Cancer Tissues Research Articles

Cancer-associated fibroblast-secreted exosomal miR-454-3p inhibits lipid metabolism and ferroptosis in breast cancer by targeting ACSL4.

Cancer-associated fibroblasts (CAFs) participate in the development of the tumor microenvironment through the secretion of exosomes. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is an essential component of ferroptosis. However, the regulatory mechanism of ACSL4 in breast cancer remains unexplored. The study aimed to determine the influence of exosomal miR-454-3p from CAFs on lipid metabolism and ferroptosis. CAF-derived exosomes (CAF-exo) were isolated from breast cancer tissue of breast cancer patients and characterized using transmission electron microscopy (TEM) and Western blot. Luciferase reporter assay and RNA immunoprecipitation (RIP) were used to demonstrate the relationship between miR-454-3p and ACSL4. Cell viability and ferroptosis-related markers were detected by CCK-8 and Western blot. Malondialdehyde (MDA), glutathione (GSH), and iron levels were detected. Reverse transcription-quantitative PCR (RT-qPCR) and fluorescence in situ hybridization (FISH) were used to assess miR-454-3p expression. miR-454-3p and ACSL4 levels were abnormally expressed in breast cancer tissues. CAF-exo significantly enhanced cell viability and GSH levels and suppressed MDA, and iron levels. CAF-exo upregulated ferroptosis suppressor protein 1 (FSP1) and glutathione peroxidase 4 (GPX4) expression, and reduced ACSL4 levels. miR-454-3p was strongly expressed in CAF-exo, and exosomal miR-454-3p suppressed lipid metabolism and ferroptosis in breast cancer cells. The effects of miR-454-3p inhibitor on lipid metabolism and ferroptosis were eliminated by ACSL4 knockdown. CAF-secreted exosomal miR-454-3p inhibited lipid metabolism and ferroptosis by targeting ACSL4 in breast cancer. This study revealed a novel molecular mechanism that offers a potential therapeutic intervention in breast cancer treatment.

Investigation of Programmed Death Ligand-1 as a New Prognostic Biomarker in Pancreatic Cancer Patients.

Pancreatic cancer is one of the most lethal and fast-growing cancers with a poor prognosis. Herein, we report the expression of programmed death ligand 1 (PD-L1) as a new prognostic biomarker in pancreatic cancer progression analysis at the clinical level. Immunohistochemistry was performed on 86 clinically proven cases of pancreatic cancer tissue microarrays (TMAs) using anti-PD-L1 antibodies. Histoscore was done, and a variety of cutoffs were identified for analyses of the results. The chi-square test and Kaplan-Meier method were used to find the association between pancreatic cancer and various clinicopathological variables and the overall survival of the patients. PD-L1 expression was associated with histological grade and recurrence of the disease for epithelial and stromal staining at 10 histoscores. In addition, PD-L1 expression was strongly associated with lymph node involvement at the stromal 20 histoscore. The tumor stage of pancreatic cancer had an association with PD-L1 expression with epithelial and stromal 20 histoscores for all comparisons. At a stromal 20 histoscore, overall survival in high-low expression of PD-L1 was 7-19 months, and at a nuclear/cytoplasmic 10 histoscore, it was 9-28 months (p = 0.0001), respectively. Overall, PD-L1 overexpression in subcellular compartments was associated with disease aggression phenotypes and poor patient survival. Overexpression of PD-L1 was directly linked to pancreatic cancer progression and a poor survival rate. Therefore, PD-L1 may be used as a prognostic biomarker in the diagnosis, treatment, and management of pancreatic cancer patients.

Alternative splicing of PBRM1 mediates resistance to PD-1 blockade therapy in renal cancer.

