Systematic optimization and evaluation of culture conditions for the construction of circulating tumor cell clusters using breast cancer cell lines.

BACKGROUND: Circulating tumor cell (CTC) clusters play a critical role in carcinoma metastasis. However, the rarity of CTC clusters and the limitations of capture techniques have retarded the research progress. In vitro CTC clusters model can help to further understand the biological properties of CTC clusters and their clinical significance. Therefore, it is necessary to establish reliable in vitro methodological models to form CTC clusters whose biological characteristics are very similar to clinical CTC clusters. METHODS: The assays of immunofluorescence, transmission electron microscopy, EdU incorporation, cell adhension and microfluidic chips were used. The experimental metastasis model in mice was used. RESULTS: We systematically optimized the culture methods to form in vitro CTC clusters model, and more importantly, evaluated it with reference to the biological capabilities of reported clinical CTC clusters. In vitro CTC clusters exhibited a high degree of similarity to the reported pathological characteristics of CTC clusters isolated from patients at different stages of tumor metastasis, including the appearance morphology, size, adhesive and tight junctions-associated proteins, and other indicators of CTC clusters. Furthermore, in vivo experiments also demonstrated that the CTC clusters had an enhanced ability to grow and metastasize compared to single CTC. CONCLUSIONS: The study provides a reliable model to help to obtain comparatively stable and qualified CTC clusters in vitro, propelling the studies on tumor metastasis.

Rosmarinic acid in combination with ginsenoside Rg1 suppresses colon cancer metastasis via co-inhition of COX-2 and PD1/PD-L1 signaling axis.

Metastasis of colorectal cancer (CRC) is a leading cause of mortality among CRC patients. Elevated COX-2 and PD-L1 expression in colon cancer tissue has been linked to distant metastasis of tumor cells. Although COX-2 inhibitors and immune checkpoint inhibitors demonstrate improved anti-tumor efficacy, their toxicity and variable therapeutic effects in individual patients raise concerns. To address this challenge, it is vital to identify traditional Chinese medicine components that modulate COX-2 and PD-1/PD-L1: rosmarinic acid (RA) exerts striking inhibitory effect on COX-2, while ginsenoside Rg1 (GR) possesses the potential to suppress the binding of PD-1/PD-L1. In this study we investigated whether the combination of RA and GR could exert anti-metastatic effects against CRC. MC38 tumor xenograft mouse model with lung metastasis was established. The mice were administered RA (100 mg·kg-1·d-1, i.g.) alone or in combination with GR (100 mg·kg-1·d-1, i.p.). We showed that RA (50, 100, 150 µM) or a COX-2 inhibitor Celecoxib (1, 3, 9 µM) concentration-dependently inhibited the migration and invasion of MC38 cells in vitro. We further demonstrated that RA and Celecoxib inhibited the metastasis of MC38 tumors in vitro and in vivo via interfering with the COX-2-MYO10 signaling axis and inhibiting the generation of filopodia. In the MC38 tumor xenograft mice, RA administration significantly decreased the number of metastatic foci in the lungs detected by Micro CT scanning; RA in combination with GR that had inhibitory effect on the binding of PD-1 and PD-L1 further suppressed the lung metastasis of colon cancer. Compared to COX-2 inhibitors and immune checkpoint inhibitors, RA and GR displayed better safety profiles without disrupting the tissue structures of the liver, stomach and colon, offering insights into the lower toxic effects of clinical traditional Chinese medicine against tumors while retaining its efficacy.

Capsaicin orchestrates metastasis in gastric cancer via modulating expression of TRPV1 channels and driving gut microbiota disorder.

