Lymphatic Endothelial Progenitor Cells Research Articles

Abstract A006: Identification of a potential lymphatic progenitor cell population in Vhlh conditional knockout mouse and in ccRCC tissue

Abstract Introduction. We aim to elucidate the role of lymphatic cells in the progression of clear-cell renal cell carcinoma (ccRCC). Objective. Inactivation of tumor suppressor gene von Hippel–Lindau (VHL) is the main cause of ccRCC, whose progression is extensively influenced by the tumor microenvironment (TME). It has been shown that tissue inflammation, with extensive infiltration of lymphocytes, is a feature of ccRCC tissue. Here, the role of the lymphatic cells in the inflammatory microenvironment was analyzed in the kidney with or without VHL inactivation. Methods. The conditional Vhlh knockout mouse (Hoxb7-Cre-GFP; Vhlh loxP/loxP) was established as a ccRCC model. Single-cell RNA sequencing (scRNA-seq) of podoplanin-positive (Pdpn+) cells revealed heterogeneity of lymphatic cells in mouse kidney. Immunostaining of 3-dimensional tissue was used to visualize lymphatic outgrowth from lymphatic vessels. Protein marker expression in mouse kidney and human ccRCC sections was detected by immunofluorescence. Relevant molecular mechanisms were quantified by qPCR, Western blot, flow cytometry, and ELISA. Results. The lymphatic vasculature is responsible for draining interstitial fluid, mediating lymphocyte recruitment, and participating in immune responses, and is a major route for metastasis. Immunofluorescence of the classical lymphatic markers such as Lyve1, Prox1, CD31, and VEGFR3 was observed in the lymphatic vessels including collecting lymphatics, lymphatic valves, and lymphatic capillaries. Moreover, immunostaining of 3-dimensional tissue showed sprouting Pdpn+ cells from lymphatic vessels, more numerous in Vhlh mutants. In Vhlh-deficient kidney tissue, the Pdpn+ cells form linearly arrayed clusters with no or small lumen and exhibit cuboidal cell shape instead of the flattened endothelial shape. Similar PDPN+ clusters of cells were also present in ccRCC tissues, compared with normal adjacent tissues. Interestingly, scRNA-seq identified a subpopulation of the progenitor marker CD133+ cells within the Pdpn+ population, providing evidence for the differentiation of novel lymphatic endothelial progenitor cells (LEPCs) in postnatal tissues in the kidney. Especially, there was a concomitant threefold increase in CD45+ lymphocyte populations in Vhlh conditional knockout mice compared to wild-type. Interestingly, the chemokine CXCl13 was highly expressed in LEPCs and the receptor CXCR5 was expressed in Vhlh-deficient tubule cells and in infiltrating immune cells. These results suggest a crosstalk between LEPCs and Vhlh-deficient cells and immune cells to modulate metastasis and immune responses. Double knockout (Hoxb7-Cre-GFP; VhlhloxP/loxP; Cxcl13-/-) will be designed for further experiments to investigate the role of CXCL13/CXCR5 axis in the progression of ccRCC. Conclusion. These investigations provide novel insights into CXCL13/CXCR5 axis that may orchestrate LEPCs and Vhlh-deficient tubule cells interactions that promote lymphocyte infiltration and potential metastasis within the tumor microenvironment. Citation Format: Tuong-Vi Nguyen, Hieu-Huy Nguyen-Tran, Thi-Ngoc Nguyen, Sheng-Chieh Lin, Tien Hsu. Identification of a potential lymphatic progenitor cell population in Vhlh conditional knockout mouse and in ccRCC tissue [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A006.

Mechanical forces in lymphatic vessel development: Focus on transcriptional regulation.

