Staining Of Tissue Microarrays Research Articles

Targeting Mesothelin Enhances Personalized Neoantigen Vaccine Induced Antitumor Immune Response in Orthotopic Pancreatic Cancer Mouse Models.

The immunosuppressive microenvironment in pancreatic cancer, characterized by low tumor-specific T cells and excessive fibrosis, limits the effectiveness of immunotherapy. Here, three datasets and multi-immunofluorescence staining of tissue microarrays in pancreatic cancer indicate that mesothelin (MSLN) expression negatively correlates with cytotoxic T cells in tumor. Anti-MSLN antibody (αMSLN) treatment of pancreatic cancer in vivo can significantly increase T cell infiltration. Meanwhile, the combination of αMSLN and neoantigen peptide vaccine identified from pancreatic cancer cell lines is demonstrated to be more effective in inducing neoantigen-specific T cell generation and infiltration at subcutaneous and orthotopic pancreatic cancer models for enhancing antitumor efficacy. Single-cell transcriptome analysis shows that the combined treatment significantly reduces the proportion of fibroblasts, improves the infiltration of IFN-γ+CD4+ and GZMK+CD8+ T cells, as well as reducesthe interaction of antigen presentation-associated ligands and receptors between antigen-presenting Cancer-Associated Fibroblasts (apCAFs) and naive CD4+ T cells. The negative correlations between apCAFs and CD8+ T cells/IFN-γ+CD4+ T cells are further confirmed in human pancreatic cancer tissues. Overall, this study demonstrates that targeting MSLN can improve neoantigen vaccine induced immune efficacy by reducing apCAFs to interrupt the conversion of naive CD4+ T cells to Tregs, and therefore increase the infiltration of tumor-specific T cells.

Hypoxia drives estrogen receptor β-mediated cell growth via transcription activation in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor with a poor prognosis. Hypoxia conditions affect multiple cellular processes promoting the adaptation and progression of cancer cells via the activation of hypoxia-inducible factors (HIF) and subsequent transcription activation of their target genes. Preliminary studies have suggested that estrogen receptor β (ERβ) might play a promoting role in the progression of NSCLC. However, the precise mechanisms, particularly its connection to HIF-1α-mediated modulation under hypoxia, remain unclear. Our findings demonstrated that the overexpression of ERβ, not ERα, increased cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. Tissue microarray staining revealed a strong correlation between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in CoCl2-induced hypoxia, 1% O2 incubation, or HIF-1α overexpressing cells. ChIP identified HIF-1α binding to a hypoxia response element in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the tumor growth, thus emphasizing the promising prospects of targeting HIF-1α and ERβ as a therapeutic approach for the treatment of NSCLC. KEY MESSAGES: ERβ, not ERα, increases cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. A strong correlation exists between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in hypoxic cells via binding to HRE in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the NSCLC tumor growth.

Integrated analysis and experiments uncover the function of disulfidptosis in predicting immunotherapy effectiveness and delineating immune landscapes in uterine corpus endometrial carcinoma.

Recently, a novel type of metabolic-regulated cell demise titled disulfidptosis has been discovered. Studies have demonstrated its importance in immune responses against cancer and its impact on the proliferation of cancer cells. Nonetheless, the precise mechanism and roles of disulfidptosis are not fully understood, particularly regarding the prognosis for individuals with uterine corpus endometrial carcinoma (UCEC). In this research, a distinctive disulfidptosis pattern was developed in UCEC, and by utilizing Non-negative Matrix Factorization (NMF) on 23 disulfidptosis related genes within the TCGA database, 3 distinct subgroups were distinguished. To collect data, we acquired gene expression profiles, somatic mutation information, copy number variation data, and corresponding clinical data from the TCGA and GEO database, specifically from UCEC patients. Cell line experiments and immunohistochemical (IHC) staining were conducted to validate the role of the LRPPRC in proliferation, migration and invasion. The genetic features and immune microenvironment of these subgroups were examined. It is worth mentioning that these subgroups offer important insights into comprehending the tumor microenvironment (TME) and the response of patients to immunotherapy and chemotherapy. Moreover, a disulfidptosis model was developed and validated, demonstrating a high level of accuracy in predicting the prognosis and outcomes of immunotherapy in UCEC patients. Additionally, a novel biomarker, LRPPRC, was identified, which can server as a promising predictor for forecasting prognosis in UCEC patients, with validation through tissue microarray staining and cell line experiments. This study has designed a classification system and a disulfidptosis model for UCEC, in addition to identifying a new biomarker, LRPPRC, for UCEC. These advancements serve as reliable and positive indicators for predicting outcomes and the efficacy of immunotherapy for each UCEC patient.

