Carcinoembryonic antigen potentiates non-small cell lung cancer progression via PKA-PGC-1ɑ axis.

Carcinoembryonic antigen (CEA) is a tumor-associated antigen primarily produced by tumor cells. It has been implicated in various biological processes such as cell adhesion, proliferation, differentiation, and metastasis. Despite this, the precise molecular mechanisms through which CEA enhances tumor cell proliferation remain largely unclear. Our study demonstrates that CEA enhances the proliferation and migration of non-small cell lung cancer (NSCLC) while also inhibiting cisplatin-induced apoptosis in NSCLC cells. Treatment with CEA led to an increase in mitochondrial numbers and accumulation of lipid droplets in A549 and H1299 cells. Additionally, our findings indicate that CEA plays a role in regulating the fatty acid metabolism of NSCLC cells. Inhibiting fatty acid metabolism significantly reduced the CEA-mediated proliferation and migration of NSCLC cells. CEA influences fatty acid metabolism and the proliferation of NSCLC cells by activating the PGC-1α signaling pathway. This regulatory mechanism involves CEA increasing intracellular cAMP levels, which in turn activates PKA and upregulates PGC-1α. In NSCLC, inhibiting the PKA-PGC-1α signaling pathway reduces both fatty acid metabolism and the proliferation and migration induced by CEA, both in vitro and in vivo. These results suggest that CEA contributes to the promotion of proliferation and migration by modulating fatty acid metabolism. Targeting CEA or the PKA-PGC-1ɑ signaling pathway may offer a promising therapeutic approach for treating NSCLC.

Evaluation of the tumor-targeting specific imaging and killing effect of a CEA-targeting nanoparticle in colorectal cancer.

INTRODUCTION: Colorectal cancer (CRC) is a prevalent digestive malignancy with significant global mortality and morbidity rates. Improving diagnostic capabilities for CRC and investigating novel therapeutic approaches are pressing clinical imperatives. Additionally, carcinoembryonic antigen (CEA) has emerged as a highly promising candidate for both colorectal tumor imaging and treatment. METHODS: A novel active CEA-targeting nanoparticle, CEA(Ab)-MSNs-ICG-Pt, was designed and synthesized, which served as a tumor-specific fluorescence agent to help in CRC near-infrared (NIR) fluorescence imaging. In cell studies, CEA(Ab)-MSNs-ICG-Pt exhibited specific targeting to RKO cells through specific antibody-antigen binding of CEA, resulting in distribution both within and around these cells. The tumor-targeting-specific imaging capabilities of the nanoparticle were determined through in vivo fluorescence imaging experiments. Furthermore, the efficacy of the nanoparticle in delivering chemotherapeutics and its killing effect were evaluated both in vitro and in vivo. RESULTS: The CEA(Ab)-MSNs-ICG-Pt nanoparticle, designed as a novel targeting agent for carcinoembryonic antigen (CEA), exhibited dual functionality as a targeting fluorescent agent. This CEA-targeting nanoparticle showed exceptional efficacy in eradicating CRC cells in comparison to individual treatment modalities. Furthermore, it exhibits exceptional biosafety and biocompatibility properties. CEA(Ab)-MSNs-ICG-Pt exhibits significant promise due to its ability to selectively target tumors through NIR fluorescence imaging and effectively eradicate CRC cells with minimal adverse effects in both laboratory and in vivo environments. CONCLUSION: The favorable characteristics of CEA(Ab)-MSNs-ICG-Pt offer opportunities for its application in chemotherapeutic interventions, tumor-specific NIR fluorescence imaging, and fluorescence-guided surgical procedures.

[The influence of knocking down the expression of low-density lipoprotein receptor associated proteins on the vascular abnormalities in hepatocellular carcinoma and its mechanisms].

