This work is supported primarily by DOD OCRP Early Career Investigator Award W81XWH-13-1-0134(GM), DOD Pilot award W81XWH-16-1-0426 (GM), DOD Investigator Initiated award W81XWH-17-OCRP-IIRA (GM), Rivkin Center for Ovarian Cancer and Michigan Ovarian Cancer Alliance. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under award number P30CA046592. CMN is supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1256260. MEB is supported by the Department of Education Graduate Assistance in Areas of National Need (GAANN) Fellowship.

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The authors have nothing to disclose.

The 384-well hanging drop plate platform for 3D spheroid formation is an easily implemented tool for any cell biology or cancer biology labs. This physiologic platform enables the study of cell lines, as well as, primary patient samples within physiologically relevant 3D cultures while allowing for high throughput drug screening. The platform also ensures that the culture conditions are highly tunable, enabling tight control over plating densities, cell&#160;co-culture ratios, extracellular components, and medium composi...

Worldwide cancer-related mortality reached a toll of 9.8 million deaths in 20181, highlighting the need for the development of improved therapeutics. Unfortunately, the cost of developing cancer drugs is increasing, with the development of a single drug costing approximately 650 million USD2 indicating the need for improved strategies to develop new anti-cancer drugs. Cancer stem cells (CSCs), which are characterized by increased chemoresistance3, the capacity to self-renew, and the ability to seed new tumors4 are thought to be responsible for tumor recurrence4, metastasis5, and chemoresistance4,6, which all contribute to the malignant capacity of the tumor and thus the high death toll. In ovarian cancer, these cells are found enriched in the malignant ascites fluid in the peritoneal cavity, a condition associated with poor clinical outcomes1. As a result of the malignant capabilities of CSCs, there has been a push to develop new CSC targeting drugs to use in conjunction with traditional chemotherapies. There are several challenges that accompany the development of CSC targeting drugs including: 1) difficulty in expanding and maintaining CSCs in vitro; 2) scarcity of patient samples; 3) physiological relevance of the culture platform; and 4) heterogeneity in drug sensitivity between patients. This protocol outlines the implementation of a high throughput 3D culture platform that can overcome each of these challenges. In particular, this system allows for rapid drug screening using small numbers of patient-derived ovarian CSCs, and is highly amenable to downstream analysis techniques. While ideal for studying ovarian cancer and CSCs, our platform is also valuable in studying other cancers and differentiated cell types in complex 3D environments.
Complex 3-dimensional (3D) models are critical in studying the tumor microenvironment (TME), which is a 3D niche made up of cancer cells, non-cancer supporting cells, and extracellular matrix (ECM) proteins4. This 3D environment results in unique cell morphology, cell-cell and cell-matrix interactions, cell differentiation, cell migration, cell density, and diffusion gradients compared to traditional 2D cell culture in vitro4. All of these factors culminate in differential drug response within 3D cultures, exhibiting increased drug resistance and physiological relevance7,8. Due to the role of the 3D TME in CSC differentiation and chemoresistance, it is vital to screen for CSC targeting drugs in physiologic microenvironments. Improving the physiological relevance of CSC drug screening platforms has the potential to improve patient specific drug screening, drug development, formulation of treatment strategies, and ultimately clinical outcomes. It is equally important that the platform used for drug screening be high-throughput and compatible with downstream analysis methods to minimize cost, time, and clinical translation time of promising drugs9.
Currently, the complex TME is best maintained for drug screening applications through in vivo models such as murine syngeneic tumor models, cell line-derived xenografts, and patient-derived xenograft (PDX) models12, as they provide physiologic conditions. However, the low-throughput nature of these models, as well as, the cost, time, and technical skill sets that they require limits their utility in rapid, high throughput drug screening applications13. As alternatives to these in vivo models, many in vitro 3D models utilizing hydrogels8, culture within microfluidic devices or &#8216;organ-on-a-chip&#8217; devices10,14, and non-adherent cultures3,8 have also been developed, due to their low barrier to entry in terms of cost, time, and required skillset.
Hydrogel culture platforms are advantageous in the fine control afforded over the matrix composition, mechanical properties, and matrix structure15; however, they can inhibit high density cell culture14. Additionally, harvesting cells from hydrogels can complicate downstream analysis, due to potentially harmful effects of harvesting methods15. Microfluidic devices, on the other hand, are microscale devices that allow for output detection within the same device and for cell culture at physiologically relevant scales with minimal consumption of reagents, decreased reaction time, minimized waste, and rapid diffusion14. These characteristics make them promising platforms for investigating drug toxicity, efficacy, and pharmacokinetics. However, the challenges of efficient, quantifiable, reproducible, and user-friendly 3D cell culture, as well as, bulky and costly pumping systems have restricted microfluidic applications in high-throughput research10. Efficient detection setups and potentially difficult implementation across fields have also hindered widespread adoption of microfluidic systems10.
Contrarily, spheroids generated in non-adherent conditions in rotating mixers (nutators), ultra-low attachment plates, and hanging droplets do not include user-defined matrix components. These methodologies are especially relevant for studying ovarian cancer as the non-adherent conditions are representative of the conditions in which spheroids grow within the peritoneal cavity5. Within these non-adherent culture methods, nutator and hanging drop spheroids have been shown to exhibit higher compaction, remodeling, and chemoresistance compared to spheroids generated in ultra-low attachment plates, suggesting increased physiological relevance16,17,18,19. Due to increased capacity for high-throughput screening from smaller well sizes and minimal required cell numbers, spheroid generation in hanging drop plates is an ideal platform for drug screening. Here, we present a tunable 3D physiologic platform in 384-well hanging drop plates, that is easy to implement and highly amenable to downstream analysis, making it ideal for high throughput drug screening of ovarian cancer and ovarian CSCs.
Our 3D physiologic platform provides all of the advantages of 3D culture, including physiological cell-cell contacts, diffusion gradients, cell densities, and naturally produced ECM proteins, which may contribute to realistic drug responses16,17,18,19. Additionally, by generating these spheroids with patient-derived CSCs, we are able to determine patient specific responses to drugs1 with many technical replicates simultaneously, to overcome heterogeneity that may be found within patient tumor samples20. Furthermore, 3D culture has been shown to enhance maintenance of CSC populations3,16 and thus is representative of enriched CSC populations in the ascites7. This combined with easy downstream analysis, including flow cytometry analysis of viability and CSC proportions allows for optimal evaluation of CSC targeting drug efficacy. Finally, this physiologic platform is compatible with imaging at multiple time points during the experiment, evaluation of cell death and proliferation, cell organization and morphology with immunohistochemistry, soluble signaling with ELISA on conditioned medium, cell phenotypes with flow cytometry, and gene expression following PCR.

