3D Tumor Cultures Research Articles

Abstract B137: Visualization and quantification of tumor-immune cell interactions in 3D cultures

Abstract Introduction: Despite the broad expansion of immunotherapies in cancer research field and drug discovery pipelines in the recent years, the actual number of patients who benefit from them is still extremely low and clinical trial failure rate is quite high. The main reason is that these treatments modalities enter the clinical space without proper understanding of mechanism of action and with a very weak rationale for combinations. However the progress in this direction is hampered by a lack of appropriate pre-clinical models, which remains one of the major bottlenecks in the immune-oncology research. Our goal is to address this issue by developing in vitro models that are both clinically relevant and suitable for routine screening of drug candidates. Experimental Procedures: To ensure the most accurate representation of the tumor biology, cancer cells are co-cultured with immune cells in a 3D setting. Tumor 3D cultures were generated from established cancer cell lines (e.g. breast, prostate) and colorectal cancer organoids (from HUB Organoid Technology) and seeded in 3D ECM-like matrix constituted from protein hydrogel. Different immune cells, such as PBMCs, purified T cells, dendritic cells or macrophages were added to the 3D tumor culture together with different immune-modulators and their effects on the infiltration of the immune cells into tumoroids and their killing was visualized using high-content imaging system. The quantification of these effects was achieved with morphometric analysis with proprietary OMinerTM software. Results: Multiparametric image-based analysis enabled the discrimination of complex immune-tumor cell interactions in 3D co-cultures. The effect of immune cell targeting on the tumor progression was clearly demonstrated by enhanced infiltration of T cells into the tumoroids and their increased killing in the presence of T cell engager. ADCC mechanism could be also depicted with this system. The reported here robust in vitro assay allows image based analysis of 3D cultures in a high-throughput manner. The 3D setting offers possibility to study active migration of immune cells towards tumoroids, infiltration of immune cells into the tumoroids and reduction of tumor volume; all of the functional read-ouds that are based on spatially resolved information. Conclusions: The developed image-based 3D platform allows for analysis of complex immunotherapy effects on different cell types that engage in a more physiologically relevant spatial setting compared to traditional 2D cultures. Preserving the third dimension also in the data analysis process provides a unique opportunity to measure spatially resolved information, not accessible by conventional monolayer cultures or biochemical assays. Visualization and quantification of tumor-immune cells interactions offer a highly powerful tool for cancer immunotherapy drug developers and brings a better understanding of the mechanism of action of novel treatments, which is ultimately translating to better clinical outcomes. Citation Format: Lidia Daszkiewicz, Gera Goverse, Nataliia Beztsinna, Kuan Yan, Emma Spanjaard, Leo Price. Visualization and quantification of tumor-immune cell interactions in 3D cultures [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B137. doi:10.1158/1535-7163.TARG-19-B137

Electrochemical live monitoring of tumor cell migration out of micro-tumors on an innovative multiwell high-dense microelectrode array

Understanding of cell migration and spreading out of tumor tissue is of great interest concerning the mechanism and causes of tumor malignancy and metastases. Although there are methods available for studying cell migration on monolayer cell cultures like transwell assays, novel techniques for monitoring cell spreading out of 3D organoids or tumor tissue samples are highly required. In this context, we developed an innovative high-dense microelectrode array for impedimetric monitoring of cell migration from 3D tumor cultures. For a proof of concept, a strongly migrating breast cancer cell line (MDA-MB-231) and two malignant melanoma cell lines (T30.6.9, T12.8.10ZII) were used for generating viable micro-tumor models. The migration propensity was determined by impedimetric monitoring over 144 hours, correlated by microscopy and validated by transwell assays. The impedimetric analysis of covered electrodes and the relative impedance maximum values revealed extended information regarding the contribution of proliferative effects. More strikingly, using reference populations of mitomycin C treated spheroids where proliferation was suppressed, distinction of proliferation and migration was possible. Therefore, our high-dense microelectrode array based impedimetric migration monitoring has the capability for an automated quantitative analysis system that can be easily scaled up as well as integrated in lab on chip devices.

The Potential Use of Electrochemotherapy in the Treatment of Uveal Melanoma: In Vitro Results in 3D Tumor Cultures and In Vivo Results in a Chick Embryo Model.

