3D Tumor Cultures Research Articles

Abstract 4200: A novel 3D culture platform, E-slice, retains intact tumor microenvironment and tumor infiltrating immune cells

Abstract Human cancer tissue is a complex ecosystem with heterogeneous cell types that communicate with each other dynamically to promote tumor progression and immune evasion. Hence, experimental systems that destroy or select for cancer cells in culture have often failed to accurately predict clinical responses of new drugs. Therefore, a technology that can retain the intact tumor microenvironment (TME) and accurately predict cancer patient’s response to anti-cancer treatments, including immunotherapies, will greatly benefit patients and dramatically reduce the cost of new drug development. E-slice is a proprietary 3D human tumor culture platform that maintains individual patient tumor’s unique ecosystem and the TME ex vivo for up to 30 days. It is generated by making thin sections of intact, fresh tumor tissues and culturing them in serum-free, defined media. As such, immune components and the TME in E-slices faithfully recapitulate human tumors ex vivo. We demonstrate that E-slices can be used to: 1) measure viability changes upon anti-cancer agent treatment longitudinally; 2) retain the native TME and tissue architecture since E-slices are never dissociated or artificially reconstituted; 3) culture any solid tumor types from patient needle biopsy cores or surgical samples, PDX, or mouse models; 4) perform high content drug screening on human tissues; 5) discover secreted biomarkers and to 6) discover or validate molecular mechanisms of action or therapy resistance; and 7) measure immunotherapy responses ex vivo from human tissues. Importantly, it has been shown to accurately predict individual patient treatment responses to chemotherapies and targeted therapies in 4-8 days, paving the way for evidence-based personalized treatment selections in a clinically actionable time frame. Furthermore, single cell RNA-sequencing analysis demonstrates that E-slices can retain the viability and molecular phenotypes of tumor infiltrating immune cells for up to 8 days ex vivo, providing a unique platform to study human tumor-immune interactions and evaluate efficacy of immune modulators on human tumor tissues ex vivo. Citation Format: Viridiana Leyva-Aranda, Thomas Gallup, Jose Maldonado, Corina Margain, Sang Yun, David Gallup, Min Kim, Kyuson Yun. A novel 3D culture platform, E-slice, retains intact tumor microenvironment and tumor infiltrating immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4200.

A PDA-Functionalized 3D Lung Scaffold Bioplatform to Construct Complicated Breast Tumor Microenvironment for Anticancer Drug Screening and Immunotherapy.

2D cell culture occupies an important place in cancer progression and drug discovery research. However, it limitedly models the "true biology" of tumors in vivo. 3D tumor culture systems can better mimic tumor characteristics for anticancer drug discovery but still maintain great challenges. Herein, polydopamine (PDA)-modified decellularized lung scaffolds are designed and can serve as a functional biosystem to study tumor progression and anticancer drug screening, as well as mimic the tumor microenvironment. PDA-modified scaffolds with strong hydrophilicity and excellent cell compatibility can promote cell growth and proliferation. After 96h treatment with 5-FU, cisplatin, and DOX, higher survival rates in PDA-modified scaffolds are observed compared to nonmodified scaffolds and 2D systems. The E-cadhesion formation, HIF-1α-mediated senescence decrease, and tumor stemness enhancement can drive drug resistance and antitumor drugscreening of breast cancer cells. Moreover, there is a higher survival rate of CD45+ /CD3+ /CD4+ /CD8+ T cells in PDA-modified scaffolds for potential cancer immunotherapy drug screening. This PDA-modified tumor bioplatform will supply some promising information for studying tumor progression, overcoming tumor resistance, and screening tumor immunotherapy drugs.

