An anti-transforming growth factor beta antibody suppresses metastasis via cooperative effects on multiple cell compartments

Overexpression of transforming growth factor beta (TGF-beta) is frequently associated with metastasis and poor prognosis, and TGF-beta antagonism has been shown to prevent metastasis in preclinical models with surprisingly little toxicity. Here, we have used the transplantable 4T1 model of metastatic breast cancer to address underlying mechanisms. We showed that efficacy of the anti-TGF-beta antibody 1D11 in suppressing metastasis was dependent on a synergistic combination of effects on both the tumor parenchyma and microenvironment. The main outcome was a highly significant enhancement of the CD8+ T-cell-mediated antitumor immune response, but effects on the innate immune response and on angiogenesis also contributed to efficacy. Treatment with 1D11 increased infiltration of natural killer cells and T cells at the metastatic site, and enhanced expression of coactivators (NKG2D) and cytotoxic effectors (perforin and granzyme B) on CD8+ T cells. On the tumor cells, increased expression of an NKG2D ligand (Rae1gamma) and of a death receptor (TNFRSF1A) contributed to enhanced immune cell-mediated recognition and lysis. The data suggest that elevated TGF-beta expression in the tumor microenvironment modulates a complex web of intercellular interactions that aggregately promote metastasis and progression. TGF-beta antibodies reverse this effect, and the absence of a major effect of TGF-beta antagonism on any one cell compartment may be critical for a good therapeutic window and the avoidance of autoimmune complications.     

Effect of inhibition of the lysophosphatidic Acid receptor 1 on metastasis and metastatic dormancy in breast cancer

Background Previous studies identified the human nonmetastatic gene 23 (NME1, hereafter Nm23-H1) as the first metastasis suppressor gene. An inverse relationship between Nm23-H1 and expression of lysophosphatidic acid receptor 1 gene (LPAR1, also known as EDG2 or hereafter LPA1) has also been reported. However, the effects of LPA1 inhibition on primary tumor size, metastasis, and metastatic dormancy have not been investigated. Methods The LPA1 inhibitor Debio-0719 or LPA1 short hairpinned RNA (shRNA) was used. Primary tumor size and metastasis were investigated using the 4T1 spontaneous metastasis mouse model and the MDA-MB-231T experimental metastasis mouse model (n = 13 mice per group). Proliferation and p38 intracellular signaling in tumors and cell lines were determined by immunohistochemistry and western blot to investigate the effects of LPA1 inhibition on metastatic dormancy. An analysis of variance-based two-tailed t test was used to determine a statistically significant difference between treatment groups. Results In the 4T1 spontaneous metastasis mouse model, Debio-0719 inhibited the metastasis of 4T1 cells to the liver (mean = 25.2 liver metastases per histologic section for vehicle-treated mice vs 6.8 for Debio-0719-treated mice, 73.0% reduction, P < .001) and lungs (mean = 6.37 lesions per histologic section for vehicle-treated mice vs 0.73 for Debio-0719-treated mice, 88.5% reduction, P < .001), with no effect on primary tumor size. Similar results were observed using the MDA-MB-231T experimental pulmonary metastasis mouse model. LPA1 shRNA also inhibited metastasis but did not affect primary tumor size. In 4T1 metastases, but not primary tumors, expression of the proliferative markers Ki67 and pErk was reduced by Debio-0719, and phosphorylation of the p38 stress kinase was increased, indicative of metastatic dormancy. Conclusion The data identify Debio-0719 as a drug candidate with metastasis suppressor activity, inducing dormancy at secondary tumor sites.     

