Name: monitoring cancer cell invasion and t-cell cytotoxicity in 3d culture
Uploaded: 2020-06-23T14:45:00
Description: Watch this Scientific Journal Video about Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture at JoVE.com

We thank Virginie Ory, PhD for helpful discussions and advice on the approach of the 3D co-culture model. We also thank Elizabeth Jones for excellent technical assistance with the IHC sectioning. This study was supported by grants from the DFG (Deutsche Forschungsgemeinschaft) to YL (LI 2547/4-1) and the National Institutes of Health to AW (R01 CA231291), to ATR (R01 CA205632), to GWP (R01 CA218670), and the Core Grant of the Cancer Center (P30 CA51008).

A list and explanation of some frequently used words throughout the protocol can be found in Supplementary File 1.
1. Generation of spheroids
<ol>
	<li>Prepare and autoclave 2% agarose in 1x PBS (e.g., 1 g agarose in 50 mL of 1x PBS) and autoclave 35- and 81-microwell rubber molds. 
	CAUTION: Avoid using low-melting agarose for generating the agarose casts for IHC processing.</li>
	<li>Prepare agarose casts 
	NOTE: 35-microwell casts are used for invasion, immunofl...

<ol>
	<li>Chen, D. S., Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elements+of+cancer+immunity+and+the+cancer-immune+set+point.">Elements of cancer immunity and the cancer-immune set point.</a> Nature. 541 (7637), 321-330 (2017).</li><li>Sharma, P., Allison, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+mechanisms+of+immune+checkpoint+therapy.">Dissecting the mechanisms of immune checkpoint therapy.</a> Nature Reviews Immunology. 20 (2), 75-76 (2020).</li><li>Blomberg, O. S., Spagnuolo, L., de Visser, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+of+metastasis:+mechanistic+insights+and+therapeutic+opportunities.">Immune regulation of metastasis: mechanistic insights and therapeutic opportunities.</a> Disease Model Mechanisms. 11 (10), (2018).</li><li>Schmeichel, K. L., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue-specific+signaling+and+organ+function+in+three+dimensions.">Modeling tissue-specific signaling and organ function in three dimensions.</a> Journal of Cell Science. 116 (12), 2377-2388 (2003).</li><li>Kramer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration+and+invasion+assays.">In vitro cell migration and invasion assays.</a> Mutation Research. 752 (1), 10-24 (2013).</li><li>xu, x., Farach-Carson, M. C., Jia, x. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+In+vitro+tumor+models+for+cancer+research+and+drug+evaluation.">Three-dimensional In vitro tumor models for cancer research and drug evaluation.</a> Biotechnology Advances. 32 (7), 1256-1268 (2014).</li><li>Lamichhane, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulating+epithelial+tumor+microenvironment+In+vitro+using+three+dimensional+tri-culture+of+human+epithelial,+endothelial,+and+mesenchymal+cells.">Recapitulating epithelial tumor microenvironment In vitro using three dimensional tri-culture of human epithelial, endothelial, and mesenchymal cells.</a> BioMed Central Cancer. 16 (581), (2016).</li><li>Klimkiewicz, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+model+of+tumour+angiogenic+microenvironment+to+monitor+hypoxia+effects+on+cell+interactions+and+cancer+stem+cell+selection.">A 3D model of tumour angiogenic microenvironment to monitor hypoxia effects on cell interactions and cancer stem cell selection.</a> Cancer Letters. 396, 10-20 (2017).</li><li>Cavo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironment+complexity+and+matrix+stiffness+regulate+breast+cancer+cell+activity+in+a+3D+In+vitro+model.">Microenvironment complexity and matrix stiffness regulate breast cancer cell activity in a 3D In vitro model.</a> Scientific Reports. 6, 35367 (2016).</li><li>Martinez, N. J., Titus, S. A., Wagner, A. K., Simeonov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+fluoresecent+imaging+approaches+for+drug+discovery+using+In+vitro+and+in+vivo+three-dimensional+models.">High throughput fluoresecent imaging approaches for drug discovery using In vitro and in vivo three-dimensional models.