Lymphatic Endothelial Markers and Tumor Lymphangiogenesis Assessment in Human Breast Cancer

Metastasis via lymphatic vessels or blood vessels is the leading cause of death for breast cancer, and lymphangiogenesis and angiogenesis are critical prerequisites for the tumor invasion–metastasis cascade. The research progress for tumor lymphangiogenesis has tended to lag behind that for angiogenesis due to the lack of specific markers. With the discovery of lymphatic endothelial cell (LEC) markers, growing evidence demonstrates that the LEC plays an active role in lymphatic formation and remodeling, tumor cell growth, invasion and intravasation, tumor–microenvironment remodeling, and antitumor immunity. However, some studies have drawn controversial conclusions due to the variation in the LEC markers and lymphangiogenesis assessments used. In this study, we review recent findings on tumor lymphangiogenesis, the most commonly used LEC markers, and parameters for lymphangiogenesis assessments, such as the lymphatic vessel density and lymphatic vessel invasion in human breast cancer. An in-depth understanding of tumor lymphangiogenesis and LEC markers can help to illustrate the mechanisms and distinct roles of lymphangiogenesis in breast cancer progression, which will help in exploring novel potential predictive biomarkers and therapeutic targets for breast cancer.

Lymphatic Thrombosis and Impaired Lymph Drainage in Cigarette Smoke-Associated Emphysema

The lymphatic vasculature is critical for lung function, but defects in lymphatic function in the pathogenesis of lung disease is understudied. In mice, lymphatic dysfunction alone is sufficient to cause lung injury that resembles human emphysema. Whether lymphatic function is disrupted in cigarette smoke (CS)-induced emphysema is unknown. In this study, we investigated lung lymphatic function in the pathogenesis of CS-induced emphysema. Analysis of human lung tissue revealed significant lung lymphatic thrombosis in patients with emphysema compared to control smokers that increased with disease severity. In vitro assays demonstrated a direct effect of CS on lymphatic endothelial cell integrity. In a mouse model, CS exposure led to lung lymphatic thrombosis, decreased lymphatic drainage, and impaired leukocyte trafficking that preceded emphysema. Proteomic analysis of lymph confirmed upregulation of coagulation and inflammatory pathways in the lymphatics of CS-exposed mice compared to control mice. These data suggest that CS exposure results in lung lymphatic dysfunction with thrombosis, impaired leukocyte trafficking, and changes in the composition of lymph. In patients with emphysema, lung lymphatic thrombosis is seen with increasing disease severity. These studies for the first time demonstrate lung lymphatic dysfunction after cigarette smoke exposure and suggest a novel component in the pathogenesis of emphysema.

Lymphatic Thrombosis and Impaired Lymph Drainage in Cigarette Smoke-Associated Emphysema

The lymphatic vasculature is critical for lung function, but defects in lymphatic function in the pathogenesis of lung disease is understudied. In mice, lymphatic dysfunction alone is sufficient to cause lung injury that resembles human emphysema. Whether lymphatic function is disrupted in cigarette smoke (CS)-induced emphysema is unknown. In this study, we investigated lung lymphatic function in the pathogenesis of CS-induced emphysema. Analysis of human lung tissue revealed significant lung lymphatic thrombosis in patients with emphysema compared to control smokers that increased with disease severity. In vitro assays demonstrated a direct effect of CS on lymphatic endothelial cell integrity. In a mouse model, CS exposure led to lung lymphatic thrombosis, decreased lymphatic drainage, and impaired leukocyte trafficking that preceded emphysema. Proteomic analysis of lymph confirmed upregulation of coagulation and inflammatory pathways in the lymphatics of CS-exposed mice compared to control mice. These data suggest that CS exposure results in lung lymphatic dysfunction with thrombosis, impaired leukocyte trafficking, and changes in the composition of lymph. In patients with emphysema, lung lymphatic thrombosis is seen with increasing disease severity. These studies for the first time demonstrate lung lymphatic dysfunction after cigarette smoke exposure and suggest a novel component in the pathogenesis of emphysema.

A short review on lymphatic endothelial cell heterogeneity

Recent single-cell RNA sequencing studies in mouse and human have clearly indicated that lymphatic endothelial cells (LECs) consist of multiple cell subsets, each expressing a unique set of genes, residing in distinct locations in the body. These studies have also revealed a conserved pattern of gene expression in LECs across animal species, as well as specialized sets of genes unique to each species. However, the extent to which this heterogeneity is adaptive to the external milieu surrounding LECs has remained unclear. The transcriptional and regulatory pathways that program the different subsets of LECs also remain unexplored.