Objectives: To investigate the effect of the expression of low-density lipoprotein receptor associated protein (LDLR) on the vascular abnormalities in hepatocellular carcinoma (HCC) and its mechanisms. Methods: Based on the information of Oncomine Cancer GeneChip database, we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen (CEA) and CD31 in hepatocellular carcinoma tissues. Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells. The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing, real-time fluorescence quantitative polymerase chain reaction, and protein immunoblotting. The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis. The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells. Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells, as well as the role of CEA in the regulation of angiogenesis by LDLR. Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues, and CEA and CD31 in 146 hepatocellular carcinoma tissues, and analyze the correlations between the expression levels of LDLR, CEA, and CD31 in the tissues, serum CEA, and alanine transaminase (ALT). Results: Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated (r=-0.64, P=0.001), whereas the expressions of CEA and CD31 in these tissues were positively correlated ( r=0.46, P=0.010). The transcriptome sequencing results showed that there were a total of 1 032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells, of which 517 genes were up-regulated and 515 genes were down-regulated. The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group. The Gene Ontology (GO) function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch, the extracellular matrix receptor interactions, and the interactions with the extracellular matrix receptors. The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43, which was higher than that of the control group (1.15±0.14, P＜0.001). The results of angiogenesis experiments showed that at 5 h, the number of main junctions, the number of main segments, and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4, 552.5±63.8, and 2 239 781.0±13 8211.9 square pixels, which were higher than those of the control group (113.3±23.5, 194.8±36.5, and 660 621.0±280 328.3 square pixels, respectively, all P＜0.01).The number of vascular major junctions, the number of major segments, and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4, 257.5±20.4, and 2 535 754.5±249 094.2 square pixels, respectively, which were all higher than those of the control group (113.3±23.5, 114.3±12.2, and 1 565 456.5±219 259.7 square pixels, respectively, all P＜0.01). In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions, the number of main segments, and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0, 286.9±12.3, and 1 966 990.0±126 249.5 spixels, which were higher than those in the control group (119.7±22.1, 202.7±33.7, and 1 421 191.0±189 837.8 square pixels, respectively). The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA, but was negatively correlated with the expression of CD31 (r=-0.167, P=0.044), the level of serum CEA (r=-0.061, P=0.032), and the level of serum ALT(r=-0.147,P=0.05). The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31 (r=0.192, P=0.020). The level of serum CEA was positively correlated with the level of serum ALT (r=0.164, P=0.029). Conclusion: Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.

SARS-Cov-2 spike induces intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9.

Background: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as a typical tumor marker, has been found to exert immunomodulatory effects in many diseases. We previously reported the clinical and molecular evidences supporting that SARS-Cov-2 infected the gastrointestinal (GI) tract and found a reduction of CEACAM5 in COVID-19 patients' feces which associated with gut dysbiosis. Yet the role of CEACAM5 in GI infection is ill-defined. Methods: Mice models were established through intraperitoneally injecting with recombinant viral spike-Fc to mimic the intestinal inflammation. We collected duodenum, jejunum, ileum and colon samples after 6h, 2 days, 4 days and 7 days of spike-Fc or control-Fc injection to perform proteomic analysis. Blood was collected from healthy donors and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation, then CD4+ T cells were isolated with magnetic beads and co-cultured with Caco-2 cells. Results: In addition to intestinal CEACAM5, the expression of tight junction and the percent of CD4+ T lymphocytes were significantly decreased in spike-Fc group compared to control (p < 0.05), accompanied with increased level of inflammatory factors. The KEGG analysis revealed differentially expressed proteins were mainly enriched in the coronavirus disease (COVID-19), tight junction, focal adhesion, adherens junction and PI3K-Akt signaling pathway. Protein-protein interaction (PPI) network analysis identified the interaction between CEACAM5 and Galectin-9 that was also verified by molecular docking and co-IP assay. We further confirmed a reduction of CEACAM5 in SARS-CoV-2 spike stimulated enterocytes could promote the expression of Galectin-9 protein in CD4+T cells. Then it gave rise to the increasing release of inflammatory factors and increased apoptosis of CD4+T cells by inhibition of PI3K/AKT/mTOR pathway. Ultimately intestinal barrier dysfunction happened. Conclusion: Our results indicated that CEACAM5 overexpression and Galectin-9 knockdown played a protective role in intestinal barrier injury upon spike-Fc stimulation. Collectively, our findings identified firstly that SARS-CoV-2 spike induced intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9. The result provides potential therapeutic targets in intestinal barrier dysfunction for treating severe COVID patients.

