Diana Moughon

Targeting distinct tumor-infiltrating myeloid cells by inhibiting CSF-1 receptor: combating tumor evasion of antiangiogenic therapy.

Tumor-infiltrating myeloid cells (TIMs) support tumor growth by promoting angiogenesis and suppressing antitumor immune responses. CSF-1 receptor (CSF1R) signaling is important for the recruitment of CD11b(+)F4/80(+) tumor-associated macrophages (TAMs) and contributes to myeloid cell-mediated angiogenesis. However, the impact of the CSF1R signaling pathway on other TIM subsets, including CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs), is unknown. Tumor-infiltrating MDSCs have also been shown to contribute to tumor angiogenesis and have recently been implicated in tumor resistance to antiangiogenic therapy, yet their precise involvement in these processes is not well understood. Here, we use the selective pharmacologic inhibitor of CSF1R signaling, GW2580, to demonstrate that CSF-1 regulates the tumor recruitment of CD11b(+)Gr-1(lo)Ly6C(hi) mononuclear MDSCs. Targeting these TIM subsets inhibits tumor angiogenesis associated with reduced expression of proangiogenic and immunosuppressive genes. Combination therapy using GW2580 with an anti-VEGFR-2 antibody synergistically suppresses tumor growth and severely impairs tumor angiogenesis along with reverting at least one TIM-mediated antiangiogenic compensatory mechanism involving MMP-9. These data highlight the importance of CSF1R signaling in the recruitment and function of distinct TIM subsets, including MDSCs, and validate the benefits of targeting CSF1R signaling in combination with antiangiogenic drugs for the treatment of solid cancers.

CSF1 Receptor Targeting in Prostate Cancer Reverses Macrophage-Mediated Resistance to Androgen Blockade Therapy

Growing evidence suggests that tumor-associated macrophages (TAM) promote cancer progression and therapeutic resistance by enhancing angiogenesis, matrix-remodeling, and immunosuppression. In this study, prostate cancer under androgen blockade therapy (ABT) was investigated, demonstrating that TAMs contribute to prostate cancer disease recurrence through paracrine signaling processes. ABT induced the tumor cells to express macrophage colony-stimulating factor 1 (M-CSF1 or CSF1) and other cytokines that recruit and modulate macrophages, causing a significant increase in TAM infiltration. Inhibitors of CSF1 signaling through its receptor, CSF1R, were tested in combination with ABT, demonstrating that blockade of TAM influx in this setting disrupts tumor promotion and sustains a more durable therapeutic response compared with ABT alone.

Immunosuppressive tumor-infiltrating myeloid cells mediate adaptive immune resistance via a PD-1/PD-L1 mechanism in glioblastoma

Background Adaptive immune resistance in the tumor microenvironment appears to attenuate the immunotherapeutic targeting of glioblastoma (GBM). In this study, we identified a tumor-infiltrating myeloid cell (TIM) population that expands in response to dendritic cell (DC) vaccine treatment. The aim of this study was to understand how this programmed death ligand 1 (PD-L1)-expressing population restricts activation and tumor-cytolytic function of vaccine-induced tumor-infiltrating lymphocytes (TILs). Methods To test this hypothesis in our in vivo preclinical model, we treated mice bearing intracranial gliomas with DC vaccination ± murine anti-PD-1 monoclonal antibody (mAb) blockade or a colony stimulating factor 1 receptor inhibitor (CSF-1Ri) (PLX3397) and measured overall survival. We then harvested and characterized the PD-L1+ TIM population and its role in TIL activation and tumor cytolysis in vitro. Results Our data indicated that the majority of PD-L1 expression in the GBM environment is contributed by TIMs rather than by tumor cells themselves. While PD-1 blockade partially reversed the TIL dysfunction, targeting TIMs directly with CSF-1Ri altered TIM expression of key chemotactic factors associated with promoting increased TIL infiltration after vaccination. Neither PD-1 mAb nor CSF-1Ri had a demonstrable therapeutic benefit alone, but when combined with DC vaccination, a significant survival benefit was observed. When the tripartite regimen was given (DC vaccine, PD-1 mAb, PLX3397), long-term survival was noted together with an increase in the number of TILs and TIL activation. Conclusion Together, these studies elucidate the role that TIMs play in mediating adaptive immune resistance in the GBM microenvironment and provide evidence that they can be manipulated pharmacologically with agents that are clinically available. Development of immune resistance in response to active vaccination in GBM can be reversed with dual administration of CSF-1Ri and PD-1 mAb.

