c-Met Expression Research Articles

Preclinical evaluation of a novel EGFR&c-Met bispecific near infrared probe for visualization of esophageal cancer and metastatic lymph nodes.

This study aimed to establish a near infrared fluorescent (NIRF) probe based on an EGFR&c-Met bispecific antibody for visualization of esophageal cancer (EC) and metastatic lymph nodes (mLNs). EGFR and c-Met expression were assessed by immunohistochemistry. EGFR&c-Met bispecific antibody EMB01 was labeled with IRDye800cw. The binding of EMB01-IR800 was assessed by enzyme linked immunosorbent assay, flow cytometry, and immunofluorescence. Subcutaneous tumors, orthotopic tumors, and patient-derived xenograft (PDX) were established for in vivo fluorescent imaging. PDX models using lymph nodes with or without metastasis were constructed to assess the performance of EMB01-IR800 in differential diagnosis of lymph nodes. The prevalence of overexpressing EGFR or c-Met was significantly higher than single marker either in EC or corresponding mLNs. The bispecific probe EMB01-IR800 was successfully synthesized, with strong binding affinity. EMB01-IR800 showed strong cellular binding to both Kyse30 (EGFR overexpressing) and OE33 (c-Met overexpressing) cells. In vivo fluorescent imaging showed prominent EMB01-IR800 uptake in either Kyse30 or OE33 subcutaneous tumors. Likewise, EMB01-IR800 exhibited superior tumor enrichment in both thoracic orthotopic esophageal squamous cell carcinoma and abdominal orthotopic esophageal adenocarcinoma models. Moreover, EMB01-IR800 produced significantly higher fluorescence in patient-derived mLNs than in benign lymph nodes. This study demonstrated the complementary overexpression of EGFR and c-Met in EC. Compared to single-target probes, the EGFR&c-Met bispecific NIRF probe can efficiently depict heterogeneous esophageal tumors and mLNs, which greatly increased the sensitivity of tumor and mLN identification.

The Receptor Tyrosine Kinase c-Met Promotes Lipid Accumulation in 3T3-L1 Adipocytes.

The receptor tyrosine kinase c-Met is elaborated in embryogenesis, morphogenesis, metabolism, cell growth, and differentiation. JNJ38877605 (JNJ) is an inhibitor of c-Met with anti-tumor activity. The c-Met expression and its role in adipocyte differentiation are unknown. Here, we investigated the c-Met expression and phosphorylation, knockdown (KD) effects, and pharmacological inhibition of c-Met by JNJ on fat accumulation in murine preadipocyte 3T3-L1 cells. During 3T3-L1 preadipocyte differentiation, strikingly, c-Met expression at the protein and mRNA levels and the protein phosphorylation on Y1234/1235 and Y1349 is crucial for inducing its kinase catalytic activity and activating a docking site for signal transducers were increased in a time-dependent manner. Of note, JNJ treatment at 20 μM that strongly inhibits c-Met phosphorylation without altering its total expression resulted in less lipid accumulation and triglyceride (TG) content with no cytotoxicity. JNJ further reduced the expression of adipogenic regulators, including CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and perilipin A. Moreover, JNJ treatment increased cAMP-activated protein kinase (AMPK) and liver kinase B-1 (LKB-1) phosphorylation but decreased ATP levels. Significantly, KD of c-Met suppressed fat accumulation and triglyceride (TG) quantity and reduced the expression of C/EBP-α, PPAR-γ, FAS, ACC, and perilipin A. Collectively, the present results demonstrate that c-Met is a novel, highly conserved mediator of adipogenesis regulating lipid accumulation in murine adipocytes.

ASAP2 interrupts c-MET-CIN85 interaction to sustain HGF/c-MET-induced malignant potentials in hepatocellular carcinoma

BackgroundSustained activation of hepatocyte growth factor (HGF)/c-MET signaling is a major driver of hepatocellular carcinoma (HCC) progression, but underlying mechanism is unclear. ArfGAP With SH3 Domain, Ankyrin Repeat And PH Domain 2 (ASAP2) can reportedly activate GTPases and promote receptor tyrosine kinase signaling. However, the exact role of ASAP2 in HCC, especially for c-MET activation, also remains elusive.MethodsASAP2 expression levels in HCC tissues and cells were quantified using qRT-PCR, western blot (WB) analysis, and immunohistochemistry staining. Cell counting kit-8 (CCK-8) and colony formation assays were performed to evaluate cell proliferation rates. Flow cytometry assays were conducted to assess apoptosis rates. Wound healing and Transwell assays were performed to determine cell migration and invasion capacities. Epithelial-mesenchymal transition (EMT)-related marker expression levels were also examined. Subcutaneous implantation and tail vein injection models were applied for in vivo growth and metastasis evaluations, respectively. Bioinformatics analyses of The Cancer Genome Atlas and STRING datasets were performed to explore ASAP2 downstream signaling. Co-immunoprecipitation and Cycloheximide chasing experiments were performed to assess protein–protein interactions and protein half-life, respectively.ResultsASAP2 had higher expression levels in HCC tissues than in normal liver, and also predicted poor prognosis. Knocking down ASAP2 significantly impaired cell proliferation, migration, and invasion capacities, but promoted apoptosis in HCC cells in vitro. However, overexpression of ASAP2 achieved the opposite effects. In vivo experiments confirmed that ASAP2 could promote HCC cell growth and facilitate lung metastasis. Interestingly, ASAP2 was essential for triggering EMT. Gene Set Enrichment Analysis demonstrated that c-MET signaling was greatly enriched in ASAP2-high HCC cases. Additionally, c-MET signaling activity was significantly decreased following ASAP knockdown, evidenced by reduced c-MET, p-AKT, and p-ERK1/2 protein levels. Importantly, ASAP2 knockdown effectively attenuated HGF/c-MET signaling-induced malignant phenotypes. c-MET and ASAP2 expression levels were positively correlated in our cohort. Mechanistically, ASAP2 can directly bind to CIN85, thereby disrupting its interaction with c-MET, and can thus antagonize CIN85-induced c-MET internalization and lysosome-mediated degradation. Notably, knocking down CIN85 can rescue the observed inhibitory effects caused by ASAP2 knockdown.ConclusionsThis study highlights the importance of ASAP2 in sustaining c-MET signaling, which can facilitate HCC progression.

