Human prostate cancer metastases target the hematopoietic stem cell niche to establish footholds in mouse bone marrow

Abstract

HSC homing, quiescence, and self-renewal depend on the bone marrow HSC niche. A large proportion of solid tumor metastases are bone metastases, known to usurp HSC homing pathways to establish footholds in the bone marrow. However, it is not clear whether tumors target the HSC niche during metastasis. Here we have shown in a mouse model of metastasis that human prostate cancer (PCa) cells directly compete with HSCs for occupancy of the mouse HSC niche. Importantly, increasing the niche size promoted metastasis, whereas decreasing the niche size compromised dissemination. Furthermore, disseminated PCa cells could be mobilized out of the niche and back into the circulation using HSC mobilization protocols. Finally, once in the niche, tumor cells reduced HSC numbers by driving their terminal differentiation. These data provide what we believe to be the first evidence that the HSC niche serves as a direct target for PCa during dissemination and plays a central role in bone metastases. Our work may lead to better understanding of the molecular events involved in bone metastases and new therapeutic avenues for an incurable disease.

Figure 1. PCa cells compete for the HSC niche and prevent HSC engraftment.

(A) Experimental model of BMT in the presence or absence of disseminated PCa cells. (B) Fewer donor HSCs (CD45.2) were able to engraft into NOD/SCID mice (CD45.1) when disseminated tumor cells (PC3 or C4-2B PCa cells) were present in BM. *P < 0.05, ^#P < 0.01 versus NMPE, Student’s t test (n = 10 per group). (C) At 4, 8, 12, and 16 weeks after transplantation, the establishment of metastases was followed by bioluminescent imaging. Data are representative of bioluminescent imaging of mice that had developed metastases at approximately 40 weeks. Dashed circles denote where the primary s.c. tumors were implanted and removed. Arrows show metastatic PCa. (D) Representative images of BM histology of mice that developed micrometastases at 16 weeks. Original magnification, ×60. Scale bars: 50 μm. (E) Osteoblast numbers were determined in the long bones. (F and G) mRNA levels of (F) CXCR4 and (G) CXCR7 in PCa cells at peripheral blood (PB) and BM. Significance of differences was determined by Kruskal-Wallis test.

Figure 2. Direct competition for the HSC niche between HSCs and PCa cells.

(A) Competitive BMT experimental model. BMT was performed in the presence or absence of PC3 or C4-2B PCa cells or NMPE control cells. TBI, total body irradiation. (B) To avoid PCa cell proliferation in vivo, NMPE control cells or PCa cells were irradiated with 8 Gy (2× 4 Gy), and the effects of irradiation on the ability of 5,000 PCa cells to form colonies of 50 cells or greater were analyzed. Nonirradiated cells served as positive controls. Irradiation inhibited the colony-forming abilities of NMPE cells and PCa cells. (C) Kaplan-Meier survival plots after BMT in the presence or absence of PC3 or C4-2B PCa cells or NMPE control cells. Survival was monitored up to 60 days. P = 0.038, PC3 versus NMPE; P = 0.041, C4-2B versus NMPE, log-rank test (n = 10 per group). (D) Representative BM histology after competitive BMT of animals in C. Original magnification, ×40. Scale bars: 50 μm. (E) Engraftment of human BM CD34⁺ cells into NOD/SCID Il2rg^–/– mice in the presence or absence of PC3 or C4-2B PCa cells or NMPE control cells. *P < 0.05, ^#P < 0.01 versus NMPE (n = 10 per group, Student’s t test). Human PCa cells prevented human HSC engraftment.

Figure 3. HSCs and PCa cells colocalize to BM niches through Runx2.

(A) To determine whether metastatic cells and HSC colocalize to the same niche, a confocal microscope was used to track prelabeled LSK HSCs (red) and prelabeled PCa cells (green) 24 hours after transplantation. Nuclei were stained with DAPI (blue). DIC, differential image contrast. (B–D) SCID mice were implanted with PCa cells. After 3 weeks, the long bones were collected. Representative elements of the BM were triple-stained with (B) anti-HLA antibodies, anti-Runx2 antibodies, and DAPI; (C) anti-CD150 antibodies, anti-lineage antibody cocktail, and anti-Runx2 antibodies; and (D) anti-CD150 antibodies, anti-lineage antibody cocktail, and anti-HLA antibodies. Arrows denote colocalization of HSCs and PCa cells (A and D), osteoblasts and PCa cells (B), or osteoblasts and HSCs (C). Original magnification, ×60. Scale bars: 10 μm.

