Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis.
ABSTRACT: 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 underinvestigated. 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 tumour-secreted factors, express CCL5 that is not expressed either in normal LECs or in cancer cells, and direct tumour dissemination into these tissues. Moreover, tumour-conditioned LECs promote angiogenesis in these organs, allowing tumour extravasation and colonization. Mechanistically, tumour cell-secreted IL6 causes Stat3 phosphorylation in LECs. This pStat3 induces HIF-1? 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.
Project description:Tumor-associated lymphatic vessels (LV) serve as a route of cancer dissemination through the prometastatic crosstalk between lymphatic endothelial cells (LECs) lining the LVs and cancer cells. Compared to blood endothelial cell-derived angiocrine factors, however, LEC-secreted factors in the tumor microenvironment and their roles in tumor metastasis are poorly understood. Here, we report that ELK3 expressed in LECs contributes to the dissemination of cancer cells during tumor growth by providing oncogenic miRNAs to tumor cells through exosomes. We found that conditioned medium from ELK3-suppressed LECs (LCM) lost its ability to promote the migration and invasion of breast cancer cells such as MDA-MB-231, Hs578T and BT20 in vitro. Suppression of ELK3 in LECs diminished the ability of LECs to promote tumor growth and metastasis of MDA-MB-231 in vivo. Exosomes derived from LECs significantly increased the migration and invasion of MDA-MB-231 in vitro, but ELK3 suppression significantly diminished the pro-oncogenic activity of exosomes from LECs. Based on the miRNA expression profiles of LECs and functional analysis, we identified miR-503-3p, miR-4269 and miR-30e-3p as downstream targets of ELK3 in LECs, which cause the above phenotype of cancer cells. These findings strongly suggest that ELK3 expressed in LECs is a major regulator that controls the communication between the tumor microenvironment and tumors to support cancer metastasis.
Project description:Angiogenesis, the formation of new blood vessels from preexisting blood vessels, is a process that supports tumor growth and metastatic dissemination. Lymphangiogenesis also facilitates metastasis by increasing dissemination through the lymphatic vessels (LVs). Even after treatment with antiangiogenic agents, breast cancer patients are vulnerable to LV-mediated metastasis. We report that a 14-amino acid peptide derived from transmembrane protein 45A shows multimodal inhibition of lymphangiogenesis and angiogenesis in breast cancer. The peptide blocks lymphangiogenic and angiogenic phenotypes of lymphatic and blood endothelial cells induced by tumor-conditioned media prepared from MDA-MB-231 breast cancer cells. The peptide delays growth of MDA-MB-231 tumor xenografts and normalizes tumor-conditioned lymph nodes (LNs). These studies demonstrate the antilymphangiogenic and antiangiogenic potential of the peptide against primary tumors and premetastatic, tumor-conditioned regional LNs. Mechanistically, the peptide blocks vascular endothelial growth factor receptors 2 and 3 (VEGFR2/3) and downstream proteins by binding to neuropilin 1/2 (NRP1/2) and inhibiting VEGFR2/3 and NRP1/2 complex formation in the presence of VEGFA/C.
Project description:Metastatic dissemination employs both the blood and lymphatic vascular systems. Solid tumors dynamically remodel and generate both vessel types during cancer progression. Lymphatic vessel invasion and cancer cells in the tumor-draining lymph nodes (LNs) are prognostic markers for breast cancer metastasis and patient outcome, and tumor-induced lymphangiogenesis likely influences metastasis. Deregulated tumor tissue fluid homeostasis and immune trafficking associated with tumor lymphangiogenesis may contribute to metastatic spreading; however, the precise functional characterization of lymphatic endothelial cells (LECs) in tumors is challenged by the lack of specific reagents to decipher their rate-limiting role in metastasis. Therefore, we generated novel transgenic mice (PDPN promoter-driven Cre recombinase transgene [PDPN-Cre] and PDPN promoter-driven thymidine kinase transgene [PDPN-tk]) that allow for the identification and genetically controlled depletion of proliferating podoplanin (Pdpn)-expressing LECs. We demonstrate that suppression of lymphangiogenesis is successfully achieved in lymphangioma lesions induced in the PDPN-tk mice. In multiple metastatic breast cancer mouse models, we identified distinct roles for LECs in primary and metastatic tumors. Our findings support the functional contribution of primary tumor lymphangiogenesis in controlling metastasis to axillary LNs and lung parenchyma. Reduced lymphatic vessel density enhanced primary tumor lymphedema and increased the frequency of intratumoral macrophages but was not associated with a significant impact on primary tumor growth despite a marked reduction in metastatic dissemination. Our findings identify the rate-limiting contribution of the breast tumor lymphatic vessels for lung metastasis.
