Overcoming Resistance to Combination Radiation-Immunotherapy: A Focus on Contributing Pathways Within the Tumor Microenvironment

Radiation therapy has been used for many years to treat tumors based on its DNA-damage-mediated ability to kill cells. More recently, RT has been shown to exert beneficial modulatory effects on immune responses, such as triggering immunogenic cell death, enhancing antigen presentation, and activating cytotoxic T cells. Consequently, combining radiation therapy with immunotherapy represents an important area of research. Thus far, immune-checkpoint inhibitors targeting programmed death-ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have been the focus of many research studies and clinical trials. The available data suggest that such immunotherapies are enhanced when combined with radiation therapy. However, treatment resistance, intrinsic or acquired, is still prevalent. Various theories as to how to enhance these combination therapies to overcome treatment resistance have been proposed. In this review, we focus on the principles surrounding radiation therapy's positive and negative effects on the tumor microenvironment. We explore mechanisms underlying radiation therapy's synergistic and antagonistic effects on immune responses and provide a base of knowledge for radio-immunology combination therapies to overcome treatment resistance. We provide evidence for targeting regulatory T cells, tumor-associated macrophages, and cancer-associated fibroblasts in combination radio-immunotherapies to improve cancer treatment

A novel preclinical model of craniospinal irradiation in pediatric diffuse midline glioma demonstrates decreased metastatic disease

BackgroundDiffuse midline glioma (DMG) is an aggressive pediatric central nervous system tumor with strong metastatic potential. As localized treatment of the primary tumor improves, metastatic disease is becoming a more important factor in treatment. We hypothesized that we could model craniospinal irradiation (CSI) through a DMG patient-derived xenograft (PDX) model and that CSI would limit metastatic tumor.MethodsWe used a BT245 murine orthotopic DMG PDX model for this work. We developed a protocol and specialized platform to deliver craniospinal irradiation (CSI) (4 Gy x2 days) with a pontine boost (4 Gy x2 days) and compared metastatic disease by pathology, bioluminescence, and MRI to mice treated with focal radiation only (4 Gy x4 days) or no radiation.ResultsMice receiving CSI plus boost showed minimal spinal and brain leptomeningeal metastatic disease by bioluminescence, MRI, and pathology compared to mice receiving radiation to the pons only or no radiation.ConclusionIn a DMG PDX model, CSI+boost minimizes tumor dissemination compared to focal radiation. By expanding effective DMG treatment to the entire neuraxis, CSI has potential as a key component to combination, multimodality treatment for DMG designed to achieve long-term survival once novel therapies definitively demonstrate improved local control

Targeting resistance to radiation-immunotherapy in cold HNSCCs by modulating the Treg-dendritic cell axis.

BACKGROUND: Numerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors, we investigate therapeutic approaches of overcoming such resistance by examining the differential microenvironmental response to RT. METHODS: We subjected two HPV-negative orthotopic mouse models of HNSCC to combination RT, regulatory T cells (Treg) depletion, and/or CD137 agonism. Tumor growth was measured and intratumorous and lymph node immune populations were compared among treatment groups. Human gene sets, genetically engineered mouse models DEREG and BATF3-/-, flow and time-of-flight cytometry, RNA-Seq, Treg adoptive transfer studies, and in vitro experiments were used to further evaluate the role of dendritic cells (DCs) and Tregs in these treatments. RESULTS: In MOC2 orthotopic tumors, we find no therapeutic benefit to targeting classically defined immunosuppressive myeloids, which increase with RT. In these radioresistant tumors, supplementing combination RT and Treg depletion with anti-CD137 agonism stimulates CD103+ DC activation in tumor-draining lymph nodes as characterized by increases in CD80+ and CCR7+ DCs, resulting in a CD8 T cell-dependent response. Simultaneously, Tregs are reprogrammed to an effector phenotype demonstrated by increases in interferonγ+, tumor necrosis factorα+, PI3K+, pAKT+ and Eomes+ populations as well as decreases in CTLA4+ and NRP-1+ populations. Tumor eradication is observed when RT is increased to an 8 Gy x 5 hypofractionated regimen and combined with anti-CD25+ anti-CD137 treatment. In a human gene set from oral squamous cell carcinoma tumors, high Treg number is associated with earlier recurrence. CONCLUSIONS: Regulating Treg functionality and DC activation status within the lymph node is critical for generating a T cell effector response in these highly radioresistant tumors. These findings underscore the plasticity of Tregs and represent a new therapeutic opportunity for reprogramming the tumor microenvironment in HNSCCs resistant to conventional radioimmunotherapy approaches

Cellular Imaging at 1.5 T: Detecting Cells in Neuroinflammation using Active Labeling with Superparamagnetic Iron Oxide

The ability to visualize cell infiltration in experimental autoimmune encephalomyelitis (EAE), a well-known animal model for multiple sclerosis in humans, was investigated using a clinical 1.5-T magnetic resonance imaging (MRI) scanner, a custom-built, high-strength gradient coil insert, a 3-D fast imaging employing steady-state acquisition (FIESTA) imaging sequence and a superparamagnetic iron oxide (SPIO) contrast agent. An “active labeling” approach was used with SPIO administered intravenously during inflammation in EAE. Our results show that small, discrete regions of signal void corresponding to iron accumulation in EAE brain can be detected using FIESTA at 1.5 T. This work provides early evidence that cellular abnormalities that are the basis of diseases can be probed using cellular MRI and supports our earlier work which indicates that tracking of iron-labeled cells will be possible using clinical MR scanners