Figure 4. HSCs and PCa cells colocalize to BM niches, and alteration of niche size regulates tumor dissemination.

(A and B) To determine whether metastatic cells and HSCs colocalize to the same niche, multiphoton imaging was used to track prelabeled LSK HSCs (red) and (A) prelabeled PCa cells (green) or (B) NMPE control cells 24 hours after transplantation. Nuclei were stained with DAPI (blue). Original magnification, ×200. (C) Statistical analyses of A. (D) SLAM HSCs and PC3 or C4-2B PCa cells colocalized to a single osteoblast in vitro, as imaged by confocal microscopy. (E) Statistical analyses of in vitro adhesion assays to Anxa2^+/+ versus Anxa2^–/– osteoblasts (see D). (F) SLAM HSCs, but not NMPE cells, in vitro localized to a single osteoblast. NMPE cells were unable to bind to Anxa2^+/+ or Anxa2^–/– osteoblasts. (G) To expand the osteoblast numbers, animals were pretreated with vehicle or PTH prior to establishing primary tumors, and the number of metastatic PC3 cells was determined at 3 weeks (n = 8 per group). *P < 0.05, #P < 0.01 versus vehicle. (H) Homing of PC3 cells to Col2.3Δ-TK versus control vossicles with or without ganciclovir (n = 8 per group). The number of disseminated PCa cells homed to vehicle-treated control vossicles was set as 100%. Significance of differences was determined by Student’s t test (C and E) or Kruskal-Wallis test (G and H). Scale bars: 10 μm (A and B); 50 μm (D and F).

Figure 5. The osteoblastic niche is critical for PCa cell growth in bone.

Luciferase-labeled PC3 cells were placed directly into Col2.3Δ-TK or control vossicles, which were subsequently implanted into the immunodeficient mice. Mice were treated with either ganciclovir or vehicle for 3 weeks to ablate the osteoblast niche. (A) Bioluminescent imaging of vossicles was performed over time, demonstrating no tumor growth in Col2.3Δ-TK vossicles in the presence of ganciclovir (n = 10 per group). (B) Cytokeratin-immunostained vossicles. Arrowheads denote cytokeratin-positive cells. (C) H&E examination of vossicles. Arrowheads denote endosteal osteoblasts. (D) Osteoblast number was quantified with H&E staining. (E) Representative TUNEL-stained vossicles. Original magnification, ×60. (F) To determine whether luciferase-labeled PC3 cells in the vossicles had undergone apoptosis, vossicles were recovered and triturated, and cells were filtered through a 40-μm cell strainer to obtain single-cell suspensions. The resulting cells were incubated first with a FITC-conjugated HLA-ABC antibody, where greater than 99% of the luciferase-labeled PC3 cells were positively stained (not shown). Thereafter, percent apoptosis of luciferase-labeled PC3 cells in the vossicles was analyzed using PE-conjugated annexin V/7-ADD by gating on HLA-ABC (n = 4 per group). Significance of differences was determined by Student’s t test. Scale bars: 50 μm (B, C, and E).

Figure 6. PCa cells target the HSC niche, and disseminated PCa cells can be mobilized from the BM niche via the CXCR4/CXCL12 axis.

