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The study establishes that the anti-tumour effects of a caloric restrictive (CR) diet, but not intermediate fasting (IF), is because of the changes in the gut microbiome. The increase of Bifidobacterium, a microbial species, and its corresponding supernatant, acetate, in the gut modulate the proliferation of CD8+ T cells in a tumor microenvironment. Balmer et al. (2016) suggests that the increase of serum acetate initiates post-translation modification in energy metabolism and enhances CD8(+) T cell production. Thus, it the effector molecule produced by a gut bacterium that is meditating this effect. This is relevant in four ways:

1.     Cancer patients usually unintentionally have CR diets because their condition alters their physical and mental reaction to food, e.g nausea, lack of hunger, increases sensitivity to certain types of food. However, not all these patients are experiencing this anti-tumour effect associated with a CR diet. Nonetheless, the researchers have isolated the effector molecule and treatment in those patients that were previously unresponsive to the diet can now benefit from its effects.

2.     Acetate did reduce tumour infract size and increases the number of CD8(+) immune cells, but it cannot be concluded that it eliminated the cancer. Hence, acetate supplementation for cancer patients can be utilized alongside current effective cancer therapies to boost success rate.

3.     The oral administration of acetate (200 uL) proved to be effective in reducing tumour infract size, indicating an accessible route of administration for treatment.

4.     The authors tested the effect on other cancer cell lines, i.e breast and lung cancer models, and concluded similar, positive results. Thus, opening a therapeutic avenue for a non-specific, board spectrum treatment to target an array of cancer types. Other publications (Miller et al., 2023, Jin et al., 2023, Comerford et al., 2014) regarding acetate and different cancer types conclud similar results with a shared mechanism of acetate impacting energy metabolism.

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Link between consumption and risk of cancer

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Link between consumption and risk of cancer

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Research has shown there is a link between consumption and the risk of cancer.

overconsumption increases the risk of cancer progression (Mao et al. 2023)

healthy diet and lifestyle reduce the risk of cancer (Zhang et al. 2022)

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Calorie Restriction (CR): a 500-750 kcal daily energy deficiency or about 30% decrease from baseline (Lien and Heiden 2019).

Intermittent Fasting (IF): limiting energy intake to specific times or taking extended gaps between meals (Lien and Heiden 2019).

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Rationale: CR/IF and Anti-tumour effects

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Rationale: CR/IF and Anti-tumour effects

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Both CR and IF have been shown to have anti-tumour effects.

CR:

tumour progression

increase cancer cell apoptosis

increased response to treatments such as chemo and radiotherapy

(Alidadi et al. 2021)

IF:

increased lymphoid progenitor cells

increased cytotoxic CD8+ tumour-infiltrating lymphocytes (TILs)

(Di Biase et al. 2016)

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Both CR and IF modulate the gut microbiota.

CR:

increases phylotypes associated with increased lifespan

decreases phylotypes associated with reduced lifespan

(Zhang et al. 2013)

IF:

leads to an elevation of acetate and lactate (fermentation products)

(Li et al. 2017)

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The gut microbiome differs between cancer patients and can affect the efficacy of cancer treatments.

composition of the gut microbiome differs between colorectal cancer patients and healthy controls (Wang et al. 2012)

commensal microbiome promotes the efficacy of checkpoint inhibitor immunotherapies (Gopalakrishnan et al. 2018)

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Does the gut microbiota mediate the anti-tumour effects observed after CR and IR?

What is the underlying mechanism behind these observed anti-tumour effects?

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To determine whether the gut microbiome mediates the anti-tumour effects of CR or IF.

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Modified from Figure 1. (B) Tumour growth kinetics of mice inoculated with MC38 cells: fed ad libitum (AL); under CR; under IF; antibiotics + fed ad libitum; under CR + antibiotics; under IF + antibiotics. (C) Images of dissected tumours and final tumour weights of mice in each treatment group on day 35.

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Mice treated with CR and IF suppressed tumour growth, compared with mice fed ad libitum.

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Mice treated with CR and IF suppressed tumour growth, compared with mice fed ad libitum.

