Slow-cycling therapy-resistant cancer cells

Abstract

Tumor recurrence after chemotherapy is a major cause of patient morbidity and mortality. Recurrences are thought to be secondary to small subsets of cancer cells that are better able to survive traditional forms of chemotherapy and thus drive tumor regrowth. The ability to isolate and better characterize these therapy-resistant cells is critical for the future development of targeted therapies aimed at achieving more robust and long-lasting responses. Using a novel application for the proliferation marker carboxyfluorescein diacetate, succinimidyl ester (CFSE), we have identified a population of slow-cycling, label-retaining tumor cells in both in vitro sphere cultures and in vivo xenograft models. Strikingly, label-retaining cells exhibit a multifold increase in ability to survive traditional forms of chemotherapy and reenter the cell cycle. Further, we demonstrate the innovative application of CFSE to live sort slow-cycling tumor cells and validate their chemoresistance and tumorigenic potential.

FIG. 1.

In vitro identification of LRCs. (A) Flow cytometry plot demonstrating HCT116 sphere dilution of CFSE over 7 days. All HCT116 cells are intensely positive directly after labeling. Over time, label is lost in rapidly proliferating cells, with a tail of more slowly cycling LRCs visible only after 3 days. (B) Florescent and bright-field image overlay of HCT116 sphere cultures and MDA231 adherent cultures 1 week after labeling (scale bar=100 μm). CFSE⁺ cells (white arrows) are visible under a fluorescent microscope. (C) Representative flow cytometry plot of digested HCT116 sphere cultures and MDA231 adherent cultures after 7 days. Black box depicts the approximate collection gate around the highest CFSE-intense cells. (D) Representative cell cycle profile of total HCT116 sphere cultures. Total cells (gray) and CFSE⁺ LRCs (black) after 1 week and adjacent quantification bar graphs. CFSE, carboxyfluorescein diacetate, succinimidyl ester; LRC, label-retaining cell.

FIG. 2.

In vivo identification of LRCs. (A) Composition of CFSE-positive cells after 2 weeks of tumor growth in HCT116 colon tumors, MDA231 breast tumors, and the primary breast sample 2597T tumors. (B) Representative flow cytometry plot of CFSE cell intensities for HCT116 tumor xenografts after 2 weeks of growth. White peak is the unanalyzed data according to CFSE intensity and cell number. Gray peaks represent predicted dilution populations calculated by the proliferation wizard of the Modfit software package. Six separate division peaks were calculated, with two small peaks of greatest CFSE intensity not visible. Black bar approximates the gate used during live sorting to collect the highest 5% intense cells. (C) Fluorescent staining of HCT116, MDA231, and 2597T xenograft tumors after 2 weeks of growth. Only rare cells retain a visible intensity of CFSE (white arrows) while less intense cells are still detectable by flow cytometry (scale bar=100 μm). (D) Cell cycle profiles for HCT116 xenografts. CFSE⁻ cells (gray) and CFSE⁺ LRCs (black) after 2 weeks and adjacent quantification bar graphs.

FIG. 3.

LRCs are colony forming and tumorigenic. (A) HCT116-labeled spheres were digested after 1-week growth (row 1) and live sorted for CFSE⁺ cells (white arrows). Fluorescent and bright-field imaging of representative HCT116 CFSE⁺ cell growth after 1-week (row 2). (B) HCT116 or 2597T CFSE-labeled tumors (column 1) were digested into single cells and live sorted for CFSE⁺ cells (EpCAM⁺ and Lin⁻). Hematoxylin and eosin images of CFSE⁺ xenograft growth (column 2). CFSE⁺ tumors are histologically similar to both parental tumor xenografts, and in the case of 2597T, similar to the primary patient tumor as well (column 3) (scale bar=100 μm).

FIG. 4.

In vitro LRCs demonstrate increased therapy resistance. (A) Timeline for in vitro HCT116 chemotherapy treatment experiments. (B) Number of surviving cells after 3 days in the presence of DMSO, 2 μM Oxaliplatin (Oxali), 250 μM 5-fluorouracil (5-FU), or the combination of Oxali and 5-FU “FOX.” (C) Percent CFSE-positive cells after 3 days in the presence of drug. Oxali-, 5-FU–, and FOX-treated samples are significantly enriched over DMSO-treated samples (n=3, *P=0.01, **P<0.0001). (D) Representative BrdU incorporation flow cytometry plots for FOX-treated sphere cultures with single color controls. DMSO, dimethyl sulfoxide.

FIG. 5.

CFSE LRCs and chemotherapy resistance model. (A) Timeline for in vivo HCT116 xenograft treatment regime. (B) Tumor masses at the end of treatment for DMSO (n=12) and FOX (n=8) (**P<0.01). (C) CFSE composition for DMSO- and FOX-treated tumors (*P=0.045). (D) Representative BrdU incorporation flow cytometry plots for FOX-treated tumors pulsed with BrdU. CFSE⁺ cells incorporated approximately equal amounts of BrdU as CFSE⁻. (E) Representative 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI)-derived cell cycle profiles for FOX-treated tumors pulsed with BrdU with adjacent quantification bar graphs.