Hepatocyte Growth Factor Receptor c-Met Research Articles

The Combination of Afatinib With Dasatinib or Miransertib Results in Synergistic Growth Inhibition of Stomach Cancer Cells.

Of various human epidermal growth factor receptor (HER) inhibitors, only the anti-HER2 monoclonal antibody (mAb) Herceptin/trastuzumab and the antibody-drug conjugate trastuzumab deruxtecan (T-Dxd) has been approved for the treatment of patients with stomach cancer. However, the duration of response may be short in many patients, with tumor heterogeneity being one contributing factor. We investigated the effect of various types of targeted agents on growth in vitro and migration of a panel of human stomach cancer cells (HSCCLs) and the impact of cell proliferation rate on the anti-tumor activities of these agents. We also investigated the association between the cell surface expression of the HER family members, hepatocyte growth factor receptor (c-Met), anaplastic lymphoma kinase (ALK)7 and cancer stem cell markers CD44 and CD133, and the response to the targeted agents. Of the 18 agents examined, the cyclin dependent kinase (CDK) 1/2/5/9 inhibitor dinaciclib was the most effective and inhibited the growth of all human HSCCLs at 50% inhibitory concentration (IC50) values between 9 nM to 23 nM. Of various HER inhibitors, the irreversible pan-HER family inhibitors (e.g., afatinib) were more effective than the reversible dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor (TKI) lapatinib and the EGFR-specific TKI erlotinib in inhibiting the growth of HSCCLs. Of agents targeting different downstream cell signaling molecules, dasatinib targeting Ab1/Src/C-Kit, trametinib targeting MERK1/2 and miransertib targeting AKT1/2/3 inhibited growth of majority of HSCCLs, with the IC50 values ranging from 2 nM to 7 µM. Many of these agents were more effective in inhibiting the growth of HSCCLs when they were proliferating at a slower rate. Treatment with neratinib, afatinib, dinaciclib, dasatinib, stattic, miransertib and paclitaxel significantly inhibited migration of stomach cancer cells. Interestingly, treatment with a combination of afatinib and dasatinib or afatinib and miransertib resulted in synergistic and additive growth inhibition of stomach cancer cells. These results suggest that treatment with a combination of these agents may be of therapeutic value in stomach cancer and warrants further investigations.

C-Met immunohistochemistry as reflex test at diagnosis for non-small cell lung cancer: a real-world experience from a monocentric case series

AimsRecent clinical trials have shown promising results with drugs targeting the hepatocyte growth factor receptor (c-Met) for advanced non-small cell lung cancers overexpressing c-Met. We assessed reflex testing of c-Met...

The SHDRA syndrome-associated gene TMEM260 encodes a protein-specific O-mannosyltransferase

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

Abstract P3-05-42: Identification of subpopulation of breast cancer patients with poor clinical outcome using CUB domain containing protein-1 (CDCP1)/CD318 and its interactive proteins SRC and the HGF axis

