Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis

Breast cancer metastasis involves lymphatic dissemination in addition to hematogenous spreading. Although stromal lymphatic vessels (LVs) serve as initial metastatic routes, roles of organ-residing LVs are under-investigated. Here we show that lymphatic endothelial cells (LECs), a component of LVs within pre-metastatic niches, are conditioned by triple-negative breast cancer (TNBC) cells to accelerate metastasis. LECs within the lungs and lymph nodes, conditioned by tumor-secreted factors express CCL5 that is not expressed either in normal LECs or cancer cells, and direct tumor dissemination into these tissues. Moreover, tumor-conditioned LECs promote angiogenesis in these organs, allowing tumor extravasation and colonization. Mechanistically, tumor cell-secreted IL6 causes Stat3 phosphorylation in LECs. This pStat3 induces HIF-1α and VEGF, and a pStat3-pc-Jun-pATF-2 ternary complex induces CCL5 expression in LECs. This study demonstrates anti-metastatic activities of multiple repurposed drugs, blocking a self-reinforcing paracrine loop between breast cancer cells and LECs.

We next checked for TCM-induced CCL5 expression in vivo, employing athymic nude mice (female, 5 weeks, NCI) to minimize the effect of T lymphocytes on CCL5 expression. CCL5 is also known as RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted), since T lymphocytes express and secrete it 10 . We injected 50 µL of serum-free media (SFM) or TCM prepared from MB231, SUM149, or MCF7 breast cancer cells subcutaneously as previously described 7,11 . Mouse lymphatic vessels (mLVs) in the lymph nodes (LNs) and lungs from the animals treated with MB231 or SUM149-TCM expressed mouse CCL5 (mCCL5), whereas the mLVs in animals treated with MCF7-TCM or SFM did not ( Fig. 1e-g). Brain tissues where LVs are absent, did not show mCCL5 expression upon MB231-TCM treatment (Fig. 1e,f). We assessed the concentration of mCCL5 in TCMtreated animals ( Supplementary Fig. 3). We did not inoculate tumor cells in these animals so that we could measure mCCL5 that was induced only by the TCM (Supplementary Fig. 3a). TCM treatment induced mCCL5 in these animals; more than 450 pg mL −1 mCCL5 was present in the mouse plasma ( Supplementary Fig. 3b).

CCL5 expressed by MB231-LECs drives metastasis
We observed that conditioned media (CM) obtained from MB231-LECs promotes MB231 cell migration ( Supplementary Fig. 7a). CCL5 can interact with CCR1/3/5 12 , so we blocked CCR1 by BX513, CCR3 by SB328437, and CCR5 by maraviroc to determine which of these receptors induces MB231 cell migration. Only maraviroc blocked MB231 cell migration (Fig. 2a,b). We confirmed that both MB231 and MDA-MB-231-luc-D3H2LN express CCR5 (Fig. 2c), suggesting that LEC-secreted CCL5 triggers chemotaxis of MB231 cells. The effect of the CCR5 inhibitor was compared with that of anti-CCR7 neutralizing antibodies in MB231 cell migration assays ( Supplementary Fig. 7b,c), because CCL21, a chemokine ligand for CCR7, is known as another inducer of lymphatic metastasis 5 . Maraviroc blocked MB231 cell migration induced by MB231-LEC CM, whereas the anti-CCR7 antibody blocked n-LEC CM induced migration, demonstrating that the CCL5-CCR5 axis is essential for tumor cell migration toward tumor-conditioned LECs rather than toward physiological LECs.
We next pre-treated animals with TCM or SFM daily for 2 weeks, followed by inoculation of 2×10 6 MDA-MB-231-luc-D3H2LN breast cancer cells into the upper inguinal mammary fat pads and treatment with maraviroc (8 mg kg −1 per day, p.o.) or vehicle ( Supplementary  Fig. 8a). At week 5, 9 out of 10 mice in the TCM-treated group had metastases while only 2 out of 10 mice in the SFM-treated group had them. In the maraviroc-treated group only 4 mice had metastases showing the anti-metastatic effect of maraviroc (Fig. 2d). Primary tumor growth was not influenced by the treatment (Fig. 2e). Maraviroc treatment inhibited metastasis in the lungs and LNs, as shown by the reduced photon flux in the organs (Fig.  2f,g). The hearts, brains, spleens, and livers did not show significant metastases ( Supplementary Fig. 8b,c). Next, the effect of maraviroc was assessed in spontaneous metastasis models without TCM pre-treatment. We showed potent prevention of lung and LN metastasis by maraviroc treatment in these models as well ( Supplementary Fig. 9). These results demonstrate that the CCL5-CCR5 axis is pivotal for lung and LN metastasis in TCM-induced and spontaneous metastasis models and that it can be targeted to inhibit metastasis.
angiogenesis. Tail-vein injection of FITC-dextran (70 kDa) visualized angiogenesis in the plugs (Fig. 3b,c). Infiltration of the host blood vessels (BVs) into the plugs was also observed (Fig. 3d). Immunostaining with anti-mCD31 (Fig. 3e,f) and anti-lectin antibodies ( Supplementary Fig. 11) showed that the recruitment of the host BVs was increased by TCM. Anti-hVEGFR3 staining was performed to detect human LECs previously included in the matrigel plugs (Fig. 3e,f).