Anoikis-related subtype and prognosis analyses based on bioinformatics, and an expression verification of ANGPTL4 based on experiments of lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is one of the most common malignant tumors with high mortality. Anoikis resistance is an important mechanism of tumor cell proliferation and migration. Our research is devoted to exploring the role of anoikis in the diagnosis, classification, and prognosis of LUAD. We downloaded the expression profile, mutation, and clinical data of LUAD from The Cancer Genome Atlas (TCGA) database. The "ConsensusClusterPlus" package was then used for the cluster analysis, and least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analyses were used to establish the prognostic model. We verified the reliability of the model using a Gene Expression Omnibus (GEO) data set. A gene set variation analysis (GSVA) was conducted to investigate the functional enrichment differences in the different clusters and risk groups. The CIBERSORT algorithm and a single-sample gene set enrichment analysis (ssGSEA) were used to analyze immune cell infiltration. The tumor mutation burden (TMB) and Tumor Immune Dysfunction and Exclusion (TIDE) scores were used to evaluate the patients' sensitivity to immunotherapy. Immunohistochemical staining of tissue microarrays was used to verify the correlation between ANGPTL4 expression and the clinicopathological characteristics and prognosis of LUAD patients. First, we screened 135 differentially expressed anoikis-related genes (ARGs) and 23 prognosis-related ARGs from TCGA-LUAD data set. Next, 494 LUAD samples were allocated to cluster A and cluster B based on the 23 prognosis-related ARGs. The Kaplan-Meier (K-M) analysis showed the overall survival (OS) of cluster B was better than that of cluster A. The clinicopathological characteristics and functional enrichment analyses revealed significant differences between clusters A and B. The tumor microenvironment (TME) analysis showed that cluster B had more immune cell infiltration and a higher TME score than cluster A. Subsequently, a LASSO Cox regression model of LUAD was constructed with ten ARGs. The K-M analysis showed that the low-risk patients had longer OS than the high-risk patients. The receiver operating characteristic curve, nomogram, and GEO data set verification results showed that the model had high accuracy and reliability. The level of immune cell infiltration and TME score were higher in the low-risk group than the high-risk group. The high-risk group had stronger sensitivity to immune checkpoint block therapy and weaker sensitivity to chemotherapy drugs than the low-risk group. ANGPTL4 expression was correlated with stage, tumor differentiation, tumor size, lymph node metastasis, and OS. We discovered novel molecular subtypes and constructed a novel prognostic model of LUAD. Our findings provide important insights into subtype classification and the accurate survival prediction of LUAD. We also identified ANGPTL4 as a prognostic indicator of LUAD.

NXPH4 mediated by m5C contributes to the malignant characteristics of colorectal cancer via inhibiting HIF1A degradation

ObjectiveColorectal cancer (CRC) is a form of malignancy that exhibits a comparatively elevated occurrence and fatality rate. Given the relatively slower progress in diagnostic and therapeutic approaches for CRC, there is a need to investigate more accurate and efficient biomarkers.MethodsCore regulatory genes were screened using the TCGA database, and the expression of neurexophilin 4 (NXPH4) and its prognostic implications were validated using tissue microarray staining. The assessment of NXPH4 functions involved a range of experiments, including cellular, organoid, and murine models. Furthermore, a regulatory network between m5C, NXPH4, and HIF1A was established through several in vitro experiments.ResultsThe overexpression of NXPH4 is associated with unfavorable prognoses in patients with CRC and hepatocellular carcinoma. Additionally, it facilitates the progression of malignant tumors both in laboratory settings and in living organisms of colorectal carcinoma. Our research also reveals that NXPH4 mRNA can avoid degradation through RNautophagy, relying on an m5C-dependent mechanism. Moreover, NXPH4 amplifies the HIF signaling pathway and stabilizes HIF1A by competitively binding to PHD4.ConclusionsNXPH4, regulated by m5C, promotes malignant tumor progression and regulates the HIF pathway. Consequently, targeting NXPH4 through molecular therapies could potentially serve as an efficacious therapeutic strategy for the management of CRC exhibiting elevated NXPH4 expression.Graphical abstract