Abstract Introduction. The CUB domain containing protein 1, also known as TRASK (Transmembrane and associated with Src kinases) or CD318, has been suggested as a biomarker for stem cells or stem like cells. The transmembrane CDCP1 protein is known to activate the Src kinase and has recently shown to play a key part in the aggressiveness of breast cancer cells including migration, invasiveness and possibly growth induced by the motogenic Hepatocyte Growth Factor (HGF) and its receptor MET (Kawase et al 2022), a process requires Rho-GEF, PI3K and STAT5. This appears to allow identification of a subset of cancer cells that are aggressive in their biological behaviours. Here, we examined the expression profile of CDCP1, SRC, the HGF/HGF receptor complex and HGF activation regulators in a cohort of breast cancer and attempt to establish if such protein axis is able to identify patients with high risk of poor clinical outcome. Methods. Using an establish breast cancer cohort which is comprised of both normal mammary tissues and breast cancer tissues freshly obtained after surgery, we quantified the gene transcripts of CDCP1/CD318 and examined its clinical and pathological relevance. Integrated analysis was conducted for CDCP1 and the expression profile of SRC, PI3K, HGF, the HGF receptor cMET, HGF activator (HGFA), HGF activation regulators including HAI-1, HAI2, matriptase-1 and matriptase-2, and STAT family members. This was done against the clinical outcome of the patients as well as the clinical and pathological factors. Results. Breast cancer tissues expressed high levels of CDCP1 compared with normal mammary tissues. CDCP1 itself had a weak yet significant value in predicting the overall survival of the patients (p=0.047). The expression levels of these CDCP1 related molecules aren’t significantly correlated with CDCP1. However, integrated analyses revealed that CDCP1, together with its pathway regulators SRC, HGF, the HGF receptor (MET) and the HGF activation regulators matriptases form a power prognostic indicator for the clinical outcome of the patients (p< 0.0001, HR=1.8 for overall survival (OS) and p=0.002, HR=1.5 for disease-free survival (DFS)). This integrated profile has also identified subgroups of patients with highly favourable and very poor clinical outcomes over a ten-year follow-up period, with the respective survival at 100% and 36% respectively. The predictive power for OS and DFS is highly independent of other clinical and pathological factors in a multivariate analysis (p=0.003 and p=0.017 respectively). The predictive power is applicable to ER negative and HER2 positive tumours. Discussion. CDCP1/CD318, a factor known to stimulate cancer cell aggressiveness, together with its pathway kinase SRC and newly identified extracellular activator HGF and the HGF regulators forms a significant independent prognostic factor. It identifies subgroups of patients with favourable and poor prognosis, allowing consideration of targeted therapies for the patients. Reference Kawase N, Sugihara A, Kajiwara K, Hiroshima M, Akamatsu K, Nada S, Matsumoto K, Ueda M, Okada M. SRC kinase activator CDCP1 promotes hepatocyte growth factor-induced cell migration/invasion of a subset of breast cancer cells. J Biol Chem, 2022, 298:101630. doi: 10.1016/j.jbc.2022.101630 Citation Format: Yiming Yang, Xuefei Dong, Tracey A. Martin, Lin Ye, Jane Lane, Andrew J. Sanders, QingPing Dou, Eleri Davies, Wen G. Jiang. Identification of subpopulation of breast cancer patients with poor clinical outcome using CUB domain containing protein-1 (CDCP1)/CD318 and its interactive proteins SRC and the HGF axis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-42.

CD82 palmitoylation site mutations at Cys5+Cys74 affect EGFR internalization and metabolism through recycling pathway.

Tetraspanin CD82 often participates in regulating the function of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met). Palmitoylation is a post-translational modification that contributes to tetraspanin web formation and affects tetraspanin-dependent cell signaling. However, the molecular mechanisms by which CD82 palmitoylation affects the localization and stability of EGFR and c-Met have not yet been elucidated. This study focuses on the expression and distribution of EGFR and c-Met in breast cancer as well as the related metabolic pathways and molecular mechanisms associated with different CD82 palmitoylation site mutations. The results show that CD82 with a palmitoylation mutation at Cys5+Cys74 can promote the internalization of EGFR. EGFR is internalized and strengthened by direct binding to CD82 with the tubulin assistance and located at the recycling endosome. After studying the recycling pathway marker proteins Rab11a and FIP2, we found that formation of the EGFR/CD82/Rab11a/FIP2 complex promotes the internalization and metabolism of EGFR through the recycling pathway and results in the re-expression of EGFR and CD82 on the cell membrane.

HGF Secreted by Mesenchymal Stromal Cells Promotes Primordial Follicle Activation by Increasing the Activity of the PI3K-AKT Signaling Pathway

Primordial follicle activation is fundamental for folliculogenesis and for the maintenance of fertility. An effective therapeutic strategy for patients with premature ovarian insufficiency (POI) is to promote the activation of residual primordial follicles. The secretome of human umbilical cord mesenchymal stromal cells (hUC-MSC-sec) contains several components that might promote the activation of primordial follicles. In the present study, we revealed that treatment with the hUC-MSC-sec significantly increased the proportion of activated primordial follicles in mouse ovaries both in vitro and in vivo. The activating effects of hUC-MSC-sec on primordial follicles were attributed to the activation of the PI3K-AKT signaling pathway by hepatocyte growth factor (HGF). While the effect of the hUC-MSC-sec was attenuated by the neutralizing antibodies against HGF, application of exogenous HGF alone also promoted the activation of primordial follicles. Furthermore, we demonstrated that HGF promoted the expression of KITL in granulosa cells by binding with the HGF receptor c-Met, thereby increasing the activity of the PI3K-AKT signaling pathway to activate primordial follicles. Taken together, our findings demonstrate that hUC-MSC-sec promotes primordial follicle activation through the functional component HGF to increase the PI3K-AKT signaling activity, highlighting the application of the hUC-MSC-sec or HGF for the treatment of POI patients.Graphical abstract

A rotaxane-based platform for tailoring the pharmacokinetics of cancer-targeted radiotracers.

