The lymphatic system is formed during embryonic development by the commitment of specialized lymphatic endothelial cells (LECs) and their subsequent assembly in primary lymphatic vessels. Although lymphatic cells are in continuous contact with mesenchymal cells during development and in adult tissues, the role of mesenchymal cells in lymphatic vasculature development remains poorly characterized. Here, we show that a subpopulation of mesenchymal cells expressing the transcription factor Osr1 are in close association with migrating LECs and established lymphatic vessels in mice. Lineage tracing experiments revealed that Osr1+ cells precede LEC arrival during lymphatic vasculature assembly in the back of the embryo. Using Osr1-deficient embryos and functional in vitro assays, we show that Osr1 acts in a non-cell-autonomous manner controlling proliferation and early migration of LECs to peripheral tissues. Thereby, mesenchymal Osr1+ cells control, in a bimodal manner, the production of extracellular matrix scaffold components and signal ligands crucial for lymphatic vessel formation.

Cancer dissemination to lymph nodes (LN) is associated with a worse prognosis, increased incidence of distant metastases and reduced response to therapy. The LN microenvironment puts selective pressure on cancer cells, creating cells that can survive in LN as well as providing survival advantages for distant metastatic spread. Additionally, the presence of cancer cells leads to an immunosuppressive LN microenvironment, favoring the evasion of anti-cancer immune surveillance. However, recent studies have also characterized previously unrecognized roles for tumor-draining lymph nodes (TDLNs) in cancer immunotherapy response, including acting as a reservoir for pre-exhausted CD8+ T cells and stem-like CD8+ T cells. In this review, we will discuss the spread of cancer cells through the lymphatic system, the roles of TDLNs in metastasis and anti-cancer immune responses, and the therapeutic opportunities and challenges in targeting LN metastasis.

Microglia are specialized immune cells that reside in the central nervous system (CNS) and play a crucial role in maintaining the homeostasis of the brain microenvironment. While traditionally regarded as a part of the innate immune system, recent research has highlighted their role in adaptive immunity. The CNS is no longer considered an immune-privileged organ, and increasing evidence suggests bidirectional communication between the immune system and the CNS. Microglia are sensitive to systemic immune signals and can respond to systemic inflammation by producing various inflammatory cytokines and chemokines. This response is mediated by activating pattern recognition receptors (PRRs), which recognize pathogen- and danger-associated molecular patterns in the systemic circulation. The microglial response to systemic inflammation has been implicated in several neurological conditions, including depression, anxiety, and cognitive impairment. Understanding the complex interplay between microglia and systemic immunity is crucial for developing therapeutic interventions to modulate immune responses in the CNS.

Microglia, the crucial immune cells inhabiting the central nervous system (CNS), perform a range of vital functions, encompassing immune defense and neuronal regulation. Microglia subsets with diverse functions and distinct developmental regulations have been identified recently. It is generally accepted that all microglia originate from hematopoiesis and depend on the myeloid transcription factor PU.1. However, a recent study reported the existence of mrc1+ microglia in zebrafish embryos, which are seemingly independent of Pu.1 and reliant on lymphatic vessels, sparking great interest in the possibility of lymphatic-originated microglia. To address this, we took advantage of a pu.1 knock-in zebrafish allele for a detailed investigation. Our results conclusively showed that almost all zebrafish embryonic microglia (~95% on average) express pu.1. Further, lineage tracing and mutant analysis revealed that these microglia neither emerged from nor depended on lymphatic vessels. In essence, our study refutes the presence of pu.1-independent but lymphatic-dependent microglia.

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Understanding the function of the nasal vasculature in homeostasis and pathogenesis of common nasal diseases is important. Here we describe an extensive network of venous sinusoids (VSs) in mouse and human nasal mucosa. The endothelium of the VSs expressed Prox1 (considered to be a constitutive marker of lymphatic endothelium) and high levels of VCAM-1 and exhibited unusual cell-to-cell junctions. VSs are supported by circular smooth muscle cells (SMCs) and surrounded by immune cells. The nasal mucosa also showed a rich supply of lymphatic vessels with distinctive features, such as the absence of the lymphatic marker LYVE1 and sharp-ended capillaries. In mouse models of allergic rhinitis or acute Coronavirus Disease 2019 (COVID-19) infection, Prox1+ VSs were regressed or compromised. However, in aged mice, the VSs lost the SMC support and were expanded and enlarged. Our findings demonstrate three-dimensional morphological and molecular heterogeneities of the nasal vasculature and offer insights into their associations with nasal inflammation, infection and aging.

