Investigation of correlation between shear wave elastography and lymphangiogenesis in invasive breast cancer and diagnosis of axillary lymph node metastasis.

BACKGROUND: Accurate evaluation of axillary lymph node metastasis (LNM) in breast cancer is very important. A large number of hyperplastic and dilated lymphangiogenesis cases can usually be found in the pericancerous tissue of breast cancer to promote the occurrence of tumor metastasis.Shear wave elastography (SWE) can be used as an important means for evaluating pericancerous stiffness. We determined the stiffness of the pericancerous by SWE to diagnose LNM and lymphangiogenesis in invasive breast cancer (IBC). METHODS: Patients with clinical T1-T2 stage IBC who received surgical treatment in our hospital from June 2020 to December 2020 were retrospectively enrolled. A total of 299 patients were eventually included in the preliminary study, which included an investigation of clinicopathological features, ultrasonic characteristics, and SWE parameters. Multivariable logistic regression analysis was used to establish diagnostic model and evaluated its diagnostic performance of LNM. The correlation among SWE values, collagen volume fraction (CVF), and microlymphatic density (MLD) in primary breast cancer lesions was analyzed in another 97 patients. RESULTS: The logistic regression model is Logit(P)=-1.878 + 0.992*LVI-2.010*posterior feature enhancement + 1.230*posterior feature shadowing + 0.102*posterior feature combined pattern + 0.009*Emax. The optimum cutoff value of the logistic regression model was 0.365, and the AUC (95% CI) was 0.697 (0.636-0.758); the sensitivity (70.7 vs. 54.3), positive predictive value (PPV) (54.0 vs. 50.8), negative predictive value (NPV) (76.9 vs. 69.7), and accuracy (65.2 vs. 61.9) were all higher than Emax. There was no correlation between the SWE parameters and MLD in primary breast cancer lesions. CONCLUSIONS: The logistic regression model can help us to determine LNM, thus providing more imaging basis for the selection of preoperative treatment. The SWE parameter of the primary breast cancer lesion cannot reflect the peritumoral lymphangiogenesis, and we still need to find a new ultrasonic imaging method.

Advances in lymphatic metastasis of non-small cell lung cancer.

Lung cancer is a deeply malignant tumor with high incidence and mortality. Despite the rapid development of diagnosis and treatment technology, abundant patients with lung cancer are still inevitably faced with recurrence and metastasis, contributing to death. Lymphatic metastasis is the first step of distant metastasis and an important prognostic indicator of non-small cell lung cancer. Tumor-induced lymphangiogenesis is involved in the construction of the tumor microenvironment, except promoting malignant proliferation and metastasis of tumor cells, it also plays a crucial role in individual response to treatment, especially immunotherapy. Thus, this article reviews the current research status of lymphatic metastasis in non-small cell lung cancer, in order to provide some insights for the basic research and clinical and translational application in this field.

Decorin suppresses tumor lymphangiogenesis: A mechanism to curtail cancer progression.

The complex interplay between malignant cells and the cellular and molecular components of the tumor stroma is a key aspect of cancer growth and development. These tumor-host interactions are often affected by soluble bioactive molecules such as proteoglycans. Decorin, an archetypical small leucine-rich proteoglycan primarily expressed by stromal cells, affects cancer growth in its soluble form by interacting with several receptor tyrosine kinases (RTK). Overall, decorin leads to a context-dependent and protracted cessation of oncogenic RTK activity by attenuating their ability to drive a prosurvival program and to sustain a proangiogenic network. Through an unbiased transcriptomic analysis using deep RNAseq, we identified that decorin down-regulated a cluster of tumor-associated genes involved in lymphatic vessel (LV) development when systemically delivered to mice harboring breast carcinoma allografts. We found that Lyve1 and Podoplanin, two established markers of LVs, were markedly suppressed at both the mRNA and protein levels, and this suppression correlated with a significant reduction in tumor LVs. We further identified that soluble decorin, but not its homologous proteoglycan biglycan, inhibited LV sprouting in an ex vivo 3D model of lymphangiogenesis. Mechanistically, we found that decorin interacted with vascular endothelial growth factor receptor 3 (VEGFR3), the main lymphatic RTK, and its activity was required for the decorin-mediated block of lymphangiogenesis. Finally, we identified that Lyve1 was in part degraded via decorin-evoked autophagy in a nutrient- and energy-independent manner. These findings implicate decorin as a biological factor with antilymphangiogenic activity and provide a potential therapeutic agent for curtailing breast cancer growth and metastasis.