Alternative pre-mRNA splicing (AS) is a biological process that results in proteomic diversity. However, implications of AS alterations in cancer remain poorly understood. Herein, we performed a comprehensive AS analysis in cancer driver gene transcripts across fifteen cancer types and found global alterations in inclusion rates of the PBAF SWI/SNF chromatin remodeling complex subunit Polybromo 1 (PBRM1) exon 27 (E27) in most types of cancer tissues compared with those in normal tissues. Further analysis confirmed that PBRM1 E27 is excluded by the direct binding of RBFOX2 to intronic UGCAUG elements. In addition, the E27-included PBRM1 isoform upregulated PD-L1 expression via enhanced PBAF complex recruitment to the PD-L1 promoter. PBRM1wild-type patients with clear cell renal cell carcinoma were resistant to PD-1 blockade therapy when they expressed low RBFOX2 mRNA levels. Overall, our study suggests targeting of RBFOX2-mediated AS of PBRM1 as a potential therapeutic strategy for immune checkpoint blockade.

Expression of histone methyltransferase WHSC1 in invasive breast cancer and its correlation with clinical and pathological data

BackgroundThe WHSC1 protein facilitates specific dimethylation of histone H3 at the K36 position, enhancing gene transcription and expression. Studies have confirmed its high expression in diverse malignant tumors. We aimed to identify novel molecular markers to assess the biological behavior of breast cancer cells. MethodsWe conducted a comprehensive analysis of mRNA expression in breast cancer and adjacent tissues based on TCGA data. We enrolled 141 breast cancer patients treated at the First Affiliated Hospital of Fujian Medical University between 2012 and 2016. Patient clinical information and pathological specimens were obtained. We utilized tissue microarray (TMA) technology. We employed the chi-square test for between-group comparisons, with p < 0.05 indicating statistical significance. Furthermore, we analyzed the associations between WHSC1 expression and clinical or pathological data. ResultsWHSC1 mRNA expression was significantly higher in breast cancer tissues than in adjacent tissues (p < 0.001). Moreover, high WHSC1 protein expression in breast cancer was associated with several important clinical parameters, such as pathological type (p = 0.007), high Ki67 expression(Ki67＞20 %) (p < 0.001), lymph node metastasis (p < 0.001), T stage (p = 0.011), N stage (p < 0.001), postoperative pathological stage (p < 0.001), premenopausal status (p = 0.004), and positive HER2 status (p < 0.001). Multivariate regression analysis showed that high WHSC1 expression, elevated Ki67 levels, and positive HER2 status were independent risk factors for axillary lymph node metastasis in breast cancer patients. ConclusionWHSC1 protein expression is upregulated in breast cancer patients and represents an independent risk factor influencing axillary lymph node metastasis, highlighting its potential significance as a strong candidate biomarker.

Acetate utilization promotes hormone therapy resistance in prostate cancer through neuroendocrine differentiation

AimsTumor fatty acid (FA) metabolic plasticity plays a pivotal role in resistance to therapy and poses limitations to anticancer strategies. In this study, our aim is to uncover the role of acetate metabolism in neurodifferentiation (NED)-mediated castration-resistant prostate cancer (CRPC). MethodsWe conducted analyses using LC-MS/MS on clinical prostate cancer tissue before and after hormone therapy. We established tumor xenograft mouse models, primary tumor cells, and human-derived organoids to detect the novel mechanism of NED and to identify potential therapies. ResultsThe hormone therapy-induced upregulation of acetate metabolism was mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2), which increased c-MYC expression for NED induction. Notably, combined treatment with an ACSS2 inhibitor and enzalutamide significantly reduced the xenograft tumor volume. ConclusionOur findings uncovered the critical role of acetate metabolism in NED-mediated CRPC and suggest that ACSS2 inhibitors may represent a novel, low-toxicity strategy when combined with hormone therapy for treating patients with NED-mediated CRPC.