Project description:During the last few years, diverse studies have shown that tumors can actively interact with the lymphatic system and promote metastases development. In order to examine the molecular mechanisms involved in this interaction, we co-cultured tumor and lymphatic endothelial cells (LEC) and subsequently analyzed the molecular alterations of LECs. Therefore, LECs were co-cultivated with either a highly or weakly metastatic breast cancer cell line using contact (mixture) and non-contact (transwell) co-cultures. mRNA profiles from LECs were subsequently analyzed for genes specifically induced by highly metastatic tumor cells ("metastatic specific"). Among the up-regulated "metastatic specific" genes, we found candidates involved in cell cycle, cell adhesion and motility (BST2, E-selectin, and HMMR), cytokines (CCL7, CXCL6, CXCL1, and CSF2) and factors of the complement system (C1R, C3, and CFB). Among the down-regulated genes, we detected the hyaluronan receptor STAB2, angiogenic factor apelin receptor (APLNR), and the glycosylation enzyme MAN1A1. In an additional prostate cancer co-culture model, we could confirm a "metastatic specific" upregulation of E-selectin and CCL7 in LECs after interaction with the prostate cancer cell lines LNCAP (highly metastatic) and DU145 (weakly metastatic). These data allowed us to identify a set of genes regulated in LECs during in vitro communication with cancer cells, which might subsequently facilitate lymphatic metastasis.
Project description:Chondrosarcoma is the second most frequently occurring type of bone malignancy that is characterized by the distant metastasis propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumor lymphangiogenesis and lymphatic metastasis. Chemokine CCL5 has been reported to facilitate angiogenesis and metastasis in chondrosarcoma. However, the effect of chemokine CCL5 on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has largely remained a mystery. In this study, we showed a clinical correlation between CCL5 and VEGF-C as well as tumor stage in human chondrosarcoma tissues. We further demonstrated that CCL5 promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium (CM) from CCL5-overexpressed cells significantly induced tube formation of human lymphatic endothelial cells (LECs). Mechanistic investigations showed that CCL5 activated VEGF-C-dependent lymphangiogenesis by down-regulating miR-507. Moreover, inhibiting CCL5 dramatically reduced VEGF-C and lymphangiogenesis in the chondrosarcoma xenograft animal model. Collectively, we document for the first time that CCL5 induces tumor lymphangiogenesis by the induction of VEGF-C in human cancer cells. Our present study reveals miR-507/VEGF-C signaling as a novel mechanism in CCL5-mediated tumor lymphangiogenesis. Targeting both CCL5 and VEGF-C pathways might serve as the potential therapeutic strategy to block cancer progression and metastasis in chondrosarcoma.
Project description:Tumor-associated lymphatic vessels (LVs) play multiple roles during tumor progression, including promotion of metastasis and regulation of antitumor immune responses by delivering antigen from the tumor bed to draining lymph nodes (LNs). Under steady-state conditions, LN resident lymphatic endothelial cells (LECs) have been found to maintain peripheral tolerance by directly inhibiting autoreactive T-cells. Similarly, tumor-associated lymphatic endothelium has been suggested to reduce antitumor T-cell responses, but the mechanisms that mediate this effect have not been clarified. Using two distinct experimental tumor models, we found that tumor-associated LVs gain expression of the T-cell inhibitory molecule PDL1, similar to LN resident LECs, whereas tumor-associated blood vessels downregulate PDL1. The observed lymphatic upregulation of PDL1 was likely due to IFN-g released by stromal cells in the tumor microenvironment. Furthermore, we found that blocking PDL1 results in increased T-cell stimulation by antigen-presenting LECs in vitro. Taken together, our data suggest that peripheral, tumor-associated lymphatic endothelium contributes to T-cell inhibition, by a mechanism similar to peripheral tolerance maintenance described for LN resident LECs. These findings may have clinical implications for cancer therapy, as lymphatic expression of PDL1 could represent a new biomarker to select patients for immunotherapy with PD1 or PDL1 inhibitors.
Project description:Lymphatic valves (LVs) are cusped luminal structures that permit the movement of lymph in only one direction and are therefore critical for proper lymphatic vessel function. Congenital valve aplasia or agenesis can, in some cases, be a direct cause of lymphatic disease. Knowledge about the molecular mechanisms operating during the development and maintenance of LVs may thus aid in the establishment of novel therapeutic approaches to treat lymphatic disorders. In this study, we examined the role of Connexin43 (Cx43), a gap junction protein expressed in lymphatic endothelial cells (LECs), during valve development. Mouse embryos with a null mutation in Cx43 (Gja1) were previously shown to completely lack mesenteric LVs at embryonic day 18. However, interpreting the phenotype of Cx43-/- mice was complicated by the fact that global deletion of Cx43 causes perinatal death due to heart defects during embryogenesis. We have now generated a mouse model (Cx43?LEC) with a lymphatic-specific ablation of Cx43 and show that the absence of Cx43 in LECs causes a delay (rather than a complete block) in LV initiation, an increase in immature valves with incomplete leaflet elongation, a reduction in the total number of valves, and altered lymphatic capillary patterning. The physiological consequences of these lymphatic changes were leaky valves, insufficient lymph transport and reflux, and a high incidence of lethal chylothorax. These results demonstrate that the expression of Cx43 is specifically required in LECs for normal development of LVs.