CRISPR-Mediated VHL Knockout Generates an Improved Model for Metastatic Renal Cell Carcinoma

Metastatic renal cell carcinoma (mRCC) is nearly incurable and accounts for most of the mortality associated with RCC. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of clear cell RCC (ccRCC) cases. Its role in regulating hypoxia-inducible factors-1α (HIF-1α) and -2α (HIF-2α) is well-studied. Recent work has demonstrated that VHL knock down induces an epithelial-mesenchymal transition (EMT) phenotype. In this study we showed that a CRISPR/Cas9-mediated knock out of VHL in the RENCA model leads to morphologic and molecular changes indicative of EMT, which in turn drives increased metastasis to the lungs. RENCA cells deficient in HIF-1α failed to undergo EMT changes upon VHL knockout. RNA-seq revealed several HIF-1α-regulated genes that are upregulated in our VHL knockout cells and whose overexpression signifies an aggressive form of ccRCC in the cancer genome atlas (TCGA) database. Independent validation in a new clinical dataset confirms the upregulation of these genes in ccRCC samples compared to adjacent normal tissue. Our findings indicate that loss of VHL could be driving tumour cell dissemination through stabilization of HIF-1α in RCC. A better understanding of the mechanisms involved in this phenomenon can guide the search for more effective treatments to combat mRCC.

Detection of immune responses after immunotherapy in glioblastoma using PET and MRI

Significance The inability to accurately monitor glioblastoma tumor progression vs. pseudoprogression has severely limited clinical treatment decisions, especially in the setting of immunotherapy. We have identified a novel noninvasive imaging combination that could distinguish intracranial immune responses from tumor progression in mice bearing orthotopic gliomas and in patients with glioblastomas. We combined the use of advanced MRI with PET imaging of deoxycytidine kinase, an enzyme overexpressed in immune cells. This combination resulted in superior differentiation between immune responses and tumors within the brain, and identified peripheral lymph nodes in which immune responses occurred after immunotherapy combinations. This combined imaging approach may provide a useful method to clinically monitor patients with glioblastomas treated with immune-based therapies, and to distinguish tumor progression from pseudoprogression. Contrast-enhanced MRI is typically used to follow treatment response and progression in patients with glioblastoma (GBM). However, differentiating tumor progression from pseudoprogression remains a clinical dilemma largely unmitigated by current advances in imaging techniques. Noninvasive imaging techniques capable of distinguishing these two conditions could play an important role in the clinical management of patients with GBM and other brain malignancies. We hypothesized that PET probes for deoxycytidine kinase (dCK) could be used to differentiate immune inflammatory responses from other sources of contrast-enhancement on MRI. Orthotopic malignant gliomas were established in syngeneic immunocompetent mice and then treated with dendritic cell (DC) vaccination and/or PD-1 mAb blockade. Mice were then imaged with [18F]-FAC PET/CT and MRI with i.v. contrast. The ratio of contrast enhancement on MRI to normalized PET probe uptake, which we term the immunotherapeutic response index, delineated specific regions of immune inflammatory activity. On postmortem examination, FACS-based enumeration of intracranial tumor-infiltrating lymphocytes directly correlated with quantitative [18F]-FAC PET probe uptake. Three patients with GBM undergoing treatment with tumor lysate-pulsed DC vaccination and PD-1 mAb blockade were also imaged before and after therapy using MRI and a clinical PET probe for dCK. Unlike in mice, [18F]-FAC is rapidly catabolized in humans; thus, we used another dCK PET probe, [18F]-clofarabine ([18F]-CFA), that may be more clinically relevant. Enhanced [18F]-CFA PET probe accumulation was identified in tumor and secondary lymphoid organs after immunotherapy. Our findings identify a noninvasive modality capable of imaging the host antitumor immune response against intracranial tumors.

