3D Cell Culture Models Research Articles

Abstract 4727: Large scale drug response comparison across different neuroblastoma cell model systems

Abstract Traditionally neuroblastoma research has predominantly used 2D cell lines, which, despite their simplicity, often fall short in mirroring the physiological complexity of actual tumors. Recently, 3D models like patient-derived tumor organoids (PDO) and patient-derived xenografts (PDX) have emerged as superior alternatives, offering more realistic predictions of patient treatment responses. This study aims to discern the differences in drug response between conventional 2D cell lines and the more advanced 3D neuroblastoma models - specifically, patient-derived organoids (nPDO) and patient-derived xenograft organoids (nPDXO). Employing high-throughput drug screening of 528 cancer drugs, we evaluated the drug responses across traditional 2D cell lines, nPDOs, and nPDXOs. Additionally, focused experiments with 29 selected drugs were conducted to determine the influence of culture topology on drug efficacy. The findings were compared to a study by Hansson et al.1 that used nPDXO. Our study discovered a pronounced difference in drug sensitivity between 2D cell lines and 3D models, with 3D models showing heightened sensitivity. Interestingly, 2D cell cultures had a more heterogenous drug response compared to 3D models. The drug response correlation between nPDOs and nPDXOs was relatively high (Spearman ~0.8), indicating consistent responses across these models. Our comparison underscores the impact of model system choice on drug response studies in neuroblastoma. The study highlights important differences in drug response between neuroblastoma 2D and 3D cell culture models. However, only subtle differences were found between PDO and PDX-derived organoid lines, despite differences in treatment centers and media compositions. This suggests that while the choice of cell culture topology greatly impacts drug response, PDO and PDX derived lines can largely be used interchangeably for drug screening experiments.

The fabrication method impacts biological properties of microfibrous three-dimensional in vitro cell culture scaffolds and their applications in drug screening

Two-dimensional (2D) cell culture models cannot accurately emulate the three-dimensional (3D) in vivo microenvironment; hence, the shift from compound screening in traditional 2D well plates to 3D cell culture models is forthcoming. New scaffold designs must comply with the 3Rs principle to reduce the use of animal-derived products, such as natural proteins. In line with this motivation, we engineered and compared various fibrous scaffolds featuring diverse fibre networks (coarse and fine fibres) and fibre surface properties (porous vs. smooth fibres), and tested them in in vitro cell culture. We fabricated coarse solid fibre scaffolds via a melt-based electrohydrodynamic process, which resulted in fibres with a diameter of 10.6 ± 1.9 µm and a porosity of 87.1 ± 0.9%. Using solution-based cryo-electrohydrodynamic process, we fabricated fine porous fibre and fine solid fibre scaffolds that exhibited fibres of 2.01 ± 0.5 µm and 1.72 ± 0.6 µm in diameter, with porosities of 96.5 ± 0.3% and 94.4 ± 0.5%, respectively. The scaffolds underwent only chemical modification via an alkaline treatment to enhance their hydrophilicity. We used mouse fibroblast L929 cells and human triple-negative breast cancer MDA-MB-231 cells as cell models, and used the MTT assay to measure their viability. After seven days of cell culture, all scaffolds were thoroughly penetrated. However, the coarse scaffolds created by the melt-based electrohydrodynamic process and the porous fibre scaffolds produced by the solution-based cryo-electrohydrodynamic process outperformed the simpler morphology of smooth fine fibres. Furthermore, the 50% half-maximum effective concentration (EC50) of doxorubicin tested in human glioblastoma (U87-MG) and human triple-negative breast cancer (MDA-MB-231) cell lines in 3D cell culture models was approximately twice as high as that achieved in conventional 2D cell cultures. Therefore, we infer that poly (ɛ-caprolactone) (PCL) microfibre scaffolds, devoid of animal-derived product surface treatment, may provide a favourable 3D milieu for various cell types for in vitro studies.

Abstract 1705: 3D growth modulates the competition between STAT3 and STAT5 in breast cancer