Smad-binding defective mutant of transforming growth factor beta type I receptor enhances tumorigenesis but suppresses metastasis of breast cancer cell lines

The role of transforming growth factor beta (TGF-beta) in carcinogenesis is complex, with tumor suppressor and pro-oncogenic activities depending on the particular tumor cell and its stage in malignant progression. We previously have demonstrated in breast cancer cell lines that Smad2/3 signaling played a dominant role in mediating tumor suppressor effects on well-differentiated breast cancer cell lines grown as xenografts and prometastatic effects on a more invasive, metastatic cell line. Our present data based on selective interference with activation of endogenous Smad2 and Smad3 by stable expression of a mutant form of the TGF-beta type I receptor (RImL45) unable to bind Smad2/3 but with a functional kinase again show that reduction in Smad2/3 signaling by expression of RImL45 enhanced the malignancy of xenografted tumors of the well-differentiated MCF10A-derived tumor cell line MCF10CA1h, resulting in formation of larger tumors with a higher proliferative index and more malignant histologic features. In contrast, expression of RImL45 in the more aggressive MCF10CA1a cell line strongly suppressed formation of lung metastases following tail vein injection. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in these cells. Using an in vitro assay, we further show that non-Smad signaling pathways, including p38 and c-Jun NH(2)-terminal kinase, cooperate with TGF-beta/Smads in enhancing migration of metastatic MCF10CA1a cells, but that, although necessary for migration, these other pathways are not sufficient for metastasis.     

Myeloid suppressor cells regulate the lung environment--letter

4T1 murine mammary carcinoma cells implanted in syngeneic Balb/c mice are increasingly being used in metastasis research, with some groups using this model to study tumor-induced accumulation of bone marrow-derived cells in metastatic target organs. Bone marrow-derived cells (including CD11b(+)Gr-1(+) myelomonocytic cells) are thought to modify the local lung microenvironment to facilitate subsequent colonization by metastatic tumor cells. While quantification of metastatic 4T1 tumor cells in various tissues can be done using ex vivo colony-forming assays, detection of metastatic 4T1 cells is often facilitated by expressing fluorescent proteins in the tumor cells prior to implantation. We found that Balb/c mice mount a potent immune response against 4T1 cells expressing green fluorescent protein (GFP) that includes the generation of anti-GFP antibodies in the circulation. Importantly, the number of bone marrow-derived CD11b(+)Gr-1(+) cells and metastatic tumor cells that accumulate in the lungs is significantly decreased in mice implanted with 4T1 cells expressing GFP compared with mice bearing wild-type 4T1 tumors. Taken together, our data caution against the use of GFP-expressing tumor cells in the Balb/c mouse strain, particularly for studying the influence of immunomodulatory cells on tumor cell metastasis.     

Tumor-induced immune suppression of in vivo effector T-cell priming is mediated by the B7-H1/PD-1 axis and transforming growth factor beta

We have generated effector T cells from tumor-draining lymph nodes (TDLN) that are efficacious in adoptive immunotherapy. We now examine the effect of concomitant tumors on the generation of effector T cells. We inoculated methylcholanthrene (MCA) 205 in the flanks of normal mice and mice bearing MCA 205 lung metastases. TDLN cells from these mice were activated and expanded in vitro, and adoptively transferred to mice bearing lung metastases. Effector T cells generated from TDLN in mice with only flank tumor mediated potent antitumor activity. However, antitumor efficacy of the effector T cells generated from TDLN in mice with pre-existent lung tumor (cTDLN) was reduced. Phenotyping studies showed that dendritic cells in cTDLN expressed higher levels of B7-H1, whereas cTDLN T cells expressed higher levels of PD-1. The levels of IFNgamma were reduced, and the levels of CD4(+)Foxp3(+) regulatory T cells were increased in cTDLN versus TDLN. The in vitro activation of cTDLN was increased by blocking B7-H1 or transforming growth factor (TGF)-beta. Importantly, we found a synergistic up-regulation of IFNgamma with simultaneous blockade of B7-H1 and TGF-beta that was much greater than observed with TDLN. In vitro activation of cTDLN with anti-B7-H1 and anti-TGF-beta and in vivo administration of these antibodies after adoptive transfer resulted in the abrogation of the suppression associated with cTDLN. These results show a major role for the B7-H1/PD-1 axis and TGF-beta as synergistic suppressive mechanisms in cTDLN. Our data have clinical relevance in the generation of effector T cells in the tumor-bearing host.     

