In-Transit Metastasis in a Dog with High-Grade Soft Tissue Sarcoma: A Case Report.

A 6 yr old male castrated American Staffordshire terrier was referred for a nonhealing wound at the site of a previously incompletely excised, high-grade soft tissue sarcoma. Physical examination revealed right popliteal lymphadenopathy and a fungating mass of the right pelvic limb at the level of the hock. Thoracic and abdominal computed tomography revealed mild lymphadenopathy of multiple iliac and inguinal lymph nodes. Right pelvic limb amputation and inguinal lymphadenectomy were performed. Histopathology was consistent of a high-grade soft tissue sarcoma with diffuse spread through the lymphatic vessels of the right pelvic limb up to the right inguinal lymph node but not affecting the lymph node itself. Doxorubicin chemotherapy was elected postoperatively as adjuvant therapy. Approximately 4 mo following initiation of chemotherapy, the patient developed a firm, tubular subcutaneous mass starting near the previous amputation site with tracking toward the thorax. Fine needle aspiration of the new mass was consistent with atypical spindle cell proliferation. Palliative care was elected, and the patient was euthanized 3 mo later because of progressive disease. In-transit metastasis is a rare behavior for soft tissue sarcomas across all species, and this is the first report of such a presentation for canine soft tissue sarcoma.

High-Frequency Irreversible Electroporation for Treatment of Primary Liver Cancer: A Proof-of-Principle Study in Canine Hepatocellular Carcinoma.

PURPOSE: To determine the safety and feasibility of percutaneous high-frequency irreversible electroporation (HFIRE) for primary liver cancer and evaluate the HFIRE-induced local immune response. MATERIALS AND METHODS: HFIRE therapy was delivered percutaneously in 3 canine patients with resectable hepatocellular carcinoma (HCC) in the absence of intraoperative paralytic agents or cardiac synchronization. Pre- and post-HFIRE biopsy samples were processed with histopathology and immunohistochemistry for CD3, CD4, CD8, and CD79a. Blood was collected on days 0, 2, and 4 for complete blood count and chemistry. Numeric models were developed to determine the treatment-specific lethal thresholds for malignant canine liver tissue and healthy porcine liver tissue. RESULTS: HFIRE resulted in predictable ablation volumes as assessed by posttreatment CT. No detectable cardiac interference and minimal muscle contraction occurred during HFIRE. No clinically significant adverse events occurred secondary to HFIRE. Microscopically, a well-defined ablation zone surrounded by a reactive zone was evident in the majority of samples. This zone was composed primarily of maturing collagen interspersed with CD3+/CD4-/CD8- lymphocytes in a proinflammatory microenvironment. The average ablation volumes for the canine HCC patients and the healthy porcine tissue were 3.89 cm3 ± 0.74 and 1.56 cm3 ± 0.16, respectively (P = .03), and the respective average lethal thresholds were 710 V/cm ± 28.2 and 957 V/cm ± 24.4 V/cm (P = .0004). CONCLUSIONS: HFIRE can safely and effectively be delivered percutaneously, results in a predictable ablation volume, and is associated with lymphocytic tumor infiltration. This is the first step toward the use of HFIRE for treatment of unresectable liver tumors.

High-frequency irreversible electroporation improves survival and immune cell infiltration in rodents with malignant gliomas.

