The collection of bacteria and other microorganisms residing in the gastrointestinal tract, termed the gut microbiota, has emerged as an important target in improving and personalizing oncology treatment. A growing body of research has shown potential roles for specific microbial taxa in the efficacy of cancer treatment, as well as in the development of associated toxicities ^[1][2][3][1,2,3]. The richness of the microbiome has also been investigated, with higher microbial diversity shown to be a key predictor of survival in people having chemoradiation for cervical cancer [4]. A seminal study by Gopalakrishnan et al. [1] clearly showed significant differences in both the diversity and composition of the gut microbiota of people who responded to PD-1 inhibitor immunotherapy for melanoma compared to non-responders. These compositional differences may lead to altered treatment response in a variety of ways, including via changes in direct drug metabolism (for example the action of beta-glucuronidase in the intestinal toxicity of irinotecan [5]), or modulation of the host immune responses ^[6][7][6,7].

With the advent of more sophisticated analytical methods, there is now more appreciation for the functional aspects of the microbiota, including the production of metabolites. Short-chain fatty acids (SCFAs) are one important class of metabolites produced by the gut microbiota. SCFAs, which include butyrate, acetate, propionate, and others (discussed below), are produced in the gut by bacterial fermentation of indigestible fibers and have a variety of functions both in the gut and distal sites including the brain and kidneys [8]. SCFAs have previously been linked to colorectal cancer development, with multiple studies showing that butyrate, propionate, and acetate induce apoptosis in colorectal cancer cells but not in healthy cells. In addition, some studies have suggested that butyrate and propionate have potent anti-neoplastic effects [9]. This has led to suggestions that SCFA manipulation may be a useful preventative or therapeutic strategy in a variety of cancers ^{[9][10][11][12][13]}[9,10,11,12,13].

Results linking the microbiota and cancer treatment outcomes have been promising but have so far failed to provide significant improvements to clinical practice [14]. It is therefore now increasingly important to move beyond simply correlating the abundance of particular bacterial taxa with a disease or phenotype, and to understand how the functional capacity of the microbiota may be critical [15]. Furthermore, in the development of microbial-based therapeutics, there is a strong rationale to investigate metabolites such as SCFAs, rather than bacteria themselves. This is because direct metabolite supplementation would remove the need to ensure microorganisms successfully colonize the host, such is the case with probiotics.

