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

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. Conclusions 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

In Vitro Modeling of Tumor-Immune System Interaction.

Immunotherapy has emerged during the past two decades as an innovative and successful form of cancer treatment. However, frequently, mechanisms of actions are still unclear, predictive markers are insufficiently characterized, and preclinical assays for innovative treatments are poorly reliable. In this context, the analysis of tumor/immune system interaction plays key roles, but may be unreliably mirrored by in vivo experimental models and standard bidimensional culture systems. Tridimensional cultures of tumor cells have been developed to bridge the gap between in vitro and in vivo systems. Interestingly, defined aspects of the interaction of cells from adaptive and innate immune systems and tumor cells may also be mirrored by 3D cultures. Here we review in vitro models of cancer/immune cell interaction and we propose that updated technologies might help develop innovative treatments, identify biologicals of potential clinical relevance, and select patients eligible for immunotherapy treatments

Establishment of the culture condition to maintain the primary Human Colorectal Cancer microenvironment by using a 3D perfusion bioreactor

The screening and assessment of new cancer drugs has for many years carried out on conventional 2D monolayer cell culture that cannot represent the complexity of a tissue. On the other hand, the generation of patient derived xenograft (PDX) determines the loss of human-cancer associated stroma and the interaction with murine environment. In this context, the use of a 3D in-vitro systems based on human specimens that maintain the complexity of the in-vivo tissue is needed. Bioreactors that use a pump system to perfuse media directly through a scaffold are known as perfusion bioreactor. The possibility of direct perfusion allows active delivery of nutrient in complex systems. By continuously removing spent media and replacing it with new media, nutrient levels are maintained for optimal growing conditions and cell waste product is removed to avoid toxicity. Moreover perfusion-based bioreactor reduces mass transfer limitations, particularly in the central part of the scaffold. Thus, active perfusion would benefit cellular proliferation and viability. Using colorectal cancer tissue (CRC), we hypothesize that a 3D perfused scaffold-based bioreactor culture system would preserve both the transformed epithelial (EpCAM+) and the non-transformed stromal (Vimentin+ and CD45+) components and, also, keep cells proliferating (Ki67+). We demonstrated the possibility of using a perfusion-based bioreactor system to maintain alive and proliferating freshly surgical excised CRC tissue fragments in porous 3D collagen type-1 scaffolds. After 10 days of culture the tissue partially maintained its original architecture with typical neoplastic disorganization. Phenotypic analysis confirmed that expanded tissues included epithelial, stromal and hematopoietic cells. Tumor cell proliferation, as provide by Ki67 staining, was assessed. Taken together, our results indicate that culture of primary tumor fragments within perfused bioreactors can be successfully achieved over a short-time period allowing the preservation of the diverse cellular components of the CRC specimens. These ex-vivo generated tissues might mirror features of the original tumor more effectively than 2D or 3D static cultures, and of PDX, thus possibly representing useful tools for drug testing and for the evaluation of sensitivity to chemotherapies for personalized medicine

Establishment of the culture condition to maintain the primary Human Colorectal Cancer microenvironment by using a 3D perfusion bioreactor

<p>The screening and assessment of new cancer drugs has for many years carried out on conventional 2D monolayer cell culture that cannot represent the complexity of a tissue. On the other hand, the generation of patient derived xenograft (PDX) determines the loss of human-cancer associated stroma and the interaction with murine environment. In this context, the use of a 3D in-vitro systems based on human specimens that maintain the complexity of the in-vivo tissue is needed. Bioreactors that use a pump system to perfuse media directly through a scaffold are known as perfusion bioreactor. The possibility of direct perfusion allows active delivery of nutrient in complex systems. By continuously removing spent media and replacing it with new media, nutrient levels are maintained for optimal growing conditions and cell waste product is removed to avoid toxicity. Moreover perfusion-based bioreactor reduces mass transfer limitations, particularly in the central part of the scaffold. Thus, active perfusion would benefit cellular proliferation and viability. Using colorectal cancer tissue (CRC), we hypothesize that a 3D perfused scaffold-based bioreactor culture system would preserve both the transformed epithelial (EpCAM+) and the non-transformed stromal (Vimentin+ and CD45+) components and, also, keep cells proliferating (Ki67+). We demonstrated the possibility of using a perfusion-based bioreactor system to maintain alive and proliferating freshly surgical excised CRC tissue fragments in porous 3D collagen type-1 scaffolds. After 10 days of culture the tissue partially maintained its original architecture with typical neoplastic disorganization. Phenotypic analysis confirmed that expanded tissues included epithelial, stromal and hematopoietic cells. Tumor cell proliferation, as provide by Ki67 staining, was assessed. Taken together, our results indicate that culture of primary tumor fragments within perfused bioreactors can be successfully achieved over a short-time period allowing the preservation of the diverse cellular components of the CRC specimens. These ex-vivo generated tissues might mirror features of the original tumor more effectively than 2D or 3D static cultures, and of PDX, thus possibly representing useful tools for drug testing and for the evaluation of sensitivity to chemotherapies for personalized medicine.</p