MB231-LECs show angiogenic phenotypes
EC proliferation, migration, adhesion, and tube formation were assessed in MB231-LEC CM (Fig. 4a,b). MB231-LEC CM promoted HUVEC proliferation, migration, and adhesion, compared to n-LEC CM (Fig. 4a). Though robust HUVEC tube formation was observed in MB231-LEC CM, LEC tube formation was relatively poor in the same CM (Fig. 4b), suggesting that the MB231-LEC CM primarily promotes angiogenesis rather than lymphangiogenesis, which is consistent with very low VEGF-C expression in MB231-LECs (Fig. 3g). We next generated growth factor depleted TCM (GF-dep-TCM) by using anti-hVEGF 165 and anti-hEGF neutralizing antibodies ( Supplementary Fig. 13a). HUVEC adhesion assays confirmed that the immunodepletion was successful ( Supplementary Fig.  13b). The immunodepletion was performed because both the TCM containing hVEGF 165 and hEGF and the TCM induced mVEGF 164 in the mouse can promote angiogenesis in vivo thus complicating interpretation of angiogenesis effects. The use of the GF-dep-TCM clarifies that "TCM-induced angiogenesis in vivo" is caused by host-derived mVEGF 164 rather than by hVEGF 165 or hEGF already present in TCM ( Supplementary Fig. 14a).
Next, the anti-mVEGF 164 antibody was tested in GF-dep-TCM induced metastasis models like the one discussed above induced by complete TCM (Fig. 2). Five weeks after tumor inoculation in the induced mice, lungs and LNs were harvested to assess metastases ex vivo (Fig. 5b). Anti-VEGF 164 antibody inhibited metastasis in the LNs and lungs (Fig. 5b,c), demonstrating that lung vascular remodeling and LN angiogenesis are initiated by GF-dep-TCM induced VEGF, and the blockade of the VEGF function prevents metastatic extravasation and colonization.

Dual inhibition of CCR5/VEGF strongly blocks metastasis
We established MB231 tumor xenografts (n=10) without TCM pre-treatment, and collected plasma at 2, 3, 4, and 5 week to estimate human tumor xenograft induced mouse VEGF and mouse CCL5 expression (Fig. 5d). Plasma samples from normal mice without tumors (n=8) were used for controls. Plasma concentration of mCCL5 and mVEGF was increased as tumors grew, compared to normal mice: mCCL5 plasma concentration was 259.2 ± 43.6 pg mL −1 , and mVEGF was 56.1 ± 4.9 pg mL −1 when the mean tumor volume was 1,232 ± 223 mm 3 (week 5). We hypothesized that dual inhibition of CCR5 and VEGF signaling would inhibit metastasis more effectively than single inhibition of each target, as the mCCL5 and mVEGF function as tumor recruitment factor and colonization factor respectively (Fig. 5e). We carried out dual inhibition of CCR5 and VEGF as described in Supplementary Fig. 15. We observed that 60% of the mice had metastases in the anti-mVEGF 164 group, 40% had metastases in the maraviroc group, and only 20% had metastases in the combination group. All the mice (100%) had thoracic metastasis in the no-treatment group (Fig. 5f).

The IL6-Stat3 axis induces CCL5 expression in LECs
We next identified key targets in tumor-conditioned LECs, which are specifically phosphorylated by TCM treatment. Among 46 kinase phosphorylation sites screened, both S727 and Y705 of Stat3 were exclusively phosphorylated in LECs by TCM treatment (Fig.  6a). The presence of phospho-Stat3 (pStat3: Y705) in TCM-treated LECs was confirmed in separate western blots (Fig. 6b). Importantly, the essential role of pStat3 in CCL5 expression in LECs was confirmed by using a small molecule, Stattic, an inhibitor of phosphorylation and dimerization of Stat3 13 (Fig. 6c,d). We next showed that IL6 and GM-CSF are exclusively expressed in TNBC cell lines (MB231 and SUM149), but not in MCF7 or LECs (Fig. 6e,f). GM-CSF was not considered as a key cytokine in the metastatic process because GM-CSF is known to phosphorylate Stat5 14 and we saw no pStat5 in TCM-treated LECs (Fig. 6a). Human IL6 in MB231/ SUM149/ MCF7 TCM and LEC CM was measured by ELISAs. High levels of IL6 were only seen in the TNBC cell lines (Fig. 6g). Only TCM containing IL6 induced pStat3 in LECs and IL6-dep-TCM (TCM immunodepleted of IL6) failed to induce phosphorylation of Stat3 in LECs (Fig. 6h, and Supplementary Fig. 16a,b,d). These data demonstrate that TNBC cell-secreted IL6 is the crucial factor for induction of Stat3 phosphorylation in LECs. We also showed that the IL6-gp130-Jak2-Stat3 axis is critical for IL6 signal transduction ( Supplementary Fig. 17). In functional assays, IL6-dep-TCM did not induce CCL5 expression in LECs compared to intact TCM but still induced some VEGF expression (Fig. 6i). These results show that CCL5 expression in MB231-LECs is totally IL6-driven, but VEGF expression can be induced by IL6 and other unknown factors in the TCM.
To establish the relevance of these results to human disease, we analyzed The Cancer Genome Atlas (TCGA) mRNA sequencing data from TNBC and ER+ PR+ HER2-tumors, and discovered higher levels of expression of IL6 and CCL5 in TNBC ( Supplementary Fig.  18a,b). Moreover, IL6 and CCL5 are significantly associated with lymph node positive breast cancer in TNBC ( Supplementary Fig. 18c,d), suggesting that the IL6-CCL5 axis that we discovered has clinical relevance for metastatic breast cancer patients.
The cAMP-responsive element (CRE) in the promoter of the CCL5 gene is known to regulate its expression in alveolar epithelial cells 15 . ATF-2 binds to the CRE 16 . Moreover, c-Jun and ATF-2 have been observed in a binary complex 17 . Importantly, Stat3 can interact with c-Jun and participate in cooperative transcriptional activation 18 . We hypothesized that the pStat3-pc-Jun-pATF-2 ternary complex would bind to the CRE site in CCL5 promoter. To test this hypothesis, we performed chromatin immunoprecipitation (ChIP) assay with ∼200-bp chromatin fragments by sonication of LECs treated with IL6 (10 ng mL −1 ) ( Fig. 7d and Supplementary Fig. 19). Three regions of the CCL5 promoter with the CRE site (−316 to −69 bp) and two distal sites (−1064 to −815 bp and −474 to −711 bp) were tested ( Supplementary Fig. 19a). We found that pStat3-pc-Jun-pATF-2 ternary complex specifically bound to only the CRE site (site 2) by real time quantitative PCR. In contrast, the distal sites (sites 1 and 3) do not show significant complex-binding capabilities ( Supplementary Fig. 19b,c). Compared to vehicle treatment, ChIPs on LECs with IL6 treatment showed specific pStat3-pc-Jun-pATF-2 ternary complex enrichment for binding to this region (Fig. 7d). Additionally, Electrophoretic Mobility Shift Assays (EMSA) were performed to show binding between the ternary complex and the CRE oligonucleotide (Fig.  7e). When LECs were treated with IL6, nuclear proteins bound to the CRE oligonucleotide, however Stattic or SP600125 treatment inhibited the binding. No binding was observed on the mutated CRE, and excess unlabeled CRE oligonucleotide competitively inhibited the binding (Fig. 7e).
VEGF expression in TCM-treated LECs can be triggered by multiple signaling pathways, since IL6 depletion did not completely inhibit VEGF expression (Fig. 6i). However, we observed that IL6 promoted the expression of HIF-1α in LECs (Fig. 7f), and this expression was blocked by Stattic but not by SP600125, demonstrating that HIF-1α expression is pStat3 dependent, but not associated with pc-Jun or p-ATF-2. Summarizing these immunoblot results (Fig. 7b,c,d,e,f) and VEGF/CCL5 expression data (Fig. 7a), we can conclude that the IL6-induced pStat3-pc-Jun-pATF-2 ternary complex is essential for CCL5 expression; VEGF expression is pStat3 induced and possibly HIF-1α associated and does not require pc-Jun or pATF-2 ( Fig. 7g).

