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><tbody><tr><td>37 &amp;micro;m Reversible Strainer, Large&amp;nbsp;</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 &amp;micro;g/mL</td></tr><tr><td>Agarose&amp;nbsp;</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-&amp;beta;-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&amp;nbsp;</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&#039;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&amp;nbsp;</td><td>Gift from Pharmacy CLB</td><td>-</td><td>stock solution, 20 mg/mL; final concentration, 100 &amp;micro;g/mL</td></tr><tr><td>iScript reverse transcriptase&amp;nbsp;</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 &amp;micro;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 &amp;micro;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&amp;nbsp;%)</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 ...

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Cancer Research

5.9K 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

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, ...

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 ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Medicine

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...