</a> Expert Opinion on Drug Discovery. 10 (12), 1347-1361 (2015).</li><li>Zanoni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tumor+spheroid+models+for+In+vitro+therapeutic+screening:+a+systematic+approach+to+enhance+the+biological+relevance+of+data+obtained.">3D tumor spheroid models for In vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained.</a> Scientific Reports. 11 (6), 19103 (2016).</li><li>Veelken, C., Bakker, G., Drell, D., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell-based+automated+quantification+of+therapy+responses+of+invasive+cancer+spheroids+in+organotypic+3D+culture.">Single cell-based automated quantification of therapy responses of invasive cancer spheroids in organotypic 3D culture.</a> Methods. 1 (128), 139-149 (2017).</li><li>Puls, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Novel+3D+Tumor-tissue+Invasion+Model+for+High-throughput,+High-content+Phenotypic+Drug+Screening.">Development of a Novel 3D Tumor-tissue Invasion Model for High-throughput, High-content Phenotypic Drug Screening.</a> Scientific Reports. 8 (1), 13039 (2018).</li><li>Jenkins, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+Vivo+Profiling+of+PD-1+Blockade+Using+Organotypic+Tumor+Spheroids.">Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids.</a> Cancer Discovery. 8 (2), 196-215 (2018).</li><li>Berens, E. B., Holy, J. M., Riegel, A. T., Wellstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cancer+Cell+Spheroid+Assay+to+Assess+Invasion+in+a+3D+Setting.">A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting.</a> Journal of Visualized Experiments. 20 (105), (2015).</li><li>Shoval, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cells+and+their+crosstalk+with+endothelial+cells+in+3D+spheroids.">Tumor cells and their crosstalk with endothelial cells in 3D spheroids.</a> Scientific Reports. 7 (1), 10428 (2017).</li><li>Giannattasio, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+and+infiltration+of+human+NK+cells+in+in+vivo-like+tumor+spheroids.">Cytotoxicity and infiltration of human NK cells in in vivo-like tumor spheroids.</a> BioMed Central Cancer. 3 (15), 351 (2015).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nature Reviews Cancer. 3 (5), 362-374 (2003).</li><li>McLennan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+migration+is+driven+by+a+few+trailblazer+cells+with+a+unique+molecular+signature+narrowly+confined+to+the+invasive+front.">Neural crest migration is driven by a few trailblazer cells with a unique molecular signature narrowly confined to the invasive front.</a> Development. 142 (11), 2014-2025 (2015).</li><li>Coffey, A., Johnson, M. D., Berry, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpOT+the+Correct+Tissue+Every+Time+in+Multi-tissue+Blocks.">SpOT the Correct Tissue Every Time in Multi-tissue Blocks.</a> Journal of Visualized Experiments. (99), e52868 (2015).</li><li>Brenner, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+inhibition+of+renal+cancer+cell+invasion+mediated+by+fibronectin,+collagen+IV+and+laminin.">Differential inhibition of renal cancer cell invasion mediated by fibronectin, collagen IV and laminin.</a> Cancer Letters. 155 (2), 199-205 (2000).</li><li>Farhat, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Extraction+from+Collagen+Gels+Using+Qiagen's+RNeasy+Kit.">RNA Extraction from Collagen Gels Using Qiagen's RNeasy Kit.</a> The Protocol Place. 2012, (2019).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> Journal of Visualized Experiments. (51), e2720 (2011).</li></ol>

The method presented here describes 3D tumor spheroid generation, which allows co-culture with T-cells, cell-based functional and molecular assays, as well as a variety of monitoring and analysis possibilities using a single device. The major advantage of our approach is that it does not necessitate transfer of the 3D culture to a separate assay and maintains the integrity of the 3D culture throughout the assays.
The workflow presented here can be modified as needed. The incubation times for s...

