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G.B., D.B., K.W., A.P., R.F., T.R.H., S.K., and J.A.A. received support from Fundacja na rzecz Nauki Polskiej (FNP) (grant ID: MAB/3/2017). D.R.G. received support from Genome Canada & Genome BC (grant ID: 264PRO). D.J.H. received support from NuCana plc (grant ID: SMD0-ZIUN05). H.A. received support from Swedish Cancer Foundation (grant ID: 211709). H.G. received support from United Kingdom Research and Innovation (UKRI) (grant ID: EP/S02431X/1). C.P. received support from Fundação para a Ciência e a Tecnologia (FCT) through LASIGE Research Unit (grant ID: UIDB/00408/2020 and UIDP/00408/2020). A.L. F.M.Z., C.P., A.R., A.P., and J.A.A. received support from European Union’s Horizon 2020 research and innovation programme (grant ID: 101017453). C.B. received support from Agence Nationale de la Recherche (ANR) through GRAL LabEX (grant ID: ANR-10-LABX-49-01) and CBH-EUR-GS 32 (grant ID: ANR-17-EURE0003). S.N.S. received support from Cancer Research UK (CRUK) and the Chief Scientist's Office of Scotland (CSO): Experimental Cancer Medicine Centre (ECMC) (grant ID: ECMCQQR-2022/100017). A.L. received support from Chief Scientist's Office of Scotland (CSO) NRS Career Researcher Fellowship. R.O.N. received support from CRUK Cambridge Centre Thoracic Cancer Programme (grant ID: CTRQQR-2021\100012). Tumor antigens can emerge through multiple mechanisms, including translation of non-coding genomic regions. This non-canonical category of antigens has recently gained attention; however, our understanding of how they recur within and between cancer types is still in its infancy. Therefore, we developed a proteogenomic pipeline based on deep learning de novo mass spectrometry to enable the discovery of non-canonical MHC-associated peptides (ncMAPs) from non-coding regions. Considering that the emergence of tumor antigens can also involve post-translational modifications, we included an open search component in our pipeline. Leveraging the wealth of mass spectrometry-based immunopeptidomics, we analyzed 26 MHC class I immunopeptidomic studies of 9 different cancer types. We validated the de novo identified ncMAPs, along with the most abundant post-translational modifications, using spectral matching and controlled their false discovery rate (FDR) to 1%. Interestingly, the non-canonical presentation appeared to be 5 times enriched for the A03 HLA supertype, with a projected population coverage of 54.85%. Here, we reveal an atlas of 8,601 ncMAPs with varying levels of cancer selectivity and suggest 17 cancer-selective ncMAPs as attractive targets according to a stringent cutoff. In summary, the combination of the open-source pipeline and the atlas of ncMAPs reported herein could facilitate the identification and screening of ncMAPs as targeting agents for T-cell therapies or vaccine development. Publisher PDF

Annotation of the COD-dipp ncMAPs.

Open search peptide-spectrum matches (PSMs) information of serine acetylation, cysteinylation, carbamidomethylation, and cysteine trioxidation.

Mapping of ncMAPs to COSMIC frameshift mutations.

Datasets and samples considered in this study.

Tumor antigens can emerge through multiple mechanisms, including translation of noncoding genomic regions. This noncanonical category of tumor antigens has recently gained attention; however, our understanding of how they recur within and between cancer types is still in its infancy. Therefore, we developed a proteogenomic pipeline based on deep learning de novo mass spectrometry (MS) to enable the discovery of noncanonical MHC class I–associated peptides (ncMAP) from noncoding regions. Considering that the emergence of tumor antigens can also involve posttranslational modifications (PTM), we included an open search component in our pipeline. Leveraging the wealth of MS-based immunopeptidomics, we analyzed data from 26 MHC class I immunopeptidomic studies across 11 different cancer types. We validated the de novo identified ncMAPs, along with the most abundant PTMs, using spectral matching and controlled their FDR to 1%. The noncanonical presentation appeared to be 5 times enriched for the A03 HLA supertype, with a projected population coverage of 55%. The data reveal an atlas of 8,601 ncMAPs with varying levels of cancer selectivity and suggest 17 cancer-selective ncMAPs as attractive therapeutic targets according to a stringent cutoff. In summary, the combination of the open-source pipeline and the atlas of ncMAPs reported herein could facilitate the identification and screening of ncMAPs as targets for T-cell therapies or vaccine development.

Supplementary Figures S1 to S7.

Legends for Supplementary tables, supplementary figures, and supplementary notes.

Supplementary notes 1 and 2.

Comparison data of COD-dipp ncMAPs with 4 other studies.

