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Nature Sub-Journal: Calorie restriction wins!

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Early studies have shown that short-term very low calorie intake, including short-term fasting (2-4 days) or dietary control of specific macronutrients, can effectively delay the growth of primary tumors
.

On the contrary, excessive consumption of animal protein will increase the risk of cancer and all-cause mortality

However, it is still unclear whether fasting time, dietary restriction levels or nutritional content are the main factors in preventing cancer
.

This study evaluated the relative effects of diet composition and low calorie intake on delaying tumor growth in a mouse model of 4T1 breast cancer

However, it is still unclear whether fasting time, dietary restriction levels or nutritional content are the main factors in preventing cancer

Low-calorie circulation slows down tumor growth and has nothing to do with diet composition

1.
Low-calorie circulation slows down tumor growth and has nothing to do with diet composition

In 16-week-old BALB/cJ female mice, the effects of diet composition and 4:10 feeding cycle on the growth rate of triple-negative breast cancer (TNBC) were studied.

The mice were implanted with homologous and highly metastatic mice in the breast.
Murine 4T1 cancer cells

In 16-week-old BALB/cJ female mice, the effects of diet composition and 4:10 feeding cycle on the growth rate of triple-negative breast cancer (TNBC) were studied.

2.
Therapeutic CR intervention can slow down tumor growth more than calorie circulation

2.
Therapeutic CR intervention can slow down tumor growth more than caloric circulation 2.

Previous studies have proven that CR is effective in delaying the growth of breast tumors
.

We tried to compare whether FMD and LCC of 4:10 cycles are as effective as daily CR

Based on the average calorie intake per 4:10 cycle of FMD/LCC, it is about 20% lower than AL

When the mice were sacrificed (the 3rd day of the second FMD/LCC cycle and then fed, that is, the 21st day of the CR cycle), various metabolic indexes were evaluated

In fact, CR mice were in a state of calorie restriction before being sacrificed, while FMD and LCC mice were sacrificed on the third day of the second refeeding period

Therefore, the increased liver mass of FMD and LCC mice (Figure 2h), and the peak of metabolic markers at the beginning of the refeeding period, may be caused by the 4-day extremely low caloric intake (70%) of FMD and LCC mice.

It is known that tumor progression is positively correlated with the promotion of splenic accumulation (splenomegaly) of tumor immune cells
.
The spleen quality of mice was significantly reduced during the daily CR and FMD or LCC cycles (Figure 21)
.
Immunity We speculate that the decrease in tumor growth may be due to the decrease in the number of immune regulatory cells that promote tumor growth
.
Immunohistochemistry ( Figure 2m) showed that no dietary intervention can reduce tumor viability
.
Therefore, regardless of dietary composition, reduced calorie intake and/or longer fasting time appear to be key drivers of the decline in tumor growth
.
Regardless of the dietary composition, reduced calorie intake and/or longer fasting periods appear to be key drivers of the decline in tumor growth
.
However, daily CR is more effective than 4:10 cycles of FMD and LCC in delaying 4T1 tumorigenesis in the body, even with a similar degree of calorie restriction
.
However, daily CR is more effective than 4:10 cycles of FMD and LCC in delaying 4T1 tumorigenesis in the body, even when receiving a similar degree of calorie restriction

3.
The preventive and therapeutic effects of CR in delaying tumor growth

3.
The preventive and therapeutic effects of CR in delaying tumor growth 3.
The preventive and therapeutic effects of CR in delaying tumor growth

To determine whether preventive CR before 4T1 implantation is more advantageous than therapeutic CR in delaying 4T1 tumor growth, an additional treatment group was included in the study shown in Figure 2 (see supplementary figure for experimental design 3a)
.
The results showed that CR was equally effective in delaying the growth of primary tumors (Supplementary Figure 3m) and reduced the overall quality of the primary tumor (Supplementary Figure 3o) and the quality of the spleen (Supplementary Figure 3p) , whether initiated before or after 4T1 cell implantation .
)
.
However, in the two groups of tumor-bearing mice, the primary tumor viability quantified by H&E-stained tumor sections did not decrease due to daily CR (Supplementary Figure 3q)
.

An additional treatment group was included in the study shown in Figure 2 (see Supplementary Figure 3a for the experimental design)
.
The results show that the results show , or both before and after starting 4T1 cell implantation, CR delay in primary tumor growth as effective in two tumor-bearing mice, the tumor sections quantification H & E stained primary tumor activity has not been The daily CR decreased (Supplementary Figure 3q)
.