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C57BL/6J and BALB/c mice were divided into six groups to evaluate tumour growth: ad libitum, CR, IF, antibiotics + ad libitum, antibiotics + CR, and antibiotics + IF

Broad spectrum oral antibiotics were fed to the mice 5 days before tumour inoculation with MC38 cells.

Mice in the CR and IF groups had significantly more suppressed tumour growth kinetics, significantly lowered tumour volume, and significantly lowered tumour weight compared to mice fed ad libitum.

When treated with antibiotics, the anti-tumour effects were significantly reduced in the CR-treated mice.

This is seen in the significantly increased tumour volume and weight, compared to the CR-treated mice without antibiotics.

This effect was not observed in IF-treated mice.

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Modified from Figure 1. (E) MC38 tumour growth kinetics of mice that received FMT treatment from donor mice in various feeding groups: FMT from mice fed ad libitum (AL); FMT from mice under CR; FMT from mice under IF. (F) Images of dissected tumours and final tumour weights of mice at the end of the FMT treatments. 

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FMT from mice treated with CR significantly reduced tumour volumes and final tumour weights.

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FMT from mice treated with CR significantly reduced tumour volumes and final tumour weights.

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Specific pathogen-free mice were pre-treated with antibiotics for 5 days to further deplete their gut microbiota.

Mice received FMT twice a week from donor mice who underwent ad libitum, CR (for 4 weeks), or IF (for 4 weeks) treatment to assess the dependency of the gut microbiota.

After 7 days, they were inoculated with MC38 cells.

Tumour growth kinetics were significantly suppressed when mice received FMT from donor mice who underwent CR treatment.

The tumour volumes and final tumour weights of mice receiving FMT from donor mice who underwent CR treatment were significantly reduced, compared to those in the FMT from donor mice who underwent IF or were fed ad libitum.

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Modified from Figure 1. (G) MC38 tumour growth kinetics in germ-free mice fed ad libitum, under CR, and under IF. (H) Images of dissected tumours and final tumour weights of germ-free mice: fed ad libitum; under CR; under IF.

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CR suppressed tumour growth in a gut microbiota-dependent manner.

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CR suppressed tumour growth in a gut microbiota-dependent manner.

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Germ-free mice were fed ad libitum or underwent either CR or IF treatment to confirm that the anti-tumour effects of CR were gut microbiota-dependent.

In a microbiota-free environment, CR lost anti-tumour effects, while IF still suppressed tumour growth, and decreased tumour volume and tumour weights.

This suggested that the anti-tumour effects of CR is dependent on the presence of gut microbiota to reduce tumour growth.

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Modified from Extended Data Figure 2. (E) Heatmap analysis showing changes in expression of genus levels of related gut microbiota in mice under ad libitum, CR, and IF treatment. Modified from Figure 2. (E) LDA analysis conducted to identify the differentially abundant bacteria between the gut microbiota of mice under CR and IF. (F) T-test conducted to identify the differentially abundant bacteria between the gut microbiota of mice under CR and IF.

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Bifidobacterium and Lactobacillus genera over-represented in mice treated with CR.

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Bifidobacterium and Lactobacillus genera over-represented in mice treated with CR.

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Fecal matter from mice treated with CR, IF, or fed ad libitum were collected to conduct 16S rRNA sequence analysis.

Linear discriminant analysis were conducted to separate between different bacterial groups in various treatments based on a linear combination of their features.

Heatmap analysis was conducted to compare microbial expression between treatment groups.

The heatmap revealed over-representation of lactobacillus and bifidobacterium in mice treated with CR.

LDA analysis and t-test further highlighted the Bifidobacterium genus as the most significantly differentiated between CR and IF treatment groups.

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Modified from Figure 3. (B) MC38 tumour growth kinetics of mice treated with five Bifidobacterium bacterial species. (E) MC38 tumour growth kinetics of mice fed ad libitum and mice fed ad libitum supplemented with B. bifidum. (F) Images of dissected tumours and final tumour weights of mice fed ad libitum and mice fed ad libitum supplemented with B. bifidum.

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Supplementation of Bifidobacterium bifidum species significantly suppressed tumour volume in antibiotics-treated mice.