Canine apocrine gland anal sac adenocarcinoma (AGASACA) is an aggressive canine tumor originating from the anal sac glands. Surgical resection, with or without adjuvant chemotherapy, represents the standard of care for this tumor, but the outcome is generally poor, particularly for tumors diagnosed at an advanced stage. For this reason, novel treatment options are warranted, and a few recent reports have suggested the activation of the immune checkpoint axis in canine AGASACA. In our study, we developed canine-specific monoclonal antibodies targeting PD-1 and PD-L1. A total of 41 AGASACAs with complete clinical and follow-up information were then analyzed by immunohistochemistry for the expression of the two checkpoint molecules (PD-L1 and PD-1) and the presence of tumor-infiltrating lymphocytes (CD3 and CD20), which were evaluated within the tumor bulk (intratumor) and in the surrounding stroma (peritumor). Seventeen AGASACAs (42%) expressed PD-L1 in a range between 5% and 95%. The intratumor lymphocytes were predominantly CD3+ T-cells and were positively correlated with the number of PD-1+ intratumor lymphocytes (ρ = 0.36; p = 0.02). The peritumor lymphocytes were a mixture of CD3+ and CD20+ cells with variable PD-1 expression (range 0–50%). PD-L1 expression negatively affected survival only in the subgroup of dogs treated with surgery alone (n = 14; 576 vs. 235 days). The presence of a heterogeneous lymphocytic infiltrate and the expression of PD-1 and PD-L1 molecules support the relevance of the immune microenvironment in canine AGASACAs and the potential value of immune checkpoints as promising therapeutic targets.

Bernese mountain dogs (BMDs), have an overall cancer incidence of 50%, half of which is comprised of an otherwise rare tumor, histiocytic sarcoma (HS). While recent studies have identified driver mutations in the MAPK pathway, identification of key predisposing genes has been elusive. Studies have identified several loci to be associated with predisposition to HS in BMDs, including near the MTAP/CDKN2A region, but no causative coding variant has been identified. Here we report the presence of a coding polymorphism in the gene encoding FANCG, near the MTAP/CDKN2A locus. This variant is in a conserved region of the protein and appears to be specific to BMDs. Canine fibroblasts derived from dogs homozygous for this variant are hypersensitive to cisplatin. We show this canine FANCG variant and a previously defined hypomorphic FANCG allele in humans impart similar defects in DNA repair. However, our data also indicate that this variant is neither necessary nor sufficient for the development of HS. Furthermore, BMDs homozygous for this FANCG allele display none of the characteristic phenotypes associated with Fanconi anemia (FA) such as anemia, short stature, infertility, or an earlier age of onset for HS. This is similar to findings in FA deficient mice, which do not develop overt FA without secondary genetic mutations that exacerbate the FA deficit. In sum, our data suggest that dogs with deficits in the FA pathway are, like mice, innately resistant to the development of FA.

A comparative canine–human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine–canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine–murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human–murine chimeric anti-CD20 antibody Rituximab.

A longstanding disconnect between the growing number of MHC Class I immunopeptidomic studies and genomic medicine hinders cancer vaccine design. We develop COD-dipp to genomically map the full spectrum of detected canonical and non-canonical (non-exonic) MHC Class I antigens from 26 cancer studies. We demonstrate that patient mutations in regions overlapping physically identified antigens better predict immunotherapy response when compared to neoantigen predictions. We suggest a vaccine design approach using 140,966 highly immune-visible regions of the genome annotated by their expression and haplotype frequency in the human population. These regions tend to be highly conserved, mutated in cancer and harbor 7.8 times more immunogenicity. Intersecting pan-cancer mutations with these immune surveilled regions revealed a potential to create off-the-shelf multi-epitope vaccines against public neoantigens. Here we release COD-dipp, a cancer vaccine toolkit as a web-application (https://www.proteogenomics.ca/COD-dipp) and open-source high-throughput resource.

Over the past few years, many molecules such as monoclonal antibodies, affibodies, nanobodies, and small compounds have been designed and tested as inhibitors of PD-1/PD-L1 complex formation. Some of them have been successfully implemented into clinical oncology practice. However, the majority of these compounds have disadvantages and limitations, such as high production price, potential for immunogenicity and/or prolonged clearance. Thus, new inhibitors of the PD-1/PD-L1 immune checkpoints are needed. Recently, peptides emerged as potential novel approach for blocking receptor/ligand interaction. In the presented studies we have designed, synthesised and tested peptides, which are potential inhibitors of the PD-1/PD-L1 axis. The amino acid sequences of the designed peptides were based on the binding sites of PD-1 to PD-L1, as determined by the crystal structure of the protein complex and also based on MM/GBSA analysis. Interactions of the peptides with PD-L1 protein were confirmed using SPR, while their inhibitory properties were studied using cell-based PD-1/PD-L1 immune checkpoint blockade assays. The characterization of the peptides has shown that the peptides PD-1(119–142) T120C-E141C, PD-1(119–142) C123-S137C and PD-1(122–138) C123-S137C strongly bind to PD-L1 protein and disrupt the interaction of the proteins. PD-1(122–138) C123-S137C peptide was shown to have the best inhibitory potential from the panel of peptides. Its 3D NMR structure was determined and the binding site to PD-L1 was established using molecular modelling methods. Our results indicate that the PD-1 derived peptides are able to mimic the PD-1 protein and inhibit PD-1/PD-L1 complex formation.