Despite significant improvements in cancer immunotherapy over the past decade, our mechanistic understanding of sensitivity and resistance to treatments are still fairly poor1. It is well-established that tumors display substantial heterogeneity, and that the dynamic interactions of the tumor cells with their microenvironment as well as with the immune cells, impact tumor cell death, invasive behavior and response to treatments that include immunotherapy1,2,3. As one arm of the adaptive immune system, T-cells execute cell-specific cytotoxicity. The analysis of T-cell recognition and response to cancer cells provides mechanistic insights into resistance and sensitivity to immune modulatory treatments.
In vitro modeling and monitoring interactions between cancer and T-cells in an appropriate environment has been challenging and so far, resulted in limited mechanistic insights. Most cell-based assays rely on a two-dimensional (2D) environment, that lacks key features that are critical for recapitulating the three-dimensional (3D) in vivo physiology4,5,6, namely spatial cell-cell interactions, contact with the extracellular matrix (ECM)7, dynamic metabolic demand, increased hypoxia due to mass growth8, and effects of the tumor microenvironment (TME)9. On the other hand, there are still a number of shortcomings with the currently used three-dimensional (3D) co-culture and invasion assay systems: (1) the time consuming nature of spheroid generation and harvest5,10, (2) the lack of control over spheroid size, shape and cell density11,12, (3) the low-throughput type assays, (4) the requirement for special equipment13,14, (5) the need to transfer the co-culture into distinct environments for different assays15,16,17. In particular, transferring of a co-culture assay often leads to disruption of spheroids and loss of the co-culture integrity. This applies especially for &#8220;loose&#8221; spheroids with reduced cell-cell adhesion. For example, most 3D invasion assays require that spheroids are harvested after their initial formation and then resuspended in ECM14,15,16. This resuspension step results in a loss of control over the distance between spheroids. Since distance between tumor spheroids impacts their invasive behavior, this loss of control introduces high inter-assay variance and reduces the reproducibility. Furthermore, the application of cell fractionation assays by consecutive centrifugation steps for assessment of the peripheral and tumor spheroid infiltrating immune cells is limited to tumor cell populations that generate more stable spheroids17.
Concept and approach
Our approach addresses the above-mentioned deficiencies using an &#8220;All-in-One&#8221;&#8212;3D spheroid co-culture model, which does not require the transfer of spheroids for subsequent assays. We adapted a spheroid formation device (see Table of Materials) to generate an assay for simultaneously monitoring invasive behavior of cancer cells and cytotoxicity of co-cultured T-cells. This method is user-friendly, inexpensive and allows for quick and easy handling in a relatively high-throughput 3D setting. Dependent on the type of device used, up to 81 large uniformly-sized spheroids can be generated in a single pipetting step with control over the individual spheroid size by modifying the number of cells seeded. Spheroid formation is forced by enhanced cell-cell interactions in scaffold-free agarose multi-well casts with U-shaped bottoms. We adapted this 3D system for dynamic cell-based functional studies as well as endpoint molecular and biochemical assays that include fluorescence activated cell sorting (FACS), immunofluorescence (IF) or immunohistochemistry (IHC) staining as well as gene expression analysis of the intact 3D co-culture.
For functional studies, embedding spheroids in type I collagen within the agarose casts results in invasion of cancer cells from equidistant spheroids and permits monitoring essential cell line-specific features, such as single cell vs. collective cell migration18,19. Furthermore, the collagen matrix is easily separated from the agarose cast, resulting in a 1&#8210;2 mm thick patch containing multiple spheroids, which can be further processed for IF-staining and imaging by confocal microscopy. This can reveal distinct cell invasion and cell-matrix interactions in a high-throughput screening. Also, cells in the collagen matrix can be isolated after collagen digestion and single cell dissociation for subsequent cell cultivation or analysis.
For IHC analysis of spheroids, after fixation and sectioning of the agarose cast, proteins or other molecules of interest are detectable whilst maintaining the geographic positions of the spheroids. In the approach described here, spheroids are directly embedded in Hydroxyethyl agarose processing gel within the agarose cast and the gel serves as a &#8220;lid&#8221; to retain the spheroids at the bottom of the microwells. After paraffin embedding of the agarose cast20, serial horizontal sectioning is performed with the bottom of the cast serving as the starting point.
This approach contrasts with conventional IHC sectioning of spheroids that requires harvesting of cells before embedding in Hydroxyethyl agarose processing gel21 and risks disruption of spheroids thus losing the spatial arrangement of cells. Also, cell fractionation by centrifugation for assessing whether immune cells infiltrated or remained peripheral to tumor spheroids17 is avoided by direct embedding.
Furthermore, 3D co-culture can be performed by admixing tumor, stromal or immune cells, and thus studying tumor cell crosstalk or recapitulating different tumor microenvironments for analyzing cell-cell interactions including co-cultures with endothelial cells16.
This 3D spheroid co-culture setting can be used to perform co-culture of different cell types present in the tumor microenvironment and to assess the effects of altered ECM elements. Besides type I collagen, other ECM components (e.g., matrigel, matrigel/collagen mixtures, fibronectin), can be used since tumor cell invasion is impacted by the abundance of different substrates22. Also, the microwells of the agarose cast are suitable for spheroid formation of primary cell lines and for cells with low cell-cell adhesion.

