Recruitment of MSCs to the primary tumor is HIF dependent, and MSCs enhance metastasis of BCCs to lungs and LNs.
MSCs exhibit homing to sites of tissue injury and tumor growth (31
). To determine whether HIF activity in the hypoxic tumor microenvironment facilitates recruitment of MSCs to the primary tumor site, MDA-231 human triple-negative BCCs (34
) were implanted in the MFP of SCID mice. When the tumors had grown to 200 mm3
, the mice received daily intraperitoneal injections of either saline or digoxin (2 mg/kg) to inhibit HIF activity (27
). After 1 week of digoxin treatment, CMFDA-labeled human MSCs were injected via tail vein, and primary tumors were harvested 16 hours later. The recruitment of MSCs (derived from a male donor) to the primary tumor (derived from a female donor) was examined by quantitative real-time PCR (qPCR) analysis of genomic DNA using SRY
gene primers (to detect Y chromosome sequences) and by FACS analysis of fluorescently labeled MSCs in cell suspensions prepared from primary tumors. Frozen tumor sections were also analyzed for the presence of fluorescently labeled MSCs. All 3 of these assays revealed significantly decreased recruitment of MSCs in tumors from digoxin- versus saline-treated mice (Figure , A–C).
MSCs are recruited to breast tumors and enhance lung and LN metastasis.
To investigate the functional consequences of interaction between MSCs and BCCs, we cocultured MSCs with MDA-231 BCCs at a 1:1 ratio for 48 hours, followed by MFP implantation of 1 × 106
of the cocultured BCCs+MSCs. Controls included mice injected with BCCs alone — either the same number of BCCs (0.5 × 106
) or the same total number of cells (1 × 106
). BCCs+MSCs did not accelerate growth of the primary tumor (Figure D). However, the lungs and ipsilateral axillary LNs of mice carrying breast tumors derived from BCCs+MSCs displayed a marked increase in metastases (Figure , E–I). The number of lung foci, as determined by H&E staining, and the total lung metastatic burden, as determined by qPCR of human-specific HK2
sequences, were significantly increased in mice injected with BCCs+MSCs (Figure , E–G). The area occupied by BCCs in the ipsilateral axillary LN was also increased in mice bearing BCCs+MSCs (Figure , H and I). In contrast, intravenous injection of BCCs+MSCs or MFP coinjection of BCCs and MSCs (without prior coculture) did not result in increased lung foci compared with injection of BCCs alone (Supplemental Figure 1; supplemental material available online with this article; doi:
). These data suggest that coculture of BCCs+MSCs increases metastasis by promoting steps prior to BCC entry into the circulation, such as tissue invasion or vascular intravasation.
HIFs regulate expression of CXCL10 in MSCs and CXCR3 in BCCs in response to coculture and hypoxia.
Based on the coculture data, we hypothesized that MSCs produce paracrine signals that induce BCCs to metastasize. To investigate crosstalk between BCCs and MSCs, an array of antibodies directed against various cytokines and chemokines was incubated with conditioned medium (CM) isolated from BCCs+MSCs that were cocultured at a 1:1 ratio for 48 hours under either nonhypoxic (20% O2) or hypoxic (1% O2) conditions. Levels differed between the 2 conditions for 13 of the 33 cytokines and chemokines analyzed (Supplemental Figure 2A). We focused on the CXCL10 and CCL5 chemokines, which were expressed at higher levels in CM from coculture of BCCs+MSCs under hypoxic conditions.
We analyzed the expression of CXCL10
mRNA by RT-qPCR using RNA prepared from coculture of BCCs+MSCs or individually cultured BCCs and MSCs under nonhypoxic or hypoxic conditions. CXCL10
expression was induced in the cocultures, and this effect was further enhanced when the cocultured cells were subjected to hypoxia (Figure A). Expression of CXCR3
, the cognate receptor for CXCL10 (36
), was induced by hypoxia in MDA-231 BCCs alone. BCCs+MSCs increased CXCR3
expression, which was further enhanced when the cocultured cells were subjected to hypoxia (Figure B). Similarly, coculture and hypoxia increased the expression of CCL5
(Figure , C and D). Our results confirmed a previous report that CCL5 expression was induced in coculture of BCCs+MSCs (31
) and extended those findings by demonstrating a synergistic effect of hypoxia.
