Adenocarcinoma Cell Research Articles

Transcriptional upregulation of MMP-9 gene under hyperglycemic conditions in AGS cells: Role of AP-1 transcription factor

Gastric cancer and diabetes are two complex and interrelated diseases having significant impact on global health. Hyperglycemic condition notably exacerbates cancer by promoting inflammation, angiogenesis, and metastasis. Elevated glucose levels can also upregulate the expression of specific matrix metalloproteinases (MMPs), especially MMP-9, which is associated with cancer cell migration and invasion. However, the molecular mechanism behind such upregulation remains unexplored. In the present study, we have identified the mechanism for hyperglycemia-induced transcriptional activation of MMP-9, in gastric adenocarcinoma (AGS) cells. Using various tools like luciferase-reporter assays with promoter deletion constructs, siRNAs, pharmacological inhibitors, and nuclear translocation experiments, we have identified that the transcriptional activation of MMP-9 under hyperglycemic conditions is predominantly governed by the MAPK pathway, via formation of the AP-1 heterodimer. The p65 NF-κB signaling pathway, although activated, plays no significant role in regulating hyperglycemia-induced MMP-9 expression. Chromatin immunoprecipitation studies indicate that the distal AP-1 binding site is responsible for hyperglycemia-induced MMP-9 transcription; whereas the proximal one accounts for both hyperglycemia-induced and basal MMP-9 transcription. Therefore, binding of AP-1 at both the proximal and distal binding sites on the MMP-9 promoter region is required for hyperglycemia-induced MMP-9 expression. Overall, our study unveils a novel mechanism of MMP-9 transcription under hyperglycemic conditions and also suggests that inhibiting the binding of the AP-1 heterodimer with its distal binding site can potentially reduce the complications developed during gastric cancer-hyperglycemia co-morbidity. A drug designed specifically to inhibit this interaction may prevent hyperglycemia-induced tumor aggressiveness to a considerable extent by impeding MMP-9 transcription.

Synthesis of Chalcones, Screening In silico and In vitro and Evaluation of Helicobacter pylori Adhesion by Molecular Docking.

We synthetized 10 hydroxylated and methoxylated chalcones and evaluated them targeting MMP-9 inhibition, looking for the rate of adhesion of H. pylori in gastric cells, and then, reduction of the inflammatory response as alternative therapeutic agents for controlling the infection. Helicobacter pylori is a Gram-negative bacterium that chronically infects the human stomach, a risk factor for the development of inflammatory gastrointestinal diseases, including cancer, and is classified as a group I carcinogen. It is estimated that it infects around 45% of the global population and that the persistence of the infection is related to the adhesion of the bacteria in the gastric epithelium. The progression of gastric lesions to cancer is connected to the activation of the NF-κB and MAPK pathways, especially in cagA+ strains, which are related to increased expression of MMP-9. The activation of these metalloproteinases (MMP's) contributes to the adhesion of the bacterium in gastric cells and the evolving stages of cancer, such as enabling metastasis. Due to the increasing resistance to the current therapy protocols, the search for alternative targets and candidate molecules is necessary. In this way, controlling adhesion seems to be a suitable option since it is a crucial step in the installation of the bacterium in the gastric environment. Synthetize ten hydroxylated and methoxylated chalcones. Assess their anti-H. pylori potential, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). Evaluate their cytotoxicity in AGS cells and selectivity with L929 cells. Analyze the results and correlate them with in silico predictions to evaluate potential anti-adhesive properties for the chalcones against H. pylori. The chalcones were synthetized by Claisen-Schmidt condensation using Ba(OH)2 or LiOH as catalysts. Predictive in silico assays in PASS Online, tanimoto similarity, ADME properties and molecular docking in MMP-9 (PDB code: 6ESM) were performed. The in vitro assays carried out were the cell viability in gastric adenocarcinoma cells (AGS) and fibroblasts (L-929) by the MMT method and anti-H. pylori, by the broth microdilution method, through the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Ten chalcones were synthesized through Claisen-Schimdt condensation with yields of 10 to 52% and characterized by 1H and 13C Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). In silico data revealed the possibility of anti-H. pylori, anti-inflammatory, and MMP-9 inhibitors for the chalcones. Chalcone 9 showed the best growth inhibition values for MIC and MBC, at 1 μg/mL and 2 μg/mL, respectively. Chalcones 14 and 15 likewise demonstrated excellent inhibitory results, being 2 μg/mL for both MIC and MBC. Additionally, 15 had the best MMP-9 inhibition score. Despite not corroborating the in silico findings, chalcones 10, 13, and 18 showed good cytotoxicity and the best selectivity indices. All compounds exhibited strong activity against H. pylori, specially 15. The predicted MMP-9 inhibition by molecular docking added to the reasonable SI and CI50 values for 15 and the satisfactory reduction in the rate of survival of the bacteria, reveals that it may be acting synergically to reduce the inflammatory response and the possibilities for developing a tumor by inhibiting both bacteria and malignant cells.

