In Vitro Models of Vascularized Bone for Studying Prostate Cancer Cell Extravasation
Wickramaarachchige, Harini (2023)
Wickramaarachchige, Harini
2023
Master's Programme in Biomedical Technology
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2023-05-17
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202304264478
https://urn.fi/URN:NBN:fi:tuni-202304264478
Tiivistelmä
Prostate cancer is one of the most prevalent malignancies impacting men globally, with most deaths related to metastatic disease. In metastases, the cancer has spread to distant organs. Cancer cells that have disseminated from the primary tumour circulate in the blood stream before extravasating and entering the surrounding tissue. Bone is the most common metastatic site of prostate cancer. The mechanisms behind cancer cell extravasation towards bone still remain poorly understood. We hypothesized that extravasation of prostate cancer cells could be modelled in a vascularized in vitro bone model containing hydroxyapatite and osteoclasts / macrophages.
We prepared two in vitro models of prostate cancer cell extravasation in AIM biotech microfluidic chips: an endothelial barrier model and a vascular network model. The endothelial barrier model was formed by a fibrin hydrogel supplemented with or without hydroxyapatite particles in the central channel flanked by monolayers of human umbilical vein endothelial cells (HUVECS), and human monocyte-derived osteoclasts / macrophages. Prostate cancer cells were added to the medium channel containing a monolayer of HUVECs and maintained for 24h.
Vasculature was formed by a coculture of HUVECs with bone marrow mesenchymal stem/stromal cells (BMSCs) for 6 days in a collagen-fibrin / fibrin hydrogel containing HA with or without osteoclasts, macrophages, or BMSC-derived osteoblasts. Functionality of the endothelial barrier and the vasculature was assessed using fluorescent microbeads. Tartrate resistant acid phosphatase (TRAcP) activity quantification assay was performed to assess the activity of osteoclasts in the cocultures. The hydrogels were immunostained for endothelial markers CD31 and VE-cadherin, osteoclast marker calcitonin receptor, and osteoblast markers osteocalcin and alkaline phosphatase.
We were able to successfully form an endothelial barrier and add osteoclasts and macrophages to the chip. We observed prostate cancer cell extravasation through the endothelial barrier after 24h. Successful vascularization could also be observed in the presence of hydroxyapatite, macrophages / osteoclasts within the hydrogel. Shrinkage of the hydrogel is a recurrent problem, hindering prostate cell extravasation experiments. The study provides promising results which benefits formation of a bone on a chip model in the future.
We prepared two in vitro models of prostate cancer cell extravasation in AIM biotech microfluidic chips: an endothelial barrier model and a vascular network model. The endothelial barrier model was formed by a fibrin hydrogel supplemented with or without hydroxyapatite particles in the central channel flanked by monolayers of human umbilical vein endothelial cells (HUVECS), and human monocyte-derived osteoclasts / macrophages. Prostate cancer cells were added to the medium channel containing a monolayer of HUVECs and maintained for 24h.
Vasculature was formed by a coculture of HUVECs with bone marrow mesenchymal stem/stromal cells (BMSCs) for 6 days in a collagen-fibrin / fibrin hydrogel containing HA with or without osteoclasts, macrophages, or BMSC-derived osteoblasts. Functionality of the endothelial barrier and the vasculature was assessed using fluorescent microbeads. Tartrate resistant acid phosphatase (TRAcP) activity quantification assay was performed to assess the activity of osteoclasts in the cocultures. The hydrogels were immunostained for endothelial markers CD31 and VE-cadherin, osteoclast marker calcitonin receptor, and osteoblast markers osteocalcin and alkaline phosphatase.
We were able to successfully form an endothelial barrier and add osteoclasts and macrophages to the chip. We observed prostate cancer cell extravasation through the endothelial barrier after 24h. Successful vascularization could also be observed in the presence of hydroxyapatite, macrophages / osteoclasts within the hydrogel. Shrinkage of the hydrogel is a recurrent problem, hindering prostate cell extravasation experiments. The study provides promising results which benefits formation of a bone on a chip model in the future.