Lack of TGFß signaling competency predicts immune poor cancer conversion to immune rich and response to checkpoint blockade.

Background: Transforming growth factor beta (TGFß) is well-recognized as an immunosuppressive player in the tumor microenvironment but also has a significant impact on cancer cell phenotypes. Loss of TGFß signaling impairs DNA repair competency, which is described by a transcriptomic score, ßAlt. Cancers with high ßAlt have more genomic damage and are more responsive to genotoxic therapy. The growing appreciation that cancer DNA repair deficits are important determinants of immune response prompted us to investigate the association of ßAlt with response to immune checkpoint blockade (ICB). We predicted that high ßAlt tumors would be infiltrated with lymphocytes because of DNA damage burden and hence responsive to ICB. Methods: We analyzed public transcriptomic data from clinical trials and preclinical models using transcriptomic signatures of TGFß targets, DNA repair genes, tumor educated immune cells and interferon. A high ßAlt, immune poor mammary tumor derived transplant model resistant to programmed death ligand 1 (PD-L1) antibodies was studied using multispectral flow cytometry to interrogate the immune system. Results: Metastatic bladder patients in IMvigor 210 who responded to ICB had significantly increased ßAlt scores and experienced significantly longer overall survival compared to those with low ßAlt scores (hazard ratio 0.62, P=0.011) . Unexpectedly, 75% of high ßAlt cancers were immune poor as defined by low expression of tumor educated immune cell and interferon signatures. The association of high ßAlt with immune poor cancer was also evident in TCGA and preclinical cancer models. We used a high ßAlt, immune poor cancer to test therapeutic strategies to overcome its inherent anti-PD-L1 resistance. Combination treatment with radiation and TGFß inhibition were necessary for lymphocytic infiltration and activated NK cells were required for ICB response. Bioinformatic analysis identified high ßAlt, immune poor B16 and CT26 preclinical models and paired biopsies of cancer patients that also demonstrated NK cell activation upon response to ICB. Conclusions: Our studies support ßAlt as a biomarker that predicts response to ICB albeit in immune poor cancers, which has implications for the development of therapeutic strategies to increase the number of cancer patients who will benefit from immunotherapy.

Dysregulation of CD4+ and CD8+ resident memory T, myeloid, and stromal cells in steroid-experienced, checkpoint inhibitor colitis.

BACKGROUND: Colitis caused by checkpoint inhibitors (CPI) is frequent and is treated with empiric steroids, but CPI colitis mechanisms in steroid-experienced or refractory disease are unclear. METHODS: Using colon biopsies and blood from predominantly steroid-experienced CPI colitis patients, we performed multiplexed single-cell transcriptomics and proteomics to nominate contributing populations. RESULTS: CPI colitis biopsies showed enrichment of CD4+resident memory (RM) T cells in addition to CD8+ RM and cytotoxic CD8+ T cells. Matching T cell receptor (TCR) clonotypes suggested that both RMs are progenitors that yield cytotoxic effectors. Activated, CD38+ HLA-DR+ CD4+ RM and cytotoxic CD8+ T cells were enriched in steroid-experienced and a validation data set of steroid-naïve CPI colitis, underscoring their pathogenic potential across steroid exposure. Distinct from ulcerative colitis, CPI colitis exhibited perturbed stromal metabolism (NAD+, tryptophan) impacting epithelial survival and inflammation. Endothelial cells in CPI colitis after anti-TNF and anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) upregulated the integrin α4ß7 ligand molecular vascular addressin cell adhesion molecule 1 (MAdCAM-1), which may preferentially respond to vedolizumab (anti-α4ß7). CONCLUSIONS: These findings nominate CD4+ RM and MAdCAM-1+ endothelial cells for targeting in specific subsets of CPI colitis patients.

Tumor-associated macrophages restrict CD8+ T cell function through collagen deposition and metabolic reprogramming of the breast cancer microenvironment.

Tumor progression is accompanied by fibrosis, a condition of excessive extracellular matrix accumulation, which is associated with diminished antitumor immune infiltration. Here we demonstrate that tumor-associated macrophages (TAMs) respond to the stiffened fibrotic tumor microenvironment (TME) by initiating a collagen biosynthesis program directed by transforming growth factor-ß. A collateral effect of this programming is an untenable metabolic milieu for productive CD8+ T cell antitumor responses, as collagen-synthesizing macrophages consume environmental arginine, synthesize proline and secrete ornithine that compromises CD8+ T cell function in female breast cancer. Thus, a stiff and fibrotic TME may impede antitumor immunity not only by direct physical exclusion of CD8+ T cells but also through secondary effects of a mechano-metabolic programming of TAMs, which creates an inhospitable metabolic milieu for CD8+ T cells to respond to anticancer immunotherapies.

Single-cell and spatial multi-omics highlight effects of anti-integrin therapy across cellular compartments in ulcerative colitis.

Ulcerative colitis (UC) is driven by immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin antibody that is effective for treating UC. VDZ is known to inhibit lymphocyte trafficking to the intestine, but its broader effects on other cell subsets are less defined. To identify the inflammatory cells that contribute to colitis and are affected by VDZ, we perform single-cell transcriptomic and proteomic analyses of peripheral blood and colonic biopsies in healthy controls and patients with UC on VDZ or other therapies. Here we show that VDZ treatment is associated with alterations in circulating and tissue mononuclear phagocyte (MNP) subsets, along with modest shifts in lymphocytes. Spatial multi-omics of formalin-fixed biopsies demonstrates trends towards increased abundance and proximity of MNP and fibroblast subsets in active colitis. Spatial transcriptomics of archived specimens pre-treatment identifies epithelial-, MNP-, and fibroblast-enriched genes related to VDZ responsiveness, highlighting important roles for these subsets in UC.

Single cell proteomics characterization of bone marrow hematopoiesis with distinct Ras pathway lesions.

Normal hematopoiesis requires constant prolific production of different blood cell lineages by multipotent hematopoietic stem cells (HSC). Stem- and progenitor- cells need to balance dormancy with proliferation. How genetic alterations impact frequency, lineage potential, and metabolism of HSC is largely unknown. Here, we compared induced expression of KRAS G12D or RasGRP1 to normal hematopoiesis. At low-resolution, both Ras pathway lesions result in skewing towards myeloid lineages. Single-cell resolution CyTOF proteomics unmasked an expansion of HSC- and progenitor- compartments for RasGRP1, contrasted by a depletion for KRAS G12D . SCENITH™ quantitates protein synthesis with single-cell precision and corroborated that immature cells display low metabolic SCENITH™ rates. Both RasGRP1 and KRAS G12D elevated mean SCENITH™ signals in immature cells. However, RasGRP1-overexpressing stem cells retain a metabolically quiescent cell-fraction, whereas this fraction diminishes for KRAS G12D . Our temporal single cell proteomics and metabolomics datasets provide a resource of mechanistic insights into altered hematopoiesis at single cell resolution.