The lymphatic system is crucial for the maintenance of interstitial fluid and protein homeostasis. It has important roles in collecting excess plasma and interstitial fluid leaked from blood vessels, lipid absorption and transportation in the digestive system, and immune surveillance and response. The development of lymphatic vessels begins during fetal life as lymphatic endothelial progenitor cells first differentiate into lymphatic endothelial cells (LECs) by expressing the master lymphatic vascular regulator, prospero-related homeobox 1 (PROX1). The lymphatic vasculature forms a hierarchical network that consists of blind-ended and unidirectional vessels. Although much progress has been made in the elucidation of the cellular and molecular mechanisms underlying the formation of the lymphatic vascular system, the causes of lymphatic vessel abnormalities and disease are poorly understood and complicated; specifically, the mechanistic basis for transcriptional dysregulation in lymphatic vessel development remains largely unclear. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms of lymphatic vascular development, including LEC differentiation, lymphangiogenesis, and valve formation, and the significance of mechanical forces in lymphatic vessels, with a focus on transcriptional regulation. We also summarize the current knowledge on epigenetic mechanisms of lymphatic gene expression.

Mafba and mafbb differentially regulate lymphatic endothelial cell migration in topographically distinct manners.

Lymphangiogenesis, formation of lymphatic vessels from pre-existing vessels, is a dynamic process that requires cell migration. Regardless of location, migrating lymphatic endothelial cell (LEC) progenitors probe their surroundings to form the lymphatic network. Lymphatic-development regulation requires the transcription factor MAFB in different species. Zebrafish Mafba, expressed in LEC progenitors, is essential for their migration in the trunk. However, the transcriptional mechanism that orchestrates LEC migration in different lymphatic endothelial beds remains elusive. Here, we uncover topographically different requirements of the two paralogs, Mafba and Mafbb, for LEC migration. Both mafba and mafbb are necessary for facial lymphatic development, but mafbb is dispensable for trunk lymphatic development. On the molecular level, we demonstrate a regulatory network where Vegfc-Vegfd-SoxF-Mafba-Mafbb is essential in facial lymphangiogenesis. We identify that mafba and mafbb tune the directionality of LEC migration and vessel morphogenesis that is ultimately necessary for lymphatic function.

Lymphatic endothelial cell fate specification in the mammalian embryo: An historical perspective

Development of the mammalian lymphatic vasculature is a stepwise process requiring the specification of lymphatic endothelial cell progenitors in the embryonic veins, and their subsequent budding to give rise to most of the mature lymphatic vasculature. In mice, formation of the lymphatic vascular network starts inside the cardinal vein at around E9.5 when a subpopulation of venous endothelial cells gets committed into the lymphatic lineage by their acquisition of Prox1 expression. Identification of critical genes regulating lymphatic development facilitated the detailed cellular and molecular characterization of some of the cellular and molecular mechanisms regulating the early steps leading to the formation of the mammalian lymphatic vasculature. A better understanding of basic aspects of early lymphatic development, and the availability of novel tools and animal models has been instrumental in the identification of important novel functional roles of this vasculature network.

Isolating and characterizing lymphatic endothelial progenitor cells for potential therapeutic lymphangiogenic applications

Lymphatic dysfunction is associated with the progression of several vascular disorders, though currently, there are limited strategies to promote new lymphatic vasculature (i.e., lymphangiogenesis) to restore lost lymphatic function. One promising approach to stimulate lymphangiogenesis involves delivering endothelial progenitor cells (EPCs), which are naturally involved in de novo blood vessel formation and have recently been identified to include a lymphatic subpopulation. However, the contribution of lymphatic EPCs in lymphangiogenesis is not clear and challenges with maintaining the activity of transplanted EPCs remain. Thus, the objective of this study was to isolate lymphatic EPCs from human umbilical cord blood and characterize their role in the initial stages of blood or lymphatic vasculature formation. Furthermore, this study also tested the applicability of alginate hydrogels to deliver lymphatic EPCs for a possible therapeutic application. We postulated and confirmed that blood and lymphatic EPC colonies could be isolated from human umbilical cord blood. Additionally, EPC populations responded to either angiogenic or lymphangiogenic growth factors and could stimulate their respective mature endothelial cells in vasculature models in vitro. Finally, lymphatic EPCs maintained their ability to promote lymphatic sprouts after prolonged interactions with the alginate hydrogel microenvironment. These results suggest EPCs have both a blood and a lymphatic population that have specific roles in promoting revascularization and highlight the potential of alginate hydrogels for the delivery of lymphatic EPCs. Statement of significanceDespite the potential therapeutic benefit of promoting lymphatic vasculature, lymphangiogenesis remains understudied. One appealing strategy for promoting lymphangiogenesis involves delivering lymphatic endothelial progenitor cells (EPCs), which are a subpopulation of EPCs involved in de novo vessel formation. Here, we investigate the role of isolated blood and lymphatic EPC subpopulations in promoting the early stages of vascularization and the utility of alginate hydrogels to deliver lymphatic EPCs. We determined that EPCs had two populations that expressed either blood or lymphatic markers, could stimulate their respective mature vasculature in tissue constructs and that alginate hydrogels maintained the therapeutic potential of lymphatic EPCs. We anticipate this work could support promising biomaterial applications of EPCs to promote revascularization, which could have many therapeutic applications.

