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.

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.

Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.

Unraveling a hidden player in lymphovascular invasion in bladder cancer.

Tumor invasion into the lymphatic vasculature represents a critical step during malignant progression of epithelial cancers. In this issue of Cancer Cell, Zheng et al. unravel how cancer-associated fibroblasts interact with lymphatic endothelial cells and the extracellular matrix to promote lymphatic tumor invasion and suggest that these processes could be treatment targets.

Quantitative multiplex immunohistochemistry reveals inter-patient lymphovascular and immune heterogeneity in primary cutaneous melanoma.

Introduction: Quantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma. Methods: We established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas. Results: Here we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates. Discussion: We describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for metastasis or immune infiltration. This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

The role of key biomarkers in lymphatic malformation: An updated review.

The lymphatic system, crucial for tissue fluid balance and immune surveillance, can be severely impacted by disorders that hinder its activities. Lymphatic malformations (LMs) are caused by fluid accumulation in tissues owing to defects in lymphatic channel formation, the obstruction of lymphatic vessels or injury to lymphatic tissues. Somatic mutations, varying in symptoms based on lesions' location and size, provide insights into their molecular pathogenesis by identifying LMs' genetic causes. In this review, we collected the most recent findings about the role of genetic and inflammatory biomarkers in LMs that control the formation of these malformations. A thorough evaluation of the literature from 2000 to the present was conducted using the PubMed and Google Scholar databases. Although it is obvious that the vascular endothelial growth factor receptor 3 mutation accounts for a significant proportion of LM patients, several mutations in other genes thought to be linked to LM have also been discovered. Also, inflammatory mediators like interleukin-6, interleukin-8, tumor necrosis factor-alpha and mammalian target of rapamycin are the most commonly associated biomarkers with LM. Understanding the mutations and genes expression responsible for the abnormalities in lymphatic endothelial cells could lead to novel therapeutic strategies based on molecular pathways.

A new lymphedema treatment using pyro-drive jet injection.

Lymphedema, resulting from impaired lymphatic drainage, causes inflammation, fibrosis and tissue damage leading to symptoms such as limb swelling and restricted mobility. Despite various treatments under exploration, no standard effective therapy exists. Here a novel technique using the pyro-drive jet injection (PJI) was used to create artificial clefts between collagen fibers, which facilitated the removal of excess interstitial fluid. The PJI was used to deliver a mixture of lactated Ringer's solution and air into the tail of animals with secondary skin edema. Edema levels were assessed using micro-CT scanning. Histopathological changes and neovascularization were evaluated on the injury-induced regenerative tissue. Regarding tissue remodeling, we focused on connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF)-C. PJI markedly diminished soft tissue volume in the experimental lymphedema animals compared to the non-injected counterparts. The PJI groups exhibited a significantly reduced proportion of inflammatory granulation tissue and an enhanced density of lymphatic vessels and α-smooth muscle actin (αSMA)-positive small vessels in the fibrous granulation tissue compared to the controls. In addition, PJI curtailed the prevalence of CTGF- and VEGF-C-positive cells in regenerative tissue. In a lymphedema animal model, PJI notably ameliorated interstitial edema, promoted lymphatic vessel growth, and bolstered αSMA-positive capillaries in fibrous granulation tissue. PJI's minimal tissue impact post-lymph node dissection indicates significant potential as an early, standard preventative measure. Easily applied in general clinics without requiring specialized training, it offers a cost-effective and highly versatile solution to the management of lymphedema.

Perturbed collagen metabolism underlies lymphatic recanalization failure in Gata2 heterozygous deficient mice.