4.
The preventive and therapeutic effects of CR in reducing metastatic burden

Next, we evaluated the impact of these dietary interventions on the metastasis and spread of 4T1 cells
.
Orthotic implantation of 4T1 cancer cells into the breast of BALB/cJ is easy to metastasize to various parts.
It provides a useful in vivo model to evaluate whether the daily CR or FMD and LCC cycle programs can delay the development and progression of lung metastasis
.
After 28 days of stimulation of the 4T1 tumor, the lung metastatic nodules of the mice undergoing CR daily (before or after 4T1 implantation) were significantly reduced (Figure 3a, b)
.
Surprisingly, the FMD and LCC regimens did not bring such benefits, because compared with the AL control group, there was no significant difference in metastases in the lungs of FMD and LCC mice (Figure 3a, b)
.
The lungs of CR (4T1 before or after implantation) mice (compared to FMD or LCC) have fewer macroscopically visible large metastases (>1.
5 mm in size) (Figure 3c) , which shows that it is more significant The metastatic growth
.
In the H&E-stained lung slices, only the daily CR (before and after 4t1 implantation) group had a significant reduction in metastases (Figure 3d)
.
In conclusion, CR has preventive and therapeutic effects on the growth and spread of TNBC mouse models
.

It is easy to transfer to various parts and provides a useful in vivo model to evaluate whether the daily CR or FMD and LCC cycle program can delay the development and progression of lung metastasis
.
After 28 days of stimulation of the 4T1 tumor, the lung metastatic nodules of the mice undergoing CR daily (before or after 4T1 implantation) were significantly reduced (Figure 3a, b)
.
Metastases in the lungs (Figure 3a, b)
.
There are fewer large metastases visible to the naked eye (Figure 3c).
In the H&E-stained lung slices, only the daily CR (4t1 before and after implantation) group had a significant reduction in metastases (Figure 3d)
.
In conclusion, CR has preventive and therapeutic effects on the growth and spread of TNBC mouse models
.
CR has preventive and therapeutic effects on the growth and spread of TNBC mouse model

5.
Preventive CR can slow down tumor growth more than calorie circulation

5.
Preventive CR can slow down tumor growth better than caloric circulation .
Preventive CR can slow down tumor growth better than caloric circulation.

Compared with the AL control group and the preventive CR group, the average body weight (Figure 4b) and caloric intake (Figure 4c) of the mice in the caloric circulation group also showed similar declines.
No lean body mass or fat was observed in either CR or caloric circulation.
Significant loss of quantity (Supplementary Figure 1c-e)
.

Metabolic markers measured at sacrifice (day 3 of the fourth FMD/LCC cycle and refeeding on day 49 of CR) showed that the glucose level of CR mice decreased significantly every day (Figure 4d)
.
No differences in insulin levels and HOMA2-IR index were observed between the treatment group and the AL control group (Figure 4e, f), although IGF-1 levels were significantly reduced in FMD and daily cr-fed mice (Figure 4g)
.
The FMD regimen resulted in a significant increase in liver weight (Figure 4h), while the FMD and LCC regimens increased kidney weight (Supplementary Figure 4f)
.
The results showed that before 4T1 implantation, the caloric cycling mice (FMD and LCC) had a metabolic "start", allowing enough time for the metabolism to adapt to severe caloric restriction and AL refeeding cycles
.

Metabolic markers measured at sacrifice (day 3 of the fourth FMD/LCC cycle and refeeding on day 49 of CR) showed that the glucose level of CR mice decreased significantly every day (Figure 4d)
.
No insulin levels were observed between the treatment group and the AL control group (Figure 4e, f), although IGF-1 levels were significantly reduced in FMD and daily cr-fed mice (Figure 4g)
.
The FMD regimen resulted in a significant increase in liver weight (Figure 4h), while the FMD and LCC regimens increased kidney weight (Supplementary Figure 4f)
.
The results showed that before 4T1 implantation, the caloric cycling mice (FMD and LCC) had a metabolic "start", allowing enough time for the metabolism to adapt to severe caloric restriction and AL refeeding cycles
.

Before 4T1 implantation, all interventions resulted in a decrease in tumor growth, especially in mice fed cr-fed daily (Figure 4i, j), tumor volume (Figure 4k) and spleen weight (Figure 4l) were simultaneously reduced
.
Daily CR is the only intervention that can significantly reduce the burden of lung metastasis (Figure 4m, n)
.
These findings indicate that daily CR is better than caloric cycling programs in reducing tumor growth rate and metastatic burden
.

