CSF1/CSF1R blockade reprograms tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in pancreatic cancer models.
ABSTRACT: Cancer immunotherapy generally offers limited clinical benefit without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment. Critical drivers of immune escape in the tumor microenvironment include tumor-associated macrophages and myeloid-derived suppressor cells, which not only mediate immune suppression, but also promote metastatic dissemination and impart resistance to cytotoxic therapies. Thus, strategies to ablate the effects of these myeloid cell populations may offer great therapeutic potential. In this report, we demonstrate in a mouse model of pancreatic ductal adenocarcinoma (PDAC) that inhibiting signaling by the myeloid growth factor receptor CSF1R can functionally reprogram macrophage responses that enhance antigen presentation and productive antitumor T-cell responses. Investigations of this response revealed that CSF1R blockade also upregulated T-cell checkpoint molecules, including PDL1 and CTLA4, thereby restraining beneficial therapeutic effects. We found that PD1 and CTLA4 antagonists showed limited efficacy as single agents to restrain PDAC growth, but that combining these agents with CSF1R blockade potently elicited tumor regressions, even in larger established tumors. Taken together, our findings provide a rationale to reprogram immunosuppressive myeloid cell populations in the tumor microenvironment under conditions that can significantly empower the therapeutic effects of checkpoint-based immunotherapeutics.
Project description:Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with an immunosuppressive microenvironment that is resistant to most therapies. IL17 is involved in pancreatic tumorigenesis, but its role in invasive PDAC is undetermined. We hypothesized that IL17 triggers and sustains PDAC immunosuppression. We inhibited IL17/IL17RA signaling using pharmacological and genetic strategies alongside mass cytometry and multiplex immunofluorescence techniques. We uncovered that IL17 recruits neutrophils, triggers neutrophil extracellular traps (NETs), and excludes cytotoxic CD8 T cells from tumors. Additionally, IL17 blockade increases immune checkpoint blockade (PD-1, CTLA4) sensitivity. Inhibition of neutrophils or Padi4-dependent NETosis phenocopies IL17 neutralization. NMR spectroscopy revealed changes in tumor lactate as a potential early biomarker for IL17/PD-1 combination efficacy. Higher expression of IL17 and PADI4 in human PDAC corresponds with poorer prognosis, and the serum of patients with PDAC has higher potential for NETosis. Clinical studies with IL17 and checkpoint blockade represent a novel combinatorial therapy with potential efficacy for this lethal disease.
Project description:Checkpoint blockade therapies that target inhibitory receptors on T cells have revolutionized clinical oncology. Antibodies targeting CTLA-4 or the PD-1/PD-L1 axis are now successfully used alone or in combination with chemotherapy for numerous tumor types. Despite the clinical success of checkpoint blockade therapies, tumors exploit multiple mechanisms to escape or subvert the anti-tumor T cell response. Within the tumor microenvironment, tumor-associated macrophages (TAM) can suppress T cell responses and facilitate tumor growth in various ways, ultimately debilitating clinical responses to T cell checkpoint inhibitors. There is therefore significant interest in identifying biologicals and drugs that target immunosuppressive TAM within the tumor microenvironment and can be combined with immune checkpoint inhibitors. Here we review approaches that are currently being evaluated to convert immunosuppressive TAM into immunostimulatory macrophages that promote T cell responses and tumor elimination. Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment that impact anti-tumor immune responses and susceptibility to checkpoint blockade. TAMs are very heterogeneous and can be either immunosuppressive or immunostimulatory. Here, Molgora and Colonna review current strategies that aim to reprogram TAMs to enhance rather than inhibit immune responses.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is a highly immune-suppressive tumor with a low response rate to single checkpoint blockade therapy. ETS homologous factor (EHF) is a tumor suppressor in PDAC. Here, we report a novel function of EHF in pancreatic cancer immune microenvironment editing and efficacy prediction for anti-PD1 therapy. Our findings support that the deficiency of tumoral EHF induced the accumulation of regulatory T (T reg) cells and myeloid-derived suppressor cells (MDSCs) and a decrease in the number of tumor-infiltrating CD8+ T cells. Mechanistically, EHF deficiency induced the conversion and expansion of T reg cells and MDSCs through inhibiting tumor TGF?1 and GM-CSF secretion. EHF suppressed the transcription of TGFB1 and CSF2 by directly binding to their promoters. Mice bearing EHF overexpression tumors exhibited significantly better response to anti-PD1 therapy than those with control tumors. Our findings delineate the immunosuppressive mechanism of EHF deficiency in PDAC and highlight that EHF overexpression may improve PDAC checkpoint immunotherapy.