Abstract Approximately 13% of women are diagnosed with invasive breast cancer. Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that is often inappropriately activated in breast cancer and is frequently associated with Triple Negative Breast Cancer (TNBC). STAT5 can also be activated and inappropriate activation of STAT5 alone or with STAT3 is generally correlated with a more favorable prognosis, a lower grade tumor, and a better response to therapies compared to inappropriate activation of STAT3 alone. Therefore, knowing the activation status of STAT5 and understanding how STAT5 activation can attenuate STAT3-driven breast cancers could help guide therapeutic choices and patient treatment. STAT3 and STAT5 can regulate a set of overlapping target genes. For example, STAT3 and STAT5 can regulate BCL6 expression, where STAT5 represses expression and STAT3 enhances it. Importantly, STAT5 outcompetes and preferentially binds in the presence of activated STAT3. This suggests that the overlapping target genes could play an important role in understanding how activation of STAT5 can lead to a more favorable breast cancer compared to activation of STAT3 alone. This previous work was conducted in a 2D cell culture model; however, 3D models can better mimic clinical settings and the use of 3D models can help bridge the gap. Therefore, understanding the relationship between STAT3 and STAT5 in 3D is important. SK-BR-3 cells (HER2+ cells with low basal STAT3 activity) and MDA-MB-231 cells (TNBC cells with constitutive STAT3 activity) are being compared between 2D cell culture and 3D media suspension. We have shown using chromatin immunoprecipitation that STAT3 DNA-binding is enhanced in 3D at certain binding sites, whereas STAT5 is not. STAT5 has reduced dominance and preferential binding over STAT3 for BCL6 in 3D compared to 2D. In other cases, STAT3 binding is decreased upon STAT5 activation in 3D but not 2D. To begin to understand these differences, we analyzed phosphorylation of these STATs in 2D and 3D. Phospho-STAT3 levels were similar between 2D and 3D in SK-BR-3 cells when activated with LIF stimulation but were higher in 3D in the MDA-MB-231 cells which contain constitutive STAT3 activity. STAT5 activation by prolactin stimulation did not directly affect STAT3 phosphorylation in 2D or 3D but does affect STAT3 binding in 3D growing cells. However, prolactin induced phospho-STAT5 seems to be reduced in 3D in both cell lines. We are currently analyzing additional binding sites to better define the differences between STAT3 and STAT5 mediated gene regulation in 2D and 3D since STAT phosphorylation does not fully address it. Overall, our data suggests that differences between 2D and 3D breast cancer models will be important for translation to patient tumors and understanding the relationship between STAT3 and STAT5 will help elucidate how STAT5 activation status can be used when treating patients with breast cancer. Citation Format: Alexandra Elizabeth Temple, Sarah R. Walker. 3D growth modulates the competition between STAT3 and STAT5 in breast cancer [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 1705.

Abstract 3167: Impact of gemcitabine modification on redox-driven responses in pancreatic cancer organoids cultured in distinct 3D microenvironments

Abstract Pancreatic ductal adenocarcinoma (PDAC) poses a formidable challenge in oncology, characterized by its high lethality and limited treatment options. The advanced stage at diagnosis, aggressive metastasis, and the constrained efficacy of conventional therapeutic drugs collectively contribute to the grim prognosis associated with PDAC. Gemcitabine, a nucleoside analog and the longstanding standard chemotherapy for pancreatic cancer, encounters a significant hurdle in its efficacy due to the development of resistance. To establish a cancer-acquired resistance model applicable to Gem-modified drugs, it is crucial to account for the dense extracellular matrix (ECM) composition characteristic of PDAC. In this investigation, we employ three-dimensional (3D) cell culture models, specifically utilizing pancreatic cancer patient-derived organoids (PDOs) within a 3D matrix with tunable density and stiffness by incorporating variations in transglutaminase crosslinking rates. PDAC cells from different patients exhibit diverse adaptations within these altered gel matrix environments, manifesting differences in morphology, doubling time, and responses to drugs. Notably, PDAC cells display pronounced stemness characteristics in the dense matrix, featuring a more rounded and compact appearance and an increase in ABC transporters.Addressing the challenge of gemcitabine resistance, modifications such as gemcitabine conjugates with stearate (Gem-S) have shown promise in preclinical studies. Exploring the effects of both Gem and Gem-S in a viscoelastic-specific ECM model derived from PDAC patient-derived organoids, distinct responses based on matrix stiffness come to light. The physical properties of the matrix exert a significant influence on cellular function, thereby impacting the effectiveness of therapeutic agents. Intriguingly, within a stiffer environment, Gem-S demonstrates higher sensitivity compared to Gem, suggesting the potential application of Gem-S in the dense pathological PDAC environment. Further investigation reveals that Gem-S treatment induces a noteworthy increase in reactive oxygen species (ROS) and HIF-1 expression compared to Gem. Surprisingly, the role of multidrug transporters in Gem-S sensitivity is limited, while the downregulation of NRF (Nrf2) after Gem-S treatment hints at distinct mechanisms underlying the development of drug resistance. This study provides valuable insights into the intricate interplay between gemcitabine resistance and the PDAC microenvironment. Understanding the matrix-specific responses opens avenues for tailored therapeutic interventions, offering the potential for improved outcomes in PDAC patients. Citation Format: Bo Han, Shuqing Zhao, Edward Agyare, Xueyou Zhu, Jose Trevino, Sherise Rogers, Enrique Velazquez, Jason Brant, Payam Eliahoo, Jonathan Barajas, Ba Xuan Hoang. Impact of gemcitabine modification on redox-driven responses in pancreatic cancer organoids cultured in distinct 3D microenvironments [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 3167.