Abstract 3614: 3D model to study migration and invasion of lung cancer

Abstract Background: In 2D cell cultures, migration of tumor cells is characterized by epithelial-to-mesenchymal transition (EMT), whereby tumor cells loose epithelial and express mesenchymal markers and very often change into a spindle cell phenotype. In contrast to 2D cultures, in 3D cultures tumor cells do not lose their differentiation. Small cell lung cancer usually moves as single cells or in small clusters, squamous cell and adenocarcinomas preferentially move in large clusters of cells. In tissue specimen, all of these carcinomas migrate in a hybrid EMT, not loosing their cytokeratin and E-cadherin expression. In AC and SCC also polarity and specification do occur, as some tumor cells act as leaders, providing orientation for the followers. However, 3D culture system might better depict real migration and come closer to the in vivo system as 2D cell culture systems. Material and Method: A 3D bioprinter (TissueLabsR) was used to spot an alveolar-mimicking lung-specific matrix on cell culture wells. Microscopic channels out of pluronicsR are created within the matrix. Pluronics is removed by cooling, leaving empty channels within the protein matrix. These channels can be filled with different interleukins, such as IL23, which will direct migration of the tumor cells. Cultured cell lines from an adenocarcinoma (A549) and small cell carcinoma (NCI-H82) are layered on top of the matrix and migration into the matrix is studied using different time points. Results and Discussion: A matrix simulating an alveolar structure was created with MatrigelR. The A549 cells were seeded on the matrix and grown for 2,5, 10, and 14 days. The cells formed cell clusters on the matrix and after one week started to invade the matrix in cell complexes. By immunohistochemistry we could show, that the A549 cells retained their epithelial differentiation. The H82 cells showed a different behavior. They did not form large clusters, but started to invade in small groups. The use of the bioprinter enabled us not only to create a 3D tumor culture system to simulate the in vivo conditions but also to create matrix structures for studying invasion and migration and to stimulate carcinoma cells with migration-activating substances. Citation Format: Melina Hardt, Kurt Zatloukal, Helmut H. Popper. 3D model to study migration and invasion of lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3614.

Combinatorial Drug Screening Based on Massive 3D Tumor Cultures Using Micropatterned Array Chips.

The establishment and application of a generalizable three-dimensional (3D) tumor device for high-throughput screening plays an important role in drug discovery and cancer therapeutics. In this study, we introduce a facile microplatform for considerable 3D tumor generation and combinatorial drug screening evaluation. High fidelity of chip fabrication was achieved depending on the simple and well-improved microcontact printing. We demonstrated the high stability and repeatability of the established tumor-on-a-chip system for controllable and massive production of 3D tumors with high size uniformity. Importantly, we accomplished the screening-like chemotherapy investigation involving individual and combinatorial drugs and validated the high accessibility and applicability of the system in 3D tumor-based manipulation and analysis on a large scale. This achievement in tumor-on-a-chip has potential applications in plenty of biomedical fields such as tumor biology, pharmacology, and tissue microengineering. It offers an insight into the development of the popularized microplatform with easy-to-fabricate and easy-to-operate properties for cancer exploration and therapy.

Assessing Polysaccharides/Aloe Vera-Based Hydrogels for Tumor Spheroid Formation.

In vitro tumor spheroids have proven to be useful 3D tumor culture models for drug testing, and determining the molecular mechanism of tumor progression and cellular interactions. Therefore, there is a continuous search for their industrial scalability and routine preparation. Considering that hydrogels are promising systems that can favor the formation of tumor spheroids, our study aimed to investigate and develop less expensive and easy-to-use amorphous and crosslinked hydrogels, based on natural compounds such as sodium alginate (NaAlg), aloe vera (AV) gel powder, and chitosan (CS) for tumor spheroid formation. The ability of the developed hydrogels to be a potential spheroid-forming system was evaluated using MDA-MB-231 and U87MG cancer cells. Spheroid abilities were influenced by pH, viscosity, and crosslinking of the hydrogel. Addition of either AV or chitosan to sodium alginate increased the viscosity at pH 5, resulting in amorphous hydrogels with a strong gel texture, as shown by rheologic analysis. Only the chitosan-based gel allowed formation of spheroids at pH 5. Among the variants of AV-based amorphous hydrogels tested, only hydrogels at pH 12 and with low viscosity promoted the formation of spheroids. The crosslinked NaAlg/AV, NaAlg/AV/glucose, and NaAlg/CS hydrogel variants favored more efficient spheroid formation. Additional studies would be needed to use AV in other physical forms and other formulations of hydrogels, as the current study is an initiation, in evaluating the potential use of AV gel in tumor spheroid formation systems.

Fluorescence intensity and lifetime imaging of lipofuscin-like autofluorescence for label-free predicting clinical drug response in cancer

Conventional techniques for in vitro cancer drug screening require labor-intensive formalin fixation, paraffin embedding, and dye staining of tumor tissues at fixed endpoints. This way of assessment discards the valuable pharmacodynamic information in live cells over time. Here, we found endogenous lipofuscin-like autofluorescence acutely accumulated in the cell death process. Its unique red autofluorescence could report the apoptosis without labeling and continuously monitor the treatment responses in 3D tumor-culture models. Lifetime imaging of lipofuscin-like red autofluorescence could further distinguish necrosis from apoptosis of cells. Moreover, this endogenous fluorescent marker could visualize the apoptosis in live zebrafish embryos during development. Overall, this study validates that lipofuscin-like autofluorophore is a generic cell death marker. Its characteristic autofluorescence could label-free predict the efficacy of anti-cancer drugs in organoids or animal models.