Project description:Breast cancer-derived vascular endothelial growth factor-C (VEGF-C) has been shown to enhance lymphangiogenesis in lymph nodes to accelerate cancer metastasis. However, the remodeling of lymph node microenvironments by VEGF-C remains elusive. By in vivo selection, we established a subline (named as "LC") with strong lymphatic tropism and high VEGF-C expression from the human MDA-MB-231 breast cancer cell line. Co-culture with LC cells or treatment with LC-conditioned medium upregulated the expression of CXC chemokines in lymphatic endothelial cells (LECs), which could be inhibited by pre-incubation with VEGF-C-neutralizing antibodies and VEGFR3 inhibitors. The chemokines produced by LECs enhanced recruitment of myeloid-derived suppressor cells (MDSCs) to tumor-draining and distant lymph nodes in tumor-bearing mice. Treatment with a CXCR2 inhibitor after tumor cell inoculation dramatically decreased the number of MDSCs in lymph nodes, suggesting the importance of the chemokine/CXCR2 signaling axis in MDSC recruitment. In addition, LEC-released chemokines also stimulated the expression of serum amyloid A1 (SAA1) in cancer cells, enhancing their lymphatic invasion by increasing VE-cadherin phosphorylation, junction disruption, and vascular permeability of LECs. Clinical sample validation confirmed that SAA1 expression was associated with increased lymph node metastasis. Collectively, we reveal a novel mechanism by which cancer cell-derived VEGF-C remodels lymphovascular microenvironments by regulating chemokine production in LECs to promote cancer invasion and MDSC recruitment. Our results also suggest that inhibition of CXCR2 is effective in treating lymphatic metastasis.
Project description:Tumour cells and stromal cells interact in the tumour microenvironment; moreover, stromal cells can acquire abnormalities that contribute to tumour progression. However, interactions between lymphatic endothelial cells (LECs) and tumour cells are largely unexamined. In this study, we aimed to determine whether tumour-specific LECs inhabit the tumour microenvironment and examine their influence on this microenvironment.We isolated normal LECs (NLECs) from a non-metastatic lymph node and tumour-associated LECs (TLECs) from cancerous lymph nodes. We examined proliferative and migratory potency, growth factor production, and gene expression of each type of LEC. Moreover, we developed a co-culture system to investigate the interactions between gastric cancer cells and LECs.When compared with NLEC, TLECs had an abnormal shape, high proliferative and migratory abilities, and elevated expression of genes associated with inflammation, cell growth, and cell migration. NLECs co-cultured with gastric cancer cells from the OCUM12 cell line acquired TLEC-like phenotypes. Also, OCUM12 cells co-cultured with TLECs expressed high levels of genes responsible for metastasis.Our results demonstrated that LECs interacted with tumour cells and obtained abnormal phenotypes that could have important roles in tumour progression.
Project description:Metastatic triple-negative breast cancer (TNBC) is a heterogeneous and incurable disease. Numerous studies have been conducted to seek molecular targets to treat TNBC effectively, but chemotherapy is still the main choice for patients with TNBC. We have previously presented evidence of the important roles of interleukin-6 (IL-6) and chemokine (C-C motif) ligand 5 (CCL5) in TNBC tumor growth and metastasis. These experiments highlighted the importance of the crosstalk between cancer cells and stromal lymphatic endothelial cells (LECs) in tumor growth and metastasis.We examined the viability and migration of MDA-MB-231-LN, SUM149, and SUM159 cells co-cultured with LECs when treated with maraviroc (CCR5 inhibitor) and tocilizumab (anti-IL-6 receptor antibody). To assess the anti-tumor effects of the combination of these two drugs in an athymic nude mouse model, MDA-MB-231-LN cells were implanted in the mammary fat pad and maraviroc (8 mg/kg, orally daily) and cMR16-1 (murine surrogate of the anti-IL-6R antibody, 10 mg/kg, IP, 3 days a week) were administrated for 5 weeks and effects on tumor growth and thoracic metastasis were measured.In this study, we used maraviroc and tocilizumab to confirm that IL-6 and CCL5 signaling are key pathways promoting TNBC cell proliferation and migration. Further, in a xenograft mouse model, we showed that tumor growth was dramatically inhibited by cMR16-1, the mouse version of the anti-IL6R antibody. The combination of maraviroc and cMR16-1 caused significant reduction of TNBC tumor growth compared to the single agents. Significantly, the combination of maraviroc and cMR16-1 abrogated thoracic metastasis.Taken together, these findings show that IL-6 and CCL5 signaling, which promote crosstalk between TNBC and lymphatic vessels, are key enhancers of TNBC tumor growth and metastasis. Furthermore, these results demonstrate that a drug combination inhibiting these pathways may be a promising therapy for TNBC patients.