Anticancer Drug Testing Research Articles

Abstract B093: The evaluation of the gene expression profile of pancreatic ductal adenocarcinomas (PDACs) in 3D cell culture vs in vivo and development of new 3D PDACs' models for an evaluation of anti-cancer drug testing

Abstract In the ever-evolving landscape of biomedical research, 3D Cell Culture (3DCC) systems have emerged as pivotal tools for mimicking in vivo environments and fostering more physiologically relevant cellular responses. Despite promises, 3DCC based drug discovery efforts didn’t fulfil the hopes. The classic way 3DCC is done, always starts with 2D cell culture (2DCC) followed by brief 3 day 3DCC and finally drug testing. Following those observations we decided to mimic much more than just brief 3DCC state but enhance the culture by constant 3DCC to 3DCC passages. We characterized the gene expression profiles and proteomic characteristics of pancreatic tumors concluding that prolonged 3DCC is the closest to an in vivo model. The protracted cultivation periods mimic the intricacies of the in vivo environment, enabling a more accurate representation of the molecular dynamics within pancreatic tumors. In our study, we identified a panel of genes characterized by similar expression levels between prolonged 3D cell culture and in vivo conditions, while displaying significant divergence between 2D cultures and short-term 3D cultures when compared to in vivo. These genes play a pivotal role in the progression and treatment of cancer, underscoring the potential of prolonged 3DCC of LifeGel® significance in understanding tumor biology and devising therapeutic strategies. Furthermore, we also investigated the implications of prolonged 3D in vitro cultures on evaluating anti-cancer drug activities. The findings highlight the significance of 3DCC in enhancing our understanding of pancreatic tumor biology and in refining drug screening methodologies for more effective therapeutic interventions. By providing a biomimetic microenvironment, our hydrogels facilitate the formation of complex cellular structures, enabling more accurate representation of tissue architecture and cellular interactions. We delivered LifeGel® platform which presents a myriad of opportunities for researchers seeking to enhance the fidelity and translatability of their in vitro studies into pre-clinial models. Citation Format: Marcin Krzykawski, David Earnshaw, Krzysztof Kus, Artur Pirog, Sachin Kote, Mark Treherne, Justyna Kocik-Krol. The evaluation of the gene expression profile of pancreatic ductal adenocarcinomas (PDACs) in 3D cell culture vs in vivo and development of new 3D PDACs' models for an evaluation of anti-cancer drug testing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B093.

A 3D-printed tumor-on-chip: user-friendly platform for the culture of breast cancer spheroids and the evaluation of anti-cancer drugs

Tumor-on-chips (ToCs) are useful platforms for studying the physiology of tumors and evaluating the efficacy and toxicity of anti-cancer drugs. However, the design and fabrication of a ToC system is not a trivial venture. We introduce a user-friendly, flexible, 3D-printed microfluidic device that can be used to culture cancer cells or cancer-derived spheroids embedded in hydrogels under well-controlled environments. The system consists of two lateral flow compartments (left and right sides), each with two inlets and two outlets to deliver cell culture media as continuous liquid streams. The central compartment was designed to host a hydrogel in which cells and microtissues can be confined and cultured. We performed tracer experiments with colored inks and 40 kDa fluorescein isothiocyanate dextran to characterize the transport/mixing performances of the system. We also cultured homotypic (MCF7) and heterotypic (MCF7-BJ) spheroids embedded in gelatin methacryloyl hydrogels to illustrate the use of this microfluidic device in sustaining long-term micro-tissue culture experiments. We further demonstrated the use of this platform in anticancer drug testing by continuous perfusion of doxorubicin, a commonly used anti-cancer drug for breast cancer. In these experiments, we evaluated drug transport, viability, glucose consumption, cell death (apoptosis), and cytotoxicity. In summary, we introduce a robust and friendly ToC system capable of recapitulating relevant aspects of the tumor microenvironment for the study of cancer physiology, anti-cancer drug transport, efficacy, and safety. We anticipate that this flexible 3D-printed microfluidic device may facilitate cancer research and the development and screening of strategies for personalized medicine.