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

Extracellular matrix stiffness mediates radiosensitivity in a 3D nasopharyngeal carcinoma model

PurposeRadiotherapy is one of the essential treatment modalities for nasopharyngeal carcinoma (NPC), however, radioresistance still poses challenges. Three-dimensional (3D) tumor culture models mimic the in vivo growth conditions of cells more accurately than 2D models. This study is to compare the tumor biological behaviors of NPC cells in 2D, On-Surface 3D and Embedded 3D systems, and to investigate the correlation between radioresistance and extracellular matrix (ECM) stiffness.MethodsThe morphology and radioresistance of the human NPC cell line CNE-1 were observed in 2D and 3D systems. The CCK-8 assay, wounding healing assays, flow cytometry, soft agar assays, and western blot analysis were used to evaluate differences in biological behaviors such as proliferation, migration, cell cycle distribution, and stem cell activity. Different ECM stiffness systems were established by co-blending collagen and alginate in varying proportions. ECM stiffness was evaluated by compressive elastic moduli measurement and colony formation assay was used to assess radioresistance of NPC cells in systems with different ECM stiffness after irradiation.ResultsCompared to 2D models, the morphology of NPC cells in 3D culture microenvironments has more in common with in vivo tumor cells and 3D cultured NPC cells exhibit stronger radioresistance. Integrin β1 but not the epithelial-to-mesenchymal transition pathway in 3D models boost migration ability. Cell proliferation was enhanced, the proportion of tumor stem cells was increased, and G1/S phase arrest occurred in 3D models. NPC cells cultured in softer ECM systems (with low alginate proportions) exhibit striking resistance to ionizing radiation.ConclusionThe tumor biological behaviors of NPC cells in 3D groups were obviously different from that of 2D. Radioresistance of NPC cells increased with the stiffness of ECM decreasing.

Microfluidics guided by deep learning for cancer immunotherapy screening

Immunocyte infiltration and cytotoxicity play critical roles in both inflammation and immunotherapy. However, current cancer immunotherapy screening methods overlook the capacity of the T cells to penetrate the tumor stroma, thereby significantly limiting the development of effective treatments for solid tumors. Here, we present an automated high-throughput microfluidic platform for simultaneous tracking of the dynamics of T cell infiltration and cytotoxicity within the 3D tumor cultures with a tunable stromal makeup. By recourse to a clinical tumor-infiltrating lymphocyte (TIL) score analyzer, which is based on a clinical data-driven deep learning method, our platform can evaluate the efficacy of each treatment based on the scoring of T cell infiltration patterns. By screening a drug library using this technology, we identified an epigenetic drug (lysine-specific histone demethylase 1 inhibitor, LSD1i) that effectively promoted T cell tumor infiltration and enhanced treatment efficacy in combination with an immune checkpoint inhibitor (anti-PD1) invivo. We demonstrated an automated system and strategy for screening immunocyte-solid tumor interactions, enabling the discovery of immuno- and combination therapies.

Flipped Well-Plate Hanging-Drop Technique for Growing Three-Dimensional Tumors.

Three-dimensional (3D) tumor culture techniques are gaining popularity as in vitro models of tumoral tissue analogues. Despite the widespread interest, need, and present-day effort, most of the 3D tumor culturing methodologies have not gone beyond the inventors’ laboratories. This, in turn, limits their applicability and standardization. In this study, we introduce a straightforward and user-friendly approach based on standard 96-well plates with basic amenities for growing 3D tumors in a scaffold-free/scaffold-based format. Hanging drop preparation can be easily employed by flipping a universal 96-well plate. The droplets of the medium generated by the well-plate flip (WPF) method can be easily modified to address various mechanisms and processes in cell biology, including cancer. To demonstrate the applicability and practicality of the conceived approach, we utilized human colorectal carcinoma cells (HCT116) to first show the generation of large scaffold-free 3D tumor spheroids over 1.5 mm in diameter in single-well plates. As a proof-of-concept, we also demonstrate matrix-assisted tumor culture techniques in advancing the broader use of 3D culture systems. The conceptualized WPF approach can be adapted for a range of applications in both basic and applied biological/engineering research.