Abstract CT185: First in human dose-escalation trial with the c-MET targeting antibody-drug conjugate BYON3521

Abstract Background: BYON3521 is a novel c-MET targeting antibody-drug conjugate (ADC) with a cleavable linker-duocarmycin (vc-seco-DUBA) payload that causes irreversible alkylation of DNA in tumor cells. BYON3521 has demonstrated potent and selective killing of c-MET expressing tumor cells in preclinical models, even at low c-MET expression levels. In addition, in vitro studies showed that the active toxin can passively permeate the cell membrane and kill neighboring, c-MET negative cells as a bystander effect. Methods: Patients with previously treated progressive locally advanced or metastatic solid tumors, c-MET positive membrane staining by immunohistochemistry and/or MET-amplified by dual in situ hybridization and/or known activating MET-mutation (excluding exon14m), ECOG performance status 0-1 and adequate organ function are eligible for the dose-escalation part of this first-in-human trial (ClinicalTrials.gov identifier: NCT05323045). An adaptive approach using the Continual Reassessment Method of Neuenschwander (N-CRM model) is used to evaluate the safety of BYON3521 and to determine the maximum tolerated dose (MTD) and recommended dose for expansion (RDE). BYON3521 is administered intravenously every three weeks until tumor progression or unacceptable toxicity. Results: Up to 01 January 2023, 8 patients (1F, 7M, median age 61 yrs) were enrolled. Tumor types were: 4 colorectal cancer, 1 NSCLC, 1 renal cell carcinoma, 1 esophageal cancer and 1 pancreatic cancer. One patient received 0.8 mg/kg, three patients each 1.6 and 3.2 mg/kg and one patient received 4.8 mg/kg. So far, no grade 3 or 4 related adverse events and no dose-limiting toxicities (DLTs) occurred. Most commonly observed related AEs were decreased appetite/weight, fatigue, abdominal pain, back pain, vomiting and chills. Typical ADC associated AEs as keratitis and pneumonitis have not been observed so far. Stable disease was the best response observed in 2 patients at a dose of 3.2 mg/kg. Human PK was in line with predicted preclinical in vivo PK. Conclusions: To date, BYON3521 is well-tolerated with no DLTs at the investigated dose levels. Patient enrollment is ongoing and updated safety, efficacy and pharmacokinetic data will be presented. After the dose-escalation phase the trial will continue with expanded cohorts of patients with specific c-MET expressing cancer types. Citation Format: N. Kotecki, C.M.L. van Herpen, C. Curigliano, M. Hendriks, T C. Vermaas, L. Corrigan, C. Belli, C. Jungels, I.M.E. Desar, N.P. Koper, J.H.M. Schellens, U. Banerji. First in human dose-escalation trial with the c-MET targeting antibody-drug conjugate BYON3521 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT185.

Design, Synthesis and Biological Evaluation of Multi-Target Anti-Cancer Agent PYR26.

This study investigates the synthesis of a new compound, PYR26, and the multi-target mechanism of PYR26 inhibiting the proliferation of HepG2 human hepatocellular carcinoma cells. PYR26 significantly inhibits the growth of HepG2 cells (p < 0.0001) and this inhibition has a concentration effect. There was no significant change in ROS release from HepG2 cells after PYR26 treatment. The mRNA expressions of CDK4, c-Met and Bak genes in HepG2 cells were significantly inhibited (p < 0.05), while mRNA expression of pro-apoptotic factors such as caspase-3 and Cyt c was significantly increased (p < 0.01). The expression of PI3K, CDK4 and pERK proteins decreased. The expression level of caspase-3 protein was increased. PI3K is a kind of intracellular phosphatidylinositol kinase. PI3K signaling pathway is involved in signal transduction of a variety of growth factors, cytokines and extracellular matrix and plays an important role in preventing cell apoptosis, promoting cell survival and influencing cell glucose metabolism. CDK4 is a catalytic subunit of the protein kinase complex and is important for G1 phase progression of the cell cycle. PERK refers to phosphorylated activated ERK, which is translocated from cytoplasm to the nucleus after activation, and then participates in various biological reactions such as cell proliferation and differentiation, cell morphology maintenance, cytoskeleton construction, cell apoptosis and cell canceration. Compared with the model group and the positive control group, the tumor volume of the nude mice in the low-concentration PYR26 group, the medium-concentration group and the high-concentration group was smaller, and the organ volume was smaller than that in the model group and the positive control group. The tumor inhibition rates of low-concentration group PYR26, medium-concentration group and high-concentration group reached 50.46%, 80.66% and 74.59%, respectively. The results showed that PYR26 inhibited the proliferation of HepG2 cells and induced apoptosis of HepG2 cells by down-regulating c-Met, CDK4 and Bak, up-regulating the mRNA expression of caspase-3 and Cyt c genes, down-regulating PI3K, pERK and CDK4 proteins and up-regulating the protein level of caspase-3. In a certain range, with the increase in PYR26 concentration, the tumor growth was slower and the tumor volume was smaller. Preliminary results showed that PYR26 also had an inhibitory effect on the tumors of Hepa1-6 tumor-bearing mice. These results suggest that PYR26 has an inhibitory effect on the growth of liver cancer cells, therefore it has potential to be developed into a new anti-liver cancer drug.

Effects of Ephedra Herb extract on the expression of EGFR-activating mutations and c-Met in non-small-cell lung cancer cell line, H1975, and its combined effects with osimertinib

We previously reported that the combined application of Ephedra Herb extract (EHE) and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), erlotinib, is effective in suppressing the growth of erlotinib-resistant non-small-cell lung cancer (NSCLC) cell line, H1993, xenograft tumor, and cell proliferation, and that EHE downregulates c-Met and wild-type EGFR in H1993 cells. However, it was unclear whether EHE could affect EGFR with active mutations. Clinically, advanced NSCLC patients who are eligible for EGFR-TKI treatment are those with detected EGFR with activating mutations. Therefore, it is important to clarify the effect of EHE on EGFR with activating mutations. H1975 cells express EGFR with activating mutations, L858R and T790M, and c-Met; this NSCLC cell line was used in the present study. EHE downregulated the expression of EGFR with activating mutations and c-Met, and inhibited autophosphorylation of c-Met. Proliferation of H1975 cells was suppressed by EHE in a concentration-dependent manner. These results suggest that EHE may be effective against NSCLC harboring EGFR with activating mutations. Considering the fact that advanced NSCLC patients, with an EGFR T790M mutation, are currently widely treated with the third-generation EGFR-TKI, osimertinib, we examined the combined effects of osimertinib and EHE on H1975 cells. The osimertinib and EHE combination downregulated the expression of these receptors and suppressed the proliferation of H1975 cells more effectively than did osimertinib alone, suggesting that this combination may be effective in treating patients with advanced NSCLC with the L858R + T790M EGFR mutation and c-Met. Graphical Abstract was created with BioRender.com.

