We acknowledge the imaging and Anipath recherche histology platforms (CRCL, CLB). We are indebted to the pharmacy of the Centre L&#233;on B&#233;rard (CLB) Hospital for the kind gift of FOLFOX and FOLFIRI. We also thank Brigitte Manship for critical reading of the manuscript. The work was supported by the FRM (Equipes FRM 2018, DEQ20181039598) and by the Inca (PLBIO19-289). MVG and LC received support from the FRM and CF received support from ARC foundation and the Centre L&#233;on B&#233;rard.

NOTE: The details of all reagents and materials are listed in the Table of Materials.
1. Spheroid formation
<ol>
	<li>Spheroid culture media
	<ol>
		<li>Prepare basal medium consisting of Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 4 mM L-alanyl-L-glutamine dipeptide.</li>
		<li>Prepare DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen/Strep) in basal medium from step 1.1.1.</li>
		<li>Prepare DMEM/basement membrane matrix medium containing 2.5% basement membrane matrix, 10% FBS, and 1% Pen/Strep in basal medium from step 1.1.1.</li>
	</ol>
	</li>
	<li>Preparation of plates for spheroid formation
	<ol>
		<li>Warm basal and DMEM/basement membrane matrix medium at room temperature (RT) for approximately 20 min.</li>
		<li>Pretreat the wells of a 24-well plate dedicated to spheroid formation by adding 500&#181;L of anti-adherence rinsing solution to each well. 
		NOTE: In these plates, each well consists of 1,200 microwells.</li>
		<li>Centrifuge the plate at 1,200 &#215; g for 5 min in a swinging bucket rotor with adaptors for plates. 
		NOTE: If only one plate is used, prepare an additional standard plate filled with water to counterbalance the weight.</li>
		<li>Rinse each well with 2 mL of warm basal medium, and aspirate the medium from the wells.</li>
		<li>Observe the plate under the microscope to ensure that bubbles have been completely removed from the microwells. If bubbles remain trapped, centrifuge again at 1,200 &#215; g for 5 min to eliminate the bubbles.</li>
		<li>Repeat the rinsing steps 1.2.4-1.2.5.</li>
		<li>Add 1 mL of warm DMEM/basement membrane matrix medium to each well.</li>
	</ol>
	</li>
	<li>Generation of spheroids
	<ol>
		<li>Grow the Caco2 cells in a 2D monolayer in DMEM medium supplemented with 10% FBS and 1% Pen/Strep at 37 &#176;C in a humidified atmosphere containing 5% CO2 (hereafter referred to as 37 &#176;C/5% CO2). 
		NOTE: The maximum number of cell passages to be used is 80.</li>
		<li>When 80% of confluency is reached, wash the cells with phosphate-buffered saline (PBS) 1x (5 mL for a 10 cm dish), add trypsin-ethylenediamine tetraacetic acid (EDTA) (2 mL for a 10 cm dish), and incubate for 2-5 min at 37 &#176;C/5% CO2.</li>
		<li>Check cell detachment under the microscope, and neutralize the trypsin by adding 4 mL of DMEM complete medium per 10 cm dish.</li>
		<li>Count cells using a hemocytometer to determine the total number of cells.</li>
		<li>Centrifuge the cell suspension at 1,200 &#215; g for 5&#160;min. Discard the supernatant, and resuspend the pellet in an appropriate volume of DMEM/basement membrane matrix medium.</li>
		<li>Refer to Table 1 to determine the number of cells required per well to achieve the desired number of cells per microwell. Alternatively, calculate the number of cells using the following formula for a 24-well plate, considering each well contains 1,200 microwells 
		Required number of cells per well = Desired number of cells per microwell &#215; 1,200</li>
		<li>Add the required volume of the cell suspension to each well to achieve the desired cell number in a final volume of 1 mL.</li>
		<li>Add 1 mL of DMEM/basement membrane matrix medium to each well to reach the final volume of 2 mL per well (see also step 1.2.7). 
		NOTE: Be careful not to introduce bubbles into the microwells.</li>
		<li>Centrifuge the plate immediately at 1,200 &#215; g for 5 min to capture the cells in the microwells. If necessary, counterbalance the centrifuge with a standard plate filled with water.</li>
		<li>Observe the plate under the microscope to verify that the cells are evenly distributed among the microwells.</li>
		<li>Incubate the plate at 37 &#176;C/5% CO2 for 48 h without disturbing the plate. 
		NOTE: According to the original protocol21, although many cell lines can form spheroids within 24 h, some require a longer incubation time. In this protocol, 48 h are sufficient for spheroid formation.</li>
