Tumor Tissue Research Articles

Tumor Tissue Acidosis as a Factor Determining the Malignant Phenotype and Tumor Resistance to Chemotherapy

Tumor Tissue Acidosis as a Factor Determining the Malignant Phenotype and Tumor Resistance to Chemotherapy

LncOCMRL1 promotes oral squamous cell carcinoma growth and metastasis via the RRM2/EMT pathway

BackgroundLong noncoding RNAs (lncRNAs) are widely involved in cancer development and progression, but the functions of most lncRNAs have not yet been elucidated. Metastasis is the main factor restricting the therapeutic outcomes of various cancer types, including oral squamous cell carcinoma (OSCC). Therefore, exploring the key lncRNAs that regulate OSCC metastasis and elucidating their molecular mechanisms will facilitate the development of new strategies for effective OSCC therapy.MethodsWe analyzed the lncRNA expression profiles of tumor tissues from OSCC patients with and without cervical lymph node metastasis, and OSCC cell lines. We revealed high expression of oral squamous cell carcinoma metastasis-related lncRNA 1 (lncOCMRL1) in OSCC patient tumor tissues with lymph node metastasis and highly metastatic OSCC cell lines. The effects of lncOCMRL1 knockdown on the invasion, migration and proliferation abilities of OSCC cells were explored through qRT-PCR, Transwell, colony formation, and cell proliferation experiments. The mechanism by which lncOCMRL1 promotes OSCC metastasis and proliferation was explored through RNA pull-down, silver staining, mass spectrometry, RIP, and WB experiments. To increase its translational potential, we developed a reduction-responsive nanodelivery system to deliver siRNA for antitumor therapy.ResultsWe determined that lncOCMRL1 is highly expressed in OSCC metastatic tumor tissues and cells. Functional studies have shown that high lncOCMRL1 expression can promote the growth and metastasis of OSCC cells both in vivo and in vitro. Mechanistically, lncOCMRL1 could induce epithelial-mesenchymal transition (EMT) via the suppression of RRM2 ubiquitination and thereby promote the proliferation, invasion, and migration of OSCC cells. We further constructed reduction-responsive nanoparticles (NPs) for the systemic delivery of siRNAs targeting lncOCMRL1 and demonstrated their high efficacy in silencing lncOCMRL1 expression in vivo and significantly inhibited OSCC tumor growth and metastasis.ConclusionsOur results suggest that lncOCMRL1 is a reliable target for blocking lymph node metastasis in OSCC.

Inhibition of Colon Cancer Xenograft Growth by Celecoxib Analog OSU-03013 Through Wnt/β-Catenin Pathway Modulation

OSU-03013 is a Celecoxib analog that dose not inhibit COX-2 expression. OSU-03013 effectively mimics the anti-tumor effects of Celecoxib, however, its exact mechanism in colon cancer (CC) is unknown. SW480 cells were injected subcutaneously into the axilla of nude mice until the volume of the transplanted tumor reached 80 mm3. SW480 cells were inoculated into the tail vein of nude mice to establish a liver metastasis model of CC. OSU-03013 was administered by gavage. The pathology of the tumor tissues was assessed using HE staining and cell apoptosis was measured using TUNEL staining. EMT markers in the tumors were determined using Western Blot, and IL-1β and TNF-α were determined using ELISA. VEGF-A and VEGFR-2 in tumor tissues were measured to assess angiogenesis using immunofluorescence. HE staining was conducted to detect the effect of OSU-03013 on liver metastasis. Western Blot assay was performed to determine the expression of Wnt2, β-catenin and GSK-3β. OSU-03013 reduced inflammatory cell infiltration and inhibited IL-1β and TNF-α production by modulating the Wnt/β-catenin pathway. OSU-03013 induced apoptosis of cancer cells in tumor tissues and possessed anti-angiogenic activity. OSU-03013 treatment reduced the number and size of metastatic nodules of cancer cells in liver tissues. OSU-03013 may inhibit tumor growth and angiogenesis through the Wnt3a/β-catenin pathway in CC nude mice.