Plasma DNA methylation detection for early screening, diagnosis, and monitoring of esophageal adenocarcinoma and squamous cell carcinoma

BACKGROUND The early diagnosis rate of esophageal cancer (EC), one of the most prevalent digestive tract cancers worldwide, remains low. AIM To investigate the utility of plasma SHOX2 , SEPTIN9 , EPO , and RNF180 methylation in the clinical diagnosis and monitoring of EC. METHODS Plasma samples were collected from 210 patients at Hubei Cancer Hospital, and TaqMan polymerase chain reaction was employed to detect plasma SHOX2 , SEPTIN9 , RNF180 , and EPO methylation. The area under the curve was used to estimate their diagnostic value for EC. Cox and logistic regression analyses were used to estimate the independent screening risk factors for patients with EC. RESULTS The sensitivity and specificity of combined assessment of plasma SHOX2 , SEPTIN9 , RNF180 , and EPO methylation for adenocarcinoma, squamous cell carcinoma (SCC), and EC detection were 66.67% and 86.27%, 77.40% and 85.29%, and 76.19% and 86.27%, respectively; the area under the curve values for diagnosing adenocarcinoma, SCC, and EC were 0.737 [95% confidence interval (CI): 0.584–0.89], 0.824 (95%CI: 0.775–0.891), and 0.864 (95%CI: 0.809–0.92), respectively. CONCLUSION According to our findings, plasma SHOX2 , SEPTIN9 , RNF180 , and EPO methylation exhibits appreciated sensitivity for diagnosing EC. The precise measurement of plasma SHOX2 , SEPTIN9 , RNF180 , and EPO methylation can improve EC diagnosis and therapy efficacy monitoring.

Development, characterization, and assessment of PLAROsomal vesicular system of curcumin for enhanced stability and therapeutic efficacy

Abstract Background Curcumin (CUR) is a natural polyphenol and one of the key phytoconstituents found in the rhizomes of Curcuma Longa. It exhibits various pharmacological properties, encompassing antioxidant, anticancer effects, antiseptic, and anti-inflammatory, among several others. A significant drawback of using CUR is its limited bioavailability, which primarily depends on gut microorganisms responsible for converting it into its bioavailable form. Therefore, the contemporary study intended to formulate a novel PLAROsomal vesicular delivery of CUR, i.e., CUR-PLAROsomes employing a design of experiments approach to examine the influence of various process parameters, such as particle size and drug percentage release. Result The prepared CUR-PLAROsomes were characterized for their physicochemical properties using various hyphenated tools. The CUR-PLAROsomes exhibited sizes ranging from 40 to 300 nm, and the optimized batch demonstrated a drug entrapment of 86.38 ± 0.22%. In-vitro anticancer studies were conducted using human colorectal adenocarcinoma cells (COLO320DM) and human breast adenocarcinoma (MCF-7). CUR-PLAROsomes exhibited significant in-vivo anti-inflammatory potential against carrageenan-induced paw edema. CUR-PLAROsomes were more potent against COLO320DM and MCF-7 cell lines, even at lower concentrations, than pure CUR. Conclusion Furthermore, based on the observations, it exhibits potential as an anti-inflammatory agent, suggesting that PLAROsomes are an effective vesicular drug delivery system. Highlights Newly introduced PLARosome is a next generation of Liposomes which have gain popularity owing to its better adaptability to overcome leakage problem of vesicular drug delivery system. This is the pioneer attempt to prepare Curcumin-loaded PLARosome as an anti-cancer and anti-inflammatory activity. Nano size of the PLAROsomes may contribute to enhance the efficacy of Curcumin as a target specific drug delivery system. Site specific delivery of phytoconstituents is possible by use of PLAROsomes as a novel drug delivery system. Graphical abstract

Abstract C026: TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis

Abstract Epithelial-to-mesenchymal transition (EMT) plays important roles during development, injury repair, cancer invasion and metastasis. The heterogeneity of EMT programs is manifest in the diverse EMT-like phenotypes occurring during tumor progression. However, little is known about the mechanistic basis and functional role of specific forms of EMT in cancer. Here we address this question in lung adenocarcinoma (LUAD) cells that enter a dormancy period in response to TGF-β upon disseminating to distant sites. Through in vivo selection, we established dormant metastasis models from human and mouse early-stage LUAD cell lines. LUAD cells with the capacity to enter dormancy are characterized by expression of SOX2 and NKX2-1 primitive progenitor markers, whereas cells derived from overt metastases express high level of the late-stage progenitor marker Sox9. Using a TGF-β signaling reporter, we showed that in the primitive progenitors, TGF-β induces growth inhibition accompanied by a classic EMT response that subsequently transitions into an atypical mesenchymal state of round morphology as the disseminated cells establish dormancy. Prolonged treatment with TGF-β in vitro recapitulates this transition in these primitive progenitors, but not in the late-stage progenitors. Given this dramatic morphological change, we focused on actin-cytoskeleton regulators and found that TGF-β induces this transition by driving the expression of the actin-depolymerizing factor gelsolin, which changes a migratory, stress fiber-rich mesenchymal phenotype into a cortical actin-rich, spheroidal state. This transition lowers the biomechanical stiffness of metastatic progenitors, protecting them from killing by mechanosensitive cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Inhibiting this actin depolymerization process clears tissues of dormant metastatic cells. Thus, LUAD primitive progenitors undergo an atypical EMT as part of a strategy to evade immune-mediated elimination during dormancy. Our results provide a mechanistic basis and functional role of this atypical EMT response of LUAD metastatic progenitors and further illuminate the role of TGF-β as a crucial driver of immune evasive metastatic dormancy. Citation Format: Zhenghan Wang, Yassmin Elbanna, Inês Godet, Lila Peters, George Lampe, Yanyan Chen, Joao Xavier, Morgan Huse, Joan Massagué. TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C026.