6.
CR effectively reduces tumor metastasis after primary tumor resection

Compared with AL-fed mice, whether or not tumors were removed, daily CR and FMD or LCC cycles were equally effective in reducing average body weight and caloric intake (Supplementary Figure 5b-d)
.
After 28 days of tumor growth, daily CR and periodic FMD or LCC resulted in a decrease in tumor volume and a smaller spleen
.
Mice undergoing CR and caloric cycles every day had smaller tumors resected after 14 days (Supplementary Figure 5e, f).
For CR and FMD, the tumor volume was significantly reduced, and the LCC group showed a downward trend (Supplementary Figure 5g)
.
The tumor was removed on the 14th day, and the spleen became smaller when sacrificed on the 28th day (Supplementary Figure 5h)
.
In tumor-removed mice, although this trend was observed in the CR group, no dietary interventions could further reduce the number of metastases
.
Daily CR is the only intervention that can limit the formation of lung metastases in tumor-burdened mice at 28 days (Supplementary Figure 5i, j)
.
These findings indicate that daily CR can inhibit lung metastasis in mice more than periodic FMD or LCC
.

7.
The degree of CR and the delay of tumor growth

7.
The degree of CR and the delay of tumor growth The degree of CR and the delay of tumor growth

Since most breast cancer cases occur in postmenopausal women, we tried to compare the benefits of different degrees of CR (10-40%) with 4:10 cycles of FMD and LCC in delaying the growth of the primary tumor (Figure 5a)
.
Compared with the AL control group, the CR level dose-dependently reduced the average body weight and food consumption of 11-month-old sterilized female mice (Figure 5b, c, Supplementary Figure 6a, b)
.
30% and 40% CR significantly reduced blood glucose levels, but insulin levels and HOMA2-IR index did not change (Supplementary Figure 6d-f)
.
Compared with young females, sterilized mice using FMD or LCC showed similar insulin levels and HOMA2-IR index to AL-fed control groups (Supplementary Figure 6e, f)
.
Only 30% of CR group had a significant decrease in IGF-1 level (Supplementary Figure 6g)
.
The unique metabolic characteristics of old and young female mice indicate that age and reproductive status rather than tumor burden itself may be a greater determinant of metabolic homeostasis
.

CR intervention (20-40%) resulted in a significant decrease in the growth rate of primary tumors and a decrease in tumor volume (Figure 5d, e)
.
The weight of the spleen in the CR group was also significantly reduced (Figure 5 g)
.
Unlike young females, FMD or LCC has no protective effect on 4T1 tumors in old female mice (Figure 5d, e)
.
The H&E staining information showed that dietary intervention did not change the viability of the tumor (Figure 5h, l)
.
Therefore, it seems that 20-40% CR, rather than caloric circulation (LCC or FMD), can delay the growth of primary tumors in women after childbirth
.

The number of metastases in mice after reproduction in the 20-40% CR group was significantly reduced (Figure 5j and Supplementary Figure 6h), while the 10% CR group and LCC group showed a downward trend
.
H&E stained lung slice histology quantitatively showed that with the increase of CR level, the total metastatic area decreased significantly (Figure 5k, l), which is consistent with the changes in metastatic load observed in young mice
.
Although the area of lung metastasis in women after reproduction is larger (Supplementary Figure 6j)
.

8.
The therapeutic effect of elevated CR on tumor growth delay

8.
The therapeutic effect of elevated CR on tumor growth delay 8.
The therapeutic effect of elevated CR on tumor growth delay

After 21 days of feeding intervention, the metabolic parameters were evaluated at the time of sacrifice
.
40% CR significantly reduced circulating glucose levels (Supplementary Figure 7e) and IGF-1 levels (Supplementary Figure 7h), while compared with the AL control group, all treatment groups had no effect on insulin levels and HOMA2-IR index ( Supplementary Figure 7f, g)
.
Therapeutic CR intervention (20-40%), like prophylactic CR treatment, resulted in a significant, dose-dependent decrease in primary tumor growth rate (Supplementary Figure 7i, j) and tumor volume (Supplementary Figure 7k)
.
Compared with AL, the spleen weight of mice in the 20-40% CR group was also significantly reduced (Supplementary Figure 71)
.
Therapeutic CR (20-40%) also resulted in a significant reduction in the number of metastases (Supplementary Figure 7m), including advanced metastases (greater than 1.
5 mm) (Supplementary Figure 7n,o)
.
Taken together, these findings indicate that CR (20-40%) is effective in delaying primary tumor growth and metastatic burden, regardless of whether it starts before or after 4t1 vaccination
.

