Tumor infiltrating lymphocyte (TIL) therapy works on the hypothesis that T cells within the tumor are specific for a multitude of different tumor associated antigens (reviewed in^[55]). TILs isolated from solid tumors and expanded ex vivo, retain their tumor-specific functionality post expansion and mediate potent anti-tumor immunity upon adoptive transfer back into the patient. Insights from transcriptomic profiling of canine CD8+ TILs showed them to be different from circulating CD8+ T cells, having increased expression of 241 genes associated with cytotoxicity and immune activation^[56]. However, exhaustion markers such as LAG3, TIM1 and 41-BB were also expressed, along with factors that tune activation signaling (NFKBIA, DUSP4, RGS2 and RGS4), those that enforce tolerance (NR4A3) and others that regulate proliferation^[56]. Ex vivo rapid expansion protocols performed in the presence of high concentrations of IL-2 help to reinvigorate TILs, and further expansion in vivo after re-infusion is aided by the concurrent administration of IL-2 to patients^[57]. In 2024, the first TIL therapy was approved by the FDA for the treatment of advanced unresectable or metastatic melanoma based on promising results in 73 human patients^[58]. This has now led to the exploration of TILs to treat canine cancers. One group recently demonstrated that TILs can be expanded from dissociated canine osteosarcoma samples to achieve clinically relevant cell numbers after a customized rapid expansion protocol ex vivo^[59]. Functional assays demonstrated that the majority of these expanded TIL products exhibited reactivity against autologous tumor cells, suggesting that TIL therapy may hold therapeutic promise for dogs with osteosarcoma. To address concerns over TILs exhibiting low TCR affinity for the self-antigens expressed on tumors, chimeric antigen receptor (CAR) technology has been explored to re-direct TILs. One group used a second generation, HER2 targeted, CAR-TIL approach in dogs with squamous cell carcinoma and melanoma which, when administered alongside tumor resection, a tyrosine kinase inhibitor and IL-2, led to transient reduction in tumor size in some cases and one year of tumor free survival in others^[60]. Additional engineering strategies to promote the activity of re-infused TILs in the tumor microenvironment are currently being investigated in human TILs^[61].

An alternative approach to provide large numbers of autologous tumor-specific T cells for adoptive transfer is to combine autologous tumor vaccination to prime T cells in vivo, with subsequent T cell harvest, ex vivo expansion and adoptive transfer back into the patient. In vivo priming with autologous tumor cells aims to increase the number of tumor-specific T cells in the patient before apheresis, thereby increasing the number in the ex vivo expanded infusion product^[5,62]. Autologous tumor vaccination prior to ACT was evaluated in two cohorts of pet dogs with osteosarcoma who underwent amputation without chemotherapy, and clinical outcomes were compared to dogs treated with standard of care amputation and carboplatin. In the first prospective pilot single arm cohort study, 10 dogs received tumor vaccination followed by apheresis and ACT, together with low‐dose systemic IL‐2 to support in vivo expansion of adoptively transferred T cells^[5]. This approach produced a median survival time of 415 days, which was comparable to that of dogs receiving standard of care amputation and carboplatin chemotherapy. Although one patient experienced severe immune-related adverse events, the therapy was otherwise well tolerated. In preliminary conference-reported data from a second small (n = 14) study of autologous tumor vaccination and ACT given after a single dose of carboplatin, a one year survival rate of 71% compared favorably to 21% achieved in a matched comparator cohort receiving standard of care carboplatin without vaccination and/or ACT^[63]. This ACT/vaccine approach (ELIAS Cancer Immunotherapy) recently received full approval from the USDA Center for Veterinary Biologics for dogs with osteosarcoma. Future prospective studies are warranted to explore predictive biomarkers of immune response that may allow for stratification of patients most likely to respond to therapy.