3D bioprinting of an in vitro hepatoma microenvironment model: Establishment, evaluation, and anticancer drug testing

Tumor behavior, including its response to treatments, is influenced by interactions between mesenchymal and malignant cells, as well as their spatial arrangement. To study tumor biology and evaluate anticancer drugs, accurate 3D tumor models are essential. Here, we developed an in vitro biomimetic hepatoma microenvironment model by combining an extracellular matrix (3DM-7721). Initially, the internal grid structure, composed of 10/6 % GelMA/gelatin loaded with SMMC-7721 cells, was printed using 3D bioprinting. The external component consisted of fibroblasts and human umbilical vein endothelial cells loaded with 10/3 % GelMA/gelatin. A control model (3DP-7721) lacked external cell loading. GelMA/gelatin hydrogels provided robust structural support and biocompatibility. The SMMC-7721 cells in the 3DM-7721 model exhibit superior tumor-associated gene expression and proliferation characteristics when compared to the 3DP-7721 model. Furthermore, the 3DM-7721 type exhibited increased resistance to anticancer agents. SMMC-7721 cells in the 3DM-7721 model exhibit significant tumorigenicity in nude mice. The 3DM-7721 model group showed pathological characteristics of malignant tumors, with a high degree of deterioration, and a significant positive correlation between malignant tumor-related gene pathways. This high-fidelity 3DM-7721 tumor microenvironment model is invaluable for studying tumor progression, devising effective treatment strategies, and discovering drugs. Statement of significance1.A hepatoma microenvironment model (3DM-7721) incorporating an extracellular matrix that is designed to establish solid tumor models in vitro.2.This model contains a 3D-printed internal tumor component of liver cancer cells loaded with 10/6 % gelatin methacrylate (GelMA)/gelatin (the 3DP-7721 model) and an external extracellular-matrix component of fibroblasts and human umbilical vein endothelial cells loaded with 10/3 % GelMA/gelatin.3.The 3DM-7721 model outperforms the 3DP-7721 model in terms of the SMMC-7721 cell proliferation characteristics and tumorigenicity, tumor-related gene expression, anticancer drug resistance, and malignant tumor characteristics.

Label-free visualization and quantification of the drug-type-dependent response of tumor spheroids by dynamic optical coherence tomography

We demonstrate label-free dynamic optical coherence tomography (D-OCT)-based visualization and quantitative assessment of patterns of tumor spheroid response to three anti-cancer drugs. The study involved treating human breast adenocarcinoma (MCF-7 cell-line) with paclitaxel (PTX), tamoxifen citrate (TAM), and doxorubicin (DOX) at concentrations of 0 (control), 0.1, 1, and 10 µM for 1, 3, and 6 days. In addition, fluorescence microscopy imaging was performed for reference. The D-OCT imaging was performed using a custom-built OCT device. Two algorithms, namely logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDSl\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_l$$\\end{document}) were used to visualize the tissue dynamics. The spheroids treated with 0.1 and 1 µM TAM appeared similar to the control spheroid, whereas those treated with 10 µM TAM had significant structural corruption and decreasing LIV and OCDSl\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_l$$\\end{document} over treatment time. The spheroids treated with PTX had decreasing volumes and decrease of LIV and OCDSl\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_l$$\\end{document} signals over time at most PTX concentrations. The spheroids treated with DOX had decreasing and increasing volumes over time at DOX concentrations of 1 and 10 µM, respectively. Meanwhile, the LIV and OCDSl\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_l$$\\end{document} signals decreased over treatment time at all DOX concentrations. The D-OCT, particularly OCDSl\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_l$$\\end{document}, patterns were consistent with the fluorescence microscopic patterns. The diversity in the structural and D-OCT results among the drug types and among the concentrations are explained by the mechanisms of the drugs. The presented results suggest that D-OCT is useful for evaluating the difference in the tumor spheroid response to different drugs and it can be a useful tool for anti-cancer drug testing.

Leveraging Patient-Derived Organoids for Personalized Liver Cancer Treatment.

Primary liver cancer (PLC) is a primary cause of cancer-related death worldwide, and novel treatments are needed due to the limited options available for treatment and tumor heterogeneity. 66 surgically removed PLC samples were cultured using the self-developed 2:2 method, and the final success rate for organoid culture was 40.9%. Organoid performance has been evaluated using comprehensive molecular measurements, such as whole-exome and RNA sequencing, as well as anticancer drug testing. Multiple organoids and their corresponding tumor tissues contained several of the same mutations, with all pairs sharing conventional TP53 mutations. Regarding copy number variations and gene expression, significant correlations were observed between the organoids and their corresponding parental tumor tissues. Comparisons at the molecular level provided us with an assessment of organoid-to-tumor concordance, which, in combination with drug sensitivity testing provided direct guidance for treatment selection. Finally, we were able to determine an appropriate pharmacological regimen for a patient with ICC, demonstrating the clinical practicality in tailoring patient-specific drug regimens. Our study provides an organoid culture technology that can cultivate models that retain most of the molecular characteristics of tumors and can be used for drug sensitivity testing, demonstrating the broad potential application of organoid technology in precision medicine for liver cancer treatment.

A 3D hanging spheroid-filter plate for high-throughput drug testing and CAR T cell cytotoxicity assay.

Tumour spheroids are widely used in immune cell cytotoxicity assays and anticancer drug testing, providing a physiologically relevant model replicating the tumour microenvironment. However, co-culture of immune and tumour cells complicates quantification of immune cell killing efficiency. We present a novel 3D hanging spheroid-filter plate that efficiently facilitates spheroid formation and separates unbound/dead cells during cytotoxicity assays. Optical imaging directly measures the cytotoxic effects of anti-cancer drugs on tumour spheroids, eliminating the need for live/dead fluorescent staining. This approach enables cost-effective evaluation of T-cell cytotoxicity with specific chimeric antigen receptors (CARs), enhancing immune cell-based assays and drug testing in three-dimensional tumour models.

Selective expansion of renal cancer stem cells using microfluidic single-cell culture arrays for anticancer drug testing.

The suboptimal prognosis associated with drug therapy for renal cancer can be attributed to the presence of stem-cell-like renal cancer cells. However, the limited number of these cells prevents conventional drug screening assays from effectively assessing the response of renal cancer stem cells to anti-cancer agents. To address this issue, the present study employed microfluidic single-cell culture arrays to expand renal cancer stem cells by exploiting the anti-apoptosis and self-renewal properties of tumor stem cells. A microfluidic chip with 18 000 hydrophilic microwells was designed and fabricated to establish the single-cell culture array. Over a 7 day culture, the large-scale single-cell culture yielded a limited quantity of single-cell-derived tumorspheres. The sphere formation rates for Caki-1, 786-O, and ACHN cells were determined to be 8.74 ± 0.53%, 12.02 ± 1.43%, and 4.98 ± 1.68%, respectively. The expanded cells exhibited stemness characteristics, as indicated by immunofluorescence, flow cytometry, serial passaging, and in vitro differentiation assays. Additionally, the comparative transcriptomic analysis showed significant differences in the gene expression patterns of the expanded cells compared to the differentiated renal cancer cells. The drug testing indicated that renal cancer stem cells exhibited reduced sensitivity towards the tyrosine kinase inhibitors sorafenib and sunitinib, compared to differentiated renal cancer cells. This reduced sensitivity can be attributed to the elevated expression levels of tyrosine kinase in renal cancer stem cells. This present study provides evidence that the utilization of microfluidic single-cell culture arrays for selective cell expansion can facilitate drug testing of renal cancer stem cells.

3D bioprinting of in vitro porous hepatoma models: establishment, evaluation, and anticancer drug testing

3D bioprinting of in vitro porous hepatoma models: establishment, evaluation, and anticancer drug testing

Label-free drug response evaluation of human derived tumor spheroids using three-dimensional dynamic optical coherence tomography

This study aims at demonstrating label-free drug-response-patterns assessment of different tumor spheroids and drug types by dynamic optical coherence tomography (D-OCT). The study involved human breast cancer (MCF-7) and colon cancer (HT-29) spheroids. The MCF-7 and HT-29 spheroids were treated with paclitaxel (Taxol; PTX) and the active metabolite of irinotecan SN-38, respectively. The drugs were applied with 0 (control), 0.1, 1, and 10 μM concentrations and the treatment durations were 1, 3, and 6 days. A swept-source OCT microscope equipped with a repeated raster scanning protocol was used to scan the spheroids. Logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDS_l) algorithms were used to visualize the tumor spheroid dynamics. LIV and OCDS_l images visualized different response patterns of the two types of spheroids. In addition, spheroid morphology, LIV, and OCDS_l quantification showed different time-courses among the spheroid and drug types. These results may indicate different action mechanisms of the drugs. The results showed the feasibility of D-OCT for the evaluation of drug response patterns of different cell spheroids and drug types and suggest that D-OCT can perform anti-cancer drug testing.

Planar Waveguide NMR Gradients (B0 and B1) for spatiotemporal Investigations of Anticancer Drug Concentration in 3D Cancer Cell Cultures

Despite the progression in cancer therapeutic approaches, cancer is still the leading cause of death worldwide with a steadily increasing incidence rate. In Jordan, CRC is the most common type of cancer among men and the second most common among women, while being one of the most accruing cancers in Germany.
 The difficulties in cancer treatments arise from the poor correlation between in vivo and in in vitro anti-cancer drug testing results and cancer cells resistance to chemotherapy, which are attributed to the heterogeneity of cancer cells, and the interaction of cancer cells with non-cancerous cells in the tumor microenvironment (TME). In addition to, the several mechanisms, that governs DNA damages repair, epigenetics, epithelial to mesenchymal transition (EMT).
 Cell culture techniques have been developed to construct in vitro multicellular cancer spheroids (MTCS) that mimic cell-cells interaction and the cellular organization in the TME. They also illustrate the proliferation gradient of solid tumors, and the nutrition and gas supply to cancer cells at different depth in the spheroid. Therefore, they are considered an excellent in vitro cancer model to study the efficiency and to investigate the penetration depth of the anti-cancer materials.
 Nuclear Magnetic Resonance (NMR) spectroscopy is a reliable analytical method that gives comprehensive and rich chemical information, along with high speciation performance. It is one of the major noninvasive tools for metabolomics, therefore it has been utilized to study the metabolic profiling of freshly isolated- non-destructed tissues, and living in vitro cellular models.
 A micro-imaging technique for real-time NMR investigations is developed to study the penetration depth, rate, and effective diffusion coefficients of anti-cancer materials through cancer spheroids. This technique is based on our patented invention of planar waveguide micro slot NMR detector with on board thermal regulator integrated and a microfluidic device. Based on this innovative development, we are able to perform real-time magnetic resonance spectroscopic imaging (MRSI) on a living synthetic tissue constructed in micrometer scale to monitor drug penetration and diffusion, while providing spectroscopic information about the production and degradation of metabolites in picomoles concentrations.

Hierarchical Vessel Network-Supported Tumor Model-on-a-Chip Constructed by Induced Spontaneous Anastomosis.

The vascular system in living tissues is a highly organized system that consists of vessels with various diameters for nutrient delivery and waste transport. In recent years, many vessel construction methods have been developed for building vascularized on-chip tissue models. These methods usually focused on constructing vessels at a single scale. In this work, a method that can build a hierarchical and perfusable vessel networks was developed. By providing flow stimuli and proper HUVEC concentration, spontaneous anastomosis between endothelialized lumens and the self-assembled capillary network was induced; thus, a perfusable network containing vessels at different scales was achieved. With this simple method, an in vivo-like hierarchical vessel-supported tumor model was prepared and its application in anticancer drug testing was demonstrated. The tumor growth rate was predicted by combining computational fluid dynamics simulation and a tumor growth mathematical model to understand the vessel perfusability effect on tumor growth rate in the hierarchical vessel network. Compared to the tumor model without capillary vessels, the hierarchical vessel-supported tumor shows a significantly higher growth rate and drug delivery efficiency.

