Treatment of agarose-agarose RENCA macrobeads with docetaxel selects for OCT4(+) cells with tumor-initiating capability

Abstract

The cancer stem cell (CSC) theory depicts such cells as having the capacity to produce both identical CSCs (symmetrical division) and tumor-amplifying daughter cells (asymmetric division). CSCs are thought to reside in niches similar to those of normal stem cells as described for neural, intestinal, and epidermal tissue, are resistant to chemotherapy, and are responsible for tumor recurrence. We recently described the niche-like nature of mouse renal adenocarcinoma (RENCA) cells following encapsulation in agarose macrobeads. In this paper we tested the hypothesis that encapsulated RENCA colonies function as an in vitro model of a CSC niche and that the majority of cells would undergo chemotherapy-induced death, followed by tumor recurrence. After exposure to docetaxel (5 µg/ml), 50% of cells were lost one week post-treatment while only one or two cells remained in each colony by 6 weeks. Surviving cells expressed OCT4 and reformed tumors at 16 weeks post-treatment. Docetaxel-resistant cells also grew as monolayers in cell culture (16-17 weeks post-exposure) or as primary tumors following transplantation to Balb/c mice (6 of 10 mice) or NOD.CB17-Prkdc(scid)/J mice (9 of 9 mice; 10 weeks post-transplantation or 28 weeks post-exposure). These data support the hypothesis that a rare subpopulation of OCT4(+) cells are resistant to docetaxel and these cells are sufficient for tumor recurrence. The reported methodology can be used to obtain purified populations of tumor-initiating cells, to screen for anti-tumor-initiating cell agents, and to investigate the in vitro correlate of a CSC niche, especially as it relates to chemo-resistance and tumor recurrence.

Keywords: OCT4; RENCA; encapsulation; macrobeads; tumor-initiating cells.

Figures

Figure 1. Cell recovery following treatment with paclitaxel or docetaxel in the RENCA macrobead. Representative H&E staining of control and treated RENCA macrobeads. Normal RENCA macrobead tumor morphology was maintained post- vehicle treatment (0.07% DMSO, top panel). Progressive loss of cell viability following paclitaxel treatment (3.5 µg/ml, center panel) restored to pre-treatment levels after 18 weeks. Extensive cell loss by week 6 post-docetaxel treatment (5.0 µg/ml, bottom panel) and viable cells in a minority of colonies at week 18 post-treatment. For all panels, original magnification = 400×, scale bar = 20 µm.

Figure 2. Viability of RENCA macrobeads following paclitaxel or docetaxel treatment. A quantitative assessment of the percent of colonies that remained viable and the average number of viable nuclei per colony in treated macrobeads. Typical levels of colony viability and the number of viable nuclei per colony were maintained in vehicle-treated macrobeads throughout the course of the study (A). When possible, an average of 3 replicate experiments was considered. Statistically significant outcome differences for paclitaxel-treated macrobeads (B) or docetaxel-treated macrobeads (C), as compared with vehicle-treatment, are denoted with an asterisk (*), P < 0.05.

Figure 3. Metabolic activity and tumor inhibitory capacity of RENCA macrobeads exposed to paclitaxel and docetaxel. Metabolic activity (as assessed by MTT assay, see Methods) of RENCA macrobeads exposed to the intermediate dose of paclitaxel (A) or docetaxel (B) as compared with untreated or vehicle treated RENCA macrobeads. Tumor inhibitory capacity of RENCA macrobeads, on exogenous tumor cells, following exposure to paclitaxel (C) or docetaxel (D) as compared with untreated or vehicle treated RENCA macrobeads.

Figure 4. Docetaxel-resistant cells cultured in vitro and after transplantation. Morphological appearance of docetaxel-resistant cells one week post-isolation from docetaxel treated macrobeads (A). Maintained in vitro for 16–17 weeks docetaxel-resistant cells developed plaques (B). These cells were passaged at week 16 post-treatment, and within 2 weeks the harvested cells assumed normal RENCA morphology (C). As a reference, RENCA monolayers demonstrating normal RENCA cell morphology (D). Representative H&E stained tissue of a Balb/cJ mouse at 56 d post-transplantation shows tumor formation inthe left kidney (E) and lung metastasis (F). Representative H&E stained tissue from tumor formation in the left kidney (G) and lung metastasis (H) of a NOD.CB17-Prkdcscid/J mouse at 66 d post-transplantation. Original magnification for all panels = 200×, scale bar: 40 µm.

Figure 5. Docetaxel-resistant RENCA cells express OCT4. OCT4 expression (green fluorescence) and nuclear staining (DAPI, blue fluorescence) is limited to a minority of the cell population composing tumor colonies in control RENCA macrobeads (A). Cells surviving in RENCA macrobeads at 6 weeks post-docetaxel treatment were positive for OCT4 expression (B–D). Original magnification of panels (A–D) = 200×, insets in panels (A and B) = 400×; scale bar = 20 µm.