Mitotic quiescence, but not unique “stemness,” marks the phenotype of bone metastasis-initiating cells in prostate cancer

Abstract

This study aimed to identify subpopulations of prostate cancer cells that are responsible for the initiation of bone metastases. Using rapidly dividing human prostate cancer cell lines, we identified mitotically quiescent subpopulations (<1%), which we compared with the rapidly dividing populations for patterns of gene expression and for their ability to migrate to the skeletons of athymic mice. The study used 2-photon microscopy to map the presence/distribution of fluorescently labeled, quiescent cells and luciferase expression to determine the presence of growing bone metastases. We showed that the mitotically quiescent cells were very significantly more tumorigenic in forming bone metastases than fast-growing cells (55 vs. 15%) and had a unique gene expression profile. The quiescent cells were not uniquely stem cell like, with no expression of CD133 but had the same level expression of other putative prostate stem cell markers (CD44 and integrins a2/b1), when compared to the rapidly proliferating population. In addition, mitotic quiescence was associated with very high levels of C-X-C chemokine receptor type 4 (CXCR4) production. Inhibition of CXCR4 activity altered the homing of quiescent tumor cells to bone. Our studies suggest that mitotic dormancy is a unique phenotype that facilitates tumor cell colonization of the skeleton in prostate

Confocal/two-photon microscopy in studying colonisation of cancer cells in bone using xenograft mouse models

“…One advantage of using these lipophilic dyes is being able to detect dormant cells as these cell membrane dyes are diluted to undetectable concentrations in proliferating cells. [20][21][22]24,25 Cancer cells are firstly washed with phosphate-buffered saline (PBS), trypsinised by 0.15% Trypsin-EDTA for 3-5 min, at 37 1C, 5% CO 2 . Cells are neutralised with appropriate media containing 10% FBS and centrifuged for 5 min at 200 g. The cell pellet is resuspended at a concentration of 1 Â 10 6 cells per ml in serum free medium for Vybrant DiD labelling or in Hanks 0 balanced salt solution (HBSS) for Vybrant CM-DiI.…”

“…As bone structures heavily scatter lights and the high collagen content generates second-harmonic signals (SHG), these advantages won two-photon microscopy increasing popularity in research of cellular activities and interactions within bone and marrow, particularly in identifying the haematopoietic stem cell niche and detecting bone metastasis-initiating cancer cells in bone. [17][18][19][20][21][22][23] In this article, using the detection of breast cancer cell bone colonisation by confocal and two-photon microscopy as a representative example, we will describe a step-by-step methodology, from sample preparation to data analyses, used to investigate cellular events in frozen and fixed/decalcified mouse bone samples ex vivo (see schematic outline, Figure 2). Advantages and limitations of this technology is also discussed to guide the reader as to which is the most appropriate for their research question.…”

The biology and clinical implications of prostate cancer dormancy and metastasis

“…Emerging evidence links the CSC hypothesis with dormancy and metastasis because both CSCs and dormant cells share features like quiescence and immune evasion [36]. On the other hand in a recent publication by Wang et al comparing mitotic quiescent subpopulations of PCa tumor cells to a rapidly dividing population showed that the quiescent cells were not stem-like, with no expression of CD133 and similar levels of CD44 and integrins α2/β1, when compared to the rapidly dividing population [37]. Their model argues that mitotic quiescence, but not unique “stemness,” marked the phenotype of bone metastasis-initiating cells in PCa [38].…”

“…A BCa clinical trial supported that adjuvant zoledronic acid treatment for those patients with DTC detected during surgery increased progression-free survival [57], further supporting the potential benefit of inhibiting bone turnover to prevent metastatic outgrowth. Another clinical study in BCa even showed that zoledronic acid contributed to the eradication of DTC [58], although in PCa a preclinical study failed to show that zoledronic acid eliminated the pre-existing DTC [37]. Nonetheless, the potential use of zoledronic acid as an adjuvant therapy to androgen deprivation therapy in minimally metastatic PCa patients will be particularly interesting – one would anticipate it may help patients preserve a more intact bone environment that is unfavorable to the outgrowth of DTC and therefore prevent the development of bone metastasis.…”

“…In PCa, reactivating dormant cells at the cellular level has been proposed by two independent groups reporting that high level of CXCR4 was associated with mitotic quiescence [37] or slow-cycling [48]. Inhibition of CXCR4 activity reactivated cell proliferation in vitro [48].…”