Supplemental Figures

Legends for Supplemental Figures and Tables

Supplemental Figure S1

A-B. Recurrence free survival in stage II (A, n = 260) or stage III (B, n = 201) patients from the GSE39582 dataset, analyzed using the R2 Genomics Analysis and Visualisation Platform (http://r2.amc.nl). High CLDN2-expressing group (red line), low CLDN2-expressing group (blue line). C. Graph summarizing the intensity and spread of claudin-2 immunostaining on a Tissue Microarray containing tumor sections from 24 CRC patients, expressed as median staining +/- 5th-95th percentiles. D. Statistical analysis of the degree of correlation between claudin-2 staining and post-treatment recurrence, using the Chi-Square test for trends on 23 of 24 patients (recurrence data was not available for one patient). E. Statistical analysis of the degree of correlation between claudin-2 sub-cellular localization and post-treatment recurrence, using the Chi-Square test for trends on 23 patients. Supplemental Figure S2

A. Representative phase contrast microphotographs illustrating differences in the number of colonospheres formed by SW480 cells made to express a human claudin-2 (CLDN2) or a control construct (CON), 7 days after seeding in ultra-low adherence plates (scale bars = 50 µm). B. Representative phase contrast microphotographs illustrating the difference in size between colonospheres formed by SW480 cells made to express a human claudin-2 (CLDN2) or a control construct (CON) and left to grow for 30 days after seeding in ultra-low adherence plates (scale bars = 100 µm). C. Representative phase contrast microphotographs illustrating the difference in size between colonospheres formed by DLD-1 cells expressing a claudin-2-selective (shCLDN2) or a control shRNA (shCON), taken 10 days after seeding (scale bar = 50 µm). D. Expression of claudin-1 mRNA was quantified using RT-qPCR in CRC cells expressing a control or claudi-2 selective siRNA (T84, DLD-1) or a control or claudin-2-encoding expression vector (CPP1), as indicated. Data is expressed as 1/DCp, n = 3. #, claudin-1 mRNA expression was not detectable in CPP1 cells.

Supplemental Figure S3

Validation of claudin-2 overexpression in the CRC patient-derived cell line CPP1 (A, B, C) and in the CMT93 mouse CRC cell line (D, E, F), transfected to overexpress claudin-2 (CLDN2) or with the corresponding control vector (CON). A, D: 100 µg of total protein were loaded and claudin-2 was detected by Western blot at 22 kD in the claudin-2 overexpressing cell line. Actin, detected at 45kD, was used as a loading control. B, E: CLDN2 mRNA overexpression was detected by qRT-PCR, transcripts were normalized against human GAPDH (CPP1) or mouse 18S RNA (CMT93) (Student t-test, ** p<0.001, mean ± SEM, n = 3). C, F: Tumour growth rates were measured weekly using a calliper after subcutaneous inoculation into NOD/SCID mice (n = 5 per group) (Student's t test, * p < 0.05; ***p<0.001, mean ± SEM).

Supplemental Figure S4

A. Incidence of liver metastasis in NOD/SCID mice 47 days after intra-caecal inoculation of Control or CLDN2-overexpressing CPP1 patient-derived CRC cells. B. Bioluminescent imaging (BLI) of mice injected with Control or CLDN2-overexpressing CPP1 cells. The top and middle panels provide representative examples of whole body imaging at day 16 and day 47 after injection, respectively. The bottom panels present the ex-vivo liver BLI of two mice from

each group. B Claudin-2 expression was quantified in Control (CT) and claudin-2-overexpressing CPP1 tumors. Data is expressed as 1/Cp, with the detected levels corresponding to a > three-fold increase in CLDN2+ tumors (**, p<0.01, t-test).

Supplemental Figure S5

A. The mRNA expression levels of CLDN2, assessed by qRT-PCR, in the ALDHLow or ALDHHigh T84 populations, 12h after transfection with siRNA down-regulating CLDN2 (siCLDN2) or control siRNA (siCON). Data are normalised on the corresponding T84 non transfected controls (Mean, representative result from 1 of 2 similar experiments). B. The mRNA expression of CLDN2 in T84 cells was quantified using qRT-PCR one day (D1), three days (D3) and six days (D6) following the transient transfection with siRNA against CLDN2. Data are normalised on the un-transfected control (D0) (Student t-test, * p<0.01, mean ± SEM, n = 3).