Abstract B039: Exploring the impact of neoantigen expression on lung tumor development

Abstract Lung adenocarcinoma (LUAD) stands as a formidable challenge in oncology, despite the emergence of immunotherapy as a promising treatment avenue. The dynamic interplay between the immune system and cancer plays a pivotal role in shaping cancer development and progression. Central to this process are neoantigens, tumor-specific antigens recognized by cytotoxic T cells, which trigger the elimination of tumor cells. It has been implicated that the presence of T cells also decreases tumor grade. However, the precise impact of neoantigen expression on LUAD development and progression remains poorly understood. Our lab previously developed a genetically engineered mouse model called the iNversion-INduced Joined neoAntigen (NINJA) system with the ability to induce expression of a fluorescent neoantigen in oncogenic KRAS, P53-null, tdTomato-expressing lung adenocarcinoma cells (KPT-NINJA). Using this system, we track tumor initiation, progression, and advanced LUAD development across 20 weeks in both immune-deficient and -sufficient settings. Further, tumors initiated in this system give rise to heterogeneous (neoantigen+ and -) tumors, enabling the study of T cell-driven tumor selection. Through multiplexed immunofluorescence, we identified that neoantigen+ tumors experience increased immune infiltration (CD3+, CD4+, CD8+, B220+) and reduced volume, ultimately leading to prolonged animal survival. Surprisingly, advanced lung tumors maintain neoantigen+ cells, indicating that endogenous lung T cells are ineffective at clearing neoantigen-expressing tumors. Yet, we detected that infiltrated tumors exhibit reduced p-ERK expression, suggesting that T cells may slow tumor progression through a non-cytotoxic mechanism. Transferring stimulated neoantigen-specific T cells to tumor-bearing mice largely eliminated neoantigen+ tumor cells. All these data together suggest complex mechanisms for the role of endogenous T cells in controlling lung tumors. Through molecular characterization of tumor progression and T cell-mediated tumor suppression, this study reveals specific pathways that LUAD cells undergo during the process of development and progression, providing valuable insights into the immune response against neoantigen-expressing tumors. The characterization of early neoantigen-expressing tumor cells is pivotal in validating its utility for studying cancer immunotherapy and identifying key molecular changes during tumor development. Ultimately, by unraveling the intricate interplay between neoantigen expression, immune surveillance, and tumor progression, this project aims to contribute significantly to our understanding of how neoantigens shape the dynamics of tumor biology. Citation Format: Jennifer L. Loza, Ishan Bansal, Brian Hunt, Kelli Connolly, Advait Jeevanandam, Srividhya Venkatesan, Nikhil Joshi. Exploring the impact of neoantigen expression on lung tumor development [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B039.

Abstract B013: Hallmarks of progression in early neoantigen-expressing lung adenocarcinoma

Abstract Lung adenocarcinoma (LUAD) is the leading cause of cancer-related mortality, largely due to its high prevalence rate and limited therapeutic options. Immunotherapy remains one of the most promising therapeutic avenues, yet further research is necessary to enhance therapy effectiveness for a broader range of patients. Central to the mechanism of immunotherapy is the detection of tumor neoantigens by cytotoxic T cells, which trigger the elimination of tumor cells. However, it is largely unknown how the early interaction between T cells and neoantigen-expressing tumor cells affects the acquisition of cancer hallmarks and tumor progression. To study this, our lab has developed the iNversion-INduced Joined neoAntigen (NINJA) genetically engineered mouse model in which we can initiate expression of a neoantigen in Kras-mutant, Trp53-null, tdTomato-expressing lung adenocarcinoma cells (KPT-NINJA). This system enables us to analyze tumor initiation, progression, and advanced LUAD development over 20 weeks in both neoantigen-expressing and non-expressing tumors. We defined cancer hallmark acquisition and tumor progression in our model by using a combination of multiplex immunofluorescence and computational algorithms to quantify tumor size, immune infiltration, metastasis, and expression of cancer molecular markers p-ERK and Ki67. We isolated tumor cell and immune cell subsets using neural networks to segment cells and tumor structures and used the colocalization of molecular markers to confirm the identity of various cell types. Our initial data validated that within immunogenic tumors there is an increase of T cell infiltration in tumors and have found immunogenic tumors are associated with overall smaller tumors at early time points. Interestingly, these tumors are associated with a decrease in p-ERK expression, suggesting immune infiltration is negatively correlated with p-ERK expression and tumor size. Together, these data indicate that increased immunogenic pressure may be disrupting early tumor proliferation and hallmark progression, shaping the trajectory of development at a very early stage. We are also correlating other molecular markers of tumor progression with immune infiltration, such as Ki67 expression, which we will colocalize with the other findings to construct a model of how tumors acquire hallmark capabilities from an immunogenic perspective. By isolating the mechanism of early tumor progression in immunogenic contexts, we can begin to predict disease trajectory. This will contribute to a better understanding of the mechanisms behind immune evasion and may uncover crucial insights to broaden immune therapy efficacy. Citation Format: Ishan Bansal, Jennifer Loza, Brian G. Hunt, Kelli Connolly, Srividhya Venkatesan, Nikhil Joshi. Hallmarks of progression in early neoantigen-expressing lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B013.