HIFs mediate coculture- and hypoxia-induced CXCL10, CXCR3, CCL5, and CCR5 expression.
To investigate the role of HIFs in these phenomena, we used a previously validated double-knockdown subclone of MDA-231 stably transfected with vectors encoding shRNAs directed against HIF1A
(MDA-231–DKD; referred to herein as DKD cells) and a control subclone stably transfected with empty vector (MDA-231–EV; referred to herein as EV cells) (26
). Expression of CXCR3
mRNA was significantly decreased in DKD+MSCs compared with EV+MSCs at both 20% and 1% O2
(Figure , E and F). CCL5
mRNA levels were also significantly decreased in DKD+MSCs versus EV+MSCs at both 20% and 1% O2
(Supplemental Figure 2, B and C). Expression of RPL13A
mRNA, which is not HIF regulated, was not changed in DKD cells and was not induced by coculture or hypoxia (Supplemental Figure 2D). In contrast to the effect of MSCs, coculture of MDA-231 BCCs with human foreskin fibroblasts did not induce increased expression of CXCL10
(Supplemental Figure 2, H and I), which indicates that the coculture effect is dependent on selective crosstalk between MDA-231 BCCs and MSCs.
To determine the cell types responsible for the production of CXCL10 and CXCR3 under coculture conditions, GFP-expressing MDA-231 BCCs were cocultured with MSCs for 48 hours under nonhypoxic or hypoxic conditions, and FACS was performed using GFP fluorescence and CD105 immunofluorescence to sort for BCCs and MSCs, respectively. RNA was isolated from the sorted cells, and RT-qPCR was performed to analyze CXCR3 and CXCL10 expression. The sorting efficiency was demonstrated using GFP primers to confirm that GFP mRNA was detected only in the BCC population (Supplemental Figure 2E). Analysis of the sorted cells revealed that CXCL10 expression was induced in MSCs, whereas CXCR3 expression was induced in BCCs (Figure , G and H). Similarly, induction of CCL5 was observed in MSCs, and CCR5 expression was induced in BCCs (Supplemental Figure 2, F and G). Taken together, the results presented in Figure and Supplemental Figure 2 indicate that HIF-dependent crosstalk between BCCs and MSCs is mediated through the CXCR3-CXCL10 and CCR5-CCL5 signaling pathways in response to coculture and hypoxia.
Acriflavine blocks hypoxia-induced CXCL10-CXCR3 and CCL5-CCR5 expression.
We next explored whether pharmacological targeting of HIF would inhibit coculture-induced gene expression. Acriflavine abrogates HIF activity by inhibiting the dimerization of HIF-1α or HIF-2α with the HIF-1β subunit (22
). BCCs+MSCs were treated with vehicle or acriflavine (1 μM) for 48 hours at 20% or 1% O2
. The effects of coculture and hypoxia on CXCR3
expression were significantly inhibited in the acriflavine-treated cocultures (Figure ).
Acriflavine blocks coculture-induced expression of CCL5, CCR5, CXCL10, and CXCR3.
Inhibiting CXCR3 or CXCL10 abrogates CXCR3-CXCL10 crosstalk.
We generated MDA-231 subclones that were stably transfected with vector encoding either of 2 different shRNAs targeting CXCR3 (shCR3-1 and shCR3-3) or a nontargeted control (NTC) shRNA (referred to herein as NTC cells). The knockdown efficiency of different subclones was determined by Western blotting (Supplemental Figure 3A). Expression of CXCR3 and, surprisingly, that of CXCL10 was abrogated when the coculture was performed with the CXCR3-knockdown MDA-231 cells (Figure , A and B). MAP kinase signaling correlated with CXCR3 expression and was inhibited when the coculture was performed with CXCR3-knockdown MDA-231 cells (Supplemental Figure 3B). We next added neutralizing antibody (NAb) against CXCL10 or control IgG to cocultures of MSCs with the parental MDA-231 BCCs. Expression of both CXCL10 and CXCR3 mRNA was significantly reduced in the presence of CXCL10 NAb compared with IgG control (Figure , C and D). The reduction of CXCL10 and CXCR3 mRNA levels caused by NAb was reversed when an excess of recombinant CXCL10 was added to the culture medium (Supplemental Figure 3, C and D).