Background: Irreversible electroporation (IRE) has been previously investigated in preclinical trials as a treatment for intracranial malignancies. Here, we investigate next generation high-frequency irreversible electroporation (H-FIRE), as both a monotherapy and a combinatorial therapy, for the treatment of malignant gliomas. Methods: Hydrogel tissue scaffolds and numerical modeling were used to inform in-vivo H-FIRE pulsing parameters for our orthotopic tumor-bearing glioma model. Fischer rats were separated into five treatment cohorts including high-dose H-FIRE (1750V/cm), low-dose H-FIRE (600V/cm), combinatorial high-dose H-FIRE + liposomal doxorubicin, low-dose H-FIRE + liposomal doxorubicin, and standalone liposomal doxorubicin groups. Cohorts were compared against a standalone tumor-bearing sham group which received no therapeutic intervention. To further enhance the translational value of our work, we characterize the local and systemic immune responses to intracranial H-FIRE at the study timepoint. Results: The median survival for each cohort are as follows: 31 days (high-dose H-FIRE), 38 days (low-dose H-FIRE), 37.5 days (high-dose H-FIRE + liposomal doxorubicin), 27 days (low-dose H-FIRE + liposomal doxorubicin), 20 days (liposomal doxorubicin), and 26 days (sham). A statistically greater overall survival fraction was noted in the high-dose H-FIRE + liposomal doxorubicin (50%, p = 0.044), high-dose H-FIRE (28.6%, p = 0.034), and the low-dose H-FIRE (20%, p = 0.0214) compared to the sham control (0%). Compared to sham controls, brain sections of rats treated with H-FIRE demonstrated significant increases in IHC scores for CD3+ T-cells (p = 0.0014), CD79a+ B-cells (p = 0.01), IBA-1+ dendritic cells/microglia (p = 0.04), CD8+ cytotoxic T-cells (p = 0.0004), and CD86+ M1 macrophages (p = 0.01). Conclusions: H-FIRE may be used as both a monotherapy and a combinatorial therapy to improve survival in the treatment of malignant gliomas while also promoting the presence of infiltrative immune cells.

High-Frequency Irreversible Electroporation (H-FIRE) Induced Blood-Brain Barrier Disruption Is Mediated by Cytoskeletal Remodeling and Changes in Tight Junction Protein Regulation.

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.

Advancements in drug delivery methods for the treatment of brain disease.

The blood-brain barrier (BBB) presents a formidable obstacle to the effective delivery of systemically administered pharmacological agents to the brain, with ~5% of candidate drugs capable of effectively penetrating the BBB. A variety of biomaterials and therapeutic delivery devices have recently been developed that facilitate drug delivery to the brain. These technologies have addressed many of the limitations imposed by the BBB by: (1) designing or modifying the physiochemical properties of therapeutic compounds to allow for transport across the BBB; (2) bypassing the BBB by administration of drugs via alternative routes; and (3) transiently disrupting the BBB (BBBD) using biophysical therapies. Here we specifically review colloidal drug carrier delivery systems, intranasal, intrathecal, and direct interstitial drug delivery methods, focused ultrasound BBBD, and pulsed electrical field induced BBBD, as well as the key features of BBB structure and function that are the mechanistic targets of these approaches. Each of these drug delivery technologies are illustrated in the context of their potential clinical applications and limitations in companion animals with naturally occurring intracranial diseases.

Companion animal models of neurological disease.

Clinical translation of novel therapeutics that improve the survival and quality of life of patients with neurological disease remains a challenge, with many investigational drug and device candidates failing in advanced stage clinical trials. Naturally occurring inherited and acquired neurological diseases, such as epilepsy, inborn errors of metabolism, brain tumors, spinal cord injury, and stroke occur frequently in companion animals, and many of these share epidemiologic, pathophysiologic and clinical features with their human counterparts. As companion animals have a relatively abbreviated lifespan and genetic background, are immunocompetent, share their environment with human caregivers, and can be clinically managed using techniques and tools similar to those used in humans, they have tremendous potential for increasing the predictive value of preclinical drug and device studies. Here, we review comparative features of spontaneous neurological diseases in companion animals with an emphasis on neuroimaging methods and features, illustrate their historical use in translational studies, and discuss inherent limitations associated with each disease model. Integration of companion animals with naturally occurring disease into preclinical studies can complement and expand the knowledge gained from studies in other animal models, accelerate or improve the manner in which research is translated to the human clinic, and ultimately generate discoveries that will benefit the health of humans and animals.

Novel ablation methods for treatment of gliomas.

Primary brain tumors are among the deadliest cancers that remain highly incurable. A need exists for new approaches to tumor therapy that can circumvent the blood brain barrier (BBB), target highly resistant tumors and cancer stem-like cells (CSCs) as well create an anti-cancer immunomodulatory environment. Successful treatments may also require a combinatory approach utilizing surgery, chemotherapy, radiation and novel ablation strategies that can both eliminate the bulk tumor and prevent any potential residual CSCs from propagating in the resected tissue. A number of thermal and non-thermal ablation methods have been developed and tested, which have gained much enthusiasm for the treatment of brain tumors. Here we review the most common primary brain tumors and the candidate ablation methods for targeting the tumor and its microenvironment.