Expression and prognostic analysis of PFKFB4 in oral squamous cell carcinoma.

Fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) is a crucial enzyme in the glycolysis pathway, possessing both kinase and phosphatase capabilities. Although it has emerged as an important oncogene in various cancer types, its function in oral squamous cell carcinoma (OSCC) is still not well understood. In our research, PFKFB4 expression was assessed via immunohistochemical (IHC) staining of tissue microarrays and OSCC patient specimens. The transcriptional expression of PFKFB4 in OSCC was analyzed by utilizing The Cancer Genome Atlas (TCGA) dataset. Correlation between PFKFB4 expression and clinicopathological features was examined using the χ2 test. Prognostic investigation of PFKFB4 was conducted via Kaplan-Meier and Cox analyses. PFKFB4 levels were notably elevated in OSCC samples in comparison to adjacent normal tissues (P < 0.001). Elevated PFKFB4 expression was associated with higher histologic grade (P = 0.0438), higher T stage (P = 0.031), and more advanced clinical stage (P = 0.0063). The ROC curve demonstrated the diagnostic potential of PFKFB4 (AUC = 0.827). Increased levels of PFKFB4 were linked to decreased overall survival (OS) (P = 0.04), poorer disease-specific survival (DSS) (P = 0.04), and shorter progression-free interval (PFI) (P < 0.001). PFKFB4 expression was identified as an independent risk factor for OS based on Cox regression analysis [hazard ratio (HR) = 1.517, P = 0.044)]. An OS nomogram was constructed with a concordance index of 0.690. Our findings reveal that upregulated PFKFB4 expression in OSCC tissues could serve as a potential prognostic biomarker.

The utility of the lineage specific immunohistochemical stains SATB2, CDX2, and villin, and the mucin glycoproteins MUC2, MUC5AC, and MUC6 to distinguish pulmonary invasive mucinous adenocarcinoma from metastatic colorectal carcinoma

ContextThe lungs are a common site of tumor metastasis. While morphology and immunophenotype can help differentiate primary from metastatic tumors, distinguishing pulmonary invasive mucinous adenocarcinoma (PIMA) from metastatic colorectal adenocarcinoma (CRC) may occasionally be challenging due to overlapping morphological and immunohistochemical features. Lineage-specific markers such as CDX2, TTF-1, and napsin A are helpful with pulmonary non-mucinous adenocarcinoma (PNMA), however they are non-specific and insensitive when applied to PIMA. SATB2 is a newer marker that distinguishes CRC from upper gastrointestinal and pancreaticobiliary tumors; its utility in distinguishing CRC from PIMA has not been fully elucidated. ObjectiveTo evaluate the performance of lineage-specific and mucin glycoprotein immunostains in distinguishing PIMA and CRC. DesignWe stained tissue microarrays comprising 34 PNMA, 31 PIMA, and 32 CRC with CK7, CK20, SATB2, CDX2, villin, TTF-1, napsin A, and gel-forming mucins MUC2, MUC5AC, and MUC6. ResultsPIMA showed significant (>50% of cells) expression of SATB2 (6%), CDX2 (6%), villin (74%), TTF-1 (13%), and napsin A (23%). However, significant CK7 expression was seen in nearly all PIMA (30/31) and none of the metastatic CRC. ConclusionOur results suggest that CK7 remains one of the most useful markers for distinguishing primary PIMA from metastatic CRC. Expression of the mucin glycoproteins MUC5AC and MUC6 and lack of expression of MUC2 favored a diagnosis of PIMA, but expression of these markers was too heterogeneous to be of clinical utility. To our knowledge this is the only study comparing the immunohistochemical profile of PIMA and metastatic CRC in lung metastasectomy specimens.

