Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

Summary

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

Abstract

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Introduction

Probiotics are the most advantageous microorganisms in the gut that improves immune homeostasis and host energy metabolism¹. Probiotics from Lactobacillus and Bifidobacterium are the most advanced of its kind found in the intestine^2,3. Previous investigations have shown that Lactobacillus has inhibitory and antiproliferative effects on several cancers, including colorectal cancer⁴. Moreover, probiotics prevent inflammatory bowel diseases, Crohn’s disease, and ulcerative colitis^5,

Protocol

1. Bacterial cell cultures and preparation of Lactobacillus  cell-free supernatant (LCFS)

NOTE: Steps 1.2 – 1.9 are conducted in an anaerobic chamber.

1. Prepare an MRS agar plate and broth containing L-cysteine and sterilize by autoclaving.
2. Pre-incubate the MRS agar plate in H₂ anaerobic chamber maintained at 37 ˚C with 20 ppm oxygen.
3. Thaw Lactobacillus bacterial stock and inoculate the agar plate with the bacterial cul.......

Discussion

The tissue microenvironment, including neighboring cells and the extracellular matrix (ECM), is fundamental to tissue generation and crucial in the control of cell growth and tissue development¹³. However, 2D cultures have several disadvantages, such as the disruption of cellular interactions, as well as alterations in cell morphology, extracellular environments, and the approach of division¹⁴. 3D cell culture systems have been rigorously studied to better reproduce in vivo.......

Materials

Name	Company	Catalog Number	Comments
10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 µl	Biorad	4561036	Pkg of 10
Applied Biosystems MicroAmp Optical Adhesive Film	Thermo Fisher Scientific	4311971	100 covers
10x transfer buffer	Intron	IBS-BT031A	1 L
10X Tris-Glycine (W/SDS)	Intron	IBS-BT014	1 L
Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/Case	Corning	SCT-200-C	500 Tubes/Pack, 10 Packs/Case
BD Difco Bacto Agar	BD	214010	500 g
BD Difco Lactobacilli MRS Broth	BD	DF0881-17-5	500 g
CellTiter-Glo 3D Cell viability assay	Promega	G9681	100μl/assay in 96-well plates
Complete Protease Inhibitor Cocktail	Sigma-Aldrich	11697498001	vial of 20 tablets
Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1	Corning	21-040-CV	500 mL
EMD Millipore Immobilon-P PVDF Transfer Membranes	fisher Scientific	IPVH00010	26.5cm x 3.75m roll; Pore Size: 0.45um
Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap	Corning	352235	25/Pack, 500/Case
Fetal Bovine Serum, certified, US origin	Thermo Fisher Scientific	16000044	500 mL
iScript cDNA Synthesis Kit, 25 x 20 µl rxns #1708890	Biorad	1708890	25 x 20 µL rxns
iTaq Universal SYBR Green Supermix	Biorad	1725121	5 x 1 mL
Lactobacillus fermentum	Korean Collection for Type Cultures	KCTC 3112
L-Cysteine hydrochloride monohydrate	Sigma-Aldrich	C6852-25G	25 g
Methyl Cellulose (3500-5600mPa·s, 2% in Water at 20°C)	TCI	M0185	500 g
MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL	Applied Biosystems	4346906	20 plates
Millex-GS Syringe Filter Unit, 0.22 µm, mixed cellulose esters, 33 mm, ethylene oxide sterilized	Millipore	SLGS033SB	250
PE Annexin V Apoptosis Detection Kit with 7-AAD	Biolegend	640934	100 tests
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122	100 mL
Propidium Iodide	Introgen	P1304MP	100 mg
RIPA Lysis and Extraction Buffer	Thermo Fisher Scientific	89901	250 mL
RNeasy Mini Kit (250)	Qiagen	74106	250
RPMI-1640	Gibco	11875-119	500 mL
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher Scientific	25200056	100 mL
Name of Materials/Equipment/Software	Company	Catalog Number	Comments/Description
anti - p-IκBα (B-9)	Santa cruze	sc-8404	200 µg/mL
anti-BclxL (H-5)	Santa cruze	sc-8392	200 µg/mL
anti-PARP 1 (C2-10)	Santa cruze	sc-53643	50 µl ascites
anti-β-actin (C4)	Santa cruze	sc-47778	200 µg/mL
BD FACSVerse	BD Biosciences		San Diego, CA, USA
Synergy HTX Multi-Mode Microplate Reader	BioT	S1LFA
CO2 incubator	Thermo fisher	HERAcell 150i
Conical tube 15 ml	SPL	50015
Conical tube 50 ml	SPL	50050
Corning Costar Ultra-Low Attachment Multiple Well Plate	Sigma-Aldrich	CLS7007
Corning Costar Ultra-Low Attachment Multiple Well Plate	Sigma-Aldrich	CLS3471
Costar 50 mL Reagent Reservoirs, 5/Bag, Sterile	Costar	4870
Countess Cell Counting Chamber Slides	Thermofisher	C10228
Countess II FL Automated Cell Counter	invitrogen	AMQAF1000
EnSpire Multimode Reader	Perkin Elmer		Enspire 2300
Eppendorf Research Plus Multi Channel Pipette, 8-channel	Eppendorf	3122000051
FlowJo software	TreeStar		Ashland, OR, USA
Goat Anti-Mouse IgG (H+L)	Jackson immunoresearch	115-035-062	1.5 mL
Goat Anti-Rabbit IgG (H+L)	Jackson immunoresearch	111-035-144	2.0 mL
GraphPad Prism 5	GraphPad Software		Inc., San Diego, CA, USA
ImageJ	NIH
ImageQuant LAS 4000 mini	Fujifilm		Tokyo, Japan
Incubated shaker	Lab companion	SIF-6000R
Multi Gauge Ver. 3.0,	Fujifilm		Tokyo, Japan
Optical density (OD)LAMBDA UV/Vis Spectrophotometers	Perkin Elmer		Waltham, MA, USA
Phase-contrast microscope	Olympus		Tokyo, Japan
SPL microcentrifuge tube 1.5mL	SPL	60015
SPL Multi Channel Reservoirs, 12-Chs, PS, Sterile	SPL	21012
StepOnePlus Real-Time PCR system	Thermo Fisher Scientific		Waltham, MA, USA
Vibra-Cell Ultrasonic Liquid Processors	SONICS-vibra cell	VC 505	500 Watt ultrasonic processor
Vinyl Anaerobic Chamber	COY LAB PRODUCTS