Lymphedema has become a global health issue following the growing number of cancer surgeries. Curative or supportive therapeutics have long been awaited for this refractory condition. Transcription factor GATA2 is crucial in lymphatic development and maintenance, as GATA2 haploinsufficient disease often manifests as lymphedema. We recently demonstrated that Gata2 heterozygous deficient mice displayed delayed lymphatic recanalization upon lymph node resection. However, whether GATA2 contributes to lymphatic regeneration by functioning in the damaged lymph vessels' microenvironment remains explored. In this study, our integrated analysis demonstrated that dermal collagen fibers were more densely accumulated in the Gata2 heterozygous deficient mice. The collagen metabolism-related transcriptome was perturbed, and collagen matrix contractile activity was aberrantly increased in Gata2 heterozygous embryonic fibroblasts. Notably, soluble collagen placement ameliorated delayed lymphatic recanalization, presumably by modulating the stiffness of the extracellular matrix around the resection site of Gata2 heterozygous deficient mice. Our results provide valuable insights into mechanisms underlying GATA2-haploinsufficiency-mediated lymphedema and shed light on potential therapeutic avenues for this intractable disease.

Toward Characterizing Lymphatic Vasculature in the Mammary Gland During Normal Development and Tumor-Associated Remodeling.

Lymphatic vasculature has been shown to promote metastatic spread of breast cancer. Lymphatic vasculature, which is made up of larger collecting vessels and smaller capillaries, has specialized cell junctions that facilitate cell intravasation. Normally, these junctions are designed to collect immune cells and other cellular components for immune surveillance by lymph nodes, but they are also utilized by cancer cells to facilitate metastasis. Although lymphatic development overall in the body has been well-characterized, there has been little focus on how the lymphatic network changes in the mammary gland during stages of remodeling such as pregnancy, lactation, and postpartum involution. In this review, we aim to define the currently known lymphangiogenic factors and lymphatic remodeling events during mammary gland morphogenesis. Furthermore, we juxtapose mammary gland pubertal development and postpartum involution to show similarities of pro-lymphangiogenic signaling as well as other molecular signals for epithelial cell survival that are critical in these morphogenic stages. The similar mechanisms include involvement of M2-polarized macrophages that contribute to matrix remodeling and vasculogenesis; signal transducer and activator of transcription (STAT) survival and proliferation signaling; and cyclooxygenase 2 (COX2)/Prostaglandin E2 (PGE2) signaling to promote ductal and lymphatic expansion. Investigation and characterization of lymphangiogenesis in the normal mammary gland can provide insight to targetable mechanisms for lymphangiogenesis and lymphatic spread of tumor cells in breast cancer.

Immediate-delayed lymphatic reconstruction after axillary lymph nodes dissection for locally advanced breast cancer-related lymphedema prevention: Report of two cases.

Approximately 60%-70% of breast cancer patients in Indonesia are diagnosed in the locally advanced stage. The stage carries a higher risk of lymph node metastasis which increases susceptibility to lymph obstruction. Hence, breast cancer-related lymphedema (BCRL) could present before axillary lymph node dissection (ALND). The purpose of this case report is to describe immediate-delayed lymphatic reconstructions with lymphaticovenous anastomosis in two subclinical lymphedema cases that present before ALND. There were 51 and 58 years old breast cancer patients with stage IIIC and IIIB, respectively. Both had no arm lymphedema symptoms, but arm lymphatic vessel abnormalities were found during preoperative indocyanine green (ICG) lymphography. Mastectomy and ALND were performed and proceeded with lymphaticovenous anastomoses (LVA) in both cases. One LVA at the axilla (isotopic) was done in the first patient. On the second patient, 3 LVAs at the affected arm (ectopic) and 3 isotopic LVAs were created. The patients were discharged on the second day without complications during the follow-up. The intensity of dermal backflow was reduced, and no subclinical lymphedema progression occurred during 11 and 9 months follow-up, respectively. Based on these cases, BCRL screening might be recommended for the locally advanced stage before cancer treatment. Once diagnosed, immediate lymphatic reconstruction after ALND should be recommended to cure or prevent BCRL progression.

Microfluidic-Based Reconstitution of Functional Lymphatic Microvasculature: Elucidating the Role of Lymphatics in Health and Disease.