	</ol>
	</li>
	<li>Harvesting the spheroids from the microwells
	<ol>
		<li>Warm the basal and DMEM/basement membrane matrix medium at RT for approximately 20 min.</li>
		<li>Using a serological pipette, remove half of the culture medium (1 mL) from each well.</li>
		<li>Add the medium back onto the surface of the well to dislodge the spheroids from the microwells. 
		NOTE: Do not touch or triturate the spheroids.</li>
		<li>Place a 37 &#181;m reversible strainer (or a 40 &#181;m standard strainer) on the top of a 15&#160;mL conical tube to collect the spheroids. 
		NOTE: If using a 40 &#181;m standard strainer, place it upside down.</li>
		<li>Gently aspirate the dislodged spheroids (from step 1.4.3), and pass the spheroid suspension through the strainer. 
		NOTE: The spheroids will remain on the filter; single cells will flow through with the medium.</li>
		<li>Using a serological pipette, dispense 1 mL of the warm basal medium across the entire surface of the well to dislodge any remaining spheroids and recover them on the strainer.</li>
		<li>Repeat this washing step 1.4.6 twice.</li>
		<li>Observe the plate under the microscope to ensure that all spheroids have been removed from the microwells. Repeat the wash if necessary (steps 1.4.6-1.4.7).</li>
		<li>Invert the strainer, and place it on a new 15&#160;mL conical tube. Collect the spheroids by washing the strainer with DMEM/basement membrane matrix medium. 
		NOTE: The collected spheroids are ready for downstream applications and analyses.</li>
	</ol>
	</li>
	<li>Long-term culture of spheroids
	<ol>
		<li>Prepare 1.5% agarose solution in basal medium, and sterilize it by autoclaving (standard cycle).</li>
		<li>While the agarose solution is warm and still liquid, coat the wells of standard culture plates or dishes, as described in Table 2. 
		NOTE: Warming the dish/plate in the oven will facilitate the coating step. Coated dishes/plates can be left at RT for up to 10 days in a sterile environment and protected from the light.</li>
		<li>Seed the harvested spheroids (from step 1.4.9) in the agarose-coated plates, and add DMEM/basement membrane matrix medium to achieve the final volume depending on the size of the plate. 
		NOTE: To avoid spheroid aggregates, seed them at the optimal density of 22 spheroids/cm2. Observe that the coating is not perfectly flat, and it rises towards the edge, creating a light concavity at the center of the plate. If the number of spheroids is too high, they are more likely to adhere to each other.</li>
		<li>Incubate the plate at 37 &#176;C/5% CO2 until recovery of the spheroids for specific analyses.</li>
	</ol>
	</li>
	<li>Treatment of spheroids with chemotherapeutic drugs
	<ol>
		<li>Plate spheroids from step 1.5.4, and grow them for 2 days. Starting from day 3 (D3), treat them with FOLFIRI (5-Fluorouracil, 50 &#181;g/mL; Irinotecan, 100 &#181;g/mL; Leucovorin, 25 &#181;g/mL) or with FOLFOX (5-Fluorouracil, 50 &#181;g/mL; Oxaliplatin, 10 &#181;g/mL; Leucovorin, 25 &#181;g/mL) chemotherapeutic regimen combinations routinely used to treat CRC patients22,23,24,25, or maintain them in (control) not-treated (NT) condition.</li>
		<li>Collect the spheroids after 3 days of treatment by using a pipette with the tip cut off (1,000 &#181;L tip), ensuring that each condition is represented by at least three replicates. Centrifuge them at 1,000 &#215; g for 3 min, and then remove the supernatant.</li>
		<li>Fix the pellets in 2% paraformaldehyde (PFA) for histological analysis (see section 3), or use the pellets for RNA extraction (see section 4).</li>
		<li>To analyze cell death, incubate the spheroids from step 1.6.1 in black culture well plates in DMEM/basement membrane matrix medium for 30 min with a nucleic acid stain (1:5000 dilution) that does not permeate live cells, but penetrates the compromised membranes of dead cells26. Measure the accumulation of fluorescence with a microplate reader.</li>
	</ol>
	</li>
</ol>
2. Monitoring spheroid growth
<ol>
	<li>Using an inverted microscope, acquire representative images of spheroids maintained under different conditions throughout the days in culture.</li>
	<li>Analyze the images by measuring three different representative diameters of each spheroid using appropriate software.</li>
	<li>Use the following formula to obtain the estimated sphere volume. 
	<img alt="Equation 1" src="/files/ftp_upload/61783/61783eq1.jpg" /> 
	 