Hypoxia augments crosstalk between BCCs and MSCs by promoting CXCL10-CXCR3 signaling.
Similar findings were observed in MDA-435 cells, another triple-negative human BCC line (37
), when CXCR3 expression was knocked down (using either of 2 different shRNAs) or when CXCL10 NAb was added to cocultures with MSCs (Supplemental Figure 4, A–D). Taken together, these data indicate that binding of CXCL10 to CXCR3 on BCCs induces BCC→MSC signaling that leads to increased CXCL10 expression in MSCs. When CXCR3 activation is blocked by CXCR3
knockdown or CXCL10 NAb, bidirectional signaling does not occur.
In contrast to its effects on CXCL10 and CXCR3 mRNA expression, CXCL10 NAb did not significantly affect the expression of CCR5 or CCL5 mRNA in cocultures of MSCs with MDA-231 or MDA-435 BCCs (Figure , E and F, and Supplemental Figure 4, E and F). These results indicate that loss of CXCL10-CXCR3 signaling did not impede CCL5-CCR5 signaling.
Hypoxia-induced CXCL10 enhances BCC migration and invasion.
We assayed CXCL10 levels in CM from BCCs cultured alone or from BCCs+MSCs. Whereas CXCL10 levels in the CM were very low when BCCs were cultured alone, coculture of EV+MSCs dramatically increased CXCL10 levels, which were further increased under hypoxic conditions (Figure A). CM from DKD+MSCs showed decreased CXCL10 levels, which were not induced by hypoxia (Figure A). Similarly, CXCL10 levels in CM from cocultures of MSCs with CXCR3-knockdown MDA-231 cells were significantly decreased and not induced by hypoxia (Figure B). The increased CXCL10 levels in DKD+MSCs compared with MSCs cultured alone suggests that HIF-independent paracrine signaling also contributes to CXCL10 expression.
CXCL10 enhances BCC migration and invasion.
CXCL10 is a major chemoattractant for activated T lymphocytes and natural killer cells (38
). We therefore evaluated the effect of CXCL10 on the migration and invasion of BCCs. CM from BCCs+MSCs at 20% O2
stimulated increased migration and invasion of naive BCCs, and the effect was augmented when CM from hypoxic cocultures was used (Figure , C and D). This increase in migration and invasion was abrogated when CXCL10 NAb was added to the CM (Figure , C and D). Treatment of control EV and NTC subclones with recombinant CXCL10 stimulated invasion through Matrigel (tumor-derived extracellular matrix), whereas this effect was significantly decreased in DKD and CXCR3
-knockdown (shCR3-1) subclones (Supplemental Figure 5, A and B). These data indicate that CXCL10 produced by MSCs stimulates the HIF-dependent migration and invasion of MDA-231 BCCs bearing the cognate receptor CXCR3.
To identify molecular mechanisms by which coculture of BCCs+MSCs may promote metastasis, we analyzed the expression of prometastatic HIF target genes (21
). Expression of matrix metalloproteinase 9 (MMP9
) and lysyl oxidase (LOX
) mRNAs was induced by coculture and hypoxia (Figure , E and F), whereas coculture did not induce expression of LOX-like 4 (LOXL4
), angiopoietin-like 4 (ANGPTL4
), or L1 cell adhesion molecule (L1CAM
) mRNAs (Supplemental Figure 5, C–E).
Coculture of HIF- or CXCR3-knockdown BCCs with MSCs inhibits metastasis.
Inhibition of HIF-1α or HIF-2α expression impairs primary breast tumor growth and lung metastasis after MFP injection (26
). To evaluate whether HIF-1α/HIF-2α expression in BCCs is required for cocultured MSCs to stimulate metastasis, EV and DKD cells were cocultured with MSCs at 20% O2
for 48 hours prior to MFP injection. Primary tumor growth was not affected by MSC coculture (Figure A). The growth rate of tumors derived from DKD cells was decreased, as previously reported (27
). When tumor volume reached 1,300 mm3
, mice were euthanized to examine metastasis to lungs and LNs. EV+MSCs, but not DKD+MSCs, significantly increased metastasis to the lungs, as determined by qPCR using human-specific primers or by counting the number of metastatic foci on H&E-stained lung sections (Figure , B and C). Human Y chromosome sequences were not detectable by qPCR analysis of genomic DNA isolated from lungs of mice that received MFP injections of BCCs+MSCs, which indicated that MSCs did not colonize the lungs. EV+MSCs, but not DKD+MSCs, also increased metastasis to the ipsilateral axillary LN, as demonstrated by immunohistochemistry using an antibody specific for human vimentin (Figure , D and E).