Uveal melanoma (UM) is the most common primary intraocular tumor that arises from neoplastic melanocytes in the choroid, iris, and ciliary body. Electrochemotherapy (ECT) has been successfully established for the treatment of skin and soft tissue metastatic lesions, deep-seated tumors of the liver, bone metastases, and unresectable pancreas lesions. The aim of this study was to evaluate the effect of ECT in vitro in 3D spheroid culture systems in primary and metastatic UM cell lines. We also investigated the chick embryo chorioallantoic membrane (CAM) as an in vivo model system for the growth and treatment of UM tumors using ECT. The cytotoxic effect of ECT in 3D spheroids was analyzed seven days following treatment by assessment of the size and MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction] assay. The cytotoxicity of ECT after intratumoral or intraarterial administration was evaluated histologically. In vitro and in vivo ECT caused a significant reduction in tumor size and viability compared to electroporation or chemotherapy in both sections of our study. The current results underline the effectiveness of ECT in the treatment of UM and prepare the way for further investigation of its potential application in UM.

Abstract 45: Patient-derived 3D tumor cultures for compound screening and pre-clinical drug development

Abstract Patient-derived xenograft (PDX) models from human cancers allow propagation, characterization and drug testing on a large panel of tumor variants. To expand the potential of these human-relevant PDX models, we sought to develop 3D ex vivo culture methods for PDX-derived tumor cells that show in vivo-like growth characteristics such as polarization and invasion, and allow measurement of response to therapeutics. By embedding dissociated PDX material in extracellular matrix enriched hydrogels we generate ex vivo cultures that form organotypic 3D tissues driven by relevant interaction with the stromal microenvironment in 384 well plates. Our automated high content screening platform with image analysis powered by OMiner software is used to measure morphologic features of the microtissues. This combination creates a unique high throughput ex vivo PDX screening platform that not only allows efficient profiling drug of responses and quantify tumor spheroid volume, apoptosis and tumor invasion, but also enables selection of the optimal PDX tumor models for subsequent validation of candidates in vivo. Results Hydrogel and growth media composition were optimized to support growth of tumor tissues in vitro from cells derived from various tumor indications, including breast, pancreas, colon, melanoma, and lung cancer PDX tumors. Tumor tissues were cultured in a 384-well format and used to test standard of care chemotherapeutics (e.g. cisplatin, 5-Fu), small molecules (e.g. lapatinib, paclitaxel), and antibodies/ADCs (Trastuzumab, T-DM-1). High content 3D image analysis and data analysis generates a tumor model specific drug profile, reporting therapeutically relevant responses such as cell growth, killing and invasion. These responses serve as a benchmark allowing comparison to novel and experimental drugs, as well as responses across tumors with different mutation profiles. We identified an absolute dependency of a pancreatic PDX model to the WNT-signaling pathway potentiator R-spondin. This allows specific stimulation and targeting of a pathway that is particularly relevant in the development pancreatic cancer. Conclusions In contrast to organoid cultures, short-term ex vivo culture of tumor captures the heterogeneity of the primary tumor as well as enabling access to large collections of well-characterized tumors. We established several PDX model-derived 3D tumor cultures in which standard-of-care, targeted therapies and novel therapeutic agents against specific pathways can efficiently be screened, based on therapeutically relevant parameters and their changing morphological profile. This method enables both the ex vivo selection of drug candidates in a pre-clinical setting as well as efficient selection of PDX models for in vivo follow-up in the same tumor. This highly translational in vitro-in vivo PDX pipeline is expected to reduce attrition and increase efficiency in early drug-discovery. Citation Format: Sander Basten, Bram Herpers, Kuan Yan, Julia Schueler, Leo S. Price. Patient-derived 3D tumor cultures for compound screening and pre-clinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 45.