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Objective: To analyze the lympho-vascular space invasion (LVSI) in different molecular subtypes of the cancer genome atlas (TCGA) molecular subtypes of endometrial cancer (EC) and to evaluate the prognostic value of LVSI in EC patients with different molecular subtypes. Methods: A total of 258 patients diagnosed EC undergoing surgery in Peking University People\'s Hospital from January 2016 to June 2022 were analyzed retrospectively. Among 258 patients, 14 cases were classified as POLE-ultramutated subtype, 43 as high-microsatellite instability (MSI-H) subtype, 155 as copy-number low (CNL) subtype, and 46 as copy-number high (CNH) subtype. Fifty-four patients were positive for LVSI, while 203 tested negative. Results: (1) The incidence of LVSI was found to be highest in the CNH subtype (32.6%,15/46), followed by the MSI-H subtype (27.9%, 12/43), the CNL subtype (16.9%, 26/154), and the POLE-ultramutated subtype (1/14), with statistically significant differences (χ2=7.79, P=0.044). (2) Staging and deep myometrial invasion were higher in the LVSI positive group than those in the LVSI negative group (all P<0.05), except for the POLE-ultramutated subtype. The grade, lymph node metastasis, and the expression of nuclear antigen associated with cell proliferation (Ki-67) were significantly higher in LVSI positive patients than those in LVSI negative EC patients with both MSI-H and CNL subtypes (all P<0.05). In CNL subtypes patients, LVSI was also associated with age, histology subtype,and progesterone receptor (PR; all P<0.05). (3) Of the 257 EC patients, 25 cases recurred during the follow-up period, with a recurrence rate of 9.7% (25/257); among them, the recurrence rate of LVSI positive patients was 22.2% (12/54), which was significantly higher than those with LVSI negative (6.4%, 13/203; χ2=12.15, P<0.001). During the follow-up period, none of the 14 patients with POLE-ultramutated had recurrence; among CNL patients, the recurrence rate was 19.2% (5/26) in LVSI positive patients, which was significantly higher than that in LVSI negative ones (5.5%, 7/128; χ2=3.94, P=0.047); where as no difference were found in both MSI-H [recurrence rates in LVSI positive and negative patients were 2/12 and 9.7% (3/31), respectively] and CNH subtype [recurrence rates between LVSI positive and negative patients were 5/15 and 9.7% (3/31), respectively] EC patients (both P>0.05). After log-rank test, the 3-year recurrence free survival (RFS) rate were significantly lower in LVSI positive patients from CNL subtype and CNH subtype than those in LVSI negative patients (CNL: 80.8% vs 94.5%; CNH: 66.7% vs 90.3%; both P<0.05). (4) Lymph node metastasis (HR=6.93, 95%CI: 1.15-41.65; P=0.034) had a significant effect on the 3-year RFS rate of EC patients with MSI-H subtype. Multivariate analysis revealed that PR expression (HR=0.04, 95%CI: 0.01-0.14;P<0.001) was significantly associated with the 3-year RFS rate of CNL subtype patients. Conclusions: LVSI has the highest positivity rate in CNH subtype, followed by MSI-H subtype, CNL subtype, and the lowest positivity rate in POLE-ultramutated subtype. LVSI is significantly associated with poor prognosis in CNL subtype patients and may affect the prognosis of CNH subtype patients. However, LVSI is not an independent risk factor for recurrence across all four TCGA molecular subtypes.
目的： 分析不同癌症基因组图谱（TCGA）分子分型子宫内膜癌（EC）中淋巴脉管间隙浸润（LVSI）的情况，并评估LVSI对不同分子分型EC患者预后的影响。 方法： 回顾性分析2016年1月—2022年6月在北京大学人民医院接受手术治疗的258例EC患者的临床病理资料。其中，TCGA分子分型为POLE超突变型14例，高度微卫星不稳定性（MSI-H）型43例，低拷贝数（CNL）型155例，高拷贝数（CNH）型46例；LVSI阳性54例，LVSI阴性203例，不详（术后失访，为CNL型）1例。 结果： （1）LVSI阳性率：不同分子分型EC患者的LVSI阳性率依次为CNH型（32.6%，15/46）、MSI-H型（27.9%，12/43）、CNL型（16.9%，26/154）、POLE超突变型（1/14），4种分子分型患者的LVSI阳性率比较，差异有统计学意义（χ2=7.79，P=0.044）。（2）临床病理资料：除POLE超突变型外，其余3种分子分型EC中LVSI阳性患者的手术病理分期、深肌层浸润比例均显著高于LVSI阴性者（P均<0.05）；在MSI-H型和CNL型EC中，LVSI阳性患者的病理分级、淋巴结转移率及细胞增殖相关核抗原（Ki-67）指数均显著高于LVSI阴性者（P均<0.05）；在CNL型EC中，LVSI状态还与患者年龄、病理类型及孕激素受体（PR）表达状态均显著有关（P均<0.05）。（3）复发率：257例EC患者（1例失访）中，随访期内复发25例，复发率为9.7%（25/257）；其中，LVSI阳性患者的复发率为22.2%（12/54），显著高于LVSI阴性者（6.4%，13/203；χ2=12.15，P<0.001）。其中，14例POLE超突变型EC患者均无复发；CNL型EC患者中，LVSI阳性患者的复发率为19.2%（5/26），显著高于LVSI阴性者（5.5%，7/128；χ2=3.94，P=0.047）；而在MSI-H型［LVSI阳性、阴性患者的复发率分别为2/12、9.7%（3/31）］和CNH型［LVSI阳性、阴性患者的复发率分别为5/15、9.7%（3/31）］EC患者中均未发现这种差异（P均>0.05）。log-rank检验显示，CNL型和CNH型EC中LVSI阳性患者的3年无复发生存（RFS）率（分别为80.8%、66.7%）均显著低于LVSI阴性者（分别为94.5%、90.3%；P均<0.05）。（4）预后影响因素：单因素分析显示，在MSI-H型EC患者中，淋巴结转移（HR=6.93，95%CI为1.15~41.65；P=0.034）显著影响患者的3年RFS率；在CNL型EC患者中，手术病理分期、病理分级、淋巴结转移、ER表达、PR表达、p53表达、Ki-67表达、手术途径、术后辅助化疗均显著影响患者的3年RFS率（P均<0.05）。多因素分析显示，PR表达（HR=0.04，95%CI为0.01~0.14；P<0.001）为影响CNL型EC患者3年RFS率的独立危险因素。 结论： LVSI在CNH型中阳性率最高，其次为MSI-H型、CNL型，在POLE超突变型中阳性率最低。LVSI与CNL型EC患者的不良预后明显相关，与CNH型EC患者的预后可能相关。但在各分子分型EC患者中，LVSI均不是影响其复发的独立危险因素。.