	NOTE: The terms, d1, d2, and d3, are the three diameters of the spheroid.</li>
</ol>
3. Immunofluorescence (IF) and histological staining
<ol>
	<li>Fixation and paraffin embedding
	<ol>
		<li>Collect the spheroids at selected time-points using a pipette with the tip cut off as described in step 1.6.2, and fix them for 30 min at RT in 2% PFA. 
		NOTE: Alternatively, store the samples at this step at 4 &#176;C until further use.</li>
		<li>For paraffin embedding, wash the spheroids 3x with PBS 1x, and resuspend them in 70% ethanol. After paraffin inclusion and sectioning, perform hematoxylin &#38; eosin (H&#38;E) staining for histological analysis.</li>
	</ol>
	</li>
	<li>Immunolabeling of paraffin sections 
	&#8203;NOTE: Use 5-&#956;m-thick sections for indirect immunostaining.
	<ol>
		<li>Incubate slides at 60 &#176;C for 2 h to melt the wax and improve deparaffinization.</li>
		<li>Wash the slides twice for 3 min in methylcyclohexane.</li>
		<li>Wash the slides for 3 min in 1:1 methylcyclohexane:100% ethanol.</li>
		<li>Wash the slides twice for 3 min in 100% ethanol. 
		NOTE: Perform all manipulations for step 3.2.2 to step 3.2.4 in a chemical hood.</li>
		<li>Wash the slides for 3 min in 90% ethanol.</li>
		<li>Wash the slides for 3 min in 75% ethanol.</li>
		<li>Wash the slides for 3 min in 50% ethanol.</li>
		<li>Wash the slides under tap water.</li>
		<li>Rehydrate the slides in distilled water for 5 min.</li>
		<li>Prepare 700 mL of 0.01 M citrate buffer, pH 6.0, and add it to a suitable container (width: 11.5 cm, length: 17 cm, height: 7 cm); submerge the slides in it. Heat the container in the microwave for 9-10 min at 700 W, and when the boiling starts, decrease the power to 400-450 W. Incubate for an additional 10 min.</li>
		<li>Let the slides cool down in the buffer to RT for approximately 30-40 min.</li>
		<li>Wash the slides twice in PBS for 5 min.</li>
		<li>Draw a circle around the sections with a marker pen to create a barrier for liquids applied to the sections.</li>
		<li>Incubate each section with 50 &#181;L of blocking buffer (10% normal goat serum, 1% bovine serum albumin (BSA), and 0.02% Triton X-100 in PBS) for at least 30 min at RT.</li>
		<li>Remove the blocking buffer, and add 50 &#181;L of primary antibodies diluted in the incubation buffer (1% normal goat serum, 0.1% BSA, and 0.02% Triton X-100 in PBS). Incubate for 2 h at RT or overnight at 4 &#176;C.</li>
		<li>Remove the primary antibodies, and wash the slides 3x in PBS for 5 min.</li>
		<li>Incubate the slides with 50 &#181;L of fluorescent secondary antibodies diluted in the incubation buffer for 1 h at RT.</li>
		<li>Remove the secondary antibodies, and wash the slides 3x in PBS for 5 min.</li>
		<li>Add 50 &#181;L of mounting medium with 4&#8242;,6-diamidino-2-phenylindole to each section, and place a glass coverslip over the section.</li>
	</ol>
	</li>
</ol>
4. RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qRT-PCR)
<ol>
	<li>Collect spheroids at different time-points, each point represented by at least three replicates. Centrifuge them at 1,000 &#215; g for 3 min, and then remove the supernatant. 
	NOTE: Pellets can be directly used for RNA extraction or stored at -20 &#176;C until further use.</li>
	<li>Isolate the total RNA using a commercial RNA isolation kit, according to the manufacturer&#39;s instructions.</li>
	<li>Reverse-transcribe 500 ng of each RNA sample into complementary DNA (cDNA) with a commercial kit according to the manufacturer&#39;s instructions.</li>
	<li>After reverse transcription, perform a PCR analysis on 1 &#181;L of cDNA to amplify a housekeeping gene with primers located in different exons. 
	NOTE: This step enables the verification of the absence of any genomic DNA contamination in the RNA preparations. For this protocol, peptidylprolyl isomerase B (PPIB) primers were used.</li>
	<li>Perform qPCR amplification on 4 &#181;L of previously diluted cDNA (1:10 in RNAse-free double-distilled water) by using primers specific for the genes of interest. In each sample, quantify specific mRNA expression by using the &#916;&#916;Ct method and values normalized against a housekeeping gene levels. 