Regulation of VEGFR Signalling in Lymphatic Vascular Development and Disease: An Update.

The importance of lymphatic vessels in a myriad of human diseases is rapidly gaining recognition; lymphatic vessel dysfunction is a feature of disorders including congenital lymphatic anomalies, primary lymphoedema and obesity, while improved lymphatic vessel function increases the efficacy of immunotherapy for cancer and neurological disease and promotes cardiac repair following myocardial infarction. Understanding how the growth and function of lymphatic vessels is precisely regulated therefore stands to inform the development of novel therapeutics applicable to a wide range of human diseases. Lymphatic vascular development is initiated during embryogenesis following establishment of the major blood vessels and the onset of blood flow. Lymphatic endothelial progenitor cells arise from a combination of venous and non-venous sources to generate the initial lymphatic vascular structures in the vertebrate embryo, which are then further ramified and remodelled to elaborate an extensive lymphatic vascular network. Signalling mediated via vascular endothelial growth factor (VEGF) family members and vascular endothelial growth factor receptor (VEGFR) tyrosine kinases is crucial for development of both the blood and lymphatic vascular networks, though distinct components are utilised to different degrees in each vascular compartment. Although much is known about the regulation of VEGFA/VEGFR2 signalling in the blood vasculature, less is understood regarding the mechanisms by which VEGFC/VEGFD/VEGFR3 signalling is regulated during lymphatic vascular development. This review will focus on recent advances in our understanding of the cellular and molecular mechanisms regulating VEGFA-, VEGFC- and VEGFD-mediated signalling via VEGFRs which are important for driving the construction of lymphatic vessels during development and disease.

When form meets function: the cells and signals that shape the lymphatic vasculature during development.

The lymphatic vasculature is an integral component of the cardiovascular system. It is essential to maintain tissue fluid homeostasis, direct immune cell trafficking and absorb dietary lipids from the digestive tract. Major advances in our understanding of the genetic and cellular events important for constructing the lymphatic vasculature during development have recently been made. These include the identification of novel sources of lymphatic endothelial progenitor cells, the recognition of lymphatic endothelial cell specialisation and heterogeneity, and discovery of novel genes and signalling pathways underpinning developmental lymphangiogenesis. Here, we review these advances and discuss how they inform our understanding of lymphatic network formation, function and dysfunction.

Decreased Lymphangiogenic Activities and Genes Expression of Cord Blood Lymphatic Endothelial Progenitor Cells (VEGFR3+/Pod+/CD11b+ Cells) in Patient with Preeclampsia.

