LOCALIZED ABLATIVE IMMUNOTHERAPY FOR THE TREATMENT OF METASTATIC PANCREATIC CANCER IN ORTHOTOPIC MOUSE MODEL

Abstract

Introduction Pancreatic cancer is one of the deadliest cancers with an overall 5-year relative survival rate of 13.7% but once the cancer metastasizes, this survival rate reduces to 3%. Despite recent advances in pancreatic cancer research, all the conventional cancer therapies, such as surgery, chemotherapy, and radiation therapy, have severe limitations in treating metastatic pancreatic cancer. Immunotherapy is a promising approach to treat advanced cancers, but the immunosuppressive nature of pancreatic cancer limits the efficacy of this therapy. Therefore, there is a need for a treatment modality that can both break the immunosuppressive nature of the tumor and control cancer metastasis. Here, a novel localized ablative immunotherapy (iLAIT), a combination of interstitial photothermal therapy (iPTT) and intratumoral administration of immunostimulant glycated chitosan (GC), was implemented to treat pancreatic tumors in mouse models. The effects of iPTT and its combination with GC on the tumor microenvironment were investigated to determine the optimal ablative effects and GC dosage. Furthermore, immunological effects in the tumor and spleen, tumor progression using IVIS, and spatial data were quantified, monitored, and validated, respectively. Methods C57BL/6 mice were orthotopically injected with metastatic pancreatic tumor cells (Panc02h7) in the tail of the pancreas. Tumors were treated with interstitial photothermal therapy (iPTT) 9 days after tumor implantation. Tumors were exposed by surgery and irradiated with 1 cm cylindrical active lens with various laser powers (0.5 W, 1.0 W, and 1.25 W) and treatment durations (5 mins, 10 mins, and 15 mins). Tumor core and surface temperatures were observed throughout the treatment duration. Additionally, ICD molecules from tumor supernatants were measured using enzyme-linked immunosorbent assay (ELISA). A longitudinal GC dosage study (0.5%, 1.0%, and 1.25%) tracked the expressions of CD4+ T helper cells and CD8+ cytotoxic T cells across all groups. Immunological studies were also conducted via flow cytometry to explore the effects of different treatments on tumor tissues and spleens. Finally, bioluminescence signals were captured using IVIS to track the tumor burden post-treatments. Survival was also monitored for both Panc02h7 cell line and transfected Panc02h7 (HDLE3) cell lines. Findings Optimization of iPTT established the optimal laser parameters to be used for the remainder of the study, and this is the combination of 1 W laser power and 10 minutes treatment duration. Dosage study showed that the combination of iPTT with 1.0% GC resulted in increased expressions of T cells 10 days after treatment as opposed to iPTT with 0.5% GC, therefore, this GC dosage was used in combination with the optimal iPTT parameter for the remainder of the study. Immunological studies to quantify immune cell infiltrations into the tumors show that iLAIT resulted in a remarkable macrophage shift toward an M1-like phenotype which plays a significant role in pro-inflammatory and anti-tumor immune responses. In addition to measuring the immune cells in the tumors, splenic cells were also quantified to investigate the toxicity or immunosuppressive effects of the treatment modalities. In the spleens, total macrophage frequency and M1-like macrophage frequency were elevated in the iLAIT group proving iLAIT’s effect in shifting the polarization of macrophages toward an anti- tumor phenotype. Expressions of CD8+ conventional dendritic cells (cDCs) also increased in the iLAIT group, and functional states of both CD4+ and CD8+ T cells also showed elevation in the iLAIT group. The lack of fluctuations in the Tregs and B cells in the spleens also rules out any systemic immunosuppression, B cell depletion, or lymphoid exhaustion. In the bioluminescence study, a non-invasive, highly sensitive IVIS system was utilized to track tumor progression and metastasis. In control and GC groups, imaging captured the highly aggressive proliferation of the primary tumor. Conversely, mice treated with iPTT or iLAIT exhibited an acute and dramatic drop, relative to the baseline control, in the bioluminescent signals. iLAIT proved its capability in reducing the tumor burden resulting in prolonged survival compared to other groups as demonstrated with the dramatic decrease in bioluminescence signals 10 days post-treatment. Finally, high-resolution spatial transcriptomics mapped the distinct spatial distribution of the dominant cell types and their corresponding gene expressions following different treatments. Laser treatments remodeled parts of the tumor into a highly active lymphoid hub as shown in the dominant cell type maps. The SpaCET deconvolution maps reveal the malignant-driven tumor sections but upon laser treatments, spatial arrangements of the immune cells and stromal cells were remodeled. The structural and vascular progressions in pancreatic cancer were also monitored and tracked using non-invasive, label-free OCT and OCTA. When laser was used, complete destruction of tumor blood vessels was observed but this was followed by the formation of normal vessels weeks after the treatment. This chapter establishes the framework for differentiating tumor phenotypes and treatment responses by capturing the baseline growth properties and vascular signatures for each tumor. By bridging therapeutic parameter optimizations and immunological validations with bioluminescence in vivo imaging and high-dimensional spatial transcriptomics, this dissertation established a highly integrated localized ablative immunotherapy that can successfully remodel the immunosuppressive tumor microenvironment into a long-lived systemic protection against advanced metastatic cancers. Significance Surgery, chemotherapy, and radiation therapy are the conventional treatments for pancreatic cancer; however, these modalities are not sufficient to treat metastatic cancers. An integrated treatment that can inhibit both the primary and metastatic tumors is needed, and in this study, we utilized the combination of photothermal therapy and immunotherapy, iLAIT. This is significant because late-stage cancers cannot be treated with conventional methods but with this synergistic approach, both the local and distant cancers can be treated. This treatment regimen presents a novel therapeutic strategy in combatting metastatic cancers.