Immunohistochemical and transcriptomic characterization of T and myeloid cell infiltrates in canine malignant melanoma.

Immune checkpoint inhibitor therapy can provide significant clinical benefit in patients with certain cancer types including melanoma; however, objective responses are only observed for a subset of patients. Mucosal melanoma is a rare melanoma subtype associated with a poor prognosis and, compared with cutaneous melanoma, is significantly less responsive to immune checkpoint inhibitors. Spontaneous canine tumours have emerged as valuable models to inform human cancer studies. In contrast to human melanoma, most canine melanomas are mucosal-an incidence that may be leveraged to better understand the subtype in humans. However, a more comprehensive understanding of the immune landscape of the canine disease is required. Here, we quantify tumour infiltrative T and myeloid cells in canine mucosal (n = 13) and cutaneous (n = 5) melanomas using immunohistochemical analysis of CD3 and MAC387 expression, respectively. Gene expression analysis using the Canine IO NanoString panel was also performed to identify genes and pathways associated with immune cell infiltration. T and myeloid cell densities were variable with geometric means of 158.7 cells/mm2 and 166.7 cells/mm2, respectively. Elevated T cell infiltration was associated with increased expression of cytolytic genes as well as genes encoding the coinhibitory checkpoint molecules PD-1, CTLA-4, TIM-3 and TIGIT; whereas increased myeloid cell infiltration was associated with elevated expression of protumourigenic cytokines. These data provide a basic characterization of the tumour microenvironment of canine malignant melanoma and suggest that, like human melanoma, inherent variability in anti-tumour T cell responses exists and that a subset of canine melanomas may respond better to immunomodulation.

Single-cell RNA sequencing reveals the cellular and molecular heterogeneity of treatment-naïve primary osteosarcoma in dogs.

Osteosarcoma (OS) is a heterogeneous, aggressive malignancy of the bone that disproportionally affects children and adolescents. Therapeutic interventions for OS are limited, which is in part due to the complex tumor microenvironment (TME). As such, we used single-cell RNA sequencing (scRNA-seq) to describe the cellular and molecular composition of the TME in 6 treatment-naïve dogs with spontaneously occurring primary OS. Through analysis of 35,310 cells, we identified 41 transcriptomically distinct cell types including the characterization of follicular helper T cells, mature regulatory dendritic cells (mregDCs), and 8 tumor-associated macrophage (TAM) populations. Cell-cell interaction analysis predicted that mregDCs and TAMs play key roles in modulating T cell mediated immunity. Furthermore, we completed cross-species cell type gene signature homology analysis and found a high degree of similarity between human and canine OS. The data presented here act as a roadmap of canine OS which can be applied to advance translational immuno-oncology research.

Antimicrobial efficacy of cyclic α- and ß-peptides incorporated in polyurethane coatings.

Microbial growth on surfaces poses health concerns and can accelerate the biodegradation of engineered materials and coatings. Cyclic peptides are promising agents to combat biofouling because they are more resistant to enzymatic degradation than their linear counterparts. They can also be designed to interact with extracellular targets and intracellular targets and/or self-assemble into transmembrane pores. Here, we determine the antimicrobial efficacy of two pore-forming cyclic peptides, α-K3W3 and ß-K3W3, against bacterial and fungal liquid cultures and their capacity to inhibit biofilm formation on coated surfaces. These peptides display identical sequences, but the additional methylene group in the peptide backbone of ß-amino acids results in a larger diameter and an enhancement in the dipole moment. In liquid cultures, ß-K3W3 exhibited lower minimum inhibitory concentration values and greater microbicidal power in reducing the number of colony forming units (CFUs) when exposed to a gram-positive bacterium, Staphylococcus aureus, and two fungal strains, Naganishia albida and Papiliotrema laurentii. To evaluate the efficacy against the formation of fungal biofilms on painted surfaces, cyclic peptides were incorporated into polyester-based thermoplastic polyurethane. The formation of N. albida and P. laurentii microcolonies (105 per inoculation) for cells extracted from coatings containing either peptide could not be detected after a 7-day exposure. Moreover, very few CFUs (∼5) formed after 35 days of repeated depositions of freshly cultured P. laurentii every 7 days. In contrast, the number of CFUs for cells extracted from the coating without cyclic peptides was >8 log CFU.

Integrated analysis of canine soft tissue sarcomas identifies recurrent mutations in TP53, KMT genes and PDGFB fusions.

