Topical application of activator protein-1 inhibitor T-5224 suppresses inflammation and improves skin barrier function in a murine atopic dermatitis-like dermatitis.

BACKGROUND: Selective activator protein (AP)-1 inhibitors are potentially promising therapeutic agents for atopic dermatitis (AD) because AP-1 is an important regulator of skin inflammation. However, few studies have investigated the effect of topical application of AP-1 inhibitors in treating inflammatory skin disorders. METHODS: Immunohistochemistry was conducted to detect phosphorylated AP-1/c-Jun expression of skin lesions in AD patients. In the in vivo study, 1 % T-5224 ointment was topically applied for 8 days to the ears of 2,4 dinitrofluorobenzene challenged AD-like dermatitis model mice. Baricitinib, a conventional therapeutic agent Janus kinase (JAK) inhibitor, was also topically applied. In the in vitro study, human epidermal keratinocytes were treated with T-5224 and stimulated with AD-related cytokines. RESULTS: AP-1/c-Jun was phosphorylated at skin lesions in AD patients. In vivo, topical T-5224 application inhibited ear swelling (P < 0.001), restored filaggrin (Flg) expression (P < 0.01), and generally suppressed immune-related pathways. T-5224 significantly suppressed Il17a and l17f expression, whereas baricitinib did not. Baricitinib suppressed Il4, Il19, Il33 and Ifnb expression, whereas T-5224 did not. Il1a, Il1b, Il23a, Ifna, S100a8, and S100a9 expression was cooperatively downregulated following the combined use of T-5224 and baricitinib. In vitro, T-5224 restored the expression of FLG and loricrin (LOR) (P < 0.05) and suppressed IL33 expression (P < 0.05) without affecting cell viability and cytotoxicity. CONCLUSIONS: Topical T-5224 ameliorates clinical manifestations of AD-like dermatitis in mice. The effect of this inhibitor is amplified via combined use with JAK inhibitors.

Persistent alteration of the skin microbiome in patients with skin rash after receiving EGFR inhibitor treatment.

The pathological mechanism responsible for EGFR inhibitor (EGFRI)-induced skin rash remains unclear. Recent studies reveal associations between skin dysbiosis and skin inflammatory diseases. This study aimed to examine whether skin dysbiosis is associated with EGFRI-induced skin rash. Bacterial swabs were taken from the forehead of 17 cancer patients at baseline and at several time points after EGFRIs initiation, as well as from 20 healthy controls. The skin microbiome was analysed using 16S rRNA sequencing. The severity of the skin rash was assessed using the rash grade. Skin surface parameters (pH, water capacitance, and sebum level) were also measured. Compared with baseline, the abundance of Cutibacterium acnes decreased in 13 of 15 cases, and that of Staphylococcus aureus, Corynebacterium spp., Staphylococcus epidermidis or Proteobacteria increased in 13 of 15 cases after EGFRIs initiation. Skin pH increased significantly in parallel with a decrease in water capacitance after EGFRI initiation. Also, the composition of the skin microbiome of patients with severe rash was significantly different from that of healthy controls. In addition, the skin dysbiosis did not return to baseline during EGFRIs treatment for >1 year. These longitudinal observations indicate that skin dysbiosis is associated with development of skin rash.

Transcriptome analysis reveals the essential role of NK2 homeobox 1/thyroid transcription factor 1 (NKX2-1/TTF-1) in gastric adenocarcinoma of fundic-gland type.