Abstract 5000: MYTX-011: A novel cMET-targeting antibody drug conjugate (ADC) engineered to increase on-target uptake in and efficacy against cMET expressing tumors

Abstract MET alterations can act as an oncogenic driver in non-small cell lung cancer (NSCLC) and elevated cMET expression occurs in many cancers. Antibody drug conjugates targeting cMET (cMET-ADCs) have been developed as a strategy to treat cMET+ tumors irrespective of dependency on cMET signaling. cMET- ADCs have shown promising clinical activity, but largely in a subset of NSCLC patients having the highest cMET levels, indicating tumor cMET levels may be limiting for efficacy. We sought to create an ADC with the potential to benefit a broader population of patients including those expressing moderate cMET levels. Here, we describe MYTX-011, an ADC incorporating the clinically validated vcMMAE linker-payload conjugated to a novel, pH-dependent anti-cMET antibody. We hypothesized that engineering the antibody to rapidly lose affinity at acidic endosomal pH would boost ADC uptake and efficacy in cMET+ tumor cells by avoiding non-productive ADC recycling. We conducted mutagenesis of anti-cMET antibodies, screening for variants that selectively lost binding under acidic conditions, and, in parallel, assessed antibody internalization in cell-based assays. The resulting lead humanized IgG1 antibody was conjugated to vcMMAE at engineered cysteine residues (DAR=2) to create MYTX-011, which exhibited rapid dissociation from cMET at pH5.4 but retained high affinity binding at pH7.4 and 6.4. MYTX-011 showed markedly higher (&amp;gt;3 fold) internalization in cMET+ tumor cells and broader, more potent cytotoxicity across a large panel of cMET+ cancer cell lines in vitro compared to a matched ADC based on the unmodified parent antibody lacking pH-dependent binding, or an in-house version of a clinical stage cMET ADC. These findings translated in vivo where MYTX-011 showed superior efficacy (&amp;gt;3 fold based on dose titration) in NSCLC xenograft models with high (EBC-1) or only moderate cMET expression levels (H1373, H1975) compared to the matched parent ADC or an in-house version of a clinical stage cMET ADC. PK and toxicity studies in cynomolgus monkeys revealed that MYTX-011 exhibited favorable PK characteristics, and a toxicity profile similar to previously described MMAE-based ADCs. Together, these findings highlight the potential of MYTX-011 as a therapeutic candidate for treating a broader range of cMET+ malignancies than other cMET-ADCs. Citation Format: Nimish Gera, Kyle Fitzgerald, Vijay Ramesh, Purvi Patel, Lena Kien, Deepak Kanojia, Simon Aoyama, Federico Colombo, Amit Deshpande, William Comb, Thomas Chittenden, Brian Fiske. MYTX-011: A novel cMET-targeting antibody drug conjugate (ADC) engineered to increase on-target uptake in and efficacy against cMET expressing tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5000.

Abstract 540: c-MET mutations sensitize to antibody-drug conjugate telisotuzumab vedotin through efficient internalization and rapid intracellular drug delivery

Abstract Background: Telisotuzumab vedotin (Teliso-V) is an antibody-drug conjugate (ADC) composed of the c-MET antibody ABT-700 and the microtubule inhibitor monomethyl auristatin E (MMAE). Patients with EGFR wildtype (WT) nonsquamous non-small cell lung cancer with high c-MET expression had an overall response rate of 54% with Teliso-V. In addition to overexpression and amplification, genetic alterations in c-MET, such as exon 14 deletion (ex14del) and missense mutations in the tyrosine kinase domain (TKD), contribute to its constitutive activation. The efficacy of Teliso-V in context of these genomic backgrounds is currently unknown. Methods: Recombinant retroviruses were used to express WT-c-MET, c-MET-TKD (D1228H/V/N and Y1230C/H mutations), c-MET-Y1003F/N (CBL E3 ubiquitin ligase binding site mutation), and c-MET-ex14del (47 aa juxtamembrane deletion) mutants in NIH3T3 and Ba/F3 cells. c-MET surface expression, intracellular signaling, ADC internalization and endolysosomal (EL) trafficking (using pHrodo Red-labeled Teliso-V), intracellular MMAE by LC/MS, and Teliso-V sensitivity were studied in 2D/3D cell cultures. Full-length c-MET protein structures were created with the ColabFold program and molecular dynamics simulation software (with/without artificial lipid bilayer) was used to predict the mechanistic basis for differential function. Results: c-MET expression was required for Teliso-V cytotoxicity and intracellular payload delivery in all cell lines. Compared with WT-c-MET, various mutants showed oncogene-driven transformation evident by elevated levels of phosphorylated c-MET/AKT, cytokine-independent colony formation, oncogene-driven proliferation, and tyrosine kinase inhibitor (TKI) resistance. Interestingly, c-MET-TKD-expressing cells were the most sensitive to Teliso-V, which was associated with faster ADC internalization and EL trafficking. An AI-generated protein model and associated molecular dynamics simulation demonstrated that all oncogenic TKD variants tested stabilized the c-MET-plexin-semaphorin-integrin domain (R592-L614), known for its ability to position the extracellular ligand-binding (ELB) site for optimal activation. Conclusions: c-MET-TKD mutations induced TKI resistance and enhanced MMAE delivery in association with robust ADC internalization and EL trafficking. Previous studies have shown that the HER2 ADC fam-trastuzumab deruxtecan-nxki was highly effective in cell lines and patients with ERBB2 kinase domain-mutant lung cancer via a similar mechanism, raising the possibility that this may be a general phenomenon across receptor tyrosine kinase-targeting ADCs. c-MET-TKD mutations are predicted to stabilize the ELB conformation to activate enhanced oncogenic signaling that may contribute to rapid endocytosis-mediated drug delivery. Citation Format: Izhar S. Batth, Bijay S. Jaiswal, Dolonchampa Maji, Robert Sparks, William Glauser, Tamar Uziel, D. Ross Camidge, Athan Vasilopoulos, Peter Ansell, PK Epling-Burnette. c-MET mutations sensitize to antibody-drug conjugate telisotuzumab vedotin through efficient internalization and rapid intracellular drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 540.