Radiolabelled monoclonal antibodies (mAbs) are a cornerstone of molecular diagnostic imaging and targeted radioimmunotherapy in nuclear medicine, but one of the major challenges in the field is to identify ways of reducing the radiation burden to patients. We reasoned that a rotaxane-based platform featuring a non-covalent mechanical bond between the radionuclide complex and the biologically active mAb could offer new ways of controlling the biophysical properties of cancer-specific radiotracers for positron emission tomography (PET). Herein, we present the photoradiosynthesis and characterisation of [89Zr]ZrFe-[4]rotaxane-azepin-onartuzumab ([89Zr]ZrFe-2), a unique rotaxane-antibody conjugate for PET imaging and quantification of the human hepatocyte growth factor receptor (c-MET). Multiple component self-assembly reactions were combined with simultaneous 89Zr-radiolabelling and light-induced bioconjugation methods to give [89Zr]ZrFe-2 in 15 ± 1% (n = 3) decay-corrected radiochemical yield, with >90% radiochemical purity, and molar activities suitable for PET imaging studies (>6.1 MBq mg-1 of protein). Cellular assays confirmed the specificity of [89Zr]ZrFe-2 binding to the c-MET receptor. Temporal PET imaging in athymic nude mice bearing subcutaneous MKN-45 gastric adenocarcinoma xenografts demonstrated specific binding of [89Zr]ZrFe-2 toward c-MET in vivo, where tumour uptake reached 9.8 ± 1.3 %ID g-1 (72 h, n = 5) in a normal group and was reduced by ∼56% in a control (blocking) group. Head-to-head comparison of the biodistribution and excretion profile of [89Zr]ZrFe-2versus two control compounds, alongside characterisation of two potential metabolites, showed that the rotaxane-radiotracer has an improved clearance profile with higher tumour-to-tissue contrast ratios and reduced radiation exposure to critical (dose-limiting) organs including liver, spleen, and kidneys. Collectively, the experimental results suggested that non-covalent mechanical bonds between the radionuclide and mAb can be used to fine-tune the pharmacokinetic profile of supramolecular radiopharmaceuticals in ways that are simply not accessible when using traditional covalent design.

In silico Modeling and Toxicity Profiling of a Set of Quinoline Derivatives as c-MET Inhibitors in the treatment of Human Tumors.

4-(2-fluorophenoxy) quinoline derivatives constitute one of the chemical classes of hepatocyte growth factor receptor (c-MET) inhibitors, a promising treatment against various human tumors. There are three aims of the present study: (1) To develop a robust and validated quantitative structure-activity relationship model to predict the c-Met kinase inhibition; (2) to examine the toxicity profiles of these compounds; (3) to design new quinoline derivatives and apply the developed model on these compounds to observe its pertinence. A multiple linear regression method was used to develop the model with calculated descriptors. State-of-the-art internal and external validation parameters were calculated. The in silico toxicity profiles including structural alerts and the lowest observed adverse effect level (LOAEL) values were evaluated using online tools. New derivatives were designed and tested on the developed model. A series of 4-(2-fluorophenoxy) quinoline derivatives was linearly modeled and vigorously validated to predict the molecules' c-MET kinase inhibition potential. Statistical metrics of the developed model showed that it was robust and able to make successful predictions for this chemical class. The mass, electronegativity, partial charges, and the structure of the molecules had an effect on the activity. Moreover, the toxicity profiles of the studied compounds were found to be adequate. Five of the synthesized compounds were observed to be auspicious for the toxicity/activity ratio. The developed model is useful in the virtual screening and in the design of new anti-tumor compounds.

Identification of other Cellular Receptors for Edema Factor of Bacillus anthracis by Independent Inhibition of Protective Antigen Evidenced by Inhibition of Embryo Growth and Angiogenesis.

Edema factor (EF) is one of the major secretory proteins of anthrax bacteria along with protective antigen (PA) and lethal factor (LF). Edema factor is a calmodulin-and calcium-dependent adenylate cyclase that increases intracellular levels of cAMP. Intracellular trafficking of EF occurs through PA by binding to ATR/CMG2 receptors, which are also involved in other physiological functions of cells. cAMP is a secondary messenger which activates multiple signaling cascades involved in the cytokinetics of actin molecules and cell junction formation. The present study evaluated the effect of EF on growth and angiogenesis patterns in chicken embryos in the in ovo model. Angiogenesis in the chorioallantoic membrane (CAM) of an embryonated chicken egg was decreased and embryo growth was delayed by EF despite the absence of trafficking moiety PA, which is required for transferring the EF molecule inside the cell. Angiogenesis inhibition and embryo growth retardation indicate the use of an alternative receptor by EF to modulate these cellular functions. Additionally, docking was performed between EF as a ligand and hepatocyte growth factor receptor (cMET) and vascular endothelial growth factor (VEGF) receptors, which are mainly involved in growth and angiogenesis. The analysis revealed a very strong binding of EF to cMET receptor (in terms of the number of hydrogen bonds and energy) compared to its ligand hepatocyte growth factor (HGF), which indicates the use of cMET receptor by EF and induction of angiogenesis and embryo growth retardation possibly by competitive inhibition of HGF ligand or receptor-mediated endocytosis.