An allosteric inhibitor of RhoGAP class-IX myosins suppresses the metastatic features of cancer cells.

Aberrant Ras homologous (Rho) GTPase signalling is a major driver of cancer metastasis, and GTPase-activating proteins (GAPs), the negative regulators of RhoGTPases, are considered promising targets for suppressing metastasis, yet drug discovery efforts have remained elusive. Here, we report the identification and characterization of adhibin, a synthetic allosteric inhibitor of RhoGAP class-IX myosins that abrogates ATPase and motor function, suppressing RhoGTPase-mediated modes of cancer cell metastasis. In human and murine adenocarcinoma and melanoma cell models, including three-dimensional spheroid cultures, we reveal anti-migratory and anti-adhesive properties of adhibin that originate from local disturbances in RhoA/ROCK-regulated signalling, affecting actin-dynamics and actomyosin-based cell-contractility. Adhibin blocks membrane protrusion formation, disturbs remodelling of cell-matrix adhesions, affects contractile ring formation, and disrupts epithelial junction stability; processes severely impairing single/collective cell migration and cytokinesis. Combined with the non-toxic, non-pathological signatures of adhibin validated in organoids, mouse and Drosophila models, this mechanism of action provides the basis for developing anti-metastatic cancer therapies.

Surgical outcome in patients with lung adenocarcinoma with mucin.

Mucin-producing adenocarcinoma is a less common variant of lung adenocarcinoma. Adenocarcinoma cells with mucin can spread through the airspace via mucus-mediated extension, leading to their implantation in distant normal lungs. Consequently, post-operative intrapulmonary recurrence frequently occurs. Mucin-producing adenocarcinomas include not only invasive mucinous adenocarcinoma but also papillary, acinar, and other subtypes. Despite increasing reports on surgical outcomes for invasive mucinous adenocarcinomas, the outcomes for total mucin-producing adenocarcinoma remain unclear. We clinically and pathologically evaluated 511 patients who underwent curative resection for lung adenocarcinoma at our institution. The patients were divided into adenocarcinoma with mucin and without mucin groups. Based on pathological findings, the adenocarcinoma with mucin was further classified into intracellular, extracellular, and mucus extension types. Additionally, the CT value of the tumor mass was analyzed using SYNAPSE VINCENT software. The 5year overall survival after surgery was 81.5% and 75.9% for patients with adenocarcinoma with versus without mucin (P = 0.774), respectively. The 5year intrapulmonary recurrence rate in patients with adenocarcinoma with mucin was 29.2%, significantly higher than 12.9% for patients without mucin. Mucus extension was a strong indicator (hazard ratio: 3.03) of intrapulmonary recurrence after surgery. According to SYNAPSE VINCENT analysis, a high volume rate of -400 HU indicated mucus extension. Our results demonstrated that intrapulmonary recurrence occurs approximately 2.3 times more often in adenocarcinoma with mucin than in adenocarcinoma without mucin. Mucus extension is a significant risk factor for intrapulmonary recurrence, and surgeons can predict it using SYNAPSE VINCENT analysis.

Antitumoral action of carvedilol–a repositioning study of the drug incorporated into mesoporous silica MCM-41

We have studied repositioning of carvedilol (an antihypertensive drug) incorporated into MCM-41 mesoporous silica. The repositioning proposes a reduction in the slow pace of discovery of new drugs, as well as toxicological safety and a significant reduction in high research costs, making it an attractive strategy for researchers and large pharmaceutical companies. We obtained MCM-41 bytemplatesynthesis and functionalized it by post-synthesis grafting with aminopropyltriethoxysilane (APTES) only or with folic acid (FA), which gave MCM-41-APTES and MCM-41-APTES-FA, respectively. We characterized the materials by scanning and transmission electron microscopy, zeta potential (ZP) measurements, Fourier transform infrared absorption spectroscopy, x-ray diffractometry, nitrogen gas adsorption, and CHNS elemental analysis. We quantified the percentage of drug that was incorporated into the MCM-41 materials by thermogravimetric analysis and evaluated their cytotoxic activity in non-tumor human lung fibroblasts and the tumor human melanoma and human cervical adenocarcinoma cell lines by XTT salt reduction (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-arboxanilide). The x-ray diffractograms of the MCM-41 materials displayed low-angle peaks in the 2θrange between 2° and 3°, and the materials presented type IV nitrogen adsorption isotherms and H2 hysteresis typical of the MCM-41hexagonal network. The infrared spectra, the charge changes revealed by ZP measurements, and the CHN ratios obtained from elemental analysis showed that MCM-41 was amino-functionalized, and that carvedilol was incorporated into it. MCM-41-APTES incorporated 23.80% carvedilol, whereas MCM-41 and MCM-41-APTES-FA incorporated 18.69% and 12.71% carvedilol, respectively. Incorporated carvedilol was less cytotoxic to tumor and non-tumor cells than the pure drug. Carvedilol repositioning proved favorable and encourages further studies aimed at reducing its cytotoxicity to non-tumor cells. Such studies may allow for larger carvedilol incorporation into drug carriers or motivate the search for a new drug nanocarrier to optimize the carvedilol antitumoral activity.

