Cellular Magnetic Resonance Imaging at 1.5T: Application to Imaging Neuroinflammation in Experimental Models of Multiple Sclerosis

The thesis herein addresses an important, yet unresolved, problem in the field of cellular MRI. The natural ability of macrophages to engulf foreign particles has made them ideal candidates for taking up cellular MR labels in vivo. In multiple sclerosis, macrophage populations in the circulation increase substantially as they home to the CNS where they cause a massive and destructive inflammatory response. Labeling macrophages in the circulation and being able detect and track them in the CNS can significantly improve the prognosis of MS and thus aid in the correct identification of a treatment plan. However, it remains unknown whether it is indeed labeled macrophages from the circulation that are detected on MR images or whether they are different cell populations actively involved in MS. In the present thesis, two novel methods are used to determine the source of the MR label in the CNS. One study utilizes a transgenic mouse where peripheral macrophages express the green fluorescent protein while other cell populations do not. Another study utilizes clodronate liposomes to deplete the’ macrophage population in the circulation prior to injection of the MR label. By using the mentioned two approaches it was determined that the source of label on MR images is largely attributed to peripheral macrophages from the circulation. The findings illustrated in this thesis will have a major impact on our understanding of neuroinflammation in multiple sclerosis and on the development of diseasemodifying therapies

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

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

PARP Inhibition Enhances Radiotherapy of SMAD4-Deficient Human Head and Neck Squamous Cell Carcinomas in Experimental Models

PurposeSMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study whether SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT).Experimental designWe analyzed HNSCC The Cancer Genome Atlas data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a phase I trial investigating olaparib were analyzed.ResultsSMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4-mutant but not SMAD4-positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4-deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared with vehicle-treated tumors. In vitro, olaparib alone or in combination with radiation caused lower clonogenic survival, more DNA damage-associated cell death, and less proliferation in SMAD4-deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage.ConclusionsSMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs