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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 intestine2,3. 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 colitis5,

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.......

Representative Results

We describe the protocol of obtaining spheroids from diverse colorectal cancer cell lines. Supplementation with methylcellulose was required to generate spheroids. We also present a method of LCFS preparation and present a model to study the correlation between probiotics and colorectal cancer. Spheroid formation and LCFS preparation protocols are schematically illustrated in Figure 1A,B. As shown in Figure 2A, methylcellulose concentration of 0.......

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.......

Acknowledgements

This research was supported by the “Establishment of measurement standards for Chemistry and Radiation”, grant number KRISS-2020-GP2020-0003, and “Development of Measurement Standards and Technology for Biomaterials and Medical Convergence”, grant number KRISS-2020-GP2020-0004 programs, funded by the Korea Research Institute of Standards and Science. This research was also supported by the Ministry of Science and ICT (MSIT), National Research Foundation of Korea (NRF-2019M3A9F3065868), The Ministry of Health and Welfare (MOHW), the Korea Health Industry Development Institute (KHIDI, HI20C0558), the Ministry of Trade, Industry & Energy (MOTI....

Materials

NameCompanyCatalog NumberComments

10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 µl
Biorad4561036Pkg of 10
Applied Biosystems MicroAmp Optical Adhesive FilmThermo Fisher Scientific4311971100 covers
10x transfer bufferIntronIBS-BT031A1 L
10X Tris-Glycine (W/SDS)IntronIBS-BT0141 L
Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/CaseCorningSCT-200-C500 Tubes/Pack, 10 Packs/Case
BD Difco Bacto AgarBD214010500 g
BD Difco Lactobacilli MRS BrothBDDF0881-17-5500 g
CellTiter-Glo 3D Cell viability assayPromegaG9681100μl/assay in 96-well plates
Complete Protease Inhibitor CocktailSigma-Aldrich11697498001vial of 20 tablets
Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1Corning21-040-CV500 mL
EMD Millipore Immobilon-P PVDF Transfer Membranesfisher ScientificIPVH0001026.5cm x 3.75m roll; Pore Size: 0.45um
Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap CapCorning35223525/Pack, 500/Case
Fetal Bovine Serum, certified, US originThermo Fisher Scientific16000044500 mL
iScript cDNA Synthesis Kit, 25 x 20 µl rxns #1708890Biorad170889025 x 20 µL rxns
iTaq Universal SYBR Green SupermixBiorad17251215 x 1 mL
Lactobacillus fermentumKorean Collection for Type CulturesKCTC 3112
L-Cysteine hydrochloride monohydrateSigma-AldrichC6852-25G25 g
Methyl Cellulose (3500-5600mPa·s, 2% in Water at 20°C)TCIM0185500 g
MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mLApplied Biosystems434690620 plates
Millex-GS Syringe Filter Unit, 0.22 µm, mixed cellulose esters, 33 mm, ethylene oxide sterilizedMilliporeSLGS033SB250
PE Annexin V Apoptosis Detection Kit with 7-AADBiolegend640934100 tests
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher Scientific15140122100 mL
Propidium IodideIntrogenP1304MP100 mg
RIPA Lysis and Extraction BufferThermo Fisher Scientific89901250 mL
RNeasy Mini Kit (250)Qiagen74106250
RPMI-1640Gibco11875-119500 mL
Trypsin-EDTA (0.25%), phenol redThermo Fisher Scientific25200056100 mL
Name of Materials/Equipment/SoftwareCompanyCatalog NumberComments/Description
anti - p-IκBα (B-9)Santa cruzesc-8404200 µg/mL
anti-BclxL (H-5)Santa cruzesc-8392200 µg/mL
anti-PARP 1 (C2-10)Santa cruzesc-5364350 µl ascites
anti-β-actin (C4)Santa cruzesc-47778200 µg/mL
BD FACSVerseBD BiosciencesSan Diego, CA, USA
Synergy HTX Multi-Mode Microplate ReaderBioTS1LFA
CO2 incubatorThermo fisherHERAcell 150i
Conical tube 15 mlSPL50015
Conical tube 50 mlSPL50050
Corning Costar Ultra-Low Attachment Multiple Well PlateSigma-AldrichCLS7007
Corning Costar Ultra-Low Attachment Multiple Well PlateSigma-AldrichCLS3471
Costar 50 mL Reagent Reservoirs, 5/Bag, SterileCostar4870
Countess Cell Counting Chamber SlidesThermofisherC10228
Countess II FL Automated Cell CounterinvitrogenAMQAF1000
EnSpire Multimode ReaderPerkin ElmerEnspire 2300
Eppendorf Research Plus Multi Channel Pipette, 8-channelEppendorf3122000051
FlowJo softwareTreeStarAshland, OR, USA
Goat Anti-Mouse IgG (H+L)Jackson immunoresearch115-035-0621.5 mL
Goat Anti-Rabbit IgG (H+L)Jackson immunoresearch111-035-1442.0 mL
GraphPad Prism 5GraphPad SoftwareInc., San Diego, CA, USA
ImageJNIH
ImageQuant LAS 4000 miniFujifilmTokyo, Japan
Incubated shakerLab companionSIF-6000R
Multi Gauge Ver. 3.0,FujifilmTokyo, Japan
Optical density (OD)LAMBDA UV/Vis SpectrophotometersPerkin ElmerWaltham, MA, USA
Phase-contrast microscopeOlympusTokyo, Japan
SPL microcentrifuge tube 1.5mLSPL60015
SPL Multi Channel Reservoirs, 12-Chs, PS, SterileSPL21012
StepOnePlus Real-Time PCR systemThermo Fisher ScientificWaltham, MA, USA
Vibra-Cell Ultrasonic Liquid ProcessorsSONICS-vibra cellVC 505500 Watt ultrasonic processor
Vinyl Anaerobic ChamberCOY LAB PRODUCTS

References

  1. Bron, P. A., Van Baarlen, P., Kleerebezem, M. Emerging molecular insights into the interaction between probiotics and the host intestinal mucosa. Nature Reviews Microbiology. 10 (1), 66-78 (2012).
  2. Ruiz, L., Delgado, S., Ruas-Madiedo, P., Sánchez, B., Margolles, A.

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