PCa cells target the HSC niche during metastasis.
To directly test whether metastatic cells compete with HSCs for the niche, disseminated PCa cells were evaluated for their ability to prevent HSC engraftment. We used a micrometastasis model to determine whether PCa cells target the HSC niche during metastasis to bone (Supplemental Methods; supplemental material available online with this article; doi:
). In this model, disseminated tumor cells shed from s.c. implanted tumors, which can be tracked by quantitative real-time PCR (QPCR), have the capacity to generate metastatic lesions over time in the BM (16
). For these studies, to establish disseminated PCa cells in the niche, NOD/SCID mice (CD45.1) were implanted s.c. with PCa tumors or with nonmetastatic transformed prostate epithelial (NMPE) cell lines as controls (Figure A and Supplemental Methods). Later, the tumors were removed, and BMT was performed using BM cells derived from CD45.2 animals (Figure A). To preserve the integrity of the niche, preparative transplant regimens (e.g., radiation and chemotherapy) were not used, and engraftment was analyzed by FACS. In all cases, greater HSC engraftment was observed in control groups (NMPE or no tumor implanted) than in the tumor-bearing animals (Figure B), which suggests that the cells shed from a primary tumor prevent HSC engraftment by occupying the HSC niche.
PCa cells compete for the HSC niche and prevent HSC engraftment.
To verify that the reduced HSC engraftment was in fact caused by the disseminated PCa cells, we used bioluminescent imaging and observed that tumors developed from the metastatic cells in these animals (4 weeks, 0 of 10; 8 weeks, 0 of 10; 12 weeks, 2 of 10; 16 weeks, 3 of 10; Figure C). Additionally, disseminated PCa cells in the BM were identified by immunohistochemistry; few or no disseminated cells were observed in either of the controls (Figure D and Supplemental Figure 1A). Furthermore, these results were not likely attributable to changes in the number or size of the niche (e.g., osteoblast number; Figure E) resulting from the primary tumor alone.
CXCL12 and its receptors, CXCR4 and CXCR7, are known to play major roles in HSC homing to the BM (17
) and in establishing PCa metastases in bone (3
). To determine whether a similar mechanism regulates PCa cell targeting to the HSC niche, mRNA levels of CXCR4
expressed by PCa cells in the niche were compared with those of circulating PCa cells in the blood. Compared with PCa cells in culture (Supplemental Figure 1, B and C) or in peripheral blood, CXCR4
expression was dramatically reduced in PCa cells isolated from the HSC niche, whereas no remarkable changes were observed in CXCR7
levels (Figure , F and G). Furthermore, time course studies tracking PCa cell dissemination into the niche along with CXCL12 BM levels (Supplemental Figure 2, A–E, and Supplemental Methods) — in conjunction with our previous work showing that CXCR4 and CXCR7 regulate metastasis (3
) — further support the concept that CXCL12 plays an important mechanism whereby PCa cells target the HSC niche.
To directly test whether disseminated tumor cells compete with HSCs for the niche, we used a competitive engraftment assay. Lethally irradiated animals were transplanted with BM cells alone or with PCa cells or NMPE control cells, and survival was evaluated as a measure of HSC engraftment (Figure A). To ensure that only occupancy of the niche occurred, rather than tumor growth, PCa and NMPE cells were irradiated to prevent proliferation (Figure B). Significantly more of the PCa cell–injected animals failed to engraft and subsequently required euthanasia than the control animals (Figure C), yet this effect was dependent on the number of PCa cells (data not shown), which suggests that PCa cells are not as efficient as HSCs in targeting the niche. BM histology demonstrated significant delays in engraftment of the animals injected with tumor cells along with their transplants (Figure D and Supplemental Figure 3), demonstrating competition between HSCs and PCa cells for the niche.
Direct competition for the HSC niche between HSCs and PCa cells.