Ficolin-3 induces apoptosis and suppresses malignant property of hepatocellular carcinoma cells via the complement pathway

AimsFicolin 3 (FCN3) has the highest complement-activating capacity through the lectin pathway and is synthesized mainly in the liver and lung. Yet, its potential molecular mechanism in hepatocarcinogenesis is not fully understood. Materials and methodsThe expression of FCN3 in hepatocellular carcinoma (HCC) tumor and non-tumor tissues was analyzed by RT-qPCR, Western blotting and immunofluorescence staining assays. Lentivector-mediated ectopic overexpression was performed to explore the role of FCN3 in vitro and in vivo. Whether FCN3 inhibited HCC cell growth and survival via complement pathway was determined with immunocytochemical staining for C3b, membrane attack complex (MAC) formation and complement killing assay using recombinant FCN3 (rFCN3) in combination with human serum with or without heat inactivation, and with C6 blocking antibody. Key findingsThe transcript and protein of FCN3 were found to be remarkably down-regulated in HCC tumor tissues. FCN3 expression was found to be associated with better survival of HCC patients. Restoration of FCN3 expression significantly inhibited proliferation, migration and anchorage independent growth of HCC cell lines, and xenograft tumor growth. FCN3 expression induced apoptosis of HCC cells. C3 and MAC formation was stimulated by FCN3 overexpression or rFCN3 treatment. rFCN3 enhanced human serum-induced complement activation and cell death. C6 blocking antibody significantly attenuated complement-mediated cell death and restored the growth of FCN3-overexpressing HCC cells. SignificanceFCN3 has a malignant suppressor role in HCC cells. Our study provides new insights into the molecular mechanisms that drive HCC progression and potential therapeutic targets for treating HCC.

MALEXNET-EUNET: MULTI-CLASS LIVER CANCER CLASSIFICATION AND SEGMENTATION USING A HYBRID DEEP LEARNING SYSTEM

Background and objective: One of the most prevalent and significant causes of cancer-related mortality worldwide is considered to be liver cancer. Techniques for automatically segmenting and classifying liver tumors are crucial for supporting medical professionals during the tumor diagnostic process. Classifying liver tumors is challenging due to noise, nonhomogeneity, and the significant appearance diversity observed in tumor tissue. Also, in recent years, most of the research has performed binary classification, but there is still a lack of research on multi-class liver cancer classification. Therefore, we perform a multi-class liver cancer classification and segmentation in this research. Methods: We propose a hybrid deep learning-based multi-class liver cancer classification and segmentation system in this research. The collected CT images are pre-processed in four stages: contrast enhancement, noise filtering, smoothing and sharpening, and liver region segmentation. Next, the binary, texture, histogram, and rotational, scalability, and translational (RST) features are extracted from the pre-processed images. Then, the average correlation (AC) and probability of error (POE) approaches are applied to selecting relevant features and excluding less significant features. After feature selection, a modified AlexNet (MAlexNet) model is used to classify the multi-class classification of liver tumors. Finally, the identified liver tumor regions are segmented using the enhanced U-Net (EUNet) model. Results: Identified liver tumors are accurately classified into three different categories using the proposed classification model: hemangioma (HEM), hepatocellular carcinoma (HCC), and metastatic carcinoma (MET), with an average accuracy of 99.19%. We have collected the multi-class liver cancer CT images from real time patients. The results of the experiments show that proposed hybrid system provides adequate overall accuracy, is less noise-sensitive, and outperforms other state-of-the-art techniques such as SVM, Faster RCNN, Mask RCNN, and SAR-U-Net on a wide range of CT images. Conclusion: Radiologists and doctors can identify liver tumors more accurately using the suggested innovative framework.

HECTD2 as a target for veratric acid in the regulation of ferroptosis in renal cell carcinoma

Function of HECTD2 in renal cell carcinoma malignant progression is undefined. Molecular mechanism behind anti-cancer effects of veratric acid (VA) from traditional Chinese medicine (TCM) is underexplored. The Cancer Genome Atlas was leveraged to study HECTD2 expression in renal cell carcinoma and its relationship with histological grading. Kaplan–Meier survival analysis was performed. HECTD2 expression was detected in cancer cells and tissues via qRT-PCR and immunohistochemistry. GPX4 and SLC7A11 expression in tumor samples with high or low HECTD2 expression was examined by immunohistochemistry, cell viability by CCK8, cell proliferation by colony formation assay, lipid ROS and mitochondrial superoxide levels by flow cytometry, Fe2+ and MDA content by assay kits, and GPX4 and SLC7A11 proteins by western blot. SeeSAR software screened TCM small molecule compounds with highest affinity to HECTD2, confirmed with cellular thermal shift assay. VA IC50 was measured by CCK8. Xenograft model was developed and treated with VA. Tumor size and weight were monitored, with immunohistochemistry to detect HECTD2 expression in tumors and assess ferroptosis-related markers. HECTD2 was overexpressed in tumor tissues and cells, which positively correlated with histological grading. HECTD2 depletion inhibited cell vitality and proliferation, raised intracellular lipid ROS, mitochondrial superoxide, Fe2+, and MDA. HECTD2 was a target with highest VA affinity. In vitro and vivo experiments concurred that VA treatment hindered malignancy of renal cell carcinoma and enhanced its susceptibility to ferroptosis. HECTD2 supports ferroptosis resistance in renal cell carcinoma, but VA, through its targeting of HECTD2, initiates ferroptosis, showcasing its anti-cancer efficacy.

