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sc-12-0380_sm_SupplData.pdf86KSupplementary Data
sc-12-0380_sm_SupplFigure1.tif1278KSupplementary Figure 1. Expression of LGR5 in normal human intestine and human colon cancer cell lines. (A): Expression of LGR5 in the crypt base cells in normal human intestine. The same section of the normal human intestine was stained with anti-LGR5 antibody (2U2E-2, 1 μg/mL) (left) or HE (right). Specific fluorescence was observed in the crypt base columnar cells (arrows). (B): Cell surface expression of LGR5 in the two colon cancer stem cell lines established in this study and six colon cancer cell lines. The cells were stained with anti-LGR5 antibody and analyzed by flow cytometry (left). Shadow, Fluorescent intensities after staining with the anti-LGR5 antibody (2L36, 2 μg/mL); Open, Fluorescent intensities after staining with control isotype antibody. The level of LGR5 mRNA was determined by RT-qPCR (right). There is a good correlation between the level of the cell surface LGR5 protein and the LGR5 mRNA.
sc-12-0380_sm_SupplFigure2.tif1641KSupplementary Figure 2. Expression of LGR5 and CK20 in the primary cells, spheroids, and adherent cells. (A): Level of the LGR5 mRNA was high in the LGR5+ cells under an adherent culture condition but was decreased under spheroid cultures and after treatment with irinotecan (drug resistant LGR5- cells). On the contrary, the level of CK20 mRNA was below the detectable level in the LGR5+ and drug resistant LGR5- cells, but was increased in the spheroid cultures of the LGR5+ cells. (B): Expressions of the LGR5, CK20, HLA-DMA and EREG proteins were analyzed by immunocytochemistry. The LGR5+ cells under spheroid cultures were fixed and treated with the specific antibodies against LGR5 (2U2E-2, 1 μg/mL), CK20 (2.5 μg/mL), HLADMA (2.5 μg/mL) and EREG (10 μg/mL). Spheroids contained LGR5+ cells, HLA-DMA+ cells, and EREG+ cells that are considered to possess tumor initiation activity. CK20+ cells that were negative for LGR5 were also present in spheroids.
sc-12-0380_sm_SupplFigure3.tif1348KSupplementary Figure 3. Expression of stem cell markers. The primary cells from the xenografts and LGR5+ cells cultured under an adherent culture condition were analyzed by flow cytometry. Scatter plots of the results corresponding to the histograms in Figure 2D are shown. The x-axis and the y-axis indicate the intensity of the fluorescence and the Side Scatter, respectively.
sc-12-0380_sm_SupplFigure4.tif1322KSupplementary Figure 4. Colony formation by the sorted LGR5+ and LGR5- cells. The sorted cells of the LGR5-/CD133-, LGR5-/low/CD133+ and LGR5+/CD133+ populations were cultured on the matrigel for 6 days. (A): Colonies formed by the sorted cells of the LGR5-/CD133-, LGR5- /low/CD133+ and LGR5+/CD133+ populations. Bar: 500 μm. (B): Phase contrast microscopy of the colonies formed by the sorted cells of the LGR5-/low/CD133+ and LGR5+/CD133+ populations. Bar: 100 μm.
sc-12-0380_sm_SupplFigure5.tif1091KSupplementary Figure 5. Effect of irinotecan on survival and growth of LGR5+ cells. (A): Numbers of viable cells before and after treatment of irinotecan. After treatment of the adherent LGR5+ cells with 10 μg/mL irinotecan for 3 days, number of viable cells was counted. The results are the mean of three independent experiments. Bars on each column indicate standard deviations. (B and C): Growth of LGR5+ cells and drug resistant LGR5- cells. 3x105 LGR5+ cells (B) or drug resistant LGR5- cells (C) were cultured for 3 days under an adherent condition, and number of viable cells was counted. Gray column: number of viable cells when seeded, Black column: number of viable cells after culturing the cells for 3 days.
sc-12-0380_sm_SupplFigure6.tif1030KSupplementary Figure 6. Expression of stem cell markers. The LGR5+ cells from the adherent cultures from PLR123 xenografts before (top) and after treatment with irinotecan (middle) were analyzed by flow cytometry. Cells re-seeded after irinotecan treatment (bottom) were also analyzed. Scatter plots of the results corresponding to the histograms in Figure 4B are shown. The x-axis and the y-axis indicate the intensity of the fluorescence and the Side Scatter, respectively.