Rapamycin enhances adenovirus-mediated cancer imaging and therapy in pre-immunized murine hosts.

Tumor-specific adenoviral vectors comprise a fruitful gene-based diagnostic imaging and therapy research area for advanced stage of cancer, including metastatic disease. However, clinical translation of viral vectors has encountered considerable obstacles, largely due to host immune responses against the virus. Here, we explored the utilization of an immunosuppressant, rapamycin, to circumvent the anti-adenovirus immunity in immunocompetent murine prostate cancer models. Rapamycin diminished adenoviral-induced acute immune response by inhibiting NF-κB activation; it also reduced the scale and delayed the onset of inflammatory cytokine secretion. Further, we found that rapamycin abrogated anti-adenovirus antibody production and retarded the function of myeloid cells and lymphocytes that were activated upon viral administration in pre-immunized hosts. Thus, the co-administration of rapamycin prolonged and enhanced adenovirus-delivered transgene expression in vivo, and thereby augmented the imaging capability of adenoviral vectors in both bioluminescent and positron emission tomography modalities. Furthermore, we showed that despite an excellent response of cancer cells to a cytotoxic gene therapeutic vector in vitro, only minimal therapeutic effects were observed in vivo in pre-immunized mice. However, when we combined gene therapy with transient immunosuppression, complete tumor growth arrest was achieved. Overall, transient immunosuppression by rapamycin was able to boost the diagnostic utility and therapeutic potentials of adenoviral vectors.

Abstract A27: Hypoxia-inducible factor 1-alpha suppression drives recruitment of metastasis-promoting granulocytes through the CXCR2 axis in renal cell carcinoma