2. Short-Chain Fatty Acids

SCFAs are small organic carboxylic acids with 1 to 6 carbon atoms, and in the intestine, are the main product of anaerobic fermentation of indigestible polysaccharides such as resistant starch, inulin, cellulose, and pectin (Figure 1) [16]. Acetate, propionate, and butyrate are the most commonly produced SCFAs in the human gut, in a roughly 3:1:1 ratio [17]. Other SCFAs include formate, isobutyrate, valerate, isovalerate, and 2-methylbutanoate. SCFAs can move from the gut via the bloodstream in differing amounts. A study using stable isotopes in healthy human subjects found that systemic availability of acetate, propionate, and butyrate was 36%, 9%, and 2%, respectively, [18]. Subsequently, SCFAs have a range of functions both in the gut and elsewhere, with differences in local and systemic effects governed by their systemic availability. These include being a key energy source for colonocytes and playing roles in G-protein coupled receptor (GPCR) binding, histone deacetylation, and immune modulation ^[16][19][16,19]. SCFA quantification in fecal samples is commonly used to quantify SCFA production. This may however not be an accurate measure, as previous research has suggested that a majority of SCFAs produced in the colon are absorbed by the gut mucosa ^[16][20][16,20]. In addition, systemic SCFA levels may be affected by gut epithelial integrity [21]. A wide range of bacteria, many of which have previously been implicated in the efficacy or toxicity of cancer treatment, can produce SCFA, with the amounts and types of SCFA produced dependent on the types of bacteria present. Acetate is the SCFA produced in the highest levels in the gut. This is due to acetate production pathways being widely distributed among multiple types of bacteria, whereas other SCFAs such as butyrate and propionate production is restricted to a small group of bacterial types [22]. The phylum Firmicutes is the main butyrate-producing taxa, particularly Faecalibacterium prausnitzii (F. prausnitzii) (Ruminococcaceae) and Roseburia spp. (Lachnospiraceae). Eubacterium and Coprococcus are also important butyrate-producers [23]. These key butyrate-producing bacteria are generally anaerobes, and therefore flourish in the low-oxygen environment of the colon. In addition, Bifidobacterium species are also able to produce acetate and lactate, and Akkermansia muciniphila, among others, produce propionate and acetate, with most propionate producers in the colon belonging to the Bacteroidota phylum ^[8][16][24][8,16,24]. Luminally, SCFAs serve to acidify their environment, and in doing so restrict the growth of pathogenic microbes by preventing cellular respiration [25]. Similarly, SCFAs such as acetate are potent nutrient sources for other commensal microbes (including butyrate producers), and this cross-feeding mechanism is critical for the maintenance of a healthy and diverse microbial ecosystem. SCFAs are absorbed by colonocytes via hydrogen or sodium-dependent monocarboxylate transporters [19], where much is used as an energy source for these cells. SCFAs, primarily butyrate and propionate, not metabolized within colonocytes are transported into the portal circulation and often then used as an energy source for hepatocytes ^[26][27][26,27]. Butyrate is a particularly important energy source for colonocytes, with previous research showing that colonizing germ-free mice with butyrate-producing bacteria increased oxidative phosphorylation and contained autophagy to normal levels in the gut [28]. Butyrate is also important in stabilizing gut epithelial barrier function, via the consumption of local oxygen molecules and subsequent stabilization of the barrier protecting transcription factor hypoxia-inducible factor (HIF) [29]. Aside from the above-mentioned monocarboxylate transporters, SCFAs can activate G protein-coupled receptors (GPCR), also known as free fatty acid receptors ^[16][30][16,30]. GPR41, GRP43, and GPR109A can be activated by multiple SCFAs and subsequently inhibit the production of cAMP [31]. These GPCRs are expressed on epithelial cells, neutrophils, and macrophages both within and outside of the gut, as well as a variety of other cell types including sympathetic neurons and pancreatic beta cells [32]. The wide range of GPCR sites shows the broad potential for SCFA-mediated effects. Enteroendocrine cells also express these receptors and respond by releasing contents that regulate neuroendocrine pathways controlling local and distant activity. Activities include appetite regulation, glucose homeostasis, and mucosal growth. See van der Hee and Wells [32] for further discussion of these effects. Butyrate and propionate can modulate inflammatory responses in the gut mucosa via histone deacetylase (HDAC) inhibition. Previous research has shown that butyrate can suppress colonic inflammation via HDAC1-dependent Fas upregulation [33]. SCFAs can also alter the immune response in numerous other ways [34]. For example, butyrate can induce Treg differentiation [35] and increase the generation of Th1 and Th17 cells [36]. Microbiota-derived SCFA has also been shown to promote the cellular metabolism of antigen-activated CD8⁺ T cells and enhance their differentiation into long-term memory T cells [37]. Combined, these functions support the central role of SCFAs in the modulation of the immune system. While it is beyond the scope of theise contents paper to describe all individual differences in metabolism and function of different SCFAs, it is important to note that each SCFA has specific functions, which may be important in understanding their role in cancer treatment outcomes. As mentioned above, pathways for acetate production are spread amongst a range of bacterial types, leading to its high production in the gut, of which a relatively high level is able to move into the bloodstream. Acetate also has specific functions systemically, including involvement in lipid synthesis and acetylation reaction [38]. Butyrate is primarily produced via butyryl CoA:acetate CoA transferase pathways, and is one of the most researched SCFAs, due to its important role as an energy source. Propionate is produced via two pathways; the succinate pathways and the propanediol pathway. See Deleu, Machiels [8] for further description of SCFA production pathways. Propionate has also been suggested to have roles in lipogenesis in hepatocytes, as well as having antiproliferative effects in colon cancer cell lines [39].