Diabetes is associated with hepatic metabolic dysfunction predisposing patients to drug-induced liver injury. Mouse models of type 2 diabetes (T2D) have dramatically reduced expression of organic anion transporting polypeptide (OATP)1A1, a transporter expressed in hepatocytes and in the kidneys. The effects of diabetes on OATP1B2 expression are less studied and less consistent. OATP1A1 and OATP1B2 both transport endogenous substrates such as bile acids and hormone conjugates as well as numerous drugs including gadoxetate disodium (Gd-EOB-DTPA). As master pharmacokinetic regulators, the altered expression of OATPs in diabetes could have a profound and clinically significant influence on drug therapies. Here, we report a method to noninvasively measure OATP activity in T2D mice by quantifying the transport of hepatobiliary-specific gadolinium-based contrast agents (GBCAs) within the liver and kidneys using dynamic contrast–enhanced MRI (DCE-MRI). By comparing GBCA uptake in control and OATP knockout mice, we confirmed liver clearance of the hepatobiliary-specific GBCAs, Gd-EOB-DTPA, and gadobenate dimeglumine, primarily though OATP transporters. Then, we measured a reduction in the hepatic uptake of these hepatobiliary GBCAs in T2D ob/ob mice, which mirrored significant reductions in the mRNA and protein expression of OATP1A1 and OATP1B2. As these GBCAs are U.S. Food and Drug Administration–approved agents and DCE-MRI is a standard clinical protocol, studies to determine OATP1B1/1B3 deficiencies in human individuals with diabetes can be easily envisioned.

Computed tomographic (CT) excretory urography is commonly used to investigate canine ureteral ectopia (UE). Modern technology allows time-resolved CT imaging (four-dimensional CT excretory urography [4D-CTEU]) over a distance exceeding the detector collimation. Objectives of this prospective, observational, diagnostic accuracy study were to evaluate the diagnostic accuracy of CT excretory urography (CTEU) and 4D-CTEU for UE in dogs with lower urinary tract signs, assess the influence of pelvis positioning, and to determine the significance of the ureterovesical junction (UVJ) angle for UE diagnosis. Thirty-six dogs, with a total of 42 normotopic ureters, 27 intramural ectopic ureters, and three extramural ectopic ureters, underwent CTEU and 4D-CTEU with randomized pelvis positioning. Randomized CTEU and 4D-CTEU studies were scored by two observers for ureteral papilla location and murality on a grading scheme. Interobserver agreement, sensitivity, and specificity for ureter topia status and diagnosis were calculated. Computed tomographic excretory urography showed moderate interobserver agreement for the left ureter and perfect for the right ureter, whereas 4D-CTEU showed bilateral nearly perfect agreement between both observers. When comparing CTEU versus confirmed diagnosis, there was a sensitivity and specificity of 73% and 90.2%, respectively, whereas 4D-CTEU showed a sensitivity and specificity of 97% and 94.6%, respectively. An obtuse UVJ angle is significantly more commonly observed in ectopic intramural than normotopic ureters and is significantly associated with increased diagnostic confidence of UE. The use of a wedge to angle the pelvis did not increase the diagnostic confidence in determining ureteral opening position. Four-dimensional CT excretory urography is an accurate and reliable diagnostic technique to investigate UE as cause of urinary incontinence in dogs that is slightly superior to CTEU

Objectives The aims of this study were to document the presence and prevalence of feline lipiduria and renal lipid deposition on CT, and to search for associations between the presence of lipiduria and sex, urinary tract abnormalities and urolithiasis. Methods The CT examinations of 252 cats were reviewed for the presence of an antigravitational hypodense bubble in the urinary bladder with density values between −180 Hounsfield units (HU) and −20 HU. To identify associations between lipiduria and sex, urinary tract abnormalities and urolithiasis, Fisher’s exact test was used. Renal cortical density measurement was performed in all cats. The Mann−Whitney test was performed to compare renal cortical density between lipiduric and unaffected cats. Results A total of 27 domestic cats (10.7%) had CT evidence of lipiduria. Lipiduric cats had a significantly lower renal cortical density than unaffected cats ( P Conclusions and relevance Lipiduria is a common physiological phenomenon in cats that can be detected on routine CT examinations. Decreased renal cortical density is associated with lipiduria. This may aid in the diagnosis of feline lipiduria and help to differentiate its presence from other pathological depositions and excretions.

Immune evasion is a major challenge for the development of successful cancer treatments. One of the known mechanisms is the expression of immune checkpoints (ICs)—proteins regulating the immune cells activation. The advent of immunotherapy using monoclonal antibodies (mAbs) to block the immune checkpoint receptor-ligand interaction brought about a landslide improvement in the treatment responses, leading to a prompt approval of such therapeutics. In recent years, it was discovered that a subset of patients receiving IC blockade treatment experienced a previously unknown pattern of treatment response called hyperprogression (HP), characterised by rapid deterioration on initialisation of the therapy. HP represents an urgent issue for clinicians and drug developers, while posing questions about the adequacy of the current clinical trial process. Here, we briefly summarise the state of knowledge and propose new directions for research into HP mechanisms, focusing on tumour-intrinsic signalling of IC proteins malignantly expressed by cancer. We also discuss the potential role of spontaneously occurring canine cancer in the assessment of immunotherapeutics, which can provide the missing link between murine and human studies.