Experimental study of early evaluation of radiosensitivity in mouse models of lung cancers using 89Zr-anti-γH2AX-TAT PET imaging.

Early evaluation of radiation sensitivity in lung cancer patients can facilitate the transition to personalized treatment strategies. To this end, we assessed the capability of 89Zr-anti-γH2AX-TAT microPET imaging in determining the radiosensitivity of lung cancer xenograft models. We prepared and conducted quality control on 89Zr-anti-γH2AX-TAT. The radiosensitivity of human non-small cell lung cancer cells (H460) and adenocarcinoma cells (A549) was analyzed through clonogenic survival experiments. Additionally, the role of γH2AX as a biomarker for radiosensitivity was validated by quantifying γH2AX foci via fluorescence staining. Subsequently, the H460 and A549 xenograft mouse models were subjected to irradiation, followed by 89Zr-anti-γH2AX-TAT microPET imaging. Concurrently, we performed immunofluorescence staining for γH2AX in tumor tissues to establish a correlation between the uptake of 89Zr-anti-γH2AX-TAT and γH2AX expression. The surviving fraction 2Gy (SF2) values of H460 and A549 indicating that A549 adenocarcinoma has higher radiosensitivity. The cell immunofluorescence experiment showed that the repair of γH2AX foci in H460 cells after irradiation was significantly higher than that in A549 cells, which also confirmed that A549 has higher radiosensitivity. The microPET imaging results showed the uptake of 89Zr-anti-γH2AX-TAT in the tumor of the A549 models after radiotherapy was higher than H460 models. The immunofluorescence staining of tumor tissue confirmed that the expression level of γH2AX was higher and the correlation with microPET imaging uptake was good. 89Zr-anti-γH2AX-TAT allows PET imaging of radiosensitivity in lung cancer xenograft models, and is expected to become an early evaluation method for lung cancer radiosensitivity.

Analysis of Characteristics of Bovine-Derived Non-Enterotoxigenic Bacteroides fragilis and Validation of Potential Probiotic Effects

Bacteroides fragilis is a new generation of probiotics, and its probiotic effects on humans and some animals have been verified. However, research on B. fragilis in cattle is still lacking. In this study, 24 stool samples were collected from two large-scale cattle farms in Wuzhong, Ningxia, including 12 diarrheal and 12 normal stools. A non-toxigenic Bacteroides fragilis (NTBF) was isolated and identified by 16S rRNA high-throughput sequencing and named BF-1153; genome composition and genome functional analyses were carried out to reflect the biological characteristics of the BF-1153 strain. A cluster analysis of BF-1153 was performed using Mega X to explore its genetic relationship. In addition, Cell Counting Kit-8 (CCK8) was used to determine the toxic effects of the strain on human ileocecal colorectal adenocarcinoma cell line cells (HCT-8), Madin-Darby bovine kidney cells (MDBK), and intestinal porcine epithelial cells (IPECs). The results showed that BF-1153 conformed to the biological characteristics of B. fragilis. BF-1153 had no toxic effects on HCT-8, MDBK, and IPEC. Animal experiments have shown that BF-1153 has no toxic effects on healthy SPF Kunming mice. Notably, the supernatant of BF-1153 enhanced cell activity and promoted cell growth in all three cell lines. At the same time, a cluster analysis of the isolated strains showed that the BF-1153 strain belonged to the same branch as the B. fragilis strain 23212, and B. fragilis strain 22998. The results of the animal experiments showed that BF-1153 had a certain preventive effect on diarrhea symptoms in SPF Kunming mice caused by a bovine rotavirus (BRV). In summary, the strain BF-1153 isolated in this experiment is NTBF, which has no toxic effect on MDBK, HCT-8, and IPEC, and has obvious cell growth-promoting effects, especially on MDBK. BF-1153 promotes the growth and development of SPF Kunming mice when compared with the control group. At the same time, BF-1153 alleviated the diarrhea symptoms caused by BRV in SPF Kunming mice. Therefore, BF-1153 has the potential to be a probiotic for cattle.

