Prostate Cancer Cells Preferentially Home to Osteoblast-rich Areas in the Early Stages of Bone Metastasis: Evidence From In Vivo Models

Abstract

It has been suggested that metastasis-initiating cells gain a foothold in bone by homing to a metastastatic microenvironment (or "niche"). Whereas the precise nature of this niche remains to be established, it is likely to contain bone cell populations including osteoblasts and osteoclasts. In the mouse tibia, the distribution of osteoblasts on endocortical bone surfaces is non-uniform, and we hypothesize that studying co-localization of individual tumor cells with resident cell populations will reveal the identity of critical cellular components of the niche. In this study, we have mapped the distribution of three human prostate cancer cell lines (PC3-NW1, LN-CaP, and C4 2B4) colonizing the tibiae of athymic mice following intracardiac injection and evaluated their interaction with potential metastatic niches. Prostate cancer cells labeled with the fluorescent cell membrane dye (Vybrant DiD) were found by twophoton microscopy to be engrafted in the tibiae in close proximity ($40 mm) to bone surfaces and 70% more cancer cells were detected in the lateral compared to the medial endocortical bone regions. This was associated with a 5-fold higher number of osteoblasts and 7-fold higher bone formation rate on the lateral endocortical bone surface compared to the medial side. By disrupting cellular interactions mediated by the chemokine (C-X-C motif) receptor 4 (CXCR4)/chemokine ligand 12 (CXCL12) axis with the CXCR4 inhibitor AMD3100, the preferential homing pattern of prostate cancer cells to osteoblast-rich bone surfaces was disrupted. In this study, we map the location of prostate cancer cells that home to endocortical regions in bone and our data demonstrate that homing of prostate cancer cells is associated with the presence and activity of osteoblast lineage cells, and suggest that therapies targeting osteoblast niches should be considered to prevent development of incurable prostate cancer bone metastases.

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.…”

“…20,27 Other bone samples (for example, ribs) can also be used for confocal/two-photon microscopy examination but extra care should be taken to maintain consistency of sample orientation while sectioning, which is important for comparison of different samples.…”

“…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.…”

Chronic stress, sympathetic activation and skeletal metastasis of breast cancer cells

“…[33][34][35] SDF1 for instance is expressed by osteoblasts and is considered a major cytokine for successful homing and survival of prostate and breast cancer cells in bone. 36,37 High CXCR4 expression has also been correlated to poor clinical outcome in patients with breast cancers. 38,39 Similarly, RANKL is expressed by osteoblasts and osteocytes (as well as Tcells and chondrocytes) in bone.…”

Chemokines and Bone

“…Using studies in which disseminated PC cells were first established in bone after which the primary tumors were removed, the investigators found there were again more circulating PC cells when animals treated with AMD3100 versus vehicle alone. Elegant studies by Wang et al [152] mapping the distribution of human PC cell lines colonizing the tibiae of athymic mice found they preferentially homed to osteoblast-rich areas. This homing was lost in the presence of AMD3100 further supports a role for the CXCR4/CXCL12 axis.…”