Targeting of IL6 and pStat3 blocks LN and lung metastasis
The mechanistic results above indicate that the IL6-Stat3 axis is a key inducer of CCL5 and VEGF expression in LECs. Thus we tried to inhibit GF-dep-TCM induced metastasis by targeting IL6 and pStat3 as described in Supplementary Fig. 20b. We generated GF/IL6dep-TCM by immunodepleting IL6 from GF-dep-TCM. Separately, we chose S3I-201, another pStat3 inhibitor with the same mechanism of action as Stattic; S3I-201 has been tested in vivo 19 . We showed that S3I-201 inhibited pStat3 levels in IL6-treated LECs ( Supplementary Fig. 20a). All mice pre-treated with GF-dep-TCM for 2 weeks prior to tumor inoculation developed metastases at 5 week; 44% of the mice (4/9) treated with S3I-201 during the pre-treatment phase developed metastases; only 22% (2/9) of the mice pre-treated with GF/IL6-dep-TCM had metastases which was less than the 33% (3/9) of mice with metastases in the SFM treated group (Fig. 8a). We observed significant reductions in lung and LN metastases by IVIS imaging, macroscopic morphology, and anti-cytokeratin immunostaining (Fig. 8b,c and Supplementary Fig. 20c). Tumor size was not influenced by these treatments (Supplementary Fig. 20d). The mechanisms presented in this study are summarized in the schematic (Fig. 8d).