Impaired meningeal lymphatic drainage in Listeria monocytogenes infection.

Previous studies have demonstrated an association between lymphatic vessels and diseases caused by bacterial infections. Listeria monocytogenes (LM) bacterial infection can affect multiple organs, including the intestine, brain, liver and spleen, which can be fatal. However, the impacts of LM infection on morphological and functional changes of lymphatic vessels remain unexplored. In this study, we found that LM infection not only induces meningeal and mesenteric lymphangiogenesis in mice, but also impairs meningeal lymphatic vessels (MLVs)-mediated macromolecules drainage. Interestingly, we found that the genes associated with lymphatic vessel development and function, such as Gata2 and Foxc2, were downregulated, suggesting that LM infection may affect cellular polarization and valve development. On the other hand, photodynamic ablation of MLVs exacerbated inflammation and bacterial load in the brain of mice with LM infection. Overall, our findings indicate that LM infection induces lymphangiogenesis and may affect cell polarization, cavity formation, and valve development during lymphangiogenesis, ultimately impairing MLVs drainage.

Tumor lymphangiogenesis index reveals the immune landscape and immunotherapy response in lung adenocarcinoma.

Background: Lymphangiogenesis (LYM) has an important role in tumor progression and is strongly associated with tumor metastasis. However, the clinical application of LYM has not progressed as expected. The potential value of LYM needs to be further developed in lung adenocarcinoma (LUAD) patients. Methods: The Sequencing data and clinical characteristics of LUAD patients were downloaded from The Cancer Genome Atlas and GEO databases. Multiple machine learning algorithms were used to screen feature genes and develop the LYM index. Immune cell infiltration, immune checkpoint expression, Tumor Immune Dysfunction and Exclusion (TIDE) algorithm and drug sensitivity analysis were used to explore the correlation of LYM index with immune profile and anti-tumor therapy. Results: We screened four lymphangiogenic feature genes (PECAM1, TIMP1, CXCL5 and PDGFB) to construct LYM index based on multiple machine learning algorithms. We divided LUAD patients into the high LYM index group and the low LYM index group based on the median LYM index. LYM index is a risk factor for the prognosis of LUAD patients. In addition, there was a significant difference in immune profile between high LYM index and low LYM index groups. LUAD patients in the low LYM index group seemed to benefit more from immunotherapy based on the results of TIDE algorithm. Conclusion: Overall, we confirmed that the LYM index is a prognostic risk factor and a valuable predictor of immunotherapy response in LUAD patients, which provides new evidence for the potential application of LYM.

Alkali injury-induced pathological lymphangiogenesis in the iris facilitates the infiltration of T cells and ocular inflammation.

Inflammatory lymphangiogenesis is intimately linked to immune regulation and tissue homeostasis. However, current evidence has suggested that classic lymphatic vessels are physiologically absent in intraocular structures. Here, we show that neolymphatic vessels were induced in the iris after corneal alkali injury (CAI) in a VEGFR3-dependent manner. Cre-loxP-based lineage tracing revealed that these lymphatic endothelial cells (LECs) originate from existing Prox1+ lymphatic vessels. Notably, the ablation of iridial lymphangiogenesis via conditional deletion of VEGFR3 alleviated the ocular inflammatory response and pathological T cell infiltration. Our findings demonstrate that iridial neolymphatics actively participate in pathological immune responses following injury and suggest intraocular lymphangiogenesis as a valuable therapeutic target for the treatment of ocular inflammation.

Fibroblasts facilitate lymphatic vessel formation in transplanted heart.