Soft tissue sarcomas (STS) are a heterogenous group of mesenchymal tumors representing over 50 distinct types with overlapping histological features and non-specific anatomical locations. Currently, localized sarcomas are treated with surgery + / - radiation in both humans and dogs with few molecularly targeted therapeutic options. However, to improve precision-based cancer therapy through trials in pet dogs with naturally occurring STS tumors, knowledge of genomic profiling and molecular drivers in both species is essential. To this purpose, we sought to characterize the transcriptomic and genomic mutation profiles of canine STS subtypes (fibrosarcoma, undifferentiated pleomorphic sarcoma, and peripheral nerve sheath tumors), by leveraging RNAseq, whole exome sequencing, immunohistochemistry, and drug assays. The most common driver mutations were in cell cycle/DNA repair (31%, TP53-21%) and chromatin organization/binding (41%, KMT2D-21%) genes. Similar to a subset of human sarcomas, we identified fusion transcripts of platelet derived growth factor B and collagen genes that predict sensitivity to PDGFR inhibitors. Transcriptomic profiling grouped these canine STS tumors into 4 clusters, one PNST group (H1), and 3 FSA groups selectively enriched for extracellular matrix interactions and PDFGB fusions (H2), homeobox transcription factors (H3), and elevated T-cell infiltration (H4). This multi-omics approach provides insights into canine STS sub-types at a molecular level for comparison to their human counterparts, to improve diagnosis, and may provide additional targets for chemo- and immuno-therapy.

Biphasic Interpositional Allograft for Rotator Cuff Repair Augmentation Is Safe in an Ovine Model.

PURPOSE: To perform a preclinical histologic assessment of a biphasic acellular interpositional cancellous allograft in an ovine model of rotator cuff repair (RCR) designed to better understand its safety profile and effects on tendon healing after RCR. METHODS: Thirty skeletally mature sheep with clinically normal shoulders with an artificially created degenerative infraspinatus tendon tear were randomized to control and treatment groups. Animals were euthanized at 3 weeks, 6 weeks, and 12 weeks. After gross dissection, rotator cuff specimens were fixed with formalin and polymerized for sectioning and staining. Blinded histologic scores evaluated inflammatory cell infiltrates, signs of degradation, particulate debris, collagen arrangement, neovascularization, and enthesis qualitative measures. RESULTS: There were no treatment specimens that exhibited histologic signs of a significant infection, inflammatory infiltrate, or foreign body reaction such as granuloma or fibrous capsule formation. Histologic scores in all categories were not significantly different at all time points, including the primary end point mean cumulative inflammatory score (control: 3.66 ± 1.21 vs treated: 4.33 ± 1.51, P = .42), when comparing the treatment and control RCR groups. In general, the degree of tendon healing and host tissue response was essentially equivalent between the 2 groups with observation of low overall levels of inflammation and progressive improvements in collagen organization, reduced tenocyte activity, and fibrocartilaginous enthesis reformation. CONCLUSIONS: This histologic study demonstrated the use of a biphasic interpositional allograft for RCR augmentation in an ovine model does not generate an inflammatory response or foreign body reaction. Use of the biphasic interpositional allograft resulted in a histological profile that was essentially equivalent to that of a standard RCR at 3-, 6-, and 12-week postoperative timepoints. These findings suggest that a biphasic interpositional allograft is safe for further clinical investigation in humans before broader clinical application. CLINICAL RELEVANCE: Patch augmentation of RCR is a popular technique that has shown clinical success in improving the likelihood of a successful repair in patients at elevated risk for retear. Newer augmentation technologies are being developed to address the biology at the interface between the bone and soft tissue where failure typically occurs.

Integrated analysis of canine soft tissue sarcomas identifies recurrent mutations in TP53, KMT genes and PDGFB fusions.