	NOTE: For this protocol, &#946;-actin (ACTB) was selected. Primers used in this protocol are listed in Table 3.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+with+basement+membrane+serves+to+rapidly+distinguish+growth+and+differentiation+pattern+of+normal+and+malignant+human+breast+epithelial+cells.">Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 89 (19), 9064-9068 (1992).</li><li>Nusse, R., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/&#946;-catenin+signaling,+disease,+and+emerging+therapeutic+modalities.">Wnt/&#946;-catenin signaling, disease, and emerging therapeutic modalities.</a> Cell. 169 (6), 985-999 (2017).</li><li>Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M. L., Stammati, A., Zucco, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caco-2+cell+line+as+a+model+of+the+intestinal+barrier:+influence+of+cell+and+culture-related+factors+on+Caco-2+cell+functional+characteristics.">The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics.</a> Cell Biology and Toxicology. 21 (1), 1-26 (2005).</li><li>Vermeulen, L., Snippert, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+dynamics+in+homeostasis+and+cancer+of+the+intestine.">Stem cell dynamics in homeostasis and cancer of the intestine.</a> Nature Reviews Cancer. 14 (7), 468-480 (2014).</li><li>van der Heijden, M., Vermeulen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+homeostasis+and+cancer+of+the+gut.">Stem cells in homeostasis and cancer of the gut.</a> Molecular Cancer. 18 (1), 66 (2019).</li><li>Barker, N., Bartfeld, S., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+adult+stem+cell+populations+of+rapidly+self-renewing+organs.">Tissue-resident adult stem cell populations of rapidly self-renewing organs.</a> Cell Stem Cell. 7 (6), 656-670 (2010).</li><li>van der Flier, L. G., Haegebarth, A., Stange, D. E., van de Wetering, M., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OLFM4+is+a+robust+marker+for+stem+cells+in+human+intestine+and+marks+a+subset+of+colorectal+cancer+cells.">OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells.</a> Gastroenterology. 137 (1), 15-17 (2009).</li><li>Potten, C. 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=Identification+of+a+putative+intestinal+stem+cell+and+early+lineage+marker;+musashi-1.">Identification of a putative intestinal stem cell and early lineage marker; musashi-1.</a> Differentiation. 71 (1), 28-41 (2003).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intestinal+crypt,+a+prototype+stem+cell+compartment.">The intestinal crypt, a prototype stem cell compartment.</a> Cell. 154 (2), 274-284 (2013).</li><li>Clark, D. W., Palle, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenases+in+cancer+stem+cells:+potential+as+therapeutic+targets.">Aldehyde dehydrogenases in cancer stem cells: potential as therapeutic targets.</a> Annals of Translational Medicine. 4 (24), 518 (2016).</li><li>Tomita, H., Tanaka, K., Tanaka, T., Hara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenase+1A1+in+stem+cells+and+cancer.">Aldehyde dehydrogenase 1A1 in stem cells and cancer.</a> Oncotarget. 7 (10), 11018-11032 (2016).</li><li>Zoetemelk, M., Rausch, M., Colin, D. J., Dormond, O., Nowak-Sliwinska, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+3D+culture+systems+of+various+complexity+for+treatment+optimization+of+colorectal+carcinoma.">Short-term 3D culture systems of various complexity for treatment optimization of colorectal carcinoma.</a> Scientific Reports. 9 (1), 7103 (2019).</li><li>Garcia-Mayea, Y., Mir, C., Masson, F., Paciucci, R., Leonart, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+new+mechanisms+and+models+of+cancer+stem+cell+multidrug+resistance.">Insights into new mechanisms and models of cancer stem cell multidrug resistance.</a> Seminars in Cancer Biology. 60, 166-180 (2020).</li><li>Marusyk, A., Janiszewska, M., Polyak, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumor+heterogeneity:+The+Rosetta+Stone+of+therapy+resistance.">Intratumor heterogeneity: The Rosetta Stone of therapy resistance.</a> Cancer Cell. 37 (4), 471-484 (2020).</li></ol>

