This work was supported by grants from the Czech Ministry of Education, Youth, and Sports (LO1304) and the Technological Agency of the Czech Republic (TE01020028). The authors would like to thank Dr. Lakshman Varanasi for taking the still images of environmental lids.

1. Preparation of Agarose-coated Plates
<ol>
	<li>Weigh 0.75 g of low-melting point agarose and add it to 100 mL of McCoy&#39;s 5A medium (with or without phenol red) without serum. Heat the solution in a microwave and swirl every 1-2 min to completely dissolve the agarose. Autoclave the solution to sterilize it.</li>
	<li>Cool the autoclaved agarose to about 70 &#176;C and filter it through a 500 mL, 0.22 &#181;m filter top by vacuum filtration in a laminar flow box. Aliquot the 0.75% filtered agarose solution (FAS) into smaller volumes if not using the entire solution at once. Store this ready-to-use agarose solution aseptically in a cold room or 4 &#176;C fridge for up to 4 weeks.</li>
	<li>Attach a small tube with a plastic- or metal-tipped dispensing cassette to a Combi reagent dispenser and prime the cassette with 70% ethanol (EtOH) and then with sterile phosphate-buffered saline (PBS) in a flow box.</li>
	<li>Prior to use, heat a stored aliquot of FAS in the microwave to melt it. Prime the dispensing cassette with agarose solution and coat 384-well, tissue culture (TC)-treated &#34;special&#34; microplates with 15 &#181;L of FAS. Allow the agarose in the plate to cool for 15-20 min before seeding the cells. Store the agarose-coated plates aseptically, wrapped in a polyethylene bag in a cold room or at 4 &#176;C and away from direct light. 
	NOTE: Avoid repeated heating of the 0.75% FAS to prevent a change in the concentration of agarose in the stock solution. Agarose-coated plates can be stored for up to 2 weeks when stored under the above-mentioned conditions. The FAS does not require heating during the coating of plates.</li>
	<li>Clean the dispensing cassette by priming it with 70-80 &#176;C sterile water to remove any remaining agarose in the cassette tips and tubes.</li>
</ol>
2. Cell Culture and MCTS Formation
<ol>
	<li>Culture human colorectal carcinoma HCT116 cells, as described previously8.</li>
	<li>Remove the required number of agarose-coated, 384-well, TC-treated microplates from cold storage and equilibrate them to room temperature (RT) for 15 min.</li>
	<li>Prepare the Combi reagent dispenser for cell seeding by priming a standard tube dispensing cassette with 70% EtOH and sterile PBS. Adjust the seeding volume to the required &#181;L and the dispensing speed to medium using the manual setting buttons on the dispenser. 
	NOTE: The entire cell seeding process is performed under sterile conditions and in a laminar flow box.</li>
	<li>Dissociate adherent cells from the tissue culture flask using a recombinant cell-dissociation enzyme. Make a cell suspension stock in a sterile beaker with seed cells at a density of 2.5 x 104 cells/mL per well in 50 &#181;L of complete growth medium. When seeding more than one 384-WP, stir the cells using a magnetic stirrer to prevent them from settling to the bottom of the beaker.</li>
	<li>Allow the plates to rest for 30 min at RT and then centrifuge for 15 min at 4 x g.</li>
	<li>Meanwhile, take an evaporation-reducing environmental lid and fill it with 8 mL of sterile H2O or 5% dimethyl sulfoxide (DMSO) in the short sides (left and right) using a 5 mL pipette (Figure 1A). First, dispense 4 mL of filling liquid into the left-side trough by sweeping the pipette tip slowly up and down. Repeat this step with the right-side trough. 
	NOTE: Ensure that the liquid added to the side troughs does not merge at the center of the lid, and leave a gap for gas exchange (Figure 1A). Adding an excess amount of H2O results in the seepage of H2O to the exterior of the lid and subsequently into the outer wells of the 384-well TC plates.</li>
	<li>Fill the liquid reservoir of the 384-well TC plate with sterile H2O and replace the regular plate lids with the liquid-filled environmental lids (Figure 2A).</li>
	<li>Place the plates in a 37 &#176;C rotary incubator with 95% humidity, 5% CO2, and 20% O2, and allow the cells to aggregate into MCTSes for 4 days. Avoid opening the incubator door for too long in the subsequent days in order to prevent the humidity level from dropping abruptly.</li>
</ol>
3. Medium Exchange Using a Robotic System
<ol>
	<li>On day 4 following MCTS formation, add 30 &#181;L of pre-warmed medium per well using an automated microplate washer dispenser. Allow the MCTSes to grow for additional 3 days. Replace the medium regularly every 3 days, until they attain the desired size for experimentation.</li>
	<li>To aspirate a defined volume of medium from each well, empirically adjust the z-height of the washer manifold. Aspirate 30 &#181;L of medium per well and replace it with 30 &#181;L of fresh, pre-warmed medium. 
	NOTE: Set the dispensing speed and the speed at which the washer manifold travels down into wells at the lowest rate to minimize turbulence in the wells.</li>
</ol>
4. High-content Imaging of MCTS and Semi-automated Image Analysis
<ol>
	<li>Image the MCTS in a high-content automated imaging system using a 4X air objective (N.A. 16).</li>
	<li>Set the exposure time to 11 ms and the binning to 4 x 4. Adjust the number and spacing of the z-stacks and the pixel binning as desired for the experiment to capture an entire MCTS per well.</li>
	<li>Process the images stepwise, as described in the &#34;readme,&#34; using an in-house routine written in programming language. 
	NOTE: The .m code and .txt readme files are available as supplementary code files (Figure 1B). The routine measures the MCTS area, major and minor axes, perimeter, and solidity from the 2D images.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55403/55403fig1.jpg" /> 
Figure 1: Preparation of environmental lids and a flowchart of the semi-automatic routine. (A) Image of an evaporation-reducing environmental lid filled with the correct (left) and excess (right) amount of filling liquid (here, 5% DMSO). The arrow indicates the short-side trough for adding liquid. The asterisk shows the gap in the middle of the lid following the addition of the correct volume of DMSO. (B) A workflow showing the steps involved in the semi-automated image analysis routine. <a href="http://cloudfront.jove.com/files/ftp_upload/55403/55403fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