Canine soft tissue sarcomas (STS) are a heterogenous group of malignant tumors arising from mesenchymal cells of soft tissues. This simplified collective of tumors most commonly arise from subcutaneous tissues, are treated similar clinically, and conventionally exclude other sarcomas with more definitive anatomical, histological, or biological features. Histologically, canine STS sub-types are difficult to discern at the light microscopic level due to their overlapping features. Thus, genomic, and transcriptomic profiling of canine STS may prove valuable in differentiating the diverse sub-types of mesenchymal neoplasms within this group. To this purpose we sought to characterize the transcript expression and genomic mutation profiles of canine STS. To delineate transcriptomic sub-types, hierarchical clustering was used to identify 4 groups with district expression profiles. Using the RNAseq data, we identified three samples carrying driver fusions of platelet derived growth factor B ( PDGFB ) and collagen genes. Sensitivity to imatinib was evaluated in a canine STS cell line also bearing a PDGFB fusion. Using whole exome sequencing, recurrent driver variants were identified in the cancer genes KMT2D (21% of the samples) and TP53 (21%) along with copy number losses of RB1 and CDKN2A. Gene amplifications and resulting transcript increases were identified in genes on chromosomes 13, 14, and 36. A subset of STS was identified with high T-cell infiltration. This multi-omics approach has defined canine STS sub-types at a molecular level for comparison to their human counterparts, to improve diagnosis, and may provide additional targets for therapy.

Improved Recovery of Captured Airborne Bacteria and Viruses with Liquid-Coated Air Filters.

The COVID-19 pandemic has revealed the importance of the detection of airborne pathogens. Here, we present composite air filters featuring a bioinspired liquid coating that facilitates the removal of captured aerosolized bacteria and viruses for further analysis. We tested three types of air filters: commercial polytetrafluoroethylene (PTFE), which is well known for creating stable liquid coatings, commercial high-efficiency particulate air (HEPA) filters, which are widely used, and in-house-manufactured cellulose nanofiber mats (CNFMs), which are made from sustainable materials. All filters were coated with omniphobic fluorinated liquid to maximize the release of pathogens. We found that coating both the PTFE and HEPA filters with liquid improved the rate at which Escherichia coli was recovered using a physical removal process compared to uncoated controls. Notably, the coated HEPA filters also increased the total number of recovered cells by 57%. Coating the CNFM filters did not improve either the rate of release or the total number of captured cells. The most promising materials, the liquid-coated HEPA, filters were then evaluated for their ability to facilitate the removal of pathogenic viruses via a chemical removal process. Recovery of infectious JC polyomavirus, a nonenveloped virus that attacks the central nervous system, was increased by 92% over uncoated controls; however, there was no significant difference in the total amount of genomic material recovered compared to that of controls. In contrast, significantly more genomic material was recovered for SARS-CoV-2, the airborne, enveloped virus, which causes COVID-19, from liquid-coated filters. Although the amount of infectious SARS-CoV-2 recovered was 58% higher, these results were not significantly different from uncoated filters due to high variability. These results suggest that the efficient recovery of airborne pathogens from liquid-coated filters could improve air sampling efforts, enhancing biosurveillance and global pathogen early warning.

A Large Animal Model for Orthopedic Foot and Ankle Research.

Trauma to the soft tissues of the ankle joint distal syndesmosis often leads to syndesmotic instability, resulting in undesired movement of the talus, abnormal pressure distributions, and ultimately arthritis if deterioration progresses without treatment. Historically, syndesmotic injuries have been repaired by placing a screw across the distal syndesmosis to provide rigid fixation to facilitate ligament repair. While rigid syndesmotic screw fixation immobilizes the ligamentous injury between the tibia and fibula to promote healing, the same screws inhibit normal physiologic movement and dorsiflexion. It has been shown that intact screw removal can be beneficial for long-term patient success; however, the exact timing remains an unanswered question that necessitates further investigation, perhaps using animal models. Because of the sparsity of relevant preclinical models, the purpose of this study was to develop a new, more translatable, large animal model that can be used for the investigation of clinical foot and ankle implants. Eight (8) skeletally mature sheep underwent stabilization of the left and right distal carpal bones following transection of the dorsal and interosseous ligaments while the remaining two animals served as un-instrumented controls. Four of the surgically stabilized animals were sacrificed 6 weeks after surgery while the remaining four animals were sacrificed 10 weeks after surgery. Ligamentous healing was evaluated using radiography, histology, histomorphometry, and histopathology. Overall, animals demonstrated a high tolerance to the surgical procedure with minimal complications. Animals sacrificed at 10 weeks post-surgery had a slight trend toward mildly decreased inflammation, decreased necrotic debris, and a slight increase in the healing of the transected ligaments. The overall degree of soft tissue fibrosis/fibrous expansion, including along the dorsal periosteal surfaces/joint capsule of the carpal bones was very similar between both timepoints and often exhibited signs of healing. The findings of this study indicate that the carpometacarpal joint may serve as a viable location for the investigation of human foot and ankle orthopedic devices. Future work may include the investigation of orthopedic foot and ankle medical devices, biologic treatments, and repair techniques in a large animal model capable of providing translational results for human treatment.