Molecular subtypes and prognostic factors of lung large cell neuroendocrine carcinoma.

Lung large cell neuroendocrine carcinoma (LCNEC) is an aggressive disease with poor prognosis and short-term survival, which lacks effective prognostic indicators. The study aims to investigate the molecular subtypes and prognostic markers of lung LCNEC. Patients diagnosed with lung LCNEC at Sun Yat-sen University Cancer Center (SYSUCC) between November 2007 and January 2021 were screened. Baseline clinical data were collected and routine blood indexes including lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and systemic immune-inflammation index (SII) were calculated. Immunohistochemistry (IHC) of ASCL1, NEUROD1, POU2F3, YAP1 were done to perform molecular subtyping, while CD56, Syn, CgA, CD3, CD8, CD20, CD68, and CD163 were also stained on tissue samples. Then prognostic factors of lung LCNEC were explored. One hundred and fifty-one lung LCNEC patients were identified, 103 of whom had complete clinical information, available routine blood and biochemical indexes were eventually included in the present study. Tumor tissue specimens were available from 64 patients. Positive expression rates of ASCL1, NEUROD1, and YAP1 were 82.8%, 50.0%, and 28.1%, respectively. No POU2F3+ cases were detected. Forty (62.5%) patients co-expressed with two or three markers. High LMR (>3.3) was an independent predictor of favorable prognosis of disease-free survival (DFS) [hazard ratio (HR), 0.391; 95% confidence interval (CI): 0.161-0.948; P=0.04] and overall survival (OS) (HR, 0.201; 95% CI: 0.071-0.574; P=0.003). Notably, high LMR was correlated with higher intra-tumoral CD3+ (P=0.004), CD8+ (P=0.01), and CD68+ (P<0.001) immune cell infiltration compared to low LMR in lung LCNEC. Our study validated molecular subtypes by IHC in lung LCNEC, and co-expression was found among different subtypes, with no prognostic effect. High blood LMR level was associated with a favorable prognosis in lung LCNEC, which might partly reflect a hot tumor tissue immune microenvironment. Our findings may benefit clinical practice, and further studies are warranted.

Dendritic Cell-Targeted Nanoparticles Enhance T Cell Activation and Antitumor Immune Responses by Boosting Antigen Presentation and Blocking PD-L1 Pathways.

Dendritic cells (DCs) within the tumor microenvironment (TME) have an insufficient capacity to activate T cells through antigen presentation. Furthermore, the programmed cell-death ligand 1 (PD-L1), abundantly expressed on tumor-associated DCs, binds the programmed cell-death 1 (PD-1)-positive T cells and suppresses their immune function. The binding of PD-L1 to CD80 (B7.1) on the same DC via cis-interactions further prevents T cell costimulation through CD28. Here, we present a strategy to simultaneously promote antigen cross-presentation and block the inhibitory interactions of PD-L1 on DCs to amplify T cell-mediated antitumor responses within the TME. Mesoporous silica nanoparticles (MSNPs) were loaded with clotrimazole (CLT) to boost MHC II-mediated antigen presentation by DCs, surface-modified with mannose to target CD206 on DCs, and then decorated with PD-L1 binding peptide (PDL1bp) to block PD-L1-mediated interactions. PDL1bp was cleaved from the mannosylated and CLT-loaded MSNPs (MSNP-MaN/CLT) under conditions simulating the TME and tethered to PD-L1 to reverse CD80 sequestration on DC2.4 cells. The blocking of PD-L1 by PDL1bp-decorated NPs (MSNP-MaN-PDL1bp) increased the cellular interactions between DC2.4 and EL4 T cells and the amount of IL-2 secretion. The MSNP-MaN/CLT were taken up rapidly by DC2.4 cells, promoted MHC II presentation of hen egg lysozyme (HEL), and increased IL-2 production from HEL antigen-primed 3A9 T cells, which was further enhanced by PDL1bp. In vivo investigation revealed that administration of the CLT-loaded and PDL1bp-functionalized MSNPs remarkably inhibited subcutaneous B16-F10 melanoma tumor growth when compared with anti-PD-L1 therapy. MSNP-MaN-PDL1bp/CLT treatment upregulated the levels of effector molecules such as granzyme B and proinflammatory cytokines (IFNγ and INFα) in the tumor tissue, indicating antitumoral T cell responses. This strategy of utilizing nanoparticles to trigger DC activation while promoting T cell stimulation can be used to amplify the antitumor T cell responses and represents a promising alternative to anti-PD-L1 immunotherapy.