Effect of MSC coculture on metastasis is lost when HIF or CXCR3 expression is inhibited in BCCs.
To further investigate the role of CXCR3 in breast cancer pathogenesis, we orthotopically implanted CXCR3-knockdown BCCs in the MFP of SCID mice. Primary tumor growth and metastasis of 2 independent subclones of CXCR3-knockdown MDA-231 cells (shCR3-1 and shCR3-3) were compared with those of NTC cells in the absence of MSCs. CXCR3 knockdown in MDA-231 cells did not affect growth of the primary tumor (Supplemental Figure 6, A and B). However, mice bearing the CXCR3-knockdown MDA-231 tumors showed a significantly decreased number of metastatic foci and total metastatic burden in the lungs compared with mice bearing NTC tumors (Supplemental Figure 6, C–E). Ipsilateral axillary LNs isolated from mice bearing CXCR3-knockdown breast tumors contained a significantly decreased number of cancer cells, as assessed by immunohistochemical staining for human vimentin (Supplemental Figure 6, F and G).
Next, we cocultured CXCR3-knockdown or NTC control MDA-231 cells with MSCs for 48 hours prior to MFP injection. Inhibition of CXCR3 expression in BCCs decreased the ability of MSCs to enhance BCC metastasis to the lungs and LNs without affecting primary tumor growth (Figure , F–J). These results indicated that CXCR3 promotes vascular and lymphatic metastasis of BCCs, which is stimulated by crosstalk between BCCs and MSCs.
Coculture enhances HIF activity.
We next analyzed HIF-1α levels in EV+MSCs and DKD+MSCs. HIF-1α expression was induced in EV+MSCs under nonhypoxic conditions, and was further enhanced with hypoxia (Figure A). HIF-1α expression was not induced in DKD+MSCs at 20% or 1% O2 (Figure A). Coculture did not induce HIF1A mRNA levels (Supplemental Figure 7). Thus, coculture of BCCs+MSCs specifically increased HIF-1α protein levels under both hypoxic and nonhypoxic conditions.
Coculture of BCCs+MSCs enhances HIF-1α expression and HIF transcriptional activity in BCCs.
To test whether coculture of BCCs+MSCs stimulates HIF transcriptional activity, MDA-231 BCCs were cotransfected with HIF-dependent reporter plasmid p2.1, which contains a hypoxia response element (HRE) from the human ENO1
gene upstream of SV40 promoter and firefly luciferase (Fluc) coding sequences (40
), and pSV-Renilla, in which Renilla
luciferase (Rluc) expression is driven by the SV40 promoter only. The ratio of Fluc/Rluc activity is a specific measure of HIF transcriptional activity. Transfected cells were cocultured with MSCs and exposed to 20% or 1% O2
for 48 hours. Coculture significantly increased HIF transcriptional activity in EV+MSCs at both 20% and 1% O2
(Figure B), consistent with the induction of HIF-1α protein expression (Figure A). Coculture of DKD+MSCs significantly reduced HIF transcriptional activity (Figure B). In contrast, the effect of coculture on HIF transcriptional activity was not lost when CXCR3 expression was knocked down in MDA-231 BCCs (Figure C). Taken together, these results indicate that coculture of BCCs+MSCs enhances HIF transcriptional activity in BCCs by a mechanism independent of CXCR3 signaling that involves increased HIF-1α protein levels.
CXCR3 is a HIF-1 target gene.