Immune blockade of inhibitory immune checkpoint (IC) mediated by Programmed cell death protein 1 (PD1) significantly increases survival of patients with advanced melanoma and other cancers. Dogs constitute a unique model for studying cancer types relevant to humans. Canine malignancies occur spontaneously and share many pathogenic pathways as well as therapy responses with their human counterparts. Dogs have a fully functional immune system, share the owners' lifestyle, environmental exposures, and their genome is more similar to the human one than the murine one is. Canine cancer research may be a superior alternative to mouse studies in the context of immunotherapy development. Unfortunately, IC-blocking antibodies are not available for companion animals. Monoclonal antibody against canine ICs has been previously developed by our group using mouse hybridoma technology. While functional, it couldn't be used clinically due to immunogenicity. To create a viable therapeutic, the antibody sequence was caninized. The murine constant regions of heavy and light chains were substituted with corresponding canine sequences. Following that, the variable regions' structural frameworks were replaced with canine sequences generated based on a canine library of antibody sequences.Additionally, we developed an antibody against the PDL1 protein using a previously established canine antibody phage library. The PD1 and PDL1 antibodies are being characterized by ELISA and cell-based blocking assays, T-cell activation assays and growth assessment of the impact on spheroid growth in patient-derived cultures.The characterized antibodies will widen the availability of canine IC blockers and pave the path for further research in comparative oncology. Citation Format: Mikolaj Kocikowski, Katarzyna Dziubek, Borek Vojtesek, David Argyle, Ted Hupp, Maciej Parys. Development of caninized monoclonal antibodies against PD1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2234.

Feline oral squamous cell carcinomas (FOSCC) are highly aggressive neoplasms with short survival times despite multimodal treatment. FOSCC are similar to squamous cell carcinomas of the head and neck (SCCHN) in humans, which also present therapeutic challenges. The current study was undertaken to identify novel chemotherapeutics using FOSCC cell lines. A high throughput drug screen using 1,952 drugs was performed to identify chemotherapeutics for further investigation. Two of the drugs identified in the drug screen, actinomycin D and methotrexate, and two drugs with similar molecular targets to drugs found to be efficacious in the screening, dinaciclib and flavopiridol, were selected for further investigation. Drug inhibition profiles were generated for each drug and cell line using an MTS assay. In addition, the effects of the drugs of interest on cell cycle progression were analyzed via a propidium iodide DNA labeling assay. Changes in caspase-3/7 activity after treatment with each drug were also determined. The findings demonstrated effectiveness of the drugs at nanomolar concentrations with sensitivity varying across cell lines. With all of the drugs except for actinomycin D, evidence for G1 arrest was found. Dinaciclib and flavopiridol were demonstrated to induce apoptosis. The results of the study suggest that the selected drugs are potential candidates for developing novel chemotherapeutic approaches to FOSCC. Through these studies, novel therapeutic strategies for the treatment of FOSCC can be developed to provide better care for affected cats which can also serve as proof of concept studies to inform translational studies in SCCHN in humans.

Background Histiocytic sarcoma is a rare disorder in humans, however it is seen with appreciable frequency in certain breeds of dogs, such as Bernese mountain dog. The purpose of this study was to fully characterize a novel canine histiocytic sarcoma cell line, and utilize it as a tool to screen for potential therapeutic drugs. Methods The histiocytic sarcoma cell line was characterized by expression of cellular markers as determined by immunohistochemistry and flow cytometry techniques. The neoplastic cells were also evaluated for their capability of phagocytizing beads particles, and their potential to grow as xenograft in an immunodeficient mouse. We investigated the in vitro cytotoxic activity of a panel of thirteen compounds using the MTS proliferation assay. Inhibitory effects of different drugs were compared using one-way ANOVA, and multiple means were compared using Tukey’s test. Results Neoplastic cells expressed CD11c, CD14, CD18, CD45, CD172a, CD204, MHC I, and vimentin. Expression of MHC II was upregulated after exposure to LPS. Furthermore, the established cell line clearly demonstrated phagocytic activity similar to positive controls of macrophage cell line. The xenograft mouse developed a palpable subcutaneous soft tissue mass after 29 days of inoculation, which histologically resembled the primary neoplasm. Dasatinib, a tyrosine kinase pan-inhibitor, significantly inhibited the growth of the cells in vitro within a clinically achievable and tolerable plasma concentration. The inhibitory response to dasatinib was augmented when combined with doxorubicin. Conclusions In the present study we demonstrated that a novel canine histiocytic sarcoma cell line presents a valuable tool to evaluate novel treatment approaches. The neoplastic cell line favorably responded to dasatinib, which represents a promising anticancer strategy for the treatment of this malignancy in dogs and similar disorders in humans. Electronic supplementary material The online version of this article (10.1186/s12885-018-4132-0) contains supplementary material, which is available to authorized users.