<table><tbody><tr><td>0.25% trypsin-EDTA</td><td>Gibco</td><td>ILT25200056</td><td></td></tr><tr><td>10 mL serological pipet</td><td>Fisher Scientific</td><td>13-678-11E</td><td></td></tr><tr><td>10,000 cSt Si oil</td><td>Millipore Sigma</td><td>63148-62-9</td><td>Used to coat spheroid array mold to facilitate removal of tissue processing gels, like Histogel, from the mold.</td></tr><tr><td>100 mm tissue culture dish</td><td>Thermo Scientific</td><td>130182</td><td></td></tr><tr><td>15 mL conical tube</td><td>Celltreat</td><td>FL4021</td><td></td></tr><tr><td>1x DMEM for Serum Free Medium</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>1x F12 for Serum Free Medium</td><td>Gibco</td><td>11765-054</td><td></td></tr><tr><td>1x phosphate buffered saline (PBS)</td><td>Gibco</td><td>ILT10010023</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>Thermo Fisher</td><td>D1306</td><td></td></tr><tr><td>40 &amp;micro;m filter</td><td>Fisher Scientific</td><td>22363547</td><td></td></tr><tr><td>6-well plate</td><td>Fisher Scientific</td><td>353046</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies Inc.</td><td>1449</td><td>A gentle cell detachment enzyme composed of proteolytic and collagenolytic enzymes.</td></tr><tr><td>ACK Lysing Buffer</td><td>Thermo Scientific</td><td>A1049201</td><td></td></tr><tr><td>alamarBlue</td><td>Invitrogen</td><td>DAL1025</td><td>Resazurin dye used to measure viability and proliferation of cells based on their ability to reduce resazurin to resorufin, which is highly fluorescent.</td></tr><tr><td>ALDEFLUOR assay kit</td><td>Stem Cell Tech</td><td>1700</td><td>Kit to identify stem and progenitor cells that express high levels of aldehyde dehydrogenase , an indicator of cancer stem cells. The kit is composed of ALDEFLUOR Reagent, DEAB, Hydrochloric Acid, Dimethylsulphoxide, and ALDEFLUOR Assay Buffer.</td></tr><tr><td>ALDEFLUOR Diethylaminobenzaldehyde (DEAB)</td><td>Stem Cell Tech</td><td>1705</td><td>Diethylaminobenzaldehyde (DEAB) is an inhibitor of ALDH isozymes, used to determine non-specific ALDH staining.</td></tr><tr><td>Andor iXon x3 CCD Camera</td><td>Oxford Instruments</td><td>-</td><td></td></tr><tr><td>Antibiotics and Antimycotics</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>APC-isotype IgG2b</td><td>Miltenyi biotec</td><td>130-092-217</td><td>Isotype control to quantify non-specific staining of IgG2b antibodies.</td></tr><tr><td>B27 Supplement</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>basic Fibroblast Growth Factor</td><td>Stem Cell Technologies</td><td>78003.1</td><td></td></tr><tr><td>BD Lo-Dose U-100 Insulin Syringes</td><td>Fisher Scientific</td><td>14-826-79</td><td></td></tr><tr><td>BioTek Synergy HT Microplate Reader</td><td>BioTek</td><td>7091000</td><td></td></tr><tr><td>CD133-APC</td><td>Miltenyi biotec</td><td>130-113-184</td><td>Fluorescent antibody targeting CD133, a cancer stem cell marker.</td></tr><tr><td>cellSens Dimension Software</td><td>Olympus</td><td></td><td></td></tr><tr><td>Cisplatin</td><td>Sigma-Aldrich</td><td>P4394</td><td>Platinum based chemotherapy agent that functions as an alkylating agent that disrupts DNA.</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>Gibco</td><td>PHG0311</td><td></td></tr><tr><td>EVOS XL Core Cell Imaging System</td><td>Life Technologies</td><td>AME3300</td><td></td></tr><tr><td>Fetal Bovine Serum - premium (FBS)</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Ficoll 400</td><td>Sigma-Aldrich</td><td>F4375</td><td></td></tr><tr><td>Hemacytometer</td><td>Hausser Scientific</td><td>1490</td><td></td></tr><tr><td>Histogel</td><td>Thermo Scientific</td><td>HG-4000-012</td><td>Tissue processing gel that can penetrate and hold the specimen within the gel while preventing discoloration around the specimen upon staining.