The association between capsaicin, the major natural pungent compound of chili peppers, and gastric cancer progression has engendered conflicting findings. In this work, we sought to explore the character of a high capsaicin diet in gastric cancer metastasis and its possible mechanism. The impact of high capsaicin consumption on gastric cancer metastasis was investigated in vivo (xenograft mouse and zebrafish models) and in vitro (biochemical and molecular assays). It was demonstrated that high diet of capsaicin gave rise to accelerate tumor metastasis, which was partially mediated by elevating the expression of transient receptor potential vanilloid 1 (TRPV1) in gastric cancer cells. Importantly, we found that genetic depletion of TRPV1 could reduce gastric cancer metastasis by diminishing the motility of tumor cells in vitro, but acted poorly in xenograft mouse model. Considering the distribution of capsaicin in vivo, 16S rRNA sequencing and fecal microbiota transplantation (FMT) were used to appraise whether the gut microbiota involved in the high capsaicin diet induced metastasis. It was demonstrated that the level of Firmicutes and Clostridiales was expressively boosted following the high consumption of capsaicin. This microbial shift contributed to the increased peripheral 5-hydroxytryptamine (5-HT) levels, yielding the aggravated metastatic burden. Collectively, our findings highlighted the potential risk of high capsaicin diet in promoting gastric cancer metastasis by virtue of modulating TRPV1 expression and gut microbiota composition, indicating the importance of controlled consumption of chili peppers for patients with gastric cancer. Video Abstract.

Ginsenoside Rh1, a novel casein kinase II subunit alpha (CK2α) inhibitor, retards metastasis via disrupting HHEX/CCL20 signaling cascade involved in tumor cell extravasation across endothelial barrier.

Tumor cell extravasation across endothelial barrier has been recognized as a pivotal event in orchestrating metastasis formation. This event is initiated by the interactions of extravasating tumor cells with endothelial cells (ECs). Therefore, targeting the crosstalk between tumor cells and ECs might be a promising therapeutic strategy to prevent metastasis. In this study, we demonstrated that Rh1, one of the main ingredients of ginseng, hindered the invasion of breast cancer (BC) cells as well as diminished the permeability of ECs both in vitro and in vivo, which was responsible for the attenuated tumor cell extravasation across endothelium. Noteworthily, we showed that ECs were capable of inducing the epithelial-mesenchymal transition (EMT) and invadopodia of BC cells that are essential for tumor cell migration and invasion through limiting the nuclear translocation of hematopoietically expressed homeobox (HHEX). The decreased nuclear HHEX paved the way for initiating the CCL20/CCR6 signaling axis, which in turn contributed to damaged endothelial junctions, uncovering a new crosstalk mode between tumor cells and ECs. Intriguingly, Rh1 inhibited the kinase activity of casein kinase II subunit alpha (CK2α) and further promoted the nuclear translocation of HHEX in the BC cells, which resulted in the disrupted crosstalk between chemokine (C-C motif) ligand 20 (CCL20) in the BC cells and chemokine (C-C motif) receptor 6 (CCR6) in the ECs. The prohibited CCL20-CCR6 axis by Rh1 enhanced vascular integrity and diminished tumor cell motility. Taken together, our data suggest that Rh1 serves as an effective natural CK2α inhibitor that can be further optimized to be a therapeutic agent for reducing tumor cell extravasation.

TRPV4 Promotes Metastasis in Melanoma by Regulating Cell Motility through Cytoskeletal Rearrangement.

The abnormal expression of Transient Receptor Potential cation channel subfamily V member 4 (TRPV4) is closely related to the progression of multiple tumors. In addition, TRPV4 is increasingly being considered a potential target for cancer therapy, especially in tumor metastasis prevention. However, the biological correlation between TRPV4 and tumor metastasis, as well as the specific role of TRPV4 in malignant melanoma metastasis, is poorly understood. In this study, we aimed to examine the role of TRPV4 in melanoma metastasis through experiments and clinical data analysis, and the underlying anticancer mechanism of Baicalin, a natural compound, and its inhibitory effect on TRPV4 with in vivo and in vitro experiments. Our findings suggested that TRPV4 promotes metastasis in melanoma by regulating cell motility via rearranging the cytoskeletal, and Baicalin can inhibit cancer metastasis, whose mechanisms reverse the recruitment of activated cofilin to leading-edge protrusion and the increasing phosphorylation level of cortactin, which is provoked by TRPV4 activation.

Thermo-Transient Receptor Potential Channels: Therapeutic Potential in Gastric Cancer.