Loss of CEACAM1 in hepatocytes causes hepatic fibrosis.

BACKGROUND: The role of insulin resistance in hepatic fibrosis in Metabolic dysfunction-Associated SteatoHepatitis (MASH) remains unclear. Carcinoembryonic Antigen-related Cell Adhesion Molecule1 protein (CEACAM1) promotes insulin clearance to maintain insulin sensitivity and repress de novo lipogenesis, as bolstered by the development of insulin resistance and steatohepatitis in AlbuminCre + Cc1fl/fl mice with liver-specific mouse gene encoding CEACAM1 protein (Ceacam1) deletion. We herein investigated whether these mice also developed hepatic fibrosis and whether hepatic CEACAM1 is reduced in patients with MASH at different fibrosis stages. METHODS: AlbuminCre + Cc1fl/fl mice were fed a regular or a high-fat diet before their insulin metabolism and action were assessed during IPGTT, and their livers excised for histochemical, immunohistochemical and Western blot analysis. Sirius red staining was used to assess fibrosis, and media transfer was employed to examine whether mutant hepatocytes activated hepatic stellate cells (HSCs). Hepatic CEACAM1 protein levels in patients with varying disease stages were assessed by ELISA. RESULTS: Hepatocytic deletion of Ceacam1 caused hyperinsulinemia-driven insulin resistance emanating from reduced hepatic insulin clearance. AlbuminCre + Cc1fl/fl livers showed inflammation, fibrosis and hepatic injury, with more advanced bridging and chicken-wire hepatic fibrosis under high-fat conditions. Media transferred from hepatocytes isolated from mutant mice activated control HSCs, likely owing to their elevated endothelin1 content. Interestingly, hepatic CEACAM1 levels were lower in the livers of patients with MASH and declined gradually with advanced fibrosis stage. CONCLUSIONS: Hepatic CEACAM1 levels declined with progression of MASH in humans. The phenotype of AlbuminCre + Cc1fl/fl mice assigned a key role to CEACAM1 loss from hepatocytes in hepatic fibrosis independently of other liver cells.

Detection and clinical significance of CEACAM5 methylation in colorectal cancer patients.

Colorectal cancer (CRC) is a globally common cancer, and the serum carcinoembryonic antigen (sCEA) is widely applied as a diagnostic and prognostic tumor marker in CRC. This study aimed to elucidate the mechanism of CEA expression and corresponding clinical features to improve prognostic assessments. In CRC cells, hypomethylation of the CEACAM5 promoter enhanced CEA expression in HCT116 and HT29 cells with 5-aza-2'-deoxycytidine (5-Aza-dC) treatment. Our clinical data indicated that 64.7% (101/156) of CRC patients had an sCEA level above the normal range, and 76.2% (77/101) of those patients showed a lower average CpG methylation level of the CEACAM5 promoter. The methylation analysis showed that both CRC cell lines and patient samples shared the same critical methylation CpG regions at -200 to -500 and -1000 to -1400 bp of the CEACAM5 promoter. Patients with hypermethylation of the CEACAM5 promoter showed features of a BRAF mutation, TGFB2 mutation, microsatellite instability-high, and preference for right-sided colorectal cancer and peritoneal seeding presentation that had a similar clinical character to the consensus molecular subtype 1 (CMS1) of colorectal cancer. Additionally, hypermethylation of the CEACAM5 promoter combined with evaluated sCEA demonstrated the worst survival among the patients. Therefore, the methylation status of the CEACAM5 promoter also served as an effective biomarker for assessing disease prognosis. Results indicated that DNA methylation is a major regulatory mechanism for CEA expression in colorectal cancer. Moreover, our data also highlighted that patients in a subgroup who escaped from inactivation by DNA methylation had distinct clinical and pathological features and the worst survival.

Study on the application of percutaneous closed pleural brushing combined with cell block technique in the diagnosis of malignant pleural effusion.

