The authors acknowledge support from the Thomas E. &#38; Pamela M. Mischell Family Foundation to S.S. and the Harold C. Schott Foundation funding of the Harold C. Schott Endowed Chair, UC College of Medicine, to S.S.

Pharmacological Targeting of Ion Channels to Treat Solid Tumors

<ol>
	<li>Prevarskaya, N., Skryma, R., Shuba, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+in+cancer:+Are+cancer+hallmarks+oncochannelopathies.">Ion channels in cancer: Are cancer hallmarks oncochannelopathies.</a> Physiological Reviews. 98 (2), 559-621 (2018).</li><li>Rao, 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=Ligand-gated+neurotransmitter+receptors+as+targets+for+treatment+and+management+of+cancers.">Ligand-gated neurotransmitter receptors as targets for treatment and management of cancers.</a> Frontiers in Physiology. 13, 839437 (2022).</li><li>Mohr, C. 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=Cancer-associated+intermediate+conductance+Ca2+-activated+K++channel+KCa3.1.">Cancer-associated intermediate conductance Ca2+-activated K+ channel KCa3.1.</a> Cancers. 11 (1), 109 (2019).</li><li>Fels, B., Bulk, E., Petho, Z., Schwab, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+TRP+channels+in+the+metastatic+cascade.">The role of TRP channels in the metastatic cascade.</a> Pharmaceuticals. 11 (2), 48 (2018).</li><li>Eil, 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=Ionic+immune+suppression+within+the+tumour+microenvironment+limits+T+cell+effector+function.">Ionic immune suppression within the tumour microenvironment limits T cell effector function.</a> Nature. 537 (7621), 539-543 (2016).</li><li>Haustrate, A., Hantute-Ghesquier, A., Prevarskaya, N., Lehen'kyi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+targeting+ion+channels+and+their+therapeutic+potential.">Monoclonal antibodies targeting ion channels and their therapeutic potential.</a> Frontiers in Pharmacology. 10, 606 (2019).</li><li>Kischel, 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=Ion+channels:+New+actors+playing+in+chemotherapeutic+resistance.">Ion channels: New actors playing in chemotherapeutic resistance.</a> Cancers. 11 (3), 376 (2019).</li><li>Tuszynski, J., Tilli, T. M., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channel+and+neurotransmitter+modulators+as+electroceutical+approaches+to+the+control+of+cancer.">Ion channel and neurotransmitter modulators as electroceutical approaches to the control of cancer.</a> Current Pharmaceutical Design. 23 (32), 4827-4841 (2017).</li><li>Kale, V. P., Amin, S. G., Pandey, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+ion+channels+for+cancer+therapy+by+repurposing+the+approved+drugs.">Targeting ion channels for cancer therapy by repurposing the approved drugs.</a> Biochimica Biophysica Acta. 1848 (10), 2747-2755 (2015).</li><li>Wickenden, A., Priest, B., Erdemli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channel+drug+discovery:+Challenges+and+future+directions.">Ion channel drug discovery: Challenges and future directions.</a> Future Medicinal Chemistry. 4 (5), 661-679 (2012).</li><li>Rocha, P. R. F., Elghajiji, A., Tosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+system+for+electrophysiology+of+cancer+cell+populations:+A+review.">Ultrasensitive system for electrophysiology of cancer cell populations: A review.</a> Bioelectricity. 1 (3), 131-138 (2019).</li><li>Sengupta, S., 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=&#945;5-GABAA+receptors+negatively+regulate+MYC-amplified+medulloblastoma+growth.">&#945;5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth.</a> Acta Neuropathologica. 127 (4), 593-603 (2014).</li><li>Jonas, O., 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=First+in+vivo+testing+of+compounds+targeting+Group+3+medulloblastomas+using+an+implantable+microdevice+as+a+new+paradigm+for+drug+development.">First in vivo testing of compounds targeting Group 3 medulloblastomas using an implantable microdevice as a new paradigm for drug development.</a> Journal of Biomedical Nanotechnology. 12 (6), 1297-1302 (2016).</li><li>Kallay, L., 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=Modulating+native+GABAA+receptors+in+medulloblastoma+with+positive+allosteric+benzodiazepine-derivatives+induces+cell+death.">Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death.</a> Journal of Neurooncology. 142 (3), 411-422 (2019).</li><li>Pomeranz Krummel, D. 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=Melanoma+cell+intrinsic+GABAA+receptor+enhancement+potentiates+radiation+and+immune+checkpoint+response+by+promoting+direct+and+T+cell-mediated+anti-tumor+activity.">Melanoma cell intrinsic GABAA receptor enhancement potentiates radiation and immune checkpoint response by promoting direct and T cell-mediated anti-tumor activity.</a> International Journal of Radiation Oncology, Biology, Physics. 109 (4), P1040-P1053 (2021).</li><li>Bhattacharya, D., 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=Therapeutically+leveraging+GABAA+receptors+in+cancer.">Therapeutically leveraging GABAA receptors in cancer.</a> Experimental Biology and Medicine. 246 (19), 2128-2135 (2021).</li><li>Mazia, D., Schatten, G., Sale, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+cells+to+surfaces+coated+with+polylysine.+Applications+to+electron+microscopy.">Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy.</a> Journal of Cell Biology. 66 (1), 198-200 (1975).