9.
CR and caloric circulation lead to unique immune characteristics

The growth and metastasis of human and bone marrow breast cancer require regulatory immune cells
.
Therefore, we tried to determine whether daily CR and 4:10 cycles of FMD and LCC resulted in unique immune characteristics
.
There is a positive correlation between FoxP3CD4 Treg cells and primary tumor area or late metastasis
.
Compared with the AL control group, the frequency of FoxP3+CD4+ cells in the peripheral blood of tumor mice with daily CR, FMD and LCC cycles was significantly reduced (Figure 6a, Supplementary Figure 8a)
.
FMD is the only program that can significantly reduce the percentage of FoxP3CD4 Tregs in the spleen (Figure 6b, Supplementary Figure 8b)
.

Compared with the AL control group, the frequency of FoxP3+CD4+ cells in the peripheral blood of tumor mice with daily CR, FMD and LCC cycles was significantly reduced (Figure 6a, Supplementary Figure 8a)
.

However, compared with the reduction in lung metastasis induced by CR (Figure 3), the progression of metastasis was not hindered by LCC and FMD , indicating that additional immune cells were involved
.
Quantifying the frequency of FoxP3CD8 Tregs showed that although the level of FoxP3+CD8+ Treg in the peripheral blood of LCC and CR mice was significantly reduced (Figure 6c, Supplementary Figure 8c), the level of FoxP3+CD8+ Treg in the spleen of CR mice was significantly reduced (Figure 6d, supplementary Figure 8d)
.
It suggests that CR-mediated 4T1 lung metastasis suppression may occur by inhibiting the activation and up-regulation of these immunosuppressive cells
.

However, compared with the reduction in lung metastasis induced by CR (Figure 3), the progression of metastasis was not hindered by LCC and FMD , indicating that additional immune cells were involved
.
Compared with CR-induced reduction in lung metastasis (Figure 3), the progression of metastasis was not hindered by LCC and FMD.
Although the levels of FoxP3+CD8+ Treg in the peripheral blood of LCC and CR mice were significantly reduced (Figure 6c, Supplementary Figure 8c), CR The level of FoxP3+CD8+ Treg in the spleen of mice was significantly reduced (Figure 6d, Supplementary Figure 8d)
.
It suggests that CR-mediated 4T1 lung metastasis suppression may occur by inhibiting the activation and up-regulation of these immunosuppressive cells
.

No changes were detected in the frequency of MDSCs in peripheral blood (Figure 6e, Supplementary Figure 8e); however, their frequency in the spleen of CR animals was significantly reduced (Figure 6f and Supplementary Figure 8f)
.
In LCC and CR mice, the ratio of FoxP3+CD8+ treg to MDSCs was significantly reduced (Supplementary Figure 9a)
.
CR also significantly increased the frequency of tumor cytotoxic effector T cells (Eff CD8 and CD4) in the spleen and surrounding blood (Figure 6 gj and Supplementary Figure 8 gj)
.
In summary, the three main immunosuppressive markers in peripheral blood and spleen are significantly down-regulated in persistent CR
.

10.
Remodeling of the tumor immune microenvironment

10, tumor immune microenvironment remodeling 10, tumor immunity remodeling microenvironment of tumor immunity

The tumor microenvironment (TME) is one of the main centers that promote tumor immune reprogramming
.
Since tumors tend to be in an immunosuppressive environment, we tried to describe the changes in immunosuppressive markers in TME
.
This study found no significant difference in the number of CD4+Foxp3+ treg (Figure 7a, Supplementary Figure 10a), which matches the previous findings in the 4T1 model with a CR45 concentration of 30%
.
However, in daily CR mice, the frequency of CD8+Foxp3+Tregs (the levels of which are correlated with poor prognosis) showed a downward trend (Figure 7b, Supplementary Figure 10b)
.

We detected a significant reduction in the total amount of MDSCs in FMD and daily CR mouse tumors (Figure 7c, Supplementary Figure 10c)
.
When exploring the MDSC subgroup, only CR mice found that the frequency of PMN-MDSCs was significantly reduced, but the frequency of M-MDSCs did not decrease (Figure 7d, e)
.
These findings are consistent with those found in tumors of breast cancer patients
.
The expression of CD103+ in CD4+ and CD8+ cells was only significantly increased in daily CR mice (Figure 7k, j and Supplementary Figure 10h, i)
.