Coating 384-Well TC Plates with Filtered Agarose
The standard practice in LOT is to use a 1-1.5% low-melting point agarose to coat the plates, which requires the agarose and/or dispensing unit to be kept heated to prevent the gelling of the agarose6. The gelling of the agarose is of potential concern while preparing multiple plates using liquid dispensing cassettes with small tubing apertures ranging between 0.2 and 0.4 mm in diameter. To overcome the potential issue of cl...

Cancer cells in tumors are physiologically arranged in a complex, 3-dimensional (3D) structure surrounded by extracellular matrix and interacting cells. As nearly all cells in tissues reside in a 3D environment, the need for more physiologically relevant in vitro tumor models that mimic tumor traits has resulted in the development of several 3D culture techniques1,2,3. These models are now becoming fundamental research tools for studying the role of the tumor microenvironment on metastasis and cell response to therapeutics in 3D2. Moreover, compared to 2-dimensional (2D) cell cultures4, 3D models allow for a better understanding of tumor-stroma interactions, which affect cell signaling pathways.
Multicellular tumor spheroids (MCTSes) of cancer cell lines are frequently used in 3D cell culture models due to their relative closeness to in vivo tumors. Out of the several techniques in use, the liquid-overlay technique (LOT) of MCTS generation on agarose-coated plates has gained significant interest for lead optimization and target validation5,6,7,8,9,10,11. This is evident from the recent studies that were successfully able to run pilot screens of compound libraries in MCTS cultures using LOT6,7. However, well-to-well variability in MCTS morphology and growth due to the evaporation-induced uneven loss of medium are common hurdles that accompany the LOT using microtiter plates (MPs). Consequently, the formation of non-uniform MCTSes compromises the significance and relevance of data from pharmacological assays8,12,13. In addition to the reproducibility issues, another practical problem that affects LOT-based high-throughput assays is the coating of the MPs with agarose when using automatic liquid dispensing units. Although the dispensing unit can be kept heated to prevent the gelling of agarose, the clogging of the dispensing cassette and tubing is a potential concern for robotic systems6.
To overcome some of these challenges, we have recently devised a few modifications in the LOT for MCTS culture8. These modifications are mainly based on possible ways to prevent uneven medium loss from the MPs using instruments that are commonly found in high-throughput screening laboratories. A detailed procedure of the modified LOT for the generation of uniformly sized and reproducible MCTSes across 384-well plates (WPs) is presented here. The manuscript also presents a semi-automated routine for the evaluation of MCTS size, particularly in partially disintegrated, drug-treated MCTSes that do not have a clearly defined boundary for the measurement of cross-sectional area.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td>Low-melting</td>
		</tr>
		<tr>
			<td>McCOY&#39;s 5A Medium</td>
			<td>Sigma-Aldrich</td>
			<td>M8403</td>
			<td></td>
		</tr>
		<tr>
			<td>&#8220;rapid&#8221; Filtermax filter</td>
			<td>TPP</td>
			<td>99505</td>
			<td>0.22 &#956;m, 500 mL</td>
		</tr>
		<tr>
			<td>Multidrop&#8482; Combi Reagent Dispenser&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Tube Dispensing cassette&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>24073295</td>
			<td>Metal tip&#160;</td>
		</tr>
		<tr>
			<td>384-well TC plate&#160;</td>
			<td>PerkinElmer</td>
			<td>6057308</td>
			<td>Plate type- CellCarrier</td>
		</tr>
		<tr>
			<td>Standard Tube Dispensing Cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>24072670</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroClime Environmental Lid</td>
			<td>Labcyte</td>
			<td>LLS-0310</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Incubator (SteriStore )</td>
			<td>HighRes Biosolutions</td>
			<td>23641</td>
			<td>Serial No.: D00384</td>
		</tr>
		<tr>
			<td>Microplate Washer Dispenser&#160;</td>
			<td>BioTek</td>
			<td>Unspecified</td>
			<td>Model: EL406&#160;</td>
		</tr>
		<tr>
			<td>High-Content Imaging System (CellVoyager )</td>
			<td>Yokogawa Electric Corporation</td>
			<td>Unspecified</td>
			<td>Model: CV7000</td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>New England Biolabs</td>
			<td>B7022S</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE&#8482; Express recombinant cell dissociation reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604021</td>
			<td>Phenol red free</td>
		</tr>
	</tbody>
</table>

evaporation-reducing culture condition increases the reproducibility of multicellular spheroid formation in microtiter plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium, particularly from peripheral wells, is a frequently encountered issue in MPs with small culture volumes. Substantially improved culture conditions, such as incubators with well-controlled temperature/humidification systems and evaporation-reducing MPs and plate lids, reduce the significant loss of medium across wells8. To measure the relative evaporation, equal volumes of Orange G (OG) were added to each well, and the change in OG absorba...

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Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

evaporation-reducing-culture-condition-increases-reproducibility

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Cancer Research

6.9K Views.  Palacky University in Olomouc. The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Video: Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

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 ...

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

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. ...