A genetic variant in the TAPBP gene enhances cervical cancer susceptibility by increasing m6A modification.

Genetic variants can affect gene expression by altering the level of N6-methyladenosine (m6A) modifications. A better understanding of the association of these genetic variants with susceptibility to cervical cancer (CC) can promote advances in disease screening and treatment. Genome-wide identification of m6A-associated functional SNPs for CC was performed using the TCGA and JENGER databases, incorporating the data from RNA-seq and MeRIP-seq. The screened risk-associated SNP rs1059288 (A>G), which is located in the 3' UTR of TAPBP, was further validated in a case-control study involving 921 cases and 1077 controls. The results revealed a significant association between rs1059288 and the risk of CC (OR 1.48, 95% CI 1.13-1.92). Mechanistically, the presence of the risk G allele of rs1059288 was associated with increased m6A modification of TAPBP compared with the A allele. This modification was facilitated by the m6A methyltransferase METTL14 and the reading protein YTHDF2. Immunohistochemical staining of tissue microarrays containing 61 CC and 45 normal tissues showed an overexpression of TAPBP in CC. Furthermore, the upregulation of TAPBP promoted the growth and migration of CC cells as well as tumor-forming ability, inhibited apoptosis, and conferred increased resistance to commonly used chemotherapeutic drugs such as bleomycin, cisplatin, and doxorubicin. Knockdown of TAPBP inhibited the JAK/STAT/MICB signaling pathway in CC cells and upregulated certain immune genes including ISG15, IRF3, PTPN6, and HLA-A. These findings offer insights into the involvement of genetic variations in TAPBP in the development and progression of CC.

High immune cell infiltration predicts improved survival in cholangiocarcinoma.

Antitumoral immune response has a crucial role in constraining cancer. However, previous studies on cholangiocarcinoma (CCA), a rare and aggressive cancer, have reported contradictory findings on the prognostic impact of tumor-infiltrating T-lymphocytes. We aimed to clarify the effect of tumor-infiltrating CD3+ and CD8+ lymphocytes and PD-1/PD-L1 expression on CCA prognosis. CD3+, CD8+, and PD-1+ lymphocyte densities, as well as PD-L1 expression rate were analyzed from stained tissue microarray samples from the tumor center and invasive margin of 47 cholangiocarcinomas. The association of CD3+ and CD8+ based Immune cell score (ICS) and its components with overall survival was evaluated, adjusting for age, sex, TNM stage, radicality of surgery, tumor location, and PD-L1 expression on immune cells. Low ICS was a strong independent prognostic factor for worse overall survival (Hazard ratio 9.27, 95% confidence interval 2.72-31.64, P<0.001). Among the ICS components, high CD8+ lymphocyte infiltration at the tumor center had the most evident impact on patient outcome. PD-1 and PD-L1 expression on immune cells did not have a significant impact on overall survival alone; however, PD-L1 positivity seemed to impair survival for ICSlow subgroup. Identifying patient subgroups that could benefit from immunotherapy with PD-1/PD-L1 pathway blockade may help improve treatment strategies for this aggressive cancer. Our findings highlight the importance of evaluating the immune contexture in cholangiocarcinoma, as ICS serves as a strong independent prognostic and selective factor for patients who might benefit from immunotherapy.

Expression of Cathepsin D in early-stage breast cancer and its prognostic and predictive value