BACKGROUND: Gastric adenocarcinoma of fundic-gland type (GA-FG) is a gastric malignancy with little relation to Helicobacter pylori. Clinical characteristics of GA-FG have been established, but molecular mechanisms leading to tumorigenesis have not yet been elucidated. METHODS: We subjected three GA-FG tumors-normal mucosa pairs to microarray analysis. Network analysis was performed for the top 30 up-regulated gene transcripts, followed by immunohistochemical staining to confirm the gene expression analysis results. AGS and NUGC4 cells were transfected with the gene-encoding NK2 homeobox 1/thyroid transcription factor 1 (NKX2-1/TTF-1) to evaluate transcriptional changes in its target genes. RESULTS: Comprehensive gene expression analysis identified 1410 up-regulated and 1395 down-regulated gene probes with ≥ two-fold difference in expression. Among the top 30 up-regulated genes in GA-FG, we identified transcription factor NKX2-1/TTF-1, a master regulator of lung/thyroid differentiation, together with surfactant protein B (SFTPB), SFTPC, and secretoglobin family 3A member 2(SCGB3A2), which are regulated by NKX2-1/TTF-1. Immunohistochemical analysis of 16 GA-FG specimens demonstrated significantly higher NKX2-1/TTF-1 and SFTPB levels, as compared to that in adjacent normal mucosa (P < 0.05), while SCGB3A2 levels did not differ (P = 0.341). Transduction of NKX2-1/TTF-1 into AGS and NUGC4 cells induced transactivation of SFTPB and SFTPC, indicating that NKX2-1/TTF-1 can function as normally in gastric cells as it can in the lung cells. CONCLUSIONS: Our first transcriptome analysis of GA-FG indicates significant expression of NKX2-1/TTF1 in GA-FG. Immunohistochemistry and cell biology show ectopic expression and normal transactivation ability of NKX2-1/TTF-1, suggesting that it plays an essential role in GA-FG development.

Utility of Comprehensive Genomic Profiling for Precise Diagnosis of Pediatric-Type Diffuse High-Grade Glioma.

In the current World Health Organization classification of central nervous system tumors, comprehensive genetic and epigenetic analyses are considered essential for precise diagnosis. A 14-year-old male patient who presented with a cerebellar tumor was initially diagnosed with glioblastoma and treated with radiation and concomitant temozolomide chemotherapy after resection. During maintenance temozolomide therapy, a new contrast-enhanced lesion developed in the bottom of the cavity formed by the resection. A second surgery was performed, but the histological findings in specimens from the second surgery were different from those of the first surgery. Although genome-wide DNA methylation profiling was conducted using frozen tissue for a precise diagnosis, the proportion of tumor cells was insufficient and only normal cerebellum was observed. We then performed comprehensive genetic analysis using formalin-fixed paraffin-embedded sections, which revealed MYCN amplification without alteration of IDH1, IDH2, or Histone H3. Finally, the patient was diagnosed with pediatric-type diffuse high-grade glioma, H3-wildtype and IDH-wildtype. In conclusion, comprehensive genetic and epigenetic analysis should be considered in pediatric brain tumor cases.

Drug repositioning of tranilast to sensitize a cancer therapy by targeting cancer-associated fibroblast.

Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment that mediate resistance of cancer cells to anticancer drugs. Tranilast is an antiallergic drug that suppresses the release of cytokines from various inflammatory cells. In this study, we investigated the inhibitory effect of tranilast on the interactions between non-small cell lung cancer (NSCLC) cells and the CAFs in the tumor microenvironment. Three EGFR-mutant NSCLC cell lines, two KRAS-mutant cell lines, and three CAFs derived from NSCLC patients were used. To mimic the tumor microenvironment, the NSCLC cells were cocultured with the CAFs in vitro, and the molecular profiles and sensitivity to molecular targeted therapy were assessed. Crosstalk between NSCLC cells and CAFs induced multiple biological effects on the NSCLC cells both in vivo and in vitro, including activation of the STAT3 signaling pathway, promotion of xenograft tumor growth, induction of epithelial-mesenchymal transition (EMT), and acquisition of resistance to molecular-targeted therapy, including EGFR-mutant NSCLC cells to osimertinib and of KRAS-mutant NSCLC cells to selumetinib. Treatment with tranilast led to inhibition of IL-6 secretion from the CAFs, which, in turn, resulted in inhibition of CAF-induced phospho-STAT3 upregulation. Tranilast also inhibited CAF-induced EMT in the NSCLC cells. Finally, combined administration of tranilast with molecular-targeted therapy reversed the CAF-mediated resistance of the NSCLC cells to the molecular-targeted drugs, both in vitro and in vivo. Our results showed that combined administration of tranilast with molecular-targeted therapy is a possible new treatment strategy to overcome drug resistance caused by cancer-CAF interaction.