7355 A Case of High-Risk Thyroid Tumor Markers Found in Subcutaneous Thyroid Implants in a Patient with Benign Thyroid Pathology

Abstract Disclosure: J. Fedorko: None. S. Fatima: None. A.P. Amblee: None. C.A. Poku: None. Background: While fine needle aspiration (FNA) remains the primary diagnostic tool for thyroid malignancies, the utilization of molecular markers has become increasingly common in the workup of thyroid nodules. They play a useful role in distinguishing between malignant and benign pathology when cytology is indeterminate and in formulating treatment plans. The vast majority of research for these genetic markers is conducted in cases with thyroid malignancy. However, there have been numerous cases where these genetic markers were identified in benign thyroid tissue. Case: A 73-year-old male with left hemithyroidectomy (benign left nodule) presented 6 years later with new superficial neck nodules along the midline, left thyroid bed and R thyroid lobe. FNA of the right thyroid nodule was benign. Three months later the patient underwent an excision of the midline neck nodules which revealed thyroid tissue arranged in nests of fibrotic tissue without definite features of papillary thyroid carcinoma. Molecular analysis (Thyroseq) was positive for HRAS, E1F1AX and TERT mutations thus metastatic follicular carcinoma was suspected. The patient underwent complete thyroidectomy. Pathology revealed normal thyroid tissue and a diagnosis of thyroid neck implants was made. Given the benign pathology, the plan was for close follow-up with neck imaging every 6 months. Discussion: Thyroid tissue implants can rarely be caused by previous surgical interventions, especially partial thyroidectomy. There is literature documenting similar clinical cases in which subcutaneous thyroid implants are found in patients after undergoing total or subtotal thyroidectomy. In one case series, there were cases in which thyroid implants were discovered 26 years post-surgery, with most of the cases reporting benign pathology. Given our patient’s presentation and clinical history, the possibility remains that his subcutaneous tissue findings were caused by his prior partial thyroidectomy. A recent article reviewed 60 publications and &amp;gt;8000 patients for the prevalence of high-risk tumor markers (RAS mutation, RET/PTC, and PAX8/PPAR-gamma chromosomal rearrangements) in benign thyroid tissue. They concluded that prevalence findings were highly variable, warning providers to exercise caution when using these genetic markers to guide diagnosis and treatment decisions. Conclusion: While certain high-risk genetic tumor markers are increasingly used to help identify thyroid malignancies, it is important to keep in mind that these genetic markers are not exclusively found in cancerous tissue, and should not be used as the sole diagnostic tool for diagnosis and treatment. While it is imperative that metastatic disease is ruled out, it is essential to consider other pathologies when evaluating recurrent thyroid and neck masses in patients with a history of benign thyroid lesions. Presentation: 6/3/2024

Machine learning and artificial intelligence: Enabling the clinical translation of atomic force microscopy-based biomarkers for cancer diagnosis

The influence of biomechanics on cell function has become increasingly defined over recent years. Biomechanical changes are known to affect oncogenesis; however, these effects are not yet fully understood. Atomic force microscopy (AFM) is the gold standard method for measuring tissue mechanics on the micro- or nano-scale. Due to its complexity, however, AFM has yet to become integrated in routine clinical diagnosis. Artificial intelligence (AI) and machine learning (ML) have the potential to make AFM more accessible, principally through automation of analysis. In this review, AFM and its use for the assessment of cell and tissue mechanics in cancer is described. Research relating to the application of artificial intelligence and machine learning in the analysis of AFM topography and force spectroscopy of cancer tissue and cells are reviewed. The application of machine learning and artificial intelligence to AFM has the potential to enable the widespread use of nanoscale morphologic and biomechanical features as diagnostic and prognostic biomarkers in cancer treatment.

Bufalin induces ferroptosis by modulating the 2,4-dienoyl-CoA reductase (DECR1)-SLC7A11 axis in breast cancer