Although vaccination prior to T cell harvest aims to enhance the potency of cellular products by increasing the frequency of tumor-specific T cells, it relies on effective in vivo T cell priming. However, defects in antigen presentation together with an imbalance in the specificity and affinity of the endogenous T cell repertoire relative to tumor antigens expressed could render vaccines ineffective at priming T cells in some cancer patients^[64]. Furthermore, down regulation of MHC molecules on the surface of tumor cells enables them to avoid detection by endogenous and adoptively transferred tumor-specific T cells. To circumvent the requirement for in vivo T cell priming and to overcome MHC down-regulation, autologous T cells can be engineered to express a CAR that enables the T cell to recognize cell surface expressed antigens^[65]. CARs are composed of the VH and VL domains of an antibody linked to a T cell co-stimulatory domain and an activating CD3 chain [Figure 2]. When expressed on the T cell surface, the CAR construct allows T cells to detect a membrane bound target antigen, without the need for MHC, activate upon CAR engagement and undergo antigen-specific proliferation, cytokine production and target cell killing. While CAR-T cell therapy has been remarkably successful in hematological malignancies in human patients, it has underperformed in the treatment of solid tumors, including osteosarcoma. Reported reasons for this include failure of trafficking into the tumor, the immunosuppressive tumor microenvironment, and target antigen downregulation and/or heterogeneity within solid tumors. These barriers to effective CAR-T cell therapy are being addressed through engineering strategies to improve trafficking, resist the hostile tumor microenvironment and circumvent epitope loss, for example through expression of bivalent CARs. Furthermore, CARs employing different antigen recognition and/or signaling domains that influence the phenotype of the CAR-T product with effects on function and persistence, are now being evaluated^[66,67].

Second generation CAR-T cells that target HER2 or B7-H3 have been developed for the treatment of human sarcomas^[68,69] and canine osteosarcoma^[43,70]. While HER2 targeting CAR-T cells show robust effector function against HER2+ osteosarcoma cell lines in vitro, they failed to show efficacy in pediatric osteosarcoma patients and have not yet been evaluated in the canine clinic. In contrast, B7-H3 targeted CAR-T (and chimeric antigen receptor invariant natural killer T (CAR-iNKT)) cells are currently being evaluated in both human (CAR-T) and canine (CAR-T and CAR-iNKT) osteosarcoma patients. B7-H3 is a checkpoint receptor that is highly expressed in many tumor types including osteosarcoma, where it correlates with a negative prognosis^[71]. B7-H3 targeting CAR-T cell approaches have demonstrated anti-tumor activity in vitro and in xenograft models of canine osteosarcoma^[42]. Safety and efficacy data is currently being obtained from a canine clinical trial of B7-H3 targeted CAR T cells and CAR-iNKT cells in metastatic and non-metastatic osteosarcoma, and parallel B7-H3 targeting CAR T cell trials in pediatric osteosarcoma are ongoing.

Tumors can downregulate or eliminate targeted antigens/epitopes when placed under selection pressure by natural immune responses or in response to targeted immunotherapy - a phenomenon known as antigen escape. In hematological malignancies, loss of the CAR-targeted epitopes is a well-documented challenge which can lead to CAR-T failure^[66,72]. Advanced engineering to generate bi-specific CARs, that target two antigens, or the use of universal CARs that target multiple antigens at once through innovative designs, are now being explored to address issues of antigen escape^[73]. Such advances are starting to be explored in canine osteosarcoma but have yet to be tested in the clinic.