Characterizing the molecular and immune landscape of canine bladder cancer.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis. Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets. We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.

Losartan Blocks Osteosarcoma-Elicited Monocyte Recruitment, and Combined With the Kinase Inhibitor Toceranib, Exerts Significant Clinical Benefit in Canine Metastatic Osteosarcoma.

PURPOSE: There is increasing recognition that progress in immuno-oncology could be accelerated by evaluating immune-based therapies in dogs with spontaneous cancers. Osteosarcoma (OS) is one tumor for which limited clinical benefit has been observed with the use of immune checkpoint inhibitors. We previously reported the angiotensin receptor blocker losartan suppressed metastasis in preclinical mouse models through blockade of CCL2-CCR2 monocyte recruitment. Here we leverage dogs with spontaneous OS to determine losartan's safety and pharmacokinetics associated with monocyte pharmacodynamic endpoints, and assess its antitumor activity, in combination with the kinase inhibitor toceranib. PATIENTS AND METHODS: CCL2 expression, monocyte infiltration, and monocyte recruitment by human and canine OS tumors and cell lines were assessed by gene expression, ELISA, and transwell migration assays. Safety and efficacy of losartan-toceranib therapy were evaluated in 28 dogs with lung metastatic OS. Losartan PK and monocyte PD responses were assessed in three dose cohorts of dogs by chemotaxis, plasma CCL2, and multiplex cytokine assays, and RNA-seq of losartan-treated human peripheral blood mononuclear cells. RESULTS: Human and canine OS cells secrete CCL2 and elicit monocyte migration, which is inhibited by losartan. Losartan PK/PD studies in dogs revealed that a 10-fold-higher dose than typical antihypertensive dosing was required for blockade of monocyte migration. Treatment with high-dose losartan and toceranib was well-tolerated and induced a clinical benefit rate of 50% in dogs with lung metastases. CONCLUSIONS: Losartan inhibits the CCL2-CCR2 axis, and in combination with toceranib, exerts significant biological activity in dogs with metastatic osteosarcoma, supporting evaluation of this drug combination in patients with pediatric osteosarcoma. See related commentary by Weiss et al., p. 571.

Canine oral squamous cell carcinoma as a spontaneous, translational model for radiation and immunology research.

Introduction: Improving outcomes for oral squamous cell carcinoma (OSCC) patients has been hindered by a lack of effective predictive animal models. Spontaneously occurring canine OSCC could help fill this gap. The objective of this study was to characterize the immune landscape of canine OSCC to advance understanding of how dogs could serve as a surrogate for human OSCC. Methods/Results: Canine OSCC contains a heterogenous tumor immune microenvironment. CD3+ T cells were the predominant tumor infiltrating immune cell population; however, there was a wide range CD3+ T cell density across samples. The most common CD3+ T cell micro-anatomical distribution was defined as "pre-existing immunity", but the remaining 20% of tumors were characterized as "immunologically ignorant" or "excluded infiltrates" patterns. When compared to normal oral mucosa, the tumor gene expression pattern suggests that canine OSCC microenvironment is highly inflamed and characterized by the presence of an anti-tumor immune response dominated by cytotoxic\effector T cells and NK cells (CD8a, GZMA, OX40, and HLA-A); however, overexpression of genes associated with effector T cell exhaustion and microenvironmental immunosuppression was also identified (PD-1, LAG3, CXCL2). Correlations between CD3+ T cell density and immune gene expression revealed key genes associated with cytotoxic anti-tumor T cell responses (GZMA, GZMB, PRF1), co-stimulation of T cells (CD27, CD28, ICOS), and other immune processes, including Type I IFN response (TNF, TNFSF10), and T cell exhaustion (CTLA4, PD-1). CD3+ T cell density in canine OSCC was significantly correlated with a cytolytic activity score (mean PRF1 and GZMA expression), suggestive of active effector CD8 T cell function. CD204+ macrophages were the second most abundant tumor infiltrating immune cell, and when comparing to normal oral mucosa, two differently expressed genes linked to tumor associated macrophages and myeloid derived suppressor cells (MDSC) were identified: CXCL2, CD70. Overexpression of CXCL2 was also identified in canine OSCC "T cell-high" tumors compared to "T cell-low" tumors. Discussion: This study identified actionable immunotherapy targets which could inform future comparative oncology trials in canine OSCC: CTLA-4, PD-1, CXCL2. These data provide a good first step towards utilizing spontaneous canine OSCC as a comparative model for human OSCC radiation and immuno-oncology research.