BACKGROUND: Atraumatic subarachnoid haemorrhage (SAH) is associated with high morbidity and mortality. Proposed mechanisms for red blood cell (RBC) clearance from the subarachnoid space (SAS) are erythrolysis, erythrophagocytosis or through efflux along cerebrospinal fluid (CSF) drainage routes. We aimed to elucidate the mechanisms of RBC clearance from the SAS to identify targetable efflux pathways.
METHODS: Autologous fluorescently-labelled RBCs along with PEGylated 40 kDa near-infrared tracer (P40D800) were infused via the cisterna magna (i.c.m.) in female reporter mice for lymphatics or for resident phagocytes. Drainage pathways for RBCs to extracranial lymphatics were evaluated by in vivo and in situ near-infrared imaging and by immunofluorescent staining on decalcified cranial tissue or dural whole-mounts.
RESULTS: RBCs drained to the deep cervical lymph nodes 15 min post i.c.m. infusion, showing similar dynamics as P40D800 tracer. Postmortem in situ imaging and histology showed perineural accumulations of RBCs around the optic and olfactory nerves. Numerous RBCs cleared through the lymphatics of the cribriform plate, whilst histology showed no relevant fast RBC clearance through dorsal dural lymphatics or by tissue-resident macrophage-mediated phagocytosis.
CONCLUSIONS: This study provides evidence for rapid RBC drainage through the cribriform plate lymphatic vessels, whilst neither fast RBC clearance through dorsal dural lymphatics nor through spinal CSF efflux or phagocytosis was observed. Similar dynamics of P40D800 and RBCs imply open pathways for clearance that do not impose a barrier for RBCs. This finding suggests further evaluation of the cribriform plate lymphatic function and potential pharmacological targeting in models of SAH.
BACKGROUND: Swiss National Science Foundation (310030_189226), SwissHeart (FF191155).

Although nanoparticle-based lymphatic drug delivery systems promise better treatment of cancer, infectious disease, and immune disease, their clinical translations are limited by low delivery efficiencies and unclear transport mechanisms. Here, we employed a three-dimensional (3D) lymphatics-on-a-chip featuring an engineered lymphatic vessel (LV) capable of draining interstitial fluids including nanoparticles. We tested lymphatic drainage of different sizes (30, 50, and 70 nm) of PLGA-b-PEG nanoparticles (NPs) using the lymphatics-on-a-chip device. In this study, we discovered that smaller NPs (30 and 50 nm) transported faster than larger NPs (70 nm) through the interstitial space, as expected, but the smaller NPs were captured by lymphatic endothelial cells (LECs) and accumulated within their cytosol, delaying NP transport into the lymphatic lumen, which was not observed in larger NPs. To examine the mechanisms of size-dependent NP transports, we employed four inhibitors, dynasore, nystatin, amiloride, and adrenomedullin, to selectively block dynamin-, caveolin-, macropinocytosis-mediated endocytosis-, and cell junction-mediated paracellular transport. Inhibiting dynamin using dynasore enhanced the transport of smaller NPs (30 and 50 nm) into the lymphatic lumen, minimizing cytosolic accumulation, but showed no effect on larger NP transport. Interestingly, the inhibition of caveolin by nystatin decreased the lymphatic transport of larger NPs without affecting the smaller NP transport, indicating distinct endocytosis mechanisms used by different sizes of NPs. Macropinocytosis inhibition by amiloride did not change the drainage of all sizes of NPs; however, paracellular transport inhibition by adrenomedullin blocked the lymphatic transport of NPs of all sizes. We further revealed that smaller NPs were captured in the Rab7-positive late-stage lymphatic endosomes to delay their lymphatic drainage, which was reversed by dynamin inhibition, suggesting that Rab7 is a potential target to enhance the lymphatic delivery of smaller NPs. Together, our 3D lymphatics-on-a-chip model unveils size-dependent NP transport mechanisms in lymphatic drug delivery.