Scholarly Works, Institute for Critical Technology and Applied Science (ICTAS)

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Research articles, presentations, and other scholarship

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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.
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.
High intensity focused ultrasound for the treatment of solid tumors: a pilot study in canine cancer patients
Carroll, Jennifer; Coutermarsh-Ott, Sheryl; Klahn, Shawna L.; Tuohy, Joanne L.; Barry, Sabrina L.; Allen, Irving C.; Hay, Alayna N.; Ruth, Jeffrey; Dervisis, Nikolaos G. (Taylor & Francis, 2022-01)
Purpose: To investigate the safety, feasibility, and outcomes of High-Intensity Focused Ultrasound (HIFU) for the treatment of solid tumors in a spontaneous canine cancer model. Methods: Dogs diagnosed with subcutaneous solid tumors were recruited, staged and pretreatment biopsies were obtained. A single HIFU treatment was delivered to result in partial tumor ablation using a commercially available HIFU unit. Tumors were resected 3-6 days post HIFU and samples obtained for histopathology and immunohistochemistry. Total RNA was isolated from paired pre and post treated FFPE tumor samples, and quantitative gene expression analysis was performed using the nCounter Canine IO Panel. Results: A total of 20 dogs diagnosed with solid tumors were recruited and treated in the study. Tumors treated included Soft Tissue Sarcoma (n = 15), Mast Cell Tumor (n = 3), Osteosarcoma (n = 1), and Thyroid Carcinoma (n = 1). HIFU was well tolerated with only 1 dog experiencing a clinically significant adverse event. Pathology confirmed the presence of complete tissue ablation at the HIFU targeted site and immunohistochemistry indicated immune cell infiltration at the treated/untreated tumor border. Quantitative gene expression analysis indicated that 28 genes associated with T-cell activation were differentially expressed post-HIFU. Conclusions: HIFU appears to be safe and feasible for the treatment of subcutaneous canine solid tumors, resulting in ablation of the targeted tissue. HIFU induced immunostimulatory changes, highlighting the canine cancer patient as an attractive model for studying the effects of focal ablation therapies on the tumor microenvironment.
High-Frequency Irreversible Electroporation (H-FIRE) Induced Blood-Brain Barrier Disruption Is Mediated by Cytoskeletal Remodeling and Changes in Tight Junction Protein Regulation
Partridge, Brittanie R.; Kani, Yukitaka; Lorenzo, Melvin F.; Campelo, Sabrina N.; Allen, Irving C.; Hinckley, Jonathan; Hsu, Fang-Chi; Verbridge, Scott S.; Robertson, John L.; Davalos, Rafael V.; Rossmeisl, John H. Jr. (MDPI, 2022-06-11)
Glioblastoma is the deadliest malignant brain tumor. Its location behind the blood–brain barrier (BBB) presents a therapeutic challenge by preventing effective delivery of most chemotherapeutics. H-FIRE is a novel tumor ablation method that transiently disrupts the BBB through currently unknown mechanisms. We hypothesized that H-FIRE mediated BBB disruption (BBBD) occurs via cytoskeletal remodeling and alterations in tight junction (TJ) protein regulation. Intracranial H-FIRE was delivered to Fischer rats prior to sacrifice at 1-, 24-, 48-, 72-, and 96 h post-treatment. Cytoskeletal proteins and native and ubiquitinated TJ proteins (TJP) were evaluated using immunoprecipitation, Western blotting, and gene-expression arrays on treated and sham control brain lysates. Cytoskeletal and TJ protein expression were further evaluated with immunofluorescent microscopy. A decrease in the F/G-actin ratio, decreased TJP concentrations, and increased ubiquitination of TJP were observed 1–48 h post-H-FIRE compared to sham controls. By 72–96 h, cytoskeletal and TJP expression recovered to pretreatment levels, temporally corresponding with increased claudin-5 and zonula occludens-1 gene expression. Ingenuity pathway analysis revealed significant dysregulation of claudin genes, centered around claudin-6 in H-FIRE treated rats. In conclusion, H-FIRE is capable of permeating the BBB in a spatiotemporal manner via cytoskeletal-mediated TJP modulation. This minimally invasive technology presents with applications for localized and long-lived enhanced intracranial drug delivery.
High-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity
Ringel-Scaia, Veronica M.; Beitel-White, Natalie; Lorenzo, Melvin F.; Brock, Rebecca M.; Huie, Kathleen E.; Coutermarsh-Ott, Sheryl; Eden, Kristin; McDaniel, Dylan K.; Verbridge, Scott S.; Rossmeisl, John H. Jr.; Oestreich, Kenneth J.; Davalos, Rafael V.; Allen, Irving C. (2019-06)
Background: Despite promising treatments for breast cancer, mortality rates remain high and treatments for metastatic disease are limited. High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation technique that utilizes high-frequency bipolar electric pulses to destabilize cancer cell membranes and induce cell death. However, there is currently a paucity of data pertaining to immune system activation following H-FIRE and other electroporation based tumor ablation techniques. Methods: Here, we utilized the mouse 4T1 mammary tumor model to evaluate H-FIRE treatment parameters on cancer progression and immune system activation in vitro and in vivo. Findings: H-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment. We further show that local treatment with H-FIRE significantly reduces 4T1 metastases. H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo. Our data indicate that the level of tumor ablation correlates with increased activation of cellular immunity. Likewise, we show that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression. Interpretation: Cell death and tumor ablation following H-FIRE treatment activates the local innate immune system, which shifts the tumor microenvironment from an anti-inflammatory state to a pro-inflammatory state. The non-thermal damage to the cancer cells and increased innate immune system stimulation improves antigen presentation, resulting in the engagement of the adaptive immune system and improved systemic anti-tumor immunity. (C) 2019 The Authors. Published by Elsevier B.V.