Abstract B011: RNA-based therapeutics to target the p53 pathway in cancer

Abstract Mutations in the TP53 gene and activation of the PI3K/AKT pathway are the two most frequent genetic alterations across multiple human tumors. We discovered a crucial mechanism of crosstalk between these two events consisting of two oncogenic lncRNAs, TROLL-2 and TROLL-3, whose expression is induced by mutant p53. We demonstrated that these lncRNAs drive tumor and metastasis formation in several orthotopic models, including breast cancer, lung adenocarcinoma, and melanoma. The investigation of these mouse models and a pan-cancer analysis of human cancers showed that these lncRNAs are markers of tumor progression and lead to the phosphorylation and subsequent activation of AKT by promoting the cytoplasmic localization of the scaffold protein WDR26. Notably, we have now proven that TROLL-2 and TROLL-3 are also involved in the response to cancer therapies. Indeed, the downregulation of these lncRNAs via siRNAs bypasses the AKT-driven resistance to chemotherapeutics (e.g. Adriamycin, docetaxel, and paclitaxel) in triple-negative breast cancer cells, to inhibitors of mutant KRAS (e.g. sotorasib) and EGFR (e.g. erlotinib) in lung adenocarcinoma cells, and to inhibitors of PARP (e.g. niraparib) in ovarian cancer cells. To target these lncRNAs in a clinically relevant manner, we designed LNA GapmeRs, also known as antisense oligonucleotides (ASOs), to downregulate these two lncRNAs directly. The efficacy of the ASOs was confirmed in ex vivo specimens of tumors and metastases of lung adenocarcinoma and ovarian cancer patients. These clinical specimens were also analyzed via single-cell RNA sequencing and phospho-proteomics to unveil the common vs. unique downstream effects of targeting each lncRNA. When administered together, the ASOs targeting TROLL-2 and TROLL-3 effectively decreased the levels of these lncRNAs, which in turn caused WDR26 sequestration in the nucleus, thus preventing the activation of the AKT pathway and the proliferation of these cancer specimens. Furthermore, we confirmed that the antitumor activities of these ASOs were also maintained in vivo, as proven by the decreased tumor proliferation observed in breast cancer xenograft models. Finally, to improve the delivery of these ASOs in vivo, the usage of non-viral systems, including lipid-based and metallic-based nanoparticles, is being tested in both ex vivo and in vivo preclinical models. Overall, our results unveiled TROLL-2 and TROLL-3 as suitable targets to counteract tumor growth and promote the efficacy of chemotherapeutics and targeted therapies in ex vivo and in vivo cancer models. Citation Format: Marco Napoli, Ashley K Lu, Avani A Deshpande, Jaden R Baldwin, Christina L Carr, Michael R Dunne, Omar K Farha, Michelle H Teplensky, Elsa R Flores. RNA-based therapeutics to target the p53 pathway in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B011.

Abstract B042: Molecular assessment of cell surface targets using integrated circulating tumor cell (CTC) RNAseq and single CTC phenotyping

Abstract Background: Androgen-targeted therapies are a mainstay of treatment for metastatic prostate cancer and have significantly improved patient outcomes. However, development of treatment resistance remains universal, occurring through androgen receptor (AR) alterations driving constitutive AR signaling, or lineage state transitions that bypass AR entirely and culminate in a small cell/neuroendocrine phenotype (NEPC). Cell surface targeted therapies, including antibody drug conjugates and targeted radioligand therapies, represent a new therapeutic class that has shown promise for treatment of androgen-resistant metastatic prostate cancer (mCRPC). Circulating tumor cells (CTCs) present an accessible source of tumor material to identify expression of known and novel targets for therapeutic development, as well as the unique potential for longitudinal profiling to understand the evolution of target expression and association with clinical resistance to cell surface targeted therapies. We have developed a CTC platform allowing for gene expression and protein profiling of cell surface targets on CTCs. Methods: Live CTCs were isolated with our automated microfluidic technology integrating negative and positive selection for CTC enrichment with a cohort of 273 samples. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA-seq, or protein staining on chip for single cell fluorescent quantification of prostate adenocarcinoma and NEPC cell surface targets including PSMA, TROP2, B7H3 and DLL3. Results: CTC RNA sequencing can be used to understand the expression of cell surface targets across different CTC phenotypes. In CTCs from patient with mCRPC, canonical prostate adenocarcinoma cell surface targets STEAP1, KLK2 and FOLH1 and epithelial cell surface target TACSTD2 had the highest expression, while prostate adenocarcinoma target STEAP2, pan-tumor target ERBB2, and tumor immune checkpoint cell surface protein CD276 (B7H3) had intermediate expression. Expression of DLL3 and SSTR2, targets associated with neuroendocrine differentiation, was lowest, though still detected in a subset of CRPC CTCs. Comparison of expression levels between CRPC and NEPC CTCs demonstrated lower expression of most adenocarcinoma cell surface targets in NEPC CTCs as expected, and a trend towards higher expression of DLL3 and SSTR2. Cell surface expression is being assessed for proteins including Trop-2, PSMA and DLL3. Evaluation of DLL3 at the protein level in a subset of patients including both CRPC (n=13) and NEPC (n=12) confirmed that DLL3 was detected in both adenocarcinoma and neuroendocrine CTCs, with a median of 45.8% DLL3-positive CTCs per sample. Conclusions: Biomarkers are needed to better predict response and resistance to targeted therapies. CTC RNAseq and single CTC phenotyping hold potential for predictive and on-treatment biomarkers of response to cell surface targeted therapies. Further study of mechanisms driving response and resistance can be evaluated with these biomarkers. Citation Format: Jamie M. Sperger, Marina N. Sharifi, Katherine R. Kaufmann, Matthew L. Bootsma, Shannon R. Reese, Viridiana Carreno, Alex H. Chang, Luke A. Nunamaker A. Nunamaker, Charlotte Linebarger, Amy K. Taylor, Katherine E Tippins, Kyle T. Helzer, Shuang G. Zhao, Joshua M Lang. Molecular assessment of cell surface targets using integrated circulating tumor cell (CTC) RNAseq and single CTC phenotyping [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B042.

