Vitamin D Controls Tumor Growth in Breast Cancer
Jun 23, 2019 22:26:23 GMT
miamianne67, tomsylver, and 2 more like this
Post by deborah on Jun 23, 2019 22:26:23 GMT
www.frontiersin.org/articles/10.3389/fimmu.2019.01307/full
Discussion
In the present study, using female mice injected with EO771 breast cancer cells and supplemented with VD, we report a beneficial role of VD on tumor growth progression and inflammation. Indeed, VD supplementation reduced tumor weight and increased CD8+ T cell infiltration into the tumor. Interestingly, these CD8+ T cells exhibited a more active central/effector memory T cell phenotype, which are considered to have increased anti-tumor properties (43). These data are in agreement with data that highlight TILs as an important predictive and prognostic biomarker in patients with breast cancer (44). Ono et al. observed an association between pathological response and TIL score in patients with triple-negative breast cancer, but not in patients with other tumor types (45). Therefore, a general consensus emerged on the central role played by effector T cells in the anti-tumor response. Similar results were obtained in colorectal cancer in which quantification of immune cell densities revealed the major positive role of cytotoxic and memory T cells for patient survival (46). The present study indeed also reports that CD8+ T cells infiltrating the tumor harbor an activated/cytotoxic phenotype eliciting PD-1/Granzyme B expression. In line with these results, several studies on cancer patients have reported that PD-1 expression is highly expressed on tumor infiltrating CD8+ T cells in various immunogenic tumors (47–49).
Surprisingly, in contrast to standard diet condition, VD supplementation of overweight-mice resulted in increased tumor progression without CD8+ T cell infiltration (illustrated in Figure 6). The impact of VD supplementation on inflammation associated with obesity has been largely described. It has been reported a beneficial role of VD supplementation on weight gain limitation in mice (50, 51). Indeed, such supplementation reduced the weight gain induced by high-fat diet and improved insulin sensitivity. The reduced weight gain in VD supplemented mice is primarily due to a limitation of fat mass accumulation (50). We have also shown in a previous study that VD displays immunoregulatory effects and reduces adipocyte inflammation in vitro and in vivo. Indeed, VD supplementation decreases the expression and secretion of a large range of inflammatory markers and macrophage infiltration in the adipose tissue of high-fat diet fed mice (38). In the present study, we report a beneficial role of VD supplementation on inflammation both in basal or overweight conditions. We have shown that VD is able to limit the expression of inflammatory markers in adipocytes (Il6, Ccl5, and Cx3cl1) and their secretion (IL-6 and CCL5) in the plasma. These results are in agreement with the previously reported anti-inflammatory effects of VD on adipocytes (38, 52). Taken together, our results reveal the influence of diet on tumor growth control and CD8+ T cell infiltration by VD supplementation. The difference observed between normal and high-fat diet conditions could be explained by a modification in the VD metabolism in adipocytes, in particular Cyp27a1 in overweight mice, which is involved in the first hydroxylation of cholecalciferol (data not shown). This enzyme is known to be increased in adipose tissue of obese mice, which suggests the possible increase of 25-hydroxylation in adipose tissue, resulting in higher local production of 25(OH)D in adipose tissue (53). Several studies about obesity have demonstrated that 25(OH)D is diluted in a higher volume in obese patients (54). We hypothesized that the increase of adipocytes in overweight-mice reduces 25(OH)D in plasma, which could have an impact on a decreased CD8+ T cell infiltration.
FIGURE 6
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Figure 6. Vitamin D controls breast cancer tumor growth and CD8+ T cells infiltrating the tumor. VD supplementation was validated in both models by the quantification of the 25(OH)D levels and inflammatory cytokines (IL-6 and CCL5) in plasma and mRNA levels of inflammatory markers (Il6, Ccl5, Ccl2, Ccl11, and Cx3cl1) in adipocytes. Tumor size was measured and CD8+ T cells were quantified in the tumor.