The abnormal development or disruption of the lymphatic vasculature has been implicated in metabolic and hypertensive diseases. Recent evidence suggests that the offspring exposed to preeclampsia (PE) in utero are at higher risk of long-term health problems, such as cardiovascular and metabolic diseases in adulthood, owing to in utero fetal programming. We aimed to investigate lymphangiogenic activities in the lymphatic endothelial progenitor cells (LEPCs) of the offspring of PE. Human umbilical cord blood LEPCs from pregnant women with severe PE (n = 10) and gestationally matched normal pregnancies (n = 10) were purified with anti-vascular endothelial growth factor receptor 3 (VEGFR3)/podoplanin/CD11b microbeads using a magnetic cell sorter device. LEPCs from PE displayed significantly delayed differentiation and reduced formation of lymphatic endothelial cell (LEC) colonies compared with the LEPCs from normal pregnancies. LECs differentiated from PE-derived LEPCs exhibited decreased tube formation, migration, proliferation, adhesion, wound healing, and 3D-sprouting activities as well as increased lymphatic permeability through the disorganization of VE-cadherin junctions, compared with the normal pregnancy-derived LECs. In vivo, LEPCs from PE showed significantly reduced lymphatic vessel formation compared to the LEPCs of the normal pregnancy. Gene expression analysis revealed that compared to the normal pregnancy-derived LECs, the PE-derived LECs showed a significant decrease in the expression of pro-lymphangiogenic genes (GREM1, EPHB3, VEGFA, AMOT, THSD7A, ANGPTL4, SEMA5A, FGF2, and GBX2). Collectively, our findings demonstrate, for the first time, that LEPCs from PE have reduced lymphangiogenic activities in vitro and in vivo and show the decreased expression of pro-lymphangiogenic genes. This study opens a new avenue for investigation of the molecular mechanism of LEPC differentiation and lymphangiogenesis in the offspring of PE and subsequently may impact the treatment of long-term health problems such as cardiovascular and metabolic disorders of offspring with abnormal development of lymphatic vasculature.

<i>Mafba</i> and  <i>Mafbb</i> Differentially Regulate Lymphatic Endothelial Cell Migration in Topographically Distinct Manners

Lymphangiogenesis is the formation of lymphatic vessels from pre-existing vessels, a dynamic process that requires cell migration. Regardless of location, lymphatic endothelial cell (LEC) progenitors probe their surroundings while migrating to form the lymphatic network. Lymphatic development regulation depends on the transcription factor MAFB in different species. Zebrafish Mafba, expressed in LEC progenitors, is essential for their migration in the trunk. However, the transcriptional mechanism that orchestrate LEC migration in different lymphatic endothelial beds remains elusive. Here, we uncover topographically different requirements of the two paralogues, Mafba and Mafbb, for lymphatic cell migration. Both mafba and mafbb are necessary for facial lymphatic development, but mafbb is dispensable for trunk lymphatic development. On the molecular level, we demonstrate a regulatory network where Vegfc-Vegfd-SoxF-Mafba-Mafbb are essential in the facial lymphangiogenesis. We identify that mafba and mafbb fine-tune the directionality of LEC migration and vessel morphogenesis that is ultimately necessary for lymphatic function.

Beyond PROX1: transcriptional, epigenetic, and noncoding RNA regulation of lymphatic identity and function.

The lymphatic vascular system acts as the major transportation highway of tissue fluids, and its activation or impairment is associated with a wide range of diseases. There has been increasing interest in understanding the mechanisms that control lymphatic vessel formation (lymphangiogenesis) and function in development and disease. Here, we discuss recent insights into new players whose identification has contributed to deciphering the lymphatic regulatory code. We reveal how lymphatic endothelial cells, the building blocks of lymphatic vessels, utilize their transcriptional, post-transcriptional, and epigenetic portfolio to commit to and maintain their vascular lineage identity and function, with a particular focus on development.