Effects of ranibizumab and amfenac on the functional abilities and radiosensitivity of uveal melanoma cells

ABSTRACT Purpose: To evaluate the effects of ranibizumab and amfenac in human uveal melanoma cell lines and to explore the ability of these compounds to sensitize uveal melanoma cells to radiation therapy. Methods: The 92.1 human uveal melanoma cell line was cultured and subjected to the proposed treatment (ranibizumab, amfenac, and a combination of both). Proliferation, migration, and invasion assays of the 92.1 uveal melanoma cell line were assessed after pretreatment with ranibizumab (125 mg/mL), amfenac (150 nM), or a combination of both. In addition, proliferation rates were assessed after treatment with ranibizumab and amfenac, and the cells were subsequently exposed to various radiation doses (0, 4, and 8 Gy). Results: Proliferation assay: cells treated with a combination of ranibizumab and amfenac had lower proliferation rates than controls (p=0.016) and than those treated with only ranibizumab (p=0.033). Migration assay: a significantly lower migration rate was observed in cells treated with amfenac than the control (p=0.014) and than those treated with ranibizumab (p=0.044). Invasion assay: there were no significant differences among the studied groups. Irradiation exposure: in the 4 Gy dose group, there were no significant differences among any groups. In the 8 Gy dose group, treatment with ranibizumab, amfenac, and their combination prior to application of the 8 Gy radiation led to a marked reduction in proliferation rates (p=0.009, p=0.01, and p=0.034, respectively) compared with controls. Conclusion: Combination of ranibizumab and amfenac reduced the proliferation rate of uveal melanoma cells; however, only amfenac monotherapy significantly decreased cell migration. The radiosensitivity of the 92.1 uveal melanoma cell line increased following the administration of ranibizumab, amfenac, and their combination. Further investigation is warranted to determine if this is a viable pretreatment strategy to render large tumors amenable to radiotherapy

Ephrin-B2 overexpression predicts for poor prognosis and response to therapy in solid tumors.

Ephrin B2 is variably expressed on tumor cells and its blockade has been shown to inhibit angiogenesis in animal models of pancreatic, colorectal, lung and head, and neck squamous cell carcinomas. However, the implications of ephrinB2 expression in cancer patients have remained elusive. In this study, we analyzed the cancer genome atlas (TCGA) for ephrinB2 expression. We report significant correlations between EFNB2 expression, overall survival and disease-free survival in head and neck squamous cell carcinoma (HNSCC, n = 519), pancreatic adenocarcinoma (n = 186), and bladder urothelial carcinoma (n = 410). In HNSCC patients, high-EFNB2 mRNA expression was associated with tumor HPV negativity, oral cavity location, alcohol intake, higher TP53 mutation, and EGFR amplification. EphrinB2 overexpression also correlated with worse response to chemotherapy and radiotherapy. The therapeutic potential of blocking ephrinB2 was validated in HNSCC patient-derived tumor xenografts and showed significant improvement in survival and tumor growth delay. Our data shows that ephrinB2 overexpression can serve as a critical biomarker for patient prognosis and response to therapy. These results should guide design of future clinical trials exploring EphrinB2 inhibition in cancer patients. © 2016 Wiley Periodicals, Inc

Combined EphB2 receptor knockdown with radiation decreases cell viability and invasion in medulloblastoma

Abstract Background Medulloblastoma is one of the most common types of pediatric brain tumor characterized by the subpopulation of cells that exhibit high invasive potential and radioresistant properties. In addition, dysregulated function and signaling by Eph family of receptors have been shown to impart pro-tumorigenic characteristics in this brain malignancy. In the current study, we investigated whether EphB2 knockdown in combination with radiation can alter invasiveness and decrease medulloblastoma tumor growth or viability in vitro. Methods The expression of EphB2 receptor was analyzed by immunohistochemistry and Western blotting. Microarray analysis and mRNA analysis was performed on medulloblastoma patient datasets and compared to the normal cerebellum. The radiosensitization effect following EphB2 knockdown was determined by clonogenic assay in human medulloblastoma cells. Effects of EphB2-siRNA in absence or presence of radiation on cell cycle distribution, cell viability, and invasion were analyzed by flow cytometry, MTT assay, trypan blue exclusion assay, xcelligence system, and Western blotting. Results We observed that EphB2 is expressed in both medulloblastoma cell lines and patient samples and its downregulation sensitized these cells to radiation as evident by decreased clonogenic survival fractions. EphB2 expression was also high across different medulloblastoma subgroups compared to normal cerebellum. The radiosensitization effect observed following EphB2 knockdown was in part mediated by enhanced G2/M cell cycle arrest. We also found that the combined approach of EphB2 knockdown and radiation exposure significantly reduced overall cell viability in medulloblastoma cells compared to control groups. Similar results were obtained in the xcelligence-based invasion assay. Western blot analysis also demonstrated changes in the protein expression of cell proliferation, cell survival, and invasion molecules in the combination group versus others. Conclusions Overall, our findings indicate that specific targeting of EphB2 receptor in combination with radiation may serve as an effective therapeutic strategy in medulloblastoma. Future studies are warranted to test the efficacy of this approach in in vivo preclinical models