This project was funded by a grant of the University of Munich (F&#246;FoLe project-no.: 789-781).

All studies shown in this manuscript, namely the use of human tumor specimens, are protected under and in consent with prior decisions from University Medicine of Mainz/University of Munich Medical Center Ethics committee. Patients have given informed consent according to national legal guidelines agreeing to scientific use of excess biological material that was obtained in the course of their treatment. Research has been performed in compliance with all institutional, national and international guidelines for human welfare.
1. Taking a Tumor Biopsy from Head and Heck Squamous Cell Carcinoma
<ol>
	<li>Perform general (propofol and/or sevoflurane, muscle relaxing agent) or local infiltration (2% ultracaine, adrenalin)/surface (xylocaine) anesthesia in the operating room or otolaryngology examination chair. Visualize the tumor mass in oral cavity/pharynx/larynx/other parts of the upper digestive and respiratory tract with standard operating instruments and, if needed, under the microscope.</li>
	<li>Take a fresh tumor biopsy from the periphery of the cancerous lesion with blunt or cutting instruments. Avoid the center of the lesion due to abundant necrosis in this area. Put the obtained tissue to a sterile container with isotonic sodium chloride solution.</li>
	<li>After the biopsy, perform hemostasis as needed, e.g., with a bipolar or monopolar coagulation device in addition to the use of vasoconstrictive substances.</li>
	<li>Bring the tumor biopsy intended to be used for cell culture experiments directly to the adjacent laboratory tract. Send other tumor biopsies to the pathologist as usual to rule out cancer.</li>
	<li>Make sure that a laboratory technician is ready to process the specimen directly.</li>
</ol>
2. Processing the Tumor Specimen
<ol>
	<li>Place the tumor specimen on a suitable and sterile surface and cut it thoroughly with a sterile single-use scalpel into as little as possible pieces. 
	Caution: Make sure the status of infectious and blood-borne diseases is well documented and the technician is in attention of the institutions standard protocols to prevent needle stick or cutting injury with potentially biohazard patient material and the protocols to follow after possible injury.</li>
	<li>After sufficient mechanical separation of the primary tissue, put the tissue into a vial containing Collagenase I/II and incubate for 1 h at 37 &#176;C. Sieve through a 70 &#181;m falcon cell strainer and wash the suspension with Hanks&#39; Balanced Salt solution (HBSS).</li>
	<li>After successful separation and subsequent washing, place the suspension containing 1-2 &#215; 106 cells into a T75 cell culture flasks (75 cm2) to grow to sub-confluency at 5% CO2 and temperature of 37 &#176;C. This step may take up to 10 days.
	<ol>
		<li>Use special keratinocyte culture medium consisting of the following: 125 mL of Dulbecco&#39;s modified eagles medium (DMEM), 250 mL of Keratinocyte complemented medium (complemented medium consisting of 500 mL of Keratinocyte SF Medium, 15 mg of bovine pituitary extract (BPE), 2.5 mL of penicillin/streptomycin, 150 ng recombinant human epithelial growth factor (EGF), 516 &#181;L of 300 mM CaCl2, stock mix to be prepared in advance), 125 mL of F12 nutrient mix, 10 mg of BPE (0.75 mL), 75 ng recombinant human EGF (2 &#181;L), 3.75 mL of 200 mM L-Alanyl-L-Glutamin-Dipeptide (Table of materials).</li>
	</ol>
	</li>
</ol>
3. Seeding the Cells into Ultra-low Adhesion Cell Culture Plates
<ol>
	<li>Confirm tumor cell growth under a microscope. Count the cells in culture (primary or cell culture) and seed 5,000 primary tumor cells or 1,000-2,000 cells of intermediate cell line/other cell line in 200-300 &#181;L of media (step 3.2.) into an ultra-low adhesion plate with concave, round bottoms (96-well).</li>
	<li>Culture the cells at 5% CO2 at 37 &#176;C and in equal parts DMEM and airway epithelial cell medium (BEGM), 10% fetal bovine serum, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% non-essential amino acids, 1% L-glutamine (step 2.3.). If cell lines are being used, media on the basis of DMEM is sufficient.
	<ol>
		<li>Perform media changes every other day. Pay attention not to aspirate the spheroid with the pipette during media changes. Culture the spheroids until the level of growth as descibed in 3.3 is reached (approximately 7-10 days).</li>
	</ol>
	</li>
	<li>Confirm spontaneous spheroid formation under the microscope by looking for three-dimensional, spheroid-shaped cell conglomerates. Exclude the wells with irregular and/or multiple spheroid formation from further investigation. 
	NOTE: Spheroids should be visible with the naked eye, too, facilitating further processing and media changes as described below.</li>
</ol>
4. Exposing Spheroids to Multimodal Standard or Experimental Tumor Therapy
<ol>
	<li>Choose a desired therapy regimen. Design sufficiently large control groups that allow comparisons of treatments to untreated spheroids or spheroids receiving only partial therapy regimens, e.g. radiation alone. The size of control groups depends on the experimental group design and cannot be defined universally.</li>
	<li>Exchange the media to media with additives, meaning media with chemotherapeutics and/or monoclonal antibodies at desired concentrations. Add Cisplatin at the concentrations of 2.5/5/10 &#181;M or 5-fluoruracil (5FU) at 30 &#181;M. 
	NOTE: For high throughput experiments or large group sizes/large number of groups, one can use an automated pipetting robot as we are further establishing in the experiments.</li>
	<li>Alternatively, radiate the cell culture plates with spheroids at 2 Gy using a suitable radiation facility. 
	Caution: Respect the institution&#39;s protocols regarding prevention of harmful radiation exposure of employees. Work only with designated and trained technicians according to radiation protection guidelines. If desired, add chemotherapeutics as described under 4.2. afterwards.</li>
	<li>Incubate the cells for 24 h in previously described culture conditions (37 &#176;C, step 3.2).</li>
	<li>After the incubation, continue the cell culture for at least 6 days with media changes every other day.</li>
</ol>
5. Assessment of Spheroid Size and Extent of Cellular Proliferation for Assay Read-out
<ol>
	<li>Measure the spheroid size in terms of area after digital photo documentation on day 6 (day 10, day 16) with a graphic software (parameter 1).</li>
	<li>After centrifugation at 520 x g for 2.5 min of the plate, remove the supernatant. Wash the cells with sufficient amount of 1x PBS and centrifuge the plate again as described, followed by removing the supernatant (PBS).</li>
	<li>Add 100 &#181;L of enzymatic cell detachment solution to each vial to allow the spheroids to dissolve. Incubate the plate for 8 min at 37 &#176;C.</li>
	<li>Check for successful dissolving of the spheroid under the microscope. Add 100 &#181;L of DMEM. Centrifuge the plate at 520 x g for 2.5 min. Remove the supernatant and suspend the cells in 100 &#181;L of DMEM.</li>
	<li>Perform a commercially available colorimetric proliferation assay, in example WST-8 assay on each vial according to the manufacturer&#39;s instructions25. Read out the assay in an enzyme-linked immunosorbent assay (ELISA) reader (parameter 2).</li>
</ol>

