1. Volume 6, Issue 6, Pages 603-615 (June 2010)
A Subpopulation of CD26+ Cancer Stem Cells with Metastatic Capacity in Human Colorectal Cancer 
Roberta Pang, Wai Lun Law, Andrew C.Y. Chu, Jensen T. Poon, Colin S.C. Lam, Ariel K.M. Chow, Lui Ng, Leonard W.H. Cheung, Xiao R. Lan, Hui Y. Lan, Victoria P.Y. Tan, Thomas C. Yau, Ronnie T. Poon, Benjamin C.Y. Wong 
Cell Stem Cell 
Volume 6, Issue 6, Pages (June 2010)
DOI: /j.stem
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1
Different Subpopulations of CSCs in Primary and Metastatic Human Colorectal Cancers
(A) Three-color flow cytometry of a representative primary colorectal cancer with 3.8% of CD133+ cells. The population was gated, further analyzed for CD26 and CD44, and was shown to express a CD133+CD26−CD44+ subpopulation (3.3%, patient P10). N, normal; T, tumor.
(B) Three-color flow cytometry of a representative primary tumor from a patient with synchronous liver metastasis (patient S3) harboring a subpopulation of CD133+CD26+CD44+ cells (8.4%). Middle panel shows the whole cell population scatter plot (left) and gating for CD45− cells (right). The percentage of cells in different subpopulations within the tumor after gating is shown in the lower panel and summarized in the prism table on the top panel. Numbers in each table cell refer to the percentage of cells isolated from the tumor after gating on CD45− to exclude hematopoietic cells.
(C) Three-color flow cytometry of the corresponding metastatic tumor from the patient in (B), with the middle panel showing the whole cell population scatter plot (left) and gating for CD45− cells (right). The percentage of cells in different subpopulations within the tumor after gating is shown in the lower panel and summarized in the prism table (top). Numbers in each table cell refer to the percentage of cells isolated from the tumor after gating on CD45−.
(D) CD26 enzymatic activity in CD26+ and CD26− cells isolated from five primary colorectal cancers and five liver metastases with CD26+ cells, showing very low enzymatic activity in the CD26− cells.
See also Tables S1 and S2.
Cell Stem Cell 2010 6, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 2
Tumorigenic Potential of Different CSC Subpopulations Derived from Patient Tumors
(A) Left: Excised subcutaneous tumors developed from Matrigel-resuspended CD133+CD26+ cells of a representative tumor (patient no. S3). Right: Two-color flow cytometry of CD133+CD26+ cells isolated from a primary tumor by MACS (98.13% purity), showing no expression of CD45 and CK20.
(B) Tumorigenic potential of 1 × 105 CD26+CD44+, CD26+CD44−, CD26−CD44+, and CD26−CD44− cells within CD133+ and CD133− cell populations, respectively, as well as unsorted tumor cells derived from five primary tumors (patient no. S3, S3M, P10, P18, and M8; flow cytometry analyses of P10, S3, and S3M were shown in Figures 1A–1C, respectively). Xenografts were established from subcutaneous injection of CD133+CD26+ cells from the five tumors, and the different cell populations were isolated from the established xenografts. Each group of mice (n = 4) was then injected with the different subpopulations from each of the five tumors. Data represent the mean ± SD of tumor volume at different time points of different groups.
(C) Tumor volumes of successive passages of xenografts grown from CD133+CD26+ cells, CD133+CD26−CD44+ cells, and CD133+CD26−CD44− cells, respectively, isolated from a representative human tumor (patient no. P1) at day 42 postinjection. The secondary xenograft was generated from injection of 1 × 103 cells of the respective populations isolated from xenografts of the preceding passage into four animals in each group. Data represent the mean ± SD of tumor volume.
(D) H&E staining of xenograft passages shows well- to moderately-differentiated adenocarcinomas that are histologically similar to the parental tumor (magnification 20×, insets show sections of 40×).
See also Table S3.
Cell Stem Cell 2010 6, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 3 Tumorigenic Capacity of Spheres Derived from CD26+ Cells
(A) Formation of spheres from CD133+CD26+ cells isolated from the primary and metastatic tumors, respectively, of a representative case of colorectal cancer with synchronous liver metastasis (patient S4), and CD133+CD26−CD44+ cells isolated from the primary and metastatic tumors of the same patient (week 4). CD133−CD26−CD44− cells isolated from the same tumors did not form spheres under the same conditions (right panels).
(B) Immunofluorescence staining of CD133+CD26+ colonic spheres isolated from a representative tumor (patient S4). Spheres were stained with CD26-FITC (left) and CD133-PE (middle), followed by merging of the two images (right).
(C) Dissociated spheres derived from CD133+CD26+, CD133+CD26−CD44+, and CD133+CD26−CD44− cells efficiently formed subspheres (secondary spheres) in serial passages. Subsphere formation efficiency is the percentage of cells present (dissociated) in a single sphere that can form secondary spheres in each passage. Each bar represents sphere formation from five patients (patients M1, M3, P2, P3, and P6) in triplicates counted at day 20 after plating.
(D) H&E staining of tumors grown by orthotopic injection of dissociated cells of spheres grown from CD133+CD26+ cells isolated from primary or metastatic tumors, and CD133+CD26−CD44+ cells isolated from the primary tumor (magnification 20×, insets show sections of 40×).
(E) Tumor formation from subcutaneous injection of 1 × 104 dissociated CD133+CD26+, CD133+CD26−CD44+, and CD133+CD26−CD44− cells from spheres of a representative case (patient M1). An illustrative H&E staining of tumor xenograft from CD133+CD26+ cells is shown (magnification 20×, insets show sections of 40×).