sc-12-0380_sm_SupplFigure7.tif2046KSupplementary Figure 7. Gene expression profiles and expression of stem cell markers. (A): Preparation of the cells used for DNA micrroaray and RT-qPCR is illustrated. DNA microarray and RT-qPCR were performed with the RNA extracted from the primary cells from the xenografts, adherent LGR5+ cells and drug resistant LGR5- cells that were prepared by treating the LGR5+ cells with irinotecan for 3 days. (B): Heat map of the well known differentiation markers, keratin 20 (KRT20), villin 1 (VIL1) and mucin 2 (MUC2). (C): Heat map of the genes that were upregulated in the LGR5+ cells. (D): Expression of the cancer stem cell markers such as LGR5, CD133, CD44, CD166 and EPCAM were examined by RT-qPCR. Note that the expression of the LGR5 mRNA was drastically decreased after irinotecan treatment, but that of CD133, CD44, CD166 and EPCAM mRNAs did not decline or even increased after the cells were treated with irinotecan.
sc-12-0380_sm_SupplFigure8.tif1314KSupplementary Figure 8. Interconversion of the LGR5+ and LGR5- states in vitro. LGR5+ cells were cultured under an adherent culture condition in the presence of 5-FU or oxaliplatin for 24 hr. The cells were further cultured without drugs for 48 hr, and expression of LGR5 was examined by immunocytochemistry. Almost all the LGR5+ cells transitioned to a LGR5- state after treatment with 5-FU or oxaliplatin. The drug resistant LGR5- cells treated with 5-FU or oxaliplatin were sorted using a MoFlo XDP cell sorter and cultured under an adherent culture condition in the absence of the drugs for 4 days, and again expression of LGR5 was examined. When the sorted drug resistant LGR5- cells were cultured without drugs, a few LGR5+ cells appeared within 4 days. To examine the expression of LGR5, the cells were stained with the anti- LGR5 antibody (2L36, 2 μg/mL). Bar: 50 μm.
sc-12-0380_sm_SupplFigure9.tif1897KSupplementary Figure 9. Pathway analysis between LGR5+ and drug resistant LGR5- cells. Genes that were differentially expressed between LGR5+ and drug resistant LGR5- cells cultured under an adherent condition were selected by t-test and used for Ingenuity Pathways Analysis. Ten pathways that were most different between the LGR5+ and drug resistant LGR5- cells are shown. (A): Pathways that were upregulated in drug resistant LGR5- cells as compared to LGR5+ cells. (B): Pathways that were downregulated in drug resistant LGR5- cells as compared to LGR5+ cells. Bars indicate significance of the differences (p-value) of each pathway between the two groups. P-values are indicated in logarithmic scale. Lines indicate ratio of the genes that were significantly different between the two groups among total number of genes in each pathway.
sc-12-0380_sm_SupplFigure10.tif1447KSupplementary Figure 10. Antigen specific binding of anti-EREG monoclonal antibody. (A and B): Antigen specificity of the anti-human EREG monoclonal antibody. Binding of anti-EREG monoclonal antibody to the antigen was confirmed by immunocytochemistry (A) and flow cytometry (B) using CHO DG44 cells transfected with the EREG cDNA. Anti-EREG antibody reacted with the cells expressing EREG but not with the parental cells. (C): Presence of EREGpositive cells in colon cancer tissues from patients. Thin sections of colon cancer tissues from patients were incubated with antibody against HLA-DMA, which is expressed in LGR5- colon cancer cells, and anti-EREG antibody. Cells positive for HLA-DMA were observed within the epithelial tumors and interstitial areas, whereas EREG-positive cells were detected only within the epithelial tumors. HLA-DMA-positive interstitial cells represent macrophages. Arrow heads and arrows indicate slow cycling CSCs and macrophages, respectively.