While autologous T cells are the most commonly used immune subset in cellular immunotherapy, intrinsic T cell dysfunction, associated with advanced disease or prior chemotherapeutics, can limit the effectiveness of T cell therapy. This, together with the high cost and labor-intensive process of generating autologous T cell products, limits their global use in human and canine patients. Together, these challenges have fueled the drive to advance allogeneic cellular immunotherapies, whereby multiple immune cell products can be generated from a single healthy donor and cryopreserved for on-demand use [Figure 3]. Key barriers to adoptive cell transfer with allogeneic T cells include graft versus host disease (GVHD) mediated by MHC mismatch between donor and recipient, and immunological rejection of allogeneic cells by the recipient^[74]. While some of these barriers are being addressed by genetic editing of T cell components including disruption of the TCR and MHC molecules, the effects of multiple edits on T cell function are not fully understood. This has led to the exploration of alternative cytotoxic immune cell types including NK and iNKT cells that can be safely transferred into MHC mismatched hosts, without causing GVHD^[75].

NK cells are innate lymphoid cells which play a critical role in the anti-tumor immune response. NK cell activation depends upon the balance of activating and inhibitory signals received through a multitude of surface receptors such as killer immunoglobulin receptors, which are stimulatory to NK cells, and interactions with self MHC molecules, which inhibit NK cell activation^[76]. Malignant cells often down-regulate MHC and are therefore vulnerable to NK cell attack. The use of autologous and allogeneic NK cells either alone or in combination with radiation and/or cytokine support has been actively explored in dogs with osteosarcoma^[77,78].

The rationale for combining adoptively transferred NK cells with radiation therapy was supported by in vitro studies suggesting that radiation increases the cytotoxicity of NK cells and enhances their homing to tumors^[79]. In a prospective pilot single-arm cohort study, autologous NK cells were administered intra-tumorally to dogs with appendicular osteosarcoma that had undergone palliative radiation. Treatment was generally well tolerated although 3/10 patients developed injection site infections requiring surgical debridement in 2 dogs. Patients experienced a significant increase in CD5+ GranzymeB+ cells in peripheral blood, and one patient had resolution of a suspected metastatic pulmonary nodule, a finding which may be consistent with systemic immune activity. Metastasis-free survival and progression-free survival compared favorably to historical controls^[79]. A follow up study by the same group revealed greater increases in CD3 and CD8 gene expression in the peripheral blood of long-term survivors compared with short term survivors amongst NK cell-treated canine patients^[80]. These findings provide preliminary evidence that NK cell therapy, in combination with radiation therapy, may potentiate anti-tumor immune responses. The study also suggested that higher serum IL-6 levels at baseline might predict shorter metastasis-free and overall survival, revealing a conflicting role for serum and intratumoral IL-6 as a potential biomarker of prognosis in canine patients^[80].

IL-15 is a critical cytokine promoting NK cell activation and expansion and has been delivered via inhalation to enhance local NK cell activity in dogs with pulmonary metastatic osteosarcoma^[81]. In a small feasibility study, treatment with inhaled recombinant human IL-15 alone was well tolerated and associated with increases in pro-inflammatory cytokines and some evidence of clinical response. To increase the effectiveness of this approach, inhaled IL-15 was supplied in combination with adoptive autologous NK cell transfer. This approach was found to be feasible, safe and well tolerated, with one out of the five dogs treated developing stable disease^[81]. To gain further insights into NK cell biology and therapeutic capabilities, canine NK cells have been extensively characterized using single-cell RNA sequencing which revealed tissue-specific canine NK cell signatures and comparability of canine NK cells to human NK cells^[82]. These studies in pet dogs have begun to provide the baseline data required to identify molecular signatures or biomarkers of NK cell fitness that could be valuable in donor selection, ensuring the best chance of success for allogeneic NK cell therapies in the future.