</td></tr><tr><td>Human Adipose-Derived Mesenchymal Stem Cells</td><td>Lonza</td><td>PT-5006</td><td></td></tr><tr><td>Human Microvascular Endothelial Cells</td><td>Lonza</td><td>CC2543</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium Supplement</td><td>Gibco</td><td>51500-056</td><td></td></tr><tr><td>Live/Dead viability kit</td><td>Invitrogen</td><td>L3224</td><td>Kit for the fluorescence based detection of live (calcein-AM) and dead cells (Ethidium Homodimer-1).</td></tr><tr><td>MEM Non-essential Amino Acids</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>MetaMorph 7.8 Software</td><td>Molecular Devices</td><td>-</td><td></td></tr><tr><td>Olympus IX81 Inverted Confocal Microscope</td><td>Olympus</td><td>-</td><td></td></tr><tr><td>Olympus IX83 Research Inverted Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Parafilm M</td><td>Thomas Scientific</td><td>7315D35</td><td>Thermoplastic polymer strips that serve to limit droplet evaporation in hanging drop plates while still allowing for gas exchange.</td></tr><tr><td>Perfecta 3D 384 Well Hanging Drop Plates</td><td>3D Biomatrix</td><td>HDP1384-8</td><td>Available through Sigma-Aldrich</td></tr><tr><td>phalloidin AlexaFluor488</td><td>Invitrogen</td><td>A12379</td><td>Phalloidin is a peptide to fluorescently label F-actin in fixed cells.</td></tr><tr><td>ProJet 3500 HD Max</td><td>3D Systems</td><td>-</td><td>3D printer</td></tr><tr><td>Sterile DI water</td><td>Fisher Scientific</td><td>353046</td><td></td></tr><tr><td>Trypan Blue</td><td>Gibco</td><td>15250061</td><td>Azo dye used to differentiate between live and dead cells based on its ability to pass through the damaged membrane of dead cells, but not the intact membrane of live cells.</td></tr><tr><td>VisiJet M3 Crystal</td><td>3D Systems</td><td>-</td><td>A biocompatible polymer material for 3D printing.</td></tr><tr><td>Yokogawa CSU-X1 Confocal Scanner Unit</td><td>Yokogawa</td><td>-</td><td></td></tr></tbody></table>

physiologic patient derived 3d spheroids for anti-neoplastic drug screening to target cancer stem cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

Spheroids formed with cell lines or patient-derived CSCs can be formed with a range of small cell numbers within hanging droplets (Figure 2A). Spheroids form reliably with as few as 10 cells per well, which allows for conservation of rare patient samples. Cells within these spheroids are surrounded by other cells in 3 dimensions as they would be in vivo, allowing for physiologic cell-cell contacts and diffusion rates. Tumor cells within the spheroids proliferate causing the spheroids to expa...

Watch this Scientific Journal Video about Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells at JoVE.com

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

physiologic-patient-derived-3d-spheroids-for-anti-neoplastic-drug

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Cancer Research

8.5K Views.  University of Michigan. This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates’ potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

Video: Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Bioengineering

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.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

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