Abstract 3090: Development and performance of the National Cancer Institute’s HTS384 NCI60 screen: A modernized platform to support drug discovery and development by the worldwide cancer research community

Abstract The NCI60 screen was established in the 1980s and introduced in the 1990s as a screening service for the worldwide cancer research community. The screen features 60 human tumor cell lines that represent 9 cancer types including non-small cell lung, colon, central nervous system, ovarian, renal, prostate and breast, as well as leukemia and melanoma. On a weekly basis, synthetic molecules, natural products, and biologics are screened against the 60-cell line panel to evaluate their anticancer potential. On average, more than 13,000 compounds are screened annually for investigators throughout the world. Cell growth and cytotoxicity were assessed by the sulforhodamine B (SRB) assay, performed in a 96-well format, that measures cellular protein content with an absorbance readout and enabled unprecedented throughput in the 1980s. Although the SRB assay supported a robust screening platform for three decades, its sensitivity and throughput are limited. Recently the NCI’s DCTD introduced the HTS384 NCI60 as a modernized screening platform. While the same 60 cell lines are evaluated, the HTS384 NCI60 is fully automated and performed in a 384-well format to enable a higher throughput. Additionally, the 48-h test agent exposure period of the SRB assay was increased to 72 h, which may result in lower 50% growth inhibition values (GI50) for some agents. A highly sensitive CellTiter-Glo luminescence readout for cell viability, based on cellular ATP content, was selected to enable comparisons of the NCI60 monolayer data to 3D cell culture models where the same assay chemistry can be applied. The HTS384 NCI60 was evaluated by screening a library of 1,003 U.S. Food and Drug Administration-approved and investigational oncology agents at five concentrations. Assay performance of the 60 cell lines was assessed from the controls of 30 - 60 microplates per cell line. The average doubling time of the 60 cell lines during the 72-h exposure period ranged between 19 h for NCI-H460 (17 - 23 h, n = 57) to 117 h for MDA-MB-468 (43 - 272 h, n = 60). The average signal-to-background ratio of all 60 cell lines was 89 (3,413 microplates) and ranged from 6 for OVCAR-5 (3 - 13, n = 54) to 229 for SF-539 (141 - 566, n = 60). The average coefficient of variation for microplate vehicle controls (n = 14) of all 60 cell lines (n = 3,413) was 5.2% and ranged from 3.2% for SK-MEL-28 (1.4 - 7.7%, n = 60) to 11.1% for HL-60(TB) (5.2 - 20.2%, n = 59). The average Z’-factor value of all 60 cell lines (n = 3,413) was 0.82 and ranged between 0.66 for HL-60(TB) (0.39 - 0.84, n = 59) to 0.89 for MALME-3M (0.79 - 0.95, n = 59), SF-295 (0.49 - 0.97, n = 59), SK-MEL-28 (0.74 - 0.95, n = 60), and UO-31 (0.8 - 0.94, n = 54). The development and performance of the HTS384 NCI60 screen will be described. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Nathan P. Coussens, Thomas S. Dexheimer, Eric M. Jones, Thomas Silvers, John R. Britt, Ronald C. Taylor, Mark W. Kunkel, James H. Doroshow, Beverly A. Teicher. Development and performance of the National Cancer Institute’s HTS384 NCI60 screen: A modernized platform to support drug discovery and development by the worldwide cancer research community [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 3090.

Abstract 1566: Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer [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 1566.

Abstract 240: Mix and match - A comprehensive pipeline for matched patient-derived PDX and PD3D® modelsfor cancer research and preclinical drug development