We next wondered if the observed contrasting effects could be due to a modification of adipogenic markers under VD supplementation. Using RT-qPCR approach, we found that VD limited adiponectin expression and a tendency for a decrease in Pparg, Pgc1a, and Cebpa without modification in high-fat condition. Our results obtained in normal diet fed-mice are in accordance with previous results reported in mouse models (55) but they cannot explain the difference between the two conditions. Indeed, the effects of VD on adipogenesis have been analyzed in several animal models, and the majority of these studies suggest that VD plays an inhibitory role in adipogenesis. The few cell culture and supplementation studies that have investigated adipogenesis in human cells indicate that, in contrast to findings from rodent studies, VD is proadipogenic (56, 57). The difference in results among mouse and human studies could be due to differences in diet, treatment periods, cell types, and/or VD treatment protocols.
Differences on CD8+ T cell infiltration into the tumor of standard diet and high-fat diet-fed mice could finally be explained by the decrease of expression levels of chemokine receptor CCR7 which is expressed on central memory T cells and involved in chemotaxis (58). Further studies are needed to investigate the molecular mechanisms involved in the regulation of CD8+ T cell migration inside the tumor under different kinds of diets.
A CD8+ T cell infiltrate is generally associated with a good cancer patient prognosis. However, other immune cell infiltrates are associated with a poor patient prognosis [infiltrates with immunosuppressive cell types such as regulatory T cells (Tregs), tumor-associated macrophages (TAMs) or myeloid-derived suppressor cells (MDSCs)] (59). In this study, only CD4+CD25+CD127low/- T cell subset has been considered (Supplemental Figure 6), as this subset could contain some Tregs. Flow cytometry analysis showed that VD did not affect the presence of CD4+CD25+CD127low/- T cells in the tumor both in basal and high-fat conditions. It would be of interest to further analyze, in the tumor microenvironment, these cell types harboring immunosuppressive properties: among immune cells but also in other cell lineages such as the cancer-associated fibroblasts (CAFs).
Adipose tissue is increasingly recognized as an active endocrine organ, which secretes several adipose tissue-specific hormones including adipokines. It plays a significant role in the regulation of homeostasis. Importantly, tumor progression does not necessarily depend on fat mass, but may be influenced more significantly by the specific metabolic state of the local adipocyte population (60). Adipose tissue is altered during inflammatory context related to obesity (61) and breast cancer (62). The factors inducing leukocyte and chemokine infiltration in adipose tissue are probably numerous. The paracrine, autocrine and endocrine signals appear to be responsible for this phenomenon (63). Indeed, adipose tissue and adipocytes in particular produce and release a variety of adipokines notably chemokines which are defined as “cytokines with selective chemoattractant properties,” coordinating leukocyte movement to sites of inflammation or injury (64). Most of the breast body is made up of adipose tissue which contains both adipocytes and immune cells. In following studies, it will be important to combine immunomonitoring to adipocyte behavior studies.
The clinical data related to the effect of VD supplementation on breast cancer are still unclear. Indeed, only a few VD supplementation trials with breast cancer have been conducted, and these have suffered from major limitations (as inappropriate dose…) (65). Another recent multicentric, randomized, placebo-controlled trial, with a two-by-two factorial design, of vitamin D3 (cholecalciferol) and omega-3 fatty acids for the prevention of cancer, was conducted in the United States. Supplementation with VD did not result in a lower incidence of invasive cancer than placebo (66). Given the interest in using VD to reduce cancer risk in lean patients, further research is needed, particularly randomized controlled trials, to demonstrate in humans whether individuals with low levels of circulating 25(OH)D are at increased risk of developing cancer (VD in prevention strategy) and whether VD supplements can reduce cancer risk and progression and improve outcome (VD in therapy). In conditions where VD favors access for cytolytic T cells to the tumor site, VD supplementation (T-cell migration inducer) could be combined with an immune checkpoint immunotherapy (T-cell activation inducer) in the treatment of breast cancers.