Abstract 6311: ABBV-400: An ADC delivering a novel topoisomerase 1 inhibitor to c-Met-positive solid tumors

Abstract Antibody-drug conjugate (ADC) technology is founded on the premise that antigen-mediated payload delivery selectively targets tumor cells and spares normal tissue. While approval of several ADCs has proven this to be a successful therapeutic approach, there remain opportunities to generate optimized ADCs with tailored selection of antigen matched with payload highly active in the target indication. To this end, ABBV-400 has been developed as an ADC that targets c-Met and topoisomerase-1 (Top1), both of which are overexpressed in solid tumors. The antibody is directed to the cell surface tyrosine kinase receptor, c-Met, a key driver of tumor cell growth and development of resistance to EGFR-targeted and other targeted therapeutics. Supporting this, Teliso-V, a c-Met-targeted ADC with MMAE payload, has been granted Breakthrough Therapy Designation in non-small cell lung cancer (NSCLC). To extend activity of a c-Met-targeted ADC to patients with tumors expressing lower c-Met levels and to indications, such as colorectal cancer (CRC), where MMAE-based ADCs have not been effective, a novel topoisomerase-1 inhibitor linker-drug was developed. Screening over 400 linker-drugs identified a unique, potent Top1 inhibitor with a cleavable valine-alanine linker and stable bromoacetamide attachment designed to minimize systemic payload release. Preclinically ABBV-400 exhibits favorable pharmacokinetics, anti-proliferative activity in vitro, and compelling efficacy in NSCLC, gastroesophageal, and CRC xenograft models. Furthermore, ABBV-400 is well tolerated by cynomolgus monkeys with bone marrow and gastrointestinal tract toxicities common to other Top1 inhibitors. This pairing of a novel linker-drug and an antibody with demonstrated clinical activity provides an exciting opportunity to treat patients with c-Met expression in indications sensitive to Top1 inhibition. ABBV-400 has progressed through dose escalation, and dose expansion is currently ongoing in a Phase 1 FIH clinical study (NCT05029882). All authors were employees of AbbVie during this research. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication Citation Format: Regina M. Reilly, Cheng Ji, Ryan P. Matuszak, Mark G. Anderson, Lora Tucker, Nadezda Klunder, Adelyn L. Lazo, Erwin R. Boghaert, Marybeth A. Pysz, Aloma D’Souza, Laura Kreckler, Milan Bruncko, Brittney J. Mills, Matthew Ravn, George Doherty, Kevin J. Freise, Andrew C. Phillips. ABBV-400: An ADC delivering a novel topoisomerase 1 inhibitor to c-Met-positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6311.

Abstract 3866: Targeting AXL can effectively inhibit c-Met-induced therapeutic resistance in renal cancer

Abstract Advanced stage renal cell carcinoma (RCC) often becomes resistant to the currently available therapeutic options. Receptor Tyrosine Kinase (RTK), c-Met, is overexpressed and hyperactivated in renal cancer; and it promotes tumor-promoting pathways following binding with its ligand, Hepatocyte Growth Factor (HGF). Cabozantinib (Cabo), an inhibitor of c-Met/few other RTKs (like, AXL and KDR), is being used for the treatment of RCC. However, acquired therapeutic resistance is a major hurdle in its clinical use. Mechanism(s) of drug resistance against c-Met inhibition is largely unknown. Although Cabo (XL184) can effectively inhibit c-Met/RTK phosphorylation, very interestingly, we found that, prolonged treatment of Cabozantinib (24 hours, in vitro) increased the expression of total c-Met and AXL in renal cancer cells in a dose-dependent manner. AXL is also overexpressed in RCC and has a novel cross-talk with c-Met. We found that silencing AXL (either by gene knock-down approach or using a specific inhibitor (TP-0903)) markedly down-regulates Cabo-mediated total c-Met expression and enhances apoptosis of human RCC cells. We have also utilized murine model(s) of renal cancer growth (both xenograft and syngeneic), and found that Cabo treatment increases the levels of c-Met and AXL in tumor tissues, and the inhibition of AXL using TP-0903 can markedly inhibit the expression levels of total c-Met and AXL. We observed that HGF-mediated c-Met phosphorylation is short-lived when AXL is silenced. We also found that a lower concentration of Cabo can mediate cancer cell survival; and either the knock-down of AXL or using AXL inhibitor in combination with Cabo can reverse these effects. We have studied the downstream signaling pathways (Akt, ERK1/2 and STAT3) of c-Met, and found that AXL inhibition is crucial to down-regulate c-Met-induced signaling molecules following Cabo treatment. Our observations indicate that Cabo-mediated increase in total c-Met in RCC cells is not regulated at the transcriptional level; however, Cabo can interfere with the receptor degradation pathway leading to increased accumulation of c-Met. We have also studied the levels of c-Met and AXL in renal tumor tissues (archived specimens) from renal cancer patients before and after Cabozantinib treatment. Interestingly, we found increased levels of total c-Met and AXL in tumor tissues following Cabo treatment. Together our data suggest that AXL plays a critical role in mediating therapeutic resistance against c-Met inhibitor; and a combination of Cabo and an AXL/other RTK inhibitor can effectively block the resistance. Citation Format: Akash Sabarwal, Laxminarayan Rawat, Johannes Wedel, Yuzuru Sasamoto, Sabina Signoretti, Toni K. Choueiri, Murugabaskar Balan, Soumitro Pal. Targeting AXL can effectively inhibit c-Met-induced therapeutic resistance in renal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3866.

