Volume 149, Issue 3, Pages 692-704 (September 2015) Maintenance of Clonogenic KIT+ Human Colon Tumor Cells Requires Secretion of Stem Cell Factor by Differentiated Tumor Cells  Szabolcs Fatrai, Susanne J. van Schelven, Inge Ubink, Klaas M. Govaert, Danielle Raats, Jan Koster, Andre Verheem, Inne H.M. Borel Rinkes, Onno Kranenburg  Gastroenterology  Volume 149, Issue 3, Pages 692-704 (September 2015) DOI: 10.1053/j.gastro.2015.05.003 Copyright © 2015 AGA Institute Terms and Conditions

Figure 1 Differentiated tumor cells increase the clonogenic capacity of colon cancer cells via a secreted factor. (A) Single cells were generated from the indicated colonosphere cultures and from DTC1. Equal numbers of colonosphere cells and DTCs were plated in Matrigel for colony-forming assay. After 2 weeks, colony formation was analyzed. The graph shows the percentage of colonies arising from 1000 plated cells. (B) The experiment was performed as in (A), but multiple DTC cultures were combined with a single colonosphere line (L145). DTC145, DTC169, DTC146, and DTC167 were established from the same tumors as the colonosphere lines with the corresponding numbers (L145, L169, L167). (C) L145 colonospheres were cultured for 3 weeks under differentiation-inducing culture conditions in the presence or absence of DTC1 cells. After 3 weeks of differentiation, both cell cultures were harvested, and 1000 single GFP+ cells were plated out to assess colony-forming potential in stem cell medium. (D) Conditioned medium was harvested from DTC1 cells or L145 colonosphere cultures and was used for colony-forming assays. (E) The assay was performed as in (D), but DTC-conditioned media were combined with the corresponding colonospheres. Each bar graph shows a representative dataset from 3 independent experiments. All experiments were performed in triplicate. Results are shown as mean ± standard error of the mean. Statistical significance was determined using the Student t test (unpaired 2-tailed). ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. Gastroenterology 2015 149, 692-704DOI: (10.1053/j.gastro.2015.05.003) Copyright © 2015 AGA Institute Terms and Conditions

Figure 2 Paracrine SCF/KIT signaling between differentiated and clonogenic tumor cells. (A) DTCs were cultured to 80% confluency. The medium was then changed to serum-free stem cell medium. Three days later conditioned medium was harvested, filtered, and used for antibody array. Stem cell medium alone was used as a control. (B) Colonospheres were harvested and used for Western blot analysis to determine KIT expression. (C) Colonospheres (n = 6) and DTCs (n = 6) were plated in stem cell medium, and the media were harvested 3 days later and were used to measure SCF secretion by enzyme-linked immunosorbent assay. The bar graph shows the means of 3 independent measurements in all 12 cultures. The mean level of SCF produced by the DTC group was significantly higher than that produced by the colonosphere group. Immunofluorescence analysis confirms that, within L145 colonospheres, SCF is predominantly expressed by differentiated CK20-positive cells. (D) Cells form colonospheres were sorted based on aldehyde dehydrogenase activity using the Aldefluor assay. Total RNA was extracted from the sorted cell populations to measure expression of KIT and SCF by RT-qPCR. The experiment was performed in triplicate. ∗∗∗P < .001; Student t test. Gastroenterology 2015 149, 692-704DOI: (10.1053/j.gastro.2015.05.003) Copyright © 2015 AGA Institute Terms and Conditions

Figure 3 Colony- and tumor-initiating capacity depends on SCF/KIT signaling. (A) Single cells were prepared from multiple colonosphere cultures and were plated in Matrigel with or without SCF (10 ng/mL). Colony formation was determined 2 weeks later. The bar graph shows the percentage of colonies arising from 1000 plated cells. (B) Single cells were prepared from L145 and CRC29 colonosphere cultures and were plated in Matrigel in DTC-conditioned medium with or without an SCF-neutralizing antibody (50 ng/mL). The experiment was performed as in (A). (C) L145 colonospheres were cultured in the presence or absence of [4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl) sulfonamide (ISCK03; 5 μM), imatinib (5 μM or anti-SCF (50 ng/mL) for the indicated periods of time. Cell death was determined by FACS analysis of sub-G1 DNA content. (D) Single cells were prepared from L145 and CRC29 colonosphere cultures and were plated in Matrigel in DTC-conditioned medium (CM, as indicated) with or without imatinib (5 μM) or ISCK (5 μM). The experiment was performed as in (A). (E) GFP-expressing L145 colonospheres were cocultured with DTC1 or human fibroblasts (control) in the presence or absence of Imatinib (5 μM for 2 weeks. GFP-expressing cells were then sorted and injected subcutaneously (10,000 live cells) into nude mice (n = 10). Tumor growth was followed by digital caliper measurements until the humane end point was reached (1000 mm3). Calculated tumor volumes at the end of the experiment are shown in the bar graph. In vitro experiments were performed 3 times in triplicate. Representative experiments are shown. Results are shown as means ± standard error of the mean. Statistical significance was determined using the Student t test (unpaired 2-tailed). ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. Gastroenterology 2015 149, 692-704DOI: (10.1053/j.gastro.2015.05.003) Copyright © 2015 AGA Institute Terms and Conditions

