Vítor E. Santo

Modelling the tumour microenvironment in long-term microencapsulated 3D co-cultures recapitulates phenotypic features of disease progression

3D cell tumour models are generated mainly in non-scalable culture systems, using bioactive scaffolds. Many of these models fail to reflect the complex tumour microenvironment and do not allow long-term monitoring of tumour progression. To overcome these limitations, we have combined alginate microencapsulation with agitation-based culture systems, to recapitulate and monitor key aspects of the tumour microenvironment and disease progression. Aggregates of MCF-7 breast cancer cells were microencapsulated in alginate, either alone or in combination with human fibroblasts, then cultured for 15 days. In co-cultures, the fibroblasts arranged themselves around the tumour aggregates creating distinct epithelial and stromal compartments. The presence of fibroblasts resulted in secretion of pro-inflammatory cytokines and deposition of collagen in the stromal compartment. Tumour cells established cell-cell contacts and polarised around small lumina in the interior of the aggregates. Over the culture period, there was a reduction in oestrogen receptor and membranous E-cadherin alongside loss of cell polarity, increased collective cell migration and enhanced angiogenic potential in co-cultures. These phenotypic alterations, typical of advanced stages of cancer, were not observed in the mono-cultures of MCF-7xa0cells. The proposed model system constitutes a new tool to study tumour-stroma crosstalk, disease progression and drug resistance mechanisms.

Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery.

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.

Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models.

Currently there is an effort toward the development of in vitro cancer models more predictive of clinical efficacy. The onset of advanced analytical tools and imaging technologies has increased the utilization of spheroids in the implementation of high throughput approaches in drug discovery. Agitation-based culture systems are commonly proposed as an alternative method for the production of tumor spheroids, despite the scarce experimental evidence found in the literature. In this study, we demonstrate the robustness and reliability of stirred-tank cultures for the scalable generation of 3D cancer models. We developed standardized protocols to a panel of tumor cell lines from different pathologies and attained efficient tumor cell aggregation by tuning hydrodynamic parameters. Large numbers of spheroids were obtained (typically 1000-1500 spheroids/mL) presenting features of native tumors, namely morphology, proliferation and hypoxia gradients, in a cell line-dependent mode. Heterotypic 3D cancer models, based on co-cultures of tumor cells and fibroblasts, were also established in the absence or presence of additional physical support from an alginate matrix, with maintenance of high cell viability. Altogether, we demonstrate that 3D tumor cell model production in stirred-tank culture systems is a robust and versatile approach, providing reproducible tools for drug screening and target verification in pre-clinical oncology research.

Drug screening in 3D in vitro tumor models: overcoming current pitfalls of efficacy read-outs

There is cumulating evidence that in vitro 3D tumor models with increased physiological relevance can improve the predictive value of pre‐clinical research and ultimately contribute to achieve decisions earlier during the development of cancer‐targeted therapies. Due to the role of tumor microenvironment in the response of tumor cells to therapeutics, the incorporation of different elements of the tumor niche on cell model design is expected to contribute to the establishment of more predictive in vitro tumor models. This review is focused on the several challenges and adjustments that the field of oncology research is facing to translate these advanced tumor cells models to drug discovery, taking advantage of the progress on culture technologies, imaging platforms, high throughput and automated systems. The choice of 3D cell model, the experimental design, choice of read‐outs and interpretation of data obtained from 3D cell models are critical aspects when considering their implementation in drug discovery. In this review, we foresee some of these aspects and depict the potential directions of pre‐clinical oncology drug discovery towards improved prediction of drug efficacy.

