found that in the epithelioid tumours, high CD4 + and CD20 + counts, and low FOXP3 + , CD68 + and NP57 + counts linked to better outcome. In the non-epithelioid group low CD8 + and low FOXP3 + counts were beneficial. On multivariate analysis low FOXP3 + remained independently associated with survival in both groups. In the epithelioid group additionally high CD4 + , high CD20 + , and low NP57 + counts were prognostic. Conclusions: Our data demonstrate for the first time, in predominately advanced disease, the association of key markers of adaptive and innate immunity with survival and the differential effect of histology. A better understanding of the immunological drivers of the different subtypes of mesothelioma will assist prognostication and disease-specific clinical decision-making.

Brain tumors in children are a devastating disease in a high proportion of patients. Owing to inconsistent results in clinical trials in unstratified patients, the role of immunotherapy remains unclear. We performed an in-depth survey of the single-cell transcriptomes and clonal relationship of intra-tumoral T cells from children with brain tumors. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with the potential to recognize tumor antigens. Such clonally expanded T cells display enrichment of transcripts linked to effector function, tissue residency, immune checkpoints and signatures of neoantigen-specific T cells and immunotherapy response. We identify neoantigens in pediatric brain tumors and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes. Notably, among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and magnitude of T cell response. Our findings suggest that characterization of intra-tumoral T cell responses may enable selection of patients for immunotherapy, an approach that requires prospective validation in clinical trials.