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Supplementation of Bifidobacterium bifidum species significantly suppressed tumour volume in antibiotics-treated mice.

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Specific pathogen-free mice were pre-treated with antibiotics, then were administered with PBS (control) or 5 isolated bifidobacterium species from a healthy donor mice.

Specifically, the 5 species were B. longum, B. breve, B. infantis, B. actobacillus, and B. bifidum.

The mice were inoculated with MC38 cells 7 days later and placed under CR treatment to observe its anti--tumour effects.

Supplementation with B. bifidum had the most significantly reduced tumour volume in microbiome-depleted mice.

B. bifidum was also supplemented in mice fed ad libitum, where significant reduction in tumour volume and weight compared to mice fed ad libitum without B. bifidum supplementation.

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Modified from Figure 4. (A) Tumour growth kinetics of mice injected with 4T1 cancer cells: fed ad libitum (AL); under CR; under IF; antibiotics + fed ad libitum; under CR + antibiotics; under IF + antibiotics. (B) Images of dissected primary 4T1 tumours of each group. (C) Final tumour weights of mice in each treatment group on day 30.

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CR and B. bifidum treatment significantly reduced 4T1 primary tumour growth.

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CR and B. bifidum treatment significantly reduced 4T1 primary tumour growth.

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Anti-tumour effects of CR and B. bifidum were further tested in the 4T1 breast cancer cell line mouse model.

BALB/c mice were injected with 4T1 cancer cells in the mammary glands.

Mice under CR and mice under antibiotics + CR + B.bifidum supplementation had significantly reduced tumour growth, volume, and weight compared to mice fed ad libitum.

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Modified from Figure 4. (D) Top, images of India ink-stained lungs from mice in each treatment, with lung metastases shown as white masses. Bottom, images of H&E staining of the lungs. (E) Total number of metastases in mice for each treatment. (F) Percentage of lung metastatic area in mice for each treatment.

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Mice treated with CR or CR plus B. bifidum supplementation had significantly reduced metastatic nodules in the lungs.

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Mice treated with CR or CR plus B. bifidum supplementation had significantly reduced metastatic nodules in the lungs.

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To simulate metastases in breast cancer, 4T1 cancer cells were injected via the tail vein into mice treated with antibiotics or antibiotics plus B. bifidum supplementation.

This was followed by CR treatment on day 5.

Mice under CR or CR plus B. bifidum treatment had significantly decreased number of lung metastatic nodules.

This further suggested B. bifidum's anti-tumour effects in CR-treated mice.

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Modified from Figure 5. (A) Heat map represents the differences in immune cell and cytokine profiles. (C)  IFNγ+CD8+ T cells in tumours were analyzed after 4 weeks of dietary treatment in all groups. (E) IFNγ+CD8+ T cells in tumours were analyzed after FMT transplantation from all mice groups.

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CR increases IFNγ+CD8+ T cells in a gut microbiota-dependent manner

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CR increases IFNγ+CD8+ T cells in a gut microbiota-dependent manner

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Analysis of immune cells was done to explore how CR treatment exerts its antitumour effects.

An increase in IFNγ+CD8+ T cells is indicative of an inflamed tumour that has active CD8+ T cells executing their effector function against tumour cells.

Not only did the abundance of IFNγ+CD8+ T cells increase under a CR diet, but also in donor mice that received an FMT from a CR donor

Overall, these results indicate the antitumour effects of CR via accumulation of IFNγ+CD8+ T cells

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Modified from Figure 6. (B) Growth of MC38 tumours in all groups. (C) Image of dissected tumours. (D) Tumour weights on day 32 from all groups

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CD8+ T cells have a critical role in the B. bifidum-mediated anti-tumour effect of CR

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CD8+ T cells have a critical role in the B. bifidum-mediated anti-tumour effect of CR

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Intraperitoneal injection of anti-CD8 antibodies was performed in all groups to determine whether CD8+ T cells were involved in the antitumour effects of B.bifidum.

Anti-CD8 treatment groups saw faster tumour growth and higher tumour volumes compared to untreated controls.

Anti-CD8 treatment significantly increased tumour weights and size.