The authors have nothing to disclose

In vitro 3D models overcome the main experimental drawbacks of 2D cancer cell cultures, as they appear to be more reliable in recapitulating typical tumoral features including microenvironment and cell heterogeneity. Commonly used 3D models of spheroids are scaffold-free (cultured in low-attachment conditions) or scaffold-based (using biomaterials to culture cells). These methods present different disadvantages as they depend on the nature of the scaffold used or give rise to spheroids that are variable in structure and ...

Colorectal cancer (CRC) remains the second leading cause of cancer-associated deaths in the world1. The development of CRC is the result of a progressive acquisition and accumulation of genetic mutations and/or epigenetic alterations2,3, including the activation of oncogenes and inactivation of tumor suppressor genes3,4. Moreover, non-genetic factors (e.g., the microenvironment) can contribute to and promote oncogenic transformation and thus participate in the evolution of CRCs5. Importantly, CRCs are composed of different cell populations, including undifferentiated CSCs and bulk tumor cells displaying some differentiation traits, which constitute a hierarchical structure reminiscent of the organization of the epithelium in a normal colon crypt6,7.
CSCs are considered to be responsible for tumor appearance8, its maintenance and growth, metastatic capacity, and resistance to conventional therapies6,7. Within tumors, cancer cells, including CSCs, display a high level of heterogeneity and complexity in terms of their distinct mutational and epigenetic profiles, morphological and phenotypic differences, gene expression, metabolism, proliferation rates, and metastatic potential9. Therefore, to better understand cancer biology, tumor progression, and acquisition of resistance to therapy and its translation into effective treatments, human preclinical models capturing this cancer heterogeneity and hierarchy are important10,11.
In vitro 2D cancer cell lines have been used for a long time and provide valuable insights into tumor development and the mechanisms underlying the efficacy of therapeutic molecules. However, their limitation with respect to the lack of the phenotypic and genetic heterogeneity found in the original tumors is now widely recognized12. Moreover, nutrients, oxygen, pH gradients, and the tumor microenvironment are not reproduced, the microenvironment being especially important for the maintenance of different cell types including CSCs11,12. To overcome these main drawbacks, several 3D models have been developed to experimentally address and reproduce the complexity and heterogeneity of cancers. In effect, these models recapitulate tumor cellular heterogeneity, cell-cell interactions, and spatial architecture, similar to those observed in vivo12,13,14. Primary tumor organoids established from fresh tumors, as well as cell line-derived spheroids, are largely employed15,16.
Spheroids can be cultured in a scaffold-free or scaffold-based manner to force the cells to form and grow in cell aggregates. Scaffold-free methods are based on the culture of cells under non-adherent conditions (e.g., the hanging-drop method or ultra-low attachment plates), whereas scaffold-based models rely on natural, synthetic, or hybrid biomaterials to culture cells12,13,14. Scaffold-based spheroids present different disadvantages as the final spheroid formation will depend on the nature and composition of the (bio)material used. Although the scaffold-free spheroid methods available so far do not rely on the nature of the substrate, they generate spheroids that vary in structure and size17,18.
This work was aimed at designing a robust and reproducible 3D culture system of spheroids, which are homogenous in size, composed of Caco2 colon adenocarcinoma cells to study CSC biology. Caco2 cells are of particular interest owing to their capacity to differentiate over time19,20, strongly suggesting a stem-like potential. Accordingly, long-term culture of the spheroids revealed the presence of different CSC populations with different responses to chemotherapy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>37 &#181;m Reversible Strainer, Large&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>27250</td>
			<td>To be used with 50 mL conical tubes</td>
		</tr>
		<tr>
			<td>5-Fluorouracil</td>
			<td>Gift from Pharmacy of the Centre Leon Berard (CLB)</td>
			<td>-</td>
			<td>stock solution, 5 mg/100 mL; final concentration, 50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggrewell 400 24-well plates</td>
			<td>STEMCELL Technologies</td>
			<td>34411</td>
			<td>1,200 microwells per well for spheroid formation and growth</td>
		</tr>
		<tr>