Project description:Triple negative breast cancer (TNBC) does not respond to checkpoint blockade immunotherapy as a result of immunosuppressive tumor microenvironment. To remodel the tumor microenvironment, we developed a liposome formulation to deliver a potential immunogenic cell death (ICD) inducing agent, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG, or tanespimycin), in a tumor targeted manner to reverse the immunosuppressive microenvironment and facilitate the checkpoint blockade immunotherapy. The 17-AAG liposomes was prepared by thin film dispersion methods. The orthotopic 4T1 murine triple negative breast cancer model was studied. 17-AAG delivered by liposome remodeled the immunosuppressive microenvironment, significantly increased tumor infiltrating T cells, lowered the hypoxia level, decreased the suppressive lymphocytes such as tumor associated macrophages and myeloid derived suppressor cells in the tumor microenvironment. In addition, real-time PCR analysis revealed that chemokines and cytokines with immunosuppressive properties were notably reduced, which further facilitated the T cell mediated immunotherapy. Despite the fact that low dose 17-AAG liposomes demonstrated a limited therapeutic effect alone on 4T1 tumor, promising efficacy was observed when 17-AAG liposomes combined with checkpoint blockade immunotherapy. Taken together, 17-AAG liposomes could remodel the immunosuppressive microenvironment of triple negative breast cancer and facilitate the checkpoint blockade immunotherapy.
Project description:Glioblastoma (GBM) is a hypervascular and malignant form of brain tumors. Anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels in clinical and preclinical studies, which resulted into marked hypoxia and recruited bone marrow derived cells (BMDCs) to the tumor microenvironment (TME). In vivo animal models to track BMDCs and investigate molecular mechanisms in AAT resistance are rare. We exploited recently established chimeric mouse to develop orthotopic U251 tumor, which uses as low as 5?×?10(6) GFP+ BM cells in athymic nude mice and engrafted >70% GFP+ cells within 14 days. Our unpublished data and published studies have indicated the involvement of immunosuppressive myeloid cells in therapeutic resistance in glioma. Similarly, in the present study, vatalanib significantly increased CD68+ myeloid cells, and CD133+, CD34+ and Tie2+ endothelial cell signatures. Therefore, we tested inhibition of CSF1R+ myeloid cells using GW2580 that reduced tumor growth by decreasing myeloid (Gr1+ CD11b+ and F4/80+) and angiogenic (CD202b+ and VEGFR2+) cell signatures in TME. CSF1R blockade significantly decreased inflammatory, proangiogenic and immunosuppressive molecular signatures compared to vehicle, vatalanib or combination. TCK1 or CXCL7, a potent chemoattractant and activator of neutrophils, was observed as most significantly decreased cytokine in CSF1R blockade. ERK MAPK pathway was involved in cytokine network regulation. In conclusion, present study confirmed the contribution of myeloid cells in GBM development and therapeutic resistance using chimeric mouse model. We identified novel molecular networks including CXCL7 chemokine as a promising target for future studies. Nonetheless, survival studies are required to assess the beneficial effect of CSF1R blockade.
Project description:<h4>Background & aims</h4>Pancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma that contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown.<h4>Methods</h4>We used a dual recombinase mouse model to delete Hif1α or Hif2α in α-smooth muscle actin-expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF HIF2α expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, reverse transcription quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, an HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade.<h4>Results</h4>CAF-specific deletion of Hif2α, but not Hif1α, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n = 21-23 mice/group, Log-rank P = .0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models.<h4>Conclusions</h4>Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.