</li><li>Wiatrak, B., Kubis-Kubiak, A., Piwowar, A., Barg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PC12+cell+line:+Cell+types,+coating+of+culture+vessels,+differentiation+and+other+culture+conditions.">PC12 cell line: Cell types, coating of culture vessels, differentiation and other culture conditions.</a> Cells. 9 (4), 958 (2020).</li><li>Baker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+in+cytochemistry+and+electron-microscopy.">Fixation in cytochemistry and electron-microscopy.</a> Journal of Histochemistry and Cytochemistry. 6 (5), 303-308 (1958).</li><li>Chung, J. Y., 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=Histomorphological+and+molecular+assessments+of+the+fixation+times+comparing+formalin+and+ethanol-based+fixatives.">Histomorphological and molecular assessments of the fixation times comparing formalin and ethanol-based fixatives.</a> Journal of Histochemistry and Cytochemistry. 66 (2), 121-135 (2018).</li><li>Crowley, L. C., Christensen, M. E., Waterhouse, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+transmembrane+potential+by+TMRE+staining.">Measuring mitochondrial transmembrane potential by TMRE staining.</a> Cold Spring Harbor Protocols. 2016 (12), (2016).</li><li>Cory, A. H., Owen, T. C., Barltrop, J. A., Cory, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+an+aqueous+soluble+tetrazolium/formazan+assay+for+cell+growth+assays+in+culture.">Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture.</a> Cancer Communications. 3 (7), 207-212 (1991).</li><li>Maro, B., Marty, M. C., Bornens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+effects+of+the+mitochondrial+uncoupler+FCCP+on+microtubules.">In vivo and in vitro effects of the mitochondrial uncoupler FCCP on microtubules.</a> EMBO Journal. 1 (11), 1347-1352 (1982).</li><li>Zheng, 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=Mechanism+for+regulation+of+melanoma+cell+death+via+activation+of+thermo-TRPV4+and+TRPV.">Mechanism for regulation of melanoma cell death via activation of thermo-TRPV4 and TRPV.</a> Journal of Oncology. 2019, 7362875 (2019).</li><li>Konno, K., Watanabe, M., Luj&#225;n, R., Ciruela, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+for+ion+channels+and+their+interacting+molecules:+Tips+for+improving+antibody+accessibility.">Immunohistochemistry for ion channels and their interacting molecules: Tips for improving antibody accessibility.</a> Receptor and Ion Channel Detection in the Brain. , (2016).</li><li>Mortensen, M., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+recording+of+ligand-gated+ion+channels.">Single-channel recording of ligand-gated ion channels.</a> Nature Protocols. 2 (11), 2826-2841 (2007).</li><li>Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J., van Bree, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+assay+of+cells+in+vitro.">Clonogenic assay of cells in vitro.</a> Nature Protocols. 1 (5), 2315-2319 (2006).</li><li>Rafehi, 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=Clonogenic+assay:+Adherent+cells.">Clonogenic assay: Adherent cells.</a> Journal of Visualized Experiments. (49), 2573 (2011).</li><li>Scudiero, D. 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=Evaluation+of+a+soluble+tetrazolium/formazan+assay+for+cell+growth+and+drug+sensitivity+in+culture+using+human+and+other+tumor+cell+lines.">Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.</a> Cancer Research. 48 (17), 4827-4833 (1988).</li><li>Wang, P., Henning, S. M., Heber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+MTT+and+MTS-based+assays+for+measurement+of+antiproliferative+activity+of+green+tea+polyphenols.">Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols.</a> PLoS One. 5, e10202 (2010).</li><li>Berridge, M. V., Tan, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+cellular+reduction+of+3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium+bromide+(MTT):+subcellular+localization,+substrate+dependence,+and+involvement+of+mitochondrial+electron+transport+in+MTT+reduction.">Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction.</a> Archives of Biochemistry and Biophysics. 303 (2), 474-482 (1993).</li><li>Plumb, J. A., Milroy, R., Kaye, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+pH+dependence+of+3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium+bromide-formazan+absorption+on+chemosensitivity+determined+by+a+novel+tetrazolium-based+assay.">Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay.</a> Cancer Research. 49 (16), 4435-4440 (1989).</li><li>Chakrabarti, R., Kundu, S., Kumar, S., Chakrabarti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+A+as+an+enzyme+that+catalyzes+the+reduction+of+MTT+to+formazan+by+vitamin+C.">Vitamin A as an enzyme that catalyzes the reduction of MTT to formazan by vitamin C.</a> Journal of Cellular Biochemistry. 80 (1), 133-138 (2000).</li><li>Dong, G. W., Preisler, H. D., Priore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+limitations+of+in+vitro+clonogenic+drug+sensitivity+assays.">Potential limitations of in vitro clonogenic drug sensitivity assays.</a> Cancer Chemotherapy and Pharmacology. 13 (3), 206-210 (1984).</li><li>Sun, 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=STIM1-+and+Orai1-Mediated+Ca2+oscillation+orchestrates+invadopodium+formation+and+melanoma+invasion.">STIM1- and Orai1-Mediated Ca2+oscillation orchestrates invadopodium formation and melanoma invasion.</a> Journal of Cell Biology. 207 (4), 535-548 (2014).</li></ol>