Cysteine allows ovarian cancer cells to adapt to hypoxia and to escape from carboplatin cytotoxicity

Ovarian cancer is the second most common gynaecologic malignancy and the main cause of death from gynaecologic cancer, due to late diagnosis and chemoresistance. Studies have reported the role of cysteine in cancer, by contributing for hydrogen sulphide (H2S) generation and as a precursor of glutathione (GSH). However, the role of cysteine in the adaptation to hypoxia and therapy response remains unclear. We used several ovarian cancer cell lines, ES2, OVCAR3, OVCAR8, A2780 and A2780cisR, to clarify cysteine relevance in ovarian cancer cells survival upon hypoxia and carboplatin. Results show that ES2 and OVCAR8 cells presented a stronger dependence on cysteine availability upon hypoxia and carboplatin exposure than OVCAR3 cells. Interestingly, the A2780 cisR, but not A2780 parental cells, benefits from cysteine upon carboplatin exposure, showing that cysteine is crucial for chemoresistance. Moreover, GSH degradation and subsequent cysteine recycling pathway is associated with ovarian cancer as seen in peripheral blood serum from patients. Higher levels of total free cysteine (Cys) and homocysteine (HCys) were found in ovarian cancer patients in comparison with benign tumours and lower levels of GSH were found in ovarian neoplasms patients in comparison with healthy individuals. Importantly, the total and S-Homocysteinylated levels distinguished blood donors from patients with neoplasms as well as patients with benign from patients with malignant tumours. The levels of S-cysteinylated proteins distinguish blood donors from patients with neoplasms and the free levels of Cys in serum distinguish blood from patients with benign tumours from patients with malignant tumours. Herein we disclosed that cysteine contributes for a worse disease prognosis, allowing faster adaptation to hypoxia and protecting cells from carboplatin. The measurement of serum cysteine levels can be an effective tool for early diagnosis, for outcome prediction and follow up of disease progression.

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.

The Volume of Three-Dimensional Cultures of Cancer Cells In Vitro Influences Transcriptional Profile Differences and Similarities with Monolayer Cultures and Xenografted Tumors

Improving the congruity of preclinical models with cancer as it is manifested in humans is a potential way to mitigate the high attrition rate of new cancer therapies in the clinic. In this regard, three-dimensional (3D) tumor cultures in vitro have recently regained interest as they have been acclaimed to have higher similarity to tumors in vivo than to cells grown in monolayers (2D). To identify cancer functions that are active in 3D rather than in 2D cultures, we compared the transcriptional profiles (TPs) of two non-small cell lung carcinoma cell lines, NCI-H1650 and EBC-1 grown in both conditions to the TP of xenografted tumors. Because confluence, diameter or volume can hypothetically alter TPs, we made intra- and inter-culture comparisons using samples with defined dimensions. As projected by Ingenuity Pathway Analysis (IPA), a limited number of signal transduction pathways operational in vivo were better represented by 3D than by 2D cultures in vitro. Growth of 2D and 3D cultures as well as xenografts induced major changes in the TPs of these 3 modes of culturing. Alterations of transcriptional network activation that were predicted to evolve similarly during progression of 3D cultures and xenografts involved the following functions: hypoxia, proliferation, cell cycle progression, angiogenesis, cell adhesion, and interleukin activation. Direct comparison of TPs of 3D cultures and xenografts to monolayer cultures yielded up-regulation of networks involved in hypoxia, TGF and Wnt signaling as well as regulation of epithelial mesenchymal transition. Differences in TP of 2D and 3D cancer cell cultures are subject to progression of the cultures. The emulation of the predicted cell functions in vivo is therefore not only determined by the type of culture in vitro but also by the confluence or diameter of the 2D or 3D cultures, respectively. Consequently, the successful implementation of 3D models will require phenotypic characterization to verify the relevance of applying these models for drug development.

Abstract A21: 3D tumor models in bioreactors recapitulate microenvironment and disease progression

