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Role of cancer associated fibroblasts in macrophage recruitment in head and neck cancer

Prajapati, Priyanka (2018) Role of cancer associated fibroblasts in macrophage recruitment in head and neck cancer. PhD thesis, University of Sheffield.

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Background: Cancer associated fibroblasts (CAFs) are known to stimulate crosstalk between stroma and cancer via paracrine signalling and promote cancer progression through matrix remodelling, angiogenesis and immunosuppression. CAFs arise from many sources and display different markers and characteristics in the tumour stroma; their presence in head and neck cancers is associated with poor prognosis. This study examines the differences in secretome of myofibroblasts and senescent fibroblasts in the context of their ability to recruit and polarise macrophages, with the aim of establishing which phenotype recruits more macrophages, which chemokine/receptor is primarily employed and which way are the macrophages polarised. Methods: Normal oral fibroblasts (NOFs) were transdifferentiated to myofibroblasts (MF) using TGF-β1, or senesced using cisplatin. MF phenotype was confirmed by expression of α-smooth muscle actin (α-SMA), fibronectin with extra domain A (FNEDA), and collagen type 1 alpha 1 (COL1A1) using RT-qPCR, western blot and immunofluorescence. Senescence was confirmed by senescence associated βgalactosidase assay, RT-qPCR for p16INK4A and p21CIP1/WAF-1. Primary monocytes were allowed to migrate towards conditioned medium (CM) from MFs, senescent fibroblasts (SFs) and patient CAFs in a Boyden chamber assay. The involvement of chemokine receptors were scrutinised using CCR2 antagonist and pertussis toxin. Macrophages were also polarised in the same CM, as assessed by qPCR. Levels of The secretome of MFs, SFs and patient-derived CAF was examined using ELISA, cytokine array and mass spectrometry. Results: CCL2 secretion correlated with monocyte migration through CCL2/CCR2 axis towards CM from SF, MF and most patient CAFs. SFs secreted more IL-6 and CCL2 than MFs, in which secretion declined with longer duration of TGF-β1 treatment. Cytokine array detected more cytokines secreted by MF than SF, while MS revealed more collagen interacting proteins secreted by SF, while both secreted several types of collagen. MF, SF and patient CAF CM also polarised macrophages towards M2 phenotype, increasing CD206 mRNA compared to normal firboblasts CM. While CM from unpolarised (M0) macrophages induced highest expression of αSMA expression. Conclusion: SF recruits more monocytes through CCL2/CCR2 axis and secreted more collagen interacting proteins than MF. However, both seem to polarisation macrophages towards M2 phenotype. Collectively the data identify differences and similarities in the secretome of MF and SF, and one or both can be potentially targeted to manipulate immune cell populations in tumour stroma to improve prognosis.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Dentistry (Sheffield)
Identification Number/EthosID: uk.bl.ethos.745700
Depositing User: Dr Priyanka Prajapati
Date Deposited: 12 Jul 2018 08:51
Last Modified: 01 Feb 2020 10:53
URI: http://etheses.whiterose.ac.uk/id/eprint/20901

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