Abstract 5737: Evaluation of the relationship between target expression and in vivo anti-tumor efficacy of AZD9592, an EGFR/c-MET targeted bispecific antibody drug conjugate

Abstract AZD9592 is a bispecific antibody drug conjugate (ADC) designed to deliver a topoisomerase 1 inhibitor (TOP1i) cytotoxic payload (AZ14170133) to tumor cells. AZD9592 selectively binds to epidermal growth factor receptor (EGFR) and c-MET, two cell surface receptors highly expressed in solid tumors including non-small-cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC). Here we evaluate the pharmacodynamic activity of the TOP1i payload delivery by AZD9592 in an NSCLC-derived xenograft model using immunohistochemistry (IHC) approaches. Treatment-induced DNA double-strand breaks (DSB) and apoptotic cell death were measured using γH2AX, phospho-RAD-50 (pRAD50), and cleaved-caspase-3 (CC3) across increasing exposure to AZD9592. Furthermore, we report in vivo antitumor efficacy of AZD9592 in a panel of NSCLC and HNSCC patient-derived xenograft (PDX) models that were characterized for somatic tumor alterations, including oncogenic driver mutations in EGFR, tumor cell expression of EGFR and c-MET by IHC and deep-learning based image analysis, and targeted proteomics by mass spectrometry. Results demonstrate dose-dependent increases in pRAD50 and γH2AX upon treatment with AZD9592, signifying induction of DNA damage. Increased CC3 and reduced tumor volume (TV) in all treatment groups compared with control groups supports that AZD9592 induces tumor cell death due to formation of DNA DSB. In PDX experiments, tumor growth inhibition (TGI), defined as ≥30% reduction in TV from baseline after a single dose of AZD9592 8 mg/kg, was observed in 73% (16/22) of EGFR mutant NSCLC models. The models evaluated included tumors with or without prior exposure to EGFR tyrosine kinase inhibitors, and harboring diverse mutational profiles and heterogeneous expression levels of EGFR and c-MET. In EGFR wildtype NSCLC and HNSCC PDX, TGI was observed in 60% (12/20) and 44% (4/9) of models, respectively. IHC demonstrated an association of target expression and response to treatment, suggesting a potential predictive feature of response in tumors with elevated antigen expression. Targeted proteomics demonstrated an association between the expression of SLFN11, a known TOP1i sensitivity marker, and treatment response. Collectively, these results support the hypothesized mechanism of action of AZD9592: TOP1i induced tumor cell death due to formation of DNA DSB, and suggest opportunities in the treatment of tumors with a range of molecular features. AZD9592 is currently in a Phase 1 clinical trial in advanced solid malignancies. Citation Format: Lara McGrath, Ying Zheng, Simon Christ, Christian C. Sachs, Sihem Khelifa, Claudia Windmüller, Steve Sweet, Yeoun Jin Kim, Daniel Sutton, Michal Sulikowski, Arthur Lewis, Ivan Inigo, Nicolas Floch, Edward Rosfjord, Fernanda Arnaldez, Frank Comer. Evaluation of the relationship between target expression and in vivo anti-tumor efficacy of AZD9592, an EGFR/c-MET targeted bispecific antibody drug conjugate. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5737.

Abstract 5087: Combination therapy of immune checkpoint inhibitor and HIF1α/c-MET peptide vaccine suppresses metastasis by modulating tumor microenvironment in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 20% of all breast cancer and has limited therapeutic option except for immunotherapy because ER, PR, and HER2 targeted for treatment are absent. It also tends to be more aggressive meaning more likely to metastasize faster to other organs such as lungs, liver, and bone, and its recurrence rate is high which leads to a poor prognosis. Immunotherapy is an option to treat TNBC such as programmed death ligand 1 (PD-L1) antibody. However, tumor cells use various immune evasion mechanisms in addition to PD-1/PD-L1 pathway to interrupt the effect of immunotherapy. Moreover, tumor microenvironment (TME) is one of the important factors for the effective anti-tumor immune responses from immunotherapy. Hypoxia is a typical event in TNBC. Hypoxia inducible factor 1-alpha (HIF1α) is a transcription factor that regulates angiogenesis by expressing VEGF, and in addition, upregulates the c-MET which increases the proliferation, migration, and survival of tumor cells. Moreover, HIF1α directly regulates the expression of CD47. CD47 is a transmembrane protein that binds to SIRPα of macrophages to inhibit the phagocytosis of macrophages. Therefore, the efficacy of immune checkpoint inhibitors decreases even if there is a higher degree of tumor-infiltrating lymphocytes (TILs). Combination therapy with immune checkpoint and tumor associated antigens (TAAs) derived peptide vaccine could inhibit metastasis. In present study, we found that HIF1α/c-MET peptide vaccination delayed metastasis in C3(1)Tag mouse. We also investigated whether combination therapy with immune checkpoint inhibitors and peptide vaccine inhibited metastasis. Next, we observed that combination strategy of HIF1α/c-MET peptide vaccine and treated anti-PD-L1 antibody or/and anti-CD47 antibody was more effective in systemically inoculated M6 cells growth in C3(1)Tag mice. Immunohistochemical analysis of the tumors showed that the protein expression of CD47, HIF1α, and c-MET were significantly decreased. On the other hand, CD4+ T cells, CD8+ T cells and M1 macrophages increased, but M2 macrophages decreased, resulting in delayed metastasis in combination therapy group compared to single therapy and control group. In addition, IFNγ ELISPOT showed that peptide vaccination significantly increased the HIF1α/c-MET specific IFNγ-secreting T cell response in splenocytes. We also assessed osteoclastogenesis in bones, which showed reduction of osteoclast in combination therapy group compared to single therapy and control group.Taken together, combination therapy with immune checkpoint inhibitor targeting PD-L1 and CD47 and peptide vaccine appears to be a promising strategy that inhibit metastasis by modulating tumor microenvironment in triple negative breast cancer. Citation Format: JinHwa Hong, Jimin Lee, Soon Young Lim, Ju Won Kim, Ah Reum Lim, Kyong Hwa Park. Combination therapy of immune checkpoint inhibitor and HIF1α/c-MET peptide vaccine suppresses metastasis by modulating tumor microenvironment in triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5087.