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The authors have nothing to disclose.

We were able to establish a protocol to generate reproducible spheroids from cell suspensions, for both cell lines and, in preliminary experiments, primary human tumor cells. We first assessed two previously described methods and identified the ULA-method, a method where culture plates with ultra-low adhesion surfaces are used, to be the safer and more reliable one for the generation of uniform three-dimensional spheroids. By combining two separate methods for assay read-out (size/area and cell viability), this multimoda...

Head and Neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with a rising incidence of mucosal human papillomavirus (HPV) infection-associated pathogenesis, next to a majority of cases caused by excessive nicotine and alcohol consumption 1,2. While smaller tumors and pre-invasive stages are usually well treatable with surgical excision, usually combined with cervical lymph node dissection, treatment for advanced-stage and recurrent HNSCC remains challenging due to aggressive tumor invasion with metastatic spread and resistance to radiation and chemotherapy protocols3,4,5,6,7,8. Recent studies suggest a high variability of cellular phenotype, and sub-characterization of circulating and disseminated tumor cells has just begun9,10. The earlier belief of a solid, uniform tumor mass had to be revised in the light of recent studies in the past years11,12,13,14. Current approaches for tumor characterization and identification of key mutations could identify several genes that seem to be associated with therapy resistance but remain a cost-intensive approach. Moreover, knowledge of genotype does not necessarily allow a reliable prediction of phenotype and its treatment response.
There have been few advances in improving overall and disease-free survival for advanced-stage and recurrent disease. For nicotine- as well as virus-associated carcinoma, current treatment options besides surgery enclose aggressive radiation and platinum-based chemotherapy regimens. There have been implications for different response rates between HPV-negative and positive carcinoma; however, this has not yet lead to a change in general therapy guidelines. Resistance towards radiation and chemotherapy is a widespread phenomenon in all tumor stages and exists for platinum-based chemotherapy as well as for targeted therapy (Anti-EGFR; epidermal growth factor-receptor) and recently emerging checkpoint inhibition15. Ineffective radiation and chemotherapy come at a high cost of significant patient morbidity in terms of dysphagia, mucositis, dry mouth and risk of decrease of renal or cardiac function among others. Predicting therapy response prior the decision of a general therapy concept for each individual patient seems to be the crucial goal, preventing unnecessary treatment concepts, side effects and costs.
We sought to establish a model to test individual patient&#39;s treatment susceptibility towards current standard chemo-radiation that could be integrated into the regular and quality-controlled oncologic treatment algorithm from a technical standing point. The far goal was to use the model without using heavily altered and aged cell lines, as they poorly represent actual human tumor cells without their variability and heterogeneity as we know now, while establishment of the protocol was done in various cell lines. To be independent only from commercially available cell lines, we recently successfully generated an intermediate cell line called &#34;PiCa&#34; from primary HNSCC cells from human tumor specimens with conserved cellular markers on its surface and limited passages16. This PiCa cell line should serve as a preparation for the development of the model on the road to then later following trials with fresh human cancer cells from tumor biopsies. It has been shown that cells in three-dimensional cell cultures react differently and more in vivo-like to administration of cancer drugs than those growing in monolayers17,18,19,20,21, mainly due to conservation of migratory and sub-differentiation properties of certain cell subsets22,23,24. Here, we describe the protocol of a spheroid-based, three-dimensional model from intermediate cell lines and primary human squamous cell carcinoma cells and ways how integrate such a model into cancer treatment of the head and neck surgeon and oncologist (Figure 1).