Effect of Ha-rasval12 on nm23 expression, tumor formation and metastasis of the transformants, and immunomodulation in tumor-bearing mice

Overexpression of the Ha-ras(val12) oncogene has frequently been detected in primary and metastatic carcinomas. NM23 is a metastasis inhibition factor and plays a suppressive role in metastasis in many types of cancer. In this study, a stable NIH/3T3 cell line harboring an inducible Ha-ras(val12) oncogene (designated as 7-4) and small interfering RNAs (siRNAs) were used to clarify the inverse correlation between nm23 and Ha-ras expression both in vitro and in vivo. A derivative 7-4/Z-3 cell line harboring a β-galactosidase reporter gene was used to trace cell metastasis in a murine tumor model. The data presented here reveal that Ha-ras(val12) is able to cause cell morphological changes, induce tumor formation, and promote metastasis of tumor cells to the lungs. In mice with metastases, the immune surveillance against tumor formation was suppressed by Ha-ras(val12) overexpression through an increase in T-reg cells and a decrease of cytotoxic T lymphocytes and natural killer cell populations. Our results suggest that the Ha-ras oncogene regulates morphogenesis, tumorigenesis, and metastasis through suppressing nm23 expression and modulation of immune cell function.     

Transforming growth factor beta subverts the immune system into directly promoting tumor growth through interleukin-17

Overexpression of the immunosuppressive cytokine transforming growth factor beta (TGF-beta) is one strategy that tumors have developed to evade effective immunesurveillance. Using transplantable models of breast and colon cancer, we made the unexpected finding that CD8+ cells in tumor-bearing animals can directly promote tumorigenesis, by a mechanism that is dependent on TGF-beta. We showed that CD8+ splenocytes from tumor-bearing mice expressed elevated interleukin (IL)-17 when compared with naive mice, and that CD8+ T cells could be induced to make IL-17 on addition of TGF-beta and IL-6 in vitro. Treatment of mice with anti-TGF-beta antibodies in vivo reduced IL-17 expression both in the tumor and the locoregional lymph nodes. Although IL-17 has not previously been shown to act as a survival factor for epithelial cells, we found that IL-17 suppressed apoptosis of several tumor cell lines in vitro, suggesting that this altered T-cell polarization has the potential to promote tumorigenesis directly, rather than indirectly through inflammatory sequelae. Consistent with this hypothesis, knockdown of the IL-17 receptor in 4T1 mouse mammary cancer cells enhanced apoptosis and decreased tumor growth in vivo. Thus, in addition to suppressing immune surveillance, tumor-induced TGF-beta may actively subvert the CD8+ arm of the immune system into directly promoting tumor growth by an IL-17-dependent mechanism.     

CXC chemokine receptor-4 antagonist blocks both growth of primary tumor and metastasis of head and neck cancer in xenograft mouse models

Squamous cell carcinoma of the head and neck (SCCHN) metastasizes to the lymph nodes and lungs. We have generated previously an orthotopic mouse model for head and neck metastasis and did in vivo selection of SCCHN cells through four rounds of serial metastases. A subpopulation of 686LN cells with high metastatic potential (686LN-Ms) was isolated. When the highly metastatic cells were compared with their low metastatic parental cells (686LN-Ps), we found that CXC chemokine receptor-4 (CXCR4) mRNA levels were significantly higher in the 686LN-Ms cells than the 686LN-Ps cells. Interestingly, the metastatic subclones had lost epithelial morphology and acquired mesenchymal features, which were maintained during cell expansion in vitro. This was featured by decreased E-cadherin and involucrin and increased vimentin and integrin beta(1). These results imply that CXCR4 and epithelial-mesenchymal transition markers can be potential biomarkers to identify the subpopulation of cells with high metastatic potential. Using the orthotopic SCCHN animal model, we showed that anti-CXCR4 treatment suppressed primary tumor growth by inhibiting tumor angiogenesis and prevented lung metastasis. Because the reduction of metastasis seen in the treated group could have resulted from 2-fold reduction in primary tumor size compared with that in the control group, we examined the effects of the CXCR4 antagonist in an experimental metastatic animal model in which 686LN-Ms cells were i.v. injected. 686LN-Ms cells failed to metastasize in the CXCR4 antagonist-treated group, whereas they metastasized to the lungs in the control group. Our data indicate that CXCR4 is an important target to inhibit tumor progression in SCCHN.     

