Multi-omics analyses of changes in the tumor microenvironment of pancreatic adenocarcinoma following neoadjuvant treatment with anti-PD-1 antibody
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ABSTRACT: Successful pancreatic ductal adenocarcinoma (PDAC) immunotherapy necessitates optimization and maintenance of activated effector T cells (Teffs). We prospectively collected and applied multi-omics analysis to paired, pre- and post-treatment PDAC specimens collected in a platform neoadjuvant study of GVAX vaccine +/- nivolumab (anti-PD-1) to uncover sensitivity and resistance mechanisms. We show that GVAX-induced tertiary lymphoid aggregates become immune-regulatory sites in response to GVAX+nivolumab. Higher densities of tumor-associated neutrophils (TANs) following GVAX+nivolumab portend poorer overall survival (OS). Increased T cells expressing CD137 associated with cytotoxic Teff signatures and correlated with increased OS. Bulk and single-cell RNA-sequencing found that nivolumab alters CD4+ T cell chemotaxis signaling in association with CD11b+ neutrophil degranulation, and CD8+ T cell expression of CD137 required for optimal T cell activation. These findings provide new insights into PD-1-regulated immune pathways in PDAC that should inform more effective therapeutic combinations that include TAN regulators and T cell activators.
Project description:Tumor-associated neutrophils (TANs) are heterogeneous; thus, their roles in tumor development could vary depending on the cancer type. Here, we showed that TANs were more detrimental to metabolic dysfunction-associated steatohepatitis hepatocellular carcinoma (MASH-related HCC) than to viral-associated HCC. We attributed this difference to the predominance of SiglecFhi TANs in MASH-related HCC tumors. Linoleic acid and GM-CSF, which are commonly elevated in the MASH-related HCC microenvironment, fostered the development of this c-Myc-driven TAN subset. Through TGFβ secretion, SiglecFhi TANs promoted HCC stemness, proliferation, and migration. Importantly, SiglecFhi TANs supported immune evasion by directly suppressing the antigen presentation machinery of tumor cells. SiglecFhi TAN removal increased the immunogenicity of a MASH-related HCC model and sensitized it to immunotherapy. Likewise, a high SiglecFhi TAN signature was associated with poor prognosis and immunotherapy resistance in HCC patients. Overall, our study highlights the importance of understanding TAN heterogeneity in cancer to improve therapeutic development
Project description:Tumor-associated neutrophils (TANs) are heterogeneous; thus, their roles in tumor development could vary depending on the cancer type. Here, we showed that TANs were more detrimental to metabolic dysfunction-associated steatohepatitis hepatocellular carcinoma (MASH-related HCC) than to viral-associated HCC. We attributed this difference to the predominance of SiglecFhi TANs in MASH-related HCC tumors. Linoleic acid and GM-CSF, which are commonly elevated in the MASH-related HCC microenvironment, fostered the development of this c-Myc-driven TAN subset. Through TGFβ secretion, SiglecFhi TANs promoted HCC stemness, proliferation, and migration. Importantly, SiglecFhi TANs supported immune evasion by directly suppressing the antigen presentation machinery of tumor cells. SiglecFhi TAN removal increased the immunogenicity of a MASH-related HCC model and sensitized it to immunotherapy. Likewise, a high SiglecFhi TAN signature was associated with poor prognosis and immunotherapy resistance in HCC patients. Overall, our study highlights the importance of understanding TAN heterogeneity in cancer to improve therapeutic development
Project description:Polymorphonuclear neutrophil (PMN) tissue accumulation is an established feature of ulcerative colitis (UC) lesions and colorectal cancer (CRC). Given the emerging evidence of PMN phenotypic and functional heterogeneity, we analyzed the transcriptomic landscape of PMNs in blood and tissue during the spatiotemporal transition from inflammatory ulceration to CRC. Based on their transcriptional programs, PMNs were effectively stratified into distinct spatial compartments of peripheral blood, inflamed colon tissue, and the tumor niche. In silico pathway overrepresentation analysis, protein-network mapping, gene signature identification, and gene-ontology scoring revealed unique enrichment of angiogenic and vasculature development pathways in tumor-associated neutrophils (TANs). Functional studies utilizing ex vivo cultures, colitis-induced murine CRC, and patient-derived xenograft models demonstrated a critical role for TANs in promoting tumor vascularization. Spp1 (OPN) and Mmp14 (MT1-MMP) were identified by unbiased -omics and by mechanistic studies to be highly induced in TANs and function to critically regulate endothelial cell chemotaxis and branched network formation. TCGA dataset and clinical specimens confirmed enrichment of SPP1 and MMP14 in high-grade CRC but not in UC patients. Importantly, pharmacological inhibition of TAN trafficking or MMP14 activity effectively reduced tumor vascular density, leading to CRC regression. The current study identified a niche-directed PMN transcriptional reprograming and functional specialization, highlighting emerging PMN plasticity. Our findings define new TAN contributions to tumor vascularization, delineating a new therapeutic framework for CRC treatment focused on TAN angiogenic properties.
