Overall trends in the data suggest that exercise influences the amount of regulatory T cells present in the tumor microenvironment and lymphoid tissue of a tumor-bearing individual.
On average, more Tregs were observed in the spleen samples than in the tumor samples. This difference was expected, as lymphocytes frequent lymphoid tissue and are recruited by the tumor microenvironment as needed. These groups are not significantly different, as the p-value of the two-tailed independent t-test comparing both groups was 0.538. Assessing the spleen samples and tumor samples of both cohorts, it was noted that sedentary rats had a larger population of Tregs in their spleens and had a larger population of Tregs in their tumors than did the exercised rats. Both comparisons are statistically insignificant, with p-values of 0.527 and 0.300 for spleen samples and tumor samples, respectively; however, the difference in Treg cell count was considerably greater between the tumors than the spleens. One potential implication of this observation is that exercise limits the recruitment of Tregs to the tumor microenvironment to a greater extent than it limits the mere presence of Tregs in lymphoid tissue. This is important because it is the population of Tregs in the tumor microenvironment that inhibits anti-tumor immune response and consequently is detrimental to the tumor-bearing individual, not merely the presence of excess Tregs circulating through blood and lymphoid tissue.
When taking into account all Tregs identified in both tumors and spleens, the sedentary group had on average 31.3333 Tregs whereas the exercised group had on average 13.6667. A two-tailed independent t-test performed at a 95% confidence interval revealed insignificant results, as the p-value of this data is 0.347. While statistical significance could not be determined at this level, and therefore the null hypothesis could not be rejected, all of the trends described above support the hypothesis that exercise reduces Treg cell count in both the tumors and spleens of individuals with cancer.
Lack of statistical significance can be attributed to, in part, the limited sample size available. Due to the time-sensitive nature of animal testing, tissue samples from only six rats could be collected and studied within the time allotted for this research. Increasing the sample size would provide more points of data and therefore enable stronger conclusions to be drawn. Additionally, working with the cancer line MATB3 would have been preferable to using the MTLn3 cancer line as MTLn3 is a more aggressive cancer. Because of this, the rats died relatively shortly after injection, whereas testing with the MATB3 cancer line would have been more comparable to the progression of cancer in humans. Moreover, a novel Foxp3 IHC protocol was designed and implemented for the rats used in this study; therefore, inherent flaws in the protocol were subject to adjustments and improvements throughout the course of this study. Moreover, the protein CD4 could not be addressed and utilized in this protocol due to a lack of CD4 antibody.
A point of interest in this research was the identification of CD25- Foxp3+ cells. Cells of this phenotype were unexpected. Although the transmembrane protein CD25 is present in many different lymphocytes, previous literature suggests CD25 to be a characteristic of Treg cells. However, the Foxp3-expressing cells did not fluoresce for CD25, indicating a relatively high population of another lymphocyte in the tumor microenvironment. The role of these cells in either the advancement or suppression of tumor growth should be considered for further study.
On average, more Tregs were observed in the spleen samples than in the tumor samples. This difference was expected, as lymphocytes frequent lymphoid tissue and are recruited by the tumor microenvironment as needed. These groups are not significantly different, as the p-value of the two-tailed independent t-test comparing both groups was 0.538. Assessing the spleen samples and tumor samples of both cohorts, it was noted that sedentary rats had a larger population of Tregs in their spleens and had a larger population of Tregs in their tumors than did the exercised rats. Both comparisons are statistically insignificant, with p-values of 0.527 and 0.300 for spleen samples and tumor samples, respectively; however, the difference in Treg cell count was considerably greater between the tumors than the spleens. One potential implication of this observation is that exercise limits the recruitment of Tregs to the tumor microenvironment to a greater extent than it limits the mere presence of Tregs in lymphoid tissue. This is important because it is the population of Tregs in the tumor microenvironment that inhibits anti-tumor immune response and consequently is detrimental to the tumor-bearing individual, not merely the presence of excess Tregs circulating through blood and lymphoid tissue.
When taking into account all Tregs identified in both tumors and spleens, the sedentary group had on average 31.3333 Tregs whereas the exercised group had on average 13.6667. A two-tailed independent t-test performed at a 95% confidence interval revealed insignificant results, as the p-value of this data is 0.347. While statistical significance could not be determined at this level, and therefore the null hypothesis could not be rejected, all of the trends described above support the hypothesis that exercise reduces Treg cell count in both the tumors and spleens of individuals with cancer.
Lack of statistical significance can be attributed to, in part, the limited sample size available. Due to the time-sensitive nature of animal testing, tissue samples from only six rats could be collected and studied within the time allotted for this research. Increasing the sample size would provide more points of data and therefore enable stronger conclusions to be drawn. Additionally, working with the cancer line MATB3 would have been preferable to using the MTLn3 cancer line as MTLn3 is a more aggressive cancer. Because of this, the rats died relatively shortly after injection, whereas testing with the MATB3 cancer line would have been more comparable to the progression of cancer in humans. Moreover, a novel Foxp3 IHC protocol was designed and implemented for the rats used in this study; therefore, inherent flaws in the protocol were subject to adjustments and improvements throughout the course of this study. Moreover, the protein CD4 could not be addressed and utilized in this protocol due to a lack of CD4 antibody.
A point of interest in this research was the identification of CD25- Foxp3+ cells. Cells of this phenotype were unexpected. Although the transmembrane protein CD25 is present in many different lymphocytes, previous literature suggests CD25 to be a characteristic of Treg cells. However, the Foxp3-expressing cells did not fluoresce for CD25, indicating a relatively high population of another lymphocyte in the tumor microenvironment. The role of these cells in either the advancement or suppression of tumor growth should be considered for further study.