DRUG TARGETING OF COLORECTAL CANCERS INCLUDING CANCER STEM CELLS
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Date
2023-03-28
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Griffith University
Abstract
For the past few decades, cancer stem cells (CSCs) have elicited substantial research interest as important targets for consideration during cancer therapy. CSCs are notoriously drug resistant and are well known for their ability to undergo self-renewal and differentiation into more mature cancer cells, as well as for their tumour-initiating ability from a relatively small number of cells. However, to date, there has been a notable dearth of investigations regarding the exact role and functions of isolated populations of CSCs. In addition, the prevalence of CSCs in malignancy is still a matter of some debate and controversy. In this research, the aim was to identify specific CSC markers and to isolate the CSC subpopulations from colon cancers for study into ways of rendering them more susceptible to chemotherapy. Further, as there has been a limited number of studies focusing on identifying those drugs that can target the CSC enriched populations as therapeutic agents, a focus of this study was to examine and evaluate specific CSC surface markers for their identification and use. Specifically, the aims also included to examine the colorectal CSCs and identify drugs targeting purified CSCs in culture as well as in pre-clinical CRC models as a means of eliminating this type of cancer cell.
SW480 and CT26 parental wild-type (WT) cells were transfected with a vector encoding the octamer-binding transcription factor 4 (OCT4) promoter site regulating expression of enhanced green fluorescent protein (EGFP). The most highly positive cell population OCT4-GFP after extensive rounds of sorting (the top ~1%–5%) were enriched by cycling involving alternating conditions of growth between normoxia and anoxia.
The cycled cell populations were also examined by immunoblotting with OCT4 antibody and by immunoperoxidase or immunofluorescent staining for levels of OCT4 protein as a marker for CSCs. Next, the CT26 WT and enriched CT26 OCT4 cells were injected intraperitoneally into BALB/c mice to compare their tumour-initiating capacity as CSCs. These experiments were also repeated using a range of different initial cell numbers. The NSAID, celecoxib was also tested for repurposing and its ability tested for inhibiting the CT26 WT and CT26 OCT4-GFP cell lines from forming colorectal tumours in vivo. The resulting tumours were examined using histological hematoxylin and eosin (H&E) staining to compare the effect on treated (celecoxib) versus untreated growths.
CT26 OCT4-GFP and SW40 OCT4-GFP showed significantly higher levels of OCT4 expression when compared with the WT cells based on Western blotting and immunofluorescence staining. Validation of the CSC phenotype was carried out using a tumourigenic colony forming assay in vivo in BALB/c mice. The results showed that the ability of the CSC subpopulation as a tumour-initiating driver was readily apparent using the mouse CT26 colon-cancer cell line to form metastases in the murine peritoneum. Compared to the WT CT26 cell line, the formation of tumours in BALB/c mice with the CT26 CSCs required relatively small initial cell populations, which is a key property of CSCs.
The CT26 OCT4-GFP enriched CSCs produced significantly greater tumour numbers with larger tumour sizes than did the CT26 WT inoculated mice. However, celecoxib treatment significantly decreased (~50% less) the colorectal tumour numbers and volume. Moreover, characterisation of the peritoneal cavities in the tumour-burdened mice after celecoxib treatment revealed the presence of significant levels of red blood cells in the untreated tumour controls but greatly inhibited angiogenesis in the celecoxib-treated tumour burdened mice.
This thesis outlines a range of studies examining colorectal cancer and CR-CSCs, providing a way forward for further research pertaining to the development of combinatorial therapies for this cancer. In particular, it highlights the potential benefits that can result from the use of drugs such as celecoxib that will target all colorectal cancer cells, including the CSCs. The study also developed a method for selectively enriching the CSC populations, and this process was also examined in vivo using a range of colorectal cell lines. In addition, a pre-clinical colorectal cancer model was developed based on the use of the CT26 cell line to establish a basis for testing the effects of drugs on cancer metastasis. A closer examination of the role of the CSC enriched population in tumour growth and metastasis and their therapeutic targeting can now be effectively conducted. The evidence obtained here should support an opportunity for celecoxib to be repurposed and used in chemosensitising cancer cells, thereby rendering them more susceptible to standard therapies. This includes commonly used drugs such as doxorubicin (DOX) or 5-fluorouracil (5-FU), although further research to explore these opportunities will be required. Such studies would contribute considerably to cancer research and will be important in ensuring improvement beyond the scope of therapies employed currently, thereby providing a greater expectation for improving the survival of cancer patients undergoing chemotherapy in the future.
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Keywords
Colorectal Cancer, Cancer Stem Cells, Drug targeting, Celecoxib chemosensitisation