Abstract 6029: Long-term three-dimensional (3D) tumor culture models using a universal well-plate platform

Abstract Generating 3D tumor spheroid in vitro has been a straightforward method not only to replicate in vivo like cytoarchitecture and also to study complex interactions to the 3D milieu. Although various approaches have been developed to date, most of the in vitro techniques were hampered by the limitations like small-volume, non-uniformity, irregular sphericity, short-term culture, labor-intensive and studies’ complexities. Here, we have developed very intuitive methods to generate 3D tumor spheroid models and scaffold-based 3D culture models in a single well of universal plate for long-term cultivation. Experimental Procedures: The prototype of 3D printed chamber was designed by Sketchup software (Trimble) and printed at Illinois MakerLab (http://makerlab.illinois.edu) for this study. The human colon cancer cell (HCT116), obtained from American Type Culture Collection (ATCC), were cultured in DMEM medium containing 10% FBS and stored in a humidified incubator of 5% CO 2 at 37 °C. Five universal 96 well plates (Corning, Thermo Scientific, CytoOne, Advangene, and Genesee Scientific), Elisa 96 well strip (Corning), Black 96-well plate (Thermo scientific) were used without any modification (Not a type of ultra-low attachment (ULA) plate). The prototype designs are flexible to be modified and will be utilized to develop a product beyond conceptual frameworks in the market. Results: Recently, microfabricated approaches have been intensively studied for micro 3D tumor models (< 300 µm in diameter) implemented with high-throughput analysis. However, the micro tumor model incubated in a short-term might not effectively develop tumor heterogeneity such as a non-perfusion core, diffusion gradient and increased interstitial pressure. We intentionally generated a tumor spheroid model incubated over 1 month with appropriate media replacement to supply nutrients. The spheroid in the system grew over 1.5mm of diameter with an excellent sphericity (> 0.95). Besides, our methodology can perform various in-situ spheroid assays in basic cancer research and also generate scaffold-free/based 3D tumor culture models in the system. To replicate in vivo like cytoarchitecture more physiologically relevant, decellularized tissue scaffold-based 3D culture was conceptually demonstrated. Conclusions: We developed a simple and effective process that generates 3D tumor culture models in millimeter size and allows long-term incubation to develop local heterogeneity. Importantly, this approach does not require additional hidden costs and any specialized instruments. We believe the present strategy satisfies all essential criteria (i.e., easiness, accessibility, reproducibility, effectiveness, and applicability) to appeal to general researchers compared to other available methods by far. Citation Format: Yoon Jeong, Ashley Tin, Joseph Irudayaraj. Long-term three-dimensional (3D) tumor culture models using a universal well-plate platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6029.

Abstract P113: Image-based quantification of immunotherapeutic effect on the tumor-immune interactions in 3D co-cultures

Abstract INTRODUCTION Despite the increasing number of immunotherapies available in preclinical cancer research and drug discovery pipelines, the number of patients benefiting from these immunotherapies remains extremely low. Moreover, the failure of immunotherapies in clinical trials remains very high to date. These high failure rates could be attributed to the lack of deep understanding the mechanism of action of drug candidates and to the absence of clinically relevant preclinical models that are suitable for drug screening purposes. To address these issues, we have developed an in vitro drug screening platform suitable for high-throughput testing in physiologically relevant 3D environment. Our preclinical drug testing platform is based on the co-culture of tumor cells with immune cells in 3D. Image analysis is applied to read out effects of drug candidates on immune cell migration, immune cell infiltration and tumoroid killing. MATERIALS AND METHODS 3D tumor cultures from human cancer cell lines (including breast, lung and colon cancer) or patient-derived colorectal organoids (from HUB Organoid Technology) were generated by embedding tumor cells in 3D ECM-like matrix constituted from protein hydrogel. Subsequently, different immune subsets, including PBMCs from healthy donors, T cells, NK cells or macrophages, were stained with cell tracker and added to the cultures. The cultures were treated with different immune-modulators (e.g. superantigens, activating antibodies, T cell engagers, CSFR1 inhibitor or STING agonist). The effect of immune-modulators on immune cell infiltration and their killing was assessed by high-content imaging and quantified after morphometric analysis with the proprietary Ominer® software. Immune cell-mediated killing was confirmed by measurement of IFNγ secretion in the culture supernatants. RESULTS Image-based analysis allowed for the dissection of complex tumor-immune cell interactions in the 3D cultures. Moreover, morphometric analysis revealed different levels of immune cell infiltration and tumoroid killing upon treatment with different immune-modulators. These effects could be reduced by inhibitory signals from the tumor microenvironment (TME) and were confirmed by the levels of IFNγ secreted by the immune cells. CONCLUSION Our in vitro platform allows quantitative image-based analysis of 3D tumor-immune cell co-cultures in a high-throughput manner, based on spatially resolved information in a more physiologically-relevant setting compared to traditional 2D cultures. This image-based analysis could be employed to dissect the effect of immunotherapy on different cell populations with the TME representing a promising tool to improve our understanding of the mechanism of action of novel treatments, treatment relapse and combination strategies, to eventually lead to a better clinical performance. Citation Format: Lidia Daszkiewicz, Gera Goverse, Nataliia Beztsinna, Marjan van de Merbel, Benjamin Visser, Tomas Veenendaal, Emma Spanjaard, Kuan Yan, Leo Price. Image-based quantification of immunotherapeutic effect on the tumor-immune interactions in 3D co-cultures [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P113.