Breast cancer (BC) is a leading cause of cancer-related mortality worldwide. 2,4-dienoyl-CoA reductase (DECR1), an auxiliary component of beta-oxidation, has been recognized for its role in enhancing lipid peroxidation and inducing ferroptosis in prostate cancer. However, its involvement in breast cancer remains largely unexplored. Our study revealed a notably elevated expression of DECR1 in breast cancer tissues, which correlated with increased malignant characteristics. Importantly, the overexpression of DECR1 significantly enhanced proliferation and migration capabilities in MDA-MB-231 cells. Through a comprehensive high-content screening approach, we identified bufalin and its derivative as potent inhibitors of DECR1 expression. Notably, bufalin demonstrated the highest binding energy during molecular docking studies and was found to promote the degradation of DECR1 via autophagy and ubiquitination. Furthermore, bufalin induced ferroptosis in MDA-MB-231 cells by modulating levels of malondialdehyde (MDA), triglycerides (TG), reactive oxygen species (ROS) and Fe2+ while downregulating the expression of hormone-sensitive lipase (HSL), ferritin heavy chain protein 1 (FPN), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4). These effects were counteracted by DECR1 overexpression. In vivo experiments demonstrated that bufalin inhibited the tumor growth, while decreasing the expression levels of HSL, FPN, SLC7A11, and GPX4, alongside increasing levels of 4-hydroxynonenal (4-HNE). Crucially, the ferroptosis effects induced by bufalin in vivo were also reversed by DECR1 overexpression. Subsequently, we discovered that SLC7A11 interacts with DECR1, inhibition of SLC7A11 led to decreased expression levels of DECR1 along with an accumulation of MDA and Fe2+, effects that were similarly reversed by DECR1 overexpression. Collectively, our findings suggest that targeted therapy against DECR1 combined with further inhibition of its downstream pathway involving SLC7A11/GPX4 may represent a promising strategy for treating breast cancer.

Pan-cancer analysis of the prognostic and immunological roles of SHP-1/ptpn6

SHP-1, a nonreceptor protein tyrosine phosphatase encoded by ptpn6, has been regarded as a regulatory protein of hematopoietic cell biology for years. However, there is now increasing evidence to support its role in tumors. Thus, the role of ptpn6 for prognosis and immune regulation across 33 tumors was investigated, aiming to explore its functional heterogeneity and clinical significance in pan-cancer. Differential expression of ptpn6 was found between cancer and adjacent normal tissues, and its expression was significantly correlated with the prognosis of tumor patients. In most cancers, ptpn6 expression was significantly associated with immune infiltration. This was further confirmed by ptpn6-related genes/proteins enrichment analysis. Additionally, genetic alterations in ptpn6 was observed in most cancers. As for epigenetic changes, it’s phosphorylation levels significantly altered in 6 tumors, while methylation levels significantly altered in 12 tumors. Notably, the methylation levels of ptpn6 were significantly decreased in 11 tumors, accompanied by its increased expression in 8 of them, suggesting that the hypomethylation may be related to its increased expression. Our results show that ptpn6 plays a specific role in tumor immunity and exerts a pleiotropic effect in a variety of tumors. It can serve as a prognostic factor for some cancers. Especially in LGG, KIRC, UCS and TGCT, the increased expression of ptpn6 is associated with poor prognosis and high immune infiltration. This aids in understanding the role of ptpn6 in tumor biology, and can provide insight into presenting a potential biomarker for poor prognosis and immune infiltration in cancers.

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway.

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent mice by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt-/-) caused a marked reduction in tumor growth in both syngeneic mice tumor models and a genetic mice colorectal cancer (CRC) model induced by mutation of the Apc gene (Apcmin). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt-/- cancer cells restored tumor growth, and this correlated with impaired CD8+ T-cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor-cell-intrinsic mechanism to repress antitumor immunity.

Aberrant cytoplasmic expression of UHRF1 restrains the MHC-I-mediated anti-tumor immune response

Immunotherapy successfully complements traditional cancer treatment. However, primary and acquired resistance might limit efficacy. Reduced antigen presentation by MHC-I has been identified as potential resistance factor. Here we show that the epigenetic regulator ubiquitin-like with PHD and ring finger domains 1 (UHRF1), exhibits altered expression and aberrant cytosolic localization in cancerous tissues, where it promotes MHC-I ubiquitination and degradation. Cytoplasmic translocation of UHRF1 is induced by its phosphorylation on a specific serine in response to signals provided by factors present in the tumor microenvironment (TME), such as TGF-β, enabling UHRF1 to bind MHC-I. Downregulation of MHC-I results in suppression of the antigen presentation pathway to establish an immune hostile TME. UHRF1 inactivation by genetic deletion synergizes with immune checkpoint blockade (ICB) treatment and induces an anti-tumour memory response by evoking low-affinity T cells. Our study adds to the understanding of UHRF1 in cancer immune evasion and provides a potential target to synergize with immunotherapy and overcome immunotherapeutic resistance.