Large inherent variability in data derived from highly standardised cell culture experiments

Cancer drug development is hindered by high clinical attrition rates, which are blamed on weak predictive power by preclinical models and limited replicability of preclinical findings. However, the technically feasible level of replicability remains unknown. To fill this gap, we conducted an analysis of data from the NCI60 cancer cell line screen (2.8 million compound/cell line experiments), which is to our knowledge the largest depository of experiments that have been repeatedly performed over decades. The findings revealed profound intra-laboratory data variability, although all experiments were executed following highly standardised protocols that avoid all known confounders of data quality. All compound/ cell line combinations with > 100 independent biological replicates displayed maximum GI50 (50% growth inhibition) fold changes (highest/ lowest GI50) > 5% and 70.5% displayed maximum fold changes > 1000. The highest maximum fold change was 3.16 × 1010 (lowest GI50: 7.93 ×10-10 µM, highest GI50: 25.0 µM). FDA-approved drugs and experimental agents displayed similar variation. Variability remained high after outlier removal, when only considering experiments that tested drugs at the same concentration range, and when only considering NCI60-provided quality-controlled data. In conclusion, high variability is an intrinsic feature of anti-cancer drug testing, even among standardised experiments in a world-leading research environment. Awareness of this inherent variability will support realistic data interpretation and inspire research to improve data robustness. Further research will have to show whether the inclusion of a wider variety of model systems, such as animal and/ or patient-derived models, may improve data robustness.

Progress of 3D Organoid Technology for Preclinical Investigations: Towards Human In Vitro Models

Review Progress of 3D Organoid Technology for Preclinical Investigations: Towards Human In Vitro Models Yingjuan Liu *, Honglin Xu, Sabu Abraham, Xin Wang, and Bernard D. Keavney* Division of Cardiovascular Sciences, Faculty of Biology, Medicine and Health, University of Manchester, M13 9PT, UK. * Correspondence: yingjuan.liu@manchester.ac.uk (Yingjuan Liu); bernard.keavney@manchester.ac.uk (Bernard D. Keavney) Received: 1 November 2022 Accepted: 24 November 2022 Published: 21 December 2022 Abstract: Currently, with an increased requirement for new therapeutic strategies, preclinical drug testing or screening platforms have rapidly evolved in recent years. In comparison to traditional 2D cell cultures, 3D organoids or spheroids with or without scaffolds improve the microenvironment of in vitro cultures, advancing the in vitro biological observation and enabling mechanistic studies of drug reactions in the human tissue-like environment. 3D organoids and spheroids are straightforward to produce, and relatively uniform in size and shape. This helps to facilitate high throughput screening requirements. Spheroids and organoids have been applied in anti-cancer drug testing, toxicity evaluations, as well as mechanism studies for variable organ systems, including the intestine, liver, pancreas, brain, and heart. Among 3D cultures of spheroids and organoids, ‘tumour spheroids’ formed by dissociated tumour tissues or cancer cell lines are relatively simple in composition and commonly applied to anticancer drug screening. The ‘healthy organoids’ differentiated from hiPSCs/hESCs are more complex in cell composition, distribution, structure and function with higher similarity to in vivo organs, and have found applications in toxicity tests, personalised medicine, and therapeutic and mechanistic studies. In most cases, the multicellular 3D organoids are more resistant and stable in reaction to stimulations or chemicals in vitro , suggesting more accurate modelling of in vivo responses. Here, we review recent progress in human-origin organoid/spheroid systems and their applications in preclinical studies.

Variants of creating heterotopic and orthotopic PDX models of human colorectal cancer

Aim. To create heterotopic and orthotopic patient-derived xenograft (PDX) models of colorectal cancer (CRC) by transplantation of patient’s tumor samples into immunodeficient BALB / c Nude mice.Materials and methods. The study was performed on 15 female BALB / c Nude mice aged 6–8 weeks weighing 21–25 g. All animals underwent transplantation of the tumor material obtained from CRC patients into the following sites: heterotopic transplantation (under the skin of the thigh and into the omentum), orthotopic transplantation (into the descending and ascending colon and into the cecum). Weight and general condition of the animals and the size of the tumor nodule had been monitored for 80 days. The success of each model was assessed by the degree of engraftment, the dynamics of tumor growth, and the reproducibility of histopathologic characteristics. At the end of the experiment, the animals were euthanized by cervical dislocation.Results. 100% survival of the animals and similar tumor growth dynamics in the xenograft models were observed throughout the experiment. The analysis of histologic specimens obtained from the xenografts and patient’s tumor showed their correspondence to moderately differentiated intestinal adenocarcinoma. The main advantages and disadvantages of different variants of PDX models were described.Conclusion. Heterotopic and orthotopic PDX models reproduce the morpho-histologic characteristics of human tumors and demonstrate stable growth dynamics. Therefore, they are a suitable tool for the development, testing, and validation of potential anticancer drugs.