INTRODUCTION: This study was to investigate the diagnostic value of percutaneous closed pleural brushing (CPBR) followed by cell block technique for malignant pleural effusion (MPE) and the predictive efficacy of pleural fluid carcinoembryonic antigen (CEA) for epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma patients with MPE. METHODS: All patients underwent closed pleural biopsy (CPB) and CPBR followed by cell block examination. MPE-positive diagnostic rates between the two methods were compared. Univariate and multivariate analyses were performed to determine factors influencing the EGFR mutations. Receiver operating characteristic (ROC) curve was used to analyze the predictive efficacy of pleural fluid CEA for EGFR mutations. RESULTS: The cumulative positive diagnostic rates for MPE after single and twice CPBR followed by cell block examination were 80.5% and 89.0%, higher than CPB (45.7%, 54.3%) (P < 0.001). Univariate analysis showed that EGFR mutation was associated with pleural fluid and serum CEA (P < 0.05). Multivariate analysis showed that pleural fluid CEA was an independent risk factor for predicting EGFR mutation (P < 0.001). The area under the curve (AUC) of pleural fluid CEA for EGFR mutation prediction was 0.774, higher than serum CEA (P = 0.043), but no difference with the combined test (P > 0.05). CONCLUSION: Compared with CPB, CPBR followed by the cell block technique can significantly increase the positive diagnostic rate of suspected MPE. CEA testing of pleural fluid after CPBR has a high predictive efficacy for EGFR mutation in lung adenocarcinoma patients with MPE, implying pleural fluid extracted for cell block after CPBR may be an ideal specimen for genetic testing.

Correlation between carcinoembryonic antigen (CEA) expression and EGFR mutations in non-small-cell lung cancer: a meta-analysis.

OBJECTIVES: The purpose of this meta-analysis was to investigate the relationship between serum carcinoembryonic antigen (CEA) expression and epidermal growth factor receptor (EGFR) mutation status in non-small cell lung cancer (NSCLC). METHODS: Databases such as PubMed, Cochrane, EMBASE and Google Scholar were systematically searched to identify studies assessing the association of serum CEA expression with EGFR mutations. Across 19 studies, 4168 patients were included between CEA expression and EGFR mutations odds ratio (OR) conjoint analysis of correlations. RESULTS: Compared with CEA-negative NSCLC, CEA-positive tumors had an increased EGFR mutation rate (OR = 1.85, 95% confidence interval: 1.48-2.32, P < 0.00001). This association was observed in both stage IIIB/IV patients (OR = 1.60, 95% CI: 1.18-2.15, P = 0.002) and stage I-IIIA (OR = 1.67, 95% CI: 1.01-2.77, P = 0.05) patients. In addition, CEA expression was associated with exon 19 (OR = 1.97, 95% CI: 1.25-3.11, P = 0.003) and exon 21 (OR = 1.51, 95% CI: 1.07-2.12, P = 0.02) EGFR mutations. In ADC pathological type had also showed the correlation (OR = 1.84, 95% CI: 1.31-2.57, P = 0.0004). CONCLUSIONS: This meta-analysis indicated that serum CEA expression was associated with EGFR mutations in NSCLC patients. The results of this study suggest that CEA level may play a predictive role in the EGFR mutation status of NSCLC patients. Detecting serum CEA expression levels can give a good suggestion to those patients who are confused about whether to undergo EGFR mutation tests. Moreover, it may help better plan of the follow-up treatment.

The role of carcinoembryonic antigen-related cell adhesion molecule 1 in cancer.

The Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), also known as CD66a, is a member of the immunoglobulin superfamily. CEACAM1 was shown to be a prognostic marker in patients suffering from cancer. In this review, we summarize pre-clinical and clinical evidence linking CEACAM1 to tumorigenicity and cancer progression. Furthermore, we discuss potential CEACAM1-based mechanisms that may affect cancer biology.

Pretreatment controlling nutritional status (CONUT) score and carcinoembryonic antigen level provide tumor progression and prognostic information in gastric cancer: A retrospective study.