Identification of targetable kinases in idiopathic pulmonary fibrosis.

BACKGROUND: Tyrosine kinase activation plays an important role in the progression of pulmonary fibrosis. In this study, we analyzed the expression of 612 kinase-coding and cancer-related genes using next-generation sequencing to identify potential therapeutic targets for idiopathic pulmonary fibrosis (IPF). METHODS: Thirteen samples from five patients with IPF (Cases 1-5) and eight samples from four patients without IPF (control) were included in this study. Six of the thirteen samples were obtained from different lung segments of a single patient who underwent bilateral pneumonectomy. Gene expression analysis of IPF lung tissue samples (n = 13) and control samples (n = 8) was performed using SureSelect RNA Human Kinome Kit. The expression of the selected genes was further confirmed at the protein level by immunohistochemistry (IHC). RESULTS: Gene expression analysis revealed a correlation between the gene expression signatures and the degree of fibrosis, as assessed by Ashcroft score. In addition, the expression analysis indicated a stronger heterogeneity among the IPF lung samples than among the control lung samples. In the integrated analysis of the 21 samples, DCLK1 and STK33 were found to be upregulated in IPF lung samples compared to control lung samples. However, the top most upregulated genes were distinct in individual cases. DCLK1, PDK4, and ERBB4 were upregulated in IPF case 1, whereas STK33, PIM2, and SYK were upregulated in IPF case 2. IHC revealed that these proteins were expressed in the epithelial layer of the fibrotic lesions. CONCLUSIONS: We performed a comprehensive kinase expression analysis to explore the potential therapeutic targets for IPF. We found that DCLK1 and STK33 may serve as potential candidate targets for molecular targeted therapy of IPF. In addition, PDK4, ERBB4, PIM2, and SYK might also serve as personalized therapeutic targets of IPF. Additional large-scale studies are warranted to develop personalized therapies for patients with IPF.

The defense response of Caenorhabditis elegans to Cutibacterium acnes SK137 via the TIR-1-p38 MAPK signaling pathway.

Cutibacterium acnes plays roles in both acne disease and healthy skin ecosystem. We observed that mutations in the tir-1/SARM1 and p38 MAPK cascade genes significantly shortened Caenorhabditis elegans lifespan upon C. acnes SK137 infection. Antimicrobial molecules were induced by SK137 in a TIR-1-dependent manner. These results suggest that defense responses against SK137 involve the TIR-1-p38 MAPK pathway in C. elegans.

Liquid Biopsy Revealed HBOC Pedigree and Led to Medical Management Among the Relatives.

A hereditary breast and ovarian cancer (HBOC) pedigree was detected via liquid biopsy, and cancer prevention was initiated for the patient's daughter, after receiving a definitive result from BRCA genetic testing. A 48-yearold woman with ovarian cancer was administered precision medicine, which used cell-free DNA from plasma. The results revealed a pathogenic variant of BRCA1 as a presumed germline pathogenic mutation. We confirmed the germline pathological variant BRCA1 c.81-1G> A and suggested treatment with a PARP inhibitor. One of her three children had the variant, was diagnosed as an unaffected pathogenic variant carrier, and was advised to initiate surveillance.

Handling of Germline Findings in Clinical Comprehensive Cancer Genomic Profiling.

Patients found to have presumed germline pathogenic variants (PGPVs) during comprehensive genomic profiling (CGP) require genetic counseling (GC) referrals. We retrospectively investigated the outcomes of patients with PGPVs. Among 159 patients who underwent CGP, we recommended GC for the 16 patients with PGPVs (3 with [FG group] and 13 without [G Group] a family/personal history of hereditary cancer) as well as for the 8 patients with no PGPVs, but a history (F group); 2 (67%), 5 (38%), and 3 (38%) patients received GC in the FG, G, and F groups, respectively. Germline testing results were positive in 1 and 2 patients of the FG and G groups, respectively. Among the patients recommended for GC, 58% did not receive GC due to lack of interest, poor performance status, or death. CGP contributes to the identification of germline variants in patients without a history of hereditary cancer. However, the proportion of patients who undergo GC should be improved.