Supplemental Figure S6

A. Purified DLD-1 ALDHHigh cells (red, top) or ALDHLow cells (blue, bottom) were independently transfected with a β-galactosidase-specific control siRNA (siCON) or with a claudin-2 specific siRNA (siCLDN2), and the percentages of ALDHHigh and ALDHLow cells were re-analyzed in each population 3 days and 6 days after transfection (Data represents one of two similar experiments). B. The mRNA expression of CLDN2 in DLD-1 cells was quantified by qRT-PCR one (D1), three (D3) and 6 days (D6) following the transient transfection with siRNA against CLDN2. Data are normalised against cells transfected with control siRNA (D0) (mean ± SEM, experiments conducted twice with similar results).

Supplemental Figure S7

CLDN2 mRNA expression levels were quantified using qRT-PCR in ALDHHigh cell populations of the CPP42 patient-derived CRC cells, 12h after transfection with siRNA down-regulating CLDN2 (siCLDN2) or control siRNA (siCON), as well as in CPP1 patient-derived CRC cells stably expressing a human claudin-2 (CLDN2+) or a control (CON) construct. Data are normalised on the corresponding non transfected controls.

Supplemental Table S1Sequences of Bgal or CLDN2-specific siRNA and shRNA, of qPCR primers, and details of miRNA mimic and inhibitors used in this study.

Supplemental Table S2

Clinical data of patients from the Tissue Microarray used for claudin-2 immunostaining (see figure 1E and Supplemental Figure S1)Supplemental Table S3

Top 10 most enriched KEGG pathways analysed on differentially expressed miRNAs, with their enrichment score and p-value.

Supplemental Table S4

Combined list of genes involved in self-renewal processes used to analyze potential enrichments for miRNA targets (Column I), identified using Gene Ontology keywords (‘Self-renewal’, columns A-B; ‘Stem cell’, columns C-D) or extracted from previous publications (Hadjimichael, World J Stem Cells 2015, column E; Merlos-Suarez, Cell Stem Cell 2011, column F; Palmer, Genome Biology 2012, column G)Supplemental Table S5

Tab 1: miRNA target enrichment scores (first tab), identifying whether each tested microRNA has statistically more potential targets within the self-renewal gene list (Suppl. Table S4) than could be expected at random. The identification of potential targets was performed using DianaMicroT, as described under supplemental methods.

Tab 2: Identified self-renewal target genes for the three selected miRNA candidates, along with their prediction score. Only those genes with a prediction score above 0.85 were included.

Supplemental Methods

Constructs and reagentsThe mouse CLDN2 cDNA was stably inserted into CMT93 cells using the TrueORF CLDN2 cDNA shuttle system (ORIGENE, #MR202768) and transfected using the Multiplier K transfection kit (Biontex K2). Inducible CLDN2 shRNA was subcloned into the pTRIPz vector (Open Biosystems) and transduced using the Trans-Lentiviral shRNA Packaging Kit (Thermo-Scientific). The CPP1 cells stably overexpressing human claudin-2 were transfected using Lipofectamine®3000 (Invitrogen) with TrueORF cDNA Vector System (RC229728, OriGene). Constitutive human and mouse claudin-2-specific and control shRNA constructs were obtained from OriGene (USA). CLDN2-selective duplexes or -galactosidase-selective siRNA (Millenium Sciences) were transiently transfected using Lipofectamine RNAiMax (Life Technologies) according to the manufacturer’s recommendations. siRNA and shRNA sequences are provided in Supplemental Table S1.

Cell lines and patient-derived tumor cells cultureCRC cell lines (T84, DLD1, SW480) were obtained from ATCC and maintained in DMEM (Gibco) with 10% FBS, or as spheroids in defined M12 media in ultra-low attachment flasks (Corning). M12 is advanced DMEM/F12 (1:1) medium (Gibco), supplemented with N2 supplement, 20 µg/mL Insulin, 3 mM Glutamine, 0.3 % Glucose, 100 U/ml Penicillin G, 100 µg/ml Streptomycin (Sigma-Aldrich), 10 ng/ml human FGF2 (R&D Systems), and 20 ng/ml human EGF (R&D Systems). Patient-derived colon cancer cells were derived as recently described (Ref 18 in the main manuscript) from CRC biopsies obtained from Centre Hospitalier Universitaire (CHU) Carémeau (Nimes, France). The protocol was approved by the CHU Institutional Ethics Committee (Human ethics agreement #2011-A01141-40, NCT01577511), and signed informed consents were obtained from patients prior to samples acquisition. CPP1 and CMT93 cells stably overexpressing Claudin-2 were maintained in normal growth medium containing 2 mg/mL or 0.8 mg/mL G418, respectively (Life Technologies).