Abstract 382: Synergistic anti-tumor effect of ABN401 in combination with KRAS inhibitor in a c-MET-altered KRASG12C non-small cell lung cancer model

Abstract ABN401, a highly selective c-MET inhibitor, is being evaluated for the treatment of non-small cell lung cancer (NSCLC) in clinical trials. NSCLC is a heterogeneous in nature and leading cause in cancer related mortality. Numerous studies are still ongoing to identify the key oncogenic mutations that promote tumor progression. Various actionable genetic mutations such as EGFR, ALK, BRAF, MET, RET, and NTRK/ROS1 have been identified, and the targeted therapies are being developed. It is known that targeted therapies can control disease progression in NSCLC patients and drug resistance due to their heterogeneity remains a clinical hurdle. Among the targets, KRAS gene mutations, an unactionable and is now considered a promising therapeutic approach for NSCLC. Currently, co-occurrence of KRASG12C mutation has been reported in NSCLC patients with MET alterations. Moreover, oncogenic KRAS protein can induce c-MET expression levels by signaling pathways. In this study, we aim to provide an alternative therapeutic strategy for NSCLC patients with KRASG12C mutation harboring MET alterations. We have performed several in vitro and in vivo efficacy studies for the combination of ABN401, c-MET inhibitor, and sotorasib, KRASG12C variant inhibitor, including a PDX model of c-MET-altered KRASG12C NSCLC. Our results suggest that the combination of ABN401 and KRAS inhibitor could be an alternative treatment option for c-MET-altered KRASG12C NSCLC patients. Citation Format: Saehyung Lee, Sunghwan Cho, Na Young Kim, Rajasekaran Nirmal, Kyung Eui Park, Jun Young Choi. Synergistic anti-tumor effect of ABN401 in combination with KRAS inhibitor in a c-MET-altered KRASG12C non-small cell lung cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 382.

Clinicopathological features and immunophenotype of Silva pattern system in endocervical adenocarcinoma.

The aim of this study was to investigate the correlation between Silva pattern system and clinicopathological features of endocervical adenocarcinoma. Moreover, itwasto find molecular markers helpful for Silva classification, andthuswe also explored the expression levels of invasion, adhesion and proliferation biomarkers in cases of Silva non-invasive and invasive types. The survival based on Silva pattern system was analysed by Kaplan-Meier survival analysis, Log-rank test and aCOX risk proportionality model. Sixty samples were chosen to detect the MMP-2, MMP-9, u-PA, E-cadherin, β-catenin, EGF, TGF-α, HDGF, c-Met and RGN expression by immunohistochemistry. Multivariate analysis showed that pattern A/pattern B/pattern C Silva pattern system providedindependent risk factors for prognosis. Our results found the levels of MMP-2, MMP-9 and u-PA were significantly higher in endocervical adenocarcinoma with destructive growth than inthe nondestructive group. The levels of E-cadherin and β-catenin were significantly lower in endocervical adenocarcinoma with destructive growth than inthenondestructive group. The levels of EGF, TGF-α and HDGF were significantly higher in endocervical adenocarcinoma with destructive growth than inthe nondestructive group. Compared with 'non-invasive/invasive Silva pattern', this study suggests 'pattern A/pattern B/pattern C Silva pattern' could be a better criteria for predicting the prognosis. Furthermore, the dual-marker combination of 'MMP-2 and u-PA' and 'E-cadherin and β-catenin' is very important in the diagnosis of Silva pattern classification.

Abstract P2-20-04: Vaccination against HIF1α/c-MET peptides overcome lapatinib resistance and inhibits tumor growth by immunomodulating in HER2 positive breast cancer

Abstract Human Epidermal growth factor Receptor 2 (HER2) is overexpressed in 20% to 30% of breast cancers and is associated with poor prognosis. Patients with advanced HER2-type breast cancer are treated with HER2-directed tyrosine kinase inhibitors as well as antibodies, but many patients finally acquire therapeutic resistance. Therefore, it is important to develop new treatment strategies that circumvent HER2 positive resistant breast cancer. Among the suggested mechanisms of resistance to HER2-targeted therapeutics, tumor hypoxia and relevant biological factors are known as important players. In this study, we aimed to explore the role of tumor hypoxia in the development of resistance to HER2-targeted therapeutics, and whether the role of an immunological therapy targeting this could contribute to overcoming resistance. To this end, we established lapatinib-resistant HER2 positive breast cancer cells in vitro (SKBR3, BT-474 for human and MMC for murine cells). First, we examined if lapatinib-sensitivity is different according to oxygen concentration using lapatinib-resistant cell lines (LR-SKBT3, LR-BT-474, LR-MMC) compared with parent cell lines. Resistant cells were resistant both in normoxic and hypoxic conditions, but parent cells were highly sensitive in both of the oxygen concentration. In the Western blot analysis, HIF1α and c-MET were overexpressed in the resistant cells compared with parent cell lines in both normoxic and hypoxic condition. Interestingly, treatment with lapatinib had reduced HIF1α and c-MET expression at normoxia in parent cell lines but not in resistant cell lines. Under the hypoxic conditions, the difference in expression of HIF1α and c-MET was more pronounced between parent cell lines and resistant cell lines treated with lapatinib. These results indicate that the expression of HIF1α and c-MET was involved in lapatinib-resistant. In a previous study, we have identified epitopes from HIF1α/c-MET and demonstrated effect of delayed bone metastasis in triple negative breast cancer model when used as peptide vaccine. Herein, we evaluated whether the immunization using HIF1α/c-MET peptide vaccine is effective in inhibition of lapatinib-resistant HER2 positive breast cancer growth in vivo. MMTVneu-transgenic mouse model was used and 6- 8 weeks old mice were injected with LR-MMC subcutaneously. Immunization using the HIF1α/c-MET peptide vaccine significantly inhibited growth of LR-MMC cells in MMTVneu-transgenic mice compared with controls. HIF1α/c-MET specific IFN-γ-secreting T cell responses in the splenocytes were significantly higher in the immunized group than controls. Immunohistochemical staining of T cells revealed that expression of tumor HIF1α and c-MET was significantly decreased, while the number of CD8+ T cell was increased in the tumors from immunized animals compared with control tumors. In conclusion, our study demonstrated that the vaccine targeting HIF1α/c-MET might be a promising cancer immunotherapy in lapatinib-resistant HER2 positive breast cancer. In vivo study to evaluate efficacy of combination therapy (lapatinib and HIF1α/c-MET vaccine) is ongoing. Citation Format: Ji Min Lee, Jin Hwa Hong, Soon Young Lim, Ju Won Kim, Ah Reum Lim, Myung Han Hyun, Kyong Hwa Park. Vaccination against HIF1α/c-MET peptides overcome lapatinib resistance and inhibits tumor growth by immunomodulating in HER2 positive breast cancer. [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 P2-20-04.