Lymphangiogenesis in renal fibrosis arises from macrophages via VEGF-C/VEGFR3-dependent autophagy and polarization

Inflammation plays a crucial role in the occurrence and development of renal fibrosis, which ultimately results in end-stage renal disease (ESRD). There is new focus on lymphangiogenesis in the field of inflammation. Recent studies have revealed the association between lymphangiogenesis and renal fibrosis, but the source of lymphatic endothelial cells (LECs) is not clear. It has also been reported that macrophages are involved in lymphangiogenesis through direct and indirect mechanisms in other tissues. We hypothesized that there was a close relationship between macrophages and lymphatic endothelial progenitor cells in renal fibrosis. In this study, we demonstrated that lymphangiogenesis occurred in a renal fibrosis model and was positively correlated with the degree of fibrosis and macrophage infiltration. Compared to resting (M0) macrophages and alternatively activated (M2) macrophages, classically activated (M1) macrophages predominantly transdifferentiated into LECs in vivo and in vitro. VEGF-C further increased M1 macrophage polarization and transdifferentiation into LECs by activating VEGFR3. It was suggested that VEGF-C/VEGFR3 pathway activation downregulated macrophage autophagy and subsequently regulated macrophage phenotype. The induction of autophagy in macrophages by rapamycin decreased M1 macrophage polarization and differentiation into LECs. These results suggested that M1 macrophages promoted lymphangiogenesis and contributed to newly formed lymphatic vessels in the renal fibrosis microenvironment, and VEGF-C/VEGFR3 signaling promoted macrophage M1 polarization by suppressing macrophage autophagy and then increased the transdifferentiation of M1 macrophages into LECs.

A dual role for Tbx1 in cardiac lymphangiogenesis through genetic interaction with Vegfr3.

The transcription factor TBX1 is the major gene implicated in 22q11.2 deletion syndrome (22q11.2DS). The complex clinical phenotype includes vascular anomalies and a recent report presented new cases of primary lymphedema in 22q11.2DS patients. We have previously shown that TBX1 is required for systemic lymphatic vessel development in prenatal mice and it is critical for their survival postnatally. Using loss-of-function genetics and transgenesis in the mouse, we show here a strong genetic interaction between Tbx1 and Vegfr3 in cardiac lymphangiogenesis. Intriguingly, we found that different aspects of the cardiac lymphatic phenotype in Tbx1-Vegfr3 compound heterozygotes were regulated independently by the two genes, with Tbx1 primarily regulating vessel numbers and Vegfr3 vessel morphology. Consistent with this observation, Tbx1Cre -activated expression of a Vegfr3 transgene rescued partially the cardiac lymphatic abnormalities in compound heterozygotes. Through time-controlled genetic experiments, we show that Tbx1 is activated and required in cardiac lymphatic endothelial cell (LEC) progenitors between E10.5 and E11.5. Furthermore, we found that it is also required later in development for the growth of the cardiac lymphatics. Finally, our study revealed a differential sensitivity between ventral and dorsal cardiac lymphatics to the effects of altered Tbx1 and Vegfr3 gene dosage, and we show that this likely results from an earlier requirement for Tbx1 in ventral cardiac LEC progenitors.

Lymphatic Endothelial Cell Progenitors in the Tumor Microenvironment.

Tumor lymphatics play a key role in cancer progression as they are solely responsible for transporting malignant cells to regional lymph nodes (LNs), a process that precedes and promotes systemic lethal spread. It is broadly accepted that tumor lymphatic sprouting is induced mainly by soluble factors derived from tumor-associated macrophages (TAMs) and malignant cells. However, emerging evidence strongly suggests that a subset of TAMs, myeloid-lymphatic endothelial cell progenitors (M-LECP), also contribute to the expansion of lymphatics through both secretion of paracrine factors and a self-autonomous mode. M-LECP are derived from bone marrow (BM) precursors of the monocyte-macrophage lineage and characterized by unique co-expression of markers identifying lymphatic endothelial cells (LEC), stem cells, M2-type macrophages, and myeloid-derived immunosuppressive cells. This review describes current evidence for the origin of M-LECP in the bone marrow, their recruitment tumors and intratumoral trafficking, similarities to other TAM subsets, and mechanisms promoting tumor lymphatics. We also describe M-LECP integration into preexisting lymphatic vessels and discuss potential mechanisms and significance of this event. We conclude that improved mechanistic understanding of M-LECP functions within the tumor environment may lead to new therapeutic approaches to suppress tumor lymphangiogenesis and metastasis to lymph nodes.