Abstract Relevant in vivo and in vitro cancer models have been invaluable for our current understanding of cancer biology, as well as for therapy development. Using appropriate models in the drug development process significantly supports reliable and swift decision making in proceeding along the value chain. In parallel, animal experiments can be designed more specific to the scientific and regulatory needs, resulting in shorter development cycles and eventually the availability of better therapies for cancer patients. Patient-derived xenografts (PDX) are generated by implantation of cancerous tissue from a patient’s tumor either under the skin (ectopic) or into the organ of tumor origin (orthotopic) of an immunocompromised mouse and are the most used in vivo model for preclinical drug development. Patient-derived 3D cell culture models (PD3D®) are gaining increasing significance as relevant in vitro cancer models that recapitulate the original tumor tissue’s biology and are suitable for high-throughput drug screening. The combination of both platform’s increases the translational relevance of the development pipeline and offers the possibility to enlarge the respective panels of tumor models. Tackling an unmet medical need, we initially created matched mesothelioma PD3D® and PDX models. Mesothelioma is an aggressive cancer with poor prognosis and limited treatment options. Development of new therapies is hampered by shortage of available relevant tumor models. We created PD3D® models from PDX mesothelioma models and vice versa and generated drug sensitivity profiles with standard of care such as Gemcitabine, platinum derivates and a number of targeted agents to confirm their suitability as matched models for future projects. We were able to prove that models express stable histological, phenotypic features and drug sensitivity profiles across the two platforms. With this proof-of-concept study, the feasibility to combine the PD3D® biobank comprising more than 500 models from CELLphenomics with the PDX bank from CRL including 700 PDX models, has gained momentum. In consideration of the changing regulatory landscape, the combination of in vitro and in vivo platforms is a crucial step towards a drug development pipeline that incorporates the principles of 3R and increases its translational value due to fully human technologies being the corner stones of the drug development process. Citation Format: Jürgen Loskutov, Eva Oswald, Lena Wedeken, Christoph J. Reinhard, Julia Schueler, Christian R. Regenbrecht. Mix and match - A comprehensive pipeline for matched patient-derived PDX and PD3D® modelsfor cancer research and preclinical drug development [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 240.

Abstract 4235: PD3D®models as jacks-of-all-trades for cancer research and therapy response prediction

Abstract Physiologically relevant in vitro tumor models are crucial in any research setting from preclinical drug development to functional precision oncology. Patient-derived 3D cell culture models (PD3D®) are validated cancer models which recapitulate the biology of the original tumor tissue. PD3D® can be used to model intratumoral heterogeneity in medium and high throughput screens. Using a reverse clinical engineering approach, PD3D® models are already applied in personalized oncology to identify treatment for an individual patient. We successfully established a living biobank of more than 500 PD3D® models, spanning from common cancers like colorectal, breast (incl. TNBC), non-small cell lung and pancreatic carcinoma, to orphan indications including various subtypes of sarcomas. Not surprisingly, PD3D® model morphology and culture requirements differ between tumor entities. Treating PD3D® models with standard of care drugs in a semi-automated high-throughput assay, we observed heterogeneity in drug sensitivity between models of the same cancer type, recapitulating clinical response of patients. Combining drug sensitivity profiles with genomic and proteomic data of the same PD3D® models, we successfully identified a biomarker for predicting chemosensitivity towards MEK-targeting drugs. For application of PD3D® in truly personalized oncology, we developed a protocol that allows us to generate a PD3D® culture and perform a drug sensitivity assay for an individual patient within a therapy-relevant timeframe. Within 38 days we identified a systemic therapy for a young patient with a relapsed metastasized synovial sarcoma. After surgery and initiation of recommended chemotherapy, the patient is now under remission. To further expand the scope of PD3D® for therapy response prediction, we established a method for irradiation of PD3D® cultures with different dosages of photon and proton radiation. We observed significant, model specific differences in radiosensitivity between PD3D® sarcoma models which also reflect clinically heterogenous responses. Additionally, PD3D® models can be employed for a tumor organoid-on-chip platform (TumOC), delivering real-time information on physiological parameters like drug response-mediated oxygen consumption in a microfluidic system. In conclusion, PD3D® models act as jacks-of-all-trades in current and future cancer research, delivering robust and reliable data that not only is of academic value, but speeds up the drug development process, and are ready for prime time in functional precision oncology. Citation Format: Lena Wedeken, Samantha Forbrig, Katja Herrera-Glomm, Juergen Loskutov, Ulrike Pfohl, Irina Piven, Michael Poehle, Manuela J. Regenbrecht, Barbara Seller, Cynthia Yapto, Sabine Finkler, Larissa Ruhe, Quirin Graf Adelmann, Christoph Reinhard, Marie Flechner, Katja Uhlig, David Kaul, Siyer Roohani, Maya Niethard, Rica Sauer, Christian R. Regenbrecht. PD3D®models as jacks-of-all-trades for cancer research and therapy response prediction [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 4235.

Organs in orbit: how tissue chip technology benefits from microgravity, a perspective.

Tissue chips have become one of the most potent research tools in the biomedical field. In contrast to conventional research methods, such as 2D cell culture and animal models, tissue chips more directly represent human physiological systems. This allows researchers to study therapeutic outcomes to a high degree of similarity to actual human subjects. Additionally, as rocket technology has advanced and become more accessible, researchers are using the unique properties offered by microgravity to meet specific challenges of modeling tissues on Earth; these include large organoids with sophisticated structures and models to better study aging and disease. This perspective explores the manufacturing and research applications of microgravity tissue chip technology, specifically investigating the musculoskeletal, cardiovascular, and nervous systems.

An Insight on Microfluidic Organ-on-a-Chip Models for PM2.5-Induced Pulmonary Complications.