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Modified from Figure 7. (B) Growth curve of MC38 tumours. (D) Tumour weight across groups. (F) Percentage of tumour-infiltrating IFNy+ CD8+ T cells in tumour tissues

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B. bifidum supernatant facilitates antitumour effects of CR 

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B. bifidum supernatant facilitates antitumour effects of CR 

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To test whether metabolites from the gut microbiota can improve antitumour effects a supernatant of B.bifidum was collected to supplement the supernatant of ABX + CR treated mice

B.bifidum supernatant alone produced similar antitumour effects as B.bifidum bacteria where a reduction in tumour volume in weight was noted

The high expression of INFy+ CD8+ T cells is indicative of the supernatant's ability to facilitate antitumour effects by enhancing the presence of TILs in the tumour microenvironment

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Modified from Figure 8. (D) MC38 tumour growth kinetics. (E) Dissected tumours and their weights on day 30. (F) Flow cytometry plots for one mouse per group following acetate treatment. (G) Percentage of tumour-infiltrating IFNy+ CD8+ T cells in tumours following acetate treatment. 

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Acetate facilitates the antitumour effect of CR

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Acetate facilitates the antitumour effect of CR

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The microbiome has shown to influence host immune responses by producing SCFA's

Quantification of SCFA's was done to identify the specific components of the B.bifidum supernatant

Acetate supplementation in CR treated mice, without gut microbiota had the greatest reduction in tumour volume and tumour weight

Suggests that acetate alone was sufficient in rescuing the antitumour effects of CR

Flow cytometry shows that acetate treatment had a significant increase in the percentage of IFNy+CD8+ T cells.

Suggests it’s critical role in CR-mediated antitumour effects through accumulation of TILs in the tumour microenvironment

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There are ongoing debates in current literature regarding the effectiveness of CR and IF diets, and which one has a stronger effect size. Most paper agree that the timing and quantity of food consumption elicit some effect on tumour growth. Both CR and IF have different mechanisms of action, as also supported by this paper, hence their effectiveness would be dependent on how those mechanisms elicit anti-tumor effects. Additionally, the microbiome composition can drastically vary in one day, and thus the timing of food consumption can impact the effect size fo the diets. In this paper, despite the focus on CR, IF did have a slightly higher effect size than CR. Despite the conclusion of the results, the lack of comparison to other treatments prevents perspective on the applicability of the treatment in real world applications. Nonetheless, the consensus in literature tends to favour the use of the diets to enhance patients’ effectiveness and tolerability to chemotherapy and other cancer treatments.

Moreover, the quality of the food is crucial in a diet. The mice were given the same chow in both CR and IF diets. However, this diet lacks to consider how nutrition is a key factor in both diet and cancer prevention. A review by Lee et al. (2023) discusses that the lack of nutritional requirements consideration in chow, and not just caloric intake, can have effects on metabolism related and physiological related processes. Hence, affecting the reproductivity and applicability of the results.

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The experimental design can be broken into two main study objectives: isolating the for the bacteria that elicits the anti-tumour effects of CR diets and the physiological effect of the bacteria.

Both Objectives:

Strengths:

The authors conducted numerous experiments with an array of comparison and sub-comparison groups. For instance, to association between CR diets and microbiome, mice were divided into both germ-free mice and ABX treated mice. Both methods are used to remove microbiome but could potentially elicit different effects. However, the same result occurred in both groups. Hence, the authors did an excellent job at controlling for confounds and had a significant amount of validation groups and measurements.

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Objective: Isolate Bacteria

Weaknesses:

While the authors did use a weighted alpha measurement to isolate the bacterial species of interest, investigating beta diversity would have provided a more comprehensive view on the dynamic changes in gut composition. They used temporal differences between the alpha diversity in samples. However, this lacks to account for the intersectional composition of the whole gut microbiome and fails to consider factors such as how other bacterial species can support or eliminate certain functions.

Additionally, the compromised gut microbiome prior to injections with the cancerous cell line had weakened immune system. The gut microbiome changes and develops with age alongside the immune system (Franceschi et al., 2018). The immune system initially was already in a weakened state before receiving cancer. Therefore, the effect size of the treatment may not be truly representative of exerting anti-tumour effects.