Rationale: Lymphangiogenesis plays a critical role in the transplanted heart. The remodeling of lymphatics in the transplanted heart and the source of newly formed lymphatic vessels are still controversial, especially the mechanism of lymphangiogenesis remains limited. Methods: Heart transplantation was performed among BALB/c, C57BL/6J, Cag-Cre, Lyve1-CreERT2;Rosa26-tdTomato and Postn(2A-CreERT2-wpre-pA)1;Rosa26-DTA mice. scRNA-seq, Elisa assay, Western blotting, Q-PCR and immunohistochemical staining were used to identify the cells and cell-cell communications of allograft heart. Cell depletion was applied to in vivo and in vitro experiments. Whole-mount staining and three-dimensional reconstruction depicted the cell distribution within transparent transplanted heart. Results: Genetic lineage tracing mice and scRNA-seq analysis have revealed that these newly formed lymphatic vessels mainly originate from recipient LYVE1+ cells. It was found that LECs primarily interact with activated fibroblasts. Inhibition of lymphatic vessel formation using a VEGFR3 inhibitor resulted in a decreased survival time of transplanted hearts. Furthermore, when activated fibroblasts were ablated in transplanted hearts, there was a significant suppression of lymphatic vessel generation, leading to earlier graft failure. Additional investigations have shown that activated fibroblasts promote tube formation of LECs primarily through the activation of various signaling pathways, including VEGFD/VEGFR3, MDK/NCL, and SEMA3C/NRP2. Interestingly, knockdown of VEGFD and MDK in activated fibroblasts impaired cardiac lymphangiogenesis after heart transplantation. Conclusions: Our study indicates that cardiac lymphangiogenesis primarily originates from recipient cells, and activated fibroblasts play a crucial role in facilitating the generation of lymphatic vessels after heart transplantation. These findings provide valuable insights into potential therapeutic targets for enhancing graft survival.

VEGFR-3 signaling in macrophages: friend or foe in disease?

Lymphatic vessels have been increasingly appreciated in the context of immunology not only as passive conduits for immune and cancer cell transport but also as key in local tissue immunomodulation. Targeting lymphatic vessel growth and potential immune regulation often takes advantage of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling to manipulate lymphatic biology. A receptor tyrosine kinase, VEGFR-3, is highly expressed on lymphatic endothelial cells, and its signaling is key in lymphatic growth, development, and survival and, as a result, often considered to be "lymphatic-specific" in adults. A subset of immune cells, notably of the monocyte-derived lineage, have been identified to express VEGFR-3 in tissues from the lung to the gut and in conditions as varied as cancer and chronic kidney disease. These VEGFR-3+ macrophages are highly chemotactic toward the VEGFR-3 ligands VEGF-C and VEGF-D. VEGFR-3 signaling has also been implicated in dictating the plasticity of these cells from pro-inflammatory to anti-inflammatory phenotypes. Conversely, expression may potentially be transient during monocyte differentiation with unknown effects. Macrophages play critically important and varied roles in the onset and resolution of inflammation, tissue remodeling, and vasculogenesis: targeting lymphatic vessel growth and immunomodulation by manipulating VEGFR-3 signaling may thus impact macrophage biology and their impact on disease pathogenesis. This mini review highlights the studies and pathologies in which VEGFR-3+ macrophages have been specifically identified, as well as the activity and polarization changes that macrophage VEGFR-3 signaling may elicit, and affords some conclusions as to the importance of macrophage VEGFR-3 signaling in disease.

Lymphatic System and the Kidney: From Lymphangiogenesis to Renal Inflammation and Fibrosis Development.

The lymphatic kidney system plays a crucial role in managing interstitial fluid removal, regulating fluid balance, and tuning immune response. It also assists in the reabsorption of proteins, electrolytes, cytokines, growth factors, and immune cells. Pathological conditions, including tissue damage, excessive interstitial fluid, high blood glucose levels, and inflammation, can initiate lymphangiogenesis-the formation of new lymphatic vessels. This process is associated with various kidney diseases, including polycystic kidney disease, hypertension, ultrafiltration challenges, and complications post-organ transplantation. Although lymphangiogenesis has beneficial effects in removing excess fluid and immune cells, it may also contribute to inflammation and fibrosis within the kidneys. In this review, we aim to discuss the biology of the lymphatic system, from its development and function to its response to disease stimuli, with an emphasis on renal pathophysiology. Furthermore, we explore how innovative treatments targeting the lymphatic system could potentially enhance the management of kidney diseases.