Three-Dimensional Culture Models to Study Innate Anti-Tumor Immune Response: Advantages and Disadvantages.

Simple SummaryThe classical approach to study the immune response against a tumor was mixing immune cells with tumor cell suspensions in several experimental settings. These models lack the appropriate tissue architecture in which the immune response takes place and do not consider other cellular and extracellular players of the tumor microenvironment essential to understand the anti-tumor immune response. Thus, to confirm in vitro data, in vivo experiments have been extensively performed, using animal models that may not fully reproduce what happens in humans. Indeed, in animal-based studies, tumors are artificially generated in a short time, and immune cell subsets and receptor-ligands pairs, involved in tumor cells recognition by the immune system, are often different from human counterparts. To reduce the number of animals used, and possibly replace animal models, alternative methods of culture have been developed. Herein, some of these approaches will be described, highlighting their advantages and disadvantages, focusing on natural killer cells as the first line of anti-tumor effector cells able to contrast tumor growth.Several approaches have shown that the immune response against tumors strongly affects patients’ clinical outcome. Thus, the study of anti-tumor immunity is critical to understand and potentiate the mechanisms underlying the elimination of tumor cells. Natural killer (NK) cells are members of innate immunity and represent powerful anti-tumor effectors, able to eliminate tumor cells without a previous sensitization. Thus, the study of their involvement in anti-tumor responses is critical for clinical translation. This analysis has been performed in vitro, co-incubating NK with tumor cells and quantifying the cytotoxic activity of NK cells. In vivo confirmation has been applied to overcome the limits of in vitro testing, however, the innate immunity of mice and humans is different, leading to discrepancies. Different activating receptors on NK cells and counter-ligands on tumor cells are involved in the antitumor response, and innate immunity is strictly dependent on the specific microenvironment where it takes place. Thus, three-dimensional (3D) culture systems, where NK and tumor cells can interact in a tissue-like architecture, have been created. For example, tumor cell spheroids and primary organoids derived from several tumor types, have been used so far to analyze innate immune response, replacing animal models. Herein, we briefly introduce NK cells and analyze and discuss in detail the properties of 3D tumor culture systems and their use for the study of tumor cell interactions with NK cells.

Simplified ARCHITECT microfluidic chip through a dual-flip strategy enables stable and versatile tumoroid formation combined with label-free quantitative proteomic analysis

Recent years, microfluidic three-dimensional (3D) tumor culture technique has made great progress in tumor microenvironment simulation and drug screening. Meanwhile, as their functionality and complexity increase, it is more difficult for current chip models to selectively collect specific-layer cells from tumoroids for further analysis. Moreover, a simplified and robust method for tumoroid formation with highly consistent size and repeatable 3D morphology is relatively ncessary. Here, we report an ARCHITECT (ARtificial CHIp for Tumor Enables Confocal Topography observation) chip, through a dual-flip strategy to implement straightforward tumoroid establishment. This platform guarantees stable batch-to-batch tumoroids formation and allows high resolution confocal imaging. Moreover, an initial cell density as low as 65 cells per chamber is efficient to deliver a tumoroid. With this ARCHITECT chip, different-layer cells of interest could be collected from tumoroid for label-free quantitative (LFQ) proteomic analysis. For application demonstration, we mainly verified this platform for lung carcinoma (A549) tumoroid construction and proteomic analysis at out layer. Our data indicate that the out-layer cells of A549 tumoroid show extensively distinct proteomic expressions compared to two-dimensional cultured A549 cells. The up-regulated proteins are mainly related to tumorigenicity, proliferation and metastasis. And the differentially expressed proteins are mainly relevant to lipid metabolism pathway which is essential to tumor progression and proliferation. This platform provides a simplified yet robust technique to connect microfluidic tumoroid construction and LFQ proteomic analysis. The simplicity of this technique should open the way to numerous applications such as discovering the innovative targets for cancer treatment, and studying the mophological and proteomic heterogeneity of different-layer cells across the tumoroid.