PurposeCathepsin D is a proteolytic enzyme that is normally localized in the lysosomes and is involved in the malignant progression of breast cancer. There are conflicting results regarding Cathepsin D significance as prognostic and predictor marker in breast cancer. This study aimed to evaluate the expression and prognostic significance of Cathepsin D in early-stage breast cancer.MethodsExpression of Cathepsin D was assessed by immunohistochemical staining of tissue microarrays, in a large well-characterized series of early-stage operable breast cancer (n = 954) from Nottingham Primary Breast Carcinoma Series between the period of 1988 and 1998 who underwent primary surgery. Correlation of Cathepsin D expression with clinicopathological parameters and prognosis was evaluated.ResultsCathepsin D expression was positive in 71.2% (679/954) of breast cancer tumours. Positive expression of Cathepsin D was significantly associated with high histological grade (p = 0.007), pleomorphism (p = 0.002), poor Nottingham Prognostic Index (NPI) score (p < 0.002), recurrence (p = 0.005) and distant metastasis (p < 0.0001). Kaplan–Meier analysis showed that Cathepsin D expression was significantly associated with shorter breast cancer-specific survival (p = 0.001), higher risk of recurrence (p = 0.001) and distant metastasis (p < 0.0001). ER-positive tumours expressing Cathepsin D and treated with tamoxifen demonstrated a significantly higher risk of distant metastasis.ConclusionCathepsin D expression significantly predicts poor prognosis in breast cancer and is associated with variables of poor prognosis and shorter outcome. The strong association of Cathepsin D with aggressive tumour characteristics and poor outcomes warrants further research of its potential as a therapeutic target The results also suggest a possible interaction between Cathepsin D and tamoxifen therapy in ER-positive breast cancer which needs further investigation to elucidate the underlying mechanisms.

Abstract 3269: APE1/NF-κB/PRDX2 activated by reflux conditions mediates chemoresistance in esophageal adenocarcinoma by suppressing ferroptosis

Abstract Background: Esophageal adenocarcinoma (EAC) stands out as one of the most lethal malignancies, characterized by a discouragingly low 5-year survival rate of less than 20%. Accumulating lines of evidence indicate that ferroptosis, a novel type of cell death, can regulate drug resistance. Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, plays an antioxidant protective role in the cells under stress by regulating redox homeostasis. We aimed to elucidate the role of PRDX2 in regulating reactive oxygen species (ROS), ferroptosis, and chemoresistance in EAC. Methods: In this investigation, we analyzed RNA sequencing data and public databases to uncover the aberrant overexpression and potential chemoresistance function of PRDX2 in EAC. We employed transient acidic bile salts exposure (ABS) and repeated ABS exposure (rABS) to mimic gastroesophageal reflux disease, the main risk factor for EAC, in in vitro cell models. EAC cells and 3D organotypic culture (OTC) served as platforms to delve into the anti-ferroptosis function and regulatory mechanisms of PRDX2. The findings were validated using patient-derived xenografts (PDX) and human EAC tissue microarrays (TMA). Results: Elevated PRDX2 level was observed in both EAC patients and cell lines exhibiting poor responsiveness to chemotherapy. Intrinsic overexpression of PRDX2 emerged as a crucial factor in the recovery from ABS-exposure-induced lipid peroxidation. Silencing PRDX2 heightened the sensitivity of EAC cells to Oxaliplatin by inhibiting ferroptosis in a GPX4-dependent manner. Mechanistically, we found ABS activate the APE1/redox/NF-kB signal axis, leading to the upregulation of PRDX2 transcription. The co-overexpression of APE1 and PRDX2 was identified through both immunofluorescent staining in cells/OTC and immunohistochemistry staining in TMA. Moreover, the APE1-redox-specific inhibitor, APX2009, significantly sensitized EAC cells to Oxaliplatin by repressing PRDX2 and inducing ferroptosis. Remarkably, the combination therapy of Oxaliplatin and APX2009 achieved a synergistic effect in EAC-PDX tumors. Conclusion: The study unveils a novel signaling axis, APE1/NF-kB/PRDX2, linking disease-associated ROS unbalance and ferroptosis-related chemoresistance. Targeting PRDX2 signaling by APE1-redox-specific inhibition provides a potential novel strategy for combined chemotherapies in refractory EAC. Citation Format: Lei Chen, Heng Lu, Farah Ballout, Dunfa Peng, Zheng Chen, Jianwen Que, Steven Chen, Oliver McDonald, Alexander Zaika, Wael El-Rifai. APE1/NF-κB/PRDX2 activated by reflux conditions mediates chemoresistance in esophageal adenocarcinoma by suppressing ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3269.