The knowledge of the blood microvasculature and its functional role in health and disease has grown significantly attributable to decades of research and numerous advances in cell biology and tissue engineering; however, the lymphatics (the secondary vascular system) has not garnered similar attention, in part due to a lack of relevant in vitro models that mimic its pathophysiological functions. Here, a microfluidic-based approach is adopted to achieve precise control over the biological transport of growth factors and interstitial flow that drive the in vivo growth of lymphatic capillaries (lymphangiogenesis). The engineered on-chip lymphatics with in vivo-like morphology exhibit tissue-scale functionality with drainage rates of interstitial proteins and molecules comparable to in vivo standards. Computational and scaling analyses of the underlying transport phenomena elucidate the critical role of the three-dimensional geometry and lymphatic endothelium in recapitulating physiological drainage. Finally, the engineered on-chip lymphatics enabled studies of lymphatic-immune interactions that revealed inflammation-driven responses by the lymphatics to recruit immune cells via chemotactic signals similar to in vivo, pathological events. This on-chip lymphatics platform permits the interrogation of various lymphatic biological functions, as well as screening of lymphatic-based therapies such as interstitial absorption of protein therapeutics and lymphatic immunomodulation for cancer therapy.

Surgical lymphedema models in the mice hindlimb-A systematic review and quality assessment.

BACKGROUND: Lymphedema constitutes a major unsolved problem in plastic surgery. To identify novel lymphedema treatments, preclinical studies are vital. The surgical mouse lymphedema model is popular and cost-effective; nonetheless, a synthesis and overview of the literature with evidence-based guidelines is needed. The aim of this review was to perform a systematic review to establish best practice and support future high-quality animal studies exploring lymphedema treatments. METHODS: We performed a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, searching four databases (PubMed, Embase, Web of Science, and Scopus) from inception-September 2022. The Animals in Research Reporting In Vivo Experiments 2.0 (ARRIVE 2.0) guidelines were used to evaluate reporting quality. Studies claiming to surgically induce lymphedema in the hindlimb of mice were included. RESULTS: Thirty-seven studies were included. Four main models were used. (1) Irradiation+surgery. (2) A variation of the surgery used by (1) + irradiation. (3) Surgery only (SPDF-model). (4) Surgery only (PLND-model). Remaining studies used other techniques. The most common measurement modality was the caliper. Mean quality coefficient was 0.57. Eighteen studies (49%) successfully induced sustained lymphedema. Combination of methods seemed to yield the best results, with an overrepresentation of irradiation, the removal of two lymph nodes, and the disruption of both the deep and superficial lymph vessels in the 18 studies. CONCLUSION: Surgical mouse hindlimb lymphedema models are challenged by two related problems: (1) retaining lymphedema for an extended period, that is, establishing a (chronic) lymphedema model (2) distinguishing lymphedema from post-operative edema. Most studies failed to induce lymphedema and used error-prone measurements. We provide an overview of studies claiming to induce lymphedema and advocate improved research via five evidence-based recommendations to use: (1) a proven lymphedema model; (2) sufficient follow-up time, (3) validated measurement methods; (4) ARRIVE-guidelines; (5) contralateral hindlimb as control.

Efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ARDS.

The lymphatic vasculature is the natural pathway for the resolution of inflammation, yet the role of pulmonary lymphatic drainage function in sepsis-induced acute respiratory distress syndrome (ARDS) remains poorly characterized. In this study, indocyanine green-near infrared lymphatic living imaging was performed to examine pulmonary lymphatic drainage function in septic mouse models. We found that the pulmonary lymphatic drainage was impaired owing to the damaged lymphatic structure in sepsis-induced ARDS. Moreover, prior lymphatic defects by blocking vascular endothelial growth factor receptor-3 (VEGFR-3) worsened sepsis-induced lymphatic dysfunction and inflammation. Posttreatment with vascular endothelial growth factor-C (Cys156Ser) (VEGF-C156S), a ligand of VEGFR-3, ameliorated lymphatic drainage by rejuvenating lymphatics to reduce the pulmonary edema and promote draining of pulmonary macrophages and neutrophils to pretracheal lymph nodes. Meanwhile, VEGF-C156S posttreatment reversed sepsis-inhibited CC chemokine ligand 21 (CCL21), which colocalizes with pulmonary lymphatic vessels. Furthermore, the advantages of VEGF-C156S on the drainage of inflammatory cells and edema fluid were abolished by blocking VEGFR-3 or CCL21. These results suggest that efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ARDS. Our findings offer a therapeutic approach to sepsis-induced ARDS by promoting lymphatic drainage function.

DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells.

Despite strong indications that interactions between melanoma and lymphatic vessels actively promote melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk, we established human primary lymphatic endothelial cell (LEC) cocultures with human melanoma cell lines. Here, we show that coculture with melanoma cells induced transcriptomic changes in LECs and led to multiple changes in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found to contribute to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the coculture with LECs, and Notch3 and WNT5B were coexpressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC coculture escaped efficiently from the primary site to the proximal tumor-draining lymph nodes, which was impaired upon WNT5B depletion. This supported the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3/WNT5B axis in melanoma. This study elucidated WNT5B as a key molecular factor mediating bidirectional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.

Impact of lymphovascular invasion on survival in surgically treated upper tract urothelial carcinoma: a nationwide analysis.

OBJECTIVES: To assess the prognostic ability of lymphovascular invasion (LVI) in upper tract urothelial carcinoma (UTUC) as a predictor of overall survival (OS) using a large North American cohort. PATIENTS AND METHODS: Our cohort included 5940 patients with clinical M0 UTUC who underwent a radical nephroureterectomy (RNU), between 2010 and 2016, within the National Cancer Database. The main variable of interest was LVI status, and its interaction with pathological nodal (pN) status. Kaplan-Meier curves were used to depict the OS also stratifying patients on LVI status. Cox regression analysis tested the impact of LVI status on OS after accounting for the available covariates. RESULTS: The median (interquartile range [IQR]) age at diagnosis was 71 (63-78) years and most patients had pathological T1 stage disease (48.6%). Nodal status was pN0, pN1 and pNx in 45.8%, 6.3% and 47.9%, respectively. Overall, 22.1% had LVI. The median (IQR) follow-up time was 32.6 (16.0-53.3) months. At the 5-year postoperative follow-up, the estimated OS rate was 28% in patients with LVI vs 66% in those without LVI (P < 0.001). When patients were stratified based on nodal status those rates were 32% vs 68% in pN0 patients (P < 0.001), 23% vs 30% in pN1 patients (P = 0.8), and 28% vs 65% in pNx patients (P < 0.001). On multivariable analysis, the presence of LVI was associated with less favourable OS (hazard ratio 1.79, 95% confidence interval 1.60-1.99; P < 0.001). CONCLUSION: Our study assessed the impact of LVI on OS in patients with UTUC in a large North American nationwide cohort. Our series, as the largest to date, indicate that LVI is associated with less favourable survival outcomes in patients with UTUC after RNU, and this variable could be used in counselling patients about their prognosis and might be a useful tool for future trials to risk-stratify patients.

Distinct mast cell subpopulations within and around lymphatic vessels regulate lymph flow and progression of inflammatory-erosive arthritis in TNF-transgenic mice.