Over the last decade, researchers have found abnormal expression of transient receptor potential (TRP) channels. In particular, members of the thermally sensitive subclass (thermo-TRPs) are involved in many disease processes. Moreover, they have a vital role in the occurrence and development of gastric cancer (GC). Accordingly, thermo-TRPs constitute a major pharmacological target, and the elucidation of the mechanisms underlying their response to physiological stimuli or drugs is key for notable advances in GC treatment. Therefore, this paper summarizes the existing literature about thermo-TRP protein expression changes that are linked to the incidence and progression of GC. The review also discusses the implication of such association to pathology and cell physiology and identifies potential thermo-TRP protein targets for diagnosis and treatment of GC.

lncRNA ZFPM2-AS1 promotes proliferation via miR-18b-5p/VMA21 axis in lung adenocarcinoma.

Lung cancer has been proved to be one of the most common kinds of cancers around the globe. Meanwhile, as the predominant type of lung cancer, lung adenocarcinoma (LUAD) has received increasing attention in cancer research. Long noncoding RNAs (lncRNAs) are known to be associated with oncogenesis and progression of various cancers. However, many lncRNAs have not been thoroughly detected in LUAD. In this study, through bioinformatics analysis we found that zinc finger protein multitype 2 antisense RNA 1 (ZFPM2-AS1) was associated with poor prognosis of LUAD patients. Also, ZFPM2-AS1 was detected to be overexpressed in LUAD tissues and cells. Furthermore, ZFPM2-AS1 could promote the proliferation of LUAD cells. Next, miR-18b-5p was found to bind with and negatively regulated by ZFPM2-AS1. VMA21, target gene of miR-18b-5p, could bind with and be negatively regulated by miR-18b-5p. More importantly, both ZFPM2-AS1 and VMA21 were found to be attached to the RNA-induced silencing complex constructed from miR-18b-5p and Ago2. Also, ZFPM2-AS1 could regulate the expression of VMA21. Therefore, ZFPM2-AS1 were confirmed to regulate VMA21 by competitively binding with miR-18b-5p. Finally, rescue assays confirmed that ZFPM2-AS1 could regulate LUAD cell proliferation via miR-18b-5p/VMA21 axis.

Relieving Sore Throat Formula Exerts a Therapeutic Effect on Pharyngitis through Immunoregulation and NF-κB Pathway.

Relieving Sore Throat Formula (RSTF) is a formula approved by the China Food and Drug Administration and has been used for the treatment of pharyngitis in clinic for many years. However, the potential pharmacological mechanism still remains unknown. We combined multiple methods including bioinformatics data digging, network pharmacology analysis, and pathway analysis to predict the potential target of RSTF. We verified our in silico prediction results with an in vivo/vitro antibacterial effect test, mouse phagocytic index test, proliferation, transformation, and migration of mouse spleen lymphocytes. Alteration of NF-κB pathway was determined by Western blotting, immunofluorescence, and PCR. The in vivo experiments demonstrated that the RSTF could significantly relieve the symptoms of pharyngitis. A rat saliva secretion test showed that RSTF can effectively relieve the xerostomia symptom. A phenol red excretion test showed that RSTF has an eliminating phlegm effect. A hot plate method and granuloma experiment proved that RSTF also have analgesic and anti-inflammatory effects. In silico prediction demonstrates that 70 active compounds of RSTF were filtered out through ADME screening and 84 putative targets correlated with different diseases. Pathway enrichment analysis showed that the candidate targets were mostly related to the response to bacteria and immunity signalling pathways, which are known contributors to pharyngitis. Experimental results confirmed that RSTF exerted therapeutic effects on pharyngitis mainly by antibacterial effect and downregulation of NF-κB activities. It is demonstrated both in silico and in vivo/vitro that RSTF exerted therapeutic effects on pharyngitis mainly through an antibiotic effect and downregulation of NF-κB signalling pathway.

Exploring the 'cold/hot' properties of traditional Chinese medicine by cell temperature measurement.

Context: It is common sense that chewing a mint leaf can cause a cooling feeling, while chewing ginger root will produce a burning feeling. In Traditional Chinese Medicine (TCM), this phenomenon is referred to as 'cold/hot' properties of herbs. Herein, it is reported that TCM with different "cold/hot" properties have different effects on the variation of cells.Objective: To explore the intrinsic 'cold/hot' properties of TCM from the perspective of cellular and molecular biology.Materials and methods: A375 cells were selected using Cancer Cell Line Encyclopaedia (CCLE) analysis and western blots. Hypaconitine and baicalin were selected by structural similarity analysis from 56 and 140 compounds, respectively. A wireless thermometry system was used to measure cellular temperature change induced by different compounds. Alteration of intracellular calcium influx was investigated by means of calcium imaging.Results: The IC50 values of GSK1016790A, HC067047, hypaconitine, and baicalin for A375 cells are 8.363 nM, 816.4 µM, 286.4 µM and 29.84 µM, respectively. And, 8 µM hypaconitine induced obvious calcium influx while 8 µM baicalin inhibited calcium influx induced by TRPV4 activation. Cellular temperature elevated significantly when treated with GSK1016790A or hypaconitine, while the results were reversed when cells were treated with HC067047 or baicalin.Discussion and conclusions: The changes in cellular temperature are speculated to be caused by the alteration of intracellular calcium influx mediated by TRPV4. In addition, the 'cold/hot' properties of compounds in TCM can be classified by using cellular temperature detection.

Beta-elemene inhibits breast cancer metastasis through blocking pyruvate kinase M2 dimerization and nuclear translocation.

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta-elemene (ß-elemene), an approved drug for complementary cancer therapy, exerted distinct anti-metastatic activity dependent on PKM2. The results indicated that ß-elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. ß-Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that ß-elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of ß-elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose-1,6-bisphosphate (FBP) and L-cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and ß-elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti-metastatic agent from natural compounds.

Zhile Capsule Exerts Antidepressant-Like Effects through Upregulation of the BDNF Signaling Pathway and Neuroprotection.

Major depressive disorder is now becoming a common disease in daily life, and most patients do not have satisfactory treatment outcomes. We herein evaluated the therapeutic effects of Zhile capsule and clarified the molecular mechanism. A rat model of chronic unpredictable mild stress-induced depression was established to assess the antidepressant-like effects of Zhile by using the sucrose preference test, open field test, forced swim test, tail suspension test and HPLC. Systems pharmacology was then performed to unravel the underlying mechanism which was confirmed by western blot, enzyme-linked immunosorbent assay, and qPCR. Zhile alleviated depression-like behaviors by upregulating the cAMP-CREB-BDNF (brain-derived neurotrophic factor) axis to exert neuroprotective effects. It may be beneficial to depressive patients in clinical practice.

Xanthatin triggers Chk1-mediated DNA damage response and destabilizes Cdc25C via lysosomal degradation in lung cancer cells.

Previous studies had shown that xanthatin, a natural xanthanolide sesquiterpene lactone, could induce mitotic arrest and apoptosis in non-small cell lung cancer (NSCLC) cells. Here, we examined whether the DNA damage response (DDR) could be a primary cytotoxic event underlying xanthatin-mediated anti-tumor activity. Using EdU incorporation assay in combination with novel imaging flow cytometry, our data indicated that xanthatin suppressed DNA replication, prevented cells from G2/M entry and increased the spot count of γH2AX nuclear foci. Given that checkpoint kinase 1 (Chk1) represents a core component in DDR-mediated cell cycle transition and the phosphorylation on Ser-345 is essential for kinase activation and function, we surprisingly found xanthatin distinctly modulated Ser-345 phosphorylation of Chk1 in A549 and H1299 cells. Further investigation on Cdc25C/CDK1/CyclinB1 signaling cascade in the absence or presence of pharmacological DDR inhibitors showed that xanthatin directly destabilized the protein levels of Cdc25C, and recovery of p53 expression in p53-deficient H1299 cells further intensified xanthatin-mediated inhibition of Cdc25C, suggesting p53-dependent regulation of Cdc25C in a DDR machinery. Moreover, exogenous expression of Cdc25C was also substantially repressed by xanthatin and partially impaired xanthatin-induced G2 arrest. In addition, xanthatin could induce accumulation of ubiquitinated Cdc25C without undergoing further proteasomal degradation. However, an alternative lysosomal proteolysis of Cdc25C was observed. Interestingly, lysosome-like vesicles were produced upon xanthatin treatment, accompanied by rapid accumulation of lysosomal associated membrane protein LAPM-1. Furthermore, vacuolar proton (V)-ATPases inhibitor bafilomycin A1 and lysosomal proteases inhibitor leupeptin could remarkably overturn the levels of Cdc25C in xanthatin-treated H1299 cells. Altogether, these data provide insight into how xanthatin can be effectively targeted DDR molecules towards certain tumors.