NR4A1 Ligands as Potent Inhibitors of Breast Cancer Cell and Tumor Growth.

Simple SummaryBis-indole derived (CDIMs) bind the orphan nuclear receptor 4A1 (NR4A1) and inhibit NR4A1-regulated cancer cell and tumor growth. In this study a subset of 3,5-disubstituted phenyl CDIM compounds that bound NR4A1 were investigated in a breast cancer model. All of these analogs were potent inhibitors of breast tumor growth in a xenograft model using MDA-MB-231 cells at doses ≤ 1 mg/kg/d.Nuclear receptor 4A1 (NR4A1, Nur77, TR3) is more highly expressed in breast and solid tumors compared to non-tumor tissues and is a pro-oncogenic factor in solid tumor-derived cancers. NR4A1 regulates cancer cell growth, survival, migration, and invasion, and bis-indole-derived compounds (CDIMs) that bind NR4A1 act as antagonists and inhibit tumor growth. Preliminary structure-binding studies identified 1,1-bis(3′-indolyl)-1-(3,5-disubstitutedphenyl)methane analogs as NR4A1 ligands with low KD values; we further investigated the anticancer activity of the four most active analogs (KD’s ≤ 3.1 µM) in breast cancer cells and in athymic mouse xenograft models. The treatment of MDA-MB-231 and SKBR3 breast cancer cells with the 3-bromo-5-methoxy, 3-chloro-5-trifluoromethoxy, 3-chloro-5-trifluoromethyl, and 3-bromo-5-trifluoromethoxy phenyl-substituted analogs decreased cell growth and the expression of epidermal of growth factor receptor (EGFR), hepatocyte growth factor receptor (cMET), and PD-L1 as well as inhibited mTOR phosphorylation. In addition, all four compounds inhibited tumor growth in athymic nude mice bearing MDA-MB-231 cells (orthotopic) at a dose of 1 mg/kg/d, which was not accompanied by changes in body weight. These 3,5-disubstituted analogs were the most potent CDIM/NR4A1 ligands reported and are being further developed for clinical applications.

Molecular mechanisms of esophageal epithelial regeneration following repair of surgical defects with acellular silk fibroin grafts

Constructive remodeling of focal esophageal defects with biodegradable acellular grafts relies on the ability of host progenitor cell populations to repopulate implant regions and facilitate growth of de novo functional tissue. Intrinsic molecular mechanisms governing esophageal repair processes following biomaterial-based, surgical reconstruction is largely unknown. In the present study, we utilized mass spectrometry-based quantitative proteomics and in silico pathway evaluations to identify signaling cascades which were significantly activated during neoepithelial formation in a Sprague Dawley rat model of onlay esophagoplasty with acellular silk fibroin scaffolds. Pharmacologic inhibitor and rescue experiments revealed that epithelialization of neotissues is significantly dependent in part on pro-survival stimuli capable of suppressing caspase activity in epithelial progenitors via activation of hepatocyte growth factor receptor (c-MET), tropomyosin receptor kinase A (TrkA), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt) signaling mechanisms. These data highlight the molecular machinery involved in esophageal epithelial regeneration following surgical repair with acellular implants.

Abstract PS17-48: Potential role of activated leukocyte cell adhesion molecule (ALCAM) in hepatocyte growth factor (HGF) signalling in vascular endothelial cells and implications in breast cancer

