Slow-cycling (dormant) cancer cells in therapy resistance, cancer relapse and metastasis

Abstract

It is increasingly appreciated that cancer cell heterogeneity and plasticity constitute major barriers to effective clinical treatments and long-term therapeutic efficacy. Research in the past two decades suggest that virtually all treatment-naive human cancers harbor subsets of cancer cells that possess many of the cardinal features of normal stem cells. Such stem-like cancer cells, operationally defined as cancer stem cells (CSCs), are frequently quiescent and dynamically change and evolve during tumor progression and therapeutic interventions. Intrinsic tumor cell heterogeneity is reflected in a different aspect in that tumors also harbor a population of slow-cycling cells (SCCs) that are not in the proliferative cell cycle and thus are intrinsically refractory to anti-mitotic drugs. In this Perspective, we focus our discussions on SCCs in cancer and on various methodologies that can be employed to enrich and purify SCCs, compare the similarities and differences between SCCs, CSCs and cancer cells undergoing EMT, and present evidence for the involvement of SCCs in surviving anti-neoplastic treatments, mediating tumor relapse, maintaining tumor dormancy and mediating metastatic dissemination. Our discussions make it clear that an in-depth understanding of the biological properties of SCCs in cancer will be instrumental to developing new therapeutic strategies to prevent tumor relapse and distant metastasis.

Keywords: Cancer stem cells; Metastasis; Quiescence; Slow-cycling cells; Therapy resistance.

Fig. 1.

Cell cycle and cell-cycle phases in normal cells. (A) Schematic depicting the 4 cell-cycle phases (G1, S, G2 and M). Depicted also is the mitotic division that has generated two different daughter cells as a result of asymmetric cell division (ACD). Cells upon mitosis may enter the early G1 or G0 phase. R indicates the Restriction point that separates the early vs. late G1. (B) The level of Rb phosphorylation dictates the G0, early G1, and late G1 phase (based on [5]). Cells in the G0 have un-phosphorylated Rb. In response to growth factor-initiated mitogenic signaling, the cyclin D/CDK4 complex may mono-phosphorylate the Rb at one of the 14 sites and push the cells into the early G1 phase. The cyclin E/CDK2 then hyper-phosphorylate the Rb in all 14 sites thus pushing the cells over the R point and irreversibly committing cells to the rest of the cell cycle (see Text). (C) A more detailed presentation of the 4 cell-cycle phases driven by cyclin/CDK activities as well as the quiescent G0 phase. M-phase represents the shortest cell-cycle phase in which the dividing cell goes through prophase, metaphase, anaphase and telophase, and ends with cytokinesis.

Fig. 2.

G0 phase. Shown are two different G0 phases, reversible and irreversible. See Text for details.

Fig. 3.

Experimental strategies to identify and purify SCCs. (A) Nucleotide analog-based pulse-chase strategy to identify cells in the S-phase (left) or the slow-cycling, long-term label-retaining cells (LRCs; right). This method does not allow purification of live cells for functional studies. (B) Dye retention-based methods to enrich and purify live SCCs (in cancer). (C) Identification and purification of SCCs based on H2B-GFP dilution. (D) FUCCI system used to separate cells in different cell-cycle phases including SCCs in G0. (E) Fluorescent reporters used to identify/purify SCCs using SCC-specific gene promoters.

Fig. 4.