Pulmonary diseases like asthma, chronic obstructive pulmonary disorder, lung fibrosis, and lung cancer pose a significant burden to global human health. Many of these complications arise as a result of exposure to particulate matter (PM), which has been examined in several preclinical and clinical trials for its effect on several respiratory diseases. Particulate matter of size less than 2.5 μm (PM2.5) has been known to inflict unforeseen repercussions, although data from epidemiological studies to back this are pending. Conventionally utilized two-dimensional (2D) cell culture and preclinical animal models have provided insufficient benefits in emulating the in vivo physiological and pathological pulmonary conditions. Three-dimensional (3D) structural models, including organ-on-a-chip models, have experienced a developmental upsurge in recent times. Lung-on-a-chip models have the potential to simulate the specific features of the lungs. With the advancement of technology, an emerging and advanced technique termed microfluidic organ-on-a-chip has been developed with the aim of identifying the complexity of the respiratory cellular microenvironment of the body. In the present Review, the role of lung-on-a-chip modeling in reproducing pulmonary complications has been explored, with a specific emphasis on PM2.5-induced pulmonary complications.

A Review on Patient-Derived 3D Micro Cancer Approach for Drug Screen in Personalized Cancer Medicine.

Precision medicine in oncology aims to identify an individualized treatment plan based on genomic alterations in a patient's tumor. It helps to select the most beneficial therapy for an individual patient. As it is now known that no patient's cancer is the same, and therefore, different patients may respond differently to conventional treatments, precision medicine, which replaces the one-size-fits-all approach, supports the development of tailored treatments for specific cancers of different patients. Patient-specific organoid or spheroid models as 3D cell culture models are very promising for predicting resistance to anti-cancer drugs and for identifying the most effective cancer therapy for high-throughput drug screening combined with genomic analysis in personalized medicine. Because tumor spheroids incorporate many features of solid tumors and reflect resistance to drugs and radiation, as in human cancers, they are widely used in drug screening studies. Testing patient-derived 3D cancer spheroids with some anticancer drugs based on information from molecular profiling can reveal the sensitivity of tumor cells to drugs and provide the right compounds to be effective against resistant cells. Given that many patients do not respond to standard treatments, patient-specific treatments will be more effective, less toxic. They will affect survival better compared to the standard approach used for all patients.

A well plate-based GelMA photo-crosslinking system with tunable hydrogel mechanical properties to regulate the PTH-mediated osteogenic fate

Versatile and efficient regulation of the mechanical properties of the extracellular matrix is crucial not only for understanding the dynamic changes in biological systems, but also for obtaining precise and effective cellular responses in drug testing. In this study, we developed a well plate-based hydrogel photo-crosslinking system to effectively control the mechanical properties of hydrogels and perform high-throughput assays. We improved cell biocompatibility by using gelatin methacryloyl (GelMA) with a visible light photo-crosslinking method. Multiple cell-laden GelMA hydrogels were simultaneously and uniformly created using multi-arrayed 520 nm light-emitting diodes in a well plate format. The elastic modulus of the hydrogels can be widely adjusted (0.5–30 kPa) using a photo-crosslinking system capable of independently controlling the light intensity or exposure time for multiple samples. We demonstrate the feasibility of our system by observing enhanced bone differentiation of human mesenchymal stem cells (hMSCs) cultured on stiffer hydrogels. Additionally, we observed that the osteogenic fate of hMSCs, affected by the different mechanical properties of the gel, was regulated by parathyroid hormone (PTH). Notably, in response to PTH, hMSCs in a high-stiffness microenvironment upregulate osteogenic differentiation while exhibiting increased proliferation in a low-stiffness microenvironment. Overall, the developed system enables the generation of multiple cell-laden three-dimensional cell culture models with diverse mechanical properties and holds significant potential for expansion into drug testing.

Development of a vestibular schwannoma tumor slice model for pharmacological testing

BackgroundOur goal was to develop a 3D tumor slice model, replicating the individual tumor microenvironment and for individual pharmaceutical testing in vestibular schwannomas with and without relation to NF2. MethodsTissue samples from 16 VS patients (14 sporadic, 2 NF2-related) were prospectively analyzed. Slices of 350 µm thickness were cultured in vitro, and the 3D tumor slice model underwent thorough evaluation for culturing time, microenvironment characteristics, morphology, apoptosis, and proliferation rates. Common drugs - Lapatinib (10 µM), Nilotinib (20 µM), and Bevacizumab (10 µg/ml) - known for their responses in VS were used for treatment. Treatment responses were assessed using CC3 as an apoptosis marker and Ki67 as a proliferation marker. Standard 2D cell culture models of the same tumors served as controls. ResultsThe 3D tumor slice model accurately mimicked VS ex vivo, maintaining stability for three months. Cell count within the model was approximately tenfold higher than in standard cell culture, and the tumor microenvironment remained stable for 46 days. Pharmacological testing was feasible for up to three weeks, revealing interindividual differences in treatment response to Lapatinib and intraindividual variability in response to Lapatinib and Nilotinib. The observed effects were less pronounced in tumor slices than in standard cell culture, indicating the model's proximity to in vivo tumor biology and enhanced realism. Bevacizumab had limited impact in both models. ConclusionThis study introduces a 3D tumor slice model for sporadic and NF2-related VS, demonstrating stability for up to 3 months, replication of the schwannoma microenvironment, and utility for individualized pharmacological testing.

