Hematopoietic Stem Cell-derived Adipocytes Promote Tumor Growth and Cancer Cell Migration

Abstract

Adipocytes, apart from their critical role as the energy storage depots, contribute to the composition of the tumor microenvironment. Our previous studies based on a single hematopoietic stem cell (HSC) transplantation model, have revealed a novel source of adipocytes from HSCs via monocyte/macrophage progenitors. Herein, we extend these studies to examine the role of HSC-derived adipocytes (HSC-Ad) in tumor progression. When cultured under adipogenic conditions, bone marrow-derived monocytic progenitors differentiated into adipocytes that accumulated oil droplets containing triglyceride. The adipokine array and ELISAs confirmed secretion of multiple adipokines by HSC-Ad. These adipocytes underwent further development in vivo when injected subcutaneously into C57Bl/6 mice. When co-injected with melanoma B16F1 cells or breast cancer E0771 cells into syngeneic C57Bl/6 mice, HSC-Ad not only accelerated both melanoma and breast tumor growth, but also enhanced vascularization in both tumors. Conditioned media from HSC-Ad supported B16F1 and E0771 cell proliferation and enhanced cell migration in vitro. Among the HSC-Ad secreted adipokines, insulin-like growth factor 1 (IGF-1) played an important role in E0771 cell proliferation. Hepatocyte growth factor (HGF) was indispensable for B16F1 cell migration, whereas HGF and platelet-derived growth factor BB (PDGF-BB) collectively contributed to E0771 cell migration. Expression levels of receptors for IGF-1, HGF, and PDGF-BB correlated with their differential roles in B16F1 and E0771 cell proliferation and migration. Our data suggest that HSC-Ad differentially regulate tumor behavior through distinct mechanisms.

Keywords: Adipocyte; Breast cancer; Hematopoietic stem cell; Melanoma; Tumor microenvironment.

Figure 1

Monocytic precursors give rise to adipocytes in vitro and in vivo-(A) Enrichment of monocytic precursor from bone marrow in the presence of interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF). FACS analysis of CD11b and F4/80 staining demonstrated the differentiation of macrophages from monocytic precursors. (B) Bone marrow-derived macrophages (BM-MΦ), and adipocytes differentiated from hematopoietic stem cell (HSC) via monocytic precursors (HSC-Ad), were stained with Oil Red O (ORO) and visualized with phase contrast imaging. (C) Triglyceride contents were extracted from BM-MΦ and HSC-Ad, and quantified. NA, not detected. (D) BM-MΦ or HSC-Ad were starved overnight and the resulting medium was used as conditioned medium, denoted as MΦ-CM or Ad-CM. MΦ-CM and Ad-CM were subjected to an Adipokine Array (R&D Systems), and the profiles of secreted adipokines were compared. (E) EGFP⁺ HSC-Ad were embedded in Matrigel and injected subcutaneously into C57Bl/6 mice. After 3 weeks, mature adipocytes were extracted, cultured and imaged. Arrows indicated the mature EGFP⁺ adipocytes from HSC origin. Scale bars=25 μm.

Figure 2

HSC-derived adipocytes accelerate melanoma (B16F1) and breast tumor (E0771) growth in vivo-Tumor cells alone ( ), HSC-Ad alone ( ), or both ( ) were injected orthotopically into C57Bl/6 mice. Tumor area was measured on a regular basis (A, D). At the end point when mice were euthanized, tumor volume (B, E) and mass (C, F) were measured. (A-C) B16F1 tumor growth. (D-F) E0771 tumor growth. ^*p≤0.05, ^**p<0.01, ^***p<0.001.

Figure 3

HSC-derived adipocytes enhance B16F1 and E0771 tumor vascularization in vivo-Tumor cells were orthotopically injected into C57Bl/6 mice, either alone or combined with HSC-Ad. (A) Tumor sections (5 μm) were stained with CD31 antibody to indicate blood vessels, and were counterstained with hemotoxylin. (B) Quantification of the number (#) of blood vessels. (C) Lumen area of blood vessels was measured and presented as fold change of co-injection vs injection of tumor cells only. Data was averaged from 3–4 high power fields (HPF, 200X) per section from 3 tumors for each group (n=3). ^***p<0.001. n.s., not significant.