Reprogramming the Canine Glioma Microenvironment with Tumor Vaccination plus Oral Losartan and Propranolol Induces Objective Responses.

Purpose: Malignant gliomas have a highly immune suppressive tumor microenvironment (TME) which renders them largely unresponsive to conventional therapeutics. Therefore, the present study evaluated a therapeutic protocol designed overcome the immune barrier by combining myeloid cell targeted immunotherapy with tumor vaccination. Experimental Design: We utilized a spontaneously occurring canine glioma model to investigate an oral TME modifying immunotherapy in conjunction with cancer stem cell (CSC) vaccination. Dogs were treated daily with losartan (monocyte migration inhibitor) and propranolol (myeloid-derived suppressor cell depleting agent) plus anti-CSC vaccination on a bi-weekly then monthly schedule. Tumor volume was monitored by MRI and correlated with patient immune responses. Results: Ten dogs with histologically confirmed gliomas were enrolled into a prospective, open-label clinical trial to evaluate the immunotherapy protocol. Partial tumor regression was observed in 2 dogs, while 6 dogs experienced stable disease, for an overall clinical benefit rate of 80%. Overall survival times (median = 351 days) and progression-free intervals (median = 163 days) were comparable to prior studies evaluating surgical debulking followed by immunotherapy. Dogs with detectable anti-CSC antibody responses had an increased overall survival time relative to dogs that did not generate antibody responses (vaccine responder MST = 500 days; vaccine non-responder MST = 218 days; p = 0.02). Conclusions: These findings suggest that combining myeloid cell targeted oral immunotherapy with tumor vaccination can generate objective tumor responses, even in the absence of conventional therapy. Overall, this approach has promise as a readily implemented therapeutic strategy for use in brain cancer patients.

Evaluation of lumbar spinal fusion utilizing recombinant human platelet derived growth factor-B chain homodimer (rhPDGF-BB) combined with a bovine collagen/ß-tricalcium phosphate (ß-TCP) matrix in an ovine model.

BACKGROUND CONTEXT: While the clinical effectiveness of recombinant human Platelet Derived Growth Factor-B chain homodimer combined with collagen and ß-tricalcium phosphate (rhPDGF-BB + collagen/ß-TCP) treatment for indications involving hindfoot and ankle is well-established, it is not approved for use in spinal interbody fusion, and the use of autograft remains the gold standard. PURPOSE: The purpose of this study was to compare the effects of rhPDGF-BB + collagen/ß-TCP treatment on lumbar spine interbody fusion in an ovine model to those of autograft bone and collagen/ß-TCP treatments using biomechanical, radiographic, and histological assessment techniques. STUDY DESIGN: Thirty-two skeletally mature Columbian Rambouillet sheep were used to evaluate the safety and effectiveness of rhPDGF-BB + collagen/ß-TCP matrix in a lumbar spinal fusion model. Interbody polyetheretherketone (PEEK) cages contained either autograft, rhPDGF-BB + collagen/ß-TCP, collagen/ß-TCP matrix, or left empty. METHODS: Animals were sacrificed 8- or 16-weeks post-surgery. Spinal fusion was evaluated via post-sacrifice biomechanical, micro-computed tomography (µCT), and histological analysis. Outcomes were statistically compared using a two-way analysis of variance (ANOVA) with an alpha value of 0.05 and a Tukey post-hoc test. RESULTS: There were no statistically significant differences between groups within treatment timepoints for flexion-extension, lateral bending, or axial rotation range of motion, neutral zone, neutral zone stiffness, or elastic zone stiffness. µCT bone volume fraction was significantly greater between treatment groups independent of timepoint where Autograft and rhPDGF-BB + collagen/ß-TCP treatments demonstrated significantly greater bone volume fraction as compared to collagen/ß-TCP (P = .026 and P = .038, respectively) and Empty cage treatments (P = .002 and P = .003, respectively). µCT mean bone density fraction was most improved in rhPDGF-BB + collagen/ß-TCP specimens at the 8 week and 16-week timepoints as compared to all other treatment groups. There were no statistically significant differences in histomorphometric measurements of bone, soft tissue, or empty space between rhPDGF-BB + collagen/ß-TCP and autograft treatments. CONCLUSIONS: The results of this study indicate that the use of rhPDGF-BB combined with collagen/ß-TCP promotes spinal fusion comparable to that of autograft bone. CLINICAL SIGNIFICANCE: The data indicate that rhPDGF-BB combined with collagen/ß-TCP promotes spinal fusion comparably to autograft bone treatment and may offer a viable alternative in large animal spinal fusion. Future prospective clinical studies are necessary to fully understand the role of rhPDGF-BB combined with collagen/ß-TCP in human spinal fusion healing.

