Browsing by Author "Nagai-Singer, Margaret A."

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    Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation
    Hendricks-Wenger, Alissa; Aycock, Kenneth N.; Nagai-Singer, Margaret A.; Coutermarsh-Ott, Sheryl; Lorenzo, Melvin F.; Gannon, Jessica; Uh, Kyungjun; Farrell, Kayla; Beitel-White, Natalie; Brock, Rebecca M.; Simon, Alexander; Morrison, Holly A.; Tuohy, Joanne L.; Clark-Deener, Sherrie; Vlaisavljevich, Eli; Davalos, Rafael V.; Lee, Kiho; Allen, Irving C. (Nature Research, 2021-04-07)
    New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients’ anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.
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    Exploration of Novel Pathways Underlying Irreversible Electroporation Induced Anti-Tumor Immunity in Pancreatic Cancer
    Imran, Khan Mohammad; Nagai-Singer, Margaret A.; Brock, Rebecca M.; Alinezhadbalalami, Nastaran; Davalos, Rafael V.; Allen, Irving C. (Frontiers, 2022-03-18)
    Advancements in medical sciences and technologies have significantly improved the survival of many cancers; however, pancreatic cancer remains a deadly diagnosis. This malignancy is often diagnosed late in the disease when metastases have already occurred. Additionally, the location of the pancreas near vital organs limits surgical candidacy, the tumor's immunosuppressive environment limits immunotherapy success, and it is highly resistant to radiation and chemotherapy. Hence, clinicians and patients alike need a treatment paradigm that reduces primary tumor burden, activates systemic anti-tumor immunity, and reverses the local immunosuppressive microenvironment to eventually clear distant metastases. Irreversible electroporation (IRE), a novel non-thermal tumor ablation technique, applies high-voltage ultra-short pulses to permeabilize targeted cell membranes and induce cell death. Progression with IRE technology and an array of research studies have shown that beyond tumor debulking, IRE can induce anti-tumor immune responses possibly through tumor neo-antigen release. However, the success of IRE treatment (i.e. full ablation and tumor recurrence) is variable. We believe that IRE treatment induces IFN gamma expression, which then modulates immune checkpoint molecules and thus leads to tumor recurrence. This indicates a co-therapeutic use of IRE and immune checkpoint inhibitors as a promising treatment for pancreatic cancer patients. Here, we review the well-defined and speculated pathways involved in the immunostimulatory effects of IRE treatment for pancreatic cancer, as well as the regulatory pathways that may negate these anti-tumor responses. By defining these underlying mechanisms, future studies may identify improvements to systemic immune system engagement following local tumor ablation with IRE and beyond.
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    Histotripsy Ablation in Preclinical Animal Models of Cancer and Spontaneous Tumors in Veterinary Patients: A Review
    Hendricks-Wenger, Alissa; Arnold, Lauren; Gannon, Jessica; Simon, Alex; Singh, Neha; Sheppard, Hannah; Nagai-Singer, Margaret A.; Imran, Khan Mohammed; Lee, Kiho; Clark-Deener, Sherrie; Byron, Christopher R.; Edwards, Michael R.; Larson, Martha M.; Rossmeisl, John H. Jr.; Coutermarsh-Ott, Sheryl; Eden, Kristin; Dervisis, Nikolaos G.; Klahn, Shawna L.; Tuohy, Joanne L.; Allen, Irving C.; Vlaisavljevich, Eli (IEEE, 2021-09-03)
    New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely noninvasive, nonthermal, and nonionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant preclinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients.
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    Improved Therapeutic Delivery Targeting Clinically Relevant Orthotopic Human Pancreatic Tumors Engrafted in Immunocompromised Pigs Using Ultrasound-Induced Cavitation: A Pilot Study
    Imran, Khan Mohammad; Tintera, Benjamin; Morrison, Holly A.; Tupik, Juselyn D.; Nagai-Singer, Margaret A.; Ivester, Hannah; Council-Troche, McAlister; Edwards, Michael; Coutermarsh-Ott, Sheryl; Byron, Christopher; Clark-Deener, Sherrie; Uh, Kyungjun; Lee, Kiho; Boulos, Paul; Rowe, Cliff; Coviello, Christian; Allen, Irving C. (MDPI, 2023-05-24)
    Pancreatic tumors can be resistant to drug penetration due to high interstitial fluid pressure, dense stroma, and disarrayed vasculature. Ultrasound-induced cavitation is an emerging technology that may overcome many of these limitations. Low-intensity ultrasound, coupled with co-administered cavitation nuclei consisting of gas-stabilizing sub-micron scale SonoTran Particles, is effective at increasing therapeutic antibody delivery to xenograft flank tumors in mouse models. Here, we sought to evaluate the effectiveness of this approach in situ using a large animal model that mimics human pancreatic cancer patients. Immunocompromised pigs were surgically engrafted with human Panc-1 pancreatic ductal adenocarcinoma (PDAC) tumors in targeted regions of the pancreas. These tumors were found to recapitulate many features of human PDAC tumors. Animals were intravenously injected with the common cancer therapeutics Cetuximab, gemcitabine, and paclitaxel, followed by infusion with SonoTran Particles. Select tumors in each animal were targeted with focused ultrasound to induce cavitation. Cavitation increased the intra-tumor concentrations of Cetuximab, gemcitabine, and paclitaxel by 477%, 148%, and 193%, respectively, compared to tumors that were not targeted with ultrasound in the same animals. Together, these data show that ultrasound-mediated cavitation, when delivered in combination with gas-entrapping particles, improves therapeutic delivery in pancreatic tumors under clinically relevant conditions.