Abstract 55: Predicting drug sensitivity in patient derived ex vivo 3D mesothelioma cultures

Abstract Background Malignant pleural mesothelioma (MPM) is almost exclusively linked to asbestos exposure with a latency period that is usually more than 30 years. Once diagnosis is made, the prognosis is poor. Systemic treatment combines an antifolate combined with platinum, resulting in modest responses for 40% of the patients. Recently, the addition of bevacizumab to this combination has shown to be of benefit. To date it is impossible to identify the patients who will respond before start of treatment. Second line treatments (either gemcitabine or vinorelbine) have shown to be of benefit in only a small percentage of patients. Tools to improve stratification for systemic treatment in the clinic would be of great value. Our technology based on image analysis of 3D tumor cultures accommodates accurate evaluation of drug sensitivity with small amounts of heterogeneous tumor material (tumor, pleural effusion). We have initiated clinical trials to establish the value of the platform for treatment response prediction for cancer patients, including mesothelioma. Based on the results from these trials we plan to develop diagnostics to predict drug responses for cancer patients. Methods 3D mesothelioma cultures embedded in a protein-rich hydrogel are generated from pleural effusion of chemo naïve patients, and exposed to standard-of-care therapies, targeted therapies and drug combinations. An automated high content screening platform measures cell and tissue morphology, and reports responses such as tumor cell killing, growth arrest, apoptosis, and local invasion. Morphological features are per drug selected as standard read-outs for the response. We correlate clinical response on standard of care drugs with drug response of patients’ tumor cultures. Results We present first results of drug sensitivity in patient derived ex vivo 3D pleural effusion cultures. Results are generated within three weeks after the pleural fluid is obtained from the patients. Take rates are >80%. Standard-of-care therapies were tested and results are compared with clinical response. Patient specific drug responses are identified. Conclusion Ex vivo 3D mesothelioma cultures enable drug sensitivity testing in weeks. Measuring patient-specific treatment responses to standard-of-care drugs, and potential novel therapeutic approaches offer opportunities to personalize treatment for mesothelioma patients. Ongoing trials will reveal the correlation of our in vitro test with treatment responses and relevant diagnostics parameters in the clinic. Keywords: predictive test, mesothelioma, ex vivo 3D tumor cultures Citation Format: Robin Cornelissen, Daphne Dumouline, Jan vd Thusen, Sander Basten, Fanny Grillet, Kuan Yan, Leo Price, Willemijn Vader. Predicting drug sensitivity in patient derived ex vivo 3D mesothelioma cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 55.

A microfluidic platform for multi-size 3D tumor culture, monitoring and drug resistance testing

The establishment of a miniaturized platform capable of microscale control for sequential, coordinated, and throughput biomimetic tumor manipulation and analysis offer a high degree of biological and clinical relevance to cancer therapy and pharmaceutical research. In this study, we developed a microfluidic system with well-established microwell arrays and simple double-layer composition that allows massive fabrication of biomimetic multi-size 3D tumors and their simultaneous manipulation and orthogonal analysis. Serial manipulations including cell localization, array-like self-assembly/cultivation, and dynamic analysis of different types of 3D tumors, were accomplished in the microfluidic device. We demonstrated that the microfluidic platform is stable and throughput cell trapping and tumor generation with quantity uniform. Furthermore, on-chip monitoring and throughput analysis of tumor phenotypes and responses to culture and different chemotherapies were also achieved in the device. The microfluidic advancement offers a new methodological approach for the development of high-performance and multifunctional 3D tumor systems and for tissue-mimicking cancer research and therapy evaluation. This microfluidic platform, which has the capability of multiple control in time and space, holds great potential for applications in the fields of tumor biology, tissue engineering, and clinical medicine.

Cocultures of human colorectal tumor spheroids with immune cells reveal the therapeutic potential of MICA/B and NKG2A targeting for cancer treatment

BackgroundImmunotherapies still fail to benefit colorectal cancer (CRC) patients. Relevant functional assays aimed at studying these failures and the efficacy of cancer immunotherapy in human are scarce. 3D tumor cultures, called tumor organoids or spheroids, represent interesting models to study cancer treatments and could help to challenge these issues.MethodsWe analyzed heterotypic cocultures of human colon tumor-derived spheroids with immune cells to assess the infiltration, activation and function of T and NK cells toward human colorectal tumors in vitro.ResultsWe showed that allogeneic T and NK cells rapidly infiltrated cell line-derived spheroids, inducing immune-mediated tumor cell apoptosis and spheroid destruction. NKG2D, a key activator of cytotoxic responses, was engaged on infiltrating cells. We thus assessed the therapeutic potential of an antibody targeting the specific ligands of NKG2D, MICA and MICB, in this system. Anti-MICA/B enhanced immune-dependent destruction of tumor spheroid by driving an increased NK cells infiltration and activation. Interestingly, tumor cells reacted to immune infiltration by upregulating HLA-E, ligand of the inhibitory receptor NKG2A expressed by CD8 and NK cells. NKG2A was increased after anti-MICA/B treatment and, accordingly, combination of anti-MICA/B and anti-NKG2A was synergistic. These observations were ultimately confirmed in a clinical relevant model of coculture between CRC patients-derived spheroids and autologous tumor-infiltrating lymphocytes.ConclusionsAltogether, we show that tumor spheroids represent a relevant tool to study tumor-lymphocyte interactions on human tissues and revealed the antitumor potential of immunomodulatory antibodies targeting MICA/B and NKG2A.