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Immune floe cytometry cannot capture the whole picture 

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Objective: Assessing Physiological Effect

Weakness:

While the authors used immune flow cytometry to concluded an increase in the CD8(+) T cell population, it does not provide insight on the morphological interaction between the acetate and the immune cell population. Further information on the cell marker interaction with acetate could provide insight on the tumor mediated activated pathway in CD8(+) T cells. Thus, demonstrating a clear communication link between the gut and the immune system.

Also, the literature asserts that acetate has a role in energy metabolism, hence it would have been beneficial for the authors to explore how acetate may cause a surplus in potentially related metabolites, such as acetyl co-A. This link to metabolism could provide insight on how the diet causes the growth of the bacteria, and if its causes of the competition in the environment or a nutritional advantage.

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The three main challenges discussed below

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A key challenge in targeting the gut microbiome for treatment is the consideration of the diverse host communicated located in the gut. There are a variety of species that have both positive and negative effects on cancer treatment. Some have a negative metabolic consequence on the cancer treatments, which would support the use of antibiotics boost the immune response. However, that would eliminate strains such as Bifidobacterium that have a positive effect. Nonetheless, because the authors concluded that the supernatant is as effective as the inclusion of the bacteria, perhaps a combinational approach would allow us to maximize the best of both worlds.

Another challenge to consider is the applicability to humans, as the human diet is vast and diverse. Even if there are calorie restrictions, the nutritional value of food is equally as important. Hence, if this were to have real world implications, it is important to consider how bacteria would continue to flourish in different type of CR diets.

Moreover, what are the ethical implications of concluding that CR diet can cause anti-tumour effects? Could this give rise to misunderstanding in individuals about the true effect size and cause implicit harm. Why chose dangerous chemotherapy, if an easily accessible CR diet that seems 'safer'.

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Visual depiction of two important variables to consider before applying to human clinical trials

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Experiments should aim to administer varying dosages of acetate and observe if there is a correlative effect between dosage and cancer markers, such as tumor infarct size or CD8(+) T cells.

Moreover, experiments should aim to administer acetate at different time points in cancer development to determine if the treatment effects remains at different cancer stages.

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Sabeeka: Introduction

Trinley: Results

Gharaza: Results

Eesha: Discussion/Future Outlook

it was amazing to work with such hardworking and dedicated peers! :)

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Alidadi M, Banach M, Guest PC, Bo S, Jamialahmadi T, Sahebkar A. The effect of caloric restriction and fasting on cancer. Seminars in Cancer Biology. 2021;73:30–44. doi:10.1016/j.semcancer.2020.09.010.

Balmer ML, Ma EH, Bantug GR, Grählert J, Pfister S, Glatter T, Jauch A, Dimeloe S, Slack E, Dehio P, Krzyzaniak MA, King CG, Burgener AV, Fischer M, Develioglu L, Belle R, Recher M, Bonilla WV, Macpherson AJ, Hapfelmeier S, Jones RG, Hess C. Memory CD8(+) T Cells Require Increased Concentrations of Acetate Induced by Stress for Optimal Function. Immunity. 2016 Jun 21;44(6):1312-24.

Clifton KK, Ma CX, Fontana L, Peterson LL. Intermittent fasting in the prevention and treatment of cancer. CA Cancer J Clin. 2021 Nov;71(6):527-546.

Comerford SA, Huang Z, Du X, Wang Y, Cai L, Witkiewicz AK, Walters H, Tantawy MN, Fu A, Manning HC, Horton JD, Hammer RE, McKnight SL, Tu BP. Acetate dependence of tumors. Cell. 2014 Dec 18;159(7):1591-602.

Di Biase S, Lee C, Brandhorst S, Manes B, Buono R, Cheng C-W, Cacciottolo M, Martin-Montalvo A, de Cabo R, Wei M, et al. Fasting-Mimicking Diet Reduces HO-1 to Promote T Cell-Mediated Tumor Cytotoxicity. Cancer Cell. 2016;30(1):136–146. doi:10.1016/j.ccell.2016.06.005

Franceschi C, Garagnani P, Parini P, Giuliani C, Santoro A. Inflammaging: a new immune-metabolic viewpoint for age-related diseases. Nat Rev Endocrinol. 2018 Oct;14(10):576-590.

Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews MC, Karpinets TV, Prieto PA, Vicente D, Hoffman K, Wei SC, et al. Gut microbiome modulates response to anti–PD-1 immunotherapy in melanoma patients. Science. 2018;359(6371):97–103. doi:10.1126/science.aan4236.

Jin F, Ni X, Yu S, Jiang X, Shi X, Zhou J, Mao D, Wang H, Liu Y, Wu F. The Ethyl acetate extract from Celastrus orbiculatus suppresses non-small-cell lung cancer by activating Hippo signaling and inhibiting YAP nuclear translocation. Phytomedicine. 2023 Jun;114:154761.

Lee J, Purello C, Booth SL, Bennett B, Wiley CD, Korstanje R. Chow diet in mouse aging studies: nothing regular about it. Geroscience. 2023 Jun;45(3):2079-2084.

Lien EC, Vander Heiden MG. A framework for examining how diet impacts tumour metabolism. Nature Reviews Cancer. 2019;19(11):651–661. doi:10.1038/s41568-019-0198-5.

Li G, Xie C, Lu S, Nichols RG, Tian Y, Li L, Patel D, Ma Y, Brocker CN, Yan T, et al. Intermittent Fasting Promotes White Adipose Browning and Decreases Obesity by Shaping the Gut Microbiota. Cell Metabolism. 2017;26(4):672-685.e4. doi:10.1016/j.cmet.2017.08.019.

Mao Y-Q, Huang J-T, Zhang S-L, Kong C, Li Z-M, Jing H, Chen H-L, Kong C-Y, Huang S-H, Cai P-R, et al. The antitumour effects of caloric restriction are mediated by the gut microbiome. Nature Metabolism. 2023;5(1):96–110. doi:10.1038/s42255-022-00716-4.

Miller KD, O'Connor S, Pniewski KA, Kannan T, Acosta R, Mirji G, Papp S, Hulse M, Mukha D, Hlavaty SI, Salcido KN, Bertolazzi F, Srikanth YVV, Zhao S, Wellen KE, Shinde RS, Claiborne DT, Kossenkov A, Salvino JM, Schug ZT. Acetate acts as a metabolic immunomodulator by bolstering T-cell effector function and potentiating antitumor immunity in breast cancer. Nat Cancer. 2023 Oct;4(10):1491-1507.

Mittelman SD. The Role of Diet in Cancer Prevention and Chemotherapy Efficacy. Annu Rev Nutr. 2020 Sep 23;40:273-297.

Thanikachalam K, Khan G. Colorectal Cancer and Nutrition. Nutrients. 2019;11(1):164. doi:10.3390/nu11010164.

Wang T, Cai G, Qiu Y, Fei N, Zhang M, Pang X, Jia W, Cai S, Zhao L. Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers. The ISME Journal. 2012;6(2):320–329. doi:10.1038/ismej.2011.109.

Zhang C, Li S, Yang L, Huang P, Li W, Wang S, Zhao G, Zhang M, Pang X, Yan Z, et al. Structural modulation of gut microbiota in life-long calorie-restricted mice. Nature Communications. 2013;4(1):2163. doi:10.1038/ncomms3163.

Zhang Q, Zhang C, Wang H, Ma Z, Liu D, Guan X, Liu Y, Fu Y, Cui M, Dong J. Intermittent Fasting versus Continuous Calorie Restriction: Which Is Better for Weight Loss? Nutrients. 2022;14(9):1781. doi:10.3390/nu14091781.

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The important part before applying to a clinical setting, is to gain a holistic understanding on the pharmacokinetics and the efficacy of the potential therapeutic. For instance, considering the minimum amount of acetate required to elicit the treatment effect.

Additionally, considering when would the supernatant be most effective, i.e timewise. CR diets in literature are often stated as a preventive measure rather than a treatment in cancer patients, however, does that also apply to acetate? Can acetate have a larger therapeutic window of action, as a supplement to current cancer therapies?