Abstract Background. Activated Leukocyte Cell Adhesion Molecule, ALCAM (also known as CD166) is a member of the immunoglobulin superfamily and a cell surface adhesion molecule involved in heterophilic and homophilic interactions during cell-cell adhesion in epithelial cells, cancer cells and endothelial cells. ALCAM appears to be linked to tumour progression in a number of cancers including breast cancer, though there are conflicting reports as to its precise prognostic implications and significance. We have previously reported that ALCAM has a tumour suppressive role in breast cancer and is inversely correlated with clinical outcome (1) and interestingly bone metastasis of breast cancer (2). We have also reported that ALCAM has a diverse role in other cell types including keratinocytes and endothelial cells. In the present study, we investigated the association between ALCAM and hepatocyte growth factor (HGF)/cMET, a signalling pathway established as a key promoter of cancer progression, influencing both the aggressive nature of cancer cells and acting as an angiogenic and lymphangiogenic factor, in breast cancer and endothelial cells. Methods. The expression pattern and correlation of ALCAM, HGF and cMET in human breast cancer were deduced from a clinical breast cancer cohort. ALCAM manipulated HECV cell lines, previously established in our laboratories, were used in conjunction with recombinant human HGF and cMET inhibitors to assess cellular functions and the implications of this relationship at a cellular level. Results. We compared the expression of ALCAM with that of HGF and cMET within the breast cancer cohort and found that ALCAM/CD166 showed a highly significant negative correlation with the HGF receptor cMET (r=-0.17, p<0.0001) and a weak negative correlation with HGF, although this was not significant. ALCAM was not found to correlate with its heterophilic partner CD6. In previously generated HECV models, ALCAM manipulation showed a marked influence on cellular function and responsiveness to HGF treatment. Discussion. ALCAM’s role in breast cancer progression and related mechanisms is complex. Our current data suggests this may involve an interaction with the cMET/HGF signalling pathway and may also impact the endothelial component within the tumour microenvironment to influence disease progression. Reference1 King JA, Ofori-Acquah SF, Stevens T, et al. Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator. Breast Cancer Res. 2004;6:R478-87. Reference2 Davies SR, Dent C, Watkins G et al. Expression of the cell to cell adhesion molecule, ALCAM, in breast cancer patients and the potential link with skeletal metastasis. Oncol Rep. 2008;19:555-61. Citation Format: Andrew James Sanders, David Guo Jiang, Jianyuan Zeng, Robert Edward Mansel, Eleri Davies, Amber Xinyu Li, Keith Gordon Harding, Wen Guo Jiang. Potential role of activated leukocyte cell adhesion molecule (ALCAM) in hepatocyte growth factor (HGF) signalling in vascular endothelial cells and implications in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-48.

Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for 85% of all lung carcinomas. The hepatocyte growth factor receptor (c-Met) has been considered as a potential therapeutic target for NSCLC. Proteasome inhibition induces cell apoptosis and has been used as a novel therapeutic approach for treating diseases including NSCLC; however, the effects of different proteasome inhibitors on NSCLC have not been fully investigated. The aim of this study is to determine a precise strategy for treating NSCLC by targeting c-Met using different proteasome inhibitors. Three proteasome inhibitors, bortezomib, MG132, and ONX 0914, were used in this study. Bortezomib (50 nM) significantly reduced c-Met levels and cell viability in H1299 and H441 cells, while similar effects were observed in H460 and A549 cells when a higher concentration (∼100 nM) was used. Bortezomib decreased c-Met gene expression in H1299 and H441 cells, but it had no effect in A549 and H460 cells. MG-132 at a low concentration (0.5 μM) diminished c-Met levels in H441 cells, while neither a low nor a high concentration (∼20 μM) altered c-Met levels in A549 and H460 cells. A higher concentration of MG-132 (5 μM) was required for decreasing c-Met levels in H1299 cells. Furthermore, MG-132 induced cell death in all four cell types. Among all the four cell lines, H441 cells expressed higher levels of c-Met and appeared to be the most susceptible to MG-132. MG-132 decreased c-Met mRNA levels in both H1299 and H441 cells. ONX 0914 reduced c-Met levels in H460, H1299, and H441 cells but not in A549 cells. c-Met levels were decreased the most in H441 cells treated with ONX 0914. ONX 0914 did not alter cell viability in H441; however, it did induce cell death among H460, A549, and H1299 cells. This study reveals that different proteasome inhibitors produce varied inhibitory effects in NSCLS cell lines.