Abstract 4644: Patient-derived 3D tumor cultures for clinical diagnostics and pre-clinical drug development

Abstract Ex vivo cultures of patient-derived tumor enable functional testing of treatment options and optimization of pre-clinical drug development. Patient biopsies or tumor from patient-derived xenografts (PDX) mouse models are a valuable source of human tumor material that can be cultured in 3D for screening of drug efficacy, drug resistance and cellular processes such as proliferation, survival and invasion. A high throughput approach using 384 well plates enables evaluation of multiple drug treatments and dose ranges in parallel. After compound exposure, cultures are fixed and stained with cellular markers. 3D image stack acquisition is followed by ultra-high content multiparametric analysis using the OMiner platform to profile drug responses and quantify tumor spheroid volume, apoptosis and tumor invasion. Dissected PDX material and tumor biopsy material, were used to establish 3D tumor cultures derived from various indications, including breast, ovarian, cervix, endometrium, stomach, pancreatic, colon, bladder and lung cancer. These were exposed to standard-of-care chemotherapeutics (e.g. 5-FU, taxanes, platinum compounds, anthracyclines, alkylating agents), small molecules (e.g. erlotinib, lapatinib, trametinib), targeted therapies (PARPi; niraparib, olaparib, rucaparib), antibodies (e.g. cetuximab, trastuzumab) and antibody-drug-conjugate (ADC, T-DM1) dose ranges. The tumor culture response was measured, generating dose-dependent profiles for relevant features. Patient-derived 3D tumor cultures were tested with standard-of-care and novel therapeutic agents and high content analysis was used to evaluate drug sensitivity. This method enables both the in vitro selection of drug candidates in a pre-clinical setting as well as efficient selection of PDX models for in vivo follow-up in the same tumor. This highly translational in vitro-in vivo PDX pipeline is expected to reduce attrition and increase efficiency in early drug-discovery. Correlation of drug sensitivity in 3D cultures from fresh patient tumor biopsies, on the other hand, can be used for development of predictive diagnostics and also provides a unique source of patient material for drug discovery and development. Citation Format: Thomas Dijkmans, Sander Basten, Bram Herpers, Kuan Yan, Torsten Giesemann, Julia Schueler, Willemijn Vader, Leo Price. Patient-derived 3D tumor cultures for clinical diagnostics and pre-clinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4644.

Abstract LB-A09: Predicting PARPi sensitivity in patient derived ex vivo 3D tumor cultures

Abstract Ex vivo culture of patient derived tumor material has broad applications including functional testing to support treatment decisions, as well as testing drugs in development. Because average therapeutic response rates are low, there is a need for clinical tools to predict treatment response. Such tools may also reduce overtreatment and provide alternatives when treatment options are exhausted. Here we present data for 3D tumor cultures focusing on standard chemotherapy in combinations with targeted therapy, namely PARPi. By embedding and optimizing growth for small amounts of heterogeneous tumor material in extracellular matrix protein hydrogels, we have generated 3D tumor cultures from various cancer indications. Initiated from both fresh and cryopreserved material, tumor cultures are seeded into 384 well plates and exposed to standard-of-care therapies, targeted therapies and drug combinations. An automated high content screening platform measures cell and tissue morphology, and reports responses such as tumor cell killing, growth arrest and local invasion. Depending on the tumor type and drug mechanism of action, morphological features are selected as standard read-outs for the response. We will present results of proof-of-concept experiments that aim to compare drug sensitivity of tumor cultures with treatment responses in the clinic. Tumor material of gastric, endometrial, cervical, and ovarian cancer patients was used to generate 3D cultures, which were subsequently tested for drug sensitivity. Patient-dependent responses are identified for treatment schedules including platinum-based drugs, taxanes, anthracyclines and 5-FU. In addition, responses to drugs that are not (yet) considered standard of care (PARP inhibitors) were measured. In conclusion, our technology can determine drug sensitivities of 3D cultured tumor tissues. This allows patient-specific treatment responses to standard-of-care drugs to be determined. In addition, this material can be used to test experimental drugs currently in development and in clinical studies. Ongoing proof-of-concept trials will reveal the correlation of our in vitro test with treatment responses in the clinic. Keywords; in vitro diagnostic; organoids; tumor cultures; drug sensitivity testing. Citation Format: Sander Basten, Bram Herpers, Kuan Yan, Willemijn Vader, Leo Price. Predicting PARPi sensitivity in patient derived ex vivo 3D tumor cultures [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A09.