Calcineurin-mediated dephosphorylation stabilizes E2F1 protein by suppressing binding of the FBXW7 ubiquitin ligase subunit

The transcription factor E2F1 serves as a regulator of the cell cycle and promotes cell proliferation. It is highly expressed in cancer tissues and contributes to their malignant transformation. Degradation by the ubiquitin-proteasome system may help to prevent such overexpression of E2F1 and thereby to suppress carcinogenesis. A detailed understanding of the mechanisms underlying E2F1 degradation may therefore inform the development of new cancer treatments. We here identified SCFFBXW7 as a ubiquitin ligase for E2F1 by comprehensive analysis. We found that phosphorylation of E2F1 at serine-403 promotes its binding to FBXW7 (F-box/WD repeat-containing protein 7) followed by its ubiquitination and degradation. Furthermore, calcineurin, a Ca2+/calmodulin-dependent serine-threonine phosphatase, was shown to stabilize E2F1 by mediating its dephosphorylation at serine-403 and thereby preventing FBXW7 binding. Treatment of cells with Ca2+ channel blockers resulted in downregulation of both E2F1 protein and the expression of E2F1 target genes, whereas treatment with the Ca2+ ionophore ionomycin induced upregulation of E2F1. Finally, the calcineurin inhibitor FK506 attenuated xenograft tumor growth in mice in association with downregulation of E2F1 in the tumor tissue. Impairment of the balance between the opposing actions of FBXW7 and calcineurin in the regulation of E2F1 abundance may therefore play an important role in carcinogenesis.

Conserved allomorphs of MR1 drive the specificity of MR1-restricted TCRs.

Major histocompatibility complex class-1-related protein (MR1), unlike human leukocyte antigen (HLA) class-1, was until recently considered to be monomorphic. MR1 presents metabolites in the context of host responses to bacterial infection. MR1-restricted TCRs specific to tumor cells have been described, raising interest in their potential therapeutic application for cancer treatment. The diversity of MR1-ligand biology has broadened with the observation that single nucleotide variants (SNVs) exist within MR1 and that allelic variants can impact host immunity. The TCR from a MR1-restricted T-cell clone, MC.7.G5, with reported cancer specificity and pan-cancer activity, was cloned and expressed in Jurkat E6.1 TCRαβ- β2M- CD8+ NF-κB:CFP NFAT:eGFP AP-1:mCherry cells or in human donor T cells. Functional activity of 7G5.TCR-T was demonstrated using cytotoxicity assays and by measuring cytokine release after co-culture with cancer cell lines with or without loading of previously described MR1 ligands. MR1 allele sequencing was undertaken after the amplification of the MR1 gene region by PCR. In vivo studies were undertaken at Labcorp Drug Development (Ann Arbor, MI, USA) or Epistem Ltd (Manchester, UK). The TCR cloned from MC.7.G5 retained MR1-restricted functional cytotoxicity as 7G5.TCR-T. However, activity was not pan-cancer, as initially reported with the clone MC.7.G5. Recognition was restricted to cells expressing a SNV of MR1 (MR1*04) and was not cancer-specific. 7G5.TCR-T and 7G5-like TCR-T cells reacted to both cancer and healthy cells endogenously expressing MR1*04 SNVs, which encode R9H and H17R substitutions. This allelic specificity could be overcome by expressing supraphysiological levels of the wild-type MR1 (MR1*01) in cell lines. Healthy individuals harbor T cells reactive to MR1 variants displaying self-ligands expressed in cancer and benign tissues. Described "cancer-specific" MR1-restricted TCRs need further validation, covering conserved allomorphs of MR1. Ligands require identification to ensure targeting MR1 is restricted to those specific to cancer and not normal tissues. For the wider field of immunology and transplant biology, the observation that MR1*04 may behave as an alloantigen warrants further study. .