D.A.P.K. is a co-founder, president, and CEO of Amlal Pharmaceuticals Inc. S.S. is a co-founder of Amlal Pharmaceuticals Inc. and sits on the Drug Safety Monitoring Board of Bexion Pharmaceuticals, Inc.

Changes in ion channel function alter intracellular signaling cascades, which can impact the overall functioning of a cell. Over the past decade, it has become increasingly clear that ion channels are important to cancer cell growth and metastasis. Importantly, many ion channels are primary targets for approved therapeutics targeting a broad range of disorders24. Investigators have probed whether ion channels could be anti-cancer targets, and the initial results are promising2</s...

Membrane transport proteins are critical for communication between cells, as well as for maintaining cellular homeostasis. Amongst the membrane transport proteins, ion channels serve to drive the growth and development of cells and to maintain the state of cells in challenging and changing environments. Ion channels have also been reported to drive and support the development of solid tumors, both systemically and in the central nervous system (CNS)1,2. For example, KCa3.1 channels are responsible for regulating membrane potential and controlling cell volume, which is important in cell-cycle regulation. Defective KCa3.1 channels have been reported to contribute to the abnormal proliferation of tumor cells3. Further, ion channels may contribute to the metastatic dissemination of cancers. Transient receptor potential (TRP) channels, for example, are involved in Ca2+ and Mg2+ influx; this influx activates several kinases and heat shock proteins that function to regulate the extracellular matrix surrounding a tumor, which is, in turn, important for initiating cancer metastasis4.
Since ion channels can contribute to the development of cancers, they may also be targets for drug-related cancer treatment. For example, resistance to treatment modalities, including chemotherapy and novel immunotherapy, is related to ion channel function dysregulation5,6,7. In addition, ion channels are emerging as important drug targets to impede the growth and development of cancers, with repurposed small molecule (FDA-approved) drugs being examined, as well as biopolymers, including monoclonal antibodies1,2,8,9. While there has been much progress on this front, ion channel cancer drug discovery remains underdeveloped. This is partly due to the unique challenges of studying ion channels in cancer cells. For example, there are technical limitations in setting up electrophysiology assays for slow-acting compounds and temporal differences in channel activation and drug action. Further, the solubility of compounds can also impede progress, as most of the automated electrophysiology systems commonly in use today utilize hydrophobic substrates, which may contribute to artifacts as a result of compound adsorption. In addition, large bioorganic molecular therapeutics such as natural products, peptides, and monoclonal antibodies are technically challenging to screen using conventional electrophysiology assays10. Finally, the bioelectrical properties of cancer cells remain poorly understood11.
Meanwhile, the immunofluorescence staining of ion channels is often challenging. This is due, in part, to the complexity of their structures and their context in the membrane, which impact the ability to both generate and employ antibodies for microscopy studies. It is especially important that the antibodies used to stain ion channels are validated for specificity, affinity, and reproducibility. Commercial antibodies for ion channels should be considered based on their validation strategy and publication record. Experiments should include negative controls to demonstrate the lack of nonspecific binding by either knockdown or knockout of the target protein. Alternatively, cell lines in which the target protein is absent or in low abundance based on mRNA or protein determinations may serve as negative controls. For example, this study shows the localization of the (GABA) receptor&#160;subunit Gabra5 in a medulloblastoma cell line (D283). D283 cells with an siRNA knockdown and Daoy&#160;cells, another cerebellar medulloblastoma cell line, were stained for Gabra5 and showed no appreciatable staining (data not shown).
Here, methods are presented to analyze and assay ion channel function, as well as the effect of ion channel modulators on cancer cells. Protocols are provided for (1) staining cells for an ion channel, (2) testing the polarized state of mitochondria, (3) establishing ion channel function using electrophysiology, and (4) in vitro drug validation. These protocols emphasize studies of the type A gamma-aminobutyric acid (GABAA) receptor2,12,13,14,15,16, a chloride anion channel and major inhibitory neurotransmitter receptor. However, the methods presented here apply to studying many other cancer cells and ion channels.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABS SpectraMax Plate Reader</td>