This study explores the role of combining the controlling nutritional status (CONUT) score and the carcinoembryonic antigen (CEA) level on predicting tumor stage and prognosis in gastric cancer (GC) patients. A total of 682 GC patients were included in this retrospective study. CONUT scores and CEA levels were combined to establish a new scoring system: CONUT-CEA score. cutoff values for distinguishing patients between stage IV and non-stage IV were established by receiver operating characteristic curves. cutoff values for predicting prognosis were determined by maximum χ2 method. The CONUT and CEA cutoff values for discriminating stage IV patients from non-stage IV patients were 2.0 and 5.58 ng/mL, respectively. Logistic regression model demonstrated that high CONUT-CEA score was related to advanced tumor stage. Among non-stage IV patients, CONUT and CEA cutoff values of 2.0 and 9.50 ng/mL predicted overall survival (OS), respectively. The Cox proportional risk model revealed that high CONUT-CEA score was notable related to decreased OS (2 vs 0: hazard ratios (HR) = 2.358, 95% confidence intervals (CI) = 1.412-3.940, P = .001) and decreased disease-free survival (2 vs 0: HR = 1.980, 95% CI = 1.072-3.656, P = .003). The CONUT-CEA score may be a good biomarker for predicting tumor stage and prognosis in GC patients.

scFv biofunctionalized nanoparticles to effective and safe targeting of CEA-expressing colorectal cancer cells.

Colorectal cancer (CRC) is one of the deadliest cancers worldwide, with the 5 year survival rate in metastatic cases limited to 12%. The design of targeted and effective therapeutics remains a major unmet clinical need in CRC treatment. Carcinoembryonic antigen (CEA), a glycoprotein overexpressed in most colorectal tumors, may constitute a promising molecule for generating novel CEA-targeted therapeutic strategies for CRC treatment. Here, we developed a smart nanoplatform based on chemical conjugation of an anti-CEA single-chain variable fragment (scFv), MFE-23, with PLGA-PEG polymers to deliver the standard 5-Fluorouracil (5-FU) chemotherapy to CRC cells. We confirmed the specificity of the developed CEA-targeted NPs on the internalization by CEA-expressing CRC cells, with an enhance of threefold in the cell uptake. Additionally, CEA-targeted NPs loaded with 5-FU induced higher cytotoxicity in CEA-expressing cells, after 24 h and 48 h of treatment, reinforcing the specificity of the targeted NPs. Lastly, the safety of CEA-targeted NPs loaded with 5-FU was evaluated in donor-isolated macrophages, with no relevant impact on their metabolic activity nor polarization. Altogether, this proof of concept supports the CEA-mediated internalization of targeted NPs as a promising chemotherapeutic strategy for further investigation in different CEA-associated cancers and respective metastatic sites.Authors: Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 1 Given name: [Maria José] Last name [Silveira]. Author 7 Given name: [Maria José] Last name [Oliveira]. Also, kindly confirm the details in the metadata are correctokAffiliations: Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary.ok.

Chemical Proteomic Approach for In-Depth Glycosylation Profiling of Plasma Carcinoembryonic Antigen in Cancer Patients.

Carcinoembryonic antigen (CEA) of human plasma is a biomarker of many cancer diseases, and its N-glycosylation accounts for 60% of molecular mass. It is highly desirable to characterize its glycoforms for providing additional dimension of features to increase its performance in prognosis and diagnosis of cancers. However, to systematically characterize its site-specific glycosylation is challenging because of its low abundance. Here, we developed a highly sensitive strategy for in-depth glycosylation profiling of plasma CEA through chemical proteomics combined with multienzymatic digestion. A trifunctional probe was utilized to generate covalent bond of plasma CEA and its antibody upon UV irradiation. As low as 1 ng/ml CEA in plasma could be captured and digested with trypsin and chymotrypsin for intact glycopeptide characterization. Twenty six of 28 potential N-glycosylation sites were well identified, which were the most comprehensive N-glycosylation site characterization of CEA on intact glycopeptide level as far as we known. Importantly, this strategy was applied to the glycosylation analysis of plasma CEA in cancer patients. Differential site-specific glycoforms of plasma CEA were observed in patients with colorectal cancers (CRCs) and lung cancer. The distributions of site-specific glycoforms were different as the progression of CRC, and most site-specific glycoforms were overexpressed in stage II of CRC. Overall, we established a highly sensitive chemical proteomic method to profile site-specific glycosylation of plasma CEA, which should generally applicable to other well-established cancer glycoprotein biomarkers for improving their cancer diagnosis and monitoring performance.

