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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.

The potential role of pathogens, particularly vector-transmitted infectious agents, as a cofactor or cause of neoplasia has not been intensively investigated. We previously reported a potential link between Bartonella spp. bacteremia and splenic hemangiosarcoma (HSA) in dogs living in the United States. The purpose of this study was to: 1/ further determine the prevalence of Bartonella spp. DNA in dogs with splenic HSA from throughout the United States; 2/ assess the impact of sample preservation methods on Bartonella spp. DNA amplification using characterized tissue samples from dogs diagnosed with HSA. In a blinded manner, we determined the presence of Bartonella spp. DNA in scrolls from biorepository formalin-fixed paraffin-embedded (FFPE) spleens from dogs living in three distant locations geographically transecting the United States. DNA extracted from non-lesional spleens (n = 249), nodular lymphoid hyperplasia spleens (n = 248), and splenic HSA (n = 330) were tested by quantitative polymerase chain reaction (qPCR), and droplet digital PCR (ddPCR). Subsequently, Bartonella PCR results from FFPE tissues and formalin-fixed tissues were compared using previously tested fresh frozen tissues from an additional 48 dogs with HSA. There was no significant difference in the proportion of Bartonella PCR positive FFPE tissues from dogs diagnosed with an alesional spleen, nodular lymphoid hyperplasia, and splenic HSA. Regardless of the histological diagnosis, the most common Bartonella species identified was B. henselae (32/38). Bartonella spp. DNA was detected in a significantly larger proportion of fresh frozen tissues compared to FFPE tissues, when tested by qPCR (22/48 versus 1/48; p <0.0001) or ddPCR (19/48 versus 1/48; p <0.0001). Using ddPCR, Bartonella DNA was more often amplified from formalin-fixed tissues compared to FFPE tissues (15/39 versus 1/39; p <0.0001). The sensitivity of qPCR on FFPE samples and formalin-fixed samples, when comparing to fresh frozen samples as the reference standard, was 4.5% and 11.8%, respectively. Due to decreased DNA amplification efficiency, FFPE scrolls should not be used for the detection of Bartonella infection in spleen samples from dogs with HSA. PCR testing of fresh frozen tissues substantially improves the detection of Bartonella spp. infection. If fresh frozen tissues are not available, formalin-fixed tissues should be tested with digital PCR to enhance Bartonella DNA detection.

Pancreatic cancer is the third leading cause of cancer deaths in the United States with more than 53,000 expected to be diagnosed with the disease in 2018. The median survival time after diagnosis is four to six months. The poor survival statistics are due in part to the fact that pancreatic cancer is typically asymptomatic until it reaches advanced stages of the disease. Although surgical resection provides the best chance of survival, pancreatic cancer is rarely detected when surgery is still possible due, in part, to lack of effective biomarkers for early detection. The goal of the research reported here was to determine if it was possible to identify metabolic biomarkers for detection of pre-cancerous pancreatic intraepithelial neoplasia (PanIN) that precede pancreatic adenocarcinoma. The transgenic Ptf1a-Cre; LSL-KrasG12D mouse strain was used as a model of pancreatic cancer progression. Nuclear magnetic resonance (NMR) spectroscopy was employed to compare metabolic profiles of urine, sera, fecal extracts, and pancreatic tissue extracts collected from control and study mice aged 5, 11, and 15 months, including 47 mice with tumors. We were able to identify the following potential biomarkers: decreased 3-indoxylsulfate, benzoate and citrate in urine, decreased glucose, choline, and lactate in blood, and decreased phenylalanine and benzoate and increased acetoin in fecal extracts. Potential biomarkers were validated by p-values, PLS-DA VIP scores, and accuracies based on area under ROC curve analyses. Essentially, all of the metabolic profiling changes could be explained as being associated with the consequences of bicarbonate wasting caused by a complete substitution of the normal pancreatic acinar tissue by tissue entirely composed of PanIN. Given the nature of the mouse model used here, our results indicate that it may be possible to use NMR-based metabolic profiling to identify biomarkers for detection of precancerous PanIN that immediately precede pancreatic cancer.

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. A single-cell RNA sequencing reference of six treatment naïve canine osteosarcoma samples. The data presented in this study reveals the presence of 41 cell types and suggests a conserved tumor microenvironment between canine and human osteosarcoma.