The high attrition rates observed in cancer drug discovery (up to 95% of failure of drugs tested in phase I trials) have raised the awareness of the scientific and industrial communities towards the need for more predictive pre-clinical models. These models should be more representative of the disease and consequently help to eliminate at pre-clinical stages drug candidates that lack efficacy or safety. Tumor microenvironment is composed by a network of fibroblasts, endothelial cells, immune-competent cells within the extracellular matrix (ECM). Interactions between these components are critical for tumor initiation, proliferation, migration and metastasis. The design of in vitro cancer cell models that recapitulate the tumor microenvironment and 3D architecture provides higher physiological relevance as they more closely resemble the in vivo cellular context. We have established methodologies for scalable generation of 3D cancer cell models in stirred-tank culture systems, and applied these to a large panel of tumor cell lines from different pathologies, including breast, colon, hepatic and lung tumor cell lines. Large numbers of spheroids were obtained per culture (typically 1000-1500 spheroids/mL) with representative characteristics of native tumors, such as morphology, proliferation and hypoxia gradients, in a cell-line dependent mode. With the aim of increasing the relevance of spheroids as tumor cell models, several aspects of tumor microenvironment were incorporated, such as the presence of stromal cells (fibroblasts and monocytes) and specific physical parameters, namely by embedding cells in a polymeric matrix. Heterotypic 3D breast and Non-Small Cell Lung Carcinoma (NSCLC) cancer models, based on co-cultures of tumor cells with stromal cells were established by using an alginate matrix to provide physical support to cells. Tumor spheroids were microencapsulated alone or with fibroblasts and monocytes, thus allowing the establishment of an epithelial tumor compartment and a stromal compartment of increasing complexity. Cultures were performed in stirred-tank vessels for 15 days with continuous monitoring. In both breast and lung tumor models, the presence of fibroblasts was associated with secretion of pro-inflammatory cytokines and accumulation of collagen in the microcapsules. Long-term culture (up to 15 days) resulted in phenotypic alterations in co-cultured breast tumor spheroids, such as loss of cell polarity, reduced cell-cell adhesions, collective cell migration and increased angiogenic potential. In contrast, the effects of fibroblasts were not as significant in NSCLC co-cultures using H1650, H1437 and H157 cell lines suggesting that the effect of tumor-stroma cross-talk is cell line dependent. Moreover, these models have also been shown as feasible tools for drug screening by assessing the effect of chemotherapeutic and specific inhibitors compounds on mono- and co-cultures. In conclusion, we have developed scalable, robust and versatile methodologies for the generation and culture of 3D cancer models, enabling long-term in vitro recapitulation of tumor-stroma crosstalk, via reconstruction of key aspects of the tumor microenvironment, allowing continuous monitoring of disease progression events in vitro. In addition, it is easily transferable to industry for feeding high-throughput systems or miniaturized bioreactors used in drug development, target validation and target identification. Acknowledgments: We acknowledge support from the Innovative Medicines Initiative Joint Undertaking (IMI grant agreement n° 115188), resources composed of financial contribution from EU - FP7 and EFPIA companies in kind contribution. iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundacao para a Ciencia e Tecnologia / Ministerio da Ciencia e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged. This research has also received support from Fundacao para a Ciencia e Tecnologia, Portugal—PTDC/BBB-BIO/1240/2012. MFE, SA and CP are the recipients of PhD fellowships from FCT (SFRH/BD/52208/2013, PD/BD/105768/2014 and SFRH/BD/52202/2013, respectively). Citation Format: Vitor E Santo, Marta F Estrada, Sofia P Rebelo, Sofia Abreu, Catarina Pinto, Elizabeth Anderson, Wolfgang Sommergruber, Paula Alves, Erwin Boghaert, Catarina Brito. 3D tumor models in bioreactors recapitulate microenvironment and disease progression. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A21.

Abstract 630: Co-injection of human fibroblasts significantly enhances tumorigenicity of orthotopically implanted human non-small cell lung cancer cells in immunocompromised mice