Oncogenic potential of human pluripotent stem cell-derived lung organoids with HER2 overexpression.

Lung adenocarcinoma (LUAD) is the most common types among lung cancers generally arising from terminal airway and understanding of multistep carcinogenesis is crucial to develop novel therapeutic strategy for LUAD. Here we used human induced pluripotent stem cells (hiPSCs) to establish iHER2-hiPSCs in which doxycycline induced the expression of the oncoprotein human epidermal growth factor receptor 2 (HER2)/ERBB2. Lung progenitors that differentiated from iHER2-hiPSCs, which expressed NKX2-1/TTF-1 known as a lung lineage maker, were cocultured with human fetal fibroblast and formed human lung organoids (HLOs) comprising alveolar type 2-like cells. HLOs that overexpressed HER2 transformed to tumor-like structures similar to atypical adenomatous hyperplasia, which is known for lung precancerous lesion and upregulated the activities of oncogenic signaling cascades such as RAS/RAF/MAPK and PI3K/AKT/mTOR. The degree of morphological irregularity and proliferation capacity were significantly higher in HLOs from iHER2-hiPSCs. Moreover, the transcriptome profile of the HLOs shifted from a normal lung tissue-like state to one characteristic of clinical LUAD with HER2 amplification. Our results suggest that hiPSC-derived HLOs may serve as a model to recapitulate the early tumorigenesis of LUAD and would provide new insights into the molecular basis of tumor initiation and progression.

CEBPγ facilitates lamellipodia formation and cancer cell migration through CERS6 upregulation.

Ceramide synthase 6 (CERS6) promotes lung cancer metastasis by stimulating cancer cell migration. To examine the underlying mechanisms, we performed luciferase analysis of the CERS6 promoter region and identified the Y-box as a cis-acting element. As a parallel analysis of database records for 149 non-small-cell lung cancer (NSCLC) cancer patients, we screened for trans-acting factors with an expression level showing a correlation with CERS6 expression. Among the candidates noted, silencing of either CCAAT enhancer-binding protein γ (CEBPγ) or Y-box binding protein 1 (YBX1) reduced the CERS6 expression level. Following knockdown, CEBPγ and YBX1 were found to be independently associated with reductions in ceramide-dependent lamellipodia formation as well as migration activity, while only CEBPγ may have induced CERS6 expression through specific binding to the Y-box. The mRNA expression levels of CERS6, CEBPγ, and YBX1 were positively correlated with adenocarcinoma invasiveness. YBX1 expression was observed in all 20 examined clinical lung cancer specimens, while 6 of those showed a staining pattern similar to that of CERS6. The present findings suggest promotion of lung cancer migration by possible involvement of the transcription factors CEBPγ and YBX1.

Best practices for the extraction of genomic DNA from formalin-fixed paraffin-embedded tumor tissue for cancer genomic profiling tests.

Recently, two cancer genomic profiling tests have been approved in Japan and implemented in routine clinical practice: the FDA-approved FoundationOne CDx test, and the OncoGuide NCC Oncopanel test. The quality and quantity of DNA significantly affects the sequencing results; therefore, preparing a sufficient amount of high-quality DNA for clinical cancer genomic profiling tests is important. We examined the best practices for the extraction of cancer genomic DNA from formalin-fixed paraffin-embedded (FFPE) tumor tissues of pancreatic, lung and colon cancer specimens. We found that the quality of cancer genomic DNA extracted from 10-µm-thick FFPE samples improved significantly, compared with that from 4-µm-thick FFPE samples, suggesting that 10-µm-thick FFPE samples are preferable for clinical cancer genomic profiling tests. For convenience, we created a quick reference table for calculating the required number of FFPE slides.

Microbiome composition comparison in oral and atherosclerotic plaque from patients with and without periodontitis.