STAT3/p-STAT3 expression is correlated with clinicopathological characteristics and prognosis in non-small cell lung cancer.

Signal transducer and activator of transcription factor 3 (STAT3)/phosphorylated STAT3 (p-STAT) play a critical role in tumorigenesis, however, there is limited information on its prognostic value in non-small cell lung cancer (NSCLC). To address this question, 239 lung cancer and 71 normal lung tissue samples were obtained in this study. Immunohistochemistry was applied to detect STAT3/p-STAT3 expression. Pearson's Chi-squared test and the Kaplan-Meier method were conducted to evaluate associations with patients' clinical characteristics and survival. According to our results, STAT3/p-STAT3 was significantly upregulated in lung cancer tissue (p<0.001). Moreover, p-STAT3 expression was significantly correlated with age (p=0.046) and pathological types (p=0.037). In survival analysis, STAT3 positivity was negatively associated with survival in patients older than 60 years (p=0.043) but failed to be an independent prognostic factor in multivariate analysis (p=0.083). Therefore, STAT3/p-STAT3 may serve as a critical biomarker in NSCLC.

RNA transcription assisted universal CRISPR/Cas12a system for programmable analysis of multiple colorectal cancer-associated microRNAs

Accurate analysis of multiple microRNA (miRNA) levels is significantly valuable for early diagnosis of colorectal cancer noninvasively considering the miRNA expression is highly relevant to the occurrence and progression of cancer. However, the low abundance and high sequence homology of miRNAs make their precise determination extremely challenging. Here, we developed a universal and programmable diagnostic strategy allowing for analyzing multiple colorectal cancer-associated miRNAs. The system combined sequentially programmable rolling circle transcription (RCT) and the CRISPR/Cas12a system with high trans-cleavage activity to achieve highly sensitive and specific detection of four target miRNAs. Owing to the remarkable performance of universal RCT-Cas12a strategy, this biosensor could detect miR-21, miR-17, miR-31 and miR-92a with a LOD of 2.1, 1.6, 3.7 and 1.0 pM, respectively. This strategy had a unique advantage in distinguishing human normal colon epithelial cells lines (NCM460) from human colon cancer cells (HT29). In particular, the designed system exhibited superior analytical capability in distinguishing paracancerous and colorectal cancer tissues from patients undergoing colorectal cancer surgery. This arbitrarily programmable, scalable, fast and specific strategy potentially offered an attractive alternative to handle varied challenges encountered with CRISPR-based systems, and held immense promise in scientific research and clinical applications.

H4K20me3 is increased in human bladder cancer tissues, and it is upregulated by reactive oxygen species in bladder cancer cell lines.

H4K20me3 is increased in human bladder cancer tissues, and it is upregulated by reactive oxygen species in bladder cancer cell lines.