Immune pathways and TP53 missense mutations are associated with longer survival in canine osteosarcoma.

Osteosarcoma affects about 2.8% of dogs with cancer, with a one-year survival rate of approximately 45%. The purpose of this study was to characterize mutation and expression profiles of osteosarcoma and its association with outcome in dogs. The number of somatic variants identified across 26 samples ranged from 145 to 2,697 with top recurrent mutations observed in TP53 and SETD2. Additionally, 47 cancer genes were identified with copy number variations. Missense TP53 mutation status and low pre-treatment blood monocyte counts were associated with a longer disease-free interval (DFI). Patients with longer DFI also showed increased transcript levels of anti-tumor immune response genes. Although, T-cell and myeloid cell quantifications were not significantly associated with outcome; immune related genes, PDL-1 and CD160, were correlated with T-cell abundance. Overall, the association of gene expression and mutation profiles to outcome provides insights into pathogenesis and therapeutic interventions in osteosarcoma patients.

Canine Non-Angiogenic, Non-Myogenic Splenic Stromal Sarcoma: a Retrospective Clinicopathological Analysis and Investigation of Podoplanin as a Marker of Tumour Histogenesis.

Splenic stromal sarcomas are rarely reported tumours that were previously grouped as non-angiomatous, non-lymphomatous mesenchymal neoplasms of the canine spleen. Highly variable survival times have been reported probably due to their heterogeneous nature. The purpose of this study was to assess the outcome and prognostic factors in dogs with splenic stromal sarcoma after treatment by splenectomy. Clinical data were collected retrospectively and histopathology was reviewed for 47 patients. Histological classification, based on morphology in haematoxylin and eosin-stained sections, in conjunction with immunolabelling of macrophage scavenger receptor-A (CD204), desmin, factor VIII-related antigen and smooth muscle actin ,yielded diagnoses of undifferentiated stromal sarcoma (n = 22), complex nodular hyperplasia (CNH, n = 9), sarcoma arising from benign complex nodular hyperplasia (n = 3), histiocytic sarcoma (n = 3), haemangiosarcoma (n = 1) and leiomyosarcoma (n = 1). Four samples were excluded from analysis due to extensive necrosis. An anti-podoplanin (PDPN) antibody was validated on canine tissue and used to assess expression of this protein as a potential indicator of the tissue of origin of the neoplasms (28/42 tumours were positive). There was a statistically significant difference in survival time between patients with stromal sarcoma (sarcoma from benign CNH and undifferentiated stromal sarcoma) and CNH (178 d versus 637 d, respectively; P = 0.027). Dogs with stromal sarcomas and high mitotic count (≥9 per 10 high-power fields) had a significantly shorter survival time (67 d versus 439 d; P = 0.01). Clinical diagnosis of splenic tumours should include evaluation for the presence of benign nodular hyperplasia morphology and immunohistochemistry to exclude more aggressive malignancies where adjuvant therapy is recommended. As in humans, PDPN may be an effective marker for stromal sarcomas of the canine spleen and immunopositivity suggests a fibroblastic reticular or follicular dendritic cell origin.

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.

Role of Periostin Expression in Canine Osteosarcoma Biology and Clinical Outcome.