Controllable and Scaffold‐Free Formation of 3D Multicellular Architectures Using a Bipolar Electrode Array

AbstractThe natural environment of cells in vivo is a 3D network in which cellular responses are determined by influences from surrounding cells on all sides. This complex system is hard to be recapitulated in a conventional way. It is highly desirable to replace 2D models with 3D cell culture models. Microfluidic technology offers a variety of advantageous approaches for the long‐term 3D cell culture. Herein, a new cell patterning approach for generating cell clusters of specified size and shape by exploiting induced charge electroosmosis (ICEO) flow at a bipolar electrode array is exploited. Cell or particle clusters can be produced at the edges or centers of bipolar electrodes (BPEs) with controlled organization, by designing electrodes with arbitrary shapes. This platform is further extended to flexibly organize biological units with different properties via the ICEO or dielectrophoresis (DEP) based assembly. Heterotypic cell clusters can be produced at the bipolar electrode (BPE) by adjusting the applied frequencies and the sample injection sequence. This hybrid approach by integrating ICEO flow vortexes and DEP properties of the cells, allows the wireless generation of high‐throughput 3D clusters or spheroids with size adjustability and biocompatibility, which shows great potential in tissue engineering, drug discovery, and tumor research.

A62 EXAMINING THE ACTIVATION OF XENOBIOTIC RECEPTORS PXR AND AHR IN COLONOIDS USING MICROBIAL METABOLITES AND CHEMICAL LIGANDS

Abstract Background The Aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) are vital xenobiotic receptors activated by foreign substances, playing pivotal roles in regulating chemical metabolism. Traditionally, these receptors have been closely linked to their roles in mediating responses to toxic compounds. However, recent findings have revealed their newfound significance in maintaining gut homeostasis and regulating inflammatory processes. Aims We investigated the dual roles of AhR and PXR activation. We started with dosage response experiments to determine optimal treatment conditions, such as treatment concentration and exposure time. Subsequently, we compared transcriptomic responses induced by chemical ligands with those from microbial metabolites, aiming to understand how AhR and PXR activation can yield both adverse and beneficial effects. Methods Using mouse 3D colonoids and 2D monolayer cultures, PXR and AhR were activated using the microbial metabolites indole-3-propionic acid (IPA) and indole-3-pyruvic acid (IPyA), respectively. Concentrations were determined through dosage response experiments. Changes to PXR and AhR target gene expression were measured using qPCR. We compared the gene induction by IPA and IPyA to responses driven by the chemical ligands pregnenolone 16α-carbonitrile (PCN) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. Results In 3D cell culture, PCN induced PXR target genes Cyp2c55 and Abcb1a, while IPA had no significant effect. Cyp3a11 was absent. Both IPyA and TCDD induced AhR genes, Cyp1a1 and Cyp1b1, with varying induction, and Cyp1a2 showed no response. In 2D cell culture, PCN significantly increased Cyp2c55 gene expression, but neither PCN nor IPA significantly induced Abcb1a. In 3D culture, TCDD significantly induced Cyp1a1 and Cyp1b1, while IPyA only induced Cyp1a1, revealing differences in AhR gene induction between 2D and 3D cell culture models. Conclusions Our research reveals contrasting gene induction patterns following AhR and PXR activation through microbial metabolites and chemical ligands. These varied responses highlight the multifaceted roles these receptors play within the gut environment. This study enhances our understanding of xenobiotic receptors' contributions to maintaining gut homeostasis and regulating inflammatory processes, shedding light on the interplay between ligands and receptors. Methods First, we isolate the crypts of mice to create 3D organoids, were we have access to the basolateral side. To capture the true biological system, we convert the 3D culture to 2D. This enables us to explore both the apical and basolateral sides, retaining the crucial polarity of the cells. Funding Agencies CIHR

Imaging Diffusion and Stability of Single-Chain Polymeric Nanoparticles in a Multi-Gel Tumor-on-a-Chip Microfluidic Device.