[Relationship between gene polymorphisms of tumor necrosis factor-α-308 and phenotypes of acute exacerbation of chronic obstructive pulmonary diseases].

OBJECTIVE: To observe the relationship between gene polymorphisms of tumor necrosis factor-α (TNF-α)-308 and phenotypes of acute exacerbation of chronic obstructive pulmonary diseases (AECOPD). METHODS: 198 AECOPD cases in the Minhang Hospital of Shanghai were recruited into the patient group, and 195 healthy people were recruited into the control group. PCR and sequencing method were used to detect the polymorphism of TNF-α-308 in all the people. RESULTS: Three genotypes with GG, GA and AA were detected in the 2 groups. The frequencies of GG/GA/AA genotype in AECOPD group were 87.4%, 10.6% and 2.0% respectively, and the control group were 95.4%, 4.6% and 0 respectively. The differences of the genotypes between AECOPD and health control were statistically significant (P<0.05). The frequencies of G and A were 92.7% and 7.3% in the AECOPD group, 97.7% and 2.3% in the control group. The differences were statistically significant (P<0.05). Dyspnea, smoke index and the increased thickness of bronchial wall in lung HRCT were the clinical features in patients with the TNF-α-308 genotype of GA/AA. CONCLUSION: There is correlation between genetic predisposition of AECOPD and TNF-α-308 genotype of GA/ AA. Clinical dyspnea, smoke index and increased thickness of bronchial wall are the chief clinical features in patients with genotype of GA/AA.

Optimization of cancer immunotherapy on the basis of programmed death ligand-1 distribution and function.

Programmed cell death protein-1 (PD-1)/programmed death ligand-1 (PD-L1) immune checkpoint blockade as a breakthrough in cancer immunotherapy has shown unprecedented positive outcomes in the clinic. However, the overall effectiveness of PD-L1 antibody is less than expected. An increasing number of studies have demonstrated that PD-L1 is widely distributed and expressed not only on the cell membrane but also on the inside of the cells as well as on the extracellular vesicles secreted by tumour cells. Both endogenous and exogenous PD-L1 play significant roles in influencing the therapeutic effect of anti-tumour immunity. Herein, we mainly focused on the distribution and function of PD-L1 and further summarized the potential targeted therapeutic strategies. More importantly, in addition to taking the overall expression abundance of PD-L1 as a predictive indicator for selecting corresponding PD-1/PD-L1 monoclonal antibodies (mAbs), we also proposed that personalized combination therapies based on the different distribution of PD-L1 are worth attention to achieve more efficient and effective therapeutic outcomes in cancer patients. LINKED ARTICLES: This article is part of a themed issue on Cancer Microenvironment and Pharmacological Interventions. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.2/issuetoc.

Targeting PKM2 signaling cascade with salvianic acid A normalizes tumor blood vessels to facilitate chemotherapeutic drug delivery.