Association between CLDN2 expression and CRC patient survival

Prospective quantification of CLDN2 mRNA expression and association analyses with survival was performed on data from a cohort of patients diagnosed with stage II or III colon cancer who underwent primary tumor surgical resection and received 5-FluoroUracil (5FU)-based adjuvant chemotherapy between 1998 and 2005. The study was approved by the Ethics Committee of the Hospital Clínic of Barcelona (1). Quantification of CLDN2 mRNAs from paraffin-embedded sections using RT-qPCR, normalization to RPL0 and 18S levels, and correlation with patient survival was performed as described (2).

The association between CLDN2 mRNA expression and disease outcome in patients with stage II/III CRC treated with 5-FU-based chemotherapy was also analyzed in two published datasets, selected based on the presence of CLDN2-specific probes, the number of patients (>150), and the availability of recurrence-free survival data. One of these dataset (GSE24551- GPL11028 (3), 160 patients) was sourced and analyzed using the SurvExpress bioresource, (http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp), with optimized risk scores determined as described in (4). The data summarizes CLDN2 expression levels (median, 5-95 percentiles) and Kaplan-Meier analysis (Hazard ratio) of recurrence-free survival, distributed in high and low risk patient subgroups. Data from the GSE39582 dataset (5) (461 patients) was analyzed using the R2: Genomics Analysis and Visualization Platform

(http://r2.amc.nl), using average expression as a selection cut-off between high and low expression groups.

In vivo mouse experimentsTo analyze the impact of claudin-2 on primary tumor initiation in vivo, CPP1 cells (50, 500 or 5000 cells) or CMT93 cells (10, 100 or 1000 cells) were embedded in Matrigel (BD, 356230) and inoculated subcutaneously into the right flank of 6-week-old NOD/SCID female mice. The frequency of tumor development and kinetics of tumor growth were monitored using twice weekly palpation and caliper measurements. Tumor volume was calculated using the following formula: (L × W2)/2, where L represents the length and W represents the width of the tumor diameter. Tumors were collected and analyzed when xenograft size reached 500 mm3. For serial-transplantation experiments, tumors were resected, pooled and tumor cells were enzymatically dissociated using the gentleMACS dissociator (#130-093-235, Miltenyi Biotec) as per manufacturer’s instructions. Viable cells were sorted by FACS and serially transplanted by sub-cutaneous injection into the flank of new NOD/SCID mice. Tumors were harvested at week 10, or upon reaching a volume of 500 mm3. To assess the role of claudin-2 on metastasis initiation, control or claudin-2 overexpressing luciferase-expressing CPP1 cells were injected into the caecum of NOD/SCID mice, and the primary tumor growth and development of metastases in the liver was monitored for 6 weeks using Bio-Luminescence Imaging (BLI). Briefly, mice were anaesthetized using 3% isoflurane and their abdomen was shaven. An incision was made through the skin and peritoneum. The caecum was externalized and 5 μL Matrigel containing 1×105 cells was injected to each mouse under a 10x surgical microscope into the sub-serosa of the caecum. The caecum was repositioned in the abdominal cavity and peritoneum and skin were sutured separately. Post-operative carprofen was administered subcutaneously and mice were monitored twice weekly. To monitor primary tumor growth and liver metastasis development, 5 min after receiving a 100 μL tail vein injection of 150 mg kg−1 D-Luciferin (CHOICE Analytical), mice were imaged for 1s using an IVIS Lumina II (Perkin Elmer) imaging system. 47 days after injection, animals were euthanized and their liver was imaged ex-vivo and snap frozen. To characterize the impact of claudin-2 on post-chemotherapy tumor relapse, NOD/SCID mice were injected subcutaneously with T84 cells expressing a control (n=18) or a claudin-2-specific (n=18) shRNA (1 x 106 cells/mouse). When tumors reached 250 +/- 25 mm3, mice were treated with FOLFIRI (40mg/kg 5-fluorouracil + 15mg/kg Irinotecan + 30mg/kg Leucovorin), given intraperitoneally twice a week for 3 weeks. 4 days after the end of treatment residual tumors were collected, tumor cells were dissociated as described for human tumor samples (cf under ‘Patient-derived organoids’). Isolated cells were incubated with DAPI and live tumor cells (DAPI-negative) were gently mixed and aliquoted towards CLDN2 RNA quantification or subcutaneous reimplantation into second generation animals at 50 (n=6 mice), 500 (n=6), or 5000 (n=6) cells per mouse. Second generation mice were examined regularly for signs of discomfort and for the presence of subcutaneous tumors until 8 weeks after reimplantation. All procedures were carried out under animal ethics agreements from the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee, Melbourne Australia (#MIPS_AEC2012.04 and #MIPS_AEC2013.01).Patient-derived organoidsFollowing overnight incubation with PBS with PenStrep/ Nystatin/ Gentamycin (250 IU/ml; 0.01mg/ml; 50ul/ml respectively), cell dissociation from primary colorectal tumor samples (stage IV, collected under human ethic agreement HREC/15/PMCC/112, Peter MacCallum Cancer Centre Human Research Ethics Committee) was performed using the Human Tumour