Thus far, our findings demonstrated that human PCa cells compete with murine HSCs for occupancy of the niche. To determine whether human PCa cells and HSCs compete for the niche in vivo, direct completion assays were performed using human CD34+ BM cells and human PCa cells in sublethally irradiated mice. Under these conditions, engraftment of the human cells was low, as expected. Significantly fewer human CD34+ cells engrafted into PCa cell–injected animals (Figure E). These data suggest that disseminated tumor cells directly compete with HSCs for occupancy of the niche.
PCa cells and HSCs colocalize to the endosteal niche.
At present, the precise cellular composition and location of the HSC niche remains controversial (5
). Recent reports have demonstrated that endosteal cells of the osteoblastic lineage contribute to the development of the HSC niche (6
). To determine whether disseminated PCa cells and HSCs colocalize to the endosteal niche, prelabeled HSCs (Lin–
, referred to herein as LSK HSCs) and prelabeled PCa cells were injected into animals simultaneously. After 24 hours, the long bones were recovered, and confocal microscopy was used to track PCa cells and HSCs after transplantation. Both cell types colocalized within a few microns of each other in the BM of recipient animals (Figure A).
HSCs and PCa cells colocalize to BM niches through Runx2.
To further characterize the interactions between PCa cells and HSCs, the long bones recovered at 3 weeks from animals implanted s.c. with human tumors and tissue sections were stained for human PCa cells using anti-human HLA antibodies, HSCs (CD150+CD41–CD48–Lin–), and osteoblastic niche cells expressing the osteoblast-specific transcription factor Runx2. Despite the rarity of both disseminated populations, both PCa cells and HSCs colocalized with Runx2-expressing cells (Figure , B and C). Similarly, disseminated PCa cells and HSCs localized close to one another (Figure D).
To further validate that the disseminated PCa cells and HSCs colocalize to the same endosteal niche, multiphoton imaging was used to track PCa cell and HSC homing to the BM after transplantation. After transplantation, both cell types colocalized within 5 cell distances of each other, whereas NMPE control cells did not colocalize with HSCs (Figure , A–C).
HSCs and PCa cells colocalize to BM niches, and alteration of niche size regulates tumor dissemination.
In vitro studies were performed to further characterize the molecular mechanisms used by PCa cells to localize to the HSC niche. HSCs were isolated using the SLAM family of receptors (Lin–
, referred to herein as SLAM HSCs; refs. 11
). In short-term adhesion assays, PCa cells were able to colocalize to a single osteoblast (Figure B and Supplemental Figure 4, A and B), which suggests that HSCs and PCa cells may localize extremely close to one another in the BM (but not NMPE control cells; Figure F). We have previously noted that Anxa2 expression by osteoblasts plays a central role in niche selection of both HSCs and PCa cells (Supplemental Methods and refs. 13
). Here, we noted that both HSCs and PCa cells bound significantly better to Anxa2-expressing osteoblasts (i.e., from Anxa2+/+
animals) than to those isolated from Anxa2–/–
animals (Figure , D and E). However, NMPE control cells did not bind to either Anxa2+/+
osteoblasts (Figure F). Together, these findings, along with the competitive engraftment data, demonstrated that PCa cells and HSCs compete for the HSC niche in BM.
Alterations of the endosteal HSC niche regulate PCa metastasis.
Changes in osteoblast numbers have been demonstrated to alter the number of HSC niches (7
). To determine whether changing the number of osteoblastic niche cells influences metastasis, mice were pretreated with PTH (50–80 μg/kg for 3 weeks), which has been shown to increase endosteal osteoblastic niches and subsequently HSC numbers in BM (Figure G; Supplemental Figure 4, C and D; and ref. 7
). To ensure no direct effect of PTH on the tumor cells themselves, the animals were rested prior to establishing s.c. primary tumors. After 3 weeks, the number of PCa cells disseminated from the primary tumors into the BM was determined. The data demonstrated that increasing the number of osteoblastic lineage cells also increased the number of metastatic PCa cells in the BM (Figure G and Supplemental Figure 4E).