DISCUSSION
According to the "seed and soil hypothesis", metastatic cancer cells function as "seeds" and a particular organ microenvironment serves as the "soil" 20 . It is difficult for cancer cells ("seeds") to survive outside their site of origin, thus they have to find a suitable location ("soil") where they can settle and grow. They also manipulate the microenvironment to optimize these pre-metastatic locations 21 . In this study, we show for the first time that tumor cell-secreted IL6 conditions LECs in the pre-metastatic organs to prime them and promotes breast cancer metastasis. Paracrine signals regulated by the IL6-Stat3 axis and operating between cancer cells and LECs play a crucial role in the induction of CCL5 and VEGF expression in LECs within pre-metastatic organs facilitating tumor cell recruitment, extravasation, and colonization (Fig. 8d).
We show that TCM containing IL6 induces lymphatic expression of CCL5 in the premetastatic organs, forming chemotactic gradients to recruit CCR5-positive cancer cells into the organs (Fig. 1,2). We measured the concentration of mCCL5 in the plasma of mice treated with TCM over a 2 week-period ( Supplementary Fig. 3). mCCL5 increased with time of TCM treatment, and the increasing trend was sustained for an additional week after stopping the TCM treatment (maximum level = 450 pg mL −1 ). Compared with the level of mCCL5 (250 pg mL −1 ) in normal tumor xenograft models without TCM treatment (Fig. 5d), TCM pre-treatment can create a dramatic CCL5 gradient in the system to facilitate tumor dissemination. TCM-induced metastasis was blocked by maraviroc, a CCR5 inhibitor (Fig.  2). We also evaluated the therapeutic effects of maraviroc in spontaneous metastasis models without TCM (Supplementary Fig. 9). Maraviroc treatment inhibited tumor metastasis, suggesting that the CCL5-CCR5 axis is also central in general and spontaneous metastasis models. The CCL5-CCR5 axis needs to be further investigated in murine tumor models as well, since our nude mouse models may have limitations with the absence of T lymphocytes that could be one of the mediators of metastasis.
To establish the clinical relevance of our findings, we evaluated the IL6-CCL5 axis by analyzing The Cancer Genome Atlas (TCGA) mRNA-sequencing datasets ( Supplementary  Fig. 18). Both IL6 and CCL5 were significantly overexpressed in TNBC tumors over ER +/PR+/HER2-tumors ( Supplementary Fig. 18a,b). This finding is consistent with our results that only TNBC cell lines (MB231 and SUM149) express IL6 and induce CCL5 expression in LECs and that the MCF7 cell line does not (Fig. 1d,6g). In addition, the expression of IL6 and CCL5 mRNAs was significantly correlated in LN-positive TNBC samples over LNnegative samples, suggesting that the IL6 and CCL5 can serve as therapeutic and prognostic markers in TNBC metastasis (Supplementary Fig. 18c). The axis needs to be further studied in other subtypes of breast cancer and other cancers to expand the application.
To expand upon the discovery that organ-residing LECs promote metastasis via CCL5 expression (Fig. 1,2), we examined whether LECs exist in the primary tumors as well to orchestrate metastasis by actually connecting the primary tumors and distant organs. LECs were detected in the MB231 tumors; moreover, LECs within the tumor expressed CCL5 (Supplementary Fig. 21). This suggests that LECs can form a CCL5 gradient even in tumor stroma, which can trigger initial recruitment of cancer cells into the lymphatic system via intratumoral and peritumoral lymphatic vessels. The presence of LECs in the tumor is due to tumor lymphangiogenesis, driven by tumor cell-secreted lymphangiogenic factors such as VEGFC/D 42,43 . To expand on the classical understanding of tumor lymphangiogenesis, we add a new concept that LECs within tumors and distal organs can create chemotactic gradients to facilitate lymphogenous metastasis via the CCL5-CCR5 axis.
We investigated mechanisms of CCL5 upregulation in LECs by TCM. In previous studies of CCL5 regulation, TNFα, not IL6/gp130, induced CCL5 expression in vascular smooth muscle cells 44 in an NFkB dependent manner; NFkB-dependent CCL5 expression has also been studied in other types of cells [44][45][46] . In this study, however, we found that IL6-induced CCL5 is not colocalized with αSMA positive cells (Supplementary Fig. 2b) and is not associated with an NFkB-Stat3 complex ( Supplementary Fig. 16c,d). Instead, pStat3 forms a ternary complex consisting of pStat3, pc-Jun, and pATF-2 in response to IL6, which regulates CCL5 expression in the lymphatic system; this mechanism has not been previously discovered. ChIP and EMSA experiments showed the binding of the ternary complex to the CRE site of the CCL5 promoter (Fig. 7d,e and Supplementary Fig. 19). We additionally tested the effect of EGF on CCL5 expression in LECs, as MB231-LECs express EGF (Fig.  3g) and EGF-derived Src pathways may contribute to activation of Stat3 pathways 29 . EGF treatment phosphorylated c-Jun and ATF-2, but not Stat3 in LECs, and did not induce CCL5 expression ( Supplementary Fig. 22). Interestingly, unlike LECs, HUVECs could not be conditioned by TCM (Fig. 3g and Supplementary Fig. 23). It has been reported that LECs express 3-4 times more gp130, compared to blood endothelial cells (BEC) 47 . Gp130 is a coreceptor of IL6 receptor and plays a role as an IL6 signal transducer 48 . We showed that the gp130-Jak2 axis is a pivotal bridge for IL6-pStat3 signaling transduction in LECs ( Supplementary Fig. 17), and consistently observed less gp130 as well as less pStat3 in BEC, and no TCM effect (Supplementary Fig. 23). Other molecular details of IL6 dependent induction of CCL5 remain to be elucidated.
We showed that tumor-conditioned LECs also promote angiogenesis (Fig. 3,4), which has not been reported before. While physiological LECs maintain angiogenic homeostasis, the TCM-treated LEC secretome is abnormal and highly angiogenic. Breast cancer involves metastasis to the lymph nodes (LNs), thus the LNs need to serve as metastatic niches 49,50 . One way tumors prime the LNs is by enhancing LN angiogenesis ensuring sufficient oxygen and nutrients around metastatic tumors 11,51,52 . We showed that LECs in the LNs are conditioned by TCM and induce LN angiogenesis (Fig. 4c,d,e). We observed that VEGF expression in TCM-induced LECs is partially IL6-driven (Fig. 6i). While IL6-depleted TCM did not induce any CCL5 in LECs, around 65% of total VEGF induced by TCM treatment was still expressed in LECs even in the absence of IL6. This demonstrates that molecules other than IL6 in the TCM, which we have not identified by our analysis, are also involved in metastasis by inducing angiogenesis; this needs to be further studied.
We additionally studied recruitment of CD45 positive leukocytes and F4/80 or Iba1 positive macrophages to the lungs and LNs of TCM treated animals ( Supplementary Fig. 4,5). Importantly, these cells were not colocalized with TCM-induced mCCL5, suggesting that the infiltration of the macrophages or leukocytes is not triggered by mCCL5, and these cells do not express mCCL5 (Supplementary Fig. 6). The recruitment of these leukocytes to the metastatic sites in TCM-treated animals could be caused by other unknown factors. We further hypothesized that macrophages can contribute to angiogenesis in the pre-metastatic organs, as macrophages are derived from monocytes that are differentiated from myeloid progenitor cells. Myeloid cells are one type of bone marrow derived cells (BMDC) that are known to promote angiogenesis in the tumor microenvironment 53 . We investigated CD33positive myeloid cells and assessed mVEGF 164 expression in the myeloid cells and LECs to dissect the relative angiogenic potentials of these cells. We found that both myeloid cells and LECs contribute to angiogenesis by expressing mVEGF 164 (Supplementary Fig. 24). However, the myeloid cells were also not associated with CCL5 ( Supplementary Fig. 24). Thus, LECs play a key role in tumor cell recruitment through the CCL5-CCR5 axis and the pro-angiogenic phenotypes in the niches can be achieved by LECs and other cells like myeloid cells.
Based on these findings, we tested several inhibitors in our metastasis models. We observed that anti-VEGF therapy prevented metastasis, and very surprisingly, the anti-VEGF treatment showed synergy with maraviroc treatment (Fig. 5f). This result suggests that current anti-angiogenic therapies can be combined with the FDA-approved anti-retroviral drug, maraviroc, which is orally available and safe for long term use, giving rise to the possibilities for therapeutic intervention for metastatic breast cancer. We also targeted IL6 and pStat3 by using anti-IL6 antibodies and S3I-201, a pStat3 inhibitor. Both IL6 depletion and S3I-201 treatment inhibited LN and lung metastasis (Fig. 8). This suggests that current anti-IL6 receptor therapy for rheumatoid arthritis (e.g., tocilizumab) may improve metastatic breast cancer outcomes. Collectively, this study proposes a complex molecular crosstalk between TNBC cells and LECs in distal organs leading to enhanced metastasis and identifies the key players, IL6-Stat3, CCL5-CCR5, and VEGF (Fig. 8d), which can be targeted in a singular or combinatorial manner using repurposed drugs. Thus, this study has significant translational relevance.