Potentiation of Bio Repositories In Personalized Medicine: Tumor Cells Establishment

The introduction of three-dimensional (3D) tumor cultures has revolutionized anticancer drug research as these cultures allow for the study of drug resistance mechanisms that cannot be explored in traditional two dimensional (2D) monolayer cultures. Discoveries in the 3D tumor culture field suggest that individualized drug sensitivity testing of solid tumor specimens through the establishment and use of 3D tumor cell cultures following tissue collection will become a routine service offered by modern tissue repositories as they expand from their traditional research role to active participation in personalized medicine. Unfortunately, most information related to 3D tumor cultures comes from studies using established tumor cell lines rather than primary tumor cultures. However, accumulation of genetic aberrations in cancer cell lines occurs with increasing number of passages severely limiting their usefulness for personalized medicine. There is only very limited information available concerning technologies and standard operating procedures for the efficient and routine isolation and processing of primary tumor cells for the establishment of 3D tumor cultures from solid tumor specimens. The purpose of this work was to review experimental data from the literature that may provide relevant information concerning the isolation and processing of primary tumor cells for the establishment of 3D tumor cultures. Information reviewed here may help bio repositories in the development and standardization of technologies and standard operating procedures related to the use of 3D tumor cultures.

Role of Biorepositories in Personalized Medicine: Isolation and Processing of Primary Tumor Cells for the initiation Tumor Cultures

The introduction of three-dimensional (3D) tumor cultures has revolutionized anticancer drug research as these cultures allow for the study of drug resistance mechanisms that cannot be explored in traditional two dimensional (2D) monolayer cultures. Discoveries in the 3D tumor culture field suggest that individualized drug sensitivity testing of solid tumor specimens through the establishment and use of 3D tumor cell cultures following tissue collection will become a routine service offered by modern tissue repositories as they expand from their traditional research role to active participation in personalized medicine. Unfortunately, most information related to 3D tumor cultures comes from studies using established tumor cell lines rather than primary tumor cultures. However, accumulation of genetic aberrations in cancer cell lines occurs with increasing number of passages severely limiting their usefulness for personalized medicine. There is only very limited information available concerning technologies and standard operating procedures for the efficient and routine isolation and processing of primary tumor cells for the establishment of 3D tumor cultures from solid tumor specimens. The purpose of this work was to review experimental data from the literature that may provide relevant information concerning the isolation and processing of primary tumor cells for the establishment of 3D tumor cultures. Information reviewed here may help bio repositories in the development and standardization of technologies and standard operating procedures related to the use of 3D tumor cultures.

The Volume of Three-Dimensional Cultures of Cancer Cells InVitro 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 5770: in vitro PDX models: 3D cultured patient-derived tumors for compound evaluation