Background: Feline idiopathic cystitis (FIC) is a common lower urinary tract disorder of domestic cats that resembles interstitial cystitis/painful bladder syndrome (IC/PBS) in humans. Diagnosis of FIC is based on clinical signs and exclusion of other disorders because of a lack of specific pathologic findings or other objective biomarkers. Cytokines are potential noninvasive biomarkers to define the presence, severity, and progression of disease, and response to treatment. Objectives: The objective of this pilot study was to determine concentrations of selected cytokines in serum from healthy cats and cats with acute FIC. Animals: Serum samples from 13 healthy cats and from 12 cats with nonobstructive acute FIC were utilized. Methods: Multiplex analysis of 19 cytokines (CCL2, CCL5, CXCL1, CXCL12, CXCL8, Flt3L, GM-CSF, IFN-c, IL-12 (p40), IL-13, IL-18, IL-1b, IL-2, IL-4, IL-6, PDGF-BB, SCF, sFas, and TNF-a) was performed with a commercially available feline-specific multiplex bead-based assay. Results: Mean serum concentrations of IL-12 (p40; P < 0.0001), CXCL12 (P = 0.002), IL-18 (P = 0.032), and Flt3L (P = 0.0024) were significantly increased in FIC cats compared to healthy cats. GM-CSF, IL-1b, IL-2, and PDGF-BB were undetectable or detected in an insufficient number of cats to allow meaningful comparisons. Conclusions and Clinical Importance: We have identified increased serum concentrations of pro-inflammatory cytokines and chemokines CXCL12, IL-12, IL-18, and Flt3L in FIC-affected cats. These findings suggest potential candidates for noninvasive biomarkers for diagnosis, staging, and therapeutic outcome monitoring of affected cats and provide additional insight into the etiopathogenesis of FIC. Key words: Feline; Interstitial; Multiplex assay; Urine.

Mesenchymal stem cells (MSC) offer a novel approach to treatment of inflammatory disorders in humans and companion animals. Cats spontaneously develop a wide variety of inflammatory disorders and may potentially benefit from MSC-based therapies. Multiple genes are involved in immunomodulation by MSC and interspecies differences between expressions of these genes exist. The goals of the study were to characterize the expression of genes known to be involved in MSC-based immunomodulation and determine the effect of MSC on proliferation of T cells in coculture experiments with peripheral blood mononuclear cells (PBMC). Unstimulated MSC expressed all immunomodulatory genes studied except for IL-10. Levels of iNOS and FASL were low or undetectable at the RNA level. INFγ stimulation resulted in significant dose-dependent upregulation of IDO1, PD-L1, IL-6, COX2, and HGF. Levels of kynurenine were increased after 3-day incubation with INFγ. TNFα stimulation increased expression of IL-6 at both RNA and protein level as well as upregulated COX2 gene expression and PTGES1.Stimulation with both INFγ and TNFα resulted in significant increase in PGE2 levels in cell culture medium. MSC significantly decreased proliferation of ConA-stimulated PBMC in coculture experiments at 1:5 ratio. Our results suggest that feline MSC have similar immunomodulatory gene expression and react to inflammatory cytokines in a manner similar to human MSC. Thus, MSC may play an important role in treatment of feline disease as well as serve as an important translational species to evaluate MSC-based therapies of diseases common to both humans and cats.

Reports of molecular and cellular imaging using computed tomography (CT) are rapidly increasing. Many of these reports use gold nanoparticles. Bismuth has similar CT contrast properties to gold while being approximately 1000-fold less expensive. Herein we report the design, fabrication, characterization, and CT and fluorescence imaging properties of a novel, dual modality, fluorescent, polymer encapsulated bismuth nanoparticle construct for computed tomography and fluorescence imaging. We also report on cellular internalization and preliminary in vitro and in vivo toxicity effects of these constructs. 40 nm bismuth(0) nanocrystals were synthesized and encapsulated within 120 nm Poly(dl-lactic-co-glycolic acid) (PLGA) nanoparticles by oil-in-water emulsion methodologies. Coumarin-6 was co-encapsulated to impart fluorescence. High encapsulation efficiency was achieved ∼70% bismuth w/w. Particles were shown to internalize within cells following incubation in culture. Bismuth nanocrystals and PLGA encapsulated bismuth nanoparticles exhibited >90% and >70% degradation, respectively, within 24 hours in acidic, lysosomal environment mimicking media and both remained nearly 100% stable in cytosolic/extracellular fluid mimicking media. μCT and clinical CT imaging was performed at multiple X-ray tube voltages to measure concentration dependent attenuation rates as well as to establish the ability to detect the nanoparticles in an ex vivo biological sample. Dual fluorescence and CT imaging is demonstrated as well. In vivo toxicity studies in rats revealed neither clinically apparent side effects nor major alterations in serum chemistry and hematology parameters. Calculations on minimal detection requirements for in vivo targeted imaging using these nanoparticles are presented. Indeed, our results indicate that these nanoparticles may serve as a platform for sensitive and specific targeted molecular CT and fluorescence imaging.