Introduction: Neutrophil infiltration into localized clear cell renal cell carcinoma (RCC) tumors has been shown to be associated with reduced survival in the clinic, though their protumorigenic role in this context is unclear. Molecularly targeted therapies for metastatic RCC, such as sunitinib and sorafenib, eventually result in resistance and disease progression concurrent with increased granulocytic infiltration. Our studies aim to elucidate the metastasis-promoting role of granulocytes in RCC. Interleukin 8 (IL-8) upregulation is one described mechanism whereby RCC tumors establish resistance to sunitinib. IL-8 is a CXCR2 ligand involved in the recruitment of granulocytes. Recent studies demonstrate that CXCR2 recruited polymorphonucler myeloid-derived suppressor cells (PMN-MDSCs), a population of immature granulocytes, promote metastasis through various mechanisms. Hypoxia-inducible factor 1-alpha (HIF-1α) suppression leads to increased expression of CXCR2 ligands, such as IL-8. These findings led us to hypothesize that suppression of the HIF-1α pathway, which is the target of current clinical therapies in RCC, stimulates the recruitment of metastasis-promoting granulocytes through upregulation of CXCR2 ligands in RCC. Experimental Procedures: The RENCA cell line was utilized for our studies, due to its propensity to spontaneously metastasize when orthotopically implanted in BALB/c renal capsules. Levels of HIF-1α were knocked down using lentivirus harboring a short hairpin RNA (shRNA) to this gene. RENCA cells were also transduced to express green fluorescent protein (GFP) and Renilla luciferase, for tracking metastases. We confirmed HIF-1α knockdown by Western blot and RT-PCR and evaluated CXCR2 ligand expression by RT-PCR. An in vitro migration assay was utilized to assess increased recruitment of granulocytes. These analyses were extended in vivo in subcutaneous, orthotopic and tail vein RENCA models. Finally, we tested for increased metastases using histology and flow cytometry. Results: Preliminary work has revealed that HIF-1α suppression by shRNA knockdown leads to a significant upregulation in gene expression of CXCR2 ligands CXCL1 and CXCL2 in the RENCA murine RCC line. This upregulation was shown to be functionally significant, as recruitment of granulocytes from peripheral blood was increased toward RENCA cells with suppressed HIF-1α as compared to control cells. This increased recruitment was validated to be CXCR2-dependent through the use of SB225002, a small molecule inhibitor of CXCR2. Tumor infiltration by granulocytes was increased in the HIF-1α knockdown RENCA tumors compared to control tumors in both subcutaneous and orthotopic models. A small orthotopic pilot study revealed increased metastatic spread of HIF-1α knockdown RENCA cells to the lungs compared to control cells, but a tail vein model of metastasis revealed a reduced tumor burden in the lungs of animals injected with HIF-1α knockdown cells compared to control cells. This indicates that increased metastasis in the HIF-1α knockdown cells might be occurring, but would have to be at the level of escape from the primary tumor. Conclusions: Our findings suggest that HIF-1α suppression does lead to increased recruitment of granulocytes in the primary tumor in a CXCR2-dependent manner, and that the step of metastasis aided by these cells is likely at the level of primary tumor escape. Future work will focus on elucidating the pathway whereby CXCR2 ligands are upregulated by HIF-1α suppression, finding how granulocytes may be contributing to increased cell motility and aggressiveness in vitro and confirming an increased metastatic phenotype in the orthotopic model. The rationale for the proposed research is that once it is known that granulocytes can contribute to disease progression, these cells can be targeted in conjunction with current clinical therapies to improve prognoses for patients with metastatic RCC. Citation Format: Shiruyeh Schokrpur, Diana Moughon, James L. Sung, Jingying Xu, Lily Wu. Hypoxia-inducible factor 1-alpha suppression drives recruitment of metastasis-promoting granulocytes through the CXCR2 axis in renal cell carcinoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A27. doi:10.1158/1538-7445.CHTME14-A27

Abstract A48: M2 macrophage inhibition reverses vascular leaks that cause malignant ascites in late-stage epithelial ovarian cancer

Introduction: Malignant ascites is a common symptom of epithelial ovarian cancer (EOC) that greatly reduces the quality of life for patients and has no good treatment options that target the source of the problem. Our study aims to find the cause of ascites in order for us to better know how to safely reverse this pathology. Experimental Procedures: Murine ID8 EOC tumor cells were implanted intraperitoneally or orthotopically into female C57BL/6 mice, and bioluminescent imaging was used to track the progress of the cancer. GW2580, a very selective colony stimulating factor 1 receptor (CSF1R) inhibitor, was given to mice for two weeks once ascites appeared. Flow cytometry, histology, and endothelial cell permeability assays were used to analyze the effects of blocking macrophages in the model. Flow cytometry and endothelial cell permeability assays were also used to analyze and characterize EOC patient ascites samples. Results: CSF1R inhibition blocked specifically M2 macrophages from the ascites and dramatically reduced ascites volume, leaving mice less anemic. When M2 macrophages numbers were reduced in the ascites, CD8 T cell numbers increased, causing the ascites microenvironment to be less immunosuppressive. Peritoneal vasculature that had grown disorganized and leaky became normalized with treatment. Ascites sera from GW2580-treated mice protected against endothelium permeability, while control mouse sera caused the endothelium to become leaky. This data agrees with our finding that patient ascites sera cause more endothelium permeability if more macrophages were present in the ascites. We also saw macrophage and myeloid cell depletion in the tumor and systemically with GW2580 treatment, along with a consistent reduction in tumor burden. Conclusions: M2 macrophages are key players that cause ascites through deregulating peritoneal vasculature. The leaky vessels are normalized once these macrophages are physically and functionally blocked from the ascites microenvironment. This finding suggests that macrophage inhibition could be a powerful tool for ascites management. VEGF inhibitors are currently in clinical trials for malignant ascites treatment with mixed, sometimes deadly, results. Targeting macrophages, the source of many vessel-deregulating proteins, might make it more difficult for resistance to build up than when just one protein, such as VEGF, is blocked, and CSF1R inhibitors have been shown to be safe and well-tolerated in the clinic. This novel approach toward ascites and EOC management should be further studied. Future work will focus on finding the specific factors that caused the vessel deregulation and normalization, looking at functional differences between macrophage from control and GW2580-treated ascites, seeing if endothelial cell-macrophage direct interaction may have any effects on the vasculature, and performing this study in genetic and human models of EOC. Citation Format: Diana Moughon, Huanhuan He, Shiruyeh Schokrpur, Ziyue Jiang, Madeeha Yaqoob, John David, Luisa Iruela-Arispe, Oliver Dorigo, Lily Wu. M2 macrophage inhibition reverses vascular leaks that cause malignant ascites in late-stage epithelial ovarian cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A48. doi:10.1158/1538-7445.CHTME14-A48