Figure 4 KIT inhibition reduces “stemness.” (A) Colonospheres were treated with imatinib (5 μM) or [4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03; 5 μM) for 3 days. Cells were harvested, total RNA was extracted and used for RT-qPCR analysis of the expression of the indicated genes. (B) Western blot analysis of OLFM4 and CK20 after 7 days of KIT inhibitor treatment. (C) Cells were treated as in (A) and the Aldefluorhigh-fraction was determined by FACS analysis. (D) Cells were treated as in (A) and the colony-forming capacity was determined in Matrigel. Experiments were performed in triplicate. Representative experiments are shown. Results are shown as means ± standard error of the mean. Statistical significance was determined using the Student t test (unpaired 2-tailed). ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. Gastroenterology 2015 149, 692-704DOI: (10.1053/j.gastro.2015.05.003) Copyright © 2015 AGA Institute Terms and Conditions

Figure 5 DTC-induced EMT requires SCF/KIT signaling. (A) Combined brightfield/fluorescence images of GFP-expressing colonospheres cocultured with human fibroblasts (left) or DTCs (right). Arrowheads show single migratory cells. (B) L145-GFP cells were cocultured with DTCs or with human fibroblasts in the presence or absence of imatinib (5 μM). After 7 days, the cocultures were harvested and, colonosphere cells were sorted based on GFP expression. RNA was isolated and expression of the indicated genes was measured by RT-qPCR. (C) The experiment was performed as in (B), but sorted cell populations were used for Western blot analysis to measure E-cadherin and Vimentin expression. (D) L145-GFP cells were cocultured with DTCs in the presence or absence of imatinib (5 μM, [4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03; 5 μM, or anti-SCF antibody (50 ng/mL). Sorting and RT-qPCR analysis was performed as in (C). (B) and (D) show representative datasets from 3 independent experiments. All experiments were performed in triplicate. Results are shown as means ± standard error of the mean. Statistical significance was determined using the Student t test (unpaired 2-tailed). ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. Gastroenterology 2015 149, 692-704DOI: (10.1053/j.gastro.2015.05.003) Copyright © 2015 AGA Institute Terms and Conditions

Figure 6 KIT co-expressed genes identify a subgroup of stem-like metastasis-prone tumors and are strongly correlated with EPAS1-co-expressed genes. (A) KIT co-expressed genes (P < e-6) were identified in 2 large cohorts5,22,23 (Supplementary Table 2) and the overlapping gene set was used to cluster the tumors of a third cohort24 into KIT-low, KIT-intermediate, and KIT-high expression subgroups. The original colon cancer subtype (CCS) classification of this cohort is shown on top of the heatmap. (B) Kaplan-Meier curve showing disease-free survival of KIT-low, KIT-intermediate, and KIT-high expression subgroups. The box plot shows that KITLG expression is significantly higher in the KIT-high group. (C) Expression of gene sets that have previously been generated to identify specific colorectal tumor subtypes5,6,24−26 in KIT-high vs KIT-low subgroups. P values of the enrichment of these gene sets in the KIT-high or KIT-low tumor subgroups were determined with the “relate two tracks” option in R2, using the Fisher exact test. (D) Differential expression of gene sets coexpressed with HIF1A, EPAS1 and platelet-derived growth factor receptor B in KIT-high and KIT-low tumor subgroups was determined as in (C). (E) Correlation between KIT and EPAS1 coexpressed genes in the AMC90 cohort (left panel), in a large panel of preclassified colorectal tumor cell lines (middle panel), and in a collection of serrated vs tubular adenomas (right panel). Mesenchymal-type tumors (AMC90; CCS324) and tumor cell lines are indicated in purple. Gastroenterology 2015 149, 692-704DOI: (10.1053/j.gastro.2015.05.003) Copyright © 2015 AGA Institute Terms and Conditions

Figure 7 Hypoxia induces KIT expression, which contributes to enhanced clonogenic capacity. (A) L145 cells were cultured under hypoxia or normoxia in the presence or absence of imatinib (5 μM). After 24 hours, the cells were used for colony-forming assays (normoxia) and for RT-qPCR analysis of KIT and OLFM4 expression. (B) L145 cells were cultured (hypoxia vs normoxia) in the presence or absence of imatinib (5 μM, [4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03; 5 μM, or anti-SCF antibody (50 ng/mL). After 24 hours, the cells were harvested and used for colony-forming assays (normoxia). (C) Tumors (L145) growing in the left liver lobe were subjected to a 45-minute clamping protocol. Twenty-four hours after clamping, the tumor tissue was harvested and used for RT-qPCR analysis of the expression of the indicated genes. (D) Immunohistochemistry analysis of the expression of KIT, SCF, and CA9 on paraffin-embedded tissue sections of colorectal tumors. L, lumen; N, necrosis; T, tumor. (E) Quantification of KIT and CA9 expression levels surrounding 30 necrotic lesions in 2 human colon tumors. Bar graphs show the means ± standard error of the mean. Statistical significance was determined using the Student t test (unpaired 2-tailed). ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. Gastroenterology 2015 149, 692-704DOI: (10.1053/j.gastro.2015.05.003) Copyright © 2015 AGA Institute Terms and Conditions