There is no conclusive evidence regarding a causal relationship between periodontitis and atherosclerosis. In this study, we examined the microbiome in the oral cavity and atheromatous plaques from atherosclerosis patients with or without periodontitis to investigate the role of oral bacteria in the formation of atheromatous plaques. We chose four patients with and without periodontitis, who had undergone carotid endarterectomy. Bacterial samples were extracted from the tongue surface, from periodontal pocket (during the oral examination), and from the atheromatous plaques (APs). We investigated the general and oral conditions from each patient and performed next-generation sequencing (NGS) analysis for all bacterial samples. There were no significant differences between both groups concerning general conditions. However, the microbiome patterns of the gingival pocket showed differences depending on the absence or presence of periodontitis, while those of the tongue surface were relatively similar. The microbiome pattern of the atheromatous plaques was entirely different from that on the tongue surface and gingival pocket, and oral bacteria were seldom detected. However, the microbiome pattern in atheromatous plaques was different in the presence or absence of periodontitis. These results suggested that oral bacteria did not affect the formation of atheromatous plaques directly.

CERS6 required for cell migration and metastasis in lung cancer.

Sphingolipids constitute a class of bio-reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non-small-cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR-101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1-positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis.

YES1 activation induces acquired resistance to neratinib in HER2-amplified breast and lung cancers.

Molecular-targeted therapies directed against human epidermal growth factor receptor 2 (HER2) are evolving for various cancers. Neratinib is an irreversible pan-HER tyrosine kinase inhibitor and has been approved by the FDA as an effective drug for HER2-positive breast cancer. However, acquired resistance of various cancers to molecular-targeted drugs is an issue of clinical concern, and emergence of resistance to neratinib is also considered inevitable. In this study, we established various types of neratinib-resistant cell lines from HER2-amplified breast and lung cancer cell lines using several drug exposure conditions. We analyzed the mechanisms of emergence of the resistance in these cell lines and explored effective strategies to overcome the resistance. Our results revealed that amplification of YES1, which is a member of the SRC family, was amplified in two neratinib-resistant breast cancer cell lines and one lung cancer cell line. Knockdown of YES1 by siRNA and pharmacological inhibition of YES1 by dasatinib restored the sensitivity of the YES1-amplified cell lines to neratinib in vitro. Combined treatment with dasatinib and neratinib inhibited tumor growth in vivo. This combination also induced downregulation of signaling molecules such as HER2, AKT and MAPK. Our current results indicate that YES1 plays an important role in the emergence of resistance to HER2-targeted drugs, and that dasatinib enables such acquired resistance to neratinib to be overcome.

Overcoming epithelial-mesenchymal transition-mediated drug resistance with monensin-based combined therapy in non-small cell lung cancer.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis and is also associated with drug resistance. Thus, controlling EMT status is a research of interest to conquer the malignant tumors. MATERIALS AND METHODS: A drug repositioning analysis of transcriptomic data from a public cell line database identified monensin, a widely used in veterinary medicine, as a candidate EMT inhibitor that suppresses the conversion of the EMT phenotype. Using TGF-ß-induced EMT cell line models, the effects of monensin on the EMT status and EMT-mediated drug resistance were assessed. RESULTS: TGF-ß treatment induced EMT in non-small cell lung cancer (NSCLC) cell lines and the EGFR-mutant NSCLC cell lines with TGF-ß-induced EMT acquired resistance to EGFR-tyrosine kinase inhibitor. The addition of monensin effectively suppressed the TGF-ß-induced-EMT conversion, and restored the growth inhibition and the induction of apoptosis by the EGFR-tyrosine kinase inhibitor. CONCLUSION: Our data suggested that combined therapy with monensin might be a useful strategy for preventing EMT-mediated acquired drug resistance.

Diameter Is a Key 3D Characteristic for Assessments of Efficient Inhibitors of Protein-Protein Interactions.

Three-dimensional (3D) molecular descriptors, including physicochemical and shape properties, for protein-protein interaction (PPI) interface inhibitors have become a topic of discussion. However, the relationships between such properties and binding free energy have not been adequately investigated. In this study, we focused on identifying key 3D molecular descriptors related to the binding free energy and/or the ligand efficiency (LE) of PPI interface inhibitors. A positive correlation was found between the binding free energy and the diameter (D) of cylindrical 3D molecules, in addition to a correlation between LE and D/heavy atom count (HAC). In addition, we showed a correlation between LE and D/HAC for macrocyclic compounds, suggesting that the present findings could be applied during assessments of the potential of macrocyclic PPI interface inhibitors in drug discovery processes.