Human tumor xenografts in immunodeficient mice have led to valuable insights into the biology of human cancer. The corresponding limitations and pitfalls of xenograft models have been described extensively. To tackle some limitations, we supplemented the murine tumor microenvironment (TME) with human stromal fibroblasts to mirror tumor-stroma cross-talk in vivo. Non-small cell lung cancer (NSCLC) cell lines Calu-1 and H1437 were co-injected with different types of fibroblasts into NOG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice in 3 independent experiments (n = 4-6/group; 106 in total). Human Dermal Fibroblasts (HDFs), lung cancer associated fibroblasts (CAFs), corresponding normal fibroblasts (NFs) and NSCLC cells were implanted into the lungs of 4-6 week old recipient mice. The influence of extracellular matrix components was evaluated by using either a matrigel/collagen mixture or alginate capsules. Tumor load was determined via overall survival (OS) and flow cytometry (FC). When mice had to be sacrificed, organs were collected and analyzed by FC and patho-histological examination. In a subsequent experiment, mice were sacrificed and tumors analyzed at defined time points and similar analyses performed. OS and metastatic pattern were similar in both cell lines when injected in mono-culture. OS of Calu-1 injected mice was not influenced when cells were co-injected with HDF but prolonged when co-injected with CAFs or NFs. In H1437 bearing mice, OS was significantly reduced when cells were co-injected with HDF or NF (Log-rank [Mantel-cox] test; p Our results demonstrate the major impact of fibroblasts on tumor cell behavior in a preclinical setting. With the successful co-cultivation of human fibroblast and NSCLC cells in vivo, it will be possible to study tumor-stroma interactions in a clinically relevant mouse model. Once validated by a compound screening approach, this model will help to reduce drug failure rates and contribute to a more efficient development of urgently needed novel anti-cancer treatments. Citation Format: Eva Oswald, Vitor E. Santo, Albin Rudisch, Catarina Brito, Wolfgang Sommergruber, Helmut Dolznig, Julia B. Schuler. Co-injection of human fibroblasts significantly enhances tumorigenicity of orthotopically implanted human non-small cell lung cancer cells in immunocompromised mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 630.

Abstract 321: In vitro recapitulation of 3D tumor microenvironment with defined oxygen and pH levels through a novel scalable bioreactor-based strategy

Drug discovery for cancer therapy has faced tremendous inefficacy at advanced stages of clinical trials. The scientific community has been trying to tackle this issue by improving preclinical models for target validation, in which the complexity and heterogeneity found in human tumours can be recapitulated. In particular, the tumour microenvironment is known to influence tumour progression and drug resistance through tumour-stroma crosstalk. Three dimensional (3D) cell models allow maintenance of cell-cell and cell-extracellular matrix (ECM) interactions, mimicking in vivo tissue organization. However, current 3D cell models are typically generated in non-scalable culture systems, with poor robustness and use undefined, biologically active matrices. In this work, we present a novel strategy for in vitro recapitulation of solid tumour microenvironments. H1650 (human NSCLC lung adenocarcinoma) and MCF7 (human ER+ breast cancer) cells were selected for the establishment of lung and breast cancer models respectively. An alginate microencapsulation strategy for co-culture of either lung or breast tumour cells with fibroblasts was developed. The constructs were then cultured in stirred-tank bioreactors, with precise definition and control of oxygen and pH levels for several weeks, and characterized by analysis of cell viability, proliferation and apoptosis along culture time, as well as metabolic profiling, phenotypic characterization and analysis of ECM deposition The strategy developed allowed the formation of stable hydrogel microcapsules, with tumour aggregates presenting an epithelial phenotype with partial polarization, surrounded by fibroblasts, recapitulating tumour-stroma organization in vivo. Co-cultures of lung or breast cancer cell aggregates with fibroblasts led to the deposition and accumulation of collagen I type inside the capsules, along with altered tumour cell phenotype, with loss of epithelial character. Perturbation studies with chemotherapeutic agents revealed differences between models established with mono-cultures of tumour cells and co-cultures of tumour cells and fibroblasts. In conclusion, we have developed a scalable, robust and versatile strategy for establishment of in vitro long-term recapitulation of tumour-stroma crosstalk and hypoxic microenvironment, providing tools for characterization of disease progression mechanisms, target validation and drug response in different cancer types. Acknowledgements: The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking (grant agreement n° 115188), resources composed of financial contribution from EU- FP7and EFPIA companies in kind contribution. M.Estrada acknowledges support from FCT, Portugal (SFRH/BD/52208/2013).The authors are also thankful to Cathrin Brisken, Heiko van der Kuip, Moshe Oren and Erwin Boghaert. Citation Format: Vitor E. Santo, Marta Estrada, Sofia Rebelo, Elizabeth Anderson, Paula M. Alves, Catarina Brito. In vitro recapitulation of 3D tumor microenvironment with defined oxygen and pH levels through a novel scalable bioreactor-based strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 321. doi:10.1158/1538-7445.AM2015-321