Engineering Lymphangiogenesis-On-Chip: The Independent and Cooperative Regulation by Biochemical Factors, Gradients, and Interstitial Fluid Flow.

Despite the crucial role of lymphangiogenesis during development and in several diseases with implications for tissue regeneration, immunity, and cancer, there are significantly fewer tools to understand this process relative to angiogenesis. While there has been a major surge in modeling angiogenesis with microphysiological systems, they have not been rigorously optimized or standardized to enable the recreation of the dynamics of lymphangiogenesis. Here, a Lymphangiogenesis-Chip (L-Chip) is engineered, within which new sprouts form and mature depending upon the imposition of interstitial flow, growth factor gradients, and pre-conditioning of endothelial cells with growth factors. The L-Chip reveals the independent and combinatorial effects of these mechanical and biochemical determinants of lymphangiogenesis, thus ultimately resulting in sprouts emerging from a parent vessel and maturing into tubular structures up to 1 mm in length within 4 days, exceeding prior art. Further, when the constitution of the pre-conditioning cocktail and the growth factor cocktail used to initiate and promote lymphangiogenesis are dissected, it is found that endocan (ESM-1) results in more dominant lymphangiogenesis relative to angiogenesis. Therefore, The L-Chip provides a foundation for standardizing the microfluidics assays specific to lymphangiogenesis and for accelerating its basic and translational science at par with angiogenesis.

S100A6 drives lymphatic metastasis of liver cancer via activation of the RAGE/NF-kB/VEGF-D pathway.

Patients diagnosed with lymph node (LN) metastatic liver cancer face an exceedingly grim prognosis. In-depth analysis of LN metastatic patients' characteristics and tumor cells' interactions with human lymphatic endothelial cells (HLECs), can provide important biological and therapeutic insights. Here we identify at the single-cell level that S100A6 expression differs between primary tumor and their LN metastasis. Of particular significance, we uncovered the disparity in S100A6 expression between tumors and normal tissues is greater in intrahepatic cholangiocarcinoma (ICC) patients, frequently accompanied by LN metastases, than that in hepatocellular carcinoma (HCC), with rare occurrence of LN metastasis. Furthermore, in the infrequent instances of LN metastasis in HCC, heightened S100A6 expression was observed, suggesting a critical role of S100A6 in the process of LN metastasis. Subsequent experiments further uncovered that S100A6 secreted from tumor cells promotes lymphangiogenesis by upregulating the expression and secretion of vascular endothelial growth factor-D (VEGF-D) in HLECs through the RAGE/NF-kB/VEGF-D pathway while overexpression of S100A6 in tumor cells also augmented their migration and invasion. Taken together, these data reveal the dual effects of S100A6 in promoting LN metastasis in liver cancer, thus highlighting its potential as a promising therapeutic target.

NKX2-1-AS1 promotes the lymphangiogenesis of lung adenocarcinoma through regulation of ERG-mediated FABP4.

Lymphatic metastasis is a common metastasis of lung adenocarcinoma (LUAD). The current study illustrated the action of lncRNA NKX2-1-AS1 in lymphangiogenesis in LUAD and the underlying mechanisms. Clinical tissue samples were collected for determining NKX2-1-AS1 expression. Then, H441 and H661 cells were selected to perform gain- and loss-of-function assays for dissecting the roles of NKX2-1-AS1 in LUAD cell proliferation and migration. Besides, H441 and H661 cell supernatant was harvested to stimulate HLECs for assessing tube formation ability. Interaction among NKX2-1-AS1, ERG, and fatty acid binding protein 4 (FABP4) was validated through luciferase and RIP assays. NKX2-1-AS1 was highly-expressed in LUAD tissues. Silencing NKX2-1-AS1 suppressed H441 and H661 cell proliferation and migration, reduced expression levels of lymphangiogenesis-related factors (LYVE-1, VEGF-C, VEGFR3, VEGF-A, VEGFR2, and CCR7), and inhibited HLEC tube formation. Interaction validation demonstrated that NKX2-1-AS1 regulated FABP4 transcription by binding to ERG. Overexpression of FABP4 could effectively block the inhibition role of NKX2-1-AS1 silencing in lymphangiogenesis in H441 and H661 cells. This study provided evidence that NKX2-1-AS1 regulated FABP4 transcription by binding to ERG to facilitate the proliferation and migration of LUAD cells and tube formation of HLECs, thus participating in lymphangiogenesis.