MET somatic activating mutations are responsible for lymphovenous malformation and can be identified using cell-free DNA next generation sequencing liquid biopsy

ObjectiveGermline mutations of either the endothelial cell-specific tyrosine kinase receptor TIE2 or the glomulin (GLMN) gene are responsible for rare inherited venous malformations. Both genes affect the hepatocyte growth factor receptor c-Met, inducing vascular smooth muscle cell migration. Germline mutations of hepatocyte growth factor are responsible for lymphatic malformations, leading to lymphedema. The molecular alteration leading to the abnormal mixed vascular anomaly defined as lymphovenous malformation has remained unknown. MethodsA group of 4 patients with lymphovenous malformations were selected. Plasma was obtained from both peripheral and efferent vein samples at the vascular malformation site for cell-free DNA extraction. When possible, we analyzed tissue biopsy samples from the vascular lesion. ResultsWe have demonstrated that in all four patients, an activating MET mutation was present. In three of the four patients, the same pathogenic activating mutation, T1010I, was identified. The mutation was found at the tissue level for the patient with tissue samples available, confirming its causative role in the lymphovenous malformations. ConclusionsIn the present study, we have demonstrated that cell-free DNA next generation sequencing liquid biopsy is able to identify the MET mutations in affected tissues. Although a wider cohort of patients is necessary to confirm its causative role in lymphovenous malformations, these data suggest that lymphovenous malformations could result from postzygotic somatic mutations in genes that are key regulators of lymphatic development. The noninvasiveness of the method avoids any risk of bleeding and can be easily performed in children. We are confident that the present pioneering results have provided a viable alternative in the future for lymphovenous malformation diagnosis, allowing for subsequent therapy tailored to the genetic defect.

Abstract 6439: CapioCyte™ technology for efficient capture and downstream analysis of circulating tumor cells from renal cell carcinoma

Abstract Objective: The objective of this work was to obtain highly sensitive purification and post-capture analysis of renal cell carcinoma (RCC) circulating tumor cells (CTCs) using CapioCyte™ technology. Background: Circulating tumor cells (CTCs) may be a highly valuable source of actionable information for the practice of precision medicine. However, CTCs are exceptionally rare in peripheral blood. The CapioCyte™ chip purifies live CTCs through a combination of biomimetic cell rolling and dendrimer-mediated multivalent immunorecognition. The CapioCyte™ platform has been previously shown to isolate CTCs with high sensitivity from peripheral blood collected from patients with head and neck cancer using a mixture of antibodies against epithelial cell adhesion molecule (EpCAM), human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor (EGFR) [Myung et al., Clinical Cancer Research 2018, 24(11):2539-2547]. However, this antibody cocktail is not universally applicable to all types of cancer. In particular, renal cell carcinoma (RCC), having no clinically actionable circulating biomarkers, often do not express HER2. To address this, we have employed a new antibody mixture targeting EpCAM, EGFR, carbonic anhydrase IX (CAIX), and hepatocyte growth factor receptor (c-MET). Our study presents enhanced capture of CTCs from renal cancer, demonstrating the modular nature of our CapioCyte™ technology. Methods: Antibody-coated beads were used to determine the functional recognition of ACHN and 786-O RCC cell lines. Flow channel experiments and atomic force microscopy (AFM) force spectroscopy measurements were used to quantify off-rate kinetics. A clinical pilot study of 7 RCC patients (pre-treatment) and 5 healthy controls was performed to validate the CapioCyte™ surface tuned for RCC capture. In recent work, CTCs recovered from the CapioCyte™ surface were sequenced using 10X Chromium system with v3 chemistry and analyzed using RaceID clustering and Gene Set Enrichment Analysis (GSEA). Results: The optimized mixture of EpCAM, CAIX, EGFR, and c-MET showed 80% enhancement in the capture of RCC cell lines compared to the mixture of EpCAM, EGFR, and HER2. Force spectroscopy measurements were used to show that dendrimers contributed to an order of magnitude enhancement in off-rate kinetics. In patient samples, the RCC capture surface isolated a total of 9.8 +/- 5.1 CTC ml-1 whole blood compared to just 1.8 +/- 2.0 CTC ml-1 with the conventional three-antibody mixture. Finally, preliminary results show the detection of oncogenic signatures within two additional peripheral blood samples. Conclusions: The prototype CapioCyte™ platform is a flexible liquid biopsy that can be tuned for various disease indications. Here, enhanced capture of RCC CTCs was demonstrated in clinical blood samples with high sensitivity. Proof-of-concept RNA sequencing shows the promise of CapioCyte™ in providing further clinically-actionable information from CTCs. Citation Format: Jiyoon Bu, Michael J. Poellmann, Marco Reyes-Martinez, Andrew Armstrong, Daniel George, Tian Zhang, Andrew Z. Wang, seungpyo hong. CapioCyte™ technology for efficient capture and downstream analysis of circulating tumor cells from renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6439.