Intravital Förster resonance energy transfer imaging reveals osteopontin‐mediated polymorphonuclear leukocyte activation by tumor cell emboli

Myeloid‐derived suppressor cells (MDSCs) cause paraneoplastic leukemoid reactions and facilitate tumor cell metastasis. However, the interaction of MDSCs with tumor cells in live tissue has not been adequately visualized. To accomplish this task, we developed an intravital imaging protocol to observe metastasized tumor cells in mouse lungs. For visualization of the activation of MDSCs, bone marrow cells derived from transgenic mice expressing a Förster resonance energy transfer biosensor for ERK were implanted into host mice. Under a two‐photon excitation microscope, numerous polymorphonuclear cells (PMNs) were found to infiltrate the lungs of tumor‐bearing mice in which 4T1 mammary tumor cells were implanted into the footpads. By Förster resonance energy transfer imaging, we found ERK activation in PMNs around the 4T1 tumor emboli in the lungs. Because antibody array analysis implied the involvement of osteopontin (OPN) in the metastasis of 4T1 cells, we further analyzed the effect of OPN knockdown. The OPN knockdown in 4T1 cells did not affect the cell growth, but markedly suppressed lung metastasis of 4T1 cells and ERK activation in PMNs in the lung. Intravenous injection of recombinant OPN restored the lung metastasis of OPN‐deficient 4T1 cells, suggesting that OPN functioned in a paracrine manner. It has been reported that ERK activation of neutrophils causes NETosis and that PMNs promote metastasis of tumor cells by NETosis. In agreement with previous reports, the NETosis inhibitor DNase I inhibited lung metastasis of 4T1 cells. These observations suggest that OPN promotes metastasis of 4T1 cells by activating PMNs and inducing NETosis.     

Conditional overexpression of active transforming growth factor beta1 in vivo accelerates metastases of transgenic mammary tumors

To address the role of transforming growth factor (TGF) beta in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-beta on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-beta1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-beta1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-beta1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-beta and increased metastases. Antisense-mediated inhibition of TGF-beta1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-beta in hosts with established TGF-beta-responsive cancers can rapidly accelerate metastatic progression.     

Silencing of transforming growth factor-beta1 in situ by RNA interference for breast cancer: implications for proliferation and migration in vitro and metastasis in vivo

PURPOSE: Overexpression of transforming growth factor (TGF)-beta has been implicated in promoting immune suppression, tumor angiogenesis, tumor cell migration, and invasion in many cancers, including carcinoma of the breast. Thus, targeted down-regulation of TGF-beta1 expression in breast cancer in situ and determination of its implications would provide new treatment approaches for disease management. EXPERIMENTAL DESIGN: Small interfering RNA constructs targeting TGF-beta1 were validated and used to develop clonal derivatives of the metastatic breast cancer cell line MDA-MB-435. The cells were used in several in vitro analyses, including migration, invasion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis, and signaling assays. A wound-healing assay was used to determine migration of the cells in culture and a Boyden chamber transwell assay was used for invasion. Further, the clones were used in an in vivo mouse model for the kinetics of tumor growth and gene expression in the primary site and in lungs upon metastasis. RESULTS: Inhibition of TGF-beta1 expression in MDA-MB-435 cells showed a 35% decrease in migration and a 55% decrease in invasion in vitro, with a 50% increase in proliferation and no effect on apoptosis. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases; however, the primary tumors did not show any difference in the growth kinetics when compared with the parental MDA-MB-435 cells. Analysis of TGF-beta signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in AKT and extracellular signal-regulated kinase activation. Interestingly, analysis of TGF-beta receptor expression showed a decrease in both receptor I and II expression in TGF-beta1 silenced cells. These results suggest that inhibition of TGF-beta1 ligand may act as a negative feedback loop to disrupt the function of all TGF-beta isoforms. CONCLUSIONS: Therapies targeting the TGF-beta signaling pathway may be more effective in late-stage disease to prevent organ metastasis but not primary tumor formation and may be combined with other tumor-targeted therapies normally limited by increased circulating TGF-beta levels.     