Analysis of the human CXCR3
gene sequence revealed candidate HREs in the 5′-flanking (HRE-1) and 3′-untranslated (HRE-2) regions that contained the HIF binding site sequence 5′-ACGTG-3′ followed by 5′-CACA-3′ (Figure A). This bipartite structure was first identified in the human EPO
) and subsequently found in other HREs, including those in the ALDOA
), and ANGPTL4
) genes (Supplemental Figure 8). To determine whether HIF-1 binds at these sites, ChIP assays were performed in MDA-231 BCCs, which demonstrated hypoxia-inducible binding of HIF-1α and HIF-1β to both HRE-1 and HRE-2 (Figure , B–E). To test whether these putative HREs are functional, a 60-bp fragment encompassing HRE-1 or HRE-2 (Figure A) was inserted downstream of SV40 promoter and Fluc coding sequences in the reporter plasmid pGL2 promoter. MDA-231 BCCs were cotransfected with pGL2–CXCR3
–HRE-1 or pGL2–CXCR3
–HRE-2 and pSV-Renilla and exposed to 20% or 1% O2
for 24 hours. Both HRE-1 and HRE-2 significantly increased Fluc activity in hypoxic MDA-231 BCCs (Figure , F and G). Mutation of 5′-CGT-3′ to 5′-AAA-3′ (Figure A), which eliminates HIF-1 binding (40
), in HRE-1 or HRE-2 significantly decreased hypoxia-induced luciferase activity (Figure , F and G). Taken together, the ChIP and reporter data indicated that HIF-1 binds to HREs present in the 5′-flanking and 3′-untranslated regions of the human CXCR3
gene and directly activates its transcription under hypoxic conditions.
HIF regulates PGF expression in BCCs and VEGFR1 expression in MSCs.
The data presented above demonstrate that production of the chemokines CXCL10 and CCL5 by MSCs enables them to communicate with BCCs, which express the cognate receptors CXCR3 and CCR5, respectively. However, the loss of CXCL10 expression by MSCs when cocultured with BCCs in which CXCR3 was inhibited suggested that BCCs must also communicate with MSCs, i.e., there must be bidirectional signaling. Furthermore, the homing of MSCs to tumors demonstrated in Figure is likely to involve a secreted factor produced by BCCs that binds to a cognate receptor on MSCs. Based on our previous studies of VEGFR1 expression in MSCs (43
), we hypothesized that BCCs may communicate with MSCs by producing placental growth factor (PGF), a ligand that binds specifically to VEGFR1. PGF expression in breast cancer biopsies is associated with an increased risk of metastasis and patient mortality (44
mRNA expression was significantly increased in hypoxic MDA-231 BCCs, and VEGFR1
mRNA expression was induced in hypoxic MSCs (Figure , A and B). Coculture of BCCs+MSCs induced the expression of PGF
mRNA. Expression of PGF
mRNA was markedly decreased in DKD+MSCs compared with EV+MSCs (Figure , C and D). Flow sorting of BCCs and MSCs after coculture showed that PGF
expression was induced in BCCs, and VEGFR1
expression was induced in MSCs (Figure , E and F).
HIF regulates PGF and VEGFR1 expression and facilitates bidirectional signaling.
We assayed secreted PGF levels in CM from BCCs cultured alone or from BCCs+MSCs. PGF levels were induced in the hypoxic CM isolated from MDA-231 or MDA-435 BCCs, which were further increased with coculture and hypoxia (Supplemental Figure 9, A and B). CM from EV+MSCs had dramatically increased PGF levels, which were further increased under hypoxic conditions, whereas PGF levels were markedly decreased in CM from DKD+MSCs (Figure G).
To determine whether PGF regulates CXCL10→CXCR3 signaling between MSCs and BCCs, we inhibited PGF expression in MDA-231 BCCs using shRNA. The knockdown efficiency of subclones (shPGF-1 and shPGF-2) was determined by ELISA (Supplemental Figure 9C). Analysis of PGF-knockdown MDA-231 cells cocultured with MSCs revealed a complete loss of CXCL10 mRNA expression and CXCL10 protein secretion (Figure H and Supplemental Figure 9D). CXCR3 expression was not changed in PGF-knockdown MDA-231 cells (Supplemental Figure 9E), which ruled out the possibility that autocrine effects of PGF on BCCs regulate CXCR3 expression.
HIF and PGF expressed by BCCs facilitate MSC migration and homing.
