Abstract

Immunotherapy is a re-emerging alternative to other invasive and debilitating cancer treatments. Monoclonal antibodies that disrupt immune checkpoint molecules, such as PD1 or CTLA4, continually demonstrate great promise. Intratumoral injections of Complete Freund’s Adjuvant (CFA) – a water-in-oil emulsion containing heat-killed mycobacteria – have also been successful in several animal models and are well tolerated by cancer patients. However, there are ongoing challenges in improving response rates, and unanswered questions remain regarding how checkpoint therapies convey a therapeutic benefit. One of the most puzzling is how the gut microbiome affects treatment outcomes. This PhD project aimed to investigate the impact of the gut microbiome on immune checkpoint therapies and to improve the efficacy our laboratory’s intratumoral CFA therapy.

Research to accomplish both aims was conducted at the Australian National University (ANU) and the Stanford University School of Medicine. We used several mouse cancer models and a tumour sampling method developed by our laboratory called fine-needle aspirate (FNA) sampling. This method allowed us to sample subcutaneous tumours to identify differences in tumour-infiltrating leukocyte (TIL) composition among animals with disparate treatment outcomes. We also used this method to investigate the gut microbiome’s effect on immune reactions following cancer treatments in mice fed antibiotics. Finally, we investigated two methods of improving CFA therapeutic efficacy: combining the treatment with systemic immune checkpoint therapies and increasing the concentration of heat-killed mycobacteria in the emulsion ten-fold (10x CFA).

We found that the P815 mastocytoma and CT26 colorectal cancer models responded well to anti-CTLA4 therapy but not anti-PD1. FNA sampling of P815-challenged mice responsive to anti-CTLA4 showed a significant increase in CD8+ T cells by day 3 post-treatment and CD4+ T cells by day 5. By day 11, the tumours of CT26-challenged animals responsive to anti-CTLA4 presented significantly increased CD8+ T cells and decreased macrophages/monocytes. Importantly, when investigating the effects of antibiotics on treatment outcomes, we found that the BALB/c and DBA/2 mouse strains do not tolerate an antibiotic cocktail used in several high-impact publications. Furthermore, while gut microbiome depletion did not impact anti-CTLA4 treatment in the CT26 model, we did find evidence suggesting a significantly impacted lymphocyte infiltration into the tumour.

Due to uncontrolled external factors, we could not progress the research as planned. Nonetheless, we successfully conducted preliminary experiments investigating methods of improving CFA therapy. We highlight a critical unanswered question regarding the impact of combining CFA with immune checkpoint therapies on metastatic disease progression in the 4T1 breast cancer model. We additionally found that 10x CFA significantly increased immune infiltration (primarily neutrophils) into the CT26 tumour, an effect not observed with standard 1x CFA.

Collectively, these findings highlight ongoing challenges and unanswered questions in the field of immunotherapy, further illustrate the potential of our laboratory’s novel CFA therapy as a cancer treatment option, and lay the groundwork for future investigations into these important topics.