Urothelial carcinoma (UC) is the fourth most common malignancy in human and the most common form of urinary bladder cancer in dogs. This neoplasia tends to be invasive at advanced stages hence novel therapeutic approaches are urgently needed. As urothelial carcinomas share many similarities between the dog and human in the immunological responses, dogs are a perfect model for studying immunotherapeutic approaches. Recently immuno-checkpoint blockade using anti-PD1/PD-L1 antibodies have shown a promising effect and durable responses in the treatment of UC. Although until recently the PD1/PD-L1 axis was considered to be mostly affecting immune cells, recent reports identified, that PD-1/PD-L1 interaction is also capable of pro-tumorigenic signaling in melanoma and other cancers. Yet, little is known regarding this phenomenon in UC. In our study, we aim at identification of the expression level of PD-1 in canine UC and subsequently characterize the effect of overexpression and CRISPR-based knock-out of PD-1 and PD-L1. Using flow cytometry and immunocytochemistry, we identified that PD-1 is expressed on the surface of canine UC cell line. Interestingly, incubation with high concentration of PD-1 antibody decreased the proliferation rate of the tumor. After transfection with a PD-1 overexpression vector, we notice a significant morphological change and decrease in proliferation and colony formation. Our result provides the first report on PD-1 intrinsic signaling play as an important role in tumor growth of canine UC and thus may also be responsive to immune checkpoint blockade. The effect identified further strengthens the dog as a potential translational model for UC immunotherapy. Citation Format: Yu Jia Wang, Katarzyna Pietrzak, Mikolaj Kocikowski, David Argyle, Ted Hupp, Maciej Parys. Investigation of tumor intrinsic PD1/PD-L1 in canine urothelial carcinomas as a spontaneous translational model for human invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3972.

Tissue engineered scaffolds (TES) hold promise for improving the outcome of cell-based therapeutic strategies for a variety of biomedical scenarios, including musculoskeletal injuries, soft tissue repair, and spinal cord injury. Key to TES research and development, and clinical use, is the ability to longitudinally monitor TES location, orientation, integrity, and microstructure following implantation. Here, we describe a strategy for using microcomputed tomography (microCT) to visualize TES following implantation into mice. TES were doped with highly radiopaque gadolinium oxide nanocrystals and were implanted into the hind limbs of mice. Mice underwent serial microCT over 23 weeks. TES were clearly visible over the entire time course. Alginate scaffolds underwent a 20% volume reduction over the first 6 weeks, stabilizing over the next 17 weeks. Agarose scaffold volumes were unchanged. TES attenuation was also unchanged over the entire time course, indicating a lack of nanocrystal dissolution or leakage. Histology at the implant site showed the presence of very mild inflammation, typical for a mild foreign body reaction. Blood work indicated marked elevation in liver enzymes, and hematology measured significant reduction in white blood cell counts. While extrapolation of the X-ray induced effects on hematopoiesis in these mice to humans is not straightforward, clearly this is an area for careful monitoring. Taken together, these data lend strong support that doping TES with radiopaque nanocrystals and performing microCT imaging, represents a possible strategy for enabling serial in vivo monitoring of TES.

Background Chronic inflammatory diseases are common in cats and mesenchymal stem cells (MSC) are a promising therapeutic approach for management of these disorders. The purpose of this study was to evaluate the safety of intraperitoneal injection of MSC in cats. Hypothesis Intrapertioneal injection of autologous MSC in cats is safe. Animals Ten healthy adult purpose-bred cats. Methods Mesenchymal stem cells were isolated from subcutaneous adipose tissue collected during ovariohysterectomy and characterized for expression of CD90, CD105 and CD44 and trilineage differentiation. Three weeks postoperatively a complete blood count, serum chemistry profile, urinalysis, and abdominal ultrasound were performed. Five cats then received 1 × 106 of autologous MSC/kg of body weight intraperitoneally with ultrasound guidance; 5 additional cats were sham injected. Cats were monitored for 6 weeks with daily physical examinations and weekly clinicopathological evaluations. Abdominal ultrasonography was repeated at weeks 1 and 5 after injection. Results Serious adverse effects were not observed in any MSC-injected cat. Two animals developed transient lethargy and decreased activity. Jejunal lymph node size was increased in MSC-injected cats compared to controls at weeks 1 (1.38 ± 0.25 versus 0.88 ± 0.25 cm2; P = .036) and 5 (1.75 ± 0.82 versus 0.79 ± 0.12 cm2; P = .047). A hyperechoic renal segmental cortical lesion was observed in 1 MSC-injected cat. Conclusions and Clinical Relevance Intraperitoneal MSC injection was well tolerated with only mild, self-limiting adverse effects being observed in 2 cats. This route provides a safe means of administration for cell-based treatment in cats.