A genome-wide genetic screen identifies CYRI-B as a negative regulator of CEACAM3-mediated phagocytosis.

Opsonin-independent phagocytosis mediated by human carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) has evolved to control a subset of human-restricted bacterial pathogens. CEACAM3 engagement triggers rapid GTP-loading of the small GTPase Rac as a master regulator of cytoskeletal rearrangements and lamellipodia-driven internalization. To identify components of the CEACAM3-initiated signaling cascade, we performed a genome-wide CRISPR/Cas9-based screen in human myeloid cells. Following infection with fluorescently labeled bacteria, cells exhibiting elevated phagocytosis (gain-of-function) as well as cells showing reduced phagocytosis (loss-of-function) were sorted and enrichment of individual single-guide RNAs (sgRNAs) was determined by next generation sequencing. Concentrating on genes whose targeting by three distinct sgRNAs consistently resulted in a gain-of-function phenotype, we identified the Rac-GTP-sequestering protein CYRI-B as a negative regulator of CEACAM3-mediated phagocytosis. Clonal HL-60 cell lines with CYRI-B knockout showed enhanced CEACAM3-downstream signaling, such as Rac GTP loading and phosphorylation of PAK kinases, leading to increased phagocytosis of bacteria. Complementation of the CYRI-B knockout cells reverted the knockout phenotype. Our results unravel components of CEACAM3-initiated opsonin-independent phagocytosis on a genome-wide level and highlight CYRI-B as a negative regulator of CEACAM3-initiated signaling in myeloid cells.

Incidence, clinicopathological features, and clinical outcomes of low HER2 expressed, inoperable, advanced, or recurrent gastric/gastroesophageal junction adenocarcinoma.

BACKGROUND: According to the DESTINY-Breast04 trial, treating patients with breast cancer and low human epidermal growth factor receptor 2 expressions (HER2-low) varies from that of those with no HER2 expression. However, it is interesting to know if HER2-low indicates for anti-HER2 therapy in the gastric or gastroesophageal junction (G/GEJ) adenocarcinoma. Hence we conducted this study to assess the incidence, clinicopathological features, and treatment outcomes of patients with HER2-low G/GEJ adenocarcinoma. PATIENTS AND METHODS: This was a single-center, retrospective observational study. Patients with previously untreated G/GEJ adenocarcinoma were classified based on their HER2 status using immunohistochemistry (IHC) with or without in situ hybridization (ISH) as follows: HER2 negative (IHC 0), HER2-low (IHC 1+ or 2+/ISH-), and HER2-positive (IHC2+/ISH+ or 3+). RESULTS: In total, 734 patients with G/GEJ adenocarcinoma were divided into three groups (HER2-negative, n = 410; HER2-low, n = 154, and HER2-positive, n = 170). The intestinal-type histology, peritoneal metastasis, and higher serum carcinoembryonic antigen (CEA) levels differed significantly among patients with negative, low, and positive HER2 statuses: intestinal-type histology (21.0%, 44.2%, and 59.8%, respectively), peritoneal metastasis (56.3%, 44.8%, and 21.8%, respectively), and higher serum CEA level (32.2%, 41.6%, and 56.5%, respectively). Improved survival was observed in the HER2-positive group than in the HER2-negative G/GEJ adenocarcinoma group [hazard ratio (HR) = 0.73, 95% confidence interval (CI) 0.59-0.89; P = 0.002]. However, the prognoses of the HER2-low and HER2-negative groups were similar (HR = 1.01, 95% CI 0.82-1.23; P = 0.843). CONCLUSIONS: Patients with HER2-low G/GEJ adenocarcinoma exhibited intermediate and distinct characteristics than those in the HER2-negative group. Similarly, the HER2-low group's prognosis was worse than that of the HER2-positive group. Therefore developing novel therapeutic strategies targeting HER2-low G/GEJ adenocarcinoma is required.