Potentiation of invasive capacity of rat ascites hepatoma cells by transforming growth factor-beta

The in vitro invasive capacity of poorly invasive cells (W1), which were cloned from rat ascites hepatoma cells (AH 130), was potentiated dose- and time-dependently by pretreating the cells with transforming growth factor-beta (TGF-beta). This potentiation of invasive capacity was completely abolished by anti-TGF-beta antibody. When the treated cells were ip inoculated into rats, the cells extensively invaded the peritoneum, and formed penetrating tumor nodules. The effect of TGF-beta was reversed by subculturing the treated cells without TGF-beta. The potentiation of invasive capacity by TGF-beta might participate in platelet-associated enhancement of tumor cell metastasis.     

Integrin-dependent response to laminin-511 regulates breast tumor cell invasion and metastasis

The basement membrane protein, laminin (LM)-511, is a potent adhesive and migratory substrate for metastatic breast tumor cells in vitro. Its expression correlates with tumor grade and metastatic potential in vivo. These observations suggest that responsiveness to autocrine or paracrine-derived LM-511 may be an important property regulating breast cancer metastasis in vivo. To address this, we compared the metastatic potential of 4T1 mammary carcinoma cells to that of 4T1 variants isolated by repeated chemotactic migration toward LM-511 in vitro (4T1LMF4) followed by serial injection into the mammary gland and recovery of spontaneous metastases from bone (4T1BM2). Variant subpopulations exhibited a distinct morphology on LM-511 and increased expression of β1 and β4 integrins compared to parental 4T1 cells. Importantly, mice inoculated with 4T1LMF4 and 4T1BM2 variants showed a 2.5- to 4-fold increase in the incidence of spontaneous metastasis to bone compared to 4T1 tumor-bearing mice. Functionally, 4T1BM2 variants were more adherent and more invasive toward LM-511 than parental 4T1 cells. Treatment of 4T1BM2 cells with lebein-1, a disintegrin with selectivity toward LM-type integrin receptors, potently inhibited their migration and invasion toward LM-511. Similarly, α3β1 integrin-dependent migration and invasion of human MDA-MB-231 breast carcinoma cells toward LM-511 were significantly inhibited by lebein-1. Taken together, these results provide strong evidence that LM-511 contributes to the metastasis of breast tumors and suggest that targeting integrin-LM-511 interactions with lebein-1 or other inhibitors of LM-511 receptors may have therapeutic potential for patients with advanced breast cancer.     

Systemic inflammation promotes lung metastasis via E-selectin upregulation in mouse breast cancer model

Systemic inflammation might modulate the microenvironment in the lungs and promotes metastasis. E-selectin, an inflammation inducible endothelial cell adhesion molecule, has been reported to play an important role in homing metastatic cancer cells. To study the effects of E-selectin expression induced by systemic inflammation on breast cancer metastasis, we first treated BALB/c mice with lipopolysaccharide (LPS) to induce systemic inflammation. Pulmonary tissues were analyzed by wet/dry ratio, hematoxylin and eosin (H&E) staining and immunohistochemistry. Then 4T1 cells were injected via tail vein. Lung surface metastasis was counted and detected by histological analysis. LPS-induced E-selectin expression and tumor cells adhesion were assessed by western blotting and immunofluorescence. The circulating levels of proinflammatory cytokines in sera were evaluated by ELISA. Our results showed that a significant increase in breast cancer metastasis to lungs was observed in LPS-treated mice vs. the PBS-treated mice, accompanying with an increased E-selectin expression in pulmonary tissue of LPS-treated mice. In vitro studies showed a significant elevation of E-selectin production in MPVECs which enhanced the adhesion activity of 4T1 cells. Treatment with anti-E-selectin antibody significantly reduced the development of metastasis in vivo, and significantly reduced the adhesion of 4T1 cells to MPVECs in vitro. Our results suggest that systemic inflammation may increase the expression of E-selectin which mediated the lung metastasis of breast cancer in mouse model.     

