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Effect of TGFβ Treatment on Fibroblast Phenotype and Deposition of Extracellular Matrix Proteins in Three Dimensional Polycaprolactone Fibre Networks

Buzzi, Irina (2020) MOBN03 20192
Degree Projects in Molecular Biology
Popular Abstract
Effects of TGFβ on the extra cellular matrix and its architects the fibroblasts

Fibroblasts are the main architects of the extra cellular matrix (ECM), the non-cellular structure, surrounding the cells throughout the body. The ECM and fibroblasts are constantly communicating with neighboring cells, and an imbalanced ECM state can have detrimental impacts in the organism. In diseases like cancer, the surrounding ECM is highly altered promoting cancer development. It is therefore important to understand these ECM changes to better comprehend disease.

To study and observe changes in the ECM, a three-dimensional (3D) setup is optimal, therefore 3D scaffolds made of polycaprolactone were used in this study. Fibroblasts, together with... (More)
Effects of TGFβ on the extra cellular matrix and its architects the fibroblasts

Fibroblasts are the main architects of the extra cellular matrix (ECM), the non-cellular structure, surrounding the cells throughout the body. The ECM and fibroblasts are constantly communicating with neighboring cells, and an imbalanced ECM state can have detrimental impacts in the organism. In diseases like cancer, the surrounding ECM is highly altered promoting cancer development. It is therefore important to understand these ECM changes to better comprehend disease.

To study and observe changes in the ECM, a three-dimensional (3D) setup is optimal, therefore 3D scaffolds made of polycaprolactone were used in this study. Fibroblasts, together with breast cancer cells were grown in the scaffolds and treated with transforming growth factor-β (TGFβ), a molecule that promotes conversion of fibroblasts into cancer-associated fibroblasts, cells that produce abnormal ECM in a cancer-promoting manner.

To observe the changes generated by TGFβ on fibroblasts and ECM, these were fluorescently labelled and observed using fluorescence microscopy. Different proteins of the ECM were observed and quantified; collagen I, fibronectin, and laminin. The secretion of cytokines IL-6 and TNF-α, signaling molecules was also studied, since these directly impact cell behavior. The cytokines were detected with ELISA a method where antibodies linked to an enzyme bind the cytokine and upon substrate addition generate different color intensities depending on the amount present in the sample.

Results
Treatment of fibroblasts with TGFβ resulted in changes in the cell morphology, the ECM components, and the presence of cytokines. Fibroblasts gained an outstretched morphology growing parallel to each other. The amount of ECM proteins was unchanged after treatment, but the organization was altered for fibronectin and collagen I where the fibers became highly aligned a known feature of these proteins in the cancer-surrounding tissue. This was also seen when fibroblasts were grown together with breast cancer cells under TGFβ treatment. Treated fibroblasts secrete higher amounts of cytokine IL-6 than untreated cells and co-cultures secrete several-fold higher amounts of IL-6 than when fibroblasts are grown on their own, however TGFβ does not affect secretion here. TNF-α was not secreted by either cell types.

The obtained results demonstrate that aspects of the tumour microenvironment can be recreated in these 3D scaffolds by growing fibroblasts together with breast cancer cells under TGFβ treatment. The use of these scaffolds can enable the recreation of a more realistic system for drug screening by covering factors that are absent in conventional two-dimensional cell culturing.

Master’s Degree Project in Molecular Biology 60 credits 2020
Department of Biology, Lund University

Advisor: Stina Oredsson and Atena Malakpour-Permlid
Zoo physiology, cell culturing group. (Less)
Please use this url to cite or link to this publication:
author
Buzzi, Irina
supervisor
organization
course
MOBN03 20192
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9028832
date added to LUP
2020-09-10 09:55:26
date last changed
2020-09-10 09:55:26
@misc{9028832,
  author       = {{Buzzi, Irina}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Effect of TGFβ Treatment on Fibroblast Phenotype and Deposition of Extracellular Matrix Proteins in Three Dimensional Polycaprolactone Fibre Networks}},
  year         = {{2020}},
}