Maintenance of primary human colorectal cancer microenvironment using a perfusion bioreactor-based 3D culture system

Manfredonia, Celeste. Maintenance of primary human colorectal cancer microenvironment using a perfusion bioreactor-based 3D culture system. 2018, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_13300

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Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide, often diagnosed in advanced stage. Chemotherapeutic regimens currently in use for human CRC show limited success rates, underlying the need of novel and personalized therapeutic schemes.
A pre-requisite for the development of tailored treatments is the possibility to predict patient responsiveness. However, a major challenge is represented by the lack of adequate in vitro models.
The heterogeneous tissue composition of CRC patients has been recognized to play a key role in response to treatment due to the interaction between cancerous and non-transformed cells within the tumor microenvironment. However, all novel experimental approaches proposed for the evaluation of tumor drug responses, including primary cell cultures or xenotransplantation of cancer specimens in immunodeficient animals, result in loss or dramatic modifications of the tumor microenvironment. Thus, the development of adequate in vitro models allowing maintenance of whole CRC microenvironment is urgently needed.
Here we have investigated the suitability of a perfusion- based bioreactor- culture system to maintain primary CRC tissues.
Freshly excised CRC specimens were cut into fragments, inserted between two collagen type I sponges in a “sandwich-like” format and cultured for three days in a perfused-based bioreactor system or under static conditions.
Fresh tissues, tissues cultured under perfusion and static conditions were weighted and subjected to histomorphological evaluation. Percentage of epithelial cells was evaluated upon hematoxylin and eosin staining. Number of stromal, hematopoietic cells and total cell nuclei were counted using CellProfiler image analysis software following staining for vimentin, CD45, and DAPI, respectively. Viability of tumor cells was assessed upon Ki67 and cleaved caspase 3 staining. The preservation of functionality of tumor-associated stromal cells in perfused cultures was evaluated by assessing release of IL-6 upon stimulation with IL-17. For assessment of immune cells, IL-2 and IFN-g release upon activation with Phytohaemagglutinin was measured. Finally, drug responsiveness of CRC tissue in perfused cultures, was evaluated by assessing proliferation and apoptosis of tumor cells to the conventional chemotherapeutic 5-Fluorouracil, upon Ki67 and cleaved caspase 3 staining, respectively.
Our results showed that CRC tissues cultured under perfusion preserve the tissue mass at higher extent as compared to static cultures. Moreover, perfused tissues maintained higher tissue cellularity in comparison to static cultures.
Tumor cells cultured under perfusion displayed an almost intact structure, as compared to the original tumors, and were viable and proliferating. In addition, stromal cells were maintained in proportions similar to those of original tumors and fully viable, as indicated by responsiveness to micro-environmental stimuli, such as IL-17. Furthermore, immune cells were also partially preserved, and were capable of releasing effector cytokines, such as IL-2 and IFN-, upon activation by mitogenic stimulation.
In contrast, in cultures performed under static conditions, fewer viable tumor and stromal cells were preserved, whereas immune cells were completely lost. In fact, in static cultures, percentages of proliferating cells were significantly reduced, whereas those of apoptotic cells were significantly increased.
Importantly, perfusion-based cultures proved suitable for testing the sensitivity of primary tumor cells to chemotherapies of current use in CRC. Indeed, following three days of treatment with 5-fluouracil (5-FU), an overall significant reduction in percentages of epithelial proliferating cells, and a significant increase in the fraction of apoptotic cells could be observed. Notably, analysis of individual samples revealed heterogeneous responses across different tumors.
Our results cumulatively suggest that primary CRC culture under perfusion preserve the microenvironment with its native tissue architecture and composition. Importantly, our culture system also preserves viability and functionality of non-transformed cells, including mesenchymal stromal cells and tumor infiltrating lymphocytes.
Moreover, bioreactor-based cultures are amenable for testing sensitivity of primary CRC tissues to currently used chemotherapies and reveal heterogeneous responsiveness across different samples.
Thanks to its capacity to maintain TME heterogeneity, our system may allow personalized drug testing within a more physiological context. Our culture system may also prove suitable for testing therapies whose efficacy is influenced by whole TME, such as drug-loaded nanoparticles and emerging stroma-targeted therapies currently under clinical investigation for CRC. Furthermore, we envisage validating its ability to predict patient-specific clinical responses in the context of follow-up studies.
Advisors:Martin, Ivan and Christofori, Gerhard M.
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel > Tissue Engineering (Martin)
05 Faculty of Science
UniBasel Contributors:Martin, Ivan and Christofori, Gerhard M.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13300
Thesis status:Complete
Number of Pages:1 Online-Ressource (verschiedene Seitenzählungen)
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Last Modified:07 Nov 2019 05:30
Deposited On:06 Nov 2019 16:13

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