<table><tbody><tr><td>Dulbeccos modified Eagles medium (DMEM)</td><td>Biochrom, Berlin, Germany</td><td>F 0425</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco Life Technologies, Paisley, UK</td><td>10500-064</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Biochrom, Berlin, Germany</td><td>A2212</td><td></td></tr><tr><td>sodium pyruvate</td><td>Biochrom, Berlin, Germany</td><td>L0473</td><td></td></tr><tr><td>non-essential amino acids</td><td>Biochrom, Berlin, Germany</td><td>K0293</td><td></td></tr><tr><td>L-Glutamine</td><td>Biochrom, Berlin, Germany</td><td>K0293</td><td></td></tr><tr><td>Liberase</td><td>Roche Life Sciences, Basel, Switzerland</td><td>5401127001</td><td></td></tr><tr><td>GravityPLUS 3D Culture and Assay Platform</td><td>InSphero, Schlieren, Switzerland</td><td>PB-CS 06-001</td><td></td></tr><tr><td>GravityTRAP plate</td><td>InSphero, Schlieren, Switzerland</td><td>PB-CS-01-001</td><td></td></tr><tr><td>Ultra-low attachment (ULA) culture plates</td><td>Corning, Corning, NY, USA</td><td>4520</td><td></td></tr><tr><td>airway epithelial cell growth medium</td><td>Promocell, Heidelberg, Germany</td><td>C-21060</td><td></td></tr><tr><td>amphotericin B</td><td>Biochrom, Berlin, Germany</td><td>A 2612</td><td></td></tr><tr><td>airway epithelial cell growth medium supplement mix</td><td>Promocell, Heidelberg, Germany</td><td>C39165</td><td></td></tr><tr><td>WST-8 test</td><td>Promocell, Heidelberg, Germany</td><td>PC PK-CA705-CK04</td><td></td></tr><tr><td>Keratinocyte SFMedium + L-Glutamine 500mL</td><td>Invitrogen</td><td>#17005-034</td><td></td></tr><tr><td>Bovine Pituitary Extract (BPE), 25mg</td><td>Invitrogen</td><td>#37000015</td><td></td></tr><tr><td>Recombinant human Epithelial Growth Factor 2.5 &amp;micro;g</td><td>Invitrogen</td><td>#37000015</td><td></td></tr><tr><td>DMEM High Glucose</td><td>Invitrogen</td><td>#21068-028</td><td></td></tr><tr><td>Penicillin Streptomycin 10000U/mL Penicillin/ 10000&amp;micro;g/mL Streptomycin</td><td>Invitrogen</td><td>#15140-122</td><td></td></tr><tr><td>F12 Nutrient Mix</td><td>Invitrogen</td><td>#21765-029</td><td></td></tr><tr><td>Glutamax (200 mM L-Alanyl-L-Glutamin-Dipeptide in NaCl)</td><td>Invitrogen</td><td>#35050087</td><td></td></tr><tr><td>HBSS (Ca, Mg)</td><td>Life Technologies</td><td>#14025-092</td><td>(no phenol red)</td></tr><tr><td>1x TrypLE Expres Enzyme</td><td>Invitrogen</td><td>#12604-013</td><td>(no phenol red)</td></tr><tr><td>Accutase (enzymatic cell detachment solution)</td><td>Innovative cell technologies</td><td>Cat# AT104</td><td></td></tr><tr><td>70 &amp;micro;m Falcon cell strainer</td><td>BD Biosciences, USA</td><td>#352350</td><td></td></tr></tbody></table>

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 were able to reproducibly generate spheroids from single cell suspensions, first from different cell lines including the proprietary PiCa cell line, later from primary human cancer cells derived from fresh tumor biopsies as described in Hagemann et al.26. We evaluated two established methods for spheroid generation; the two being the so-called hanging drop (HD) method and the ultra-low adhesion (ULA) method, the latter being the more effective and safe ...

Watch this Scientific Journal Video about Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma at JoVE.com

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

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 ...

therapy-testing-spheroid-based-3d-cell-culture-model-for-head-neck

Research

JoVE Journal

Cancer Research

9.7K Views.  Johannes-Gutenberg University Medical Center. We beschrijven de evolutie van een sferoïde gebaseerde, driedimensionale in vitro model waarmee we kunnen testen van de huidige standaard voor experimentele therapie regimes voor hoofd en nek plaveiselcelcarcinoom op cellijnen, die gericht zijn op het evalueren van therapie gevoeligheid en weerstand op primaire cellen van menselijke specimens in de toekomst.

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

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 ...

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 ...

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. ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Medicine

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

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 ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

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

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