Project description:Background: Targeting tumor-associated macrophages through C-C chemokine receptor type 2 (CCRs) in pancreatic ductal adenocarcinoma (PDAC) improves the efficacy of chemotherapy and restores T cell immunity. Methods: We conducted a phase I/II single institution study (NCT03496662) combining chemotherapy gemcitabine and nab-paclitaxel (GnP), an oral CCR2/5 inhibitor BMS-813160 and nivolumab for four 28-day cycles as a neoadjuvant therapy for patients with borderline resectable (BR) or locally advanced (LA) PDAC. The recommended phase 2 dose (RP2D) of BMS-813160 was established in the standard 3+3 design. Primary end points were safety and objective response rate (ORR). Secondary end points included resection rate, progression-free survival (PFS) and overall survival (OS). Results: 8 patients were treated with GnP alone (control arm) and 31 patients (29 response evaluable) were treated at RP2D. No grade 3 or 4 toxicities attributed to nivolumab or BMS-813160 were identified. After all 4 cycles of study treatment (N=26), ORR was 35.7%, 16.7% among BR- and LA-PDAC patients, compared to 0% of control arm patients. 78.6% BR- and 16.7% of LA-PDAC patients who completed study treatment underwent surgical resection. For intent-to-treat analyses, BR-PDAC patients had a mPFS and mOS of 14.6 and 20.4 months respectively; and LA-PDAC patients had a mPFS and mOS of 14.7 and 17 months respectively. Single cell RNAseq analysis showed enhanced proliferating CD4 and CD8 T cells and gene signatures indicative of effector function. Conclusions: Neoadjuvant BMS-813160/nivolumab/GnP was well tolerated and appears to achieve higher ORR and resectability than historical data, warranting a larger randomized phase II study.
Project description:Intratumoral microbiota can impact the development and progression of many types of cancer, including gastric cancer (GC). A better understanding of the precise mechanisms by which microbiota support GC could lead to improved therapeutic approaches. Here, we investigated the effect of intratumoral microbiota on the tumor immune microenvironment (TIME) during GC malignant progression. Analysis of human GC tissues with 16S rRNA amplicon sequencing revealed that Fusobacterium nucleatum (F. nucleatum) was significantly enriched in GC tissues with lymph node metastasis and correlated with a poor prognosis. F. nucleatum infection spontaneously induced chronic gastritis and promoted gastric mucosa dysplasia in mice. Furthermore, GC cells infected with F. nucleatum showed accelerated growth in immunocompetent mice compared to immunodeficient mice. Single-cell RNA sequencing uncovered that F. nucleatum recruited tumor-associated neutrophils (TANs) to reshape the tumor immune microenvironment. Mechanistically, F. nucleatum invaded GC cells and activated IL-17/NF-κB/RelB signaling, inducing TAN recruitment. F. nucleatum also stimulated TAN differentiation into the pro-tumoral subtype and subsequent promotion of PD-L1 expression, further facilitating GC immune evasion while also enhancing the efficacy of anti-PD-L1 antibody therapy. Together, this data uncovers mechanisms by which F. nucleatum affects GC immune evasion and immunotherapy efficacy, providing insights for developing effective treatment strategies.
Project description:Successful pancreatic ductal adenocarcinoma (PDAC) immunotherapy requires therapeutic combinations that induce quality T cells. Tumor microenvironment (TME) analysis following therapeutic interventions can identify response mechanisms to guide design of more effective combinations. We provide a reference single-cell dataset from PDAC-infiltrating T cell and monocyte subsets from a human neoadjuvant clinical trial comparing the granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting allogeneic PDAC vaccine GVAX alone, in combination with anti-PD1, or with both anti-PD1 and a CD137 agonist. Tumor infiltrating leukocytes analyzed two weeks post neoadjuvant therapy revealed shifts in CD8+ T cell activation as well as cytoskeletal and extracellular matrix (ECM)-interacting components with Cy/GVAX and anti-PD1. Addition of a CD137 agonist was associated with increased abundance of activated and clonally expanded CD8+ T cells with distinct perturbations in cytoskeletal and ECM components. Furthermore, a new computational method to compare ligand-receptor networks between patients from different treatment arms found that CD137 agonism is associated with immunosuppressive TREM2 signaling in tumor associated macrophages (TAMs) that corresponds to changes in cell metabolism, function, and ECM interactions. Altogether these findings associate therapy with GVAX, anti-PD1, and CD137 agonist with enhanced CD8+ T cell function while inducing alternative immunosuppressive pathways in patients with PDAC.
Project description:Neutrophils are the most abundant circulating leucocytes and constitute an essential component of innate immunity. Although their role in cancer development is still poorly defined, pro- or anti-tumor properties have been attributed to tumor-associated neutrophils (TANs), suggesting major functional diversities. In this study, we focused on the mechanisms involved in neutrophil accumulation within the lung tumor mass. We first identified G-CSF as an inducer of the high-affinity glucose transporter Glut1 in neutrophils, increasing their survival ex vivo. In a genetically engineered mouse model of lung adenocarcinoma, we report that TANs have an increased Glut1 expression and glucose metabolism compared to normal neutrophils. To elucidate the impact of glucose uptake on TANs, we used an in vivo strategy based on two recombinases, Flp to initiate lung tumors, and Cre to delete Glut1 specifically in neutrophils. We demonstrate that the loss of Glut1 decreases the SiglecFhigh TAN subpopulation by accelerating neutrophil turnover in tumors through reduced survival and augmented recruitment. Accelerated TAN turnover led to a decreased tumor growth and synergized with radiotherapy. Altogether, our results demonstrate the importance of Glut1 for neutrophil turnover, which directly affects the pro- versus anti-tumor balance within the tumor. These results also suggest that metabolic vulnerabilities can be exploited to target tumor-supportive neutrophil populations.