			<td>Anti Caspase3 - Rabbit</td>
			<td>Cell Signaling</td>
			<td>9661</td>
			<td>dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti Musashi-1 (14H1) - Rat</td>
			<td>eBioscience/Thermo Fisher</td>
			<td>14-9896-82</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution x 100 mL</td>
			<td>STEMCELL Technologies</td>
			<td>07010</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD133 (13A4) - Rat</td>
			<td>Invitrogen</td>
			<td>14-133-82</td>
			<td>dilution 1:100</td>
		</tr>
		<tr>
			<td>Anti-CD44 -Rabbit</td>
			<td>Abcam</td>
			<td>ab157107</td>
			<td>dilution 1:2000</td>
		</tr>
		<tr>
			<td>Anti-PCNA - Mouse</td>
			<td>Dako</td>
			<td>M0879</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Anti-&#946;-catenin - Mouse</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7963</td>
			<td>dilution 1:50</td>
		</tr>
		<tr>
			<td>Black multiwell plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>237108</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid Monohydrate</td>
			<td>Sigma</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>CLARIOstar apparatus&#160;</td>
			<td>BMG Labtech</td>
			<td></td>
			<td>microplate reader</td>
		</tr>
		<tr>
			<td>Dako pen</td>
			<td></td>
			<td></td>
			<td>marker pen to mark circles on slides for creating barriers for liquids</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21202</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10037</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10042</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) Glutamax (L-alanyl-L-glutamine dipeptide)</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorogel mounting medium with DAPI</td>
			<td>Interchim</td>
			<td>FP-DT094B</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11077</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td></td>
			<td></td>
			<td>Spheroid image analysis</td>
		</tr>
		<tr>
			<td>Irinotecan&#160;</td>
			<td>Gift from Pharmacy CLB</td>
			<td>-</td>
			<td>stock solution, 20 mg/mL; final concentration, 100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>iScript reverse transcriptase&#160;</td>
			<td>Bio-Rad</td>
			<td>1708891</td>
			<td></td>
		</tr>
		<tr>
			<td>Leucovorin</td>
			<td>Gift from Pharmacy CLB</td>
			<td>-</td>
			<td>stock solution, 50 mg/mL; final concentration, 25 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>Nucleospin RNA XS Kit</td>
			<td>Macherey-Nagel</td>
			<td>740902 .250</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxaliplatin</td>
			<td>Gift from Pharmacy CLB</td>
			<td>-</td>
			<td>stock solution, 100 mg/20 mL;final concentration, 10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140130</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR qPCR Premix Ex Taq II (Tli RNaseH Plus)</td>
			<td>Takara</td>
			<td>RR420B</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX- Green</td>
			<td>Thermo Fisher Scientific</td>
			<td>S7020</td>
			<td>nucleic acid stain; dilution 1:5000</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05&#160;%)</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss-Axiovert microscope</td>
			<td></td>
			<td></td>
			<td>inverted microscope for acquiring images of spheroids</td>
		</tr>
	</tbody>
</table>

a three-dimensional model of spheroids to study colon cancer stem cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

As the lack of homogeneity in the size of spheroids is one of the main drawbacks of currently available 3D spheroid culture systems13, the aim of this work was to set up a reliable and reproducible protocol to obtain homogenous spheroids. First, to establish ideal working conditions, different numbers of Caco2 cells were tested, ranging from 50 to 2,000 cells per microwell/spheroid using dedicated plates (Table 1). In effect, each well in these pla...

Watch this Scientific Journal Video about A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells at JoVE.com

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

a-three-dimensional-model-spheroids-to-study-colon-cancer-stem

Research

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Cancer Research

5.8K Views.  Centre de Recherche en Cancérologie de Lyon, INSERM U1052, CNRS UMR5286, Université de Lyon, Université Lyon 1, Centre Léon Bérard. This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Video: A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

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 ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

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 ...

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

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.

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 ...