HIF-1 is required for VEGFR1 expression and chemotactic responses to PGF in mouse MSCs (43
). To study whether PGF secretion from human BCCs stimulates the motility of human MSCs, we isolated CM from NTC, shPGF-1, and shPGF-2 MDA-231 subclones. Compared with CM from NTC cells, CM from shPGF-1 or shPGF-2 knockdown cells induced significantly less MSC migration (Figure , A and B). Furthermore, CM from hypoxic NTC cells markedly increased the chemotactic activity of MSCs, whereas CM from hypoxic shPGF-1 or shPGF-2 cells had no stimulatory effect.
HIF and PGF expressed by BCCs are required for MSC migration and homing.
To determine whether PGF secretion from BCCs promotes recruitment of MSCs to the primary tumor site, NTC, shPGF-1 knockdown, and shPGF-2 knockdown MDA-231 cells were implanted in the MFP of SCID mice. When the tumors had grown to 250 mm3, human MSCs were injected via tail vein, and primary tumors were harvested 16 hours later. Recruitment of MSCs to the primary tumor was examined by qPCR analysis of genomic DNA using SRY gene primers. The recruitment of MSCs was significantly decreased in tumors derived from PGF-knockdown MDA-231 cells compared with NTC-derived tumors (Figure C).
To study migration in a coculture assay, CMFDA-labeled MDA-231 BCCs and CMTPX-labeled MSCs were cocultured in a LiveAssay 2-chamber device coated with fibronectin and allowed to attach overnight. Time-lapse photomicroscopy (Figure D), which was performed for 12 hours at 20% or 1% O2, revealed that the migration of MSCs and BCCs toward each other was significantly increased under hypoxic conditions (Figure , E–H). Coculture with DKD cells or shPGF-1 cells led to decreased migration of MSCs compared with those cocultured with control BCCs (Figure , E and G).
PGF is a HIF-1 target gene.
Analysis of the human PGF gene sequence revealed 2 candidate HIF-1 binding sites in the 5′ flanking region. In PGF, HRE-1 and HRE-2 are located 200 and 2,000 bp upstream of the transcription start site, respectively (Figure A). To determine whether HIF-1 binds to these sites, ChIP assays were performed in MDA-231 BCCs, which demonstrated hypoxia-inducible binding of HIF-1α and HIF-1β to both HRE-1 and HRE-2 (Figure , B–E), providing evidence for direct regulation of PGF gene transcription by HIF-1. To test whether these putative HREs in the PGF gene are functional, a 60-bp fragment encompassing HRE-1 or HRE-2 (Figure A) was inserted downstream of SV40 promoter and Fluc coding sequences in the reporter plasmid pGL2 promoter. MDA-231 BCCs were cotransfected with pGL2–PGF–HRE-1 or pGL2–PGF–HRE-2 and pSV-Renilla and exposed to 20% or 1% O2 for 24 hours. Both HRE-1 and HRE-2 significantly increased luciferase activity in hypoxic MDA-231 BCCs (Figure , F and G). Mutation of 5′-CGT-3′ to 5′-AAA-3′ in HRE-1 or HRE-2 significantly decreased hypoxia-induced luciferase activity (Figure , F and G). Taken together, the ChIP and reporter data indicated that HIF-1 binds to HREs present in the 5′-flanking region of the human PGF gene and directly activates its transcription under hypoxic conditions.
PGF promotes the metastasis of breast cancer cells.
To further investigate the role of PGF in breast cancer pathogenesis, PGF-knockdown BCCs were orthotopically implanted in the MFP of SCID mice. Primary tumor growth and metastasis of the independent shPGF-1 and shPGF-2 MDA-231 subclones were compared with those of NTC control cells. PGF knockdown in MDA-231 BCCs modestly inhibited growth of the primary tumor (Figure , A and B). However, mice bearing the PGF-knockdown MDA-231 tumors showed a significantly decreased number of metastatic foci and total metastatic burden in the lungs compared with NTC tumor–bearing mice (Figure , C–E). Ipsilateral axillary LNs isolated from mice bearing PGF-knockdown breast tumors contained a significantly decreased number of cancer cells, as assessed by immunohistochemical staining for human vimentin (Figure , F and G). Thus, PGF plays a significant role in promoting breast cancer metastasis.
PGF promotes lung and LN metastasis of BCCs.