Epigenetic priming by Dot1l in lymphatic endothelial progenitors ensures normal lymphatic development and function

Proper functioning of the lymphatic system is required for normal immune responses, fluid balance, and lipid reabsorption. Multiple regulatory mechanisms are employed to ensure the correct formation and function of lymphatic vessels; however, the epigenetic modulators and mechanisms involved in this process are poorly understood. Here, we assess the regulatory role of mouse Dot1l, a histone H3 lysine (K) 79 (H3K79) methyltransferase, in lymphatic formation. Genetic ablation of Dot1l in Tie2(+) endothelial cells (ECs), but not in Lyve1(+) or Prox1(+) lymphatic endothelial cells (LECs) or Vav1(+) definitive hematopoietic stem cells, leads to catastrophic lymphatic anomalies, including skin edema, blood–lymphatic mixing, and underdeveloped lymphatic valves and vessels in multiple organs. Remarkably, targeted Dot1l loss in Tie2(+) ECs leads to fully penetrant lymphatic aplasia, whereas Dot1l overexpression in the same cells results in partially hyperplastic lymphatics in the mesentery. Genetic studies reveal that Dot1l functions in c-Kit(+) hemogenic ECs during mesenteric lymphatic formation. Mechanistically, inactivation of Dot1l causes a reduction of both H3K79me2 levels and the expression of genes important for LEC development and function. Thus, our study establishes that Dot1l-mediated epigenetic priming and transcriptional regulation in LEC progenitors safeguard the proper lymphatic development and functioning of lymphatic vessels.

Enhancement of cardiac lymphangiogenesis by transplantation of CD34+VEGFR-3+ endothelial progenitor cells and sustained release of VEGF-C

Impairment of cardiac lymphatic vessels leads to cardiac lymphedema. Recent studies have suggested that stimulation of lymphangiogenesis may reduce cardiac lymphedema. However, effects of lymphatic endothelial progenitor cells (LEPCs) on cardiac lymphangiogenesis are poorly understood. Therefore, this study investigated effectiveness of LEPC transplantation and VEGF-C release with self-assembling peptide (SAP) on cardiac lymphangiogenesis after myocardial infarction (MI). CD34+VEGFR-3+ EPCs isolated from rat bone marrow differentiated into lymphatic endothelial cells after VEGF-C induction. VEGF-C also stimulated the cells to incorporate into the lymphatic capillary-like structures. The functionalized SAP could adhere with the cells and released VEGF-C sustainedly. In the condition of hypoxia and serum deprivation or abdominal pouch assay,the SAP hydrogel protected the cells from apoptosis and necrosis. At 4 weeks after intramyocardial transplantation of the cells and VEGF-C loaded with SAP hydrogel in rat MI models, cardiac lymphangiogenesis was increased, cardiac edema and reverse remodeling were reduced, and cardiac function was improved significantly. Delivery with SAP hydrogel favored survival of the engrafted cells. VEGF-C released from the hydrogel promoted differentiation and incorporation of the cells as well as growth of pre-existed lymphatic vessels. Cardiac lymphangiogenesis was beneficial for elimination of the inflammatory cells in the infarcted myocardium. Moreover, angiogenesis and myocardial regeneration were enhanced after reduction of lymphedema. These results demonstrate that the combined delivery of LEPCs and VEGF-C with the functionalized SAP promotes cardiac lymphangiogenesis and repair of the infarcted myocardium effectively. This study represents a novel therapy for relieving myocardial edema in cardiovascular diseases.