CCL28-Deficient Mice Have Reduced IgA Antibody-Secreting Cells and an Altered Microbiota in the Colon.

CCL28 induces the migration of IgA Ab-secreting cells (ASCs) via CCR10 and also displays a potent antimicrobial activity in vitro. To explore the role of CCL28 in vivo, we generated CCL28-deficient mice. The mice exhibited a significant reduction and abnormal distribution of IgA ASCs in the lamina propria of the colon. The concentrations of total and Ag-specific IgA in the fecal extracts of CCL28-deficient mice were also drastically reduced. The average amount of IgA secreted by a single IgA ASC derived from the colon was also substantially reduced in CCL28-deficient mice. Furthermore, CCL28 was found to significantly increase the average amount of IgA secreted by a single IgA ASC derived from the colon in vitro. In contrast, the generation of IgA ASCs in Peyer's and cecal patches was not significantly impaired in CCL28-deficient mice. We also found a relative increase in the Class Bacilli in the fecal extracts of CCL28-deficient mice and demonstrated a potent antimicrobial activity of CCL28 against Bacillus cereus and Enterococcus faecalis, both of which belong to Class Bacilli. Thus, CCL28 may also suppress the outgrowth of some bacterial species by its direct antimicrobial activity. Finally, CCL28-deficient mice exhibited a highly aggravated dextran sodium sulfate-induced colitis that was ameliorated by pretreatment with antibiotics. Collectively, CCL28 plays a pivotal role in the homing, distribution, and function of IgA ASCs in the colon and may also affect the intestinal microbiota through its direct antimicrobial activity.

Newly developed anti-S100A8/A9 monoclonal antibody efficiently prevents lung tropic cancer metastasis.

The metastatic dissemination of cancer cells to remote areas of the body is the most problematic aspect in cancer patients. Among cancers, melanomas are notoriously difficult to treat due to their significantly high metastatic potential even during early stages. Hence, the establishment of advanced therapeutic approaches to regulate metastasis is required to overcome the melanoma disease. An accumulating mass of evidence has indicated a critical role of extracellular S100A8/A9 in melanoma distant metastasis. Lung S100A8/A9 is induced by melanoma cells from distant organs and it attracts these cells to its enriched lung environment since melanoma cells possess several receptors that sense the S100A8/A9 ligand. We hence aimed to develop a neutralizing antibody against S100A8/A9 that would efficiently block melanoma lung metastasis. Our protocol provided us with one prominent antibody, Ab45 that efficiently suppressed not only S100A8/A9-mediated melanoma mobility but also lung tropic melanoma metastasis in a mouse model. This prompted us to make chimeric Ab45, a chimera antibody consisting of mouse Ab45-Fab and human IgG2-Fc. Chimeric Ab45 also showed significant inhibition of the lung metastasis of melanoma. From these results, we have high hopes that the newly produced antibody will become a potential biological tool to block melanoma metastasis in future clinical settings.

exSSSRs (extracellular S100 soil sensor receptors)-Fc fusion proteins work as prominent decoys to S100A8/A9-induced lung tropic cancer metastasis.

Within the "seed and soil" theory of organ tropic cancer metastasis is a growing compilation of evidence that S100A8/A9 functions as a soil signal that attracts cancer cells to certain organs, which prove beneficial to their growth. S100A8/A9-sensing receptors including Toll-like receptor 4 (TLR4), advanced glycation end products (RAGE), and also important receptors we recently succeeded in identifying (EMMPRIN, NPTNß, MCAM, and ALCAM) have the potential to become promising therapeutic targets. In our study, we prepared extracellular regions of these novel molecules and fused them to human IgG2-Fc to extend half-life expectancy, and we evaluated the anti-metastatic effects of the purified decoy proteins on metastatic cancer cells. The purified proteins markedly suppressed S100A8/A9-mediated lung tropic cancer metastasis. We hence expect that our novel biologics may become a prominent medicine to prevent cancer metastasis in clinical settings through cutting the linkage between "seed and soil".