(A) Experimental model of HSC mobilization out of the niche via AMD3100 treatment to open the HSC niche. (B) PCa cell number in BM after i.c. injection of 1 × 10⁶ cells after AMD3100 mobilization of HSCs. *P < 0.05, ^#P < 0.01 versus vehicle. (C) Experimental model to determine whether AMD3100 mobilizes disseminated PCa cells from BM (n = 8 per group). (D) Peripheral blood levels of PC3 cells mobilized with AMD3100 or vehicle, evaluated by QPCR. (E) SLAM HSC numbers in the BM after AMD3100 treatment were enumerated by FACS. (F and G) mRNA levels of (F) CXCR4 and (G) CXCR7 in HSCs at peripheral blood and BM with or without AMD3100 treatment. (H) Peripheral blood levels of PC3 cells mobilized with G-CSF or vehicle, evaluated by QPCR. (I) Number of HSCs in BM after G-CSF treatment, enumerated by FACS. (J) BM mRNA levels of MMP2 and MMP9 after G-CSF treatment, determined by QPCR. *P < 0.05, ^#P < 0.01 versus vehicle. (K) CXCL12-immunostained BM. Original magnification, ×60. Scale bars: 50 μm. Significance of differences was determined by Kruskal-Wallis test (B, D, F–H, and J) or Student’s t test (E and I).

Figure 7. Mechanisms regulating niche competition between PCa cells and HSCs: competition for binding to osteoblasts.

(A) Competition binding assays to murine osteoblasts between 10⁴ LSK HSCs and 0–10⁵ PCa cells or NMPE control cells. (B) A fixed number of labeled NMPE control cells and PCa cells (10⁴ cells) and 0–10⁵ HSCs were layered onto murine osteoblasts. The binding ability of NMPE control cells and PCa cells to osteoblasts in the presence of HSCs was evaluated using a fluorescent plate reader. (C) A fixed number of fluorescently labeled HSCs (10⁴ cells) was layered onto murine osteoblasts. At the same time, cultures were treated with 0–1 μg/μl of medium alone or conditioned medium (CM) derived from NMPE control cells and PCa cells. The binding ability of HSCs was measured by fluorescent plate reader. (D) Competition binding assays between 10⁴ HSCs and 10³ CD133⁺CD44⁺ or CD133^–CD44^– PCa cells. Data are from 3 independent experiments. (E–H) mRNA levels of (E) CXCR4, (F) CXCR7, (G) CCND1, and (H) CCNA1 in CD133⁺CD44⁺ or CD133^–CD44^– PCa cells. Significance of differences was determined by Student’s t test (A–D) or Kruskal-Wallis test (E–H).

Figure 8. Mechanisms regulating niche competition between PCa cells and HSCs: PCa cells drive HSCs out from the HSC niche.

(A–G) SCID mice were implanted either NMPE cells or PCa cells (PC3 or C4-2B) (n = 5 per group). After 3 weeks, the BM cells were collected. (A) Expression of stem cell survival and cell-to-cell adhesion genes in SLAM HSCs was evaluated by quantitative real-time RT-PCR (n = 5 per group). *P < 0.05, ^#P < 0.01 versus NMPE. (B) SLAM HSC numbers in BM were counted by FACS, and (C) HPC numbers were determined using colony-forming assays. (D and E) mRNA levels of (D) CCND1 and (E) CCNA2 in HSCs. (F) Cell cycle (Ki-67–positive cells) and (G) apoptotic state (PE-conjugated annexin V/7-ADD) of HPCs were analyzed by gating on Lin^–Sca-1⁺ populations. (H) Peripheral blood was collected from subjects with local PCa disease (n = 18, 61.6 ± 9.2 years of age) and disseminated PCa disease (n = 39, 68.1 ± 11.1 years of age). The number of hematopoietic colonies was compared with that in healthy controls (young age, n = 13, 34.3 ± 5.6 years; age-matched, n = 11, 62.4 ± 7.1 years). Significance of differences was determined by Kruskal-Wallis test (A, D, and E) or Student’s t test (B, C, and F–H); P values in B–F are versus NMPE.

Figure 9. Competition between disseminated PCa cells and HSCs for the endosteal niche facilitates metastasis.

The endosteal (or osteoblast) niche is thought to maintain HSCs quiescence and regulate differentiation. It is also appreciated that many solid tumors metastasize to the bone. Our hypothesis is that metastatic cells shed from a primary tumor compete with HSCs to engage the endosteal niche, which suggests that solid tumors use the HSC niche as metastatic niche. Once in the niche, disseminated cells may remain in a quiescent /dormant state for extended periods of time. Eventually, however, metastatic growth exceeds the niche’s regulatory capacity, and clinically relevant disease occurs.