The performance of single-chain polymeric nanoparticles (SCPNs) in biomedical applications highly depends on their conformational stability in cellular environments. Until now, such stability studies are limited to 2D cell culture models, which do not recapitulate the 3D tumor microenvironment well. Here, a microfluidic tumor-on-a-chip model is introduced that recreates the tumor milieu and allows in-depth insights into the diffusion, cellular uptake, and stability of SCPNs. The chip contains Matrigel/collagen-hyaluronic acid as extracellular matrix (ECM) modelsand is seeded with cancer cell MCF7 spheroids. With this 3D platform, it is assessed how the polymer's microstructure affects the SCPN's behavior when crossing the ECM, and evaluates SCPN internalization in 3D cancer cells. A library of SCPNs varying in microstructure is prepared. All SCPNs show efficient ECM penetration but their cellular uptake/stability behavior depends on the microstructure. Glucose-based nanoparticles display the highest spheroid uptake, followed by charged nanoparticles. Charged nanoparticles possess an open conformation while nanoparticles stabilized by internal hydrogen bonding retain a folded structure inside the tumor spheroids. The 3D microfluidic tumor-on-a-chip platform is an efficient tool to elucidate the interplay between polymer microstructure and SCPN's stability, a key factor for the rational design of nanoparticles for targeted biological applications.

Human Coronary Artery Endothelial Cell Response to Porphyromonas gingivalis W83 in a Collagen Three-Dimensional Culture Model.

P. gingivalis has been reported to be an endothelial cell inflammatory response inducer that can lead to endothelial dysfunction processes related to atherosclerosis; however, these studies have been carried out in vitro in cell culture models on two-dimensional (2D) plastic surfaces that do not simulate the natural environment where pathology develops. This work aimed to evaluate the pro-inflammatory response of human coronary artery endothelial cells (HCAECs) to P. gingivalis in a 3D cell culture model compared with a 2D cell culture. HCAECs were cultured for 7 days on type I collagen matrices in both cultures and were stimulated at an MOI of 1 or 100 with live P. gingivalis W83 for 24 h. The expression of the genes COX-2, eNOS, and vWF and the levels of the pro-inflammatory cytokines thromboxane A2 (TXA-2) and prostaglandin I2 (PGI2) were evaluated. P. gingivalis W83 in the 2D cell culture increased IL-8 levels at MOI 100 and decreased MCP-1 levels at both MOI 100 and MOI 1. In contrast, the 3D cell culture induced an increased gene expression of COX-2 at both MOIs and reduced MCP-1 levels at MOI 100, whereas the gene expression of eNOS, vWF, and IL-8 and the levels of TXA2 and PGI2 showed no significant changes. These data suggest that in the collagen 3D culture model, P. gingivalis W83 induces a weak endothelial inflammatory response.

Differentiation States of Phenotypic Transition of Melanoma Cells Are Revealed by 3D Cell Cultures.

Melanoma is characterized by high metastatic potential favored by the epithelial-to-mesenchymal transition (EMT), leading melanoma cells to exhibit a spectrum of typical EMT markers. This study aimed to analyze the expression of EMT markers in A375 and BLM melanoma cell lines cultured in 2D monolayers and 3D spheroids using morphological and molecular methods. The expression of EMT markers was strongly affected by 3D arrangement and revealed a hybrid phenotype for the two cell lines. Indeed, although E-cadherin was almost undetectable in both A375 and BLM cells, cortical actin was detected in A375 2D monolayers and 3D spheroids and was strongly expressed in BLM 3D spheroids. The mesenchymal marker N-cadherin was significantly up-regulated in A375 3D spheroids while undetectable in BLM cells, but vimentin was similarly expressed in both cell lines at the gene and protein levels. This pattern suggests that A375 cells exhibit a more undifferentiated/mesenchymal phenotype, while BLM cells have more melanocytic/differentiated characteristics. Accordingly, the Zeb1 and 2, Slug, Snail and Twist gene expression analyses showed that they were differentially expressed in 2D monolayers compared to 3D spheroids, supporting this view. Furthermore, A375 cells are characterized by a greater invasive potential, strongly influenced by 3D arrangement, compared to the BLM cell line, as evaluated by SDS-zymography and TIMPs gene expression analysis. Finally, TGF-β1, a master controller of EMT, and lysyl oxidase (LOX), involved in melanoma progression, were strongly up-regulated by 3D arrangement in the metastatic BLM cells alone, likely playing a role in the metastatic phases of melanoma progression. Overall, these findings suggest that A375 and BLM cells possess a hybrid/intermediate phenotype in relation to the expression of EMT markers. The former is characterized by a more mesenchymal/undifferentiated phenotype, while the latter shows a more melanocytic/differentiated phenotype. Our results contribute to the characterization of the role of EMT in melanoma cells and confirm that a 3D cell culture model could provide deeper insight into our understanding of the biology of melanoma.