Project description:Neutrophils are the most abundant circulating leucocytes and constitute an essential component of innate immunity. Although their role in cancer development is still poorly defined, pro- or anti-tumor properties have been attributed to tumor-associated neutrophils (TANs), suggesting major functional diversities. In this study, we focused on the mechanisms involved in neutrophil accumulation within the lung tumor mass. We first identified G-CSF as an inducer of the high-affinity glucose transporter Glut1 in neutrophils, increasing their survival ex vivo. In a genetically engineered mouse model of lung adenocarcinoma, we report that TANs have an increased Glut1 expression and glucose metabolism compared to normal neutrophils. To elucidate the impact of glucose uptake on TANs, we used an in vivo strategy based on two recombinases, Flp to initiate lung tumors, and Cre to delete Glut1 specifically in neutrophils. We demonstrate that the loss of Glut1 decreases the SiglecFhigh TAN subpopulation by accelerating neutrophil turnover in tumors through reduced survival and augmented recruitment. Accelerated TAN turnover led to a decreased tumor growth and synergized with radiotherapy. Altogether, our results demonstrate the importance of Glut1 for neutrophil turnover, which directly affects the pro- versus anti-tumor balance within the tumor. These results also suggest that metabolic vulnerabilities can be exploited to target tumor-supportive neutrophil populations.
Project description:Pre-treatment tumor expression of PD-L1 has been shown to correlate with favorable clinical outcomes following PD-1 blocking therapies. However, the majority of patients with PD-L1+ tumors do not respond to treatment. With the goal of obtaining insights into the mechanisms underlying the response to anti-PD-1 targeted therapies in patients with renal cell carcinoma (RCC) with positive tumor expression of PD-L1, microarray expression data was obtained for pre-treatment tumors from patients with RCC who did or did not have a positive response to anti-PD-1 (nivolumab) therapy. Insight into these mechanisms may lead to improved rationally designed therapies, and biomarkers for selecting patients who are more likely to benefit from these treatments. Gene expression profiling was performed on total RNA extracted from 11 formalin-fixed paraffin-embedded (FFPE) RCC specimens, 4 of which were from patients who had a positive response (objective tumor regression) to the anti-PD-1 (nivolumab) immunotherapy, and 7 of which were from patients who did not have a response. Details of the design, and the gene signatures found are given in the paper associated with this GEO Series: Maria Libera Ascierto, Tracee L. McMiller, Alan E. Berger, Ludmila Danilova, Robert A. Anders, George J. Netto, Haiying Xu, Theresa S. Pritchard, Jinshui Fan, Chris Cheadle, Leslie Cope, Charles G. Drake, Drew M. Pardoll, Janis M. Taube, and Suzanne L. Topalian, The Intratumoral Balance between Metabolic and Immunologic Gene Expression Is Associated with Antiâ??PD-1 Response in Patients with Renal Cell Carcinoma, published online in Cancer Immunology Research on 4 August 2016, doi: 10.1158/2326-6066.CIR-16-0072 http://cancerimmunolres.aacrjournals.org/content/early/2016/07/28/2326-6066.CIR-16-0072.full.pdf+html
Project description:Lysine (K)-specific demethylase 6A (KDM6A) is a frequently mutated tumor suppressor gene in pancreatic ductal adenocarcinoma (PDAC). However, how KDM6A loss impacts PDAC tumor immune microenvironment is not known. Tumor-associated neutrophils (TANs) and neutrophil extracellular traps (NETs) in the tumor microenvironment contribute to PDAC progression. This study used genetically engineered pancreas-specific Kdm6a-knockout PDAC mouse model and human PDAC tissue samples to demonstrate that KDM6A loss correlates with increased TANs and NETs formation. Genome-wide Bru-seq analysis showed that the expression of many chemotactic cytokines, especially CXC motif chemokine ligand 1 (CXCL1), were upregulated in KDM6A-knockout PDAC cells. We confirmed that KDM6A-deficient PDAC cells secreted higher levels of CXCL1 protein, which in turn recruits neutrophils. Furthermore, the CXCL1 neutralizing antibody blocked the chemotactic and NETs-promoting property of KDM6A-deficient PDAC cells and tumor growth in a syngeneic orthotopic xenograft mouse model, confirming that CXCL1 was the main mediator of chemotaxis and PDAC growth in this model. These findings shed light on how KDM6A regulates tumor immune microenvironment and PDAC progression and suggest that the CXCL1-CXCR2 axis may be a candidate target for treating PDACs with KDM6A loss.