Human cancer cell spheres and prostate cancer xenografts harbor LT-LRCs (SCCs). (A-B) Human cancer cell spheres harbor LT-LRCs (i.e., SCCs) with distinct levels of quiescence. Human PCa (LAPC4 and LNCaP), glioblastoma (U373), breast cancer (MCF7) and melanoma (YVM562) cell-derived spheres were pulsed with 10 μM BrdU for 43 h, after which BrdU was washing off and sphere cultures chased for the days (d) indicated. The endpoint spheres were harvested and embedded in paraffin, and sections used in BrdU staining. At least 6 sections were analyzed and a total of 1,500 – 3,000 cells counted under an epifluorescence microscope for each cell type. The BrdU⁺ (i.e., label-retaining) cells were presented as bar graphs (mean ± S.D). (C) Human PCa xenografts have raie SCCs (LT-LRCs) in vivo. To identify LRCs in xenograft prostate tumors, 100 pi of BrdU solution (20 mM stock) was i.p injected (4x over 43 h) into the NOD/SCID mice bearing Du145, LAPC4, or LAPC9 xenograft tumors. Tumors were then “chased” for 45, 68 or 20 days (d), respectively, until harvest. Tumors were embedded in paraffin and serial sections of 5 pm were cut and used in immunohistochemical staining of BrdU. At least 6 sections were analyzed and counted for each tumor type and the BrdU ‘ cells scored under a microscope.

Fig. 5.

Autochthonous TRAMP tumors harbor LT-LRCs. Eight-week old TRAMP tumors in C57/FVB mice were continuously pulsed with 20 mg/ml BrdU using the slow-releasing Alzet pumps for 19 days (i.e., from Dec. 22 of 2005 to Jan. 10 of 2006) and then the pumps were removed (i.e., termination of pulse; indicated by *). Prostates chased for 1 day, 6 or 8 weeks (wks), or 3 or 6 months were harvested and embedded in paraffin. Serial sections of 5 μm were cut and used in BrdU IHC staining. At least 5 sections were analyzed and BrdU⁺ cells counted. Shown are the average BrdU⁺ cells in the dorsal prostate (DP) and ventral prostate (VP), respectively.

Fig. 6.

Adapting the inducible H2B-GFP labeling/chasing system to identify and study slow-cycling luminal progenitor cells in the mouse prostate. (A) Chase time-dependent decrease in GFP fluorescence intensity and in GFP⁺ cells. Shown on top are micro-dissected mouse prostate alveolar-ductal tree structure in relation to the urethra (U). Distal (secretory) alveoli and proximal (Prox) ducts close to the urethra are indicated. Shown below are the corresponding GFP images in non-chased (left; 20-week), 6-week chased (middle) and 12-week chased prostates. Note that the no-chase and 6-week chase images were adapted from [54] with permission. (B) The non-chased prostate in (A; the arrow) is shown in higher magnifications, to illustrate the homogeneous GFP labeling in the proximal, distal and medial regions of the mouse prostate. (C) Schematic of the model (see Text). (D) Analysis of a 12-week non-chased anterior prostate shows that the GFP⁺ cells are largely K8⁺ luminal epithelial cells. (E) Analysis of a 12-week chased ventral prostate reveals significantly reduced numbers of GFP⁺ cells, which are mostly AR⁻.

Fig. 7.

Human PCSCs are generally slow-cycling. (A-B) The CD44⁺ PCSCs have intermediate slow-cycling properties. The LAPC4 spheres were either pulsed with BrdU for 2 or 16 h without chase or pulsed for 48 h and then chased for up to 33 days. The BrdU-retaining cells, i.e., LRCs, were quantified (A). In B, the CD44 and BrdU double-positive cells were quantified from serial sections obtained from at least 12 spheres. The bars are the mean ± SEM obtained from three independent experiments. Data were from [24] with permission. (C) CD44^+/hi PCa cells are relatively quiescent. Shown are two Du 145 cell holoclones pulsed with BrdU for 4 h followed by BrdU (red) and CD44 (green) staining. As can be seen, most CD44^+/hi Du145 cells in the holoclones are BrdU-negative. Taken from [26] with permission. (D) The PSA^−/lo PCSCs are quiescent compared to differentiated PSA⁺ PCa cells. Shown are time-lapse images of one PSA⁺ (green) LNCaP cell recorded for 180 h and one PSA^−/lo LNCaP cell recorded for 230 h (taken from [33] with permission). (E-F) PSA^−/lo LNCaP cells have much longer cell-cycle transit time (E) and lower population doublings (F) compared to the corresponding PSA⁺ LNCaP cells. Adapted from [40] with permission.