Dissociation Kit (#130-095-929, MACS Miltenyi Biotec) with the gentleMACS Dissociator (#130-093-235, MACS Miltenyi Biotec) as per manufacturers’ protocol, followed by washing in Red Blood Cells lysis buffer (10 mM KHCO3, 150 mM NH4Cl, 0.1 mM EDTA Na2). Tumour cells were then embedded in Matrigel (BD Matrigel Growth Factor Reduced, In Vitro Technologies, #FAL356230), and grown in tumour organoid medium, prepared as follows: Advanced DMEM/F12 (D6421, SIGMA) complemented with antibiotics (as above), Glutamax (#35050-061, Life Technologies), A83-01(500 nM, SIGMA), B27 (undiluted, #17504044, Gibco), EGF (50 ng/ml, #130-097-750, MACS Militenyi), Gastrin (1 µg/ml, G9145, SIGMA), SB202190 (10 µM, S7067, SIGMA), SB431542 (4 mg/mL, RDS16141, SIGMA), YP-27632 (10 µM, SIGMA). Tumour-derived organoids were grown under 2% O2 at 37°C and medium was renewed twice a week. Wells were photographed daily from seeding. Organoid initiation was quantified using the ImageJ. software (organoids with a diameter ≥ 30 µm were considered as positive).

CLDN2 Immunohistochemistry on human CRC tissue samplesFormalin–fixed and paraffin-embedded tumor sections from 24 CRC patient samples were provided by the Victorian Cancer Biobank (University of Melbourne Human Ethics Sub-Committee for Medicine and Dentistry, agreement # 1648273.2) as a Tissue Macro Array after quality control by Biobank histo-pathologists. Samples were collected prior to medical treatment from patients with Stage II and III tumors who later received adjuvant 5-FU-based chemotherapy. To account for intra-tumor heterogeneity, core biopsies were performed from two different areas of each tumor on the TMA slides. De-identified recurrence data was available for a period of >5 years for all except two patients, with one other dating back 2 years and the other unavailable.For claudin-2 staining, antigen retrieval was performed in citrate buffer pH 6, endogenous peroxidase was quenched, and blocking was performed overnight in Tris Buffered Saline (TBS) containing 0.5% BSA, 0.05% Tween 20 and 0.5% Triton. The primary anti-claudin-2 antibody (1/100, #51-6100, Invitrogen) or isotype control were incubated overnight at 4C. Biotinylated secondary antibody (DAKO E0432) was added before using the Tyramide Signal Amplification (PerkinElmer, B40951) and developed using 3-3′-diaminobenzidine tetrahydrochloride (DAB, Vector). Slides were counterstained with haematoxylin, and coverslipped with DPX. TMA slides were scanned using a high throughput scanner (PMCC imaging facility, Melbourne) and images were then visualized using Image Viewer. Primary scoring for claudin-2 immuno-positivity was performed according to the intensity and area of tumor tissue containing positive staining across 2 independent cores for each patient sample.