Osteosarcoma (OS) is the most common primary tumor of the bone in both humans and dogs. The dog, with its spontaneously occurring OS, shared pathogenic pathways and active immune system is one of the best models for studying novel immunotherapeutic approaches to human OS. Checkpoint blockade using antibodies against PD-1 has revolutionized the current approach to treatment of melanoma and other cancers. Recent reports have also shown that tumor intrinsic signalling plays a potential role in the success of PD-1 blockade in melanoma although studies in OS have not been published to date. PD-1/PD-L1 signalling has been reported to have role in epithelial to mesenchymal transition. In this study, we wanted to characterize intrinsic PD-1 signalling in canine osteosarcoma. We have created a canine specific PD-1 monoclonal antibody and we identified that PD-1 is intrinsically expressed in the cell line tested- OSA31. To further characterize the effects of PD-1 expression in this cell line we cloned canine PD-1 into a pcDNA 3.1 expression vector and transfected the cell line. Transfection of the cell line with PD-1 resulted in phenotypic changes characteristic for mesenchymal to epithelial transition. Interestingly overexpression of PD-1 as well as blockade of this receptor using the created antibody, resulted in decrease in cellular proliferation. To our knowledge this is the first report to identify role of PD-1 in mesenchymal to epithelial transition. These findings also suggest that PD-1 may be a promising therapeutic target in canine OS and warrant future clinical trials. Citation Format: Maciej Parys, Marta Nekulová, Bořivoj Vojtěšek, Jakub Faktor, Ana Lima, Theodore Hupp. Tumor intrinsic PD-1/PD-L1 signalling induces mesenchymal to epithelial transition in canine osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-115.

Histiocytic diseases encompass a spectrum of proliferative diseases in humans and dogs. While these orphan malignancies are rare in the human, histiocytic diseases are relatively frequent in certain breeds of dog. In the dog, these diseases range from benign histiocytoma to malignant histiocytic sarcoma (HS). Disseminated HS has a poor prognosis and lacks effective treatment options in both humans and dogs. The overall goal of this study is to unravel the mechanisms of tumorigenesis in histiocytic diseases and identify better treatments. Our studies have focused on microRNAs (miRNAs), which are master regulators of gene expression and may have a significant role in tumorigenesis in histiocytic diseases. MiRNA profiling of canine histiocytic diseases was conducted on cases of reactive histiocytosis, HS, and hemophagocytic HS. These samples were compared to normal canine histiocytes derived from peripheral blood or peritoneal fluid. Several miRNAs were identified to be upregulated in the disease samples and were selected for validation by qRT-PCR. From these results, two miRNA targets were selected for further evaluation. We are probing the significance of the upregulation of these miRNAs by inhibiting their function in canine HS cell lines using chemical miRNA inhibitors and sponge constructs designed to competitively bind the upregulated miRNAs, thus preventing them from acting on their targets. Our results to date indicate that inhibiting these miRNAs in the canine HS cell line, DH82, decreases growth rate by as much as 39%. Studies into other aspects of tumorigenicity potentially affected by the inhibition of these miRNAs are underway. Our preliminary data suggests these miRNAs themselves may be effective therapeutic targets for histiocytic diseases. Additionally, further analysis of the pathways regulated by these miRNAs may identify critical gene targets in HS tumorigenesis and thus increase our understanding of these malignancies and discover novel and effective therapies. Comparative studies have the potential to provide opportunities for advancement in these orphan malignancies, and in this case, improve clinical outcome in dogs and humans. Citation Format: Emmalena J. Gregory-Bryson, Maciej Parys, Matti Kiupel, Vilma Yuzbasiyan-Gurkan. Effects of inhibition of upregulated microRNAs in canine histiocytic sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4383. doi:10.1158/1538-7445.AM2014-4383

Histiocytic diseases encompass a spectrum of proliferative diseases in humans and dogs. While relatively rare in the human, histiocytic diseases are quite prevalent in certain breeds of dog. In the dog, these diseases range from benign histiocytoma to the malignancy, histiocytic sarcoma (HS). Disseminated HS has a poor prognosis and lacks effective treatment options. The goal of this study is to unravel the mechanisms of tumorigenesis in histiocytic diseases by analyzing the differential expression of microRNAs (miRNAs) along the spectrum of canine histiocytic diseases. As regulators of gene expression, miRNAs will provide insight into the pathways for tumorigenesis in histiocytic malignancies as compared to benign histiocytic diseases. MiRNA profiling of canine histiocytic diseases was conducted on cases of reactive histiocytosis, histiocytic sarcoma, and hemophagocytic histiocytic sarcoma. These samples were compared to normal canine histiocytes derived from peripheral blood. Based upon analysis of the profiling data, several miRNAs were selected for verification and quantification by quantitative reverse transcription PCR. The expression of the known canine miRNAs was evaluated by miRNA array. Data analysis with unsupervised clustering resulted in miRNA expression pattern clusters that were similar to the disease groups. The miRNA profiling data revealed three miRNA targets that were significantly upregulated in all cases as compared to normal canine histiocytes. Functional studies of these miRNAs are underway. These findings support the hypothesis that each histiocytic disease entity has a unique miRNA profile. These miRNAs and/or genes in the pathways they regulate may be targeted for novel and effective therapies. Comparative studies have the potential to elucidate HS tumorigenesis, and improve clinical outcome in dogs and humans. Citation Format: Emmalena Gregory-Bryson, Maciej Parys, Matti Kiupel, Vilma Yuzbasiyan-Gurkan. Identification of critical microRNA regulated pathways in histiocytic diseases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5288. doi:10.1158/1538-7445.AM2013-5288