In conclusion, our results show that VD modulates tumor growth progression and the infiltration of the cytotoxic CD8+ T cells in the tumor of mice depending on the type of diet. If our data can be confirmed in human in randomized clinical trials, VD supplementation could represent a new adjuvant in the treatment of breast cancers.
Discussion
In the present study, using female mice injected with EO771 breast cancer cells and supplemented with VD, we report a beneficial role of VD on tumor growth progression and inflammation. Indeed, VD supplementation reduced tumor weight and increased CD8+ T cell infiltration into the tumor. Interestingly, these CD8+ T cells exhibited a more active central/effector memory T cell phenotype, which are considered to have increased anti-tumor properties (43). These data are in agreement with data that highlight TILs as an important predictive and prognostic biomarker in patients with breast cancer (44). Ono et al. observed an association between pathological response and TIL score in patients with triple-negative breast cancer, but not in patients with other tumor types (45). Therefore, a general consensus emerged on the central role played by effector T cells in the anti-tumor response. Similar results were obtained in colorectal cancer in which quantification of immune cell densities revealed the major positive role of cytotoxic and memory T cells for patient survival (46). The present study indeed also reports that CD8+ T cells infiltrating the tumor harbor an activated/cytotoxic phenotype eliciting PD-1/Granzyme B expression. In line with these results, several studies on cancer patients have reported that PD-1 expression is highly expressed on tumor infiltrating CD8+ T cells in various immunogenic tumors (47–49).
Surprisingly, in contrast to standard diet condition, VD supplementation of overweight-mice resulted in increased tumor progression without CD8+ T cell infiltration (illustrated in Figure 6). The impact of VD supplementation on inflammation associated with obesity has been largely described. It has been reported a beneficial role of VD supplementation on weight gain limitation in mice (50, 51). Indeed, such supplementation reduced the weight gain induced by high-fat diet and improved insulin sensitivity. The reduced weight gain in VD supplemented mice is primarily due to a limitation of fat mass accumulation (50). We have also shown in a previous study that VD displays immunoregulatory effects and reduces adipocyte inflammation in vitro and in vivo. Indeed, VD supplementation decreases the expression and secretion of a large range of inflammatory markers and macrophage infiltration in the adipose tissue of high-fat diet fed mice (38). In the present study, we report a beneficial role of VD supplementation on inflammation both in basal or overweight conditions. We have shown that VD is able to limit the expression of inflammatory markers in adipocytes (Il6, Ccl5, and Cx3cl1) and their secretion (IL-6 and CCL5) in the plasma. These results are in agreement with the previously reported anti-inflammatory effects of VD on adipocytes (38, 52). Taken together, our results reveal the influence of diet on tumor growth control and CD8+ T cell infiltration by VD supplementation. The difference observed between normal and high-fat diet conditions could be explained by a modification in the VD metabolism in adipocytes, in particular Cyp27a1 in overweight mice, which is involved in the first hydroxylation of cholecalciferol (data not shown). This enzyme is known to be increased in adipose tissue of obese mice, which suggests the possible increase of 25-hydroxylation in adipose tissue, resulting in higher local production of 25(OH)D in adipose tissue (53). Several studies about obesity have demonstrated that 25(OH)D is diluted in a higher volume in obese patients (54). We hypothesized that the increase of adipocytes in overweight-mice reduces 25(OH)D in plasma, which could have an impact on a decreased CD8+ T cell infiltration.
FIGURE 6
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Figure 6. Vitamin D controls breast cancer tumor growth and CD8+ T cells infiltrating the tumor. VD supplementation was validated in both models by the quantification of the 25(OH)D levels and inflammatory cytokines (IL-6 and CCL5) in plasma and mRNA levels of inflammatory markers (Il6, Ccl5, Ccl2, Ccl11, and Cx3cl1) in adipocytes. Tumor size was measured and CD8+ T cells were quantified in the tumor.