If increasing the number of HSC niches in BM leads to increased number of metastatic PCa cells in BM, then it would be predicted that decreasing the number of niches in BM would decrease the number of metastatic cells in BM. To test the possibility that decreasing the number of osteoblasts (e.g., by decreasing niche size) reduces metastasis, s.c. metastasis assays were performed in which vertebral bodies (vossicles) derived from wild-type mice or mice expressing the Col2.3Δ-TK transgene (Figure H) served as the metastatic target. Activation of the osteoblast-specific Col2.3Δ-TK transgene results in loss of osteoblasts in the presence of ganciclovir (Supplemental Methods and ref. 15
). There were no changes in PCa cells derived from the primary tumor in wild-type or Col2.3Δ-TK vossicles without osteoblastic niche cell ablation (stimulated with 3–8 mg/kg ganciclovir for 3 weeks; Figure H and Supplemental Figure 4F). There were also no changes in disseminated PCa cells in wild-type vossicles treated with ganciclovir or vehicle alone (Figure H and Supplemental Figure 4F). Importantly, fewer disseminated metastatic PCa cells were recovered from the Col2.3Δ-TK vossicles in animals treated with ganciclovir (Figure H and Supplemental Figure 4F). While the possibility remains that ganciclovir treatment of Col2.3Δ-TK vossicles affects other cells of the BM microenvironment, these data suggest that metastasis depends on the number of osteoblastic niche in bone.
To determine whether tumor growth in BM is dependent on the osteoblastic HSC niches, tumor cells were directly injected into vossicles prior to implantation (13
). Without ganciclovir, the tumors grew at similar rates in wild-type and Col2.3Δ-TK vossicles. In contrast, tumors did not grow in ganciclovir-treated Col2.3Δ-TK vossicles, while having no negative effects on primary tumor growth (Figure , A and B, and Supplemental Figure 5). Ganciclovir treatment ablated osteoblastic lineage cells in Col2.3Δ-TK tissues, but had no effect on the number of osteoblastic lineage cells in vehicle-treated Col2.3Δ-TK or wild-type vossicles (Figure , C and D). Importantly, ganciclovir alone had no effect on PCa cell growth in wild-type vossicles, but decreased PCa cell growth in Col2.3Δ-TK vossicles by inducing apoptosis (Figure , E and F), which suggests that osteoblastic niche cells are critical for PCa cell growth in bone. Together, these data suggest that PCa metastasis and tumor growth in bone is dependent upon the endosteal osteoblastic niche.
The osteoblastic niche is critical for PCa cell growth in bone.
Removing HSCs from the niche increases metastasis.
In steady-state conditions, HSCs and hematopoietic progenitor cell (HPCs) circulate in the blood at low frequency, but increase in response to myelosuppressive chemotherapy or growth factors (e.g., G-CSF; ref. 25
) and when CXCR4/CXCL12 signaling is interrupted (18
). If PCa cells and HSCs compete for the niche, then it should be possible to increase the number of metastatic cells in the niche by vacating HSCs from the niche. To explore this possibility, mechanistic studies were designed to explore whether disseminated PCa cells use the CXCR4/CXCL12 pathway to gain entrance and egress of the HSC niche (18
). Here, experimental animals were pretreated for 5 days with 5 mg/kg AMD3100, an antagonist of CXCR4 that mobilizes HSCs into the peripheral blood to “open” the HSC niche (18
). Subsequently, PCa cells were inoculated into the animals by intracardiac (i.c.) injection to establish disseminated tumor cells (Figure A). More PCa cells had homed to the BM 24 hours later in the bones of the AMD3100-pretreated animals than in the vehicle-treated animals (Figure B and Supplemental Figure 6A). These data suggest that HSC occupancy of the niche limits metastasis.