Mesenchymal stem cell-derived extracellular vesicles exert pro-angiogenic and pro-lymphangiogenic effects in ischemic tissues by transferring various microRNAs and proteins including ITGa5 and NRP1.

Mesenchymal stem cells/stromal cells (MSCs)-derived extracellular vesicles (EVs) mediate pro-regenerative effects in damaged ischemic tissues by regulating angiogenesis. MSCs-EVs modulate functions of cells including endogenous mature cells, progenitors and stem cells, resulting in restoration of blood flow. However, the mechanisms underlying such MSC-EV activity still remain poorly understood. The present study analyzes biological effects of bone marrow (BM) MSC-EVs on endothelial cells (ECs) in ischemic tissues both in in vitro and in vivo conditions and elucidates the molecular mechanisms underlying the tissue repair. MSC-EVs were isolated from murine BM-derived MSCs and their morphological, antigenic and molecular composition regarding protein and microRNA levels were evaluated to examine their properties. Global proteomic analysis demonstrated the presence in MSC-EVs of proteins regulating pro-regenerative pathways, including integrin α5 (Itgα5) and neuropilin-1 (NRP1) involved in lymphangiogenesis. MSC-EVs were also enriched in microRNAs regulating angiogenesis, TGF-ß signaling and processes guiding cellular adhesion and interactions with extracellular matrix. The functional effects of MSC-EVs on capillary ECs in vitro included the increase of capillary-like tube formation and cytoprotection under normal and inflammatory conditions by inhibiting apoptosis. Notably, MSC-EVs enhanced also capillary-like tube formation of lymphatic ECs, which may be regulated by Itgα5 and NRP1. Moreover, in a mouse model of critical hind limb ischemia, MSC-EVs increased the recovery of blood flow in ischemic muscle tissue, which was accompanied with increased vascular density in vivo. This pro-angiogenic effect was associated with an increase in nitric oxide (NO) production via endothelial NO-synthase activation in ischemic muscles. Interestingly, MSC-EVs enhanced lymphangiogenesis, which has never been reported before. The study provides evidence on pro-angiogenic and novel pro-lymphangiogenic role of MSC-EVs on ECs in ischemic tissue mediated by their protein and miRNA molecular cargos. The results highlight Itgα5 and NRP1 carried by MSC-EVs as potential therapeutic targets to boost lymphangiogenesis.

Small extracellular vesicles promote the formation of the pre-metastatic niche through multiple mechanisms in colorectal cancer.

Colorectal cancer (CRC) ranks among the most prevalent global malignancies, posing significant threats to human life and health due to its high recurrence and metastatic potential. Small extracellular vesicles (sEVs) released by CRC play a pivotal role in the formation of the pre-metastatic niche (PMN) through various mechanisms, preparing the groundwork for accelerated metastatic invasion. This review systematically describes how sEVs promote CRC metastasis by upregulating inflammatory factors, promoting immunosuppression, enhancing angiogenesis and vascular permeability, promoting lymphangiogenesis and lymphatic network remodeling, determining organophilicity, promoting stromal cell activation and remodeling and inducing the epithelial-to-mesenchymal transition (EMT). Furthermore, we explore potential mechanisms by which sEVs contribute to PMN formation in CRC and propose novel insights for CRC diagnosis, treatment, and prognosis.

NAT10-mediated ac4C-modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear-cell renal cell carcinoma by attenuating YWHAE-driven cytoplasmic retention of YAP1.