We next wondered if the observed contrasting effects could be due to a modification of adipogenic markers under VD supplementation. Using RT-qPCR approach, we found that VD limited adiponectin expression and a tendency for a decrease in Pparg, Pgc1a, and Cebpa without modification in high-fat condition. Our results obtained in normal diet fed-mice are in accordance with previous results reported in mouse models (55) but they cannot explain the difference between the two conditions. Indeed, the effects of VD on adipogenesis have been analyzed in several animal models, and the majority of these studies suggest that VD plays an inhibitory role in adipogenesis. The few cell culture and supplementation studies that have investigated adipogenesis in human cells indicate that, in contrast to findings from rodent studies, VD is proadipogenic (56, 57). The difference in results among mouse and human studies could be due to differences in diet, treatment periods, cell types, and/or VD treatment protocols.
Differences on CD8+ T cell infiltration into the tumor of standard diet and high-fat diet-fed mice could finally be explained by the decrease of expression levels of chemokine receptor CCR7 which is expressed on central memory T cells and involved in chemotaxis (58). Further studies are needed to investigate the molecular mechanisms involved in the regulation of CD8+ T cell migration inside the tumor under different kinds of diets.
A CD8+ T cell infiltrate is generally associated with a good cancer patient prognosis. However, other immune cell infiltrates are associated with a poor patient prognosis [infiltrates with immunosuppressive cell types such as regulatory T cells (Tregs), tumor-associated macrophages (TAMs) or myeloid-derived suppressor cells (MDSCs)] (59). In this study, only CD4+CD25+CD127low/- T cell subset has been considered (Supplemental Figure 6), as this subset could contain some Tregs. Flow cytometry analysis showed that VD did not affect the presence of CD4+CD25+CD127low/- T cells in the tumor both in basal and high-fat conditions. It would be of interest to further analyze, in the tumor microenvironment, these cell types harboring immunosuppressive properties: among immune cells but also in other cell lineages such as the cancer-associated fibroblasts (CAFs).
Adipose tissue is increasingly recognized as an active endocrine organ, which secretes several adipose tissue-specific hormones including adipokines. It plays a significant role in the regulation of homeostasis. Importantly, tumor progression does not necessarily depend on fat mass, but may be influenced more significantly by the specific metabolic state of the local adipocyte population (60). Adipose tissue is altered during inflammatory context related to obesity (61) and breast cancer (62). The factors inducing leukocyte and chemokine infiltration in adipose tissue are probably numerous. The paracrine, autocrine and endocrine signals appear to be responsible for this phenomenon (63). Indeed, adipose tissue and adipocytes in particular produce and release a variety of adipokines notably chemokines which are defined as “cytokines with selective chemoattractant properties,” coordinating leukocyte movement to sites of inflammation or injury (64). Most of the breast body is made up of adipose tissue which contains both adipocytes and immune cells. In following studies, it will be important to combine immunomonitoring to adipocyte behavior studies.
The clinical data related to the effect of VD supplementation on breast cancer are still unclear. Indeed, only a few VD supplementation trials with breast cancer have been conducted, and these have suffered from major limitations (as inappropriate dose…) (65). Another recent multicentric, randomized, placebo-controlled trial, with a two-by-two factorial design, of vitamin D3 (cholecalciferol) and omega-3 fatty acids for the prevention of cancer, was conducted in the United States. Supplementation with VD did not result in a lower incidence of invasive cancer than placebo (66). Given the interest in using VD to reduce cancer risk in lean patients, further research is needed, particularly randomized controlled trials, to demonstrate in humans whether individuals with low levels of circulating 25(OH)D are at increased risk of developing cancer (VD in prevention strategy) and whether VD supplements can reduce cancer risk and progression and improve outcome (VD in therapy). In conditions where VD favors access for cytolytic T cells to the tumor site, VD supplementation (T-cell migration inducer) could be combined with an immune checkpoint immunotherapy (T-cell activation inducer) in the treatment of breast cancers.
In conclusion, our results show that VD modulates tumor growth progression and the infiltration of the cytotoxic CD8+ T cells in the tumor of mice depending on the type of diet. If our data can be confirmed in human in randomized clinical trials, VD supplementation could represent a new adjuvant in the treatment of breast cancers.