PCa cells target the HSC niche, and disseminated PCa cells can be mobilized from the BM niche via the CXCR4/CXCL12 axis.
PCa cells can be mobilized out of the HSC niche and into the blood using HSC mobilizing agents.
If PCa cells target the HSC niche, then it should also be possible to induce PCa cells to reenter the peripheral circulation by interfering with CXCR4/CXCL12 signaling, as has been shown for HSCs. To explore this possibility, disseminated PCa cells were first established in bone after tumor implantation (Figure C). After removal of the primary tumors, the animals were rested and then treated with AMD3100 or vehicle. Blood was collected 24 hours after the last AMD3100 injection, and the number of circulating PCa cells determined (Figure C). More circulating PCa cells were found in blood after treatment with AMD3100 versus vehicle (Figure D and Supplemental Figure 6B). AMD3100 mobilized the HSCs from the BM by inhibiting mRNA expression of both CXCR4 and CXCR7 in the HSCs (Figure , E–G). These data suggest that disseminated PCa cells target the HSC niche through the CXCR4/CXCL12 pathway.
G-CSF is another agent that is used clinically to mobilize HSCs out of the niche and into the peripheral blood to improve stem cell collection prior to BMT (25
). Here, the mobilization studies were repeated using recombinant G-CSF. G-CSF mobilized PCa cells from the HSC niche and into the peripheral blood with higher frequency than in vehicle-treated control animals (Figure H and Supplemental Figure 6C). To exclude a direct effect of G-CSF on PCa cells, we also confirmed that PCa cells did not express G-CSF receptors, nor did G-CSF induce PCa cell proliferation (Supplemental Figure 6, D–G). As expected, G-CSF mobilized the HSCs from the BM (Figure I), which suggests that PCa cells use the same mechanisms as HSCs and HPCs to enter the peripheral circulation.
Mobilization of HSCs and HPCs by G-CSF is thought to occur through the induction of 2 pathways. The first is through the production of enzymes (e.g., CD26, cathepsin G, elastase, MMP2, and MMP9) that degrade CXCL12 (25
). To determine whether loss of CXCL12 could be responsible for the mobilization of PCa cells into the peripheral blood once in the HSC niche, levels of MMP2
, and CXCL12 were examined after G-CSF treatment. As expected, G-CSF induced increases in the expression of MMP2
in the BM of G-CSF–treated tumor-bearing mice and resulted in a substantial decrease in the levels of CXCL12 (Figure , J and K). A second major pathway believed to regulate HSC mobilization is through the induction of osteoclastic bone resorption (26
). Therefore, we next explored the role of osteoclasts in mobilization of PCa cells from the niche. Interestingly, G-CSF induced osteoclastogenesis in the presence of disseminated PCa cells (Supplemental Figure 6, H and I). Conversely, AMD3100 — which does not mobilize HSCs by activating osteoclastogenesis, but rather by interfering directly with CXCR4/CXCL12 binding — did not active osteoclasts to mobilize PCa cells, as predicted (Supplemental Figure 6, J and K; and ref. 27
). Together, these data suggest that disseminated PCa cells compete with HSCs for the niche using the same molecular mechanisms as do HSCs to gain access and egress of the niche (e.g., CXCR4/CXCL12).
Direct cell-to-cell competition for niche binding favors PCa cells.
A second molecular mechanism for the competition between HSCs and PCa cells for the niche may be that disseminated PCa cells can displace or outcompete HSCs for adhesion of niche-binding elements. Accordingly, we performed competitive binding assays between HSCs and PCa cells. PCa cells blocked HSC binding to osteoblasts, whereas NMPE control cells were less able to prevent HSC binding to osteoblasts (Figure A). Recently, we demonstrated that Anxa2 expressed by osteoblasts is a critical molecule used by both HSCs and PCa cells for binding to osteoblasts (13
). The competitive binding assays were therefore repeated using Anxa2 as the binding target. As expected, PCa cells blocked the binding of HSCs to Anxa2 better than did the NMPE control cells (Supplemental Figure 7A). Conversely, HSCs more efficiently prevented the binding of NMPE cells to osteoblasts and Anxa2 than they blocked PCa cell binding (Figure B and Supplemental Figure 7B). These effects required direct cell-to-cell interactions, as soluble factors present in the conditioned medium of PCa cells alone were unable to alter HSC binding to osteoblasts (Figure C).