Analysis of erythrocyte signalling pathways during Plasmodium falciparum infection identifies targets for host-directed antimalarial intervention

Intracellular pathogens mobilize host signaling pathways of their host cell to promote their own survival. Evidence is emerging that signal transduction elements are activated in a-nucleated erythrocytes in response to infection with malaria parasites, but the extent of this phenomenon remains unknown. Here, we fill this knowledge gap through a comprehensive and dynamic assessment of host erythrocyte signaling during infection with Plasmodium falciparum. We used arrays of 878 antibodies directed against human signaling proteins to interrogate the activation status of host erythrocyte phospho-signaling pathways at three blood stages of parasite asexual development. This analysis reveals a dynamic modulation of many host signalling proteins across parasite development. Here we focus on the hepatocyte growth factor receptor (c-MET) and the MAP kinase pathway component B-Raf, providing a proof of concept that human signaling kinases identified as activated by malaria infection represent attractive targets for antimalarial intervention.

Increase in CD4+FOXP3+ regulatory T cell number and upregulation of the HGF/c-Met signaling pathway during the liver metastasis of colorectal cancer.

Colorectal cancer (CRC) is the third and second most common type of cancer diagnosed in males and females, respectively, and is the fourth leading cause of cancer-associated mortality worldwide. Liver metastasis is the primary cause of mortality in patients with CRC, and therefore requires therapeutic focus. Regulatory T cells (Tregs) and hepatic stellate cells (HSCs) are potentially involved in regulating the immune response during liver metastasis. The aim of the present study was to evaluate the influence of CD4+ forkhead box p3 (Foxp3)+ Tregs and the HGF/c-Met signaling pathway in the liver metastasis of CRC. A model of the latter was established using Balb/c mice via splenic injection of human CRC cells (CT-26 line). The mice were monitored for 3 weeks after being injected, and the spleens and livers were removed on day 22 for further analysis. Moreover, the single-cell suspensions were labeled with CD4 and Foxp3 antibodies, and were analyzed using flow cytometry. Expression levels of α-smooth muscle actin (SMA), hepatocyte growth factor (HGF) and hepatocyte growth factor receptor (c-Met) were analyzed using immunohistochemistry. Mice injected with CT-26 cells exhibited signs of illness and significant weight loss, compared with the control mice (P=0.013), and they also developed liver metastases, at an average of 20.5 tumors per mouse. Pathological evaluation using hematoxylin and eosin staining confirmed the tumors as liver metastases of CRC. The numbers of CD4+ T cells were significantly decreased in the spleen (P<0.001) and liver (P=0.003) of tumor-bearing mice, while the proportions of CD4+FOXP3+ Tregs increased significantly in the spleen (P<0.001) and liver (P=0.026) compared with that in the controls. Additionally, α-SMA, HGF and c-Met levels increased significantly during metastatic growth in the liver. In conclusion, CD4+FOXP3+ Treg levels increased and the HGF/c-Met pathway was upregulated during the liver metastasis of CRC in mice, indicating the presence of potential therapeutic targets.

Design, Synthesis and Biological Evaluation of Novel 4-phenoxypyridine Derivatives Containing Semicarbazones Moiety as Potential c-Met Kinase Inhibitors.

The Hepatocyte Growth Factor Receptor (HGFR) c-Met is over-expressed and/or mutated in various human tumor types. Dysregulation of c-Met/HGF signaling pathway affects cell proliferation, survival and motility, leading to tumor growth, angiogenesis, and metastasis. Therefore, c-Met has become an attractive target for cancer therapy. This study is aimed to evaluate a new series of 4-phenoxypyridine derivatives containing semicarbazones moiety for its cytotoxicity. A series of novel 4-phenoxypyridines containing semicarbazone moieties were synthesized and evaluated for their in vitro cytotoxic activities against MKN45 and A549 cancer cell lines and some selected compounds were further examined for their inhibitory activity against c-Met kinase. In order to evaluate the mechanism of cytotoxic activity of compound 24, cell cycle analysis, Annexin V/PI staining assay, AO/EB assay, wound-healing assay and docking analysis with c-Met were performed. The results indicated that most of the compounds showed moderate to good antitumor activity. The compound 28 showed well cytotoxic activity against MKN45 and A549 cell lines with IC50 values of 0.25μM and 0.67μM, respectively. Compound 24 showed good activity on c-Met and its IC50 value was 0.093μM. Their preliminary Structure-Activity Relationships (SARs) studies indicated that electronwithdrawing groups on the terminal phenyl rings are beneficial for improving the antitumor activity. Treatments of MKN45 cells with compound 24 resulted in cell cycle arrest in G2/M phase and induced apoptosis in a dose-dependent manner. In addition, AO/EB assays indicated 24 induced dose-dependent apoptosis of A549 and MKN45 cells. Wound-healing assay results indicated that compound 24 strongly inhibited A549 cell motility.