Abstract 5403: Meeting the Physical Activity Guidelines is associated with increased CD8+ T-cell density in the tumor microenvironment of breast cancer patients

Abstract Background: Our current understanding of how physical activity (PA) is associated with immunity in breast cancer (BC) patients is based on pre-clinical evidence gleaned from mouse models or immune cells in the circulating blood, which may not be reflective of the tumor immune microenvironment (TIME). Whether self-reported PA is associated with immune cell infiltration in the breast TIME, the most relevant site for prognosis, remains a fundamental gap in knowledge. Herein, we examined the association of self-reported PA in the year before BC diagnosis with CD8+ T-cell density in the breast TIME. As prior evidence from healthy adults suggests the relationship between exercise and immunity is not linear, we hypothesized that patients reporting the highest PA level would not have the highest CD8+ T-cell density in their tumor. Methods: We leveraged data and tumor samples from 685 women with BC (548 self-identified Black and 137 self-identified White) enrolled in the Women’s Circle of Health Study and the Women’s Circle of Health Follow-Up Study. CD8+ T-cell density per square millimeter of tumor tissue was derived from IHC staining of tissue microarrays. Weekly PA in the year before diagnosis was categorized as inactive; low active (&amp;lt;7.5 MET hours/week); moderately active [7.5-14.9 MET hours/week, the equivalent of meeting the minimum PA Guidelines (PAG)]; or high active (≥15.0 MET-hours/week, exceeding the minimum PAG). Log transformed mean CD8+ T-cell densities were compared by PA level using t-tests and multivariable linear regression with adjustment for age, race, estrogen receptor status, and tumor grade. Results: Meeting the minimum PA Guidelines was associated with significantly higher CD8+ T-cell density in the breast TIME [120.4 (353.6)] in comparison to inactivity [62.0 (154.5), p=0.02]. Although there were also distinct differences in T-cell density between moderate and low-active patients [120.4 (353.6) versus 69.5 (172.6), p=0.10] and moderate and high-active patients [120.4 (353.6) versus 80.7 (223.3), p=0.051], associations were shy of significance. Multivariable linear regression analyses confirmed that moderately active patients had significantly higher CD8+ T-cell density in comparison to inactive patients, overall (p=0.029), and among Black patients (p=0.021). Conclusions: In comparison to inactivity, BC patients meeting the PAG (moderate activity) had significantly increased CD8+ T-cell density in their tumor, which is associated with better survival outcomes. This new translational knowledge suggests exercise-induced immunomodulation may be an important molecular mechanism underlying the association of PA with improved BC survival. As the immune system is a key player in treatment response and efficacy, further study of the role that PA ancillary to therapy may play in improving treatment and survival outcomes is warranted. Citation Format: Rikki Cannioto, Angela Omilian, Evan Davis, Lucas Mendicino, Scott Abrams, Bo Qin, Chi-Chen Hong, Elisa Bandera, Song Yao, Christine Ambrosone. Meeting the Physical Activity Guidelines is associated with increased CD8+ T-cell density in the tumor microenvironment of breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5403.

Basic Transcription Factor 3 Like 4 Enhances Malignant Phenotypes through Modulating Tumor Cell Function and Immune Microenvironment in Glioma

Recent investigations into the tumor microenvironment have provided insights into the limited response of glioma progression to immunotherapy. However, the specific involvement of Basic Transcription Factor 3 Like 4 (BTF3L4) in glioma progression and its correlation with immune cell infiltration remained areas of uncertainty that require further exploration. In the present study, BTF3L4 expression was delineated using gene-expression-profiling-interactive-analysis and multiplex-immunohistological staining of tissue microarrays. Then, the prognostic value of BTF3L4 was assessed using Cox regression models and Kaplan-Meier methods and conducted in vitro experiments to investigate how BTF3L4 protein impacts the proliferation, migration, and invasion capabilities of glioma cells. Furthermore, the Cibersort and Estimate methods were employed to quantify immune cells that correlate to BTF3L4 expression and applied multiplex-immunohistological staining to investigate its correlation with infiltrated immune cells in glioma tissues. These findings revealed a higher BTF3L4 expression in glioma tissues compared to non-tumor brain tissues, which correlated with clinical characteristics and worse patient prognosis. Furthermore, the downregulation of BTF3L4 protein in glioma cell line had a detrimental effect on cell migration, invasion, and proliferation. Additionally, the association between BTF3L4 and key immune molecules in glioma, particularly with the infiltration of CD66B+ neutrophils and PD-L1 expression was identified. These results highlight the prognostic significance of BTF3L4 and propose BTF3L4 as a potential target for glioma immune therapy.