			<td>Molecular Devices</td>
			<td>ABS</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Invitrogen</td>
			<td>00-4555-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Flor 488</td>
			<td>Invitrogen</td>
			<td>A32723</td>
			<td>Goat Anti-Rabbit</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td>Lacks vitamin A</td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>LABCONCO</td>
			<td>302381101</td>
			<td>Class II, Type A2</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>BP1606-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Fisher Scientific</td>
			<td>13-998-211</td>
			<td>Heracell VIOS 160i</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C7902</td>
			<td>Dihydrate</td>
		</tr>
		<tr>
			<td>Cell Culture Dishes, 150 mm</td>
			<td>Fisher Scientific</td>
			<td>12-600-004</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Cell Culture Flasks, 75 cm2</td>
			<td>Fisher Scientific</td>
			<td>430641U</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Cell Culture Plates, 6 well</td>
			<td>Fisher Scientific</td>
			<td>353046</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Cell Culture Plates, 96 well</td>
			<td>Fisher Scientific</td>
			<td>353072</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>EP-5804R</td>
			<td>Refrigerated</td>
		</tr>
		<tr>
			<td>Corning CoolCell</td>
			<td>Fisher Scientific</td>
			<td>07-210-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-553-450</td>
			<td>Corning brand</td>
		</tr>
		<tr>
			<td>D283 Med</td>
			<td>ATCC</td>
			<td>HTB-185</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO Mounting Media</td>
			<td>EMS</td>
			<td>17989-97</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Life Sciences</td>
			<td>D9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>DMEM/F12, base media</td>
			<td>Fisher Scientific</td>
			<td>11330-032</td>
			<td>With phenol red</td>
		</tr>
		<tr>
			<td>DMEM/F12, phenol red free</td>
			<td>Fisher Scientific</td>
			<td>21041-025</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>STEMCELL</td>
			<td>78006.1</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Abcam</td>
			<td>AB120081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Qualified</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor, Basic</td>
			<td>Millipore</td>
			<td>GF003</td>
			<td></td>
		</tr>
		<tr>
			<td>GARBA5 Antibody</td>
			<td>Aviva</td>
			<td>ARP30687_P050</td>
			<td>Rabbit Polyclonal</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Mounting Medium</td>
			<td>EMS</td>
			<td>17989-60</td>
			<td>With DAPI+DABCO</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Millipore Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>STEMCELL</td>
			<td>7980</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>HyClone</td>
			<td>SH3023701</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Open platform</td>
			<td></td>
			<td>With Fiji plugins</td>
		</tr>
		<tr>
			<td>Immuno Mount DAPI</td>
			<td>EMS</td>
			<td>17989-97</td>
			<td></td>
		</tr>
		<tr>
			<td>KRM-II-08</td>
			<td></td>
			<td></td>
			<td>experimental compounds not available from a commercial source</td>
		</tr>
		<tr>
			<td>Leica Application Suite X</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor</td>
			<td>Novus</td>
			<td>N276314100U</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>M9272</td>
			<td>Hexahydrate</td>
		</tr>
		<tr>
			<td>Microscope, Confocal</td>
			<td>Leica</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope, Light</td>
			<td>VWR</td>
			<td>76382-982</td>
			<td>DMiL Inverted</td>
		</tr>
		<tr>
			<td>MTS - Promega One Step</td>
			<td>Promega</td>
			<td>G3581</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipette, 0.5-10 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683914</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipette, 10-100 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683930</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipette, 30-300 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683957</td>
			<td></td>
		</tr>
		<tr>
			<td>Nest-O-Patch</td>
			<td>Heka</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>Gibco</td>
			<td>10888022</td>
			<td>Without vitamin A</td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>Gibco</td>