Antitumor Potential of Guttiferone E Combined With Carboplatin Against Osimertinib-resistant H1975 Lung Cancer Through Apoptosis.

Low selectivity and high frequency of side-effects are the major problems of currently used chemotherapeutics. Among natural compounds, the polyprenylated acylphloroglucinol, guttiferone E, isolated from Brazilian red propolis, has attracted attention due to its marked anticancer properties and was evaluated here for its role against osimertinib-resistant H1975 cells (with double mutations of epidermal growth factor receptor: EGFR L858R/T790M). Guttiferone E was obtained from red propolis using established extraction procedures. Guttiferone E was tested using the H1975 cell line in in vitro (2D and 3D) cell cultures and in vivo in BALB/c athymic nude mice. Live/dead assay was also performed to support the results. Tumor tissues obtained from in vivo studies were used for western blotting. Guttiferone E reduced H1975 cell viability in a concentration-dependent manner. The IC50 values in 2D and 3D cell lines were 2.56±0.12 μM and 11.25±0.34 μM. Furthermore, at 10 mg/kg intraperitoneally, guttiferone E significantly reduced the tumor volume in tumor xenografts when used alone and in combination with carboplatin. Guttiferone E and carboplatin displayed synergistic inhibition of H1975 cells and animal tumors. Co-treatment of guttiferone E with carboplatin induced more prominent apoptosis than treatment with either drug alone. Guttiferone E treatment induced cleavage of poly-ADP ribose polymerase and induced apoptosis by significantly reducing levels of mammalian target of rapamycin, sirtuin 1, sirtuin 7, superoxide dismutase, programmed death-ligand 1, and programmed cell death 1 in tumor tissues. Our results show guttiferone E to be a promising, novel and potent antitumor drug candidate for osimertinib-resistant lung cancer with EGFR L858R/T790M mutations.

Methyl Gallate Suppresses Canine Mammary Gland Tumors by Inducing Apoptosis and Anti-angiogenesis.

Methyl gallate (MG), a plant phenolic compound, has known anticancer properties. However, its effects on canine mammary gland tumors (CMTs) are unclear. This study evaluated the impact of MG on cell viability, migration, and apoptosis in two CMT cell lines. CMT-U27 and CF41.mg cells were used. In vitro experiments included MTT and scratch assays, Annexin-V/propidium iodide double staining, immunocytochemistry, and western blot analyses. An in vivo CMT xenograft mouse model was also used to observe the effects of MG on tumor growth and vasculature. Immunohistochemistry was performed to analyze vessel density and apoptosis in tumor tissues. Cell migration and tube formation assays with canine aortic endothelial cells assessed the anti-angiogenic effects of MG. Data showed a significant decrease in cell viability and migration in both CMT cell lines after 24 h exposure to various MG concentrations. MG treatment induced dose-dependent apoptotic cell death and elevated cleaved caspase-3 expression. In vivo experiments confirmed tumor growth suppression 21 days post-treatment with 40 mg/kg MG. Tumor tissues displayed increased cleaved caspase-3 and reduced vessel density. MG also inhibited cell migration and disrupted tube formation in canine endothelial cells. MG has potential as an anticancer drug for CMTs by promoting apoptotic cell death and reducing angiogenesis, highlighting its therapeutic promise.

Hyaluronan-Based Hydrogels for 3D Modeling of Tumor Tissues.

Although routine two-dimensional (2D) cell culture techniques have advanced basic cancer research owing to their simplicity, cost-effectiveness, and reproducibility, they have limitations that necessitate the development of advanced three-dimensional (3D) tumor models that better recapitulate the tumor microenvironment. Various biomaterials have been used to establish these 3D models, enabling the study of cancer cell behavior within different matrices. Hyaluronic acid (HA), a key component of the extracellular matrix (ECM) in tumor tissues, has been widely studied and employed in the development of multiple cancer models. This review first examines the role of HA in tumors, including its function as an ECM component and regulator of signaling pathways that affect tumor progression. It then explores HA-based models for various cancers, focusing on HA as a central component of the 3D matrix and its mobilization within the matrix for targeted studies of cell behavior and drug testing. The tumor models discussed included those for breast cancer, glioblastoma, fibrosarcoma, gastric cancer, hepatocellular carcinoma, and melanoma. The review concludes with a discussion of future prospects for developing more robust and high-throughput HA-based models to more accurately mimic the tumor microenvironment and improve drug testing. Impact Statement This review underscores the transformative potential of hyaluronic acid (HA)-based hydrogels in developing advanced tumor models. By exploring HA's dual role as a critical extracellular matrix component and a regulator of cancer cell dynamics, we highlight its unique contributions to replicating the tumor microenvironment. The recent advancements in HA-based models provide new opportunities for more accurate studies of cancer cell behavior and drug responses. Looking ahead, these innovations pave the way for high-throughput, biomimetic platforms that could revolutionize drug testing and accelerate the discovery of effective cancer therapies.