Pet dogs with metastatic osteosarcoma are also being employed to advance the development of adoptive cell therapy (ACT) using a rare subset of T cells known as iNKT cells. iNKT cells comprise less than 1% of peripheral T cells and express an invariant T cell receptor specific for glycolipid antigens presented in the context of the monomorphic non-classical MHCI molecule^[74]. Similar to NK cells, allogeneic iNKT cells can be adoptively transferred without causing GVHD and produce cytokines which potentiate cytotoxic anti-tumor immune responses while regulating excessive or toxic immune related adverse events^[75,83]. Non-terminal studies in 2 healthy research dogs demonstrated that adoptive transfer of ex-vivo expanded allogeneic, MHC mismatched iNKT cells was feasible, safe and resulted in the persistence of donor iNKT cells within recipients for over 3 months^[75]. In this preclinical study, single cell RNA sequencing of ex vivo expanded canine iNKT cells identified significant inter-donor differences in iNKT cell phenotype (e.g., central memory versus effector memory), cytotoxicity and replicative capacity, and these differences were validated using in vitro assays. Therefore, the study provided important information regarding donor variability and the effect of donor selection on allogeneic iNKT products. Engineered allogeneic CAR-iNKT cells are now being evaluated in dogs with metastatic osteosarcoma and as neoadjuvant therapy in primary appendicular osteosarcoma, in NCI supported clinical trials^[84]. Serial integrated immunologic analyses of canine patients receiving allogeneic CAR-iNKT cells is ongoing and should help to clarify any immune modulatory and anti-tumor effects of CAR-iNKT cells in this setting.

Multiple studies in human osteosarcoma have shown that macrophage infiltrates and M2 phenotypes positively correlate with outcome^[95,96]. In the previously referenced retrospective transcriptomic study^[85], CIBERSORT was used to deconvolute immune populations in canine osteosarcoma tissue, and the authors unexpectedly found that M2 subsets were enriched in the favorable outcome group whereas M0 macrophages were associated with a poor prognosis^[85]. Furthermore, a retrospective pathology study of 24 canine osteosarcomas used immunostaining to show that the % area of CD204+ (M2 like) macrophage staining within the primary tumor positively correlated with CD3+ T cell and FOXP3+ infiltrates, and with disease-free interval but not with overall survival time^[26]. Thus, in both human and canine osteosarcoma, and in contrast to other tumor types, specific subsets of tumor associated macrophages (TAMs) appear to be beneficial, and this has formed the basis of employing macrophage-activating agents therapeutically. One such agent is liposomal muramyl tripeptide phosphatidyl ethanolamine (L-MTP-PE), which in a prospective randomized double-blind placebo-controlled clinical trial was found to extend survival when administered to dogs with appendicular osteosarcoma following amputation and cisplatin chemotherapy^[97]. A similar benefit was identified in non-metastatic pediatric osteosarcoma patients, treated with mifamurtide (Mepact), a muramyl-dipeptide derivative, following definitive surgery and in combination with maintenance chemotherapy^[98]. Mifamurtide is now approved by the European Medicines Agency for treatment of high-grade, resectable, non-metastatic osteosarcoma in pediatric and young adult cohorts^[99]. Although the initial studies showing survival benefit using mifamurtide were performed in dogs, it is not FDA approved for veterinary use.

