Screening Ion Channels in Cancer Cells

Summary

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.

Abstract

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.

Introduction

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. Defect....

Protocol

1. Immunolabeling ion channels in cultured cells

1. Preparing the cells and experimental set-up
   1. Maintain the cells as an actively growing culture in 75 cm² culture flasks. Passage the cells once until they become 50%-90% confluent, depending on the doubling time of the cell line being used.
      NOTE: For the present study, D283 cells, a Group 3 medulloblastoma cell line, were used.
   2. Collect the cells from the culture flask into a centrifuge tube (15 mL or 50 mL), and add 2 mL.......

Discussion

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

Materials

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