Abstract P5-06-04: The prognostic value of c-MET monitoring by using c-MET-enriched circulating tumor cells in HR-positive HER2-negative metastatic breast cancer

Abstract [Background] As the development of endocrine resistance and late recurrences are the major clinical concerns in hormone receptor (HR)-positive/HER2-negative metastatic breast cancer (MBC) patients, biomarkers to predict the occurrence of endocrine resistance or disease progression are crucial for improving patient outcomes. Aberrant HGF/c-MET signaling pathway has been reported to play a role in various cellular processes in cancer. Estrogen Receptor 1 (ESR1) mutations, encoding estrogen receptor α, are associated with endocrine resistance in HR+ breast cancer. PIK3CA hotspot mutations that induce hyperactivation of the PI3K are found in 30-40% of HR+ advanced breast cancers. In this context, we evaluated the prognostic values of c-MET-enriched CTC, ESR1 mutations, PIK3CA mutations, and cfDNA concentrations detected in the blood of HR+HER2- MBC patients. [Methods] MBC patients were prospectively enrolled during standard treatments at Samsung Medical Center (IRB No.2019-08-119). Circulating tumor cells (CTCs) were isolated using the GenoCTC® with c-MET-enriched or EpCAM-enriched CTC isolation kits (Genobio Corp., South Korea) from 4mL of blood each. PIK3CA and ESR1 hotspot mutations were analyzed by droplet digital PCR kits (Gencurix Inc., South Korea). cfDNA concentrations were calculated using ESR1 gene copy numbers from plasma. To compare the proportion of c-MET overexpression between primary breast tumors and metastatic sites in HR+HER2- breast cancer patients, primary breast (n=358) and metastatic sites (n=27) were independently collected. c-MET expression was evaluated by an immunohistochemistry assay using an anti-total c-MET (SP44) antibody with a Ventana Discovery XY automated system according to the manufacturer’s instruction. c-MET overexpression was defined if the staining was scored as 2+ or 3+. Progression-free survival (PFS) was defined as the time from blood draw to the first of either disease progression or death during standard therapy. [Results] Out of 93 patients with HR+ MBC, analysis was performed in 63 HR+HER2- MBC patients. Seventeen patients (27%) had one or more EpCAM-enriched CTCs, and fourteen patients (22%) had one or more c-MET-enriched CTCs detected in their blood. The median follow-up time and median time to censoring were 8.4 months and 18.7 months, respectively. According to the Kaplan-Meier survival analysis by log-rank test, c-MET-enriched CTCs, cfDNA concentrations, and ESR1 mutations were significantly associated with PFS (p=0.0026, 0.0064, and 0.011, respectively). However, PIK3CA mutations and EpCAM-enriched CTCs were not statistically significant with PFS (p=0.38 and 0.86, respectively). Multivariate analysis showed that both c-MET-enriched CTCs (HR=3.5, p=0.014) and cfDNA concentrations (HR=2.2, p=0.031) were independent predictors for PFS in HR+HER2- MBC. The proportion of c-MET overexpression was significantly higher in metastatic sites (22.2%) than in primary breast tumors (4.7%) in HR+HER2- breast cancer patients (p=0.00002). As c-MET-enriched CTCs and cfDNA concentrations were independent predictors of disease progression, patients were divided into two groups depending on the result of c-MET-enriched CTCs and cfDNA concentration. When patients with low c-MET-enriched CTC and cfDNA concentrations were classified as a low-risk group and other patients into a high-risk group, the high-risk group had a shorter PFS than the low-risk group (p=0.003). [Conclusion] This study provided c-MET-enriched CTCs and cfDNA concentrations calculated by ESR1 copy numbers in patient blood were significant independent predictors of disease progression in HR+HER2- MBC. The poor prognosis in the c-MET-enriched CTC-high group and the difference in the c-MET overexpression rate between the primary breast and metastatic sites suggested the importance of monitoring c-MET-enriched CTCs in the blood of HR+HER2- MBC patients. Citation Format: Jieun Park, Eun Sol Chang, Ji-Yeon Kim, Chaithanya Chelakkot, Minjung Sung, Ji-Young Song, Kyungsoo Jung, Na Young Kim, Hyegyeong Lee, Mi-Ran Kang, Yeon Hee Park, Young Kee Shin, Yoon-La Choi. The prognostic value of c-MET monitoring by using c-MET-enriched circulating tumor cells in HR-positive HER2-negative metastatic breast cancer [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 P5-06-04.

Co-expression of PD-L1 and oncogenic receptor tyrosine kinases HER2 and cMET is low in metastatic urothelial carcinoma.

533 Background: Activation of HER2 and cMET signaling has been implicated in the pathogenesis of urothelial carcinoma subsets, and the use of anti-PD-(L)1 agents is well established in this disease. Novel therapeutic combinations targeting both immune checkpoints and the cMET or HER2 pathways are currently under investigation in metastatic urothelial carcinoma (mUC). Co-expression of these molecular targets has not been characterized in mUC. We hypothesized that the rate of PD-L1 co-expression with cMET and HER2 may be low. Therefore, we sought to define tumor protein co-expression of PD-L1, HER2 and cMET in a cohort of mUC patients. Methods: Patients with mUC were identified from an institutional database. FFPE tumor specimens from metastatic sites and available paired primary tumors underwent immunohistochemical staining for PD-L1 (SP-142), cMET, and HER2 expression. High expression was defined using the following thresholds: PD-L1: &gt;=5% of infiltrated immune cells; cMET: &gt;=50% of tumor cells; HER2: score=3+ (complete membrane staining that is intense and &gt;10% of tumor cells). Simple and Weighted Cohen’s kappa Statistics (κ) were utilized to assess the agreement in expression and co-expression between paired primary and metastatic samples. Cohen’s interpretation of the κ results is provided. Results: Specimens from 143 mUC patients were analyzed, including 79 with available paired primary tumors. In primary tumors (N=79), high expression of PD-L1, cMET, and HER2 was observed in 15.2%, 34.2%, and 12.7%, respectively. In metastatic samples (N=143), high expression of PD-L1, cMET and HER2 was detected in 9.8%, 41.3%, and 9.8%, respectively. Expression agreement rates between primary and metastatic specimens (n=79) were PD-L1: 79.7% (κ=0.09, slight agreement), cMET: 69.6% (κ=0.35, fair agreement), HER2: 84.8% (κ=0.17, slight agreement) and were driven by high prevalence of low expression. High PD-L1/cMET co-expression was observed in only 5.1% (n = 4) of primary and 4.9% (n =7) of metastatic specimens. High co-expression of PD-L1/HER2 occurred in only 3.8% (n=3) of primary samples and in no metastatic samples. The overall co-expression agreement rate between paired primary and metastatic samples was 55.7% (κ = 0.22, fair agreement) for PD-L1/cMET and 67.1% (κ = 0.06, slight agreement) for PD-L1/HER2. Importantly, however, agreements rates for high co-expression between primary and metastatic samples were very low (2.5% for PD-L1/cMET and 0% for PD-L1/HER2). Conclusions: Tumor co-expression of high cMET or HER2 and PD-L1 is low in this cohort of metastatic urothelial carcinoma patients. Furthermore, agreement of high co-expression between primary and metastatic sites is rare. These results suggest that investigational strategies combining immune checkpoint inhibition with either cMET or HER2 targeted agents in mUC may have limited synergistic activity.