Conversely, several canine and human studies also support a tumor-promoting role of macrophages in osteosaroma. Transcriptomic studies have shown that CCL2 (also known as monocyte chemoattractant protein-1, MCP-1), a protein that plays a central role in monocyte recruitment to areas of tissue damage and inflammation, is highly expressed in primary and metastatic canine osteosarcoma lesions^[25]. In murine and human preclinical models, CCL2 was associated with intra-tumoral enrichment of CCR2+ myeloid cells, angiotensin-II driven upregulation of TGF-β signaling, macrophage recruitment and collagen deposition/fibrosis, contributing to the pre-metastatic niche^[100]. Drug library screening identified the angiotensin II receptor blocker, losartan, as an inhibitor of CCL2 that might block or retard development of an immunosuppressive metastatic niche. This hypothesis led to mouse studies and canine clinical trials in which treatment with high doses of losartan was associated with partial responses in 4/16 dogs and stable disease in 4/16 dogs^[93]. At the highest dose of losartan, 95% and 66% increases in median progression-free survival and overall survival respectively were observed. Losartan treatment was also associated with reduced circulating monocytes in a subset of dogs in this study, who experienced prolonged progression-free survival compared to those with no changes in monocytes in response to therapy^[93]. Losartan treatment did not affect serum TGF-β concentrations, suggesting that its primary mode of action was inhibition of monocyte recruitment to osteosarcoma lesions. The combination of losartan and toceranib is now widely employed in the treatment of dogs with metastatic osteosarcoma by veterinary oncologists. These promising results in pet dogs led to an ongoing study of losartan and sunitinib in human patients with metastatic osteosarcoma, exemplifying that findings from canine studies are being used to inform clinical trials in human osteosarcoma patients^[101]. Canine studies have now been extended to include the CXCR1 and CXCR2 inhibitor ladarixin, in combination with losartan and toceranib. Preliminary reports indicate that this regimen has produced objective responses and clinical benefit in some dogs with pulmonary osteosarcoma metastases, although the final results are pending^[102]. Early gene expression profiling data generated from the PBMC of dogs treated in the triple combination therapy trial, has so far shown therapy-induced alterations in genes and pathways with obvious relevance to anti-tumor immunity e.g., downmodulation of the checkpoint inhibitor cytotoxic T-lymphocyte associated protein 4 (CTLA-4), and upregulation of CCL19 (a chemokine ligand involved in lymphocyte recruitment)^[102]. Given the promising results in the initial trial, losartan/toceranib/ladarixin is now being evaluated in the adjuvant setting in dogs post-amputation, in the absence of systemic chemotherapy.

Since myeloid cells are heterogeneous, identifying specific subsets and establishing their role in tumor progression and metastasis is a key focus of current research^[103]. Collaborations between veterinary and human research groups, including analyses of clinical cohorts across humans^[94] and dogs^[102], may inform therapies that eliminate immunoregulatory macrophage subsets whilst preserving those that support anti-tumor immune responses.

CAFs are also thought to play a fundamental role in stromal conditioning and immunosuppression in canine osteosarcoma. In one study, canine CAFs isolated from the tumors of dogs with malignant epithelial tumors, were analyzed using immunostaining and ELISA and were shown to produce CXCL12^[104]. CAF derived TGF-β augmented this effect, acting in an autocrine manner to upregulate CXCL12 secretion. CXCL12 is a cytokine which may encourage CXCR4-expressing T cells to sequester in the peritumoral stroma rather than infiltrating the tumor, and CAF derived TGF also functioned in an endocrine fashion to increase CXCR4 expression by T cells^[104]. Overall, this canine study provided evidence in support of the CAF-mediated CXCL12-CXCR4 axis contributing to immune exclusion in solid tumors. Spatial transcriptomic analysis has also shown that the CXCR4 axis is important in the peritumoral region of human osteosarcoma metastases^[88]. Further analysis of CAFs in canine osteosarcoma could support a renewed drive to target fibroblasts for therapeutic gain.

In order to target CAFs, markers of immunosuppressive CAF subsets must be defined. Amongst human cohorts, fibroblast activating protein (FAP) is expressed by CAFs and has been targeted with radionucleotide therapy and CAR-T cells, leading to CAF depletion and control of tumor growth in clinical trials of multiple cancers, including osteosarcoma^[105]. However, markers of immunosuppressive CAFs have previously been lacking in dogs. Canine-specific omics and immunostaining analyses have now allowed canine CAF subsets to be better characterized, and populations expressing SFRP2 and PRSS23 at the gene level^[106], and FAP at the protein level^[107] have been shown to be associated with a negative prognosis in pet dogs. Subsequently, cross-reactive anti-FAP antibodies have been identified, paving the way for FAP-targeted antibodies and CAR-T cells to enter the canine clinic. Current canine trials are employing FAP-directed CAR-T cells in combination with telmisartan and toceranib, to collectively address the immunosuppressive TME. The clinical outcome data of these studies together with immune correlates of response to therapy are eagerly awaited^[108].