Scalable Formation of Highly Viable and Functional Hepatocellular Carcinoma Spheroids in an Oxygen-Permeable Microwell Device for Anti-Tumor Drug Evaluation.

For high-throughput anti-cancer drug screening, microwell arrays may serve as an effective tool to generate uniform and scalable tumor spheroids. However, microwell arrays are commonly anchored in non-oxygen-permeable culture plates, leading to limited oxygen supply for avascular spheroids. Herein, a polydimethylsiloxane (PDMS)-based oxygen-permeable microwell device is introduced for generating highly viable and functional hepatocellular carcinoma (HCC) spheroids. The PDMS sheets at the bottom of the microwell device provide a high flux of oxygen like in vivo neighboring hepatic sinusoids. Owing to the better oxygen supply, the generated HepG2 spheroids are larger in size and exhibit higher viability and proliferation with less cell apoptosis and necrosis. These spheroids also exhibit lower levels of anaerobic cellular respiration and express higher levels of liver-related functions. In anti-cancer drug testing, spheroids cultured in PDMS plates show a significantly stronger resistance against doxorubicin because of the stronger stem-cell and multidrug resistance phenotype. Moreover, higher expression of vascular endothelial growth factor-A produces a stronger angiogenesis capability of the spheroids. Overall, compared to the spheroids cultured in conventional non-oxygen-permeable plates, these spheroids can be used as a more favorable model for early-stage HCCs and be applied in high-throughput anti-cancer drug screening.

Patient-derived cancer models: Valuable platforms for anticancer drug testing.

Molecularly targeted treatments and immunotherapy are cornerstones in oncology, with demonstrated efficacy across different tumor types. Nevertheless, the overwhelming majority metastatic disease is incurable due to the onset of drug resistance. Preclinical models including genetically engineered mouse models, patient-derived xenografts and two- and three-dimensional cell cultures have emerged as a useful resource to study mechanisms of cancer progression and predict efficacy of anticancer drugs. However, variables including tumor heterogeneity and the complexities of the microenvironment can impair the faithfulness of these platforms. Here, we will discuss advantages and limitations of these preclinical models, their applicability for drug testing and in co-clinical trials and potential strategies to increase their reliability in predicting responsiveness to anticancer medications.

Disulfiram increases the efficacy of 5-fluorouracil in organotypic cultures of colorectal carcinoma

Drug efficacy determined in preclinical research is difficult to transfer to clinical practice. This is mainly due to the use of oversimplified models omitting the effect of the tumor microenvironment and the presence of various cell types participating in the formation of tumors in vivo. In this study, we used robust three-dimensional models including spheroids grown from colon cancer cell lines and organotypic cultures prepared from the colorectal carcinoma tissue to test novel therapeutic strategies. We developed a multi-modal approach combining brightfield and fluorescence microscopy for evaluating drug effects on organotypic cultures. Combined treatment with 5-fluorouracil and disulfiram/copper efficiently eliminated cancer cells in these 3D models. Moreover, disulfiram/copper down-regulated the expression of markers associated with 5-fluorouracil resistance, such as thymidylate synthase and CD133/CD44. Thus, we propose combined therapy of 5-fluorouracil and disulfiram/copper for further testing as a treatment for colorectal carcinoma. In addition, we show that organotypic cultures are suitable models for anti-cancer drug testing.

Ordered micropattern arrays fabricated by lung-derived dECM hydrogels for chemotherapeutic drug screening.