Generation and in vivo characterization of a novel high-affinity human antibody targeting carcinoembryonic antigen.

There are no effective treatment options for most patients with metastatic colorectal cancer (mCRC). mCRC remains a leading cause of tumor-related death, with a five-year survival rate of only 15%, highlighting the urgent need for novel pharmacological products. Current standard drugs are based on cytotoxic chemotherapy, VEGF inhibitors, EGFR antibodies, and multikinase inhibitors. The antibody-based delivery of pro-inflammatory cytokines provides a promising and differentiated strategy to improve the treatment outcome for mCRC patients. Here, we describe the generation of a novel fully human monoclonal antibody (termed F4) targeting the carcinoembryonic antigen (CEA), a tumor-associated antigen overexpressed in colorectal cancer and other malignancies. The F4 antibody was selected by antibody phage display technology after two rounds of affinity maturation. F4 in single-chain variable fragment format bound to CEA in surface plasmon resonance with an affinity of 7.7 nM. Flow cytometry and immunofluorescence on human cancer specimens confirmed binding to CEA-expressing cells. F4 selectively accumulated in CEA-positive tumors, as evidenced by two orthogonal in vivo biodistribution studies. Encouraged by these results, we genetically fused murine interleukin (IL) 12 to F4 in the single-chain diabody format. F4-IL12 exhibited potent antitumor activity in two murine models of colon cancer. Treatment with F4-IL12 led to an increased density of tumor-infiltrating lymphocytes and an upregulation of interferon γ expression by tumor-homing lymphocytes. These data suggest that the F4 antibody is an attractive delivery vehicle for targeted cancer therapy.

Electrochemiluminescence imaging of a membrane carcinoembryonic antigen at single tissue sections.

Histopathological molecular testing of tissue sections is an essential step in tumor diagnosis; however, the commonly used immunohistochemical methods have problems such as low specificity and the subjective bias of the observer. Here, we report an electrochemiluminescence (ECL) imaging method to detect a membrane carcinoembryonic antigen (CEA) at the single tissue sections of cancer patients. By permeabilizing the tissue attached to a glassy carbon electrode, Ru(bpy)32+ tagged at the membrane CEA of the tissue could electrochemically react with TPrA in solution to emit ECL that has near-zero background and an extremely high signal-to-background ratio. Using the established ECL method, the expression differences and distribution characteristics of the CEA protein in the carcinoma and paracancerous tissues of pancreatic ductal carcinoma (PDAC) and lung adenocarcinoma (LUAD) patients are investigated. The images reveal that CEA proteins are mostly distributed in the acini and surrounding areas both in PDAC and LUAD tissues. Therefore, the presented approach could be able to provide a new molecular recognition method for the diagnosis of adenocarcinoma and other tumors.

Establishment and characterization of a new human ampullary carcinoma cell line, DPC-X1.

BACKGROUND: An in-depth study of the pathogenesis and biological characteristics of ampullary carcinoma is necessary to identify appropriate treatment strategies. To date, only eight ampullary cancer cell lines have been reported, and a mixed-type ampullary carcinoma cell line has not yet been reported. AIM: To establish a stable mixed-type ampullary carcinoma cell line originating from Chinese. METHODS: Fresh ampullary cancer tissue samples were used for primary culture and subculture. The cell line was evaluated by cell proliferation assays, clonal formation assays, karyotype analysis, short tandem repeat (STR) analysis and transmission electron microscopy. Drug resistances against oxaliplatin, paclitaxel, gemcitabine and 5-FU were evaluated by cell counting kit-8 assay. Subcutaneous injection 1 × 106 cells to three BALB/c nude mice for xenograft studies. The hematoxylin-eosin staining was used to detect the pathological status of the cell line. The expression of biomarkers cytokeratin 7 (CK7), cytokeratin 20 (CK20), cytokeratin low molecular weight (CKL), Ki67 and carcinoembryonic antigen (CEA) were determined by immunocytochemistry assay. RESULTS: DPC-X1 was continuously cultivated for over a year and stably passaged for more than 80 generations; its population doubling time was 48 h. STR analysis demonstrated that the characteristics of DPC-X1 were highly consistent with those of the patient's primary tumor. Furthermore, karyotype analysis revealed its abnormal sub-tetraploid karyotype. DPC-X1 could efficiently form organoids in suspension culture. Under the transmission electron microscope, microvilli and pseudopods were observed on the cell surface, and desmosomes were visible between the cells. DPC-X1 cells inoculated into BALB/C nude mice quickly formed transplanted tumors, with a tumor formation rate of 100%. Their pathological characteristics were similar to those of the primary tumor. Moreover, DPC-X1 was sensitive to oxaliplatin and paclitaxel and resistant to gemcitabine and 5-FU. Immunohistochemistry showed that the DPC-X1 cells were strongly positive for CK7, CK20, and CKL; the Ki67 was 50%, and CEA was focally expressed. CONCLUSION: Here, we have constructed a mixed-type ampullary carcinoma cell line that can be used as an effective model for studying the pathogenesis of ampullary carcinoma and drug development.