% tumor cellsIntensity 1

(<33%)2 (33-66%)

3 (>66%)

0 (Negative)

0 0 0

1 (Weak) 1 2 32 (Moderate)

2 4 6

3 (Strong) 3 6 9For statistical analysis, tumors were then divided into three categories with either Negative/Weak (corresponding to scores 0-1), Moderate (scores 2-4) or Strong (above 4) claudin-2 immunostaining. A Chi-square test for trend was run to assess the degree of correlation between claudin-2 staining and recurrence after chemotherapy.The cellular localization of claudin-2 staining was also recorded and samples were separated into three categories depending on whether claudin-2 was detected predominantly at the cell

membrane, in the cytoplasm or the nucleus, or at various locations within most cells. A Chi-square test for trend was run to assess the degree of correlation between claudin-2 localization and recurrence after chemotherapy.

RNA extraction and real-time PCRTotal RNA was extracted using the RNeasy mini kit (Qiagen) and treated with DNAse-1. The first strand cDNA was synthetized using Superscript II (Invitrogen) and random hexamers, and gene expression was measured by real-time PCR as previously reported (Refs 14 and 18 in the main manuscript). Primer sequences are provided in Supplemental Table S1.For the RT-qPCR validation of the miRNAs, 25 ng of RNA was converted to cDNA (TaqMan MicroRNA Reverse Transcription Kit, Applied Biosystems) according to the manufacturers’ protocol with a primer pool containing 6 miRNA assays (TaqMan microRNA assays, 5x, Applied Biosystems). cDNA samples were pre-amplified (TaqMan PreAmp Master Mix Kit, Applied Biosystems) and RT-qPCR (TaqMan Fast Advanced Master Mix, Applied Biosystems) was performed using individual miRNA assays (TaqMan microRNA assays, 20x, Applied Biosystems) and run on the ViiA™ 7 Real-Time PCR System. Reverse transcription and pre-amplification no template controls using primer pools and individual assays were also prepared to ensure there was no background amplification of miRNA assays. Normalization of Ct values of each gene and determination of fold differences in gene expression was calculated by the 2-∆∆Ct method.

miRNA quantificationRNA including miRNAs were isolated using the miRNeasy mini kit (QIAGEN) and RNA quality was analyzed on the Agilent 2100 Bioanalyzer™ using RNA6000 and small RNA assays (Agilent Technologies, Australia). Small RNA libraries were constructed using 50 ng of RNA using the Ion Total RNA-Seq Kit V2 (Life Technologies, Australia) and ligated to adapters containing a unique index barcode (Ion Xpress™ RNA-Seq Barcode 1-16 Kit, Life Technologies, Australia) according to the manufacturers’ protocol. The yield and size distribution of the small RNA libraries were assessed using the Agilent 2100 Bioanalyzer™ instrument with the high sensitivity DNA chip (Agilent Technologies). Equally pooled libraries were prepared for deep sequencing using the Ion OneTouch™ system (Life Technologies) and sequenced on the Ion Torrent PGM™ using Ion™ 318 V2 chips (Life Technologies) and the Ion PGM™ 200 V2 Sequencing Kit (Life Technologies). Pre-processing of reads, removal of adapters and barcodes were performed using the Torrent Suite (v.3.4.1). Sequences were analyzed for quality control (FASTQC), aligned to the Human genome (HG19) using the Torrent Suite and files transferred to Partek Genomic Suite and Flow (Partek Incorporated, Singapore) for mapping against miRBase V.21 and Ensembl Release 75 to identify miRNA, non-coding and coding RNA species. Reads were normalized to reads per million reads (RPM). miRNAs identified with at least 10 reads were used for further analysis on Partek Genomic suite which included statistical analysis and hierarchical clustering. Targeted quantification of miR-222-3p was performed using RT-qPCT using the Taqman microRNA assay #002276 (Applied Biosystems), with quantification of U6 RNA (Assay ID: 001973) used to standardize results.