sc-12-0380_sm_SupplFigure11.tif822KSupplementary Figure 11. Changes in the numbers of LGR5+ and LGR5- cells within tumors after irinotecan treatment in vivo. The adherent LGR5+ cells of PLR123 were subcutaneously injected into NOG mice and the mice were administered irinotecan (120 mg/kg/day) 12, 15 and 18 days after the inoculation of tumor cells. Thin sections of the xenotransplanted tumor tissues were treated with anti-LGR5 (2U2E-2, 1 μg/mL) and anti-HLA-DMA (2.5 μg/mL) antibodies, and then the LGR5+ and HLA-DMA+ cells were counted. Values represent the sum of counted cells in each group (n=3).
sc-12-0380_sm_SupplFigure12.tif1991KSupplementary Figure 12. Effect of anti-EREG antibody on the growth of tumors from LGR5+ cells. (A): Expression of EREG in LGR5+ and drug resistant LGR5- cells. LGR5+ and drug resistant LGR5- cells cultured under an adherent condition were incubated with anti-EREG antibody and analyzed by flow cytometry. The anti-EREG antibody bound both LGR5+ and drug resistant LGR5- cells, demonstrating the cell surface expression of EREG. (B): ADCC activity of anti-EREG antibody (mouse-human chimeric antibody). LGR5+ and drug resistant LGR5- cells of PLR123 were incubated with anti-EREG antibody and human PBMC at the ET ratio of 40. Note that the anti-EREG antibody elicited ADCC activity against both LGR5+ and drug resistant LGR5- cells. (C): Effect of anti-EREG antibody on cell growth. The LGR5+ and drug resistant LGR5- cells of PLR123 were incubated with the anti-EREG antibody and cultured for 3 days. Percentages of the surviving cells as compared to those of untreated control cells are shown. Note that the anti-EREG antibody per se did not affect on the growth of the LGR5+ and the drug resistant LGR5- cells. (D): Effect of anti-EREG antibody (mouse-human chimeric antibody) against tumors after irinotecan treatment. The LGR5+ cell of PLR123 were subcutaneously injected into SCID mice and the mice were administered irinotecan (100 mg/kg/day) 6, 10 and 13 days after the inoculation of tumor cells. Then, the mice received intravenous injections of the anti-EREG antibody (10 mg/kg/day) 17 and 24 days after the inoculation of tumor cells. Each value represents mean ± SD (n=4).
sc-12-0380_sm_SupplFigure13.tif874KSupplementary Figure 13. Expression of BMI1, LRIG1, TERT and HOPX in the LGR5+ cells and drug resistant LGR5- cells. Drug resistant LGR5- cells were prepared by treating the adherent LGR5+ cells with irinotecan for 3 days. Expression of the BMI1, LRIG1, TERT and HOPX mRNA was determined by RT-qPCR. Note that expression of the TERT mRNA was drastically decreased after irinotecan treatment, but that of BMI1 and LRIG1 did not decline or even increased after the cells were treated with irinotecan. The HOPX mRNA was below a detectable level both in the LGR5+ and drug resistant LGR5- cells.
sc-12-0380_sm_SupplTable1.pdf55KSupplementary Table 1. Tumor initiating activity of the cells from xenografts of PLR123
sc-12-0380_sm_SupplTable2.pdf50KSupplementary Table 2. Tumor initiating activity of the cells from primary, spheroid, and adherent cultures
sc-12-0380_sm_SupplTable3.pdf56KSupplementary Table 3. Tumor initiating activity of LGR5+ cells after many passages in vitro
sc-12-0380_sm_SupplTable4.pdf49KSupplementary Table 4. Quantification of LGR5+ and drug resistant LGR5- cells with or without irinotecan treatment
sc-12-0380_sm_SupplTable5.pdf45KSupplementary Table 5. Tumor initiating activity of drug resistant LGR5- cells
sc-12-0380_sm_SupplTable6.pdf61KSupplementary Table 6. Quantification of LGR5+ and drug resistant LGR5- cells in the single cell assay
sc-12-0380_sm_SupplTable7.pdf58KSupplementary Table 7. Selection of antibodies for immunohistochemical staining of the gene products upregualted in the LGR5- cells
sc-12-0380_sm_SupplTable8.pdf63KSupplementary Table 8. Presence and localization of LGR5+ and LGR5- cells in colon cancer tissues from patients
sc-12-0380_sm_SupplTable9.pdf12KSupplementary Table 9. Primers used for quantitative real time PCR analysis

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