Figure 4

HSC-derived adipocytes support B16F1 and E0771 cell proliferation through activated ERK and AKT pathways in vitro-(A) B16F1 and (B) E0771 cells were cultured in either serum-free αMEM ( )or Ad-CM ( ) for 3 days and cell numbers (#) were quantified daily and averaged from multiple experiments. ^*p<0.05, ^***p<0.001. (C) Tumor cells were pretreated with DMSO or the indicated inhibitors (UO126, 10 μM; MK2206, 1 μM) overnight, followed by the addition of Ad-CM at 37°C for 1h. Cell lysates were subject to SDS-PAGE and blotted with the indicated antibodies. (D) B16F1 ( ) and E0771 ( ) cells were cultured for 3 days in either Ad-CM or Ad-CM supplemented with different concentrations of UO126 or MK2206. Cell numbers were quantified on day 3. Data was averaged from two independent experiments and presented as percentage of Ad-CM.

Figure 5

Insulin-like growth factor 1 (IGF-1)/IGF-1 receptor (IGF-1R) signal axis is responsible for HSC-Ad-stimulated E0771 cell proliferation in vitro-(A) Tumor cells were cultured for 3 days in either serum-free MEM or αMEM supplemented with 100 ng/mL of recombinant insulin growth factor-1 (rIGF-1). Cell numbers were quantified on day 3 and averaged from multiple experiments. Data was presented as percentage of αMEM. (B) Tumor cells were cultured for 3 days in either Ad-CM or Ad-CM supplemented with 1 μg/mL of IGF-1 neutralizing antibodies. Cell numbers were quantified on day 3 and averaged from multiple experiments. Data was presented as percentage of Ad-CM. (C) IGF-1R levels in B16F1 and E0771 cells were compared by western blot, β-actin served as loading control. (D) B16F1( ) and E0771 ( ) cells were cultured for 3 days in either Ad-CM or Ad-CM supplemented with different concentrations of IGF-1R inhibitor OSI-906. Cell numbers were quantified on day 3 and averaged from three independent experiments. Data was presented as percentage of Ad-CM. ^*p<0.05. n.s., not significant.

Figure 6

Hepatocyte growth factor (HGF)/c-Met is important for B16F1 and E0771 cell migration-(A) B16F1 cells and (B) E0771 cells, untreated or treated with 1 μM c-Met inhibitor PHA-665752, were exposed to the indicated medium for 4 h. Migrated cells were counted and cell # were averaged from 10 HPFs from multiple experiments. Ad-CM/del HGF, immune-depletion of HGF from Ad-CM. (C) Tumor cells were exposed to 100 ng/mL of recombinant HGF (rHGF) for 4 h. Migrated cells were counted and averaged from multiple experiments. (D) Concentrations of HGF in the indicated medium were quantified and averaged from multiple experiments with HGF ELISA kit (R&D Systems). NA, not detected. (E) Surface c-Met levels (solid lines) of tumor cells were compared by FACS analysis, isotype IgG (dash lines) served as control. (F) E0771 cells, untreated or treated with 1 μM PHA-665752, were exposed to 100 ng/mL of rHGF for 4 h. Migrated cells were counted and averaged from multiple experiments. ^*p≤0.05, ^**p<0.01, ^***p<0.001. n.s., not significant.

Figure 7

Platelet-derived growth factor-BB (PDGF-BB) mediates E0771 cell migration-(A) PDGF receptor α/β (PDGFRα/β) levels in B16F1 and E0771 cells were compared with western blot, β-actin served as loading control. (B) Tumor cells were exposed to αMEM or 100 ng/mL of recombinant PDGF-BB (rPDGF-BB) for 4 h. Migrated cells were counted and averaged from multiple experiments. (D) Concentrations of PDGF-BB in three independent Ad-CM preparations were quantified and averaged with PDGF-BB ELISA kit (R&D Systems). NA, not detected. (D) E0771 cells, untreated or treated with 100 nM PDGFRα/β inhibitor CP-673451 or a combination of 1 μM PHA-665752 and 100 nM CP-673451, were exposed to the indicated medium for 4 hr. Migrated cells were counted and averaged from multiple experiments. ^**p<0.01, ^***p<0.001. n.s., not significant.