BACKGROUND: Lymphatic metastasis is one of the most common metastatic routes and indicates a poor prognosis in clear-cell renal cell carcinoma (ccRCC). N-acetyltransferase 10 (NAT10) is known to catalyze N4-acetylcytidine (ac4C) modification of mRNA and participate in many cellular processes. However, its role in the lymphangiogenic process of ccRCC has not been reported. This study aimed to elucidate the role of NAT10 in ccRCC lymphangiogenesis, providing valuable insights into potential therapeutic targets for intervention. METHODS: ac4C modification and NAT10 expression levels in ccRCC were assessed using public databases and clinical samples. Functional investigations involved manipulating NAT10 expression in cellular and mouse models to study its role in ccRCC. Mechanistic insights were gained through a combination of RNA sequencing, mass spectrometry, co-immunoprecipitation, RNA immunoprecipitation, immunofluorescence, and site-specific mutation analyses. RESULTS: We found that ac4C modification and NAT10 expression levels increased in ccRCC. NAT10 promoted tumor progression and lymphangiogenesis of ccRCC by enhancing the nuclear import of Yes1-associated transcriptional regulator (YAP1). Subsequently, we identified ankyrin repeat and zinc finger peptidyl tRNA hydrolase 1 (ANKZF1) as the functional target of NAT10, and its upregulation in ccRCC was caused by NAT10-mediated ac4C modification. Mechanistic analyses demonstrated that ANKZF1 interacted with tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) to competitively inhibit cytoplasmic retention of YAP1, leading to transcriptional activation of pro-lymphangiogenic factors. CONCLUSIONS: These results suggested a pro-cancer role of NAT10-mediated acetylation in ccRCC and identified the NAT10/ANKZF1/YAP1 axis as an under-reported pathway involving tumor progression and lymphangiogenesis in ccRCC.

A phosphodiesterase pathway essential for lymphangiogenic growth arrest and maturation.

In developing embryos, downregulation of lymphatic endothelial proliferation is needed for maturation of lymphatic vessels into a hierarchical network. In this issue of Developmental Cell, Carlantoni discover that phosphodiesterase2A controls lymphatic endothelial growth arrest and maturation via regulation of cGMP, p38 MAP kinase, and Notch pathway.

The cytoskeleton adaptor protein Sorbs1 controls the development of lymphatic and venous vessels in zebrafish.

BACKGROUND: Lymphangiogenesis, the formation of lymphatic vessels, is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in the zebrafish. RESULTS: We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of the trunk lymphatic vessel network. Interestingly, we show that Sorbs1 interacts with the BMP pathway and could function outside of Vegfc signaling. Mechanistically, Sorbs1 controls FAK/Src signaling and subsequently impacts on the cytoskeleton processes regulated by Rac1 and RhoA GTPases. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contacts rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties. CONCLUSIONS: Overall, using in vitro and in vivo assays, we identify Sorbs1 as an important regulator of venous and lymphatic angiogenesis independently of the Vegfc signaling axis. These results provide a better understanding of the complexity found within context-specific vascular and lymphatic development.

Lymphangiogenesis: A new strategy for heart disease treatment (Review).

Heart disease remains a global health challenge, contributing notably to morbidity and mortality. The lymphatic vasculature, an integral component of the cardiovascular system, plays a crucial role in regulating essential physiological processes, including fluid balance, transportation of extravasated proteins and immune cell trafficking, all of which are important for heart function. Through thorough scientometric analysis and extensive research, the present review identified lymphangiogenesis as a hotspot in cardiovascular disease research, and the mechanisms underlying impaired cardiac lymphangiogenesis and inadequate lymph drainage in various cardiovascular diseases are discussed. Furthermore, the way used to improve lymphangiogenesis to effectively regulate a variety of heart diseases and associated signaling pathways was investigated. Notably, the current review also highlights the impact of Traditional Chinese Medicine (TCM) on lymphangiogenesis, aiming to establish a clinical basis for the potential of TCM to improve cardiovascular diseases by promoting lymphangiogenesis.