Bacteria-based cancer immunotherapy: A re-emerging strategy with contemporary implications

Abstract

Immunotherapy is a re-emerging alternative to other invasive and debilitating cancer treatments. Monoclonal antibodies that disrupt immune checkpoint molecules, such as programmed cell death protein 1 (PD1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA4), have been paradigm-shifting advances in medical oncology. However, therapy response rates need improvement, and important questions remain regarding the mechanisms by which checkpoint therapies promote their therapeutic effects. One intriguing factor is how the presence and composition of the gut microbiome influence treatment outcomes. I hypothesised that checkpoint therapies allow gut bacteria to colonise extraintestinal tumours, and this theory relates to our laboratory's intratumoural Complete Freund's Adjuvant (CFA) therapy. CFA is a water-in-oil emulsion containing heat-killed mycobacteria, has successfully improved survival in several animal cancer models, and is well tolerated by cancer patients. Thus, the primary aims of this project were to explore the impact of the gut microbiome on checkpoint therapies and enhance the efficacy of our laboratory's CFA therapy.

The research was conducted at the Australian National University (ANU) and the Stanford University School of Medicine. I used six mouse cancer models across three mouse strains and a tumour sampling method developed by our laboratory called fine-needle aspirate (FNA) sampling. FNA sampling with flow cytometry enables the longitudinal comparison of tumour-infiltrating leukocytes (TILs) among animals with disparate treatment outcomes without sacrificing them for data acquisition. This method was also used to investigate how depleting the gut microbiome with antibiotics would impact intratumoural immune reactions following treatment. Lastly, two methods of improving CFA therapeutic efficacy were investigated: (1) combining the treatment with checkpoint therapies and (2) increasing the concentration of heat-killed mycobacteria in the emulsion ten-fold (10x CFA).

Despite the challenges posed by external factors, including the global coronavirus disease 2019 (COVID-19) pandemic, I successfully progressed towards both aims. Preliminary experiments highlighted critical unanswered questions regarding the consequences of combining CFA with checkpoint therapies on the metastatic disease progression of the 4T1 mammary adenocarcinoma model. I also found that 10x CFA significantly increased immune infiltration (primarily neutrophils) into the CT26 colorectal cancer model, an effect not observed with standard 1x CFA therapy that may have critical implications on therapeutic efficacy.

While examining checkpoint therapies, I found that the P815 mastocytoma and CT26 cancer models responded well to anti-CTLA4 but not anti-PD1. P815-challenged mice responsive to anti-CTLA4 showed significant increases in tumour-infiltrating CD8^+^ T cells by days 3, 5, and 7 post-treatment and CD4^+^ T cells by days 5 and 7. CT26-challenged animals responsive to anti-CTLA4 had significantly more tumour-infiltrating CD8^+^ T cells by day 11 post-treatment. While investigating how gut microbial depletion affects treatment outcomes in CT26-challenged animals, I found that BALB/c (and DBA/2) mice do not tolerate a common antibiotic cocktail used in several high-impact publications. Regardless, depletion was successfully achieved, and while anti-CTLA4 therapy was not impacted, I did find evidence of an altered immune state among antibiotic-fed animals.

Collectively, these findings highlight ongoing challenges and unanswered questions in the field of immunotherapy and lay the groundwork for future investigations into these exciting topics. Show more