New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheroids induced by purified colon tumor-initiating cells

Abstract

Background: Growing evidence suggests that the majority of tumors are organized hierarchically, comprising a population of tumor-initiating, or cancer stem cells (CSCs) responsible for tumor development, maintenance and resistance to drugs. Previously we have shown that the CD133high/CD44high fraction of colon cancer cells is different from their bulk counterparts at the functional, morphological and genomic levels. In contrast to the majority of colon cancer cells expressing moderate levels of CD133, CD44 and CD166, cells with a high combined expression of CD133 and CD44 possessed several characteristic stem cell features, including profound self-renewal capacity in vivo and in vitro, and the ability to give rise to different cell phenotypes. The present study was undertaken for two aims: a) to determine stem cell-related genomic characteristics of floating 3D multicellular spheroids induced by CD133high/CD44high colon cancer cells; and b) to evaluate CSC-specific alterations induced by new-generation taxoid SB-T-1214.

Results: Selected CSC phenotype was isolated from three independent invasive colon cancer cell lines, HCT116, HT29 and DLD-1. A stem cell-specific PCR array assay (SABiosciences) revealed that colonospheres induced by purified CD133high/CD44high expressing cells display profound up-regulation of stem cell-related genes in comparison with their bulk counterparts. The FACS analysis has shown that the 3D colonospheres contained some minority cell populations with high levels of expression of Oct4, Sox2, Nanog and c-Myc, which are essential for stem cell pluripotency and self-renewal. Single administration of the SB-T-1214 at concentration 100 nM-1 microM for 48 hr not only induced growth inhibition and apoptotic cell death in these three types of colon cancer spheroids in 3D culture, but also mediated massive inhibition of the stem cell-related genes and significant down-regulation of the pluripotency gene expression. PCR array and FACS data were confirmed with western blotting. Importantly, viable cells that survived this treatment regimen were no longer able to induce secondary floating spheroids and exhibited significant morphological abnormalities.

Conclusions: We report here that a new-generation taxoid SB-T-1214 possesses significant activity against colon cancer spheroids induced by and enriched with drug resistant tumorigenic CD133high/CD44high cells and efficiently inhibited expression of the majority of stem cell-related genes. Our data indicates that the previously observed long-term efficacy of SB-T-1214 against drug resistant colon tumors in vivo may be explained by the down-regulation of multiple stem cell-related genes in the tumorigenic cell population, in addition to its known efficacy as a mitotic poison against proliferating cancer cells.

Figure 1

Histopathological, immunohistochemical and FACS analyses of the mice tumor xenografts. Tumors recovered from the control (vehicle treated; A-D) and SB-T-1214 treated (E-G) mice were sectioned and stained with hematoxylin and eosin (A, E), human epithelial adhesion molecule hEpCAM-FITC; B, F) and CD133-FITC (C, G). The tissue section from vehicle treated mouse show large area of living tumor positive for human EpCAM-FITC (B). Cluster of the CD133-positive cells from the outer tumor area (C). FACS analysis of the dissociated and immunomagnetically sorted for hEpCAM untreated mice tumor xenograft shows the presence of the minor population of cancer cells with high combined expression of CD133-APC and CD44-PE (dashed square; D). Complete loss of the tumor cells (E) and hEpCAM- (F) and CD133-positivity (G) after three consequents treatments with SB-T-1214.

Figure 2

The CD133^high/CD44^high phenotypic cell population has high sphere-forming capacity in the three independent colon cancer cell lines HCT116, HT29 and DLD-1. Cells grown at standard culture conditions possessed the minority phenotypic subpopulations with high combined levels of expression of CD133 and CD44 (left panel; outlined by dashed squares). The sphere-forming efficiency of these cells was 1 of 66 for HCT116 cell line, 1 of 175 for HT29, and 1 of 118 for DLD-1 cell line. In contrast, bulk cancer cells could induce only a few loose flat colonies under non-adherent culture conditions. Induced spheroids retained original cell phenotypes (right panel).

Figure 3

Cytotoxic effects of SB-T-1214 on colon CSC-enriched cell populations grown on type I collagen-coated surfaces. FACS sorted CD133^high/CD44^high cells derived from colon cancer cell lines (HCT116 is shown) and peritoneal fluid of patient with metastatic colon cancer were plated for a short time on collagen type I-coated plates in the MSCB medium. Although a majority of these cells were killed after 48 hours incubation with 100 nM of SB-T-1214, some percentage of the cells survived treatment (A-C, D-F). The cells which survived treatment expressed multiple abnormalities, including greatly enlarged size (G-I, K), multiple nuclei (E, G-I), significant increase in the number of long (J), and knobby (F, I) projections and severe vacuolization (K). A-C: magnification 10×; D-K: magnification 40×.

Figure 4

Cytotoxic effects of the SB-T-1214 against colonospheres grown in 3D cultures. Ten day-old colonospheres grown from FACS sorted CD133^high/CD44^high HCT116 cells on the ultra-low attachment 6-well plates (Corning) were treated with 100 nM SB-T-1214 for 48 hours, then drug-containing medium was replaced with regular MSCBM and analyzed after 24 hours. Treatment with drug induced a profound loss of sphere integrity (A, C) and apoptosis in the majority of the sphere cells (D-E; FACS analysis with the annexin V-FITC and propidium iodide). The FITC-conjugated drug showed efficient penetration into spheroid after 30 min of incubation (B). To evaluate possible alterations in sphere-forming capacity, control and drug treated spheroids/cells were dissociated to a single cell suspension and an equal number of viable cells (400 cells) were plated on ULA 6-well plates in MSCB medium containing 10% Matrigel. The number of secondary spheroids was counted ten days after plating (F).

Figure 5

Drug-induced alteration in the stem cell-related gene expression profiles (PCR Array assay). A majority of the stemness genes were up-regulated in floating spheroids grown from CD133^high/CD44^high cells derived from HCT116, HT29 and DLD-1 cell lines in comparison with their corresponding bulk counterparts (left panel). Treatment of colonospheres with 100 nM SB-T-1214 induced significant down-regulation of a majority of the stemness genes (treated colonospheres were analyzed compared to untreated ones).

Figure 6

Drug-induced alterations in the expression of the markers of pluripotency. FACS analysis shows the presence of minor subpopulations of colon cancer cells within the 3D spheroids induced by CD133^high/CD44^high cells (A), which express the three key pluripotency genes (Sox2, Oct4, and c-Myc). Western blot analysis shows the SB-T-1214-induced changes in the levels of protein expression of these pluripotency markers (B). Untreated spheroids (Con 1 and Cont 2); treated with drug (SB 1 and SB 2).