Aberrant tumor blood vessels are prone to propel the malignant progression of tumors, and targeting abnormal metabolism of tumor endothelial cells emerges as a promising option to achieve vascular normalization and antagonize tumor progression. Herein, we demonstrated that salvianic acid A (SAA) played a pivotal role in contributing to vascular normalization in the tumor-bearing mice, thereby improving delivery and effectiveness of the chemotherapeutic agent. SAA was capable of inhibiting glycolysis and strengthening endothelial junctions in the human umbilical vein endothelial cells (HUVECs) exposed to hypoxia. Mechanistically, SAA was inclined to directly bind to the glycolytic enzyme PKM2, leading to a dramatic decrease in endothelial glycolysis. More importantly, SAA improved the endothelial integrity via activating the ß-Catenin/Claudin-5 signaling axis in a PKM2-dependent manner. Our findings suggest that SAA may serve as a potent agent for inducing tumor vascular normalization.

Internet of things-based management versus standard management of home noninvasive ventilation in COPD patients with hypercapnic chronic respiratory failure: a multicentre randomized controlled non-inferiority trial.

Background: Effective monitoring and management are crucial during long-term home noninvasive positive pressure ventilation (NPPV) in patients with hypercapnic chronic obstructive pulmonary disease (COPD). This study investigated the benefit of Internet of Things (IOT)-based management of home NPPV. Methods: This multicenter, prospective, parallel-group, randomized controlled non-inferiority trial enrolled patients requiring long-term home NPPV for hypercapnic COPD. Patients were randomly assigned (1:1), via a computer-generated randomization sequence, to standard home management or IOT management based on telemonitoring of clinical and ventilator parameters over 12 months. The intervention was unblinded, but outcome assessment was blinded to management assignment. The primary outcome was the between-group comparison of the change in health-related quality of life, based on severe respiratory insufficiency questionnaire scores with a non-inferiority margin of -5. This study is registered with Chinese Clinical Trials Registry (No. ChiCTR1800019536). Findings: Overall, 148 patients (age: 72.7 ± 6.8 years; male: 85.8%; forced expiratory volume in 1 s: 0.7 ± 0.3 L; PaCO2: 66.4 ± 12.0 mmHg), recruited from 11 Chinese hospitals between January 24, 2019, and June 28, 2021, were randomly allocated to the intervention group (n = 73) or the control group (n = 75). At 12 months, the mean severe respiratory insufficiency questionnaire score was 56.5 in the intervention group and 50.0 in the control group (adjusted between-group difference: 6.26 [95% CI, 3.71-8.80]; P < 0.001), satisfying the hypothesis of non-inferiority. The 12-month risk of readmission was 34.3% in intervention group compared with 56.0% in the control group, adjusted hazard ratio of 0.56 (95% CI, 0.34-0.92; P = 0.023). No severe adverse events were reported. Interpretation: Among stable patients with hypercapnic COPD, using IOT-based management for home NPPV improved health-related quality of life and prolonged the time to readmission. Funding: Air Liquide Healthcare (Beijing) Co., Ltd.

The association between the respiratory tract microbiome and clinical outcomes in patients with COPD.

Though it has been widely accepted that infections of the respiratory tract is associated with aetiology of acute exacerbation of chronic obstructive pulmonary disease (AECOPD), more recent techniques have shown emerging evidence on the importance of alterations of diversity and composition of microbiota itself in the disease process. Specifically, these alterations is widely present in COPD patients from a variety of populations, and is associated with severity of disease, frequency of acute exacerbation, as well as prediction of exacerbation. In addition, the microbiota from respiratory tract contributes to disease mechanisms, and more recently have been shown to interact with gut microbiota in a bidirectional way. Therefore, updating progress in the field is crucial as it not only reveals potential underlying mechanisms of the disease, but also highlights the potential utilisation of microbiota as a biomarker for disease prediction and as a target for treatment. In this narrative review, we summarize current updates on microbiota dysbiosis in COPD, including techniques for sampling and analysis of microbiota, recent findings on the presence of microbiota dysbiosis and its correlation with clinical prediction and prognosis of the disease, as well as its potential roles in disease mechanisms. In addition, how gut-lung axis contributes to COPD progression is also discussed. Finally, we addressed the utilisation of prebiotic and probiotic treatment for COPD. Together, we hope to provide useful information to advocate the use of microbial parameters as important tools for diagnosis, treatment and long-term follow-up for COPD patients.

Inducing vascular normalization: A promising strategy for immunotherapy.