Abstract Background. Patient-derived xenograft (PDX) models in immune-compromised mice allow propagation of and compound testing in human-derived tumors in vivo. To expand the potential of these human-relevant PDX models, we sought to develop 3D in vitro culture methods for PDX-derived tumor cells that show in vivo-like growth characteristics, invasion and responses to therapeutics. In combination with advanced 3D image analysis methods, we created a unique high throughput in vitro PDX screening platform that not only allows efficient identification of active and selective molecules but also enables selection of the optimal PDX tumor models for subsequent validation of candidates in vivo. Results. Each PDX model has its own unique growth characteristics. Hydrogel and growth media composition were optimized to support growth of tumor tissues in vitro from cells derived from bladder, stomach, breast, pancreas, colon and lung cancer PDX tumors. Tumor tissues were cultured in a 384-well format and used to test chemotherapeutics (e.g. 5-FU, doxorubicin, paclitaxel, cisplatin), small molecules (e.g. erlotinib, lapatinib, trametinib, everolimus), antibodies (e.g. cetuximab, trastuzumab) and antibody-drug-conjugate (ADC, T-DM1) dose ranges. Using OcellO’s 3D image analysis platform, Ominer, tumoroid growth, cell proliferation, apoptosis, invasion, cell polarity, differentiation and other aspects of cell and tissue architecture were analyzed and the effects of compound exposure on tumoroids was determined. By performing feature training based on reference compounds, we selected ±10 morphological features (out of more than 500) to generate a phenotypic signature that described the unique phenotypic change induced by each compound. Different compounds that target the same molecule were found to induce a similar morphological change whereas compounds with off-target effects could be discriminated. This approach enabled a high resolution evaluation and comparison of compound activity in an automated manner. Conclusions. We established several PDX model-derived 3D tumor cultures in which standard-of-care and novel therapeutic agents (small molecules, antibodies and ADCs) can efficiently be screened, based on therapeutically relevant parameters and their changing morphological profile. This method enables both the in vitro selection of promising compounds in a pre-clinically relevant setting and the selection of optimum PDX tumor models for follow-up in vivo studies. This highly translational in vitro-in vivo PDX pipeline is expected to reduce attrition and increase efficiency in early drug-discovery. Citation Format: Sander Basten, Bram Herpers, Julia Schueler, Torsten Giesemann, Leo S. Price. in vitro PDX models: 3D cultured patient-derived tumors for compound evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5770. doi:10.1158/1538-7445.AM2017-5770

Abstract LB-044: A 384-well in vitro PDX culture platform for compound screening and PDX model selection

Abstract Background. There is very high attrition of compounds in early stage anti-cancer drug discovery. This is partly due to the failure of early stage drug testing platforms to represent human biology. 2D cultures of human cells lack the realistic complexity of an organism, leading to overestimation of compound potency. Animal models do retain complexity, but are limited in throughput and frequently fail to reflect human tumor biology. Patient-derived xenograft (PDX) models in immune-compromised mice allow propagation of and compound testing in human-derived tumors. To expand the potential of these human-relevant PDX models, we sought to develop 3D in vitro culture methods for PDX-derived tumor cells that show in vivo-like growth characteristics, invasion and responses to therapeutics. In combination with advanced 3D image analysis methods, we created a unique high throughput in vitro PDX screening platform that not only allows efficient identification of active and selective molecules but also enables selection of the optimal PDX tumor models for subsequent validation of candidates in vivo. Results. Each PDX model has its own unique growth characteristics. Hydrogel and growth media composition were optimized to support growth of tumor tissues in vitro from cells derived from bladder, gastric, breast, colon and lung cancer PDX tumors. Tumor tissues were cultured in a 384-well format and used to screen chemotherapeutics, small molecules, antibodies and antibody-drug-conjugates (ADCs). Using OcellO's 3D image analysis platform, Ominer, tumoroid growth, cell proliferation, apoptosis, invasion, cell polarity, differentiation and other aspects of cell and tissue architecture were analyzed and the effects of compound exposure on tumoroids was determined. By performing feature training based on reference compounds, we selected ±10 morphological features (out of more than 500) to generate a phenotypic signature that described the unique phenotypic change induced by each compound. Different compounds that target the same molecule were found to induce a similar morphological change whereas compounds with off-target effects could be discriminated. This approach enabled a high resolution evaluation and comparison of compound activity in an automated manner. Conclusions. We established several bladder, gastric, breast, colon and lung cancer PDX model-derived 3D tumor cultures in which standard-of-care and novel therapeutic agents (small molecules, antibodies and ADCs) can efficiently be screened, based on therapeutically relevant parameters and their changing morphological profile. This method enables both the in vitro selection of promising compounds in a pre-clinically relevant setting and the selection of optimum PDX tumor models for follow-up in vivo studies. This highly translational in vitro-in vivo PDX pipeline is expected to reduce attrition and increase efficiency in early drug-discovery. Citation Format: Bram Herpers, Lidia Daszkiewicz, Torsten Giesemann, Leo Price. A 384-well in vitro PDX culture platform for compound screening and PDX model selection. [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 LB-044.

