The scale of the problem is considerable. Canine oral tumors account for up to 12 percent of all cancers seen in dogs, with melanoma, squamous cell carcinoma, acanthomatous ameloblastoma, and fibrosarcoma representing the most common diagnoses of the oral cavity and pharynx. When these tumors are caught early, wide surgical excision remains the most effective local treatment. But many oral cancers are diagnosed at a locally advanced stage, when complete surgical resection is no longer possible. Radiation and chemotherapy become the fallback options, and while radiation can produce high response rates in tumors such as oral melanoma and squamous cell carcinoma, one-year survival rates for some of the most common oral cancers fall below 50 percent. Novel adjuvant therapies are urgently needed to make those responses last.

The drug at the heart of the study, CB101, is a proprietary hydrogel-based injectable formulation of resiquimod, a molecule known as a toll-like receptor 7/8 agonist. Toll-like receptors sit on the surface or within certain immune cells and act as sentinels for danger signals. In humans, TLR8 is uniquely expressed on myeloid dendritic cells, monocytes, and natural killer cells, making it an attractive lever for pulling the immune system into a fight against cancer. Activating these receptors may disrupt the immunosuppressive microenvironment that tumors build around themselves. Resiquimod itself has never been approved by the FDA, but topical formulations have been tested in human clinical trials for skin diseases and as a vaccine adjuvant, generating an extensive body of toxicology and pharmacology data supporting its safety profile.

The rationale for pairing this immune agonist with radiation rests on a phenomenon called immunogenic cell death. High-dose radiation, such as the 8 Gy per fraction used in some hypofractionated protocols, can kill tumor cells in a way that makes them more visible to the immune system, releasing tumor antigens that dendritic cells can engulf and cross-present to CD8-positive cytotoxic T lymphocytes. Research suggests that doses around 8 Gy per fraction are particularly well suited to synergizing with immunotherapeutics. In principle, this cascade can generate an adaptive, tumor-specific immune response capable of affecting not just the irradiated tumor but also distant, non-irradiated lesions, the so-called abscopal effect. Preliminary mouse data showed that the combination of radiation and CB101 improved local tumor control compared with either treatment alone and elicited such abscopal responses. Injecting the drug directly into the tumor is designed to limit systemic exposure and minimize off-target immune side effects.

Three dogs with histologically confirmed head and neck cancers were prospectively enrolled in the pilot study. Each animal received a baseline CT scan for radiation planning, followed by palliative radiation therapy delivered as four weekly 8 Gy fractions over weeks one through four. CB101 was administered intratumorally at a fixed dose of 10 micrograms in 500 to 1,000 microliters of volume at one-week intervals during weeks two through five, with the first dose given immediately before the second radiation fraction. Treatment planning relied on CT-based software, and dogs were anesthetized and immobilized in vacuum cushions with bite blocks to ensure reproducible setup. A follow-up CT scan at week 12 assessed tumor response, and serial thoracic radiographs at week 24 monitored for distant metastasis. All owners provided informed consent under institutional animal care protocols.

Feasibility proved to be the study’s clearest success. Little to no difficulty occurred during the intratumoral injections, and leakage of the formulation was negligible. Tumors arising from or containing bone were more challenging to infuse; one dog with mandibular fibrosarcoma required both intratumoral and peritumoral administration because of the density of the tumor and its proximity to the normal mandible. Injections were generally performed under general anesthesia for patient comfort, and no pain was noted after the procedure. The volume of each injection was chosen based on the expected ability of the hydrogel to diffuse into the tumor and surrounding tissue, guided by physical examination, CT evidence of bone infiltration, and three-dimensional tumor measurements.

Safety outcomes were equally encouraging. Toxicity was graded using the Veterinary Cooperative Oncology Group criteria and a veterinary radiation morbidity scoring scheme. Only one adverse event was attributable to CB101 itself: a case of grade 1, mild pain during a fourth injection performed under light sedation rather than general anesthesia. Every other adverse event documented was an expected side effect of palliative radiation, and no dog experienced toxicity higher than grade II. One dog developed grade I skin and mucosal toxicity along with grade II ocular toxicity requiring eye drops and antibiotics, an outcome anticipated from the radiation dose distribution, which included a large portion of the left eye. Another dog experienced no toxicity at all.

Tumor responses varied, as expected in such a small and histologically diverse cohort. The dog with oronasal squamous cell carcinoma showed a partial response at week 12 but developed distant progressive disease, first in lymph nodes and then in the lungs, between weeks 12 and 24, and was euthanized 198 days after enrollment. The dog with oral melanoma experienced local progression at week 12 and pulmonary metastasis by week 24, surviving 254 days. The dog with oral fibrosarcoma had stable disease at the week 12 scan, with eventual progression outside the radiation field at week 26 and a survival time of 371 days. All three dogs were free of distant metastases when treatment began.

Intriguingly, serum cytokine analysis revealed negligible changes in the immune signaling molecules the team measured, including TNF-alpha, IL-6, IL-15, interferon-alpha, IL-1 beta, and IL-12p40. The researchers suspect the sampling schedule missed any transient cytokine peaks, which could be expected within hours to three days after dosing rather than a full week later. The absence of clinical signs of cytokine release suggests the weekly 10 microgram dose was well tolerated but may have fallen below a therapeutic threshold. The starting dose was chosen conservatively because a human trial using 12.5 micrograms intralesionally had reportedly triggered cytokine storm and hypotension in some patients. Other veterinary researchers have used far higher resiquimod doses, with a University of Kansas group treating dogs with cutaneous mast cell tumors using doses ranging from 0.07 to 1.36 milligrams and observing one complete response and three partial responses among six dogs.

The Penn team acknowledges several confounders, including the use of NSAIDs in two of the three dogs, which could have either blunted the innate immune response or improved outcomes through COX-2 inhibition, and antimicrobial treatment in one dog, which in human melanoma patients has been associated with worse survival on immunotherapy. Future studies, the authors say, should standardize these medications, sample blood at multiple early time points after injection, and consider analyzing tumor tissue to capture local immune effects within the microenvironment. A dose escalation study appears warranted, and the researchers envision eventually combining TLR agonists with checkpoint inhibitors, an approach that could shift immunologically cold tumors toward hot ones. As academic and industry groups work toward commercializing canine checkpoint inhibitors, this modest pilot study in three dogs may mark the first step of a translational path that runs from the veterinary clinic to human oncology and back again.

Subject of Research: Combining intratumoral resiquimod immunotherapy with hypofractionated radiation therapy in canine head and neck cancer

Article Title: Preliminary evaluation of the safety and feasibility of toll-like receptor ligand CB101 combined with hypofractionated radiation therapy in canine head and neck cancer: a pilot study

Article References: Ghanian, A., DiBona, J., Duda, L., Xu, X., Lukens, J. N., Pearce, T., Ehrhardt, M., Rook, A., Durham, A., Maity, A., & Flesner, B. (2025). Preliminary evaluation of the safety and feasibility of toll-like receptor ligand CB101 combined with hypofractionated radiation therapy in canine head and neck cancer: a pilot study. Veterinary Oncology, 2(1), Article 18. https://doi.org/10.1186/s44356-025-00031-6

Image Credits: AI Generated

DOI: 10.1186/s44356-025-00031-6

Keywords: canine cancer, head and neck cancer, radiation therapy, resiquimod, CB101, toll-like receptors, immunotherapy, intratumoral injection, veterinary oncology, dendritic cells, abscopal effect, pilot study