Muscle regeneration in gilthead sea bream: Implications of endocrine and local regulatory factors and the crosstalk with bone.

Fish muscle regeneration is still a poorly known process. In the present study, an injury was done into the left anterior epaxial skeletal muscle of seventy 15g gilthead sea bream (Sparus aurata) juveniles to evaluate at days 0, 1, 2, 4, 8, 16 and 30 post-wound, the expression of several muscle genes. Moreover, transcripts' expression in the bone (uninjured tissue) was also analyzed. Histology of the muscle showed the presence of dead tissue the first day after injury and how the damaged fibers were removed and replaced by new muscle fibers by day 16 that kept growing up to day 30. Gene expression results showed in muscle an early upregulation of igf-2 and a downregulation of ghr-1 and igf-1. Proteolytic systems expression increased with capn2 and ctsl peaking at 1 and 2 days post-injury, respectively and mafbx at day 8.A pattern of expression that fitted well with active myogenesis progression 16 days after the injury was then observed, with the recovery of igf-1, pax7, cmet, and cav1 expression; and later on, that of cav3 as well. Furthermore, the first days post-injury, the cytokines il-6 and il-15 were also upregulated confirming the tissue inflammation, while tnfα was only upregulated at days 16 and 30 to induce satellite cells recruitment; overall suggesting a possible role for these molecules as myokines. The results of the bone transcripts showed an upregulation first, of bmp2 and ctsk at days 1 and 2, respectively; then, ogn1 and ocn peaked at day 4 in parallel to mstn2 downregulation, and runx2 and ogn2 increased after 8 days of muscle injury, suggesting a possible tissue crosstalk during the regenerative process. Overall, the present model allows studying the sequential involvement of different regulatory molecules during muscle regeneration, as well as the potential relationship between muscle and other tissues such as bone to control musculoskeletal development and growth, pointing out an interesting new line of research in this group of vertebrates.

Engineering c-Met-CAR NK-92 cells as a promising therapeutic candidate for lung adenocarcinoma

Mesenchymal-epithelial transition factor (C-Met) has been acknowledged as a significant therapeutic target for treating lung adenocarcinoma (LUAD). However, the potential application of chimeric antigen receptors (CAR)-modified natural killer (NK) cells targeting c-Met in LUAD is rarely explored. In this study, bioinformatic databases were searched and a tissue microarray (TMA) was enrolled to investigate expression status and prognostic role of c-Met in LUAD. Then, four types of c-Met-CAR structures were designed and prepared. The engineering CAR-NK cells containing c-Met-CARs were transfected, verified and characterized. The tumor-inhibitory role of c-Met-CAR-NK cells was finally evaluated in vitro and in vivo. The results demonstrated that c-Met expression elevated and confirmed that high c-Met expression was significantly associated with unfavorable prognosis in LUAD. Then, C-Met-CAR-NK cells were successfully constructed and DAP10 designed in CAR structure was a favorable stimulator for NK cell activation. CCN4 containing DAP10 co-stimulator exhibited the strongest cytotoxicity compared with other CAR-NK cells. Furthermore, CCN4 cells also exerted the prominent tumor-inhibitory effect on xenograft tumor growth. Collectively, this study suggests that DAP10 is a potent stimulator in CAR structure for NK cell activation, and CCN4-based immunotherapy may represent a promising strategy for the treatment of c-Met-positive LUAD.

EXPRESSION OF HEPATOCYTE GROWTH FACTOR AND C-MET RECEPTOR IN STROMAL FIBROBLASTS AND TUMOR CELLS OF ENDOMETRIAL CARCINOMA.

HGF/c-Met is one of the main signaling pathways that ensure communication between epithelial cells and components of the tumor microenvironment determining the invasive and metastatic potential of many cancers. However, the significance of HGF and c-Met in endometrial carcinoma (ECa) progression remains unclear. To evaluate copy number variations as well as expression of the c-Met receptor and its ligand HGF in endometrial carcinomas considering the clinical and morphological characteristics of ECa. The study was conducted on ECa samples of 57 patients, among which 32 had lymph nodes and/or distant metastasis. The copy number of c-MET gene was estimated by qPCR. The expression of HGF and c-Met in tissue samples was determined by the immunohistochemical method. Amplification of c-MET gene was detected in 10.5% of the ECa cases. In most carcinomas, a combined expression pattern of HGF and c-Met was established, in which co-expression of these markers was observed in tumor cells, and the content of HGF+ fibroblasts increased in the stroma. The expression of HGF in tumor cells was associated with the tumor differentiation grade and was higher in G3 ECa (p = 0.041). The number of HGF+ fibroblasts in the stromal component increased in the ECa cases with metastasis compared to the cases without metastasis (p = 0.032). The content of stromal c-Met+ fibroblasts was higher in deeply invasive carcinomas of patients with metastases than in tumors with invasion of < 1/2 myometrium (p = 0.035). Increased expression of HGF and c-Met in stromal fibroblasts of endometrial carcinomas is associated with metastasis in patients with ECa and deep invasion of the tumor into the myometrium, and can contribute to the aggressive course of the disease.