Conditioned media
When MDA-MB-231, MCF7 and SUM149 cells reached confluence in T175 tissue culture flasks, the normal growth media was replaced with 8 mL serum-free media (SFM). After 24 h incubation, the supernatant was centrifuged and filtered through 0.2 µm syringe filters (Corning). The resulting tumor-conditioned media (TCM) was stored in aliquots at −80°C. When LECs/HUVECs reached 30-40% confluence in T75 tissue culture flasks, EGM was replaced with 30% TCM (TCM:EGM=3:7) to allow the TCM to condition the LECs/ HUVECs. LECs/HUVECs were allowed to grow in the media for 3-4 days then the media was replaced with 3 mL SFM with 2% FBS (not supplemented with bullet kit). After 48 h, the supernatant was centrifuged and filtered. The resulting tumor-conditioned LEC/HUVEC conditioned media (MB231-LECs or MB231-HUVECs) was stored in aliquots at −80°C to avoid multiple freeze thaws.

Platypus cell migration assays
Cancer cell migration was assessed by using the Oris™ cell migration kit (Platypus) 54 . MDA-MB-231 cells were pre-labeled with Cell Tracker Green (Invitrogen) according to the manufacturer's protocol. 50,000 labeled cancer cells in complete media (RPMI-1640) were added to each well of a 96-well plate containing stoppers to prevent the cells from settling in the center region of the wells. Cells were allowed to adhere for 4 h, after which the stoppers were carefully removed. MB231-LEC CM with or without inhibitors was added, and the cells that migrated to the center of the well were quantified by measuring the fluorescence at 485/530 nm on a Victor V plate reader (PerkinElmer). The migrated cells were visualized by imaging on the Eclipse T-100 fluorescence microscope (Nikon). 20 µM of BX513 (Tocris), Maraviroc (R&D Systems), SB328437 (Tocris), or anti-CCR7 antibodies (R&D systems, 30 µg mL −1 ) was used as inhibitors.

ACEA cell migration/adhesion assays
HUVEC migration was assessed, using CIM-plates (Roche) and the RTCA system (ACEA Bioscience); adhesion was assessed using E-plate (Roche) in the RTCA system 55 . Briefly, the membrane of the top chamber of a CIM-plate was coated with fibronectin by adding 40 µL of 20 µg mL −1 fibronectin dissolved in PBS and incubating at 37°C for 30 min. 180 µL of EGM-2 (complete media for HUVECs) or EBM (serum free media) or MB231-LEC CM was added to the bottom chambers. The equilibrated plate was removed from the incubator and 100 µL of the trypsinized cells (45,000 HUVECs per well) with or without inhibitors were added to the top chamber. After 30 min incubation at room temperature, the stabilized chamber was loaded in the RTCA machine and the cell index was measured continuously for 20 h. Cell indices at 20 h were selected for analysis. ACEA E-plates (Roche Diagnostics) were used to measure the extent of HUVEC adhesion. Briefly, HUVECs (25,000 cells per well) in 100 µL of EGM-2 (complete media for HUVECs) or EBM (serum free media) or MB231-LEC CM were added. After equilibrating at room temperature for 30 min, the Eplate was loaded into the RTCA personal system. Cell indices at 3 h were analyzed.