Objective: Inflammatory-erosive arthritis is exacerbated by dysfunction of joint-draining popliteal lymphatic vessels (PLVs). Synovial mast cells are known to be pro-inflammatory in rheumatoid arthritis (RA). In other settings they have anti-inflammatory and tissue reparative effects. Herein, we elucidate the role of mast cells on PLV function and inflammatory-erosive arthritis in tumor necrosis factor transgenic (TNF-tg) mice that exhibit defects in PLVs commensurate with disease progression. Methods: Whole mount immunofluorescent microscopy, toluidine blue stained histology, scanning electron microscopy, and in silico bioinformatics were performed to phenotype and quantify PLV mast cells. Ankle bone volumes were assessed by µCT, while corresponding histology quantified synovitis and osteoclasts. Near-infrared indocyanine green imaging measured lymphatic clearance as an outcome of PLV draining function. Effects of genetic MC depletion were assessed via comparison of 4.5-month-old WT, TNF-tg, MC deficient KitW-sh/W-sh (cKit-/-), and TNF-tg x cKit-/- mice. Pharmacological inhibition of mast cells was assessed by treating TNF-tg mice with placebo or cromolyn sodium (3.15mg/kg/day) for 3-weeks. Results: PLVs are surrounded by MCT+/MCPT1+/MCPT4+ mast cells whose numbers are increased 2.8-fold in TNF-tg mice. The percentage of peri-vascular degranulating mast cells was inversely correlated with ICG clearance. A population of MCT+/MCPT1-/MCPT4- mast cells were embedded within the PLV structure. In silico single-cell RNA-seq (scRNAseq) analyses identified a population of PLV-associated mast cells (marker genes: Mcpt4, Cma1, Cpa3, Tpsb2, Kit, Fcer1a & Gata2) with enhanced TGFß-related signaling that are phenotypically distinct from known MC subsets in the Mouse Cell Atlas. cKit-/- mice have greater lymphatic defects than TNF-tg mice with exacerbation of lymphatic dysfunction and inflammatory-erosive arthritis in TNF-tg x cKit-/- vs. TNF-Tg mice. Cromolyn sodium therapy stabilized PLV mast cells, increased TNF-induced bone loss, synovitis, and osteoclasts, and decreased ICG clearance. Conclusions: Mast cells are required for normal lymphatic function. Genetic ablation and pharmacological inhibition of mast cells exacerbates TNF-induced inflammatory-erosive arthritis with decreased lymphatic clearance. Together, these findings support an inflammatory role of activated/degranulated peri-PLV mast cells during arthritic progression, and a homeostatic role of intra-PLV mast cells, in which loss of the latter dominantly exacerbates arthritis secondary to defects in joint-draining lymphatics, warranting investigation into specific cellular mechanisms.

Lymphangiogenesis after nonvascularized lymph node transplantation: Lymphangiographic findings in mice and minipigs.

The establishment of new connections after NVLNT (non-vascularized lymph node transplantation) is still poorly understood. The purpose of this study was to investigate lymphatic connections after NVLNT using lymphangiography. In a mice model, 40 mice were allocated to undergo NVLNT or sham surgery. On day 21 after NVLNT, the lymphatic vessels were observed on near-infrared fluorescence imaging with indocyanine green. In a minipig model, 12 minipigs underwent NVLNT. On day 14 after NVLNT, the transplanted lymph node and donor site were checked by ultrasound, and minipigs with viable transplanted LNs were allocated to lipiodol lymphangiography or MR lymphangiography groups. Transplanted LN engraftment was examined with immunohistochemical staining. After NVLNT in mice, the signal intensities in the popliteal region at 3 minutes and 5 minutes were higher in the transplanted side than the control side (21.3 ± 8.1 vs. 11.0 ± 4.6 at 3 minutes, 26.7 ± 6.8 vs. 19.7 ± 5.9 at 5 minutes), while in the sham group, there were no significant differences between sides. In minipigs, lipiodol lymphangiography (n = 5) showed Lipiodol accumulation in transplanted LNs with innumerable newly formed lymphatic vessels and lymphovenous shunts. MR lymphangiography (n = 5) showed higher enhancement on the transplanted side compared to the control side. Histology showed successful engraftment of transplanted LNs in 16 out of 20 (80%) mice and 9 out of 12 (75%) minipigs. Omnidirectional lymphangiogenesis forming a dense lymphatic network and spontaneous formation of lymphovenous shunts were shown after NVLNT.

YAP1/Piezo1 involve in the dynamic changes of lymphatic vessels in UVR-induced photoaging progress to squamous cell carcinoma.