Project description:<b>Background:</b> Pancreatic ductal adenocarcinoma (PDAC) is dominated by an immunosuppressive microenvironment, which makes immune checkpoint blockade (ICB) often non-responsive. Understanding the mechanisms by which PDAC forms an immunosuppressive microenvironment is important for the development of new effective immunotherapy strategies. <b>Methods:</b> This study comprehensively evaluated the cell-cell communications between malignant cells and immune cells by integrative analyses of single-cell RNA sequencing data and bulk RNA sequencing data of PDAC. A Malignant-Immune cell crosstalk (MIT) score was constructed to predict survival and therapy response in PDAC patients. Immunological characteristics, enriched pathways, and mutations were evaluated in high- and low MIT groups. <b>Results:</b> We found that PDAC had high level of immune cell infiltrations, mainly were tumor-promoting immune cells. Frequent communication between malignant cells and tumor-promoting immune cells were observed. 15 ligand-receptor pairs between malignant cells and tumor-promoting immune cells were identified. We selected genes highly expressed on malignant cells to construct a Malignant-Immune Crosstalk (MIT) score. MIT score was positively correlated with tumor-promoting immune infiltrations. PDAC patients with high MIT score usually had a worse response to immune checkpoint blockade (ICB) immunotherapy. <b>Conclusion:</b> The ligand-receptor pairs identified in this study may provide potential targets for the development of new immunotherapy strategy. MIT score was established to measure tumor-promoting immunocyte infiltration. It can serve as a prognostic indicator for long-term survival of PDAC, and a predictor to ICB immunotherapy response.
Project description:The epithelial-to-mesenchymal transition, which conveys epithelial (E) carcinoma cells to quasi-mesenchymal (qM) states, enables them to metastasize and acquire resistance to certain treatments. Murine tumors composed of qM mammary carcinoma cells assemble an immunosuppressive tumor microenvironment (TME) and develop resistance to anti-CTLA4 immune-checkpoint blockade (ICB) therapy, unlike their E counterparts. Importantly, minority populations of qM cells within a tumor can cross-protect their more E neighbors from immune attack. The underlying mechanisms of immunosuppression and cross-protection have been unclear. We demonstrate that abrogation of qM carcinoma cell-derived factors (CD73, CSF1, or SPP1) prevents the assembly of an immunosuppressive TME and sensitizes otherwise refractory qM tumors partially or completely to anti-CTLA4 ICB. Most strikingly, mixed tumors in which minority populations of carcinoma cells no longer express CD73 are now sensitized to anti-CTLA4 ICB. Finally, loss of CD73 also enhances the efficacy of anti-CTLA4 ICB during the process of metastatic colonization. SIGNIFICANCE: Minority populations of qM carcinoma cells, which likely reside in human breast carcinomas, can cross-protect their E neighbors from immune attack. Understanding the mechanisms by which qM carcinoma cells resist antitumor immune attack can help identify signaling channels that can be interrupted to potentiate the efficacy of checkpoint blockade immunotherapies.<i>This article is highlighted in the In This Issue feature, p. 995</i>.
Project description:For decades, tumor-bearing murine models established using tumor cell lines have been the most commonly used models to study human cancers. Even though there are several studies reported that implant sites caused disparities in tumor behaviors, few of them illuminated the positional effect on immunotherapy. Herein, we describe surgical techniques for a novel orthotopic implantation of syngeneic pancreatic ductal adenocarcinoma (PDAC) tissue slices. This method has a high success modeling rate and stable growth kinetics, which makes it useful for testing novel therapeutics. Pathological examination indicated that the orthotopic tumor displayed poor vascularization, desmoplastic stromal reaction, and a highly immunosuppressive tumor microenvironment. This unique microenvironment resulted in limited response to PD1/CTLA4 blockade therapy and anti-MUC1 (αMUC1) CAR-T transfer treatment. To reverse the suppressive tumor microenvironment, we developed gene modified T-cells bearing a chimeric receptor in which activating receptor NKG2D fused to intracellular domains of 4-1BB and CD3ζ (NKG2D CAR). The NKG2D CAR-T cells target myeloid-derived suppressor cells (MDSCs), which overexpress Rae1 (NKG2D ligands) within the TME. Results indicated that NKG2D CAR-T cells eliminated MDSCs and improved antitumor activity of subsequently infused CAR-T cells. Moreover, we generated a bicistronic CAR-T, including αMUC1 CAR and NKG2D CAR separated by a P2A element. Treatment with the dual targeted bicistronic CAR-T cells also resulted in prolonged survival of orthotopic model mice. In summary, this study describes construction of a novel orthotopic PDAC model through implantation of tissue slices and discusses resistance to immunotherapy from the perspective of a PDAC microenvironment. Based on the obtained results, it is evident that elimination MDSCs by NKG2D CAR could rescue the impaired CAR-T cell activity.
Project description:Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) dramatically limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME thus rendering tumors more responsive to immunotherapy. We treated KP orthotopic tumor-bearing mice with vehicle and FAK inhibitor (FAKi) for 14 days, then extracted total RNA from tumor tissues.