AimsThis study aims to evaluate ECM-coated micropattern arrays derived from decellularization of native porcine lungs as a novel three-dimensional cell culture platform.MethodsECM derived from decellularization of native porcine lungs was exploited to prepare hydrogels. Then, dECM-coated micropattern arrays were fabricated at four different diameters (50, 100, 150 and 200 ​μm) using polydimethylsiloxane (PDMS). Two lung cancer cell lines, A549 and H1299, were tested on a dECM-coated micropattern array as a novel culture platform for cell adhesion, distribution, proliferation, viability, phenotype expression, and drug screening to evaluate the cytotoxicity of paclitaxel, doxorubicin and cisplatin.ResultsThe ECM derived from decellularization of native porcine lungs supported cell adhesion, distribution, viability and proliferation better than collagen I and Matrigel as the coated matrix on the surface. Moreover, the optimal diameter of the micropattern arrays was 100–150 ​μm, as determined by measuring the morphology, viability, proliferation and phenotype of the cancer cell spheroids. Cell spheroids of A549 and H1299 on dECM-coated micropattern arrays showed chemoresistance to anticancer drugs compared to that of the monolayer. The different distributions of HIF-1α, MCL-1 (in the center) and Ki-67 and MRP2 (in the periphery) of the spheroids demonstrated the good establishment of basal-lateral polarity and explained the chemoresistance phenomenon of spheroids.ConclusionsThis novel three-dimensional cell culture platform is stable and reliable for anticancer drug testing. Drug screening in dECM-coated micropattern arrays provides a powerful alternative to existing methods for drug testing and metabolic profiling in the drug discovery process.

Culture of cancer spheroids and evaluation of anti-cancer drugs in 3D-printed miniaturized continuous stirred tank reactors (mCSTRs)

Cancer continues to be a leading cause of mortality in modern societies; therefore, improved and more reliable in vitro cancer models are needed to expedite fundamental research and anti-cancer drug development. Here, we describe the use of a miniaturized continuous stirred tank reactor (mCSTR) to first fabricate and mature cancer spheroids (i.e. derived from MCF7 cells, DU145 cells, and a mix of MCF7 cells and fibroblasts), and then to conduct anti-cancer drug assays under continuous perfusion. This 3 ml mCSTR features an off-center agitation system that enables homogeneous chaotic laminar mixing at low speeds to support cell aggregation. We incubated cell suspensions for 3 d in ultra-low-attachment plates to allow formation of discoid cell aggregates (∼600 µm in diameter). These cell aggregates were then transferred into mCSTRs and continuously fed with culture medium. We characterized the spheroid morphology and the expression of relevant tumor biomarkers at different maturation times for up to 4 weeks. The spheroids progressively increased in size during the first 5–6 d of culture to reach a steady diameter between 600 and 800 µm. In proof-of-principle experiments, we demonstrated the use of this mCSTR in anti-cancer drug testing. Three drugs commonly used in breast cancer treatment (doxorubicin, docetaxel, and paclitaxel) were probed at different concentrations in MCF7-derived spheroids. In these experiments, we evaluated cell viability, glucose consumption, spheroid morphology, lactate dehydrogenase activity, and the expression of genes associated with drug resistance (ABCB1 and ABCC1) and anti-apoptosis (Bcl2). We envision the use of this agitated system as a tumor-on-a-chip platform to expedite efficacy and safety testing of novel anti-cancer drugs and possibly in personalized medicine applications.

Optimization of tumor spheroid model in mesothelioma and lung cancers and anti-cancer drug testing in H2052/484 spheroids.

Advanced lung cancers and mesothelioma remain incurable diseases. Despite some promising new therapy strategies, predicting whether an individual patient will be sensitive to a given therapy is challenging. The purpose of this study is to establish and evaluate the efficiency of a three-dimensional spheroid model of human thoracic cancer in predicting the efficacy of drugs.Human mesothelioma and lung tumor spheroids were established from cell lines and primary cells derived from the patient. The growth kinetics and cell viability of microtumors were assessed using spheroid size and intracellular ATP level. The sensitivity of the mesothelioma spheroids to the cisplatin or cisplatin/pemetrexed combination was determined.We determined that studying the kinetics of the spheroid growth for 15 days after seeding 1000 cells/well in a 96-well plate was optimal. Monitoring the growth kinetic and intracellular ATP of spheroids allowed the identification of early changes in spheroid viability. Finally, we validated this model by measuring a dose-dependent reduction in the cell viability of mesothelioma H2052/484 spheroids treated with both first-line treatments, cisplatin and the cisplatin/pemetrexed combination. In conclusion, we have developed a three-dimensional spheroid model of thoracic tumor cells useful for tailoring the medical treatment to the specific characteristics of each patient.