This study represents the first successful Foxp3 IHC protocol in rats. This protocol was utilized to identify, as well as quantify, the presence of Tregs in tumor and spleen tissue. Overall, the data consistently demonstrates that the tissue of sedentary rats contained, on average, increased counts of Tregs as compared to the tissue of exercised rats. The trends, indicating a decrease in the Treg population of exercised tumor and spleen tissue, suggest that exercise regulates the quantity of Tregs, thereby limiting the extent to which Tregs can suppress the anti-tumor immune response in cancer patients. Because prior literature determines elevated levels of Tregs in patients to correspond to poor prognoses, exercise can improve the prognosis of cancer patients by way of modulating the proliferation of Tregs, and consequently diminishing protumorigenic activity in the tumor microenvironment.
The implementation of the protocol itself is significant, as it provides new methodology for continuing this line of research; nevertheless, changes in the methods should be employed in furthering this study. In continuing this research, samples sizes for both the sedentary and exercised cohorts should be increased, as collecting more data points would enable the significance and implications of the data to be more accurately analyzed. Applying CD24 antibodies to tissue samples would be a beneficial addition to the protocol, as it would help to differentiate between Tregs and Bregs (regulatory B cells). Additionally, antigen retrieval via Sudan Black B was initially attempted to minimize background fluorescence in the fluorescence microscopy images. However, the use of Sudan Black B was eliminated from the protocol thereafter, as it resulted in the absence of all fluorescence. Modifying the concentration and amount of Sudan Black B applied to the tissue samples in efforts to successfully implement it into the protocol would be beneficial in producing more clear images from the fluorescence microscope. In addition to fluorescence microscopy, alternative forms of data collection may be considered. Confocal microscopy for IHC would provide clearer, more magnified views of the cells to more accurately locate CD25+ Foxp3+ cells. Flow cytometry would enable greater amounts of quantitative data to be collected, allowing for more comprehensive and accurate analysis to be conducted. Moreover, the CD25- Foxp3+ cells identified in the tissue samples could be further observed to explore their presence, activity, and function in the tumor microenvironment in order to assess how they promote or inhibit the progression of cancer.
The reduction of Tregs in the tumor microenvironment observed in this study can be considered and further explored as a mechanism by which exercise impacts cancer. Examination of the effect of exercise on specific lymphocytes is critical, as better understanding of the interactions between cancerous cells and lymphocytes at the molecular level can lead to the subsequent development of more efficient, more successful cancer treatment strategies. It is imperative to explore the potential of exercise to regulate, deter, and even regress cancer as well as reduce recurrence and mortality rates through its effect on lymphocytes, as the implications of this research would help the millions of people continuing to die from cancer every year.
The implementation of the protocol itself is significant, as it provides new methodology for continuing this line of research; nevertheless, changes in the methods should be employed in furthering this study. In continuing this research, samples sizes for both the sedentary and exercised cohorts should be increased, as collecting more data points would enable the significance and implications of the data to be more accurately analyzed. Applying CD24 antibodies to tissue samples would be a beneficial addition to the protocol, as it would help to differentiate between Tregs and Bregs (regulatory B cells). Additionally, antigen retrieval via Sudan Black B was initially attempted to minimize background fluorescence in the fluorescence microscopy images. However, the use of Sudan Black B was eliminated from the protocol thereafter, as it resulted in the absence of all fluorescence. Modifying the concentration and amount of Sudan Black B applied to the tissue samples in efforts to successfully implement it into the protocol would be beneficial in producing more clear images from the fluorescence microscope. In addition to fluorescence microscopy, alternative forms of data collection may be considered. Confocal microscopy for IHC would provide clearer, more magnified views of the cells to more accurately locate CD25+ Foxp3+ cells. Flow cytometry would enable greater amounts of quantitative data to be collected, allowing for more comprehensive and accurate analysis to be conducted. Moreover, the CD25- Foxp3+ cells identified in the tissue samples could be further observed to explore their presence, activity, and function in the tumor microenvironment in order to assess how they promote or inhibit the progression of cancer.
The reduction of Tregs in the tumor microenvironment observed in this study can be considered and further explored as a mechanism by which exercise impacts cancer. Examination of the effect of exercise on specific lymphocytes is critical, as better understanding of the interactions between cancerous cells and lymphocytes at the molecular level can lead to the subsequent development of more efficient, more successful cancer treatment strategies. It is imperative to explore the potential of exercise to regulate, deter, and even regress cancer as well as reduce recurrence and mortality rates through its effect on lymphocytes, as the implications of this research would help the millions of people continuing to die from cancer every year.