In summary, a key finding of this study is that daily CR can significantly reduce the growth of primary tumors than calorie circulation
.
Daily CR and other less stringent forms of CR can limit tumor progression
.

Another key observation is to recognize immune remodeling in the peripheral tissues of daily CR
.
Daily CR can cause CD8 and CD4 cells to increase in peripheral tissues
.
In the TME of CR-fed mice, the number of CD4 or CD8 cells was not affected, but the cytotoxic killing potential was increased
.
Therefore, CR can transform TME to inhibit the growth of malignant cells, while enhancing the proliferation and cytotoxicity of T cells
.
Another key observation is to recognize immune remodeling in the peripheral tissues of daily CR
.
Daily CR can cause CD8 and CD4 cells to increase in peripheral tissues
.
In the TME of CR-fed mice, the number of CD4 or CD8 cells was not affected, but the cytotoxic killing potential was increased
.
Therefore, CR can transform TME to inhibit the growth of malignant cells, while enhancing the proliferation and cytotoxicity of T cells
.
Daily CR can cause CD8 and CD4 cells to increase in peripheral tissues
.
In the TME of CR-fed mice, the number of CD4 or CD8 cells was not affected, but the cytotoxic killing potential was increased
.
Therefore, CR can transform TME to inhibit the growth of malignant cells, while enhancing the proliferation and cytotoxicity of T cells
.

The reduction in the effect of caloric circulation (LCC or FMD) on tumor progression in elderly women is another important finding of this study
.
An increase in the daily CR level (20% or higher), whether before or after the 4T1 injection, will result in a significant reduction in the tumor burden of elderly women, while the tumor growth rate in the caloric circulation group is still increasing
.
The reduction in the effect of caloric circulation (LCC or FMD) on tumor progression in elderly women is another important finding of this study
.
An increase in the daily CR level (20% or higher), whether before or after the 4T1 injection, will result in a significant reduction in the tumor burden of elderly women, while the tumor growth rate in the caloric circulation group is still increasing
.
The reduction in the effect of caloric circulation (LCC or FMD) on tumor progression in elderly women is another important finding of this study
.
An increase in the daily CR level (20% or higher), whether before or after the 4T1 injection, will result in a significant reduction in the tumor burden of elderly women, while the tumor growth rate in the caloric circulation group is still increasing
.

Even a short-term daily CR, from 20% to 40%, can significantly reduce the transfer burden, while the caloric cycle (LCC or FMD) is ineffective
.
In addition, after the resection of the primary tumor, heat circulation (LCC or FMD) cannot prevent the progression of lung metastasis, while CR shows a downward trend
.
Even a short-term daily CR, from 20% to 40%, can significantly reduce the transfer burden, while the caloric cycle (LCC or FMD) is ineffective
.
In addition, after the resection of the primary tumor, heat circulation (LCC or FMD) cannot prevent the progression of lung metastasis, while CR shows a downward trend
.
Even a short-term daily CR, from 20% to 40%, can significantly reduce the transfer burden, while the caloric cycle (LCC or FMD) is ineffective
.
In addition, after the resection of the primary tumor, heat circulation (LCC or FMD) cannot prevent the progression of lung metastasis, while CR shows a downward trend
.

In general, CR is undoubtedly the most effective non-pharmacological intervention for induced and spontaneous cancers
.
The results of this study show that compared with daily CR, the 4:10 feeding cycle is less effective and cannot prevent lung metastasis, regardless of dietary composition or when treatment is initiated (before or after 4t1 injection)
.
What is important is that daily CR triggers a unique immune activation feature, which significantly reduces the number of tumor-promoting immune cells (CD11b+Gr1+), and at the same time up-regulates anti-tumor (CD8+ and CD4+) immune cells
.
These findings indicate that in the 4T1 mouse breast cancer model, the duration and degree of CR are the most critical factors determining the protection against cancer progression
.
In general, CR is undoubtedly the most effective non-pharmacological intervention for induced and spontaneous cancers
.
What is important is that daily CR triggers a unique immune activation feature, which significantly reduces the number of tumor-promoting immune cells (CD11b+Gr1+), and at the same time up-regulates anti-tumor (CD8+ and CD4+) immune cells
.
These findings indicate that in the 4T1 mouse breast cancer model, the duration and degree of CR are the most critical factors determining the protection against cancer progression
.

Original source:

Laura CD Pomatto-Watson, et al.
Daily caloric restriction limits tumor growth more effectively than caloric cycling regardless of dietary composition.

NATURE COMMUNICATIONS | (2021) 12:6201 | https://doi.
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