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.

Trinley: Results

Gharaza: Results

Eesha: Discussion/Future Outlook

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

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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.

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

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

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.

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

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?

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)

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.

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'.

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)

• 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)

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

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.

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.