Mechanisms regulating niche competition between PCa cells and HSCs: competition for binding to osteoblasts.
When PCa cells expressing a putative PCa stem cell phenotype (CD133+CD44+) were isolated from culture and used in competitive adhesion assays with HSCs, they were better able to block HSC binding to osteoblasts compared with CD133–CD44– cells (Figure D). These CD133+CD44+ cells expressed higher levels of CXCR4, but lower CXCR7 (Figure , E and F), and progressed through the cell cycle slower than did PCa cells expressing a CD133–CD44– phenotype, based on expression of CCNA1 and CCND1 (encoding cyclin A1 and cyclin D1, respectively; Figure , G and H). Intriguingly, although the CD133+CD44+ cells represented a very small fraction of the total cells found in culture (0.86% ± 0.52% of PC3 and 1.47% ± 0.74% of C4-2B in vitro), the frequency of this population was significantly enriched in BM 24 hours after i.c. injection (Table ).
Frequency of CD133+CD44+ and CD133–CD44– PCa cells
PCa cell occupation of the HSC niche alters the phenotype of HSCs and displaces HSCs from the niche.
A third mechanism that may assist PCa cells in the competition for the HSC niche may be that tumor cells themselves have direct and indirect effects on HSCs. PCa cells may be able to drive HSCs to maturity and into HPC populations so that they eventually vacate the niche. To explore this possibility, mice were implanted with metastatic PCa cells or NMPE control cells to establish disseminated tumors in the BM. HSCs isolated from animals with disseminated PCa cells expressed lower levels of the niche adhesion molecules (NOTCH1
, ref. 7
, ref. 9
) and transcription factors known to regulate HSC self-renewal and proliferation (BMI1
; Figure A and refs. 28
). These results suggest that disseminated PCa cells reduce HSC numbers by altering HSC self-renewal. Consistent with these observations, fewer HSCs were found in the tumor-bearing animals (Figure B). One mechanism to explain this reduction is that HSCs are driven into HPCs pools. Indeed, more HPCs were recovered from the BM of tumor-bearing animals than from that of controls (Figure C). As expected, HSCs and HPCs derived from tumor-bearing animals were induced into a cell-cycling state (Figure , D–F), while there was no effect of PCa cells on HPC apoptosis (Figure G). Although it is not presently clear what molecules are responsible for these activities, HSCs were chemotactic toward PCa cells themselves, but PCa cells did not have similar effects on HSCs, and HSCs were able to enhance the growth of PCa cells (Supplemental Figure 8, A–C).
Mechanisms regulating niche competition between PCa cells and HSCs: PCa cells drive HSCs out from the HSC niche.
If PCa cells compete for the niche with HSCs and HPCs and drive these cells into the circulation, then it would be expected that more HSCs or HPCs would be present in the circulation of men with metastatic bone disease than in age-matched controls. Therefore, HPC assays were performed on peripheral blood collected from subjects with disseminated PCa bone disease. As expected, more circulating HPCs were found in subjects with disseminated disease compared with local disease (defined as PCa with no imaging evidence of metastases and PSA <15 ng/ml) and age-matched controls (Figure H). Although other explanations are possible (e.g., inflammatory cytokines or cachexia), these data are consistent with the hypothesis that disseminated tumor cells target the HSC niche and displace HPCs into the peripheral blood. Together, these data strongly suggest that PCa cells target the HSC niche during metastasis.