Sorafenib plus memory-like natural killer cell immunochemotherapy boosts treatment response in liver cancer

BackgroundHeterogeneity of hepatocellular carcinoma (HCC) presents significant challenges for therapeutic strategies and necessitates combinatorial treatment approaches to counteract suppressive behavior of tumor microenvironment and achieve improved outcomes. Here, we employed cytokines to induce memory-like behavior in natural killer (NK) cells, thereby enhancing their cytotoxicity against HCC. Additionally, we evaluated the potential benefits of combining sorafenib with this newly developed memory-like NK cell (pNK) immunochemotherapy in a preclinical model.MethodsHCC tumors were grown in SD rats using subcapsular implantation. Interleukin 12/18 cytokines were supplemented to NK cells to enhance cytotoxicity through memory activation. Tumors were diagnosed using MRI, and animals were randomly assigned to control, pNK immunotherapy, sorafenib chemotherapy, or combination therapy groups. NK cells were delivered locally via the gastrointestinal tract, while sorafenib was administered systemically. Therapeutic responses were monitored with weekly multi-parametric MRI scans over three weeks. Afterward, tumor tissues were harvested for histopathological analysis. Structural and functional changes in tumors were evaluated by analyzing MRI and histopathology data using ANOVA and pairwise T-test analyses.ResultsThe tumors were allowed to grow for six days post-cell implantation before treatment commenced. At baseline, tumor diameter averaged 5.27 mm without significant difference between groups (p = 0.16). Both sorafenib and combination therapy imposed greater burden on tumor dimensions compared to immunotherapy alone in the first week. By the second week of treatment, combination therapy had markedly expanded its therapeutic efficacy, resulting in the most significant tumor regression observed (6.05 ± 1.99 vs. 13.99 ± 8.01 mm). Histological analysis demonstrated significantly improved cell destruction in the tumor microenvironment associated with combination treatment (63.79%). Interestingly, we observed fewer viable tumor regions in the sorafenib group (38.9%) compared to the immunotherapy group (45.6%). Notably, there was a significantly higher presence of NK cells in the tumor microenvironment with combination therapy (34.79%) compared to other groups (ranging from 2.21 to 26.50%). Although the tumor sizes in the monotherapy groups were similar, histological analysis revealed a stronger response in pNK cell immunotherapy group compared to the sorafenib group.ConclusionsExperimental results indicated that combination therapy significantly enhanced treatment response, resulting in substantial tumor growth reduction in alignment with histological analysis.

Gene Expression Aberrations in Alcohol-Associated Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer, ranking as the sixth most common cancer worldwide and the fourth leading cause of cancer-related deaths. Most HCC cases originate from cirrhotic livers, typically due to chronic liver diseases, such as hepatitis B (HBV) and hepatitis C (HCV) infections, and alcoholism. HCC cells often harbor numerous somatic mutations that are implicated in HCC development, but epigenetic factors, such as miRNA interference, can also affect HCC initiation and progress. miRNA-221 has been explored as a factor affecting HCC development in HCC of viral etiology, but little is known about its effects on gene expression in alcohol-associated HCC. This study aimed to explore potentially similar gene expression aberrations underlying viral and alcohol-induced HCC. We analyzed available transcriptome data from non-tumor hepatocytes and viral-induced HCC tissues. The most notable differences in gene expression associated with miRNA-221 between non-tumor hepatocytes and viral-induced HCC involved NTF-3 and MYBL1 genes. To assess these data in alcohol-induced HCC, we examined 111 tissue samples: tumor tissue and cirrhotic tissue samples from 37 HCC patients and 37 samples from non-tumor liver tissue using RT-Q PCR. We found no significant difference in NTF-3 expression, but MYBL1 expression was significantly lower in HCC tissue compared to non-tumor hepatocytes and cirrhotic tissue. Our findings highlight the importance of the MYBL1 gene in HCC development and emphasize the need for diverse approaches in evaluating tumor mechanisms.