•Vaccination against maxadilan (MAX) protect mice from aggravated Leishmania infection.•Vaccination against MAX increases the number of IFN-γ-producing cells in draining lymph nodes.•The peptide MAX-CLDC vaccine improves host immunity against MAX-mediated immune modulation. Leishmaniasis is an arthropod vectored disease causing considerable human morbidity and mortality. Vaccination remains the most realistic and practical means to interrupt the growing number and diversity of sand fly vectors and reservoirs of Leishmania. Since transmission of Leishmania is achieved exclusively by sand fly vectors via immune-modulating salivary substances, conventional vaccination requiring an unmodified host immune response for success are potentially destined to fail unless immunomodulatory factors are somehow neutralized. Using cationic liposome DNA complexes (CLDC) as an adjuvant system along with Lu. longipalpis sand fly salivary component maxadilan (MAX) as antigen (Ag), we show that mice are protected from the MAX-induced exacerbation of infection with Leishmania major (Lm). The CLDC adjuvant and alum were comparable in terms of lesion induration and decreased parasite burden, however the alum adjuvant imposed more inflammation at the injection site. BALB/c, C3H and C57BL/6 mice vaccinated with MAX-CLDC containing either the full-length MAX or peptides spanning the N- and C-terminal regions of MAX are protected against footpad challenges with Lm co-injected with MAX. When compared to unvaccinated controls, all strains of mice immunized with CLDC containing either peptides encompassing the first 20 N-terminal AA or those spanning the last 15 AA of the C-terminal domain of MAX demonstrated decreased parasite burden after 9 or 18 weeks post challenge with Lm + MAX. MAX-CLDC immunized mice showed increased IFNγ-secreting and decreased IL-4-secreting CD4+ cells in footpad-draining lymph nodes. Antisera from C-terminal peptide (P11) MAX-CLDC-vaccinated animals was capable of recognizing FL-MAX and its C-terminal domain and also blocked MAX-mediated reprogramming of bone marrow-derived dendritic cells (BM-DC) in vitro. This peptide vaccine targeting sand fly MAX, improves host immunity against MAX-mediated immunomodulation.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading forms of cancer related deaths in the United States. With limited treatment options and unreliable diagnostic methods, long-term survival rates following a diagnosis of pancreatic cancer remain poor. Pancreatic intraepithelial neoplasia (PanIN) are precancerous lesions that precede progression towards PDAC. PanIN occur in increasing complexity as the disease progresses and the description of PanIN plays a critical role in describing, staging and diagnosing PDAC. Inconsistencies in PanIN classifications exist even amongst leading pathologists. This has led to debate and confusion among researchers and pathologists involved in pancreatic cancer research, diagnosis and treatment. We have sought to initiate a discussion with leading pathologists with a goal of increasing consensus in the interpretation of PanIN and associated structures within the precancerous pancreas. Toward achieving this goal, we are in the process of conducting an extensive study of over 1000 male and female pancreata in varying stages of PanIN progression isolated from the Ptf1aCre/+;LSL-KrasG12D/+ transgenic mouse model of pancreatic cancer. Using this extensive database, we have established the Mouse Model of Pancreatic Cancer Atlas (MMPCA) to serve as a platform for meaningful and interactive discussion among researchers and pathologists who study pancreatic disease. We hope that the MMPCA will be an effective tool for promoting a more consistent and accurate consensus of PanIN classifications in the future.

Current approaches for the fluorescent labelling of extracellular vesicles (EVs) have been reported to produce widely variable and controversial results, highlighting a significant need for validated, reproducible labelling methods to advance the field of EV research. Lipophilic membrane dyes are commonly used but have been shown to produce non‐specific fluorescent particles that are indistinguishable from labelled EVs, confounding experimental results. We aimed to distinguish conditions that can either promote or reduce the formation of non‐specific dye particles when using the prototypical lipophilic membrane dye PKH26. We optimised a labelling approach that minimises the production of non‐specific dye particles by altering buffer conditions during staining and validated this method across cell‐based and in vivo systems of EV biodistribution. To do this, we specifically isolated small EVs using ultrafiltration and size exclusion chromatography and validated sample purity and post‐isolation processing steps. We then used single‐EV spectral flow cytometry and transmission electron microscopy to investigate the impact of four different buffer conditions on PKH26 non‐specific particle formation. We also determined the extent to which non‐specific PKH26 particles were detectable in cell‐based assays and in vivo within mouse lymph nodes using flow cytometry, immunofluorescence, and intravital imaging. By optimising buffer conditions to eliminate additional protein, we were able to minimise the formation of dye aggregates while maintaining efficient EV labelling, producing a much higher signal‐to‐noise ratio both in vitro and in vivo. We also demonstrate that failure to include proper vehicle controls can have significant implications on experimental results, leading to false positive data. This work emphasizes the importance of adequately benchmarking EV labelling approaches as it is essential for accurate evaluation of EV trafficking in physiologic and pathologic states. Lipophilic membrane dyes, like PKH26, have been associated with the production of non‐specific fluorescent dye particles that can confound experimental results. We aimed to investigate conditions that can minimise these non‐specific signals while maintaining efficient labelling of true extracellular vesicles. We identified that the addition of protein during the staining process can lead to substantial formation of dye‐protein aggregates that cannot be removed using traditional approaches. We validated an optimised PKH26 staining approach across both in vitro and in vivo systems.

Thyroid tumors represent 1–4% of cancers in both dogs and humans. Most canine tumors are follicular (FTC) or medullary carcinomas (MTC), unlike humans, where only 10–15% are FTC and 2% are MTC, with BRAF/NRAS or RET mutations, respectively. Here, we conduct histological and molecular analyses of canine thyroid tumors. Transcriptionally, elevated ERBB2 expression characterizes FTC tumors, whereas MTC tumors show upregulated RET signaling. Elevated HER2 protein-staining and larger tumor size associate with shorter progression-free survival. Recurrent mutations are rarely observed with potential driver variants in MEN1 (10%), KRAS (7%), and TSHR (3%), among others. Notably, mutations in DNA repair pathway genes are the most consistently shared across tumors, occurring in 60% of cases. Thus, the genomic profile of canine FTC differs significantly from that of humans, with limited reliance on RAS/RAF signaling for oncogenic progression. Conversely, RET signaling likely underlies tumorigenesis in both canine and human MTC. Comprehensive molecular analysis of canine thyroid cancer (TC) uncovers distinct oncogenic pathways: ERBB2/HER2 activation in follicular TC, and RET signaling in medullary TC, mirroring human TC and highlighting species-specific mechanisms.

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.Sunetra Das et al. combine genetic and immunohistochemistry data to characterize mutation and expression profiles of canine osteosarcoma and its association with clinical outcomes. Their results provide further insight into the pathogenesis and potential future therapeutic targets for osteosarcoma.