Learning to predict prostate cancer recurrence from tissue images

Roughly 30% of men with prostate cancer who undergo radical prostatectomy will suffer biochemical cancer recurrence (BCR). Accurately predicting which patients will experience BCR could identify who would benefit from increased surveillance or adjuvant therapy. Unfortunately, no current method can effectively predict this. We develop and evaluate PathCLR, a novel semi-supervised method that learns a model that can use hematoxylin and eosin (H&E)-stained tissue microarrays (TMAs) to predict prostate cancer recurrence within 5 years after diagnosis. The learning process involves 2 sequential steps: PathCLR (a) first employs self-supervised learning to generate effective feature representations of the input images, then (b) feeds these learned features into a fully supervised neural network classifier to learn a model for predicting BCR. We conducted training and evaluation using 2 large prostate cancer datasets: (1) the Cooperative Prostate Cancer Tissue Resource (CPCTR) with 374 patients, including 189 who experienced BCR, and (2) the Johns Hopkins University (JHU) prostate cancer dataset of 646 patients, with 451 patients having BCR. PathCLR’s (10-fold cross-validation) F1 score was 0.61 for CPCTR and 0.85 for JHU. This was statistically superior (paired t-test with P <.05) to the best-learned model that relied solely on clinicopathological features, including PSA level, primary and secondary Gleason Grade, etc. We attribute the improvement of PathCLR over models using only clinicopathological features to its utilization of both learned latent representations of tissue core images and clinicopathological features. This finding suggests that there is essential predictive information in tissue images at the time of surgery that goes beyond the knowledge obtained from reported clinicopathological features, helping predict the patient’s 5-year outcome.

ImmunoPET imaging of Trop2 expression in solid tumors with nanobody tracers.

The high expression of the transmembrane glycoprotein trophoblast cell-surface antigen 2 (Trop2) was strongly associated with the progression of solid tumors, including pancreatic and gastric cancers. Our study aimed to construct Trop2-specific immuno-positron emission tomography (immunoPET) probes and assess the diagnostic abilities in preclinical pancreatic and gastric cancer models. The expression of Trop2 in pancreatic cancer was determined by single-cell sequencing and immunohistochemistry on tissue microarray (TMA). Flow cytometry was used to screen the expression of Trop2 in pancreatic cancer cell lines. Two nanobodies (i.e., RTD98 and RTD01) targeting Trop2 were developed and labeled with gallium-68 (68Ga, T1/2 = 1.1 h) to construct immunoPET imaging probes. The agents were researched in cell-derived pancreatic and patient-derived gastric cancer models expressing varying Trop2. Single-cell sequencing results showed high expression of Trop2 in pancreatic ductal cells as well as acinar cells and immunohistochemical staining of TMA from pancreatic cancers showed significantly higher expression of Trop2 in cancerous than in paracancerous tissues. ImmunoPET utilizing [68Ga]Ga-NOTA-RTD98 could clearly delineate subcutaneous tumors, both in cell-derived pancreatic cancer models and patient-derived gastric cancer models, superior to imaging using [18F]-FDG or a non-specific probe [68Ga]Ga-NOTA-RTD161. Another probe with improved pharmacokinetics targeting Trop2, [68Ga]Ga-NOTA-RTD01, was further prepared and showed advantageous diagnostic capabilities in preclinical pancreatic cancer models. In the work, we reported two nanobody tracers targeting human Trop2 which may facilitate better use of Trop2-targeted therapeutics by noninvasively displaying expression dynamics of the target.