Evaluation of GARP immunohistochemical expression in papillary thyroid carcinoma

BackgroundGlycoprotein A repetitions predominant (GARP) is a novel transmembrane protein highly expressed on the surface of regulatory T cells (Tregs), which are a subset of immunosuppressive T lymphocytes that play a major role in inhibiting the antitumor immune response. Many studies documented increased GARP expression in various tumors, which is related to a poorer prognosis, and only one single paper investigated its expression in thyroid tumors.AimTo evaluate the immunohistochemical expression of GARP in differentiated thyroid carcinomas and their tumor-infiltrating lymphocytes (TILs) in comparison to its expression in other benign and low-risk lesions.MethodsSixty-nine cases of different thyroid lesions were subgrouped into 37 cases of malignant thyroid neoplasms, 25 cases of benign thyroid lesions, and 7 cases of low-risk neoplasms collected from the Pathology Department Laboratories of Ain Shams University Hospitals during the period from January 2017 to December 2021 and stained immunohistochemically for GARP. Immunohistochemical (IHC) results were evaluated in thyroid epithelial cells and TILs. The expression of GARP was correlated with the different clinicopathological parameters.ResultsGARP expression discloses a significant statistical difference between the three studied groups (P < 0.001). High GARP expression was detected in 89.19% of the malignant cases and in 28.57% of low-risk neoplasms, while all benign lesions exhibited low GARP expression. High GARP expression of TILs was detected in 60% of the malignant cases. Synchronous high GARP expression in tumor tissue and in the surrounding TILs was detected in 63.16% of the malignant cases, yet these results did not reach statistical significance.ConclusionGARP is a marker of Tregs, whose high expression is increased in malignant over benign and low-risk lesions. It might be a potential novel target for anticancer immunotherapy.

Codonopsis pilosula polysaccharide suppresses the progression of non-small cell lung cancer by triggering NLRP3/GSDMD-dependent pyroptosis

BackgroundNon-small cell lung cancer (NSCLC) represents a prevalent and challenging malignancy, often posing difficulties in treatment due to late-stage diagnosis and limited therapeutic options.ObjectiveThis study aims to elucidate the impact of Codonopsis pilosula polysaccharide (CPP) on NSCLC, exploring its potential anticancer molecular mechanisms.Materials and methodsPurchase A549 cells and treat them with varying concentrations of CPP, dividing them into groups. Use CCK-8 assays for cell proliferation and TUNEL assays for apoptosis detection. Perform Western blot (WB) to detect NF-κB p65, pNF-κB p65, and pyroptosis-related proteins. Measure ROS, inflammatory factors, and LDH levels using kits. Observe pyroptosis morphology under a microscope. Inoculate A549 cells subcutaneously into nude mice to create a xenograft model and treat them with daily CPP injections. Collect tumor tissues, measure tumor volume and mass. Perform TUNEL immunohistochemistry for apoptosis and H&E staining for tumor histology. Detect pyroptosis-related proteins with Western blot and immunohistochemistry, and assess inflammatory factors using ELISA and immunohistochemistry.ResultsThe research reveals a concentration-dependent inhibitory effect of CPP on A549 cell viability, with optimal efficacy observed at 40 μmol/L CPP. CPP demonstrates the capacity to induce apoptosis, mediate NF-κB activation, and accumulate reactive oxygen species (ROS). Notably, CPP promotes pyroptosis in A549 cells and in vivo, upregulating pyroptosis-related proteins. In vivo administration of CPP significantly inhibits tumor growth, accompanied by notable morphological and histological alterations. Additionally, both in vitro and in vivo studies confirm elevated levels of IL-1β and IL-18 in tumor tissues following CPP treatment.Discussion and conclusionIn conclusion, CPP, as a multifaceted agent, suppresses the progression of NSCLC by inducing NLRP3/GSDMD-dependent pyroptosis. This research provides valuable insights into the therapeutic potential of CPP and stimulates further exploration for its integration into future NSCLC treatments.

Pentacyclic Triterpenoid Content in Cranberry Raw Materials and Products

Cranberry fruit extracts have been shown to inhibit expression of pro-inflammatory cytokines in THP-1 cells and reduce colonic tumor burden and tissue inflammation in a mouse model of colitis. These activities are attributed to both the triterpenoid and polyphenol constituents of the fruit. The pentacyclic triterpenoids ursolic acid (UA), oleanolic acid (OA), corosolic acid (CA), maslinic acid (MA), and esters of UA and OA occur in the waxy layer of cranberry peel, and their content in cranberry products is likely to vary with the fruit source and processing methods. UPLC-MS (ultra performance liquid chromatography-mass spectrometry) was applied to determine the four triterpenoid acids and their esters in cranberry products and raw materials. Cranberry pomace, a side stream in juice production, was a rich source at 64,090 µg total triterpenoids/g DW. Cranberry juice beverages ranged from 0.018 to 0.26 µg/g of product, fruit samples ranged from 6542 to 17,070 µg/g DW, and whole berry products contained up to 2665 µg/g DW. Free UA was the most plentiful triterpenoid in all samples. These analyses illustrate the potential value of an underutilized side stream in cranberry juice production and highlight potential benefits of whole fruit products.

