title: The role of Salmonella Typhimurium and microbiome-derived metabolites in tumour regression
Creators: name: Alfaqeer, Najla Qalit
Publisher: name: University of Glasgow
Year: 2023

description: Cancer is a significant global health challenge, being the second leading cause of death worldwide with nearly 10 million deaths attributed in 2020, a rate of one in six deaths worldwide. Despite the variety of cancer treatments available, most existing treatments are not sufficiently effective to prevent progression of the disease, and many treatment options linked to unpleasant and intolerable long-term or short-term side effects. While surgery, chemotherapy, and radiotherapy form the backbone of most common treatment options, recent advances including targeted immunotherapy and hormonal therapy are improving outcomes and offer opportunities to reduce side effects. In this study, we have considered whether bacteria and bacterial-derived metabolites, offer any potential as therapies in cancer treatment.

The first part of this study aimed to investigate the capacity of S. Typhimurium strains to selectively target and kill cancer cells. To this end, we evaluated the anti-tumour properties of several strains of S. Typhimurium, aiming to identify the strain with the highest activity for further optimization and use in cancer therapy. In terms of motility, virulence, and antitumour efficacy, the wild-type strain SL1344 and its attenuated SL7207 (SL1344ΔaroA), SL1344ΔsipB, SL1344ΔavrA, SL1344ΔsipA, and VV341 strains were evaluated in vitro using a 3D spheroid melanoma model. After infection, three mutant strains, SL1344, ΔavrA, ΔsipA, and VV341, in addition to the double mutant strain ΔaroA/ΔavrA, were highly invasive and suppressed tumour growth, with SL1344 and ΔsipA restricting tumour growth by almost 36% and VV341and ΔavrA by 76%. Interestingly the highest reduction in tumour growth, approximately 89%, was observed when the tissue was infected by the double deletion mutant ΔaroA/ΔavrA.

These results demonstrate that attenuated S. Typhimurium possess anti-tumour activity, which could be further improved to allow these bacteria to be used therapeutically individually or synergetically with other therapies.

The second part of this study focused on the interaction between microbiome metabolites and cancer. Specifically, we examined two microbiome-derived metabolites produced by bacteria of the Lachnospiraceae family. These compounds 4-(trimethylammonio) pentanoate (4-TMAP), and 3-methyl-4-(trimethylammonio) butanoate (3M-4-TMAB) are thought to interfere with fatty acid β oxidation pathway within the mitochondria. Using these metabolites to induce mitochondrial dysfunction and block fatty acid oxidation, offers an opportunity to suppress tumour growth and prevent cancer progression. To test our hypothesis, three cancer cell lines were selected, which are highly dependent on fatty acid oxidation for progression and metastasis: breast cancer (BT549), prostate cancer (PC-3) and the melanoma cell line B16F10 which has previously been used to evaluate anti-cancer activity in Salmonella. We evaluated the tumour suppressive activity of the metabolites, 4-TMAP and 3M-4-TMAB separately and in combination with conventional tumour drug treatments (Docetaxel and MCL-1 inhibitor) using cell viability and the ability of cancer cells to proliferate and produce colonies, as readouts for efficacy. This showed that the metabolites some suppressed efficacy on cell viability when used as individual agents compared with the conventional drug treatments Docetaxel and Mcl-1 inhibitor (which were very efficacious showing a dramatic reduction of viability by 50% and 48% by Docetaxel and MCL-1 respectively). Furthermore, the sensitivity of tumour colony formation to metabolites varied between cell lines. However, 4-TMAP provided the highest inhibition percentage (41 %) in BT549 and B16F10 cells, while 3M-4-TMAB showed the highest inhibition (40 %) in PC-3 cells. In terms of cell metastasis evaluation, our results demonstrated that 4-TMAP and 3M-4-TMAB suppress tumour growth and cell migration of BT549 and B16F10 cells but did not prevent PC-3 migration. Our results, which examined the influence of metabolites on ATP production in the absence of glucose, showed an approximately 45% inhibition with 3M-4-TMAB in all cell lines.

Finally, a metabolomics analysis study of the impact of treatment of the breast cancer cell line BT549 with 3M-4-TMAB and 4-TMAP demonstrated downregulation of several compounds related to carnitine compared to control. However, it was challenging using this data to determine the specific pathways affected by these treatments. In contrast, RNA-Seq data generated from the same cell line treated with the same metabolites revealed differential gene expression in genes that were mostly related to immune activity. Interestingly, CXCL1, a gene implicated in promoting tumour progression and metastasis, was downregulated after 3M-4TMAB treatment.

Collectively, our results highlight a potential therapeutic application for targeting tumours with two different approaches; utilizing S. Typhimurium attenuated strains on melanoma and metabolites derived from Lachnospiracea family on three different cancer cell lines BT-549, PC-3, and B16F10.