Myeloid-Derived Lymphatic Endothelial Cell Progenitors Significantly Contribute to Lymphatic Metastasis in Clinical Breast Cancer

Lymphatic metastasis is a high-impact prognostic factor for mortality of breast cancer (BC) patients, and it directly depends on tumor-associated lymphatic vessels. We previously reported that lipopolysaccharide-induced inflammatory lymphangiogenesis is strongly promoted by myeloid-derived lymphatic endothelial cell progenitors (M-LECPs) derived from the bone marrow (BM). As BC recruits massive numbers of provascular myeloid cells, we hypothesized that M-LECPs, within this recruited population, are specifically programmed to promote tumor lymphatics that increase lymph node metastasis. In support of this hypothesis, high levels of M-LECPs were found in peripheral blood and tumor tissues of BC patients. Moreover, the density of M-LECPs and lymphatic vessels positive for myeloid marker proteins strongly correlated with patient node status. It was also established that tumor M-LECPs coexpress lymphatic-specific, stem/progenitor and M2-type macrophage markers that indicate their BM hematopoietic-myeloid origin and distinguish them from mature lymphatic endothelial cells, tumor-infiltrating lymphoid cells, and tissue-resident macrophages. Using four orthotopic BC models, we show that mouse M-LECPs are similarly recruited to tumors and integrate into preexisting lymphatics. Finally, we demonstrate that adoptive transfer of invitro differentiated M-LECPs, but not naïve or nondifferentiated BM cells, significantly increased metastatic burden in ipsilateral lymph nodes. These data support a causative role of BC-induced lymphatic progenitors in tumor lymphangiogenesis and suggest molecular targets for their inhibition.

Abstract 1967: Nab-paclitaxel chemotherapy of breast tumors upregulates chemokines that increase chemotaxis of myeloid-lymphatic progenitors

Abstract Introduction: Lymph node metastasis is the main prognostic indicator for poor survival of breast cancer patients. Metastasis is promoted by tumor-induced lymphangiogenesis mediated, in part, through recruitment of myeloid-derived lymphatic endothelial cell progenitors (M-LECP). We previously showed that differentiation of M-LECP is induced by activated Toll-like receptor-4 (TLR4). The goal of this study was to identify TLR4-induced chemokines produced by tumors alone or in response to nab-paclitaxel (nab-PXL) chemotherapy that enhance bone marrow (BM) differentiation and tumor recruitment of M-LECP. Methods: Profile of BM composition for lymphatic-like, myeloid and progenitor cells was assessed by flow cytometry in normal and tumor-bearing mice treated either with saline or nab-PXL. We used two TLR4 overexpressing tumor models: MDA-MB-231 and HCC1806-TLR4+. RT-qPCR was used to determine differences in expression of 130 cytokines in cultured tumor cells and tumors from mice treated either with saline or nab-PXL. Targets with >2-fold upregulation were analyzed for protein expression by ELISA. ELISA-validated cytokines were tested for their ability to induce migration of M-LECP. Results: Both MDA-MB-231 or HCC1806-TLR4+ tumors significantly increased the levels of differentiated M-LECP in the BM, and that was further enhanced by treatment with taxane chemotherapy. Expression of progenitor markers CD117, CD33, and Sca-1 was increased up to 7-fold in both tested tumor models, and further upregulated by nab-PXL treatment by additional 3 to 9-fold. Out of 23 cytokines measured in tumor lysates by ELISA, 40% were upregulated by nab-PXL. Migration assay showed that several of these cytokines increased chemotaxis of M-LECP with CXCL1 being the strongest. Neutralization of CXCL1 in the conditioned medium of nab-PXL-treated MDA-MB-231 cells inhibited migration of lymphatic progenitors by 75%. Conclusion: Taxane chemotherapy acting presumably as a ligand for TLR4 significantly expanded M-LECP population in the BM. Our analysis suggests that these lymphatic precursors can mobilize to tumors by chemokines such as CXCL1 that are highly upregulated in TLR4-positive tumor cells, particularly when activated by paclitaxel. These data suggest that CXCL1 plays an important role in recruiting myeloid-lymphatic progenitors to breast tumors, which, in turn, promotes lymphatic vessel formation and metastasis. Blocking this cytokine might have clinical utility for prevention of tumor mobilization of M-LECP, and by extension, breast cancer metastasis to lymph nodes. Citation Format: Lisa D. Volk-Draper, Sophia Ran. Nab-paclitaxel chemotherapy of breast tumors upregulates chemokines that increase chemotaxis of myeloid-lymphatic progenitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1967.

