Probing the role of regulatory T cells in tumor selective chemotherapy used in combination with in vivo dendritic cell-targeted genetic immunization

Biswas, Diptomit (2021) Probing the role of regulatory T cells in tumor selective chemotherapy used in combination with in vivo dendritic cell-targeted genetic immunization. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Diptomit Biswas (16MS144)) 16MS144_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (2MB)

Abstract

Intra-tumoral T-regulatory cells play a vital role in the unchecked growth and proliferation of cancerous tissue by shielding them from elimination by the anti-tumor effector T cells1. This sort of protection from the immune system and the inherent ability of tumors to mutate and adapt due to selective pressure caused by chemotherapy/radiotherapy makes it very difficult to completely eliminate them from the body. Therefore, novel cancer therapies are focusing on breaking this immunosuppressive shield and making cancer cells more vulnerable to other forms of cytotoxic treatments that are being used in combination with it2–4. This sort of combination of two or more therapies is showing better efficacy in treating cancers5–7 since it is difficult for cancer cells to become resistant to both treatments simultaneously. To reduce the side effects of such cancer therapies, targeted delivery of therapeutic load is also an option that scientists are adopting. Surface functionalized cationic liposomes are a safe and effective delivery vehicle that some cancer therapies have adopted to deliver therapeutic molecules to target cells. A study by Barui, S. et al. (Unpublished work from the PhD Thesis of Barui, S) from our laboratory came up with a novel combination approach involving in vivo dendritic cell-targeted genetic immunization and tumor-selective chemotherapy and tested it in established melanoma bearing mice. Two different types of receptor-selective cationic liposomes were used to deliver the therapeutic load to the target cell. The treatment caused complete regression of established melanoma tumors. While investigating the causes behind such a dramatically positive therapeutic outcome, a very initial flow cytometric experiment using only four mice indicated a reduced population of the CD4+ CD25+ T regulatory cells in the tumor microenvironment of mice treated with the above-mentioned combination approach when compared to that for other control treatment group. Constant high expression of CD25 is a well-established marker present in most suppressive FOXP3+ T-regulatory cells, but CD25 can also be expressed in other TCR stimulated CD4+ T cells. Our current study aims to establish the exact nature and phenotype of the above-mentioned CD4+ CD25+ T cells that are being depleted because of the treatment using a statistically significant number of mice in the in vivo experiments. Another aim is to investigate how the depletion of these cells is occurring. The hypothesis is that the combination therapy is causing the depletion of intra-tumoral suppressive CD4+ CD25+ FOXP3+ T-regulatory cells by hindering their migration into the tumor and also by inhibiting their suppressive nature. To test this, I had planned to use flow cytometry experiments to characterize the population of intra-tumoral CD4+ CD25+ FOXP3+ T regulatory cells present in melanoma bearing mice treated with different therapies (including the combination therapy). Several other experiments like T-regulatory suppression assay and T-regulatory induction assay in the presence and absence of activated dendritic cells (formed after transfection with tumor antigen encoded plasmid DNA) were also planned. Two types of receptor-selective cationic liposomes made of CGKRK lipopeptides and shikimoyl containing cationic lipid were required to carry this out. I was able to synthesize both of these cationic lipids. However, due to the paucity of time caused by extensive COVID-19 lockdowns, I could not proceed further with the planned work.

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