Technical Development and In Silico Implementation of SyntheticMR in Head and Neck Adaptive Radiation Therapy: A Prospective R-IDEAL Stage 0/1 Technology Development Report.

The purpose of this study was to investigate the technical feasibility of integrating the quantitative maps available from SyntheticMR into the head and neck adaptive radiation oncology workflow. While SyntheticMR has been investigated for diagnostic applications, no studies have investigated its feasibility and potential for MR-Simulation or MR-Linac workflow. Demonstrating the feasibility of using this technique will facilitate rapid quantitative biomarker extraction which can be leveraged to guide adaptive radiation therapy decision making. Two phantoms, two healthy volunteers, and one patient were scanned using SyntheticMR on the MR-Simulation and MR-Linac devices with scan times between four to six minutes. Images in phantoms and volunteers were conducted in a test/retest protocol. The correlation between measured and reference quantitative T1, T2, and PD values were determined across clinical ranges in the phantom. Distortion was also studied. Contours of head and neck organs-at-risk (OAR) were drawn and applied to extract T1, T2, and PD. These values were plotted against each other, clusters were computed, and their separability significance was determined to evaluate SyntheticMR for differentiating tumor and normal tissue. The Lin's Concordance Correlation Coefficient between the measured and phantom reference values was above 0.98 for both the MR-Sim and MR-Linac. No significant levels of distortion were measured. The mean bias between the measured and phantom reference values across repeated scans was below 4% for T1, 7% for T2, and 4% for PD for both the MR-Sim and MR-Linac. For T1 vs. T2 and T1 vs. PD, the GTV contour exhibited perfect purity against neighboring OARs while being 0.7 for T2 vs. PD. All cluster significance levels between the GTV and the nearest OAR, the tongue, using the SigClust method was p < 0.001. The technical feasibility of SyntheticMR was confirmed. Application of this technique to the head and neck adaptive radiation therapy workflow can enrich the current quantitative biomarker landscape.

GDF15 and LCN2 for early detection and prognosis of pancreatic cancer

BackgroundThe prognosis of pancreatic ductal adenocarcinomas (PDAC) remains very poor, emphasizing the critical importance of early detection, where biomarkers offer unique potential. Although growth differentiation factor 15 (GDF15) and Lipocalin 2 (LCN2) have been linked to PDAC, their precise roles as biomarkers are uncertain. MethodsCirculating levels of GDF15 and LCN2 were examined in human PDAC patients, heathy controls, and individuals with benign pancreatic diseases. Circulating levels of IL-6, CA19-9, and neutrophil-to-lymphocyte ratio (NLR) were measured for comparisons. Correlations between PDAC progression and overall survival were assessed. A mouse PDAC model was employed for comprehensive analyses, complementing the human studies by exploring associations with various metabolic and inflammatory parameters. Sensitivity and specificity of the biomarkers were evaluated. FindingsOur results demonstrated elevated levels of circulating GDF15 and LCN2 in PDAC patients compared to both healthy controls and individuals with benign pancreatic diseases, with higher GDF15 levels associated with disease progression and increased mortality. In PDAC mice, circulating GDF15 and LCN2 progressively increased, correlating with tumor growth, behavioral manifestations, tissue and molecular pathology, and cachexia development. GDF15 exhibited highly sensitive and specific for PDAC patients compared to CA19-9, IL-6, or NLR, while LCN2 showed even greater sensitivity and specificity in PDAC mice. Combining GDF15 and LCN2, or GDF15 and CA19-9, enhanced sensitivity and specificity. InterpretationOur findings indicate that GDF15 holds promise as a biomarker for early detection and prognosis of PDAC, while LCN2 could strengthen diagnostic panels.

DNA methylation analysis of the SDC2, SEPT9 and VIM genes in fecal DNA for colorectal cancer diagnosis.