WISP-3 Stimulates VEGF-C-Dependent Lymphangiogenesis in Human Chondrosarcoma Cells by Inhibiting miR-196a-3p Synthesis

Chondrosarcoma is a malignant bone tumor with high metastatic potential. Lymphangiogenesis is a critical biological step in cancer metastasis. WNT1-inducible signaling pathway protein 3 (WISP-3) regulates angiogenesis and facilitates chondrosarcoma metastasis, but the role of WISP-3 in chondrosarcoma lymphangiogenesis is unclear. In this study, incubation of chondrosarcoma cells with WISP-3 increased the production of VEGF-C, an important lymphangiogenic factor. Conditioned medium from WISP-3-treated chondrosarcoma cells significantly enhanced lymphatic endothelial cell tube formation. WISP-3-induced stimulation of VEGF-C-dependent lymphangiogenesis inhibited miR-196a-3p synthesis in the ERK, JNK, and p38 signaling pathways. This evidence suggests that the WISP-3/VEGF-C axis is worth targeting in the treatment of lymphangiogenesis in human chondrosarcoma.

Downregulation of Exosomal miR-26a-5p Promotes Lymphangiogenesis and Lymphatic Metastasis in Endometrial Cancer

Background: Endometrial cancer (EC) patients with lymph node (LN) metastasis have poor prognosis. However, the potential biomarkers that predict LN metastasis and the molecular mechanism of tumor-induced peritumoral lymphangiogenesis have not been well explored. Cancer-secreted exosomal miRNAs are emerging mediators of cell-cell communication in the tumor environment.Methods: Exosomes were isolated with a differential centrifugation method and confirmed by Transmission electron microscopy, NanoSight analysis, and Western blot. MicroRNA (miRNA) sequencing of exosomes derived from EC patients and healthy donors were performed. FISH and qRT-PCR were used to detect the indicated miRNA expression. Exosomal miRNA transferred to cells were confirmed by immunofluorescence and confocal microscope. siRNA and plasmid transfections as well as viral infection were performed to manipulate gene expression. A series of in vitro and in vivo phenotype experiments (tube formation, migration, and popliteal LN metastasis model) were performed to investigate the role of indicated miRNA in EC. RNA sequencing was used to select the underlying transcription factor, and luciferase activity assay and chromatin immunoprecipitation were performed to elucidate the molecular mechanisms. Results: Our data showed that serum exosomal miR-26a-5p was significantly reduced in EC patients, and its level was positively associated with LN metastasis. Loss of miR-26a-5p promoted the migratory and invasive abilities of EC cells, and miR-26a-5p could be transferred from EC cells-secreted exosomes into human lymphatic endothelial cell (HLEC). Mechanistically, miR-26a-5p could regulate LEF1/c-myc/VEGFA axis via binding to its direct downstream target lymphoid enhancer binding factor 1 (LEF1), consequently promoting HLEC tube formation and migration in vitro, facilitating lymphangiogenesis and LN metastasis in vivo. Re-expression and knockdown of LEF1 could respectively promote and rescue the effects induced by exosomal miR-26a-5p. Moreover, we demonstrated that transcriptional factor EB (TFEB) directly induced miR-26a-5p expression.Conclusions: Our results show that exosomal miR-26a-5p/LEF1/c-myc/VEGFA axis is dysregulated and plays a critical role in LN metastasis and exosomal miR-26a-5p may be used as a blood-based biomarker for EC patients with LN metastasis.

Abstract MP52: Hypertensive Stimuli Indirectly Stimulate Mouse Mesometrial Lymphangiogenesis Through Immune Cells

We previously reported increased renal lymphatic density in multiple mouse models of hypertension, and further augmenting renal lymphatics lowers blood pressure. However, whether interstitial levels of hypertensive stimuli have a direct effect on lymphatics or an indirect effect through secreted immune cell factors has not been examined. We hypothesized that hypertensive stimuli directly increases lymphatic endothelial cell (LEC) proliferation and increases sprouting of mouse mesometrial lymphatic vessels. Murine LECs were cultured and treated with angiotensin II (angII), salt, and asymmetric dimethylarginine (ADMA) for 24 hours. To mimic the in vivo environment, a lymphatic-specific reporter mouse (Prox1-tdTomato) mesometrium tissue explant was treated with either the same hypertensive stimuli or with hypertensive conditioned media for 8 days. Mesometrial vascular beds were cultured in DMEM supplemented with 20% fetal bovine serum to induce lymphatic sprouting and this was replenished every day. The conditioned media was made by treating murine splenocytes for 24 hours with the same hypertensive stimuli. These stimuli had no effect on murine LEC proliferation. Hypertensive stimuli significantly decreased mesometrial lymphatic vessel sprout length (SL) and sprout number (SN) compared to controls (control SL in pixels by ImageJ analysis: 34.0 ± 2.6, angII: 3.7 ± 2.6, salt: 2.67 ± 2.18, ADMA: 9.06 ± 5.12, all p<0.05; control SN: 7 ± 3, angII: 0 ± 0, salt: 0 ± 0, ADMA: 1 ± 1, all p<0.05). Conditioned media treatment normalized SL and SN by day 8 for all hypertensive stimuli except salt. In conclusion, hypertensive stimuli directly inhibit mesometrial lymphangiogenesis, but this was mitigated by hypertensive stimuli induced immune cell secreted factors.