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.
Histotripsy Ablation of Bone Tumors: Feasibility Study in Excised Canine Osteosarcoma Tumors
Arnold, Lauren; Hendricks-Wenger, Alissa; Coutermarsh-Ott, Sheryl; Gannon, Jessica; Hay, Alayna N.; Dervisis, Nikolaos G.; Klahn, Shawna L.; Allen, Irving C.; Tuohy, Joanne L.; Vlaisavljevich, Eli (Elsevier, 2021-12)
Osteosarcoma (OS) is a primary bone tumor affecting both dogs and humans. Histotripsy is a non-thermal, non-invasive focused ultrasound method using controlled acoustic cavitation to mechanically disintegrate tissue. In this study, we investigated the feasibility of treating primary OS tumors with histotripsy using a 500-kHz transducer on excised canine OS samples harvested after surgery at the Veterinary Teaching Hospital at Virginia Tech. Samples were embedded in gelatin tissue phantoms and treated with the 500-kHz histotripsy system using one- or two-cycle pulses at a pulse repetition frequency of 250 Hz and a dosage of 4000 pulses/point. Separate experiments also assessed histotripsy effects on normal canine bone and nerve using the same pulsing parameters. After treatment, histopathological evaluation of the samples was completed. To determine the feasibility of treating OS through intact skin/soft tissue, additional histotripsy experiments assessed OS with overlying tissues. Generation of bubble clouds was achieved at the focus in all tumor samples at peak negative pressures of 26.2 ± 4.5 MPa. Histopathology revealed effective cell ablation in treated areas for OS tumors, with no evidence of cell death or tissue damage in normal tissues. Treatment through tissue/skin resulted in generation of well-confined bubble clouds and ablation zones inside OS tumors. Results illustrate the feasibility of treating OS tumors with histotripsy. CORRIGENDUM: The authors regret that errors were present in the above article. The legend for Figure 5 on page 3441 should read “Fig. 5. Normal, healthy, non-neoplastic bone was excised from amputated canine limbs and subjected to histology. No histological differences were noted between untreated (a: magnification 4 x, b: magnification 40 x) and treated samples (c: magnification 4 x, d: magnification 40 x).” Also, the final section heading on page 3439 should read “Histotripsy ablation of ex vivo bone and nerve specimens.” Finally, the reference after the last complete sentence on page 3437 is incomplete and should read “Focal pressure waveforms for the 500-kHz transducer were measured using a custom-built fiberoptic hydrophone (FOPH) in degassed water at the focal point of each transducer (Parsons et al. 2006).” The authors would like to apologise for any inconvenience caused.
Pulmonary Exposure to Magnéli Phase Titanium Suboxides Results in Significant Macrophage Abnormalities and Decreased Lung Function
McDaniel, Dylan K.; Ringel-Scaia, Veronica M.; Morrison, Holly A.; Coutermarsh-Ott, Sheryl; Council-Troche, McAlister; Angle, Jonathan W.; Perry, Justin B.; Davis, Grace; Leng, Weinan; Minarchick, Valerie; Yang, Yi; Chen, Bo; Reece, Sky W.; Brown, David A.; Cecere, Thomas E.; Brown, Jared M.; Gowdy, Kymberly M.; Hochella, Michael F. Jr.; Allen, Irving C. (Frontiers, 2019-11-28)
Coal is one of the most abundant and economic sources for global energy production. However, the burning of coal is widely recognized as a significant contributor to atmospheric particulate matter linked to deleterious respiratory impacts. Recently, we have discovered that burning coal generates large quantities of otherwise rare Magnéli phase titanium suboxides from TiO2 minerals naturally present in coal. These nanoscale Magnéli phases are biologically active without photostimulation and toxic to airway epithelial cells in vitro and to zebrafish in vivo. Here, we sought to determine the clinical and physiological impact of pulmonary exposure to Magnéli phases using mice as mammalian model organisms. Mice were exposed to the most frequently found Magnéli phases, Ti6O11, at 100 parts per million (ppm) via intratracheal administration. Local and systemic titanium concentrations, lung pathology, and changes in airway mechanics were assessed. Additional mechanistic studies were conducted with primary bone marrow derived macrophages. Our results indicate that macrophages are the cell type most impacted by exposure to these nanoscale particles. Following phagocytosis, macrophages fail to properly eliminate Magnéli phases, resulting in increased oxidative stress, mitochondrial dysfunction, and ultimately apoptosis. In the lungs, these nanoparticles become concentrated in macrophages, resulting in a feedback loop of reactive oxygen species production, cell death, and the initiation of gene expression profiles consistent with lung injury within 6 weeks of exposure. Chronic exposure and accumulation of Magnéli phases ultimately results in significantly reduced lung function impacting airway resistance, compliance, and elastance. Together, these studies demonstrate that Magnéli phases are toxic in the mammalian airway and are likely a significant nanoscale environmental pollutant, especially in geographic regions where coal combustion is a major contributor to atmospheric particulate matter.

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