Abstract C183: Inhibition of CSF1 receptor signaling targets tumor associated macrophages in a murine ovarian cancer model.

Rationale: CSF1 receptor (CSF1R) mediated signaling is important for the recruitment of CD11b + F4/80 + tumor-associated macrophages (TAMs). TAMs support tumor growth by several mechanisms including suppressing anti-tumor immune responses via the M2 macrophage subpopulation. Macrophages are found in the ascites and tumor tissue of patients with ovarian cancer and therefore present a suitable target for therapy. This study evaluates the effect of pharmacologic CSF1R inhibition on the profile of intraperitoneal macrophages in a murine model of ovarian cancer. In addition, we assessed the direct effect of CSF1R inhibition on several established human ovarian cancer cell lines. Methods: To establish an orthotopic ovarian cancer model, we injected murine ID8 epithelial ovarian cancer cells into the ovarian bursa of immuno-competent C57BL6 mice. ID8 cells were labeled with the luciferase gene for bioluminescence. Upon establishment of tumor disease and ascites, mice were treated with daily oral doses of the selective CSF-1R inhibitor GW2580. Ascites and tumor tissue was assessed for the presence of CD11b + Gr-1 lo Ly6C hi mononuclear myeloid-derived suppressor cells (MDSCs) and CD11b + F4/80 + tumor-associated macrophages (TAMs). The human ovarian cancer cell lines OVCAR5, IGROV1 and SKOV3 were treated with CSF1R inhibitors and cell proliferation assessed. Results: In vivo gene imaging showed ID8 tumor growth initially confined to the ovary with subsequent metastatic spread to the peritoneal cavity closely mimicking the growth pattern of human ovarian cancer. In animals with established metastatic tumor disease and ascites, GW2580 treatment caused a significant reduction in the ascites volume to about 25% of the level in untreated animals. GW2580 induced a drastic reduction in TAMs and MDSCs in ascites and intraperitoneal tumor tissue. Both MHCII+ and MHCII- TAM subpopulations were reduced equally. OVCAR5, IGROV1 and SKOV3 showed a decrease in cell proliferation when treated with CSF1R inhibitors albeit at higher doses (>10 M). Conclusion: Inhibition of CSF1R signaling in vivo reduces immune-suppressive TAMs and MDSCs in an intraperitoneal murine ovarian cancer model. In addition, CSF1 decreases ovarian cancer cell proliferation. This suggests that CSF1R inhibition might have dual therapeutic effects by modulating the intraperitoneal microenvironment and directly targeting ovarian cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C183.