Optimization of Viable Glioblastoma Cryopreservation for Establishment of Primary Tumor Cell Cultures.

Background: Technologies related to the establishment of primary tumor cell cultures from solid tumors, including glioblastoma, are increasingly important to oncology research and practice. However, processing of fresh tumor specimens for establishment of primary cultures on the day of surgical collection is logistically difficult. The feasibility of viable cryopreservation of glioblastoma specimens, allowing for primary culture establishment weeks to months after surgical tumor collection and freezing, was demonstrated by Mullins et al. in 2013, with a success rate of 59% that was not significantly lower than that achieved with fresh tumor tissue. However, research targeting optimization of viable glioblastoma cryopreservation protocols for establishment of primary tumor cultures has been limited.Objectives: The objective of this study was to optimize glioblastoma cryopreservation methods for viable cryobanking and to determine if two-dimensional (2D) or three-dimensional (3D) culture conditions were more supportive of glioblastoma growth after thawing of frozen tumor specimens.Methods: Portions of eight human glioblastoma specimens were cryopreserved by four different protocols differing in the time of enzymatic digestion (before or after cryopreservation), and in the type of cryopreservation media (CryoStor CS10 or 10% dimethyl sulfoxide and 90% fetal calf serum). After 1 month, frozen tissues were thawed, enzymatically digested, if not digested before, and used for initiation of 2D or 3D primary tumor cultures to determine viability.Results: Among the tested cryopreservation and culturing protocols, the most efficient combinations of cryopreservation and culture were those associated with the use of CryoStor CS10 cryopreservation medium, enzymatic digestion before freezing, and 2D culturing after thawing with a successful culture rate of 8 out of 8 cases (100%). Two-dimensional cultures were in general more efficient for the support of tumor cell growth after thawing than 3D cultures.Conclusions: This study supports development of evidence-based viable glioblastoma cryopreservation methods for use in glioblastoma biobanking and research.

Abstract 810: Beyond genomics - A precision oncology approach utilizing multiplex protein profiling for tumor and tumor organoid samples

Abstract Precision medicine's goal of achieving a better response rate by avoiding ineffective therapies has sparked new approaches, including the testing of patient-derived 3d (PD3Ds) tumor cultures for modeling individual patient response, and the use of various molecular pathology techniques for advanced tumor profiling. An intrinsic limitation of well-established genomics methodologies is, however, that they cannot directly assess cell-signaling activity within the tumor cells defined by the phosphorylation status of cellular signaling pathway networks. Here we present the development of a robust protein profiling strategy utilizing the DigiWest immuno-assay platform, to obtain data on the activation status of key cellular signaling networks implicated in cancer, and on proteins targeted by FDA-approved drugs. This includes targeted cancer therapies for e.g. EGFR, HER2, PI3K, mTOR, ALK and AKT. Accordingly, we compiled a list of relevant pathway nodes and their phosphorylation sites that yield activity information on RAS/RAF/ERK, PI3K/AKT and mTOR signaling pathways. Based on this, we validated 242 total and phospho antibodies in a pre-set oncoproteomic DigiWest panel that yields information on the activity of all major signaling pathways from the receptor level down to transcription factors, apoptosis and proliferation. This oncoproteomic panel can be utilized for elucidating drug response in pre-clinical cell models, in PD3D organoid models or in clinical tumor samples. Exemplary, we show differential activation states in cancer cell lines representing the most prevalent cancer types and the impact of pathway activation in different models. Finally, we tested this panel in PD3D organoids that were subjected to screening against common targeted therapies. While the initial results are promising, further work on evaluating how such an oncoproteomic panel profiling might contribute to the rationale for personalized therapy decisions is required. Disclosure Statement: NMI TT Pharmaservices offers DigiWest protein profiling as a service. CELLphenomics offers PD3D cell cultures and drug testing as a service. Citation Format: Gerrit Erdmann, Przemylaw Dudys, Anja Arndt, Christian Regenbrecht, Markus Templin, Christoph Sachse. Beyond genomics - A precision oncology approach utilizing multiplex protein profiling for tumor and tumor organoid samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 810.