Abstract 1506: Lymph node metastasis in clinical breast cancer is significantly promoted by myeloid-derived lymphatic endothelial cell progenitors

Abstract Introduction: Lymph node (LN) metastasis, a key prognostic factor for poor survival of breast cancer (BC) patients, directly depends on tumor lymphangiogenesis. We previously showed in orthotopic BC models that lymphangiogenesis is strongly promoted by tumor-mobilized myeloid-derived lymphatic endothelial cell (LEC) progenitors (M-LECP) and that M-LECP density correlates with lymphatic metastasis. The goals of this study were to determine the blood levels of M-LECP, their tumor density and correlation with metastasis in clinical human BC. Methods: Circulating CD14+ cells from BC patients (N=25) and healthy donors (N=24) were analyzed for expression of lymphatic-specific markers. Specimens from BC patients (N=104) along with normal mammary tissues (N=6) were co-stained with antibodies against myeloid and lymphatic markers including CD68, CD14, CD18 as well as LYVE1, podoplanin, and VEGFR-3. Sections were also double-stained to identify LYVE1+ cells expressing lymphoid, M2-type macrophage, and stem cell markers. Densities of tumor CD68+ macrophages, LYVE1+ vessels, and CD68+/LYVE1+ double-stained M-LECP were quantified and correlated with node status and other parameters. Pro-metastatic potential of experimentally produced mouse M-LECP was determined by injecting in vitro differentiated myeloid-lymphatic cells into mice bearing syngeneic EMT6 breast tumors. Results: 92% of BC patients had monocytes expressing at least one lymphatic marker compared 2% of healthy donors. More than 70% of breast tumors contained M-LECP with density ranging from 2 to 50 cells per high-power field. Tumor-recruited M-LECP express specific markers of myeloid but not lymphoid lineages as well as M2-type, and stem cell markers. Tumors with high density (>20 cells/field) of M-LECP were significantly more likely to belong to aggressive sub-groups (HER2+ and TNBC) and present with lymph node metastasis (P-value <0.02). Conclusion: These data show for the first time that human breast tumors recruit significant amount of immature myeloid cells with lymphatic phenotype and vessel-promoting properties. Such cells are largely absent from normal breast tissues. Statistical analyses show that M-LECP can significantly contribute to formation of new lymphatic vessels leading to increased nodal metastasis. Citation Format: Lisa D. Volk-Draper, Radhika Patel, Nihit Bhattarai, Jie Yang, Sophia Ran. Lymph node metastasis in clinical breast cancer is significantly promoted by myeloid-derived lymphatic endothelial cell progenitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1506.

Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation.

SummaryExogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies.

A blood capillary plexus-derived population of progenitor cells contributes to genesis of the dermal lymphatic vasculature during embryonic development.

Despite the essential role of the lymphatic vasculature in tissue homeostasis and disease, knowledge of the organ-specific origins of lymphatic endothelial progenitor cells remains limited. The assumption that most murine embryonic lymphatic endothelial cells (LECs) are venous derived has recently been challenged. Here, we show that the embryonic dermal blood capillary plexus constitutes an additional, local source of LECs that contributes to the formation of the dermal lymphatic vascular network. We describe a novel mechanism whereby rare PROX1-positive endothelial cells exit the capillary plexus in a Ccbe1-dependent manner to establish discrete LEC clusters. As development proceeds, these clusters expand and further contribute to the growing lymphatic system. Lineage tracing and analyses of Gata2-deficient mice confirmed that these clusters are endothelial in origin. Furthermore, ectopic expression of Vegfc in the vasculature increased the number of PROX1-positive progenitors within the capillary bed. Our work reveals a novel source of lymphatic endothelial progenitors employed during construction of the dermal lymphatic vasculature and demonstrates that the blood vasculature is likely to remain an ongoing source of LECs during organogenesis, raising the question of whether a similar mechanism operates during pathological lymphangiogenesis.