An Emerging Role of Biorepositories In Personalized Medicine: Isolation and Processing of Primary Tumor Cells For the Establishment of 3d Tumor Cultures From Solid Tumor Specimens

The introduction of three-dimensional (3D) tumor cultures has revolutionized anticancer drug research as these cultures allow for the study of drug resistance mechanisms that cannot be explored in traditional two dimensional (2D) monolayer cultures. Discoveries in the 3D tumor culture field suggest that individualized drug sensitivity testing of solid tumor specimens through the establishment and use of 3D tumor cell cultures following tissue collection will become a routine service offered by modern tissue repositories as they expand from their traditional research role to active participation in personalized medicine. Unfortunately, most information related to 3D tumor cultures comes from studies using established tumor cell lines rather than primary tumor cultures. However, accumulation of genetic aberrations in cancer cell lines occurs with increasing number of passages severely limiting their usefulness for personalized medicine. There is only very limited information available concerning technologies and standard operating procedures for the efficient and routine isolation and processing of primary tumor cells for the establishment of 3D tumor cultures from solid tumor specimens. The purpose of this work was to review experimental data from the literature that may provide relevant information concerning the isolation and processing of primary tumor cells for the establishment of 3D tumor cultures. Information reviewed here may help biorepositories in the development and standardization of technologies and standard operating procedures related to the use of 3D tumor cultures.

Heterotypic 3D tumor culture in a reusable platform using pneumatic microfluidics.

The construction of a micro-platform capable of microscale control for continuous, dynamic, and high-throughput biomimetic tumor manipulation and analysis plays a significant role in biological and clinical research. Here, we introduce a pneumatic microstructure-based microfluidic platform for versatile three-dimensional (3D) tumor cultures. The manipulative potential of pneumatic microstructures in a fabrication-optimized microfluidic device can be stimulated to achieve ultra-repetitive (tens of thousands of times) and persistent (over several months) microfluidic control. We demonstrated that the microfluidic platform is reusable (dozens of times) for stable, reproducible, and high-throughput generation of tumors with uniform size. Various heterotypic and homotypic 3D tumor arrays can be produced successfully in the device based on robust pneumatic control. On-chip monitoring and analysis of tumor phenotypes and responses to different culture conditions and chemotherapies were also achieved in real-time in the microfluidic platform. The results indicate that fibroblasts cocultured with tumor cells positively promote the phenotypical appearance of heterotypic tumors. This microfluidic advancement offers a new methodological approach for the development of high-performance and non-disposable 3D culture systems and for tissue-mimicking cancer research. We believe that it could be valuable for various tumor-related research fields such as oncology, pharmacology, tissue engineering, and bioimaging.

Monitoring tumor response to anticancer drugs using stable three-dimensional culture in a recyclable microfluidic platform.

The development and application of miniaturized platforms with the capability for microscale and dynamic control of biomimetic and high-throughput three-dimensional (3D) culture plays a crucial role in biological research. In this study, pneumatic microstructure-based microfluidics was used to systematically demonstrate 3D tumor culture under various culture conditions. We also demonstrated the reusability of the fabrication-optimized pneumatic device for high-throughput cell manipulation and 3D tumor culture. This microfluidic system provides remarkably long-term (over 1 month) and cyclic stability. Furthermore, temporal and high-throughput monitoring of tumor response to evaluate the therapeutic efficacy of different chemotherapies, was achieved based on the robust culture. This advancement in microfluidics has potential applications in the fields of tissue engineering, tumor biology, and clinical medicine; it also provides new insight into the construction of high-performance and recyclable microplatforms for cancer research.