Identification of Hepatocellular Carcinoma-Specific Targets for Imaging and Therapy

Despite advances in the diagnosis and treatment of hepatocellular carcinoma (HCC), there are no tumor-selective agents that are clinically approved in the US for this disease. Here, we aimed to identify and validate molecules that are overexpressed on HCC plasma membrane compared to normal tissues, which could be facilitate the design, engineering, and testing of tumor-selective imaging and therapeutic agents. We analyzed next-generation sequencing (NGS) public datasets (TCGA and TIGER-LC) and NCI single cell RNA-sequencing datasets to identify overexpressed plasma membrane molecules and aimed to validate these targets using immunohistochemical staining (IHC) of patient tissue microarrays (TMAs), and flow cytometry using liver cancer cell lines (Huh7, HepG2, and Hep3B). NGS data identified GPC3, EGFR, MET, MUC13, and ROBO1 molecules overexpressed in HCC relative to non-tumor tissues. In HepG2 cell line, EGFR (p<0.05) and MET (p<0.01) demonstrated statistically significant increased median fluorescence intensity (MFI) relative to controls in flow cytometry. In the Hep3B cell line, MET, GPC3, and EGFR demonstrated an increased MFI relative to the control (p<0.01). No statistically significant difference was observed in Huh7 cell lines. IHC staining of TMAs for GPC3, MET, MUC14, and ROBO1 showed statistically significantly higher staining relative to the normal tumor tissue (p<0.001). We identified and validated plasma membrane molecules overexpressed in HCC compared to non-tumor tissue. Because GPC3, a well-known HCC-specific marker that is expressed in 75% of HCC, was identified using our approach, we are confident that that additional molecules may also represent promising HCC-selective targets. This work could facilitate the design, engineering, and testing of novel precision oncology imaging and therapeutic agents for HCC.

JS08.5.A VEGFR2-SPECIFIC CAR T CELLS WITH ANTI-GLIOMA AND ANTI-ANGIOGENIC ACTIVITY AGAINST GLIOBLASTOMA

Abstract BACKGROUND Chimeric antigen receptor (CAR) T cell therapy has emerged as a potent immunotherapy against hematological malignancies. In glioblastoma, however, limited infiltration of the tumor by CAR T cells, tumor heterogeneity, antigen escape and an immunosuppressive tumor microenvironment remain significant obstacles. Vessel-targeting CAR T cells have been shown to infiltrate solid tumors more efficiently and have the additional benefit of co-targeting the tumor vasculature with genomic stability. Therefore, vascular endothelial growth factor receptor 2 (VEGFR2)-CAR T cells may represent a promising strategy by targeting the tumor vasculature as well as VEGFR2-expressing tumor cells. Here, we explored the efficacy of VEGFR2-specific CAR T cells against experimental gliomas as well as the contribution of anti-tumor and anti-vasculature-dependent effects of this strategy. METHODS Tissue microarrays of glioblastoma patients (n=113) were stained for VEGFR2 expression. Human CAR T cells were generated by lentiviral transduction to express a second generation CAR construct against either mouse or human VEGFR2 (mVEGFR2 or hVEGFR2). Their activity was assessed in co-culture assays in vitro against murine endothelial and human glioma cells, respectively. Several orthotopic xenograft mouse glioma models were used to test the in vivo activity of the newly generated CAR T cells. RESULTS We confirmed high VEGFR2 expression on endothelial cells in glioblastoma tissue of stained tissue microarrays as well as in 20.3% of tissue samples also on tumor cells. In co-culture assays, hVEGFR2-CAR T cells were exclusively active against human glioma cells and mVEGFR2-CAR T cells against mouse endothelial cells, respectively. The specificity of hVEGFR2-CAR T cells was confirmed by a CRISPR/Cas9-mediated knockout of hVEGFR2 in a human glioma cell line. In all three in vivo glioma models, intratumoral treatment of hVEGFR2-CAR T cells significantly prolonged the survival of glioma-bearing mice and cured a substantial fraction of these animals in one model. Additionally, we found that survival was prolonged after mVEGFR2-CAR T cell treatment in one glioma model, which correlated with high vascularization of these tumors. CONCLUSION Our dataset demonstrates that VEGFR2-CAR T cells prolong the survival of glioma-bearing mice through anti-glioma and anti-glioma vasculature activity. The results suggest that the magnitude of the vasculature