<table>
	<tbody>
		<tr>
			<td>3D Petri Dishes</td>
			<td>Microtissues Inc</td>
			<td>Z764019 &#38; Z764051</td>
			<td>referred to as &#34;rubber molds&#34; in the protocols; 81-microwell &#38; 35-microwell molds</td>
		</tr>
		<tr>
			<td>8-well Chamber Slides</td>
			<td>Lab-Tek</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose Type I, low EEO</td>
			<td>Sigma-Aldrich</td>
			<td>A6013</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit-HRP conjugated secondary antibody</td>
			<td>Agilent</td>
			<td>K4003</td>
			<td>ready to use</td>
		</tr>
		<tr>
			<td>Collagen Type I, Rat Tail, 100 mg</td>
			<td>Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type 4, 1 g</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, fetal bovine serum</td>
			<td>ThermoFisher</td>
			<td>11965092, 16000044</td>
			<td>referred to as &#34;cell culture medium&#34; in the protocols</td>
		</tr>
		<tr>
			<td>Harris hematoxylin</td>
			<td>ThermoFisher</td>
			<td>SH30-500D</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoGel</td>
			<td>ThermoFisher</td>
			<td>HG-4000-012</td>
			<td>referred to as &#34;Hydroxyethyl agarose processing gel&#34; in the protocols</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Life Technologies</td>
			<td>H1399</td>
			<td>1/1000 dilution</td>
		</tr>
		<tr>
			<td>Phalloidin 546</td>
			<td>Invitrogen</td>
			<td>486624</td>
			<td>1/200 dilution</td>
		</tr>
		<tr>
			<td>rabbit anti-CD8 antibody</td>
			<td>Cell Signaling</td>
			<td>98941</td>
			<td>1/25 dilution</td>
		</tr>
		<tr>
			<td>rat anti-keratin 8</td>
			<td>DSHB</td>
			<td>TROMA-I AB_531826</td>
			<td>1/500 dilution</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>referred to as &#34;RNA extraction kit&#34; in the protocols</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>ThermoFisher</td>
			<td>11875093</td>
			<td>for T-cell culture medium</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BioRad</td>
			<td>1610407</td>
			<td>referred to as &#34;Octoxynol&#34; in the protocols</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>ThermoFisher</td>
			<td>15596026</td>
			<td>referred to as &#34;guanidinium thiocyanate with phenol&#34; in the protocols</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>referred to as &#34;polysorbate 20&#34; in the protocols</td>
		</tr>
		<tr>
			<td>TypLE</td>
			<td>ThermoFisher</td>
			<td>12604013</td>
			<td>referred to as &#34;cell dissociation enzymes solution&#34; in the protocols</td>
		</tr>
	</tbody>
</table>

monitoring cancer cell invasion and t-cell cytotoxicity in 3d culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The 3D co-culture model allows for different assays shown in Figure 1A, which can be combined or modified as needed. In our established experimental setup, tumor and T-cells are co-cultured for 2 days followed by initiation of the invasion assay for selection of invasive and/or resistant tumor cells (Figure 1B). On day 4 the quantitation of invasion is performed and &#8220;survivor&#8221; cells are isolated from the collagen matrix or directly processed for RNA<...

Watch this Scientific Journal Video about Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture at JoVE.com

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Research

JoVE Journal

Cancer Research

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

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 ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

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. ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...