Periostin is a matricellular protein important in regulating bone, tooth, and cardiac development. In pathologic conditions, periostin drives allergic and fibrotic inflammatory diseases and is also overexpressed in certain cancers. Periostin signaling in tumors has been shown to promote angiogenesis, metastasis, and cancer stem cell survival in rodent models, and its overexpression is associated with poor prognosis in human glioblastoma. However, the role of periostin in regulating tumorigenesis of canine cancers has not been evaluated. Given its role in bone development, we sought to evaluate mRNA and protein expression of periostin in canine osteosarcoma (OS) and assess its association with patient outcome. We validated an anti-human periostin antibody cross-reactive to canine periostin via western blot and immunohistochemistry and evaluated periostin expression in microarray data from 49 primary canine OS tumors and 8 normal bone samples. Periostin mRNA was upregulated greater than 40-fold in canine OS tumors compared to normal bone and was significantly correlated with periostin protein expression based on quantitative image analysis. However, neither periostin mRNA nor protein expression were associated with time to metastasis in this cohort. Gene Set Enrichment Analysis demonstrated significant enhancement of pro-tumorigenic pathways including canonical WNT signaling, epithelial-mesenchymal transition, and angiogenesis in periostin-high tumors, while periostin-low tumors demonstrated evidence of heightened antitumor immune responses. Overall, these data identify a novel antibody that can be used as a tool for evaluation of periostin expression in dogs and suggest that investigation of Wnt pathway-targeted drugs in periostin overexpressing canine OS may be a potential therapeutic target.

Transcriptional regulation of alcohol induced liver fibrosis in a translational porcine hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) is the 5th most common and 2nd deadliest cancer worldwide. HCC risk factors include alcohol induced liver cirrhosis, which prompts hepatic inflammation, cell necrosis, and fibrosis deposition. As 25% of HCC cases are associated with alcohol induced liver disease, understanding the effects of the cirrhotic liver microenvironment on HCC tumor biology and therapeutic responses are critical. This study utilized the Oncopig Cancer Model-a transgenic pig model that recapitulates human HCC through induced expression of KRASG12D and TP53R167H driver mutations-to investigate the molecular mechanisms underlying alcohol induced liver disease. Oncopigs (n = 5) underwent fibrosis induction via infusion of ethanol and ethiodized oil (1:3 v/v dosed at 0.75 mL/kg) into the hepatic arterial circulation. Eight-weeks post induction, liver tissue samples from fibrotic and age-matched control (n = 5) Oncopigs were collected for histological evaluation and transcriptional profiling. Increased hepatic inflammation and fibrosis was observed in fibrotic Oncopigs via pathological assessment. Transcriptional profiling (RNA-seq) resulted in the identification of 4387 differentially expressed genes between Oncopig fibrotic and control livers. GO term enrichment analysis identified pathway alterations associated with cirrhosis progression in humans, including cell proliferation, angiogenesis, extracellular matrix deposition, and oxidation-reduction. Key alterations include activation of hepatic stellate cells, increased matrix metalloproteinase production, and altered expression of ABC and SLC transporter genes involved in transport of anticancer drugs.These results demonstrate Oncopig liver fibrosis recapitulates transcriptional hallmarks of human cirrhosis, making the Oncopig an ideal model for studying the effects of the cirrhotic liver microenvironment on HCC tumor biology and therapeutic response.

First-in-Class Inhibitors of Oncogenic CHD1L with Preclinical Activity against Colorectal Cancer.

Since the discovery of CHD1L in 2008, it has emerged as an oncogene implicated in the pathology and poor prognosis of a variety of cancers, including gastrointestinal cancers. However, a mechanistic understanding of CHD1L as a driver of colorectal cancer has been limited. Until now, there have been no reported inhibitors of CHD1L, also limiting its development as a molecular target. We sought to characterize the clinicopathologic link between CHD1L and colorectal cancer, determine the mechanism(s) by which CHD1L drives malignant colorectal cancer, and discover the first inhibitors with potential for novel treatments for colorectal cancer. The clinicopathologic characteristics associated with CHD1L expression were evaluated using microarray data from 585 patients with colorectal cancer. Further analysis of microarray data indicated that CHD1L may function through the Wnt/TCF pathway. Thus, we conducted knockdown and overexpression studies with CHD1L to determine its role in Wnt/TCF-driven epithelial-to-mesenchymal transition (EMT). We performed high-throughput screening (HTS) to identify the first CHD1L inhibitors. The mechanism of action, antitumor efficacy, and drug-like properties of lead CHD1L inhibitors were determined using biochemical assays, cell models, tumor organoids, patient-derived tumor organoids, and in vivo pharmacokinetics and pharmacodynamics. Lead CHD1L inhibitors display potent in vitro antitumor activity by reversing TCF-driven EMT. The best lead CHD1L inhibitor possesses drug-like properties in pharmacokinetic/pharmacodynamic mouse models. This work validates CHD1L as a druggable target and establishes a novel therapeutic strategy for the treatment of colorectal cancer.

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFß signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFß signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis in vivo without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.