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    NLRX1 Deficiency Alters the Gut Microbiome and Is Further Exacerbated by Adherence to a Gluten-Free Diet
    Morrison, Holly A.; Liu, Yang; Eden, Kristin; Nagai-Singer, Margaret A.; Wade, Paul A.; Allen, Irving C. (Frontiers, 2022-04-28)
    Patients with gluten sensitivities present with dysbiosis of the gut microbiome that is further exacerbated by a strict adherence to a gluten-free diet (GFD). A subtype of patients genetically susceptible to gluten sensitivities are Celiac Disease (CeD) patients, who are carriers of the HLA DR3/DQ2 or HLA DR4/DQ8 haplotypes. Although 85-95% of all CeD patients carry HLA DQ2, up to 25-50% of the world population carry this haplotype with only a minority developing CeD. This suggests that CeD and other gluten sensitivities are mediated by factors beyond genetics. The contribution of innate immune system signaling has been generally understudied in the context of gluten sensitivities. Thus, here we examined the role of NOD-like receptors (NLRs), a subtype of pattern recognition receptors, in maintaining the composition of the gut microbiome in animals maintained on a GFD. Human transcriptomics data revealed significant increases in the gene expression of multiple NLR family members, across functional groups, in patients with active CeD compared to control specimens. However, NLRX1 was uniquely down-regulated during active disease. NLRX1 is a negative regulatory NLR that functions to suppress inflammatory signaling and has been postulate to prevent inflammation-induced dysbiosis. Using Nlrx1(-/-) mice maintained on either a normal or gluten-free diet, we show that loss of NLRX1 alters the microbiome composition, and a distinctive shift further ensues following adherence to a GFD, including a reciprocal loss of beneficial microbes and increase in opportunistic bacterial populations. Finally, we evaluated the functional impact of an altered gut microbiome by assessing short- and medium-chain fatty acid production. These studies revealed significant differences in a selection of metabolic markers that when paired with 16S rRNA sequencing data could reflect an overall imbalance and loss of immune system homeostasis in the gastrointestinal system.
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    NLRX1 Is a Multifaceted and Enigmatic Regulator of Immune System Function
    Nagai-Singer, Margaret A.; Morrison, Holly A.; Allen, Irving C. (2019-10-11)
    Over the last decade, significant progress has been achieved in defining mechanisms underlying NLR regulation of immune system function. However, several NLR family members continue to defy our best attempts at characterization and routinely exhibit confounding data. This is particularly true for NLR family members that regulate signaling associated with the activation of other pattern recognition receptors. NLRX1 is a member of this NLR sub-group and acts as an enigmatic regulator of immune system function. NLRX1 has been shown to negatively regulate type-I interferon, attenuate pro-inflammatory NF-kappa B signaling, promote reactive oxygen species production, and modulate autophagy, cell death, and proliferation. However, the mechanism/s associated with NLRX1 modulation of these pathways is not fully understood and there are inconsistencies within the field. Likewise, it is highly likely that the full repertoire of biological functions impacted by NLRX1 are yet to be defined. Recent mouse studies have shown that NLRX1 significantly impacts a multitude of diseases, including cancer, virus infection, osteoarthritis, traumatic brain injury, and inflammatory bowel disease. Thus, it is essential that the underlying mechanism associated with NLRX1 function in each of these diseases be robustly defined. Here, we summarize the current progress in understanding mechanisms associated with NLRX1 function. We also offer insight into both unique and overlapping mechanisms regulated by NLRX1 that likely contribute to disease pathobiology. Ultimately, we believe that an improved understanding of NLRX1 will result in better defined mechanisms associated with immune system attenuation and the resolution of inflammation in a myriad of diseases.