Exploration of the possible role of microRNA(s) and small extracellular vesicles in chemotherapy-induced bystander effects

Ude, Arinzechukwu

Abstract

Advances in modern chemotherapeutic regimens have led to an increase in the overall survival of leukaemia patients in recent years. However, haematopoietic stem cell transplantation (HSCT) remains the curative option for these patients. In HSCT, high dose chemotherapy is often given pre-transplant to remove the leukaemic stem cells (LSC) within the bone marrow (BM) microenvironment of the patients prior to infusion of donor stem cells to reconstitute haematopoiesis. Despite its success, HSCT can also lead to development of de novo primary malignancy as chemotherapy can also exert deleterious effects on actively dividing cells whilst targeting LSC. Donor cell leukaemia (DCL) is a type of de novo primary malignancy whereby transplanted stem cells become malignant in the recipient whilst the donor remains healthy. Despite the growing incidence of DCL due to advancement in genetic testing, the aetiology and mechanism of DCL remain unknown. Therefore, using DCL as a pathological pivot, this thesis explored the possible roles of microRNA (miRNAs) and small extracellular vesicles (sEVs) in chemotherapy-induced bystander effects (CIBE). Bystander effect occurs when toxic signals induce biological effects in unexposed cells, which are in close proximity to the directly exposed cells, via intercellular communication. A co-culture bystander model, which promotes cell-to-cell communication between cell compartments in vitro was utilised to investigate the concept of CIBE thus mimicking the BM microenvironment. HS-5 stromal cells and TK6 lymphoblast cells were co-cultured using culture inserts, which allowed isolation of bystander TK6 cells to detect cytotoxicity and genotoxicity following indirect exposure to drugs. The drugs used in this study were alkylating agents (chlorambucil and carmustine), and topoisomerase inhibitors (etoposide and mitoxantrone), which have been shown to induce CIBE in a previous study. Microarray analysis was performed using bystander cells’ RNA to identify candidate miRNAs that may be involved in CIBE. Drug-treated HS-5 cells were fixed with 2% paraformaldehyde, negatively stained with uranyl acetate and examined by transmission electron microscopy (TEM) to visualise sEVs release. Furthermore, sEVs derived from drug-treated HS-5 cells were isolated, characterised and tracked for uptake and internalization by bystander cells. Expression of candidate miRNAs was validated in treated HS-5 cells, conditioned medium (CM), isolated sEVs and bystander cells by qRT-PCR following sEVs uptake.

All drugs promoted genotoxicity in the bystander cells whilst maintaining good viability. However, only mitoxantrone produced statistically significant cytotoxic and genotoxic events in the bystander cells. These events persisted within the bystander cells over five days thus suggesting that the ‘safe period’ given to transplant patients to recover from the effects of pre-transplant chemotherapy may not be safe after all. Further examination of the CM illustrated that chemotherapy promotes the release of sEVs into the CM by HS-5 cells in comparison to untreated HS-5 cells. These sEVs were internalized by the bystander cells in a time-dependent manner and once internalized, released their cargo into the cells to elicit bio-molecular effects. Differential miRNA signatures were also found in the treated HS-5 cells and bystander TK6 cells. The expression levels of hsa-miR-146a-5p, hsa-miR-16-5p, hsa-miR-20a-5p, hsa-miR-17-5p were all upregulated in treated HS-5 cells but repressed in bystander TK6 cells. However, the expression level of hsa-miR-30d-5p was repressed in treated HS-5 cells and upregulated in bystander TK6 cells. These candidate miRNA signatures were also found in CM and sEVs. However, only hsa-miR-17-5p was found in treated sEVs thus implying hsa-miR-17-5p may play a crucial role in CIBE. These candidate miRNAs regulate genes involved in cellular signalling pathways, cell division and cell survival. Therefore, these suggest that the miRNAs are selectively sorted and packaged into EVs, and subsequently trafficked to the bystander cells wherein they may determine the fate of these cells. Collectively, these analyses provide a novel finding that soluble factors such as miRNAs and sEVs, released by treated stromal cells within the BM microenvironment may play a vital role in the propagation of CIBE signals to the incoming donor cells thereby eliciting deleterious effects. However, these may not tell the whole story as there may be an interplay between complex signals in CIBE. Thus, further investigation needs to be done to fully understand the mechanisms involved.

• Thesis Type: Thesis
• Deposit Date: Jan 11, 2021
• Publicly Available Date: Apr 15, 2021
• Public URL: https://uwe-repository.worktribe.com/output/6971684
• Award Date: Apr 15, 2021