Abstract B015: Toxin-producing bacterial therapy limits tumor growth in autochthonous mouse models of pancreatic ductal adenocarcinoma

Abstract Treating pancreatic ductal adenocarcinoma (PDAC) with systemic chemotherapeutic drugs has remained a challenge, due in part to the hypovascularized and poorly perfused nature of PDAC tumors, impeding the accumulation of systemically delivered drugs. Several clinical trials aimed at improving drug delivery in PDAC, through targeting of ECM components (HALO-301) or stromal angiogenic signaling (IPI-926-03) have unfortunately not been effective. However, the features that have interfered with systemic therapy in PDAC are potential advantages for the use of bacterial therapies, as bacteria can actively migrate through tissues, thrive in hypoxic microenvironments, and benefit from local immune suppression. Recent developments in the field of synthetic biology have made it possible to engineer complex logic circuits into bacteria, enabling the production of anticancer therapies directly within the tumor parenchyma. Furthermore, live bacteria, once colonized within the tumor niche, are capable of providing a stable source of anticancer compounds directly, rather than relying on repeated systemic doses. We have therefore worked to develop novel bacterial strains and demonstrate preclinical efficacy of a novel strain of therapeutic bacteria for targeting PDAC. We began by testing a range of bacteria-produced toxins and identified the pore-forming protein theta toxin as having the greatest effect in both 2D cell culture and PDAC explant (tissue slice) models. We then engineered a non-toxic probiotic bacteria, E. coli Nissle 1917, to produce either theta toxin or GFP. To assess preclinical efficacy, we performed intratumoral injections of live GFP- and theta-expressing bacteria into the “KPC” genetically engineered mouse model (Kras LSL.G12D/+; Tp53 LSL.R172H/+; PdxCre tg/+). While GFP-producing bacteria did not induce a change in tumor growth kinetics, treatment with theta toxin-producing bacteria demonstrated prolonged stabilization of tumor growth, increasing the median survival to 34 days compared to ~12 days for vehicle- or gemcitabine-treated controls and 18.5 days for GFP-producing bacteria without additional therapy. Strikingly, while there was minimal spread of bacteria to non-tumor tissues, we observed translocation of the bacteria to macro- and micro- metastatic lesions and distant papillomas following injection of the primary pancreatic tumor, indicating a mechanism for targeting both known and unknown metastases following local administration. Histological analyses demonstrated that diffuse populations of bacteria co-localized with regions of cell death, but that bacterial presence and evidence of increased cell death was not observed in healthy tissues, such as the lung, liver, intestine, and diaphragm. Additionally, bacterial treatment, independent of the payload, led to a modest influx of anti-tumor immune populations, suggesting potential synergy with immunotherapies. Together these data demonstrate potent preclinical activity of cytotoxic bacterial therapy as a novel strategy to circumvent the challenges of systemic treatment of PDAC. Citation Format: Amanda R. Decker-Farrell, Stephen A. Sastra, Tetsuhiro Harimoto, Marie C. Hasselluhn, Fangda Li, Rosa Vincent, Carmine F. Palermo, Edward R. Ballister, Tal Danino, Kenneth P. Olive. Toxin-producing bacterial therapy limits tumor growth in autochthonous mouse models of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B015.

Decellularized porcine kidney-incorporated hydrogels for cell-laden bioprinting of renal cell carcinoma model

More than 90% of kidney cancers are attributed to renal cell carcinoma (RCC), which is however highly resistant to traditional chemotherapy. The challenges met in treating RCC signify an imperative to accelerate the development of new and effective drugs. Preclinical testing has served as a foundation for evaluating potential effectiveness of new drugs, but this endeavor is deeply restricted by the current generation of in vitro two-dimensional culture models, which cannot accurately mimic the tumor microenvironment (TME). Therefore, new in vitro three-dimensional (3D) cell culture models that can better mimic the components and architecture of TME have been developed for preclinical testing, but only a few existing 3D cell culture models can simulate the TME of RCC, representing a limitative obstacle impeding the development of novel drugs for RCC. In this study, we prepared a bioink by mixing porcine kidney decellularized extracellular matrix (dECM) powders with gelatin methacryloyl (GelMA) to bioprint an in vitro 3D cell culture model for RCC. We found that GelMA stability, mechanical properties, and printability were all significantly improved following the addition of the dECM powder. Moreover, cell cultures using ACHN cells suggested that kidney dECM powders significantly improved the cellular proliferation and metastasis via upregulation of markers related to epithelial– mesenchymal transition, along with activation of several cancer progression-related signaling pathways. More importantly, ACHN cells also demonstrated higher resistance to sunitinib under the stimulation of kidney dECM, indicating that GelMA-kidney dECM hydrogels may be an appropriate preclinical model to be used for building an in vitro RCC platform for drug screening and development.