Cell proliferation assays
HUVEC proliferation assays were performed using the WST-1 reagent (Roche) 55 in HUVEC/LEC conditioned media, EGM, or TCM. 2,000 cells per well were plated in 96well plates and allowed to adhere overnight. On the following day the media was exchanged with HUVEC/LEC conditioned media, EGM, or TCM. Three days later the media was replaced with serum-free media (EBM-2) containing WST-1 reagent and the plates were incubated for four hours. Changes in color due to the formazan dye resulting from the cleavage of the tetrazolium salt WST-1 by the mitochondrial succinate-tetrazolium reductase were read on a Victor V fluorescence plate reader (Perkin Elmer, MA) by measuring the absorbance at 450 nm.

Tube formation assays
HUVEC/LEC tube formation assays were performed 55 . 50 µL matrigel (growth factor reduced, BD Biosciences), thawed on ice at 4°C overnight, was loaded in each well of a precooled 96-well plate, and the plate was incubated at 37°C for 30 min. Fifteen thousand HUVECs and LECs in 100 µL LEC conditioned media were added on top of the matrix in the 96-well plate. For controls, LECs and HUVECs in endothelial cell growth media or serum-free media were also loaded. The plate was then incubated at 37°C, and the wells were imaged using a Nikon microscope at 20 h (Nikon).

TCM-induced metastasis models
Animal protocols described in this study were approved by the Institutional Care and Use Committee at the Johns Hopkins Medical Institutions. Before tumor inoculation, we pretreated athymic nude mice (female, 5 weeks, NCI) by injecting 50 µL tumor-conditioned media (TCM) or growth factor-depleted TCM (GF-dep-TCM), or GF/IL6-dep-TCM, or serum-free media (SFM) subcutaneously for 2 weeks daily as described previously 54 . After 2 weeks of induction, luc-MB231 cells (2×10 6 per mouse, 100 µL of 50% matrigel solution) were injected into the upper inguinal mammary fat pad of the animals under anesthesia (50 mg kg −1 ketamine and 5 mg kg −1 acepromazine). The tumor size was measured by using a caliper, and the volume was calculated, using the formula: V = 0.52 × (length) × (width) 2 . We imaged animals every week to track anterior tumor metastases, using the IVIS Xenogen 200 optical imager (Xenogen) after i.p. injection of D-luciferin (Caliper, 150 mg kg −1 ). After 5 weeks, organs were harvested and bathed in D-luciferin solution for 5-10 min and placed in the IVIS imager to detect metastases ex vivo. Luciferase-mediated photon flux was quantified by using Living Image® 3D Analysis (Xenogen). Maraviroc (8 mg kg −1 , R&D systems) was administered orally daily; anti-hVEGF 165 or anti-mVEGF 164 (5 mg kg −1 , R&D systems) was administered intraperitoneally every 4 days; and S3I-201 (5 mg kg −1 , Calbiochem) was administered intravenously every 2 days. One hundred microliter of blood was collected from the tail-vein, and EDTA plasma was prepared to perform ELISAs.

Spontaneous metastasis models without TCM treatment
We established MB231 xenograft models as described above without TCM pre-treatment. We initiated systemic administration of a CCR5 inhibitor immediately after tumor cell inoculation. The tumor size was measured as described above. We imaged animals every week to track anterior tumor metastases up to 7 weeks, using the IVIS Xenogen 200 optical imager (Xenogen) after i.p. injection of D-luciferin (Caliper, 150 mg kg −1 ). After 7 weeks, organs were harvested and bathed in D-luciferin solution for 5-10 min and placed in the IVIS imager to detect metastases ex vivo.

LEC-included matrigel plug assays
High-concentrated matrigel (500 µL, BD Biosciences) containing LECs or HUVECs (2×10 6 per gel) and heparin (10 units per gel) was injected subcutaneously on the ventral side of both flanks of a nude mouse. TCM or SFM (50 µL per injection) was subcutaneously administered daily for 10 days, the mice were euthanized, and the gel plugs were excised and analyzed. For visualizing blood vessels, FITC-dextran (70 kDa, 80 mg kg −1 , Santa-Cruz) was injected through the tail-vein 1h before sacrifice.

Duration of TCM effect in vivo
We assessed time-dependent changes in the concentrations of CCL5 in plasma to understand the duration of the TCM effect in vivo. We treated mice for 2 weeks with TCM or serumfree media (SFM). From week 0 (before the TCM induction), we collected mouse blood samples using the retro-orbital bleeding method, every week up to 7 weeks (4 animals per group). We centrifuged the collected blood samples for 20 min at 2000xg within 30 min of collection, after which the supernatant (EDTA plasma sample) was obtained and stored at −20°C avoiding repeated freeze-thaw cycles. The samples were analyzed using mCCL5 or mVEGF ELISAs.

Histology
LNs and lungs were fixed, frozen and sectioned as above. After blocking with 5% goat serum in PBST for 1 h, at RT, the sections were treated with mouse anti-cytokeratin antibodies (1:500, Sigma) overnight at 4°C. The rest of the 3,3' diaminobenzidine (DAB) procedure was performed according to regular protocols 54 .