See also Table S4.
Cell Stem Cell 2010 6, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 4 Development of Metastasis from CD133+CD26+ Cells
(A) Orthotopic implantation of CD133+CD26+ cells from a primary tumor with synchronous liver metastasis (patient S2) in SCID mice led to primary tumor development at the site of injection (left) and metastasis in the liver (right).
(B) H&E staining of the patient's tumor and primary and secondary xenografts from a colorectal liver metastasis (patient M12) showing similar histology (magnification 20×, insets show sections of 40×).
(C) Left: Metastases to the colon from orthotopic implantation of CD133+CD26+ cells from a primary colorectal cancer (patient 9). Right: Liver metastasis developed from CD133+CD26+ cells isolated from a primary colorectal cancer (patient 21).
(D) Top: Flow cytometry showing CD133+CD26+CD44+ cells in the portal vein blood from a mice at week 6 after orthotopic implantation of CD133+CD26+CD44+ cells isolated from a patient tumor. CD45− cells were gated for further analysis of CD133+, CD26+, and CD44+ expression. Bottom: Liver metastasis developed from intraportal vein injection of CD133+CD26+CD44+ cells (week 9).
(E) Invasive and migratory capacities of CD133+CD26+ and CD133+CD26−CD44+ tumor cells in Boyden chamber assay (left) and transwell migration assay (right). Bars represent the mean ± SD of invaded/migrated cells isolated from established tumors of five different animals for each of the patient sample shown.
(F) Adhesive capacity of different cell populations to the ECM. Percent adhesion is calculated as the number of adhesive cells/adhesive cells+nonadhesive cells. Data are expressed as the percent of adhesive cells determined over three fields per assay and expressed as an average of triplicate determinations.
Cell Stem Cell 2010 6, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 5 Functional Effects of CD26 Downregulation in CD26+ CSCs
(A) Immunoblotting of EMT proteins from lysates of CSCs isolated from primary or metastatic tumors of three different patients. CD133+CD26+ CSCs showed significantly dysregulated EMT proteins compared with CD133+CD26−CD44+ CSCs. Graph represents band density ratio of the respective protein to actin (n = 3 for each protein).
(B) Depletion of CD26 expression by transient transfection of siCD26 oligo. Analysis of mRNA expression by RT-PCR (left) and protein expression by flow cytometry (right) at 48 hr posttransfection.
(C) Invasive capacity of CD26+ cells transfected with nonsense control oligo (NS) or siCD26 oligo. Data represent the mean numbers of invaded cells per well and performed in triplicates.
(D) CD26+ cells isolated from a primary colorectal tumor transiently transfected with CD 26 siRNA showed decreased adhesion to fibronectin, type I collagen, or laminin. Percent adhesion is calculated as the number of adhesive cells/adhesive cells+nonadhesive cells. Data represent the mean of three different tumors (two primary colorectal and one liver metastasis tumors).
(E) CD26+ cells preincubated with blocking antibodies against integrin β1 (4B4), but not isotype control IgG, showed decreased adhesion to fibronectin and collagen. Data represent the mean of three different tumors (two primary colorectal and one liver metastasis tumors). Decreased expression of phosphorylated integrin β1 at Ser785 was observed in CD26 siRNA, but not their corresponding nonsense control (NS) transfectants.
(F) Immunoblotting of EMT proteins from lysates of CD26+ cells transfected with nonsense control oligo (NS) or siCD26 oligo. CD26+ cells were isolated from a colorectal cancer patient with liver metastasis.
(G) Tumor formation efficiency of CD133+CD26+CD44+, CD133+CD26+, and CD133−CD26−CD44− cells derived from three primary patient tumors (patients P1, P2, and P5) in the presence/absence of HIF. Data represent the means of the three patients for each subpopulation. Metastatic capacity of CD133+CD26+ cells derived from a patient (S2) with (right) and without (left) preinjection of HIF.
See also Table S5.
Cell Stem Cell 2010 6, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions

7. Figure 6
Enrichment of CD133+CD26+ Cells by Chemotherapeutic Treatments
(A) Treatment of tumor cells with 5-FU in vitro decreased cell viability as shown by MTT assay. Data represent mean ± SD of tumor cells isolated from six different patients in triplicates.
(B) Decreased cell viability was accompanied with enhanced apoptosis (upper panel) and enrichment of the CSC-positive subpopulation (lower panel).
(C) Treatment of tumor cells with 5-FU in vivo led to tumor shrinkage (left) markedly enriched the CD133+CD26+ subpopulation (right). Tumors from the control group (saline) exceeded the maximum allowable sizes approved by Animal Ethics Committee and therefore were sacrificed beyond day 56.
(D) Treatment of tumor cells with oxaliplatin in vivo led to inhibition of tumor growth (left), but enrichment of the CD133+CD26+ subpopulation (right). Each point in the tumor growth curve represents the mean ± SD of five animals. Each treatment group consisted of 30 animals, and three animals were sacrificed at each of the indicated time points for analysis of the CD133+CD26+ subpopulation. Each bar in the right panel represents the mean percentage ± SD of viable cells from the three excised tumors at the respective time point after treatment with oxaliplatin. Propidium iodine staining was performed to exclude dead cells from the dissociated tumor by flow cytometry. Tumors from the control group (saline) exceeded the maximum allowable sizes approved by Animal Ethics Committee and therefore were sacrificed beyond day 42.
Cell Stem Cell 2010 6, DOI: ( /j.stem )
Copyright © 2010 Elsevier Inc. Terms and Conditions