Colorectal cancer is one of the most common cancers worldwide. DNA methylation sites may serve as a new gene signature for colorectal cancer diagnosis. The search for representative DNA methylation sites is urgently needed. This study aimed to systematically identify a methylation gene panel for colorectal cancer diagnosis via tissue and fecal samples. A total of 181 fecal and 50 tumor tissue samples were collected. They were obtained from 83 colorectal cancer patients and 98 healthy subjects. These samples were evaluated for DNA methylation of 9 target genes via quantitative bisulfite next-generation sequencing. We employed the rank-sum test to screen the colorectal cancer-specific methylation sites in the tissue and fecal cohorts. A data model was subsequently constructed and validated via the dedicated validation dataset. Compared with the fecal and negative control samples, the colorectal cancer tissue samples presented significantly higher methylation rates for all the selected gene sites. The methylation rates of the tissue and preoperative fecal samples showed the same high and low rates at the same sites. After screening, a panel of 29 loci in the SDC2, SEPT9, and VIM genes proved to be reliable biomarkers for colorectal cancer diagnosis in fecal samples. Logistic regression models were then constructed and validated using this panel. The sensitivity of the model was 91.43% (95% CI = [89.69, 93.17]), the specificity was 100% (95% CI = [100,100]), and the AUC value is 99.31% (95% CI = [99,99.62]). The diagnostic accuracy of the model for stage I and stage II colorectal cancer was 100% (11/11) and 91.3% (21/23), respectively. Overall, this study confirms that the gene locus panel and the model can be used to diagnose colorectal cancer effectively through feces. Our study identified a set of key methylation sites for colorectal cancer diagnosis from fecal samples, highlighting the importance of using tissue and fecal samples to accurately assess DNA methylation levels to screen for methylation sites, and developing an effective diagnostic model for colorectal cancer.

The mechanism of L1 cell adhesion molecule interacting with protein tyrosine kinase 2 to regulate the focal adhesion kinase–growth factor receptor-bound protein 2–son of sevenless–rat sarcoma pathway in the identification and treatment of type I high-risk endometrial cancer

Objective: The objective of this study was to investigate how L1 cell adhesion molecule (L1CAM) interacting with protein tyrosine kinase 2 (PTK2) affects endometrial cancer (EC) progression and determine its association with the focal adhesion kinase (FAK)–growth factor receptor-bound protein 2 (GRB2)–son of sevenless (SOS)–rat sarcoma (RAS) pathway. EC is a female cancer of major concern in the world, and its incidence has increased rapidly in recent years. L1CAM is considered a reliable marker of poor prognosis in patients with EC. Material and Methods: A single-center and prospective study was conducted using data from the Cancer Genome Atlas and samples from normal and EC tissues to explore the differential expression of L1CAM. Additional experimental models included human immortalized endometrial epithelium cells (hEECs) and EC cell lines such as KLE, RL95-2, and Ishikawa. L1CAM expression was regulated using lentiviruses designed for either overexpression or interference, and PTK2/focal adhesion kinase (FAK) signaling was inhibited with PF431396. Transfected KLE cells were injected into mice, and tumor growth was monitored over 14 days. Cellular proliferation and survival were assessed using cell counting kit, colony formation, and terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate (dUTP) nick-end labeling assays. Metastatic behavior was evaluated through Transwell assays for cell migration and invasion. The expression levels of matrix metallopeptidase (MMP) 2 and MMP9 were determined by Western blot. In addition, the activation of the FAK–GRB2–SOS–RAS pathway was examined by assessing the protein levels of FAK, GRB2, SOS, and RAS. Results: There was a significant difference in L1CAM expression between EC tumor tissues and normal tissues, and L1CAM messenger RNA (1.85-fold) and L1CAM protein (2.59-fold) were significantly more expressed in EC tissues (P &lt; 0.01) than in normal tissues. The tumor growth of L1CAM overexpressing EC cells was faster than that of negative control EC cells (6.43 fold; P &lt; 0.001). L1CAM promoted the expression of FAK (1.43-2.72-fold; P &lt; 0.001); enhanced EC cell proliferation (P &lt; 0.01), survival and motility (P &lt; 0.001), migration (P &lt; 0.001), and invasion (P &lt; 0.001); and activated the FAK–GRB2–SOS–RAS pathway, all of which were reversed when FAK expression was not upregulated (P &lt; 0.001). Conclusion: By upregulating PTK2 and its encoded protein FAK, L1CAM was found to promote tumor progression and increase the activation of the FAK–GRB2–SOS–RAS pathway. These findings establish L1CAM and PTK2 as reference genes for poor prognostic prediction in EC and as targets for EC therapy, providing a valuable basis for distinguishing between benign and malignant endometrial conditions and justifying the necessity of targeted therapeutic approaches.