Cellular Context Dictates the Suppression or Augmentation of Triple-Negative Mammary Tumor Metastasis by NLRX1.

Prior studies have defined multiple, but inconsistent, roles for the enigmatic pattern recognition receptor NLRX1 in regulating several cancer-associated biological functions. In this study, we explore the role of NLRX1 in the highly metastatic murine 4T1 mammary tumor model. We describe a functional dichotomy of NLRX1 between two different cellular contexts: expression in healthy host cells versus expression in the 4T1 tumor cells. Using Nlrx1-/- mice engrafted with 4T1 tumors, we demonstrate that NLRX1 functions as a tumor suppressor when expressed in the host cells. Specifically, NLRX1 in healthy host cells attenuates tumor growth and lung metastasis through suppressing characteristics of epithelial-mesenchymal transition and the lung metastatic niche. Conversely, we demonstrate that NLRX1 functions as a tumor promoter when expressed in 4T1 tumor cells using gain- and loss-of-function studies both in vitro and in vivo. Mechanistically, NLRX1 in the tumor cells augments 4T1 aggressiveness and metastasis through regulating epithelial-mesenchymal transition hallmarks, cell death, proliferation, migration, reactive oxygen species levels, and mitochondrial respiration. Collectively, we provide critical insight into NLRX1 function and establish a dichotomous role of NLRX1 in the 4T1 murine mammary carcinoma model that is dictated by cellular context.

Successful In Situ Targeting of Pancreatic Tumors in a Novel Orthotopic Porcine Model Using Histotripsy.

OBJECTIVE: New therapeutic strategies and paradigms are direly needed to treat pancreatic cancer. The absence of a suitable pre-clinical animal model of pancreatic cancer is a major limitation to biomedical device and therapeutic development. Traditionally, pigs have proven to be ideal models, especially in the context of designing human-sized instruments, perfecting surgical techniques and optimizing clinical procedures for use in humans. However, pig studies have typically focused on healthy tissue assessments and are limited to general safety evaluations because of the inability to effectively model human tumors. METHODS: Here, we establish an orthotopic porcine model of human pancreatic cancer using RAG2/IL2RG double-knockout immunocompromised pigs and treat the tumors ex vivo and in vivo with histotripsy. RESULTS: Using these animals, we describe the successful engraftment of Panc-1 human pancreatic cancer cell line tumors and characterize their development. To illustrate the utility of these animals for therapeutic development, we determine for the first time, the successful targeting of in situ pancreatic tumors using histotripsy. Treatment with histotripsy resulted in partial ablation in vivo and reduction in collagen content in both in vivo tumor in pig pancreas and ex vivo patient tumor. CONCLUSION: This study presents a first step toward establishing histotripsy as a non-invasive treatment method for pancreatic cancer and exposes some of the challenges of ultrasound guidance for histotripsy ablation in the pancreas. Simultaneously, we introduce a highly robust model of pancreatic cancer in a large mammal model that could be used to evaluate a variety biomedical devices and therapeutic strategies.

Improved Therapeutic Delivery Targeting Clinically Relevant Orthotopic Human Pancreatic Tumors Engrafted in Immunocompromised Pigs Using Ultrasound-Induced Cavitation: A Pilot Study.

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.

NLRX1 functions as a tumor suppressor in Pan02 pancreatic cancer cells.

Pancreatic cancer is a deadly malignancy with limited treatment options. NLRX1 is a unique, understudied member of the Nod-like Receptor (NLR) family of pattern recognition receptors that regulates a variety of biological processes that are highly relevant to pancreatic cancer. The role of NLRX1 in cancer remains highly enigmatic, with some studies defining its roles as a tumor promoter, while others characterize its contributions to tumor suppression. These seemingly contradicting roles appear to be due, at least in part, to cell type and temporal mechanisms. Here, we define roles for NLRX1 in regulating critical hallmarks of pancreatic cancer using both gain-of-function and loss-of-function studies in murine Pan02 cells. Our data reveals that NLRX1 increases susceptibility to cell death, while also suppressing proliferation, migration, and reactive oxygen species production. We also show that NLRX1 protects against upregulated mitochondrial activity and limits energy production in the Pan02 cells. Transcriptomics analysis revealed that the protective phenotypes associated with NLRX1 are correlated with attenuation of NF-κB, MAPK, AKT, and inflammasome signaling. Together, these data demonstrate that NLRX1 diminishes cancer-associated biological functions in pancreatic cancer cells and establishes a role for this unique NLR in tumor suppression.

NLRX1 Deficiency Alters the Gut Microbiome and Is Further Exacerbated by Adherence to a Gluten-Free Diet.

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.

Exploration of Novel Pathways Underlying Irreversible Electroporation Induced Anti-Tumor Immunity in Pancreatic Cancer.

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 IFNγ 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.

Methods to evaluate virus-mediated acute lung inflammation.