Chromatin Immunoprecipitation (ChIP)
ChIP was carried out using the EZChip kit (Millipore) according to the manufacturer's protocol. Five million LEC were seeded into 15 cm plates and grown to 90% confluency in EGM-2MV media. Then they were treated with 10 ng mL −1 IL6 or vehicle overnight. The cells were crosslinked with 1% formaldehyde for 10 min and sonicated with a Covaris S220 (20% duty cycle, 5 intensity, 200 burst per cycle, 30 cycles of 30 sec) for 30 min on ice. The immunoprecipitations were performed using anti-pATF-2, anti-pc-Jun, and anti-pStat3 antibodies (all from Cell Signaling) or control IgG and the ChIP DNA in the complex was amplified using the primers for the CCL5 promoter regions. Three regions of the CCL5 promoter with the CRE site (−316 to −69 bp, site 2) and two distal sites (−1064 to −815 bp, site 1; and −474 to −711 bp, site 3) were tested. The primer sequences for Site 1, Site 2, and Site3 are GGGTTCTGATCCCAACTCTG (forward)/ AGCGCGTGTCAACTCATTTA (reverse); ACTGCCACTCCTTGTTGTCC (forward)/ GCATTGGCCGGTATCATAAG (reverse); TCTGACTCATGCCTGTCAGC (forward)/ GTGCCAAAATCAGCACAATG (reverse), respectively. PCR products were analyzed by agarose gel electrophoresis and by real time quantitative PCR.

Electrophoretic Mobility Shift Assays (EMSA)
The 2 strands of the wild type CRE oligonucleotide (5'-3', AAAGAGGAAACTGATGAGCTCACTCTAGAT) and of mutated CRE (5'-3', AAAGAGGAAACTGATACAGCCACTCTAGAT), conjugated with biotin at the 5' end were synthesized (Invitrogen). Equal amounts of both strands in 0.5 M NaCl, 10 mM Tris pH7.5 were annealed by boiling for 5 min at 95°C, and very gradually cooled on a hot plate. A DNA retardation gel (6%, Novex) was pre-run at 120 V for 50 min at 4°C. For the binding reaction, 3 µg nuclear extract, and 0.5 µg poly(dI-dC) with or without excess unlabeled CRE oligonucleotide were incubated in binding buffer (10 mM Tris, 1 mM EDTA, 50 mM KCl, 10 mM MgCl 2 , 5 µg mL −1 BSA, 0.1 mM DTT) for 10 min at RT, after which oligonucleotide-biotin was added (finally 40 pM) and incubated for 30 min at RT. Ten microliter of binding sample was mixed with 1 µL 5X TBE sample buffer (Invitrogen), loaded on the gel, and run for 1 h at 120 V in 0.5X TBE running buffer. The gel was transferred to a DNA transfer stack (Invitrogen), using the iBlot transfer module (Program 8, 7 min). The nylon membrane was dried and cross-linked under a UV source (305 nm) for 15 min, then probed by the Chemiluminescent Nucleic Acid Detection Module (Pierce).

Coimmunoprecipitation (Co-IP)
LECs (2×10 6 ) treated with Stattic, SP600125, IL6 or EGM, were used to prepare cell lysates or nuclear extracts. Five hundred microliters of cell lysates or 200 µL nuclear extracts were incubated overnight at 4°C with antibodies suitable for IP (1:100 diluted): pStat3, pc-Jun, pATF-2, pNFkB, and NFkB (Cell Signaling). Ten microliters of Protein A/G Plus Agarose (Santa Cruz Biotech) was added and incubated for 3 h at 4°C. The beads were rinsed 3 times with 500 µL cell lysis buffer for IP (Pierce) supplemented with the protease inhibitor and phosphatase inhibitor cocktail 2/3 (Sigma). The protein complex was reduced and separated by SDS-PAGE and probed with the following antibodies in a Western assay: pStat3, pc-Jun, pATF-2, pNFkB, and NFkB (Sigma). All the original gel images of immunoblot analyses are presented in Supplementary Fig. 25.

Lung vascular permeability assays
After 2 weeks of TCM or SFM treatment with or without anti-VEGF antibodies, FITCdextran was injected i.v. through the tail-vein (70 kDa, 80 mg kg −1 ) 1h before sacrifice. Harvested lungs were stained with mCD31 and observed under the LSM-510 confocal microscope.

TCGA data analyses
All analyses of The Cancer Genome Atlas (TCGA) primary breast cancer tissues 56 were performed in R (3.0.1). TCGA Level 3, RSEM v2 gene expression measurements from RNA-sequencing for CCL5 and IL6 were obtained from the cBioPortal with the CRAN cgdsr package 57 . ER and PR status were obtained from cross-platform summaries, and HER2 status from IHC measurements in the TCGA clinical data, with a total of 99 triple negative samples and 326 ER+ PR+ HER2−. Samples were called lymph node positive if at least one lymph node was positive by either IHC or H&E staining, consistent with pathological staging in the TCGA clinical annotations (Supplementary Methods). There were 50 lymph node negative and 37 lymph node positive samples within the triple negative breast cancer subtype. We compared expression between subtypes with one-sided t-tests on log transformed RSEM values. Correlation coefficients and corresponding p-values were computed with Pearson's correlation. All the codes and supplementary data used are described in the Supplementary Methods.