			<td>12348-017</td>
			<td>Phenol red free</td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>NOR-QH-II-66</td>
			<td></td>
			<td></td>
			<td>experimental compounds not available from a commercial source</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>50-998-944</td>
			<td>4 inch width</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>EMS</td>
			<td>RT-15710</td>
			<td></td>
		</tr>
		<tr>
			<td>PATHCHMASTER</td>
			<td>Heka</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion System</td>
			<td>Nanion</td>
			<td>4000120</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>EMS</td>
			<td>RT-15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Bufered Saline</td>
			<td>Fisher Scientific</td>
			<td>AAJ75889K2</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Fisher Scientific</td>
			<td>A3890401</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma Life Sciences</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Port-a-Patch</td>
			<td>Nanion</td>
			<td>21000072</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Life Sciences</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Antibody</td>
			<td>Invitrogen</td>
			<td>MA5-34653</td>
			<td>Rabbit Monoclonal</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fisher Scientific</td>
			<td>NC0758676</td>
			<td>1 mL ampule</td>
		</tr>
		<tr>
			<td>QH-II-66</td>
			<td></td>
			<td></td>
			<td>experimental compounds not available from a commercial source</td>
		</tr>
		<tr>
			<td>Reagent Reservoirs</td>
			<td>VWR</td>
			<td>89094-664</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Slides, 75 x 25 mm</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td>Frosted one side</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Corning</td>
			<td>25-035-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Synth-a-Freeze Medium</td>
			<td>Gibco</td>
			<td>R00550</td>
			<td>Cryopreservation</td>
		</tr>
		<tr>
			<td>TMRE</td>
			<td>Fisher Scientific</td>
			<td>50-196-4741</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>TMRE Kit</td>
			<td>Abcam</td>
			<td>AB113852</td>
			<td>Kit</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>NC0704309</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Gibco</td>
			<td>15-250-061</td>
			<td>Solution, 0.4%</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td>Solution, 0.25%</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>VWR</td>
			<td>97043-562</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Filter Paper</td>
			<td>Fisher Scientific</td>
			<td>09-927-841</td>
			<td></td>
		</tr>
	</tbody>
</table>

screening ion channels in cancer cells

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.

Above are select procedures that can be employed to characterize ion channels in cancerous cells. The first protocol highlights the staining of an ion channel. As detailed, there are many challenges when staining an ion channel or, for that matter, any protein that is present in the extracellular membrane. Shown in Figure 1 is the staining for a subunit of the pentameric GABAA receptor. The second protocol highlights the results of testing the polarized state of mitochondria in ca...

Watch this Scientific Journal Video about Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches at JoVE.com

Author Spotlight: Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches 

The pharmacological targeting of ion channels is a promising approach to treating solid tumors. Detailed protocols are provided for characterizing ion channel function in cancer cells and assaying the effects of ion channel modulators on cancer viability.

Screening Ion Channels in Cancer Cells

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development ...

Research

JoVE Journal

Cancer Research

2.0K Views.  University of Cincinnati College of Medicine. The pharmacological targeting of ion channels is a promising approach to treating solid tumors. Detailed protocols are provided for characterizing ion channel function in cancer cells and assaying the effects of ion channel modulators on cancer viability. 

Video: Author Spotlight: Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches 

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 ...

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

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 ...

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

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

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

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

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...