Lu, M.
(2022)
Human rather than mouse mesenchymal stem cells in 3D co-culture models boost proliferative and migratory characteristics of colorectal cancer cells through paracrine signalling pathways, highlighting species disconnects.
PhD thesis, University of Nottingham.
Abstract
Colorectal cancer (CRC) is one of the most prevalent cancers in the world, especially in western countries. It is a multifactorial malignant disease that involves multiple genetic mutations and epigenetic alterations. A large number of patients present with metastasis at the first diagnosis and the five-year survival rate is not ideal. Only a small percentage of the therapeutic agents have been proven to be efficacious for patients after phase III clinical trials. The reason for the disconnection between the preclinical testing and clinical application is in part due to the limitations of experimental models.
To date, various models have been used in colorectal cancer research, including cell line in vitro models and animal in vivo models; however, the former cannot form 3-dimensional (3D) spatial architecture which is closer to the real human physical structure while the latter lacks human stroma which is a crucial component in the tumour microenvironment (TME). Tumour heterogeneity arises not only from the tumour cells but also from the TME which plays a critical role in the whole process of oncogenesis, tumour infiltration and metastasis. In this project, 3D co-culture colorectal cancer models are established to explore the interactions between colorectal cancer cells (CRCs) and mesenchymal cells. Mesenchymal stem cells (MSCs) were used because they are known to localise to tumours from the bone marrow and differentiate into cancer-associated fibroblasts, facilitating tumour growth.
Based on previous microarray and upstream Ingenuity Pathway analysis, 24 growth factor-encoding genes were identified to drive the signalling pathways that were downregulated in the patient-derived xenografts (PDXs) compared with colorectal cancer patient tissues. These down-regulated pathways were hypothesised to be pathways that can only be maintained by signals provided by human stroma and are lost when human stroma is replaced by mouse stroma. To compare the impact of human MSCs (hMSCs) and mouse (mMSCs) on CRCs, initially in our project, 3D spheroids of CRC monoculture and co-culture with hMSCs/mMSCs were formed. Live and dead staining was used to optimise the cell density for the 3D monoculture and co-cultures, ensuing 3D spheroids were formed without a necrotic core. In the study of phenotypes, a 3D luciferase assay was used firstly to investigate the proliferation of luciferase-labelled CRCs in the co-culture models compared with monoculture. Then, a series of 3D invasion assays, in which spheroids were formed up in the standard medium and transferred into different concentrations of Basement Membrane Extract (BME), were applied to observe the invasive tendency of monoculture and co-culture spheroids. A 3D migration assay was conducted next to explore the migratory capacity of CRCs either cultured alone or indirectly co-cultured with hMSCs/mMSCs. Moreover, immunohistochemistry and in situ hybridization were also used to detect specific protein and mRNA expression in the spheroids. In the target investigation and validation, Human phospho-receptor tyrosine kinase (p-RTK) arrays based on direct co-culture and indirect co-culture models were utilised to identify differentially phosphorylated RTKs in CRCs from monoculture and co-cultures. Afterwards, Western blot was carried out to confirm the protein expression of the targets as well as their phosphorylation in monoculture and indirect co-cultures (ico-cultures) in which the cell-cell contact between CRCs and MSCs was absent. Lastly, inhibitors of IR/IGF-1R and Met were used in the intervention studies to uncover the inhibitory effect on CRCs when hMSCs or mMSCs were present.
It was identified that hMSCs promoted the proliferation of HCT116, HT29 and HCT15 CRCs in either the direct or indirect co-culture models, whereas mMSCs did not show marked effects in both ways. Besides, the immortalized hMSCs (ihMSCs) failed to enhance the proliferation of CRCs as the wild-type hMSCs did. Moreover, cancer cell invasion with different growth patterns was observed in co-cultures but not in monoculture. Additionally, enhanced migration of CRCs indirectly co-cultured with hMSCs was observed compared with the monoculture and mMSCs ico-culture. Phosphorylation of Insulin receptor (IR), Met, EphA5 and EphA7 was significantly elevated in hMSC ico-culture compared with monoculture and mMSC ico-culture.
Application of the 3D co-culture models may provide a system in which the potential disconnects between human tumour cells and mouse stromal cells, present in standard animal models, can be investigated. The 3D models also can be useful in studying paracrine pathways between human tumour cells and human stromal cells, which cannot be readily achieved in animal models. The altered p-RTKs identified by the 3D co-culture models may serve as promising targets for novel drug development in CRC treatment.
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