Integrin β8 Facilitates Macrophage Infiltration and Polarization by Regulating CCL5 to Promote LUAD Progression.

The tumor microenvironment (TME) influences cancer progression and metastasis. Integrin β8 (ITGβ8), a member of the integrin family, is upregulated in various cancers. In this study, it is determined as a key factor that mediates the interaction between lung adenocarcinoma (LUAD) cells and macrophages. Increased expression levels of ITGβ8 are associated with increased numbers of CD163+ macrophages and poor prognosis in LUAD patients. The overexpression of ITGβ8 in LUAD cells promotes the polarization of THP-1 macrophages toward the M2 phenotype. In contrast, TCM (conditioned medium from the co-culture system) from THP-1 macrophages and ITGβ8-overexpressing A549 cells promoted the proliferation and invasion of A549 cells. Mechanistically, chemokine (C-C motif) ligand 5 (CCL5) plays an important role in mediating ITGβ8-induced macrophage polarization, and the phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase (AKT)/interferon regulatory factor 9 (IRF9) pathway is involved in this process. Moreover, interleukin 8 (IL8) and interleukin 10 (IL10) produced by M2-like macrophages regulate the expression of ITGβ8 in LUAD cells through the spi-1 proto-oncogene (SPI1). This study elucidates the feedback mechanism of ITGβ8 between LUAD cells and macrophages.

OGC O06 - Socioeconomic Health Inequality in Oesophago-gastric Cancer in a Single Tertiary Centre: On Behalf of Greater Manchester OG Unit

Abstract Background Oesophagogastric cancer (OGC) 5-year survival is poor (15%). Only 34% of patients are considered for potentially curative treatment, which is associated with a 41% chance of survival. In other cancer, deprivation is associated with poor outcomes. This study aims to determine the association between deprivation, tumour and treatment intent (TI), and 5-year all-cause mortality (M) in OGC. Method This retrospective cohort study analysed patients newly diagnosed with OGC (adenocarcinoma or squamous cell carcinoma) across Greater Manchester between January 2018 and January 2023. TI was defined as the treatment pathway, at completion of staging investigations, either curative (pathway to resection or definitive chemoradiotherapy) or palliative (best supportive care, palliative chemotherapy or radiotherapy). Statistical analysis encompassed methods such as binary logistic regression, Cox proportional hazards models and Kaplan-Meier survival curves with adjustments made for age, gender, and clinical stage. IMD scores were grouped from deciles into quintiles leaving 5 groups, with IMD1 representing the most deprived. Results 3027 patients were included (2102, 69.4% males). Patients in IMD1 were diagnosed 4 years younger than in IMD5 (most affluent) (median age 70 (IQR=17) vs 74 (IQR=16), p<0.001). There was a higher proportion of deprived patients (32.9% IMD1 vs 15.3% IMD5, p<0.001). The proportion of stage 4b disease in IMD1 compared with IMD5 was not different (406 (40.7%) vs 174 (37.5%), p=0.081). The median survival was lower in IMD1 compared with IMD5 (0.81 vs 0.90 years, p=0.074). Following adjustment for confounders IMD1 was associated with a poorer overall survival when compared with IMD5 (HR 1.16, CI[1.02-1.32],p=0.020). Conclusion This study highlights a significant health inequality in the outcomes of OGC. Poorer outcomes in the most deprived group were independent of tumour stage, gender and age. Urgent measures to address socioeconomic health inequalities need to be instigated.

SOCS1 Inhibits IL-6-Induced CD155 Overexpression in Lung Adenocarcinoma

CD155, also known as the poliovirus receptor (PVR), is a crucial molecule in the development and progression of cancer, as its overexpression favors immune evasion and resistance to immunotherapy. However, little is known about the mechanisms that regulate its overexpression. Proinflammatory factors produced by various cellular components of the tumor microenvironment (TME) have been associated with CD155 expression. We analyzed the effect of interleukin (IL)-6 on CD155 expression in lung adenocarcinoma. We found a positive relationship between mRNA and protein levels. This correlation was also observed in bioinformatics analysis and in biopsies and serum from patients with lung adenocarcinoma. Interestingly, lung adenocarcinoma cell lines expressing suppressor of cytokine signaling 1 (SOCS1) did not show increased CD155 levels upon IL-6 stimulation, and SOCS1 silencing reverted this effect. IL-6 and SOCS1 are critical regulators of CD155 expression in lung adenocarcinoma. Further basic and clinical studies are needed to define the role of these molecules during tumor development and to improve their clinical impact as biomarkers and targets for predicting the efficacy of immunotherapies. This study deepens the understanding of the intricate regulation of the immune checkpoints mediated by soluble factors and allows us to devise new ways to combine conventional treatments with the most innovative anticancer options.