Abstract 5167: Identification and validation of a novel immuno-oncology target and selection of a therapeutic antibody candidate with a pharmacologically beneficial activity profile

Abstract Identification of novel targets in cancer immunotherapy is needed to address the significant number of patients that either do not respond to current therapies or encounter unacceptable toxicities. The first two generations of immuno-oncology drugs have been antagonist antibodies against immune checkpoint proteins, such as cytotoxic T lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Moving forward, there has been progress in targeting co-stimulatory receptors like inducible T cell co-stimulator (ICOS), OX40 and CD137 with agonist antibodies. At Oxford BioTherapeutics, in-depth expression profiling of membrane proteins from intact tumors collected in the proprietary OGAP database revealed novel IO targets in primary tumor-derived lymphocytes (TILs). Proteomic and flow cytometry analysis of TILs and PBMCs establish that OX003R is a novel co-stimulatory IO target. It is expressed on naïve T and B cells; however, higher expression is observed in TILs and activated or exhausted T cells. OX003R expression is observed by immunohistochemistry in infiltrating lymphocytes in a variety of solid tumor types. A Fab phage display library was screened by FACS for binding to target on the cell surface. All the Fabs were also profiled by an interferon gamma release assay for T cell activation. Five best binders demonstrating T cell activation were reformatted into full-length chimeric mAbs and expressed in mammalian Expi 293 cells. Recombinant antibodies were extensively screened for T cell activation in an ex vivo 3D tumor culture system developed in-house using fresh non-small cell lung and colorectal carcinomas. Interferon gamma release was assessed by ELISpot assay and expression of the target was confirmed by immunohistochemistry on the corresponding tumor samples. Chimeric antibody 1B3 robustly activated T cells in most of the tumor samples in a dose-dependent manner as compared to isotype control and was chosen as the lead therapeutic antibody for humanization. The lead therapeutic antibody was tested for the propensity to facilitate the undesirable cytokine storm in whole blood and did not induce the release of dangerous levels of cytokines. Conclusion: OX003R is a validated immuno-oncology target and chimeric 1B3 is being developed as a promising therapeutic antibody with agonistic TIL activity, specifically in the tumor microenvironment. Citation Format: Arnima Bisht, Angelo Kaplan, Livija Deban, Chander Sekhar Peddaboina, Murray Cox, Lindsey Hudson, James Ackroyd, Nickolas Attanasio, San Lin Lou, Jason Allen, Martin Barnes, Robert Boyd, Eugene Zhukovsky, Abderrahim Fandi, Christian Rohlff. Identification and validation of a novel immuno-oncology target and selection of a therapeutic antibody candidate with a pharmacologically beneficial activity profile [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5167.

Abstract 4159: Ovarian cancer ex vivo 3D tumor cultures predict patient specific drug sensitivity

Abstract Introduction While systemic treatment has been quite static in the past decade in ovarian cancer, the challenge for the coming years is to stratify patients for optimal and new treatment strategies, such as targeting DNA damage response pathways, or immune modulation. Patient primary tumor from resections, biopsy or ascites can be cultured such that tumor heterogeneity and resident immune cells are preserved. These can be used to predict treatment response in patients or to test new cancer therapies. Here we present a high throughput short-term 3D ex-vivo culture platform combined with high content image-based analysis. This platform allows visualization and quantification of standard of care therapy and new drugs on fresh and cryopreserved patient derived ovarian cancer tumoroids. Methods Patients with advanced primary or relapsing ovarian carcinoma are included in ongoing multi-center clinical trials in the Netherlands. Ovarian cancer derived tumoroids, freshly isolated or cryopreserved, are embedded in a protein-rich hydrogel and exposed to up to 20 drugs during a short term 3D culture. The automated high content imaging analysis platform measures a large panel of tumoroid morphological features, and responses such as tumor cell killing, growth arrest and local invasion are measured to define the response for each drug. In addition, clinical response data from the ongoing trials are collected to be correlated to clinical outcome of patients treated with systemic therapy. Results We present results of drug sensitivity testing in patient-derived primary tumor cells of fresh and cryopreserved ascites and solid tumor. Patient-specific drug sensitivity is identified for up to 20 drugs including standard of care (e.g. carboplatin, paclitaxel) and novel treatment strategies (e.g. PARP inhibitors). Activity of immunomodulatory drugs were also monitored successfully in vitro. Accurate response evaluation demonstrates the feasibility of high-throughput drug screening on patient primary material within OcellO's platform. Our results show a high reproducibility in testing freshly isolated or cryopreserved material, highlighting the potential of a cryopreserved collection of patient derived primary material to test new drugs. Conclusion OcellO's advanced 3D image analysis of patient primary material represents a rapid and biologically relevant approach to test various candidate compounds (e.g. antibodies, antibody-drug conjugates and small molecules) for ovarian cancer. The availability of drug response data combined with patient clinical data, including BRCA mutation and cytology/histology pathologist evaluation is expected to support drug discovery, improve the efficiency of clinical trials and establish predictive testing in the clinical setting. Citation Format: Fanny Grillet, Abbas Jariani, Juul Overkamp, Lidia Daszkiewicz, Kuan Yan, Leo S. Price, Willemijn Vader. Ovarian cancer ex vivo 3D tumor cultures predict patient specific drug sensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4159.

