A study of the effects of immunomodulators 6-mercaptopurine and 6-thioguanine on the crosstalk between human colon cancer cells and macrophages in vivo

Mahmood, Arfa (2023) A study of the effects of immunomodulators 6-mercaptopurine and 6-thioguanine on the crosstalk between human colon cancer cells and macrophages in vivo. Masters by Research thesis (MPhil), Manchester Metropolitan University.

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Abstract

Colon cancer is one of the most diagnosed cancers in the world, with early onset cases rising in recent years in the western world. This could be related to an increased consumption of high fat diets and sedentary lifestyle choices. Risk factors such as Inflammatory bowel disease (IBD) can lead to colon cancer, due to development of lesions in the colon. IBD can be treated with a range of immunomodulators. Although treatment has been established for IBD, little is known about the effects of immunomodulator on tumour progression. The aims of this study were to determine the effects of the immunomodulators, 6-MP and 6-TG on the crosstalk between human colon cancer cells and macrophages in vitro, particularly in relation to the alterations of the phenotypes of tumour associated macrophage (TAM) and colon cancer cells. THP-1 and U937 cell derived macrophages and colon cancer cells were co-cultured together to allow cell-to-cell interactions to be studied. Light and scanning electron microscopy were used to visualise the change in morphology of monocyte to macrophages. Flow cytometry was to confirm the phenotypic marker expression of macrophages after differentiation. qRT-PCR assays were used to analyse the changes of cytokine and chemokine gene expressions by co-cultured cell lines after treatment with immunomodulators. ELISA were conducted to evaluate the levels of IL-6 in the media of the co-cultured cells. The treatment of 1μM 6MP and 3μM 6-TG resulted in significant (P<0.05) increases in the mRNA expressions of M1 surface markers such as CD80, CD86, CD11, and CD14 by THP-1 cells after co-culture and treatment with 3μM 6-TG. The flow cytometry data demonstrated a significant (P<0.05) increase in CD16 after treatment with 10μM 6MP and 30μM 6-TG indicating a potential anti-tumoral effect induced by immunomodulation. Additionally, ELISA analysis showed IL-6 levels in differentiated U937-HT29 co-cultured media significantly elevated (P<0.05) following 1μM 6MP and 30μM 6-TG treatment compared to negative controls. This further highlights the impact of the treatment on the THP1-/ U937-differentiated macrophages and HT29 cell communication. The combined evidence strongly suggests a notable alteration in the cellular immune response following 6MP or 6-TG treatment in vitro, hinting at an increased anti-tumour potential due to the immunomodulator. This study suggests that treatment with immunomodulators has a pro-inflammatory but anti-tumoral effect on the crosstalk between the two cell types. However, further investigation is necessary to provide a comprehensive and detailed understanding the effects of immunomodulator treatments.