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* These authors contributed equally
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.
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.
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,
1. Bacterial cell cultures and preparation of Lactobacillus cell-free supernatant (LCFS)
NOTE: Steps 1.2 – 1.9 are conducted in an anaerobic chamber.
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.......
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.......
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....
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 |
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