In solid tumors, the vasculature is highly abnormal in structure and function, resulting in the formation of an immunosuppressive tumor microenvironment by limiting immune cells infiltration into tumors. Vascular normalization is receiving much attention as an alternative strategy to anti-angiogenic therapy, and its potential therapeutic targets include signaling pathways, angiogenesis-related genes, and metabolic pathways. Endothelial cells play an important role in the formation of blood vessel structure and function, and their metabolic processes drive blood vessel sprouting in parallel with the control of genetic signals in cancer. The feedback loop between vascular normalization and immunotherapy has been discussed extensively in many reviews. In this review, we summarize the impact of aberrant tumor vascular structure and function on drug delivery, metastasis, and anti-tumor immune responses. In addition, we present evidences for the mutual regulation of immune vasculature. Based on the importance of endothelial metabolism in controlling angiogenesis, we elucidate the crosstalk between endothelial cells and immune cells from the perspective of metabolic pathways and propose that targeting abnormal endothelial metabolism to achieve vascular normalization can be an alternative strategy for cancer treatment, which provides a new theoretical basis for future research on the combination of vascular normalization and immunotherapy.

Kaempferol impairs aerobic glycolysis against melanoma metastasis via inhibiting the mitochondrial binding of HK2 and VDAC1.

Metastasis is the leading cause of death in melanoma patients. Aerobic glycolysis is a common metabolic feature in tumor and is closely related to cell growth and metastasis. Kaempferol (KAM) is one of the active ingredients in the total flavonoids of Chinese traditional medicine Sparganii Rhizoma. Studies have shown that it interferes with the cell cycle, apoptosis, angiogenesis and metastasis of tumor cells, but whether it can affect the aerobic glycolysis of melanoma is still unclear. Here, we explored the effects and mechanisms of KAM on melanoma metastasis and aerobic glycolysis. KAM inhibited the migration and invasion of A375 and B16F10 cells, and reduced the lung metastasis of melanoma cells. Extracellular acidification rates (ECAR) and glucose consumption were obviously suppressed by KAM, as well as the production of ATP, pyruvate and lactate. Mechanistically, the activity of hexokinase (HK), the first key kinase of aerobic glycolysis, was significantly inhibited by KAM. Although the total protein expression of HK2 was not significantly changed, the binding of HK2 and voltage-dependent anion channel 1 (VDAC1) on mitochondria was inhibited by KAM through AKT/GSK-3ß signal pathway. In conclusion, KAM inhibits melanoma metastasis via blocking aerobic glycolysis of melanoma cells, in which the binding of HK2 and VDAC1 on mitochondria was broken.

Manipulation of the crosstalk between tumor angiogenesis and immunosuppression in the tumor microenvironment: Insight into the combination therapy of anti-angiogenesis and immune checkpoint blockade.

Immunotherapy has been recognized as an effective and important therapeutic modality for multiple types of cancer. Nevertheless, it has been increasing recognized that clinical benefits of immunotherapy are less than expected as evidenced by the fact that only a small population of cancer patients respond favorably to immunotherapy. The structurally and functionally abnormal tumor vasculature is a hallmark of most solid tumors and contributes to an immunosuppressive microenvironment, which poses a major challenge to immunotherapy. In turn, multiple immune cell subsets have profound consequences on promoting neovascularization. Vascular normalization, a promising anti-angiogenic strategy, can enhance vascular perfusion and promote the infiltration of immune effector cells into tumors via correcting aberrant tumor blood vessels, resulting in the potentiation of immunotherapy. More interestingly, immunotherapies are prone to boost the efficacy of various anti-angiogenic therapies and/or promote the morphological and functional alterations in tumor vasculature. Therefore, immune reprograming and vascular normalization appear to be reciprocally regulated. In this review, we mainly summarize how tumor vasculature propels an immunosuppressive phenotype and how innate and adaptive immune cells modulate angiogenesis during tumor progression. We further highlight recent advances of anti-angiogenic immunotherapies in preclinical and clinical settings to solidify the concept that targeting both tumor blood vessels and immune suppressive cells provides an efficacious approach for the treatment of cancer.