Targeting tumor hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors

Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1-inducible protein that regulates intra- and extracellular pH under hypoxic conditions and promotes tumor cell survival and invasion in hypoxic microenvironments. Interrogation of 3,630 human breast cancers provided definitive evidence of CAIX as an independent poor prognostic biomarker for distant metastases and survival. shRNA-mediated depletion of CAIX expression in 4T1 mouse metastatic breast cancer cells capable of inducing CAIX in hypoxia resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. Stable depletion of CAIX in MDA-MB-231 human breast cancer xenografts also resulted in attenuation of primary tumor growth. CAIX depletion in the 4T1 cells led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. Treatment of mice harboring CAIX-positive 4T1 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without inhibitory effects on CAIX-negative tumors. Similar inhibitory effects on primary tumor growth were observed in mice harboring orthotopic tumors comprised of lung metatstatic MDA-MB-231 LM2-4(Luc+) cells. Our findings show that CAIX is vital for growth and metastasis of hypoxic breast tumors and is a specific, targetable biomarker for breast cancer metastasis.     

Anti‐endoglin monoclonal antibodies are effective for suppressing metastasis and the primary tumors by targeting tumor vasculature

Anti‐metastatic activity of an antitumor agent is exceedingly important because metastasis is the primary cause of death for most solid cancer patients. In this report, we show that 3 anti‐endoglin (ENG) monoclonal antibodies (mAbs) SN6a, SN6j and SN6k which define individually distinct epitopes of ENG of tumor vasculature are capable of suppressing tumor metastases in the multiple metastasis models. The metastasis models were generated by i.v., s.c. (into flank) or mammary gland fat pad injection of 4T1 murine mammary carcinoma cells and splenic injection of two types of colon26 murine colorectal carcinoma cells. Individual mAbs were injected i.v. via the tail vein of mice. SN6a and SN6j effectively suppressed the formation of metastatic colonies of 4T1 in the lung in all of the three 4T1 metastatic models. In addition, these mAbs were effective for suppressing the primary tumors of 4T1 in the skin and mammary fat pad. These mAbs effectively suppressed microvessel density and angiogenesis in tumors as measured by the Matrigel plug assay in mice. No significant side effects of the administered mAbs were detected. Furthermore, SN6a and SN6j extended survival of the tumor‐bearing mice. SN6j, SN6k and their immunoconjugates with deglycosylated ricin A‐chain were all effective for suppressing hepatic metastasis of colon26. The findings in the present study are clinically relevant in view of the ongoing clinical trial of a humanized (chimerized) form of SN6j. © 2009 UICC     

Targeting lysophosphatidic acid receptor type 1 with Debio 0719 inhibits spontaneous metastasis dissemination of breast cancer cells independently of cell proliferation and angiogenesis

Metastasis is the main cause of death for cancer patients. Targeting factors that control metastasis formation is a major challenge for clinicians. Lysophosphatidic acid (LPA) is a bioactive phospholipid involved in cancer. LPA activates at least six independent G protein-coupled receptors (LPA1-6). Tumor cells frequently co-express multiple LPA receptors, puzzling the contribution of each one to cancer progression. All three receptors, LPA1, LPA2 and LPA3, act as oncogenes and prometastatic factors in the mouse mammary gland. The competitive inhibitor of LPA1 and LPA3 receptors, Ki16425, inhibits efficiently breast cancer bone metastases in animal models. We showed here that Debio 0719, which corresponds to the R-stereoisomer of Ki16425 exhibited highest antagonist activities at LPA1 (IC50=60 nM) and LPA3 (IC50=660 nM) than Ki16425 [IC50=130 nM (LPA1); IC50=2.3 μM (LPA3)]. In vitro, Debio 0719, inhibited LPA-dependent invasion of the 4T1 mouse mammary cancer cells. In vivo, early but not late administration of Debio 0719 (50 mg/kg p.o. twice daily) to BALB/c mice during the course of orthotopic 4T1 primary tumor growth reduced the number of spontaneously disseminated tumor cells to bone and lungs without affecting the growth of primary tumors and tumor-induced angiogenesis. We found that increased LPA1 mRNA expression in primary tumors of breast cancer patients correlated significantly with their positive lymph node status (p<0.001). Altogether, our results suggest that LPA1 controls early events of metastasis independently of cell proliferation and angiogenesis. Therefore, targeting this receptor with Debio 0719 has a high therapeutic potential against metastasis formation for breast cancer patients.