Intratumoral injection of a novel TLR4 agonist elicits infiltration by immune effector cells in a canine soft tissue sarcoma: An Immunologic Case Study

Background Immunostimulatory adjuvants used in vaccines to protect against infectious disease have demonstrated efficacy in stimulating anti-cancer immunity. The most commercially advanced ones activate Toll-Like Receptor-4 (TLR4), a transmembrane signaling molecule expressed by macrophages and dendritic cells triggering innate immune responses. Lipopolysaccharide, the first identified TLR4 agonist, induces toxic, unregulated immune activation. Mimetics of monophosphoryl lipid A, the stimulatory component in lipopolysaccharide, reduced immunotoxicity while retaining immunostimulatory properties. Intratumoral injection of formulated TLR4 agonists can stimulate in situ antitumor immune responses by recruitment of immune cells and production of inflammatory cytokines that exert antitumor effects via a variety of mechanisms – including direct cancer cell death and recruitment of effector cells. Human clinical cancer trials have shown efficacy - both locally and through abscopal effects employing this approach. Methods We hypothesized that injection of a novel TLR4 agonist, EmT4™, into a canine soft tissue sarcoma (STS) could alter the tumor microenvironment by attracting and activating immune cells in situ . With the dog owner’s interest and written consent, a 3-cm soft tissue mass on the right forelimb of an 8-year-old female spayed Boston terrier received two intratumoral injections of EmT4™, two weeks apart. There was transient lethargy on the day of the first injection that resolved within hours. The tumor was excised 4 weeks after the second injection. Histopathology, immunohistochemistry, and in situ RNA hybridization were utilized to explore immune cell populations in the tumor microenvironment. Results Histopathology revealed grade 2 STS with large numbers of densely packed perivascular immune cells disseminated within the tumor. Immunohistochemistry for immune cell markers showed heterogeneous positive staining within cell clusters – CD3 (25%), CD20 (57%), FOXP3 (8%), CD204 (5%), and Iba-1 (36%). In situ hybridization performed on serial STS sections with RNAscope™ identified transcripts for CD4 (29%), TNF-α (24%), CD8 (3.2%), and interferon-g (1.3%) in lymphocyte clusters. Conclusions EmT4™ may have elicited an innate immune response that attracted and activated immune effector cells intratumorally. Clinical circumstances prevented acquisition of a pre-EmT4™ biopsy hampering a definitive conclusion although cell infiltrates observed are unusual in canine STS. This case is foundational for continued EmT4™ investigations for canine cancer immunotherapy.