Antioxidant activity of Micractinium sp. (Chlorophyta) extracts against H2O2 induced oxidative stress in human breast adenocarcinoma cells.

In response to the growing demand for high-value bioactive compounds, microalgae cultivation has gained a significant acceleration in recent years. Among these compounds, antioxidants have emerged as essential constituents in the food, pharmaceutical, and cosmetics industries. This study focuses on Micractinium sp. ME05, a green microalgal strain previously isolated from hot springs flora in our laboratory. Micractinium sp. cells were extracted using six different solvents, and their antioxidant capacity, as well as total phenolic, flavonoid, and carotenoid contents were evaluated. The methanolic extracts demonstrated the highest antioxidant capacity, measuring 7.72 and 93.80µmol trolox equivalentsg-1 dry weight (DW) according to the DPPH and FRAP assays, respectively. To further characterize the biochemical profile, reverse phase high-performance chromatography (RP-HPLC) was employed to quantify twelve different phenolics, including rutin, gallic acid, benzoic acid, cinnamic acid, and β-carotene, in the microalgal extracts. Notably, the acetone extracts of Micractinium sp. grown mixotrophically contained a high amount of gallic acid (469.21 ± 159.74µgg-1DW), while 4-hydroxy benzoic acid (403.93 ± 20.98µgg-1DW) was the main phenolic compound in the methanolic extracts under heterotrophic cultivation. Moreover, extracts from Micractinium sp. exhibited remarkable cytoprotective activity by effectively inhibiting hydrogen peroxide-induced oxidative stress and cell death in human breast adenocarcinoma (MCF-7) cells. In conclusion, with its diverse biochemical composition and adaptability to different growth regimens, Micractinium sp. emerges as a robust candidate for mass cultivation in nutraceutical and food applications.

XRCC1 is linked to poor prognosis in adenocarcinoma of the esophagogastric junction after radiotherapy: transcriptome and alternative splicing events analysis.

This study aimed to (i) investigate the relationship between X-ray repair cross-complementing protein 1 gene (XRCC1) and prognosis in patients with adenocarcinoma of the esophagogastric junction (AEG), and (ii) analyze the roles of XRCC1 in human gastric adenocarcinoma (AGS) cells following X-ray radiation. A total of 46 AEG patients were enrolled and examined for XRCC1 protein by immunohistochemistry. XRCC1 was knocked down in AGS cells by transfection, and AGS cells were subsequently exposed to 6Gy of X-ray radiation. XRCC1 mRNA and protein expression was examined via quantitative real-time PCR (qRT-PCR) and Western blot analysis. The apoptosis of AGS cells was examined by flow cytometer. RNA-sequencing technology was used to identified differentially expressed genes and alternative splicing events following XRCC1 knockdown and radiation exposure. XRCC1 positivity was strongly associated with distant metastasis, pathological tumor-node-metastasis (pTNM) classification, and radiotherapy resistance in AEG patients. A significant difference in progression-free survival was observed between AEG patients with low and high XRCC1 protein expression. The knockdown of XRCC1 notably exacerbated the effects of X-ray radiation on apoptosis in AGS cells. Additionally, X-ray radiation modified the expression of genes related to apoptosis and immune response in XRCC1-knockdown AGS cells. Furthermore, the generation of splice variants was influenced by XRCC1 knockdown in AGS cells. XRCC1 may serve as a key oncogene that elucidates the role of alternative splicing events in the progression of AEG following X-ray treatment.

EPCO-42. SINGLE-CELL MULTIOMIC ANALYSIS OF BRAIN METASTASES ACROSS MULTIPLE PRIMARY TUMOR TYPES

Abstract Brain metastases are the most common form of intracranial tumor in adults, however the prognosis remains dismal due to lack of effective treatments and limited understanding of the underlying physiology allowing them to thrive in the foreign cell type environment of the brain. Here we characterized the transcriptomic and epigenomic landscape of 35 human brain metastases obtained from surgical resection using combined scRNA-seq and ATAC-seq spanning the most prevalent primary tumor types including lung adenocarcinoma (n=16), melanoma (n=14), breast (n=10), and renal cell carcinoma (n=6). Varying molecular subtypes across primary tumors were included allowing for a comprehensive analysis between and within primary tumor types. Approximately 200,000 cells total were profiled to a sequencing depth of 50,000 mean reads per cell with 2,500 median genes per cell. ATAC-seq libraries were sequenced to a depth of 25,000 reads per cell with 10-15,000 median fragments per cell. Unbiased clustering and integrated analysis identified various cell types within the tumor, immune microenvironment, and normal brain tissue. Tumor cell identities were confirmed throughout multiple clusters via inferred copy number alteration analyses. Integration with primary tumor atlases highlight differences between cell states of primary and metastatic tumors, revealing shifts of enhancer function in transcriptional regulation of metastatic cells. Overall, our findings describe the genetic and epigenetic states of metastatic cells contributing to the understanding of CNS metastatic colonization.