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Development of a 3D in vitro model to study the impact of COL4A1 expression in Pericytes on Glioblastoma migration by CRISPR/Cas9 knockout

Chitale, Gayatri Girish (2023) MOBN03 20222
Degree Projects in Molecular Biology
Abstract
Glioblastoma (GBM) is the most aggressive type of primary brain tumours in adults and accounts for 49.1% of the cases. Most of the cases are recorded in elderly patients, but GBM affects people of all age groups. Till date no cure has been found. Previous data has suggested that pericytes might influence tumour invasion by interacting with tumour cells by the secretion of or interaction with extracellular matrix proteins. The aim of this project was to establish a 3D in vitro model to study the effects of specific genes overexpressed by pericytes on GBM cell migration. Based on the previous work, COL4A1 was selected as a gene to study. We knocked-out COL4A1 from pericytes by lentivirus mediated CRISPR/Cas9. We induced GFP expression in U87... (More)
Glioblastoma (GBM) is the most aggressive type of primary brain tumours in adults and accounts for 49.1% of the cases. Most of the cases are recorded in elderly patients, but GBM affects people of all age groups. Till date no cure has been found. Previous data has suggested that pericytes might influence tumour invasion by interacting with tumour cells by the secretion of or interaction with extracellular matrix proteins. The aim of this project was to establish a 3D in vitro model to study the effects of specific genes overexpressed by pericytes on GBM cell migration. Based on the previous work, COL4A1 was selected as a gene to study. We knocked-out COL4A1 from pericytes by lentivirus mediated CRISPR/Cas9. We induced GFP expression in U87 cells (GBM cell line) and FACs sorted the GFP+ U87 cells. Gelatin-methacrylate (GelMa) hydrogel was synthesized as a matrix for the 3D model. Endothelial cells, astrocytes, knockout or wild type pericytes and a spheroid of GFP+ U87 cells were embedded in the GelMa. A U87-only group lacking other cell types was included as control. In the co-culture groups cells assembled to form vascular networks mimicking the conditions in the brain. We imaged the setup on days 1, 4 and 7 and designed protocols for analysing the GBM migration. The U87 only group showed a considerably higher migration rate than the co-culture groups. Significant difference was not observed in the migration rates of wildtype and knockout groups likely due to non-optimal cell numbers and analysis methods that are currently used. In this project we successfully setup a 3D in vitro model for studying pericyte-tumour interactions in GBM and progress has been made to optimize the approaches for studying the impact of pericyte genes on migration of GBM cells, however further optimization is required in order to draw meaningful biological conclusions. (Less)
Popular Abstract
Growing brain tumours in a plate

Glioblastoma (GBM) is the most aggressive type of brain tumours in adults. The current treatment procedure is surgery followed by radiotherapy and chemotherapy. Even with the best possible treatment options, there is no cure for GBM. The tumour grows fast and spreads quickly to other parts of the brain. It is one of the deadliest tumours and you don’t want it in your brain! GBMs form new blood vessels for themselves. Pericytes are cells that closely wrap around blood vessels and capillaries. They provide structural support to the blood vessels and have a role in immune functions. Pericytes have important roles in the healthy brain as well as in tumours. Extracellular matrix is a network of molecules that... (More)
Growing brain tumours in a plate

Glioblastoma (GBM) is the most aggressive type of brain tumours in adults. The current treatment procedure is surgery followed by radiotherapy and chemotherapy. Even with the best possible treatment options, there is no cure for GBM. The tumour grows fast and spreads quickly to other parts of the brain. It is one of the deadliest tumours and you don’t want it in your brain! GBMs form new blood vessels for themselves. Pericytes are cells that closely wrap around blood vessels and capillaries. They provide structural support to the blood vessels and have a role in immune functions. Pericytes have important roles in the healthy brain as well as in tumours. Extracellular matrix is a network of molecules that surround and give structural support to cells. Based on the work previously done in our lab, we thought that pericytes might be helping tumours to grow deeper into the brain by interacting with tumour cells through extracellular matrix proteins like collagen 4A1 (COL4A1). The aim of my project was to establish a 3D model to investigate the effects of these pericytes proteins on GBM invasion. We selected the gene for COL4A1 for this purpose.