Novel inflammation-combined prognostic index to predict survival outcomes in patients with gastric cancer.

BACKGROUND: We focused on the lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) and devised an inflammation-combined prognostic index (ICPI) as a prognostic marker of cancer-specific survival (CSS). METHODS: We reviewed the clinicopathological data of 480 patients with gastric cancer undergoing curative laparoscopic gastrectomy between 2009 and 2019. This study examined the significance of LMR, NLR, PLR, and ICPI as cancer-specific prognostic markers. RESULTS: In univariate analysis, tumor diameter, histological differentiation, pathological tumor-node-metastasis (pTNM) stage, LMR, NLR, PLR, C-reactive protein (CRP) level, carcinoembryonic antigen (CEA), and postoperative chemotherapy were significantly associated with CSS. In multivariate analysis, pTNM stage and CEA were the independent risk factors for CSS, although LMR, NLR, and PLR were not the independent risk factors for CSS. The ICPI formula was constructed using hazard ratios for three inflammation-based biomarkers with worse prognosis identified in the univariate analysis: LMR <4.315, NLR ≥2.344, and PLR ≥212.01, which were each scored as 1, with all remaining values pointed at 0. ICPI was calculated as follows: ICPI = 2.9 × LMR + 2.8 × NLR + 2.8 × PLR. The optimal cutoff value of ICPII was 2.9. On multivariate analysis, pTNM stage, CEA, and ICPI were independent prognostic factors for CSS. In the Kaplan-Meier survival analysis, CSS in the high ICPI group was significantly worse than that in the low ICPI group. CONCLUSION: ICPI was devised as a novel predictive index for prognosis, and its usefulness was clarified.

Neutrophil CEACAM1 determines susceptibility to NETosis by regulating the S1PR2/S1PR3 axis in liver transplantation.

Neutrophils, the largest innate immune cell population in humans, are the primary proinflammatory sentinel in the ischemia-reperfusion injury (IRI) mechanism in orthotopic liver transplantation (OLT). Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CC1, or CD66a) is essential in neutrophil activation and serves as a checkpoint regulator of innate immune-driven IRI cascade in OLT. Although CC1 alternative splicing generates two functionally distinct short and long cytoplasmic isoforms, their role in neutrophil activation remains unknown. Here, we undertook molecular and functional studies to interrogate the significance of neutrophil CC1 signaling in mouse and human OLT recipients. In the experimental arm, we employed a mouse OLT model to document that ablation of recipient-derived neutrophil CC1-long (CC1-L) isotype aggravated hepatic IRI by promoting neutrophil extracellular traps (NETs). Notably, by regulating the S1P-S1PR2/S1PR3 axis, neutrophil CC1-L determined susceptibility to NET formation via autophagy signaling. In the clinical arm, liver grafts from 55 transplant patients selectively enriched for neutrophil CC1-L showed relative resistance to ischemia-reperfusion (IR) stress/tissue damage, improved hepatocellular function, and clinical outcomes. In conclusion, despite neutrophils being considered a principal villain in peritransplant tissue injury, their CC1-L isoform may serve as a regulator of IR stress resistance/NETosis in human and mouse OLT recipients.