Single-Nuclei Multiome (ATAC + Gene Expression) Sequencing of a Primary Canine Osteosarcoma Elucidates Intra-Tumoral Heterogeneity and Characterizes the Tumor Microenvironment.

Osteosarcoma (OSA) is a highly aggressive bone tumor primarily affecting pediatric or adolescent humans and large-breed dogs. Canine OSA shares striking similarities with its human counterpart, making it an invaluable translational model for uncovering the disease's complexities and developing novel therapeutic strategies. Tumor heterogeneity, a hallmark of OSA, poses significant challenges to effective treatment due to the evolution of diverse cell populations that influence tumor growth, metastasis, and resistance to therapies. In this study, we apply single-nuclei multiome sequencing, encompassing ATAC (Assay for Transposase-Accessible Chromatin) and GEX (Gene Expression, or RNA) sequencing, to a treatment-naïve primary canine osteosarcoma. This comprehensive approach reveals the complexity of the tumor microenvironment by simultaneously capturing the transcriptomic and epigenomic profiles within the same nucleus. Furthermore, these results are analyzed in conjunction with bulk RNA sequencing and differential analysis of the same tumor and patient-matched normal bone. By delving into the intricacies of OSA at this unprecedented level of detail, we aim to unravel the underlying mechanisms driving intra-tumoral heterogeneity, opening new avenues for therapeutic interventions in both human and canine patients. This study pioneers an approach that is broadly applicable, while demonstrating significant heterogeneity in the context of a single individual's tumor.

Development of OX40 agonists for canine cancer immunotherapy.

Recent breakthroughs in cancer immunotherapy have provided unprecedented clinical benefits to human cancer patients. Cancer is also one of the most common causes of death in pet dogs. Thus, canine-specific immune therapies targeting similar signaling pathways can provide better treatment options for canine cancer patients. Here, we describe the development and characterization of two canine-specific anti-OX40 agonists to activate OX40 signaling. We show that canine OX40, like human OX40, is not expressed on resting T cells, and its expression is markedly increased on canine CD4 T cells and Tregs after stimulation with concanavalin A (Con-A). cOX40 is also expressed on tumor-infiltrating lymphocytes (TILs) in canine osteosarcoma patients. The canine-specific OX40 agonists strongly activates cPBMCs by increasing IFN-γ expression and do not require Fc receptor-mediated cross-linking for OX40 agonism. Together, these results suggest that cFcOX40L proteins are potent OX40 agonists and have the potential to enhance antitumor immunity in canine cancer patients.

Canine models of human cancer: Bridging the gap to improve precision medicine.

Dogs are remarkable, adaptable, and dependable creatures that have evolved alongside humans while contributing tremendously to our survival. Our canine companions share many similarities to human disease, particularly cancer. With the advancement of next-generation sequencing technology, we are beginning to unravel the complexity of cancer and the vast intra- and intertumoral heterogeneity that makes treatment difficult. Consequently, precision medicine has emerged as a therapeutic approach to improve patient survival by evaluating and classifying an individual tumor's molecular profile. Many canine and human cancers share striking similarities in terms of genotypic, phenotypic, clinical, and histological presentations. Dogs are superior to rodent models of cancer because they are a naturally heterogeneous population in which tumors occur spontaneously, are exposed to similar environmental conditions, and show more similarities in key modulators of tumorigenesis and clinical response, including the immune system, drug metabolism, and gut microbiome. In this chapter, we will explore various canine models of human cancers and emphasize the dog's critical role in advancing precision medicine and improving the survival of both man and man's best friend.

Transcriptomic Analysis of Canine Osteosarcoma from a Precision Medicine Perspective Reveals Limitations of Differential Gene Expression Studies.

Despite significant advances in cancer diagnosis and treatment, osteosarcoma (OSA), an aggressive primary bone tumor, has eluded attempts at improving patient survival for many decades. The difficulty in managing OSA lies in its extreme genetic complexity, drug resistance, and heterogeneity, making it improbable that a single-target treatment would be beneficial for the majority of affected individuals. Precision medicine seeks to fill this gap by addressing the intra- and inter-tumoral heterogeneity to improve patient outcome and survival. The characterization of differentially expressed genes (DEGs) unique to the tumor provides insight into the phenotype and can be useful for informing appropriate therapies as well as the development of novel treatments. Traditional DEG analysis combines patient data to derive statistically inferred genes that are dysregulated in the group; however, the results from this approach are not necessarily consistent across individual patients, thus contradicting the basis of precision medicine. Spontaneously occurring OSA in the dog shares remarkably similar clinical, histological, and molecular characteristics to the human disease and therefore serves as an excellent model. In this study, we use transcriptomic sequencing of RNA isolated from primary OSA tumor and patient-matched normal bone from seven dogs prior to chemotherapy to identify DEGs in the group. We then evaluate the universality of these changes in transcript levels across patients to identify DEGs at the individual level. These results can be useful for reframing our perspective of transcriptomic analysis from a precision medicine perspective by identifying variations in DEGs among individuals.

Characterization of Canine Adenovirus Type 2 Virus Infection Pattern in Canine and Human Cell Lines.

Canine adenovirus type 2 (CAV2) is a nonhuman adenovirus with a known ability to infect human and canine cells. The cell surface receptors involved in CAV2 transduction are still unknown. Identification of these would provide valuable information to develop enhanced gene delivery tools and better understand CAV2 biology. CAV2 is erroneously grouped with Ad5 based on the knowledge that CAV2 may transduce using CAR. Therefore, we have evaluated CAV2 and Ad5 (CAV2GFP, Ad5G/L) infection patterns in various canine and human cell lines to determine their different tropisms. Our research demonstrates that CAV2 can successfully infect cells that Ad5 does not infect, and CAV2 infections do not correlate with CAR expression. CAV2 can infect cells that have a low or minimal expression of CAR. Our data suggest that CAV2 transduction is not dependent on the CAR receptor, and thus, it is crucial to find novel CAV2 receptors.

In vitro functional genetic modification of canine adenovirus type 2 genome by CRISPR/Cas9.

Genetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.