We prepared a gelatin-based biomaterial called GelMa to be used as the matrix for the 3D model and provide structural support to the cells embedded in it. We performed tests in order to verify that the matrix was not toxic for the cells and to understand what chemical properties it had. Next, we treated GBM cells to express green fluorescent protein (GFP), so that they were easily identified. These cells were grown together to form a spheroid structure which resembles the tumours in the patient. We cut out the COL4A1 gene in pericytes using a technique called CRISPR/Cas9 to stop the production of the COL4A1 protein. The technique is based on the fact that Cas9 protein can cut the DNA in very specific sections. It was introduced in the cells to cut the DNA using a virus that we produced in our lab. Next, we mixed the cell types that form or help the blood vessels (vascular cells) and a GBM spheroid in GelMa. The model had multiple groups depending on the cell types included in the GelMa. One group contained only brain tumour cells and the other groups consisted of all the cell types with either COL4A1 expressing or COL4A1 non-expressing pericytes.

We took images of the matrix by microscopy 1, 4 and 7 days after the cells were put together. The vascular cells assembled to form a blood vessels-similar networks. This showed that the model was effective in imitating the tumour environment in the brain. Pericytes seemed to surround the immediate boundaries of the spheroids in all the groups. Our observations further highlighted the importance of pericytes in association with the tumour cells. We designed multiple analysis methods by which we compared the presence of tumour cells at different distances for the spheroid centre, the rate at which the cells migrate out of the spheroids and the total area covered by the growing spheroids and the cells migrating out of them. Based on our preliminary results, tumour cells surrounded by pericytes that do not express COL4A1 did not migrate faster or further than the tumour cells surrounded by normal pericytes. However, the methods need to be further improved in order to draw meaningful biological conclusions.

Overall, in this thesis project we successfully set up a 3D model for studying pericyte-tumour interactions in GBM and progress has been made to optimize the approaches for understanding the impact of pericyte genes on GBM migration.

Master’s Degree Project in Molecular Biology 60 credits 2023
Department of Biology, Lund University
Supervisor: Gesine Paul-Visse Co-supervisor: Carolina Buizza
Department of Clinical Sciences, Lund University (Less)
Please use this url to cite or link to this publication:
author
Chitale, Gayatri Girish
supervisor
organization
course
MOBN03 20222
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9135693
date added to LUP
2023-08-30 15:25:30
date last changed
2023-08-30 15:25:30
@misc{9135693,
  abstract     = {{Glioblastoma (GBM) is the most aggressive type of primary brain tumours in adults and accounts for 49.1% of the cases. Most of the cases are recorded in elderly patients, but GBM affects people of all age groups. Till date no cure has been found. Previous data has suggested that pericytes might influence tumour invasion by interacting with tumour cells by the secretion of or interaction with extracellular matrix proteins. The aim of this project was to establish a 3D in vitro model to study the effects of specific genes overexpressed by pericytes on GBM cell migration. Based on the previous work, COL4A1 was selected as a gene to study. We knocked-out COL4A1 from pericytes by lentivirus mediated CRISPR/Cas9. We induced GFP expression in U87 cells (GBM cell line) and FACs sorted the GFP+ U87 cells. Gelatin-methacrylate (GelMa) hydrogel was synthesized as a matrix for the 3D model. Endothelial cells, astrocytes, knockout or wild type pericytes and a spheroid of GFP+ U87 cells were embedded in the GelMa. A U87-only group lacking other cell types was included as control. In the co-culture groups cells assembled to form vascular networks mimicking the conditions in the brain. We imaged the setup on days 1, 4 and 7 and designed protocols for analysing the GBM migration. The U87 only group showed a considerably higher migration rate than the co-culture groups. Significant difference was not observed in the migration rates of wildtype and knockout groups likely due to non-optimal cell numbers and analysis methods that are currently used. In this project we successfully setup a 3D in vitro model for studying pericyte-tumour interactions in GBM and progress has been made to optimize the approaches for studying the impact of pericyte genes on migration of GBM cells, however further optimization is required in order to draw meaningful biological conclusions.}},
  author       = {{Chitale, Gayatri Girish}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Development of a 3D in vitro model to study the impact of COL4A1 expression in Pericytes on Glioblastoma migration by CRISPR/Cas9 knockout}},
  year         = {{2023}},
}