As more infectious viruses emerge that result in respiratory illness, there is a significant need to standardize airway harvests and maximize data acquisition. Animal models of respiratory viral infections have been outlined to allow for the analysis of the host immune response and viral pathogenesis kinetics. This chapter outlines two separate tissue harvest protocols following the intranasal infection of mice to investigate both the host immune response and viral pathogenesis. These protocols combine standard laboratory techniques for the analysis of the samples, making it easily integrable for labs without the need for specialized training. In offering two separate yet parallel tissue collection techniques, investigators can ultimately decide which technique will yield the best data for their particular research questions and can maximize data from each animal study.

Employing Novel Porcine Models of Subcutaneous Pancreatic Cancer to Evaluate Oncological Therapies.

Immunocompromised mice are commonly utilized to study pancreatic cancer and other malignancies. The ability to xenograft tumors in either subcutaneous or orthotopic locations provides a robust model to study diverse biological features of human malignancies. However, there is a dire need for large animal models that better recapitulate human anatomy in terms of size and physiology. These models will be critical for biomedical device development, surgical optimization, and drug discovery. Here, we describe the generation and application of immunocompromised pigs lacking RAG2 and IL2RG as a novel model for human xenograft studies. These SCID-like pigs closely resemble NOD scid gamma mice and are receptive to human tumor tissue, cell lines, and organoid xenografts. However, due to their immunocompromised nature, these immunocompromised animals require housing and maintenance under germfree conditions. In this protocol, we describe the use of these pigs in a subcutaneous tumor injection study with human PANC1 cells. The tumors demonstrate a steady, linear growth curve, reaching 1.0 cm within 30 days post injection. The model described here is focused on subcutaneous injections behind the ear. However, it is readily adaptable for other locations and additional human cell types.

Histotripsy Ablation in Preclinical Animal Models of Cancer and Spontaneous Tumors in Veterinary Patients: A Review.

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.

Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation.

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.

Evaluation of Myogenin and MyoD1 as Immunohistochemical Markers of Canine Rhabdomyosarcoma.

Canine rhabdomyosarcoma (RMS) presents a diagnostic challenge due to its overlapping histologic features with other soft tissue sarcomas. The diagnosis of RMS currently relies on positive immunohistochemical (IHC) labeling for desmin; however, desmin expression is also observed in non-RMS tumors. Myogenin and MyoD1 are transcription factors reported to be sensitive and specific IHC markers for human RMS, but they are not widely used in veterinary oncology. The goals of this study were to develop an IHC protocol for myogenin and MyoD1, evaluate myogenin and MyoD1 labeling in canine RMS, and report clinical outcomes. Sixteen cases of possible RMS were retrospectively evaluated. A diagnosis of RMS was confirmed in 13 cases based on histological features and immunolabeling for myogenin and MyoD1, with the aid of electron microscopy in 2 cases. Desmin was negative in 3 cases of RMS. Two cases were of the sclerosing variant. The median age of dogs with RMS was 7.2 years. Anatomic tumor locations included previously reported sites such as bladder, larynx, heart, and orbit, as well as other locations typical of soft tissue sarcomas. Survival ranged from 47 to 1480 days for 5 dogs with available data. This study demonstrated that MyoD1 and myogenin should be included with desmin as part of a diagnostic IHC panel for canine RMS. Utilization of these antibodies to improve the accuracy of canine RMS diagnosis will ultimately allow for better characterization of the biological behavior and clinical outcomes of this disease, providing the groundwork for future comparative investigations in canine RMS.

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

To protect or adversely affect? The dichotomous role of the NLRP1 inflammasome in human disease.

NLRP1 is an inflammasome forming pattern recognition receptor (PRR). When activated by pathogen- and damage- associated molecular patterns (PAMPS/DAMPS), NLRP1 inflammasome formation leads to inflammation through the production of proinflammatory cytokines IL-18 and IL-1ß. As with other inflammasome forming NLR family members, NLRP1 also regulates cell death processes, termed pyroptosis. The domain structure of NLRP1 differs between mice and humans, making it possible for the function of the inflammasome to differ between species and adds complexity to the study of this NLR family member. In humans, mutations in both coding and non-coding regions of the NLRP1 gene are linked to a variety of diseases. Likewise, interruption of NLRP1 inhibitors or changes in the prevalence of NLRP1 activators can also impact disease pathobiology. Adding to its complexity, the NLRP1 inflammasome plays a dichotomous role in human diseases, functioning to either attenuate or augment miscellaneous biological processes in a tissue specific manner. For example, NLRP1 plays a protective role in the gastrointestinal tract by modulating the microbiome composition; however, it augments neurological disorders, cardio-pulmonary diseases, and cancer through promoting inflammation. Thus, it is critical that the role of NLRP1 in each of these disease processes be robustly defined. In this review, we summarize the current research landscape to provide a better understanding of the mechanisms associated with NLRP1 function and dysfunction in human disease pathobiology. We propose that a better understanding of these mechanisms will ultimately result in improved insight into immune system dysfunction and therapeutic strategies targeting inflammasome function in multiple human diseases.

NLRX1 Is a Multifaceted and Enigmatic Regulator of Immune System Function.

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-κB 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.