Effect of silencing hepatocyte growth factor receptor c-Met expression on biological characteristics of colon cancer cells

Objective: To investigate the effect of silencing hepatocyte growth factor receptor (c-Met) expression on the biological characteristics of HCT116 colon cancer cells. Methods: Cellular model of c-Met transient transfection was established by using small interfering RNA (siRNA), the expression of c-Met in colon cancer cells was detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot. The apoptosis assay, cell invasion assay, cell migration and other experiments were conducted to observe the effects of silencing c-Met on the biological characteristics of colon cancer cells. Results: RT-qPCR results showed that the relative expression levels of c-Met mRNA in siRNA-Met group, blank control group and siRNA negative control (siRNA-NC) group were 0.32±0.26, 1.01±0.03 and 1.05±0.23, respectively, and the difference was statistically significant (P<0.05). Western blot analysis showed that the expression level of c-Met protein in the siRNA-Met group was 0.24±0.03, significantly lower than 1.23±0.06 in the blank control group and 1.18±0.11 in the siRNA-NC group (P<0.05). The cell counting kit-8 (CCK8) results showed that the 72-hour absorbance (A) values of the siRNA-Met group, blank control group and the siRNA-NC group were 1.13±0.05, 1.48±0.08 and 1.53±0.07, respectively, and the difference was statistically significant (P<0.01). Cell cycle results showed that the proportion of cells in G(2)/M phase was (14.65±1.41)% in siRNA-Met group , (5.07±0.70)% in blank control group and (5.63±0.71)% in siRNA-NC group, and the difference was statistically significant (P<0.05). The expression levels of cell cycle regulatory proteins Cdc25c and cyclin B1 in siRNA-Met group were significantly decreased. The apoptotic rate in siRNA-Met group was (5.85±0.35)%, significantly higher than (1.00±0.17)% in blank control group and (0.91±1.14)% in siRNA-NC group (P<0.05). The expression level of apoptosis-related protein Bcl-2 in the siRNA-Met group was significantly decreased while Bcl-2 associated X protein (BAX) expression level was significantly increased. The cell scratching result showed that the cell migration abilities of the siRNA-Met group, blank control group and the siRNA-NC group were (51.33±8.62)%, (100.00±3.72)% and (102.33±6.43)%, respectively, and the difference was statistically significant (P<0.05). The number of cell penetrating into the basement membrane of the siRNA-Met group, blank control group and the siRNA-NC group were 47.50±10.60, 100.00±5.33 and 102.50±10.61, respectively, and the difference was statistically significant (P<0.05). The expressions of invasion related proteins including MMP-2 and MMP-9 in siRNA-Met group were decreased significantly. Conclusions: c-Met plays an important role in maintaining the biological characteristics of colon cancer cells. Inhibition of c-Met may have important values in the treatment of colon cancer.

Surface engineering for efficient capture of circulating tumor cells in renal cell carcinoma: From nanoscale analysis to clinical application

Sensitive detection of circulating tumor cells (CTCs) from patients' peripheral blood facilitates on-demand monitoring of tumor progression. However, clinically significant capture of renal cell carcinoma CTCs (RCC-CTCs) remains elusive due to their heterogenous surface receptor expression. Herein, a novel capture platform is developed to detect RCC-CTCs through integration of dendrimer-mediated multivalent binding, a mixture of antibodies, and biomimetic cell rolling. The nanoscale binding kinetics measured using atomic force microscopy reveal that dendrimer-coated surfaces exhibit an order of magnitude enhancement in off-rate kinetics compared to surface without dendrimers, which translated into cell capture improvements by ~60%. Selectin-induced cell rolling facilitates surface recruitment of cancer cells, further improving cancer cell capture by up to 1.7-fold. Lastly, an antibody cocktail targeting four RCC-CTC surface receptors, which included epithelial cell adhesion molecule (EpCAM), carbonic anhydrase IX (CA9), epidermal growth factor receptor (EGFR), and hepatocyte growth factor receptor (c-Met), improves the capture of RCC cells by up to 80%. The optimal surface configuration outperforms the conventional assay solely relying on EpCAM, as demonstrated by detecting significantly more CTCs in patients’ samples (9.8 ± 5.1 vs. 1.8 ± 2.0 CTCs mL-1). These results demonstrate that the newly engineered capture platform effectively detects RCC-CTCs for their potential use as tumor biomarkers.