Canine histiocytic sarcoma is an aggressive and uniformly-fatal round-cell neoplasm. The localized, peri-articular form of the disease (PAHS) appears to have statistically significant prolonged survival, when compared to the non - peri-articular form of the disease (non-PAHS). Knowledge of the molecular basis for the aggressiveness of the canine histiocytic sarcoma is foundational for developing effective treatments. The aim of this study was to characterize the molecular expression profiles of the different sub-types of the disease. Canine patients, with spontaneously-arising histiocytic sarcoma, were recruited for the study. Tumor tissue samples and peripheral blood were obtained and used for primary cell line development and gene expression analysis. Eight PAHS and 8 non-PAHS tumor samples were selected. RNA extraction, amplification and labeling were performed using standard techniques. Raw gene expression values were obtained using the Affymetrix GeneChip Canine Genome 2.0 Array. Background correction, normalization, PM correction, summarization, and expression analysis were performed using R/affy/limma. Gene set analysis was performed using BRB-ArrayTools, and the Molecular Signatures Database (MSigDB), to investigate gene ontology groups of genes whose expression was differentially regulated. Primary cell lines from tumor biopsies and peripheral blood samples were developed and characterized based on cellular morphology, growth characteristics, and immunophenotyping. In our gene expression array experiments, we identified specific gene transcripts that are more likely to represent true and biologically meaningful differences in expression levels between PAHS and non-PAHS. More specifically, metallopeptidase genes, and homeobox domain gene expression levels were enriched in the PAHS group, while ATP-binding cassette genes, and interleukin 1 receptor signaling gene expression levels were enriched in the non-PAHS group. Our results suggest presence of gene expression signatures that can provide insight in the diverse biologic behavior of the disease and highlight canine spontaneous tumors in understanding tumor biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5092. doi:1538-7445.AM2012-5092

Dogs are used as pharmacokinetic and preclinical models, but no study has yet evaluated polymorphisms in a large dog population. We hypothesize that genetic variation exists in the coding region of the canine cytochrome P-450 homologue genes CYP1A1, CYP1A2, CYP2E1, and CYP2B6. The canine CYP1A1, CYP1A2, CYP2E1, and CYP2B11 (homologue to CYP2B6) genomic sequences were identified using the UCSC Genome Browser. Intronic primers, flanking individual exons were designed using Primer3. Amplification of all exons was carried out by PCR, products were electrophoresed in agarose gel, and visualized using ethidium bromide. Genomic DNA from 20 dogs (10 breeds, 2 dogs/breed) from our lab's genomic DNA bank was used as template. Direct sequencing of the PCR amplicons was performed and the results were analyzed using the DNASTAR Lasergene 7. PCR restriction fragment length polymorphism (RFLP) assay was designed for each polymorphism identified by sequencing. PCR-RFLP assays were used to evaluate identified polymorphic sites in dogs of 10 different breeds (10 dogs/breed). The potential effect of each polymorphism to the protein function was estimated using PolyPhen and SIFT software. In CYP1A1 a total of 6 SNPs were identified: three SNPs in exon 1, and three SNPs in exon 6. One SNP was a synonymous change (c.1362T>A), while five were nonsynonymous (c.53C>G → p.A18G; c.146G>T → p.W49L; c.345C>G → p.F115L; c.C1305C>A → p.F435L; and c.1561T>C → p.S521P). In CYP1A2, A total of 6 SNPs were identified. Four of these SNPs have been already described (c.1117C>T; c.1173C>G; c.1299C>T; and c.1303G>A), while 2 novel SNPs were identified (c.1165A>G; and c.1451A>G). Of the novel SNPs, one is a synonymous change (1451A>G), and one is nonsynonymous (c.1165A>G → p.N389D). A potential InDel was identified at the 6th exon in 7/13 Siberian huskies initially assayed. Sequencing of the cloned potential InDel - containing exon revealed a 10 base long duplication (1464_1473dupCCCCATCTAT). All 7 dogs harboring the duplication, appeared to be heterozygotes for it, hence we expanded our screening of the duplication to a set of a total of 274 Siberian husky genomic DNA. We identified 75 homozygotes for the wild type, and 199 dogs that appeared heterozygotes for the duplication. When we assayed 28 Alaskan malamutes for the duplication identified in the Siberian huskies, we failed to identify any dog positive for this duplication. In CYP2E1, a total of 2 SNPs in the coding region and 2 intronic SNPs were identified. The 2 exonic SNPs are nonsynonymous (c.85C>T → p.R29C; c.1453T>C → p.Y485H). The two intronic SNPs are located at the 5th (IVS5+5G>A), and the 6th intron, (IVS6+27C>T). In CYP2B11 a total of 3 SNPs were identified. Two SNPs were synonymous (c123G>A; c966G>A) and 1 was nonsynonymous (c220C>T → p.R74C). Genetic variation in the canine CYP1A1, CYP1A2, CYP2E1, and CYP2B11 genes exists, and may have significant implications in the use of dogs as preclinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2661.