BACKGROUND: UV-induced cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers. The constant alterations of the lymphatic-centered immune microenvironment are essential in transforming from photoaging to cSCC. Studying the mechanism will be beneficial for new targets exploration to the early prediction of cSCC. AIMS: To investigate the dynamic changes and mechanism of the lymphatic-centered immune microenvironment in transforming from photoaging to cSCC induced by ultraviolet irradiation (UVR). METHODS: TIMER2.0 was used to analyze whether YAP1/VEGFC signaling pathway is involved in lymphangiogenesis in head and neck squamous cell carcinoma (HNSCC). Meanwhile, lymphatic-centered immune microenvironments alterations and the related cumulative survival time were also analyzed. With the accumulated UVR, skin photoaging developed and gradually progressed into actinic keratosis and cSCC on SKH-1 hairless mice. The skin lymphatic-centered immune microenvironment was evaluated at the 0th, 8th, 12th, 16-18th, and 20-24th week of UVR. Skin phenotype was assessed using optical coherence tomography (OCT) and skin image. H&E and Masson's trichrome staining evaluated epidermis and dermis. The structure of lymphatic vessels (LVs), blood vessels, and different types of T cells were evaluated by immunohistochemistry staining. The expression of Piezo1 whose deletion in adult lymphatics led to substantial valve degeneration, VE-cadherin that maintained the permeability of LVs, and YAP1 were evaluated by immunohistochemistry staining as well. Besides, the drainage function of LVs was assessed by Evans Blue assay in vivo. RESULTS: The lymphatic function and immune cell infiltration underwent adaptive changes under continuous UVR. TIMER2.0 analysis indicated that VEGFC genes high expressed in HNSCC. YAP1 gene expression was positive correlated with VEGFC in HNSCC. LV density increased in human cSCC. More LVs in HNSCC were beneficial to prolong the survival time. VEGFC gene overexpression was positive correlated to CD8+T cell infiltration. More CD8A+T cells and CD8B+T cell infiltration in HNSCC extended survival time. When YAP1 gene overexpression and high infiltration of endothelial cells took place simultaneously might prolong the survival time of HNSCC patients. And high infiltration of CD8+T cells prolonged the survival time as well. In animal studies, UVR-induced eight weeks (photoaging) and 16-18 weeks (precancerous) were two turning points. The density of LVs in UV-8w was the least. When photoaged skin developed into AK lesions (UV-16-18w), LV slightly exceeded healthy skin and proliferated sharply in cSCC (UV-20-24w). YAP1 expression was almost consistent with LV but rose after the photoaging stage. The drainage of cSCC mice induced by UVR was better than that of photoaged skin and worse than that of health skin. The dynamic alterations of LVs number, Piezo1 expression, and collagen might be reasons for it. The expression of Piezo1 was in the highest point after 8 weeks of UVR, then gradually descended to the platform. The total T cells increased slowly, but the infiltration of CD4+T cells increased, and CD8+T cells decreased after eight weeks of UVR. The CD8+T cells and CD4+T cells increased sharply in UV-16-18w and UV-20-24w groups. CONCLUSION: The lymphatic-centered immune microenvironment underwent adaptive changes under continuous UVR via regulating YAP1/VEGFC and Piezo1. During the formation of cSCC, there are two turning points, eight weeks (photoaging) and 16-18 weeks (precancerous). YAP1, Piezo1, LVs, and immune cells constantly changed with the skin state induced by UVR. According to these changes the process of cSCC can be identified in advance and intervene timely.

Potential significance of podoplanin immunohistochemical expression in papillary thyroid carcinoma.

Podoplanin (PDPN) is a lymphatic endothelial marker expressed by a range of human malignancies in which it has been shown to contribute to tumor progression and metastasis. However, there is a lack of the studies, examining the function of PDPN in thyroid cancer. The current study was performed to explore the possible diagnostic value of PDPN expression in papillary thyroid cancer (PTC) and to evaluate the marker's potential for prediction of regional lymph node metastasis. Lymphatic vascular density (LVD) and the stromal/cancer-associated fibroblasts (CAFs), labeled by PDPN, were examined in PTC compared to the other thyroid lesions. The current study included 50 cases of PTC and 50 cases of non-PTC thyroid lesions. Immunohistochemical staining was performed using monoclonal PDPN antibodies. Podoplanin expression was scored as positive and negative. Podoplanin expression was found in 36% of PTC cases, but it was not found in benign, low risk (borderline), or malignant lesions other than PTC. Furthermore, lymph node metastasis was significantly correlated with PDPN expression, LVD and CAFs (p-values < 0.00001, < 0.001 and 0.0002 respectively). These findings support the diagnostic utility of PDPN expression in PTC and its predictive value for LN metastasis.