Introduction

Immunotherapy has been heralded as the 4th pillar of cancer treatment and promises to improve outcomes for immunogenic tumors like canine oral malignant melanoma (cOMM). Multiple factors contribute to impactful immunotherapeutic responses including the immune profile of a tumor with “immune inflamed” phenotypes associated with superior outcomes. Immunogenic cell death (ICD) is a form of specialized programmed death that promotes immune “awakening” and generation of “immune inflamed” profiles through damage associated molecular patterns (DAMPS; calreticulin, ATP, HMBG1) induced by bona fide ICD chemotherapeutics. The evaluation of ICD-inducing agents and characterization of immune profiles in cOMM would generate provocative findings and promote the rational design of chemoimmunotherapeutic strategies for improving outcomes in pet dogs.

Methods

Three putative ICD-inducing agents (Doxorubicin, Oxaliplatin, and Bortezomib) were evaluated in 3 immortalized cOMM cell lines for inducing canonical markers of ICD, being calreticulin membrane translocation, ATP and HMGB1 release via confocal microscopy, luminescent ATP assay, and western blot, respectively. The immune profile of 50 cOMM samples were characterized using immunohistochemistry for Iba1, PAX5, CD3, and FOXP3.

Results

Doxorubicin and Oxaliplatin promoted ICD in cOMM cell lines as demonstrated by calreticulin translocation (ER to plasma membrane), as well as HMGB1 release. Immune profiling of cOMM samples identified robust macrophage infiltration within all samples, with lesser and heterogeneous quantities of TILs and minimal Tregs.

Conclusion

Doxorubicin and Oxaliplatin are bona fide ICD inducing agents in cOMM and should be included into future designed chemoimmunotherapeutic protocols. Overall, cOMM samples possess inflamed profiles with predominance of macrophage infiltration.

Funding Information

Morris Animal Foundation