A simple microsphere-based mold to rapidly fabricate microwell arrays for multisize 3D tumor culture.

Three-dimensional (3D) tumor has been considered as the best in vitro model for cancer research. In recent years, various methods have been developed to controllable prepare multisize 3D tumors. Nonetheless, reported technologies are still problematic and difficult to produce 3D tumors with highly uniform size and cell content. Here, a novel and simple microsphere-based mold approach is proposed to rapidly fabricate spherical microwell arrays for multisize 3D tumors formation, culture, and recovery. Larger amounts of HepG2 3D tumors with excellent quality and uniformity can be efficiently generated using this method. In addition, the tumor size can also be simply controlled by adjusting the diameter of the microwell arrays. All experimental results indicated that the proposed method offers a promising platform to generate and recover highly controlled multisize 3D tumors for various cell-based biomedical research.

Abstract B139: Patient specific drug sensitivity to novel treatment approaches in ovarian cancer ex vivo 3D tumor cultures

Abstract Background: Many new drugs and treatment strategies are in development for ovarian cancer. While systemic treatment has been quite static in the past decade, the challenge for the coming years is to stratify ovarian cancer patients for optimal treatment strategies, like targeting DNA damage response pathways or immune modulatory approaches. Patient primary tumor material collection offers cryopreserved viable primary tumor cell clusters, containing the whole heterogeneity found in the patient ascites (including potential immune cells) to predict treatment response in patients or to test new cancer therapies. Here we present a high throughput short-term 3D ex-vivo culture platform combined with high content image-based analysis. This platform allows visualization and quantification of standard of care therapy and new drugs on fresh and cryopreserved patient derived ovarian cancer tumoroids. Methods: Patients with ovarian carcinoma are included in ongoing multi-center clinical trials in the Netherlands. Ovarian cancer derived primary tumoroids, freshly isolated or cryopreserved, are embedded in a protein-rich hydrogel and exposed to up to 20 drugs during a short term 3D culture. The automated high content imaging analysis platform measures a large panel of tumoroid morphological features, and responses such as tumor cell killing, growth arrest and local invasion are measured to define the response for each drug. In addition, clinical response data from the ongoing trials will be correlated to clinical outcome of patients treated with systemic therapy. Results:We present first results of drug sensitivity in patient-derived primary tumor cells of fresh and cryopreserved ascites. Patient-specific drug sensitivity is identified for up to 20 drugs including standard of care (e.g. carboplatin, paclitaxel) and novel treatment strategies (e.g. PARP inhibitors). Accurate response evaluation demonstrates the feasibility of high-throughput drug screening on patient primary material within OcellO’s platform. Our results show a high reproducibility in testing freshly isolated or cryopreserved material, highlighting the potential of a cryopreserved collection of patient derived primary material to test new drugs. Conclusion: OcellO’s advanced 3D image analysis of patient primary material represents a rapid and biologically relevant approach to test various candidate compounds (e.g. antibodies, antibody-drug conjugates and small molecules) for ovarian cancer. The availability of drug response data combined with patient clinical data, including BRCA mutation and cytology/histology pathologist evaluation is expected to support drug discovery, improve the efficiency of clinical trials and establish predictive testing in the clinical setting. Citation Format: Fanny Grillet, Abbas Jariani, Juul Overkamp, Lidia Daszkiewicz, Kuan Yan, Leo Price, Willemijn Vader. Patient specific drug sensitivity to novel treatment approaches in ovarian cancer ex vivo 3D tumor 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 B139. doi:10.1158/1535-7163.TARG-19-B139