Microfluidic 3D HepG2 cell culture: Reproducing hepatic tumor gene and protein expression in in vitro scaffolds

As for 3D cell culture in general, it is also true for tumor cells that 3D conditions create a spatial microenvironment that is stimulated by concentration gradients of nutrients and gases. Cellular vitality and metabolic activity were examined in 2D and scaffold‐based 3D cultures of HepG2 cells. We compared gene and protein expression levels of tumor‐ and hepato‐specific markers such as alpha‐fetoprotein, albumin, integrin beta 1, insulin‐like growth factor , vascular endothelial growth factor A, and matrix metalloproteinase 11. Although similar biological functions were influenced under 3D static and perfused conditions, their gene expression levels differed. Immunofluorescence stainings detected increased integrin beta 1 expression while alpha‐fetoprotein was downregulated both in static and perfused 3D cultures of HepG2 cells. We hypothesized that improved mass flow in the bioreactors enables the cells to retain their nutrient supply, which is indicated by moderate or even downregulated expression of genes related to angiogenesis and apoptosis. Potential hypoxic stress in static 3D cultures of HepG2 cells resulted in upregulated vascular endothelial growth factor A expression and enhanced caspase 3 activity. Tuning the mass flow in 3D tumor cultures may lead to promising tools to investigate the metabolism and differentiation state of tumor tissues in vitro.

A novel 96-well multielectrode array based impedimetric monitoring platform for comparative drug efficacy analysis on 2D and 3D brain tumor cultures.

Aggressive cancer entities like neuroblastoma and glioblastoma multiforme are still difficult to treat and have discouraging prognosis in malignant stage. Since each tumor has its own characteristics concerning the sensitivity towards different chemotherapeutics and moreover, can obtain resistance, the development of novel chemotherapeutics with a broad activity spectrum, high efficacy and minimum side effects is a continuous process. Sophisticated in vitro assays for comprehensive prediction of in vivo drug efficacy and side effects represent an actual bottleneck in the drug development process. In this context, we developed a novel in vitro 2D and 3D multiwell-multielectrode device for drug efficacy monitoring based on direct real-time impedance spectroscopy measurement in combination with our unique 96-well multielectrode arrays and microcavity arrays. For demonstration, we used three neuro- and glioblastoma cell lines that were cultured as monolayer and multicellular tumor spheroids for recapitulating in vivo conditions. Using our novel 96-well multielectrode array based system it was possible to detect time and concentration dependent responses concerning treatment with doxorubicin, etoposide and vincristine. While all tested chemotherapeutics revealed high potency for apoptosis induction in neuroblastoma cells, etoposide was ineffective for glioblastoma cell lines. Determination of IC50 values allowed us to compare drug efficacy in 2D and 3D culture models and moreover, revealed chemotherapeutic and tumor cell line specific activity patterns. These pharmacokinetic patterns are of great interest in the context of preclinical drug development. Thus, impedance spectroscopy based monitoring systems could be used for the fast in vitro based in vivo prediction of novel anti-tumor drugs.

Engineering Tissue Model for Anti-Breast Cancer Pharmacotherapy

Breast cancer is the leading cause of death among women worldwide in both developed and developing countries. Over the years we see an explosion of treatments in the forms of surgical, biological, hormonal, radiation, and chemo therapies for breast cancer patients. Recent advances in tissue engineering herald a new approach to fight breast cancer. This review will begin with an introduction on breast cancer oncology and its pharmacotherapeutic issues, followed by an overview of the current landscape of breast cancer treatments, and then the description of the applications of tissue engineering in breast cancer treatment. Of particular interest is the development of tumor 3D culture models in breast cancer research as a platform for pharmacotherapy. Besides being important in shedding light on breast cancer mechanisms of tumorigenesis, metastasis, microenvironment, signaling pathways, and cancer treatment, in vitro 3D cell model can be used as an assessment tool in anti-breast cancer drug therapy. A part of this review will focus on some of the key constituents of 3D cell model, including the consideration of cell source, biomaterial biocompatibility, the generation of crucial microenvironmental cues, the optimal design of constructs and scaffold for the cells. Finally, this review closes with a brief discussion on the various perfusion configurations currently used for 3D cell culturing systems. Tissue engineering provides a promising mean to further understand breast cancer and most importantly to derive potentially effective treatments for this deadly disease. Keywords: 3D tumor culture model, breast cancer, pharmacotherapy, tumorigenesis, microenvironmental, pathophysiological, pathophysiological, serine/threonine kinase 11, chemotherapy, trinucleotide.