Selection of candidate miRNAs for experimental validationUsing DianaMicroT (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index), miRNA-target interactions with a score >=0.85 were selected. From this set of relationships, we used a Chi Squared test to determine which miRNAs were significantly enriched in a manually curated set of 770 genes (Supplemental Table S4)

identified as playing a role in the regulation of stem cells and self-renewal was selected from previous publications (6-8) and from the Gene Ontology Website (‘Stem Cells’ and ‘Self-renewal’ searches, http://geneontology.org/). 1721 miRs were found to target at least one of the 777 genes contained within the list, with all 9 CLDN2-related miRNAs positioned in the top 10% of those. We then performed the reverse test, taking candidate miRNA from our experimental results and testing to determine which were significantly associated with self-renewal (Supplemental Table S5). From the list of 9 CLDN2-regulated miRNA identified in this study, we selected 3 candidate miRNAs for further exploration based on the number of their putative gene targets within the self-renewal list (>10) and on the level of statistical significance of their target enrichment (Chi-Squared test, p value at or below 0.05). Two of these miRNAs (miR-371b-3p and miR-222-3p) were among those found to be upregulated in ALDHhigh cells upon claudin-2 down-regulation, while miR-589 is down-regulated upon exposure of ALDHhigh CRC cells to CLDN2-specific siRNA.

miRNA rescue experimentsTo determine whether selected miRNAs played a causal role in the regulation of self-renewal by claudin-2, we performed siRNA-mediated downregulation of claudin-2 expression in ALDHhigh DLD-1 CRC cells in the presence or not of a miRNA-589-3p mimic or of miRNA-371b-3p or miR-222-3p inhibitors, in comparison with their respective controls (see Supplemental Table S1 for assay numbers). CLDN2 siRNA transfection was performed simultaneously with miRNA mimic or inhibitor treatment. 24h after transfection cells were lysed for RNA extraction or seeded into an ultra-low adherence 96-well plate at 3, 10, 30, 100 or 1000 cells/well to perform an Extreme Limiting Dilution Assay (ELDA) as described elsewhere.

Western blotCells were lysed with RIPA buffer in the presence of protease inhibitors (Roche). For Western blotting, samples were subjected to 10% SDS-PAGE, transferred to PVDF membrane (GE Healthcare) and scanned with a ChemiDoc XRS+ (BIORAD). The following antibodies were used: beta ACTIN (13E5, Cell Signaling), CLAUDIN2 (#51-6100, Invitrogen). Bands intensities were measured using the Image Lab software (BIORAD, J, NIH).

Aldefluor assay-based fluorescence-activated cell sorting (FACS)The Aldefluor assay (Stem Cell Technologies) was performed according to the manufacturer’s instructions (Stem Cell Technologies) as described previously (Ref 14 and 18 in the main manuscript). ALDHHigh cells (ALDH+) were identified in CRC cell lines and xenografts by comparing the same sample with and without the ALDH inhibitor diethylaminobenzaldehyde (DEAB). FACS gating of ALDH activity was set at 0.1% in presence of DEAB. Cells were analyzed and sorted using FACSAria and Summit 6.0 or Cyflogic softwares. Dead cells were excluded based on light scatter characteristics.Immunostaining of mouse tumorTumors were harvested and fixed for 2 hours with PFA 4% and were dehydrated with serial ethanol baths followed by xylene dehydration series before being embedded in paraffin. 6µm sections were cut using a microtome (Microm, HM335E). Embedded tumor slides were dewaxed by heating at 56°C and immersing in serial xylene and graded ethanol baths. Antigen retrieval was performed in citrate buffer pH 6. Blocking was performed overnight in Tris Buffered Saline (TBS) containing 0.5% BSA, 0.05% Tween20 and 0.5% Triton. The primary antibody CLAUDIN2 (1/100, #51-6100, Invitrogen) was incubated overnight. Secondary antibodies used for immunohistochemistry were developed using 3-3′-

diaminobenzidine tetrahydrochloride (DAB, Vector). Slides were counterstained with haematoxylin, and coverslipped with DPX. The slides were visualized under a Leica DM-IRB.

Sphere formation assaysThe sphere-forming percentage was determined after plating 100 cells/well in M12 medium in ultra-low attachment 96-well plates (Corning). Spheroids with a diameter exceeding 50 µM were counted. The frequency of cancer cells with in vitro self-renewal ability was determined using the Extreme Limiting Dilution Analysis (ELDA). Spheres were dissociated into single cell suspension, and were seeded at 1, 10, 100 or 1,000, in 100 μL per well in M12 media onto ultra-low attachment 96-well plate (#3474, Corning) and maintained for at least 7 days. Spheres were scored as previously described (Ref 14 and 18 in the main manuscript). For YAP pathway characterization experiments, treatment with Verteporfin (0.5 µg/ml) or Simvastatin (4 µM) was performed once at the time of cell seeding into ELDA assays, and sphere quantification was performed 7-10 days later.

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