Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry

Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimise and validate image segmentation strategies and automate the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. With these methods we interrogate the remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, highlighting the infiltration and activation of antigen presenting cells and effector cells.

Pharmacological rescue of tumor intrinsic STING expression and immune response in LKB1-mutant lung cancer via the IAP-JAK regulatory axis

Harnessing the power of the immune system to treat cancer has become a core clinical approach. However, rewiring of intrinsic circuitry enables tumor cells to escape immune attacks, leading to therapeutic failure. Pharmacological strategies to reverse tumor genotype-dictated therapeutic resistance are urgently needed to advance precision immunotherapy. Here, we identify antagonists of Inhibitor of Apoptosis Protein (IAP) as potent sensitizers that restore immune-dependent killing of LKB1-mutant lung cancer cells. Mechanistic studies reveal an LKB1-IAP-JAK trimolecular complex that bridges the LKB1-mutant genotype with IAP-dependency and a STING-deficiency-mediated immune resistance phenotype. Ultimately, inhibition of IAP re-establishes JAK-regulated STING expression and DNA sensing pathway as well as enhanced cytotoxic immune cell infiltration and selective immune-dependent anti-tumor activity in an LKB1-mutant immune-competent mouse model. Thus, IAP-JAK-modulatory strategies, like IAP inhibitors, offer promising immunotherapy adjuvants to re-establish the responsiveness of "immunologically-cold" LKB1-mutant tumors to immune checkpoint inhibitors or STING-directed therapies.

Immunological and Pathological Landscape of Dengue Serotypes 1-4 Infections in Immune-Competent Mice

Dengue virus (DENV), a Flavivirus, causes a broad spectrum of disease in humans with key clinical signs including thrombocytopenia, vascular leakage and hemorrhaging. A major obstacle to understanding DENV immunity has been the lack of a validated immune-competent mouse model. Here, we report the infection profiles of human clinical isolates of DENV serotypes 1-4 in an immune-competent mouse model. We detected replicating DENV in the peritoneal cells, liver and the spleen that was generally resolved within 2 weeks. The DENV target cell types for infection were monocytes/macrophages, dendritic cells, endothelial cells, and we identified a novel DENV cellular target, fibroblast reticular cells of the spleen. We observed gross pathologies in the spleen and liver that are consistent with dengue disease, including hemorrhaging as well as transcriptional patterns suggesting that antiviral responses and tissue damage were induced. Key clinical blood parameters that define human DENV disease such as hemoconcentration, leukopenia and reduced number of platelets were also observed. Thus, immune-competent mice sustain replicating infection and experience signs, such as hemorrhaging, that define DENV disease in humans. This study thoroughly characterizes DENV1-4 infection in immune-competent mice and confirms the wild-type mouse model as a valid and reproducible system for investigating the mechanisms of DENV pathogenesis.

Evaluation of DNA-Launched Virus-Like Particle Vaccines in an Immune Competent Mouse Model of Chikungunya Virus Infection

Chikungunya virus (CHIKV) infection can result in chronic and debilitating arthralgia affecting humans in tropical and subtropical regions around the world, yet there are no licensed vaccines to prevent infection. DNA launched virus like particle (VLP) vaccines represent a potentially safer alternative to traditional live-attenuated vaccines; however, fully characterized immunocompetent mouse models which appropriately include both male and female animals for preclinical evaluation of these, and other, vaccine platforms are lacking. Utilizing virus stocks engineered to express mutations reported to enhance CHIKV virulence in mice, infection of male and female immunocompetent mice was evaluated, and the resulting model utilized to assess the efficacy of candidate DNA launched CHIKV VLP vaccines. Results demonstrate the potential utility of DNA launched VLP vaccines in comparison to a live attenuated CHIKV vaccine and identify gender differences in viral RNA loads that impact interpretation of vaccine efficacy and may have important implications for future CHIKV vaccine development.

Characterisation of tumour immune microenvironment remodelling following oncogene inhibition in preclinical studies using an optimised imaging mass cytometry workflow

Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), novel highly multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimised and validated image segmentation strategies and automated the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. Incorporating user-specific plugins, imcyto can be flexibly tailored to a wide range of segmentation needs. With these methods we interrogated the dramatic remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, showcasing their potential as key discovery tools to enhance understanding of the interplay between tumour, stroma, and immune cells in the spatial context of the tissue.

Immune Profiling of Syngeneic Murine and Patient GBMs for Effective Translation of Immunotherapies

Immunotherapy for brain tumors remains elusive, unlike many other cancer types for which it is one of the most promising therapeutic options. Recent studies have comprehensively profiled the immune-landscape of the highly malignant brain tumor, glioblastoma (GBM) in patients and identified novel immune-modulatory targets. However, given that pre-clinical exploration of potential novel therapeutics is primarily performed in immune-competent mice, it is vital to compare the immune-profiling data obtained from syngeneic mouse GBM models with GBM patient samples. This will pave the way for utilizing appropriate clinically relevant mouse GBM models for evaluating novel immune-therapies in pre-clinical settings. Recent brain tumor immune-profiling studies using state-of-the-art time of flight cytometry (CyTOF) analysis compared different human and mouse GBM types and reported immunological distinctions amongst these mouse models. These studies also contrast the immune phenotype of brain tumor patients with commonly used pre-clinical immune-competent mouse models. In this perspective, we provide the outcomes of very recent brain tumor immune-profiling studies and their implications on designing and translating unique, tumor-subtype specific therapeutics.

Characterisation of tumour immune microenvironment remodelling following oncogene inhibition in preclinical studies using an optimised imaging mass cytometry workflow

Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), novel highly multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimised and validated image segmentation strategies and automated the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. Incorporating user-specific plugins, imcyto can be flexibly tailored to a wide range of segmentation needs. With these methods we interrogated the dramatic remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, showcasing their potential as key discovery tools to enhance understanding of the interplay between tumour, stroma, and immune cells in the spatial context of the tissue.

Development of multi-drug loaded PEGylated nanodiamonds to inhibit tumor growth and metastasis in genetically engineered mouse models of pancreatic cancer

Schematic representation demonstrating the fabrication and in vivo evaluation of an immune-modulatory nano-formulation consisting of irinotecan and curcumin in immune-competent mouse models of pancreatic adenocarcinoma.