Statistical Analysis
Error bars correspond to s.e.m, unless otherwise stated. Differences between two groups are regarded as significant when P is less than 0.05 using the Student's t-test.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  (a) MB231 cells were pre-labeled with Cell Tracker Green and their migration was assessed using the Oris™ cell migration kit. 50,000 labeled MB231 cells in complete media were added to each well of a 96-well plate containing stoppers to prevent the cells from settling in the center region of the wells. Cells were allowed to adhere for 4 h, after which the stoppers were carefully removed. MB231-LEC CM with or without inhibitors was added, and the cells that migrated to the center of the well were quantified by measuring the fluorescence at 485/530 nm (n=4). Maraviroc, a CCR5 inhibitor potently blocked MB231 cell migration in the presence of MB231-LEC CM at 18 h. Scale bars, 500 µm. (b) Fluorescent signal from the migrated cells from (a) was measured at 485/530 nm and quantified (*P = 0.013, n=4). (c) Human CCR5 levels in 300,000 MB231 and luc-MB231 were measured by western blotting. GAPDH was used as a loading control. (d) Athymic nude mice (4-5 weeks, female, NCI, n=10) were pre-treated with TCM or SFM (50 µL) for 2 weeks before inoculation with luc-MB231 tumor cells and initiation of maraviroc (8 mg kg −1 per day, p.o.) or vehicle treatment. Five weeks later the maraviroc-treated group showed approximately 50% inhibition of metastasis, compared to vehicle-treated group. Red circles represent thoracic metastasis observed with the IVIS imager. (e) Tumor volume was measured using a caliper (n=10), and the volume was calculated using the formula: V = 0.52 × (length) × (width) 2 . (f) Quantification of (g), luciferase-mediated photon flux from the lungs (n=9-10) and the LNs (n=35-39) were obtained by using Living Image® 3D Analysis (Xenogen) (**P = 0.008, *P = 0.042). (g) Representative organ images under the IVIS imager. Data (b,e,f) are reported as mean ± s.e.m. Original gel images of data (c) are presented in Supplementary Fig. 25. LECs or HUVECs (2×10 6 per gel) and heparin (10 units per gel) was injected subcutaneously on the ventral side of both flanks of nude mice. TCM or SFM (50 µL) was subcutaneously administered daily for 10 days, the mice were euthanized, and the gel plugs were excised and analyzed. Scale bar, 10 mm. (b) One hour before sacrifice, FITC-dextran (70 kDa) was injected through the tail-vein to visualize blood vessels in the gel plugs. Plugs were homogenized and the intensity of FITC was measured and normalized to the volume of (a) In the proliferation assays, 2,000 HUVECs per well were plated in 96-well plates and allowed to adhere overnight. On the following day the media was exchanged with LEC conditioned media, EGM, or SFM. Three days later proliferating cells were quantified at 450 nm by using the WST-1 reagent (*P = 0.039, n=6). In the migration assays, 180 µL of EGM-2 or SFM or MB231-LEC CM was added to the bottom chambers (CIM-plates), HUVECs (45,000 cells per well) were added to the top chamber. The bottom and top chambers were combined, loaded in the RTCA system, and the cell index was measured Anti-mVEGF 164 and maraviroc treatment inhibits LN and lung metastasis. (a) Nude mice were treated with GF-dep-TCM or SFM for 2 weeks. Additionally, anti-hVEGF 165 orand anti-mVEGF 164 antibodies (i.p. injection, 5 mg kg −1 , at day 1, 5, 10, 14). FITC-dextran perfusion showed that lung vascular permeability was increased by GF-dep-TCM treatment. The increased permeability was normalized not by anti-hVEGF 165 but by anti-mVEGF 164 . Scale bars, 1,000 µm. (b) Based on the result in (a), we administered anti-mVEGF 164 antibodies to inhibit GF-dep-TCM induced metastasis. Nude mice were treated with SFM or GAPDH was used as a loading control. (d) CCL5 levels were assessed by ELISA following 5 µM Stattic treatment (**P < 0.01, ***P = 0.0008, n=4). (e) Reverse western assays with which is essential for CCL5 expression. pStat3 promotes HIF-1α expression and separately induces VEGF expression. Upon Stattic treatment, pStat3 and the ternary complex disappear, resulting in no expression of CCL5 and VEGF; the pc-Jun-pATF-2 binary complex that remains upon Stattic treatment does not induce either CCL5 or VEGF expression. SP600125 dissociates both ternary and binary complexes, but pStat3 separately induces HIF-1α and VEGF expression. Data (a,d) are reported as mean ± s.e.m. Original gel images of data (b,c,f) are presented in Supplementary Fig. 25. After two weeks of these treatments, inguinal primary tumor was established, and thoracic metastasis was monitored for 5 weeks (as described in Supplementary Fig. 20b). The number of mice in each group with thoracic metastasis is shown. (b) Representative images of live animals in the IVIS imager, and images of harvested lungs. Black arrows represent tumor nodules. (c) Immunohistochemistry with anti-cytokeratin antibodies on the lungs and LNs (cortex and medulla). Metastatic colonies are delineated with red-dotted curves. Scale bars, 500 µm. (d) Graphical summary of the whole study. Breast cancer cells secrete IL6 that interacts with IL6 receptor on LECs within the lungs and LNs. Activated IL6 receptors transduce the signals through glycoprotein 130 (gp130), phosphorylating lymphatic Stat3. pStat3 translocates into the nucleus to form the pStat3-pc-Jun-pATF-2 ternary complex, which is essential for CCL5 expression by targeting the CRE region in its promoter. pStat3 also induces HIF-1α to ultimately express VEGF in LECs. LEC-secreted CCL5 recruits CCR5-positive breast cancer cells into the lymphatic system. The secreted VEGF enhances lung vascular permeability and induces LN angiogenesis to promote metastatic extravasation and colonization. The four possible targets in the overall mechanism were blocked as follows: by anti-IL6 antibody (to target IL6), by maraviroc (to interrupt the CCL5-CCR5 axis), by anti-VEGF antibody (to block LN angiogenesis and lung vascular leakiness), and by Stattic/S3I-201 (to inhibit pStat3). Treatments are marked with red and green circles, which represent "tested in vivo" and "tested in vitro" respectively.