TRLR-02 TARGETING GLIOBLASTOMA MICROENVIRONMENT TO UNLEASH IMMUNE FORCES

Abstract Brain cancer has persistently eluded therapeutic advances over the past three decades. Here, we aim to raise awareness of the potential differences between the cancer microenvironment within the brain vs. peripheral organ cancers, which may contribute to the therapeutic setbacks observed in brain cancer treatment. While the blood-brain barrier has long been implicated in drug impermeability, its impact on brain cancer may extend far beyond mere drug exclusion. Studies have shown that soluble factors such as VEGF are more concentrated in brain cancer than in any other malignancies. Moreover, recent studies revealed that VEGF, TWEAK, and others can also press on the immune brakes. Therefore, we hypothesize that normalizing the tumor microenvironment could pave the way for effective therapeutic interventions, especially immunotherapy. Our in vitro studies confirmed abundant production of VEGF by glioblastoma 1 (GBM1) cell line, which is exceptionally well scavenged by the anti-VEGF antibody – bevacizumab. Moreover, in vitro, co-culture of T cells and GBM1 cell line without vascular component demonstrated no therapeutic activity of bevacizumab or typical immunotherapeutic drug – anti-PD1 checkpoint inhibitor – nivolumab given in monotherapies. However, a combination of both antibodies revealed therapeutic efficacy in a dose-dependent manner in our experimental system. Currently, intravenous delivery of bevacizumab is the standard of care in patients with glioblastoma recurrence, but its effectiveness has not been proven in this group of patients. Intravenous nivolumab failed in clinical trials. Based on our observations and the negligible penetration of antibodies to the brain, we hypothesize that transiently opening the blood-brain barrier may be necessary to enhance antibody delivery to the tumor microenvironment, alleviate immune suppression, and unlock immune cell activity. Therefore, achieving high antibody concentrations in brain cancer tissue, coupled with a combination of classic checkpoint inhibitors with scavengers of soluble immunosuppressive molecules, could herald a new era of brain cancer therapy.

Pressure loading regulates the stemness of liver cancer stem cells via YAP/BMF signaling axis.

Cancer stem cells (CSCs) are considered the major cause of the occurrence, progression, chemoresistance/radioresistance, recurrence, and metastasis of cancer. Increased interstitial fluid pressure (IFP) is a key feature of solid tumors. Our previous study showed that the distribution of liver cancer stem cells (LCSCs) correlated with the mechanical heterogeneity within liver cancer tissues. However, the regulation of liver cancer's mechanical microenvironment on the LCSC stemness is not fully understood. Here, we employed a cellular pressure-loading device to investigate the effects of normal IFP (5 mmHg), as well as increased IFP (40 and 200 mmHg) on the stemness of LCSCs. Compared to the control LCSCs (exposure to 5 mmHg pressure loading), the LCSCs exposed to 40 mmHg pressure loading exhibited significantly upregulated expression of CSC markers (CD44, EpCAM, Nanog), enhanced sphere and colony formation capacities, and tumorigenic potential, whereas continuously increased pressure to 200 mmHg suppressed the LCSC characteristics. Mechanistically, pressure loading regulated Yes-associated protein (YAP) activity and Bcl-2 modifying factor (BMF) expression. YAP transcriptionally regulated BMF expression to affect the stemness of LCSCs. Knockdown of YAP and overexpression of BMF attenuated pressure-mediated stemness and tumorgenicity, while YAP-deficient and BMF-deletion recused pressure-dependent stemness on LCSCs, suggesting the involvement of YAP/BMF signaling axis in this process. Together, our findings provide a potential target for overcoming the stemness of CSCs and elucidate the significance of increased IFP in cancer progression.

B-245 Novel monoclonal antibodies validated specifically for immunohistochemistry by performance assessment on tissue microarrays

Abstract Background Immunohistochemistry (IHC) is a laboratory immunoassay that is a pillar of diagnostic pathology, particularly in the identification and characterization of neoplasms and other diseases/disorders. Though performed routinely, rigorous SOP protocols must be determined empirically for each molecular target, beginning with antibody selection. Both traditional polyclonal and hybridoma-based monoclonal antibodies have been employed in the past, though monoclonal, and particularly recombinant monoclonal, antibodies are preferred. However, any reagent used for diagnostic purposes must be meticulously validated to minimize, if not eliminate, erroneous interpretations of IHC assays. Thus, the extent of validation is the pivotal aspect for establishing an antibody as a reliable diagnostic tool for IHC applications. Methods GeneTex has assembled an extensive catalog of rabbit and mouse monoclonal antibodies validated specifically for IHC. These antibodies are tested on both human normal tissue and cancer tissue microarrays, with upwards of 76 normal and numerous tumor tissues included. Images of the staining are available for all the tested tissues. This broad spectrum of tissues allows a researcher to assess the performance of the antibody on known positive and negative samples, thereby minimizing the risk of false positive or false negative readouts (Gown, 2016). In addition, the data images are of sufficient resolution to determine proper cellular localization of the signal. This special catalog of IHC-optimized reagents are listed under the “HistoMAX” designation. New antibodies will be added continuously, many of them being fully recombinant rabbit monoclonals manufactured using a multi-parameter FACS-based methodology to select antigen-specific IgG+ memory B cells from an immunized rabbit with subsequent cloning of the matched IgG heavy and light chains (Starkie et al., 2016). These recombinant antibodies will be validated following the same approach described above. Results Presently more than 130 mouse or rabbit monoclonal antibodies are available in the HistoMAX line of IHC-validated products and are directed against a spectrum of important targets. This includes antibodies for the commonly analyzed cytokeratins 5, 6, 7, 18, 19, and 20, among many others. Antibodies for CTLA4 and PD-L1 headline products for immunology and immunotherapy research, while other reagents are directed against various cadherins. Hormone receptor antibodies are also represented, with antibodies against estrogen receptor alpha, progesterone receptor, Her2/ErbB2, and the androgen receptor. There are also antibodies against various key “Cluster of Differentiation” (CD) proteins as well as other membrane proteins such as ACE2. Many antibodies are relevant to the study of human malignancies, such as CD66e/CEACAM5/CEA, MSH6, p63, MUC1, Collagen IV, EpCAM, PAX6, PAX8, GP2, TRIM29, S100, and GAD65. All antibodies were evaluated as described above. Conclusions Antibodies are application-specific reagents, and the best antibodies for IHC are validated through systematic staining of a multitude of normal and abnormal tissues. This is the only way that an antibody’s performance for IHC can be critically assessed. The HistoMAX reagents consist only of those monoclonal antibodies that have generated expected signals, or the lack of a signal, in a wide range of tissues in a manner consistent with the literature.

Identification of 10 differentially expressed genes involved in the tumorigenesis of cervical cancer via next-generation sequencing.

The incidence and mortality of cervical cancer remain high in female malignant tumors worldwide. There is still a lack of diagnostic and prognostic markers for cervical carcinoma. This study aimed to screen differentially expressed genes (DEGs) between normal and cervical cancer tissues to identify candidate genes for further research. Uterine cervical specimens were resected from our clinical patients after radical hysterectomy. Three patients' transcriptomic datasets were built by the next generation sequencing (NGS) results. DEGs were selected through the edgeR and DESeq2 packages in the R environment. Functional enrichment analysis, including GO/DisGeNET/KEGG/Reactome enrichment analysis, was performed. Normal and cervical cancer tissue data from the public databases TCGA and GTEx were collected to compare the expression levels of 10 selected DEGs in tumor and normal tissues. ROC curve and survival analysis were performed to compare the diagnostic and prognostic values of each gene. The expression levels of candidate genes were verified in 15 paired clinical specimens via quantitative real-time polymerase chain reaction. There were 875 up-regulated and 1,482 down-regulated genes in cervical cancer samples compared with the paired adjacent normal cervical tissues according to the NGS analysis. The top 10 DEGs included APOD, MASP1, ACKR1, C1QTNF7, SFRP4, HSPB6, GSTM5, IGFBP6, F10 and DCN. GO, DisGeNET and Reactome analyses revealed that the DEGs were related to extracellular matrix and angiogenesis which might influence tumorigenesis. KEGG enrichment showed that PI3K-Akt signaling pathway might be involved in cervical cancer tumorigenesis and progression. The expression levels of selected genes were decreased in tumors in both the public database and our experimental clinical specimens. All the candidate genes showed excellent diagnostic value, and the AUC values exceeded 0.90. Additionally, APOD, ACKR1 and SFRP4 expression levels could help predict the prognosis of patients with cervical cancer. In this study, we selected the top 10 DEGs which were down-regulated in cervical cancer tissues. All of them had dramatically diagnostic value. APOD, ACKR1 and SFRP4 were associated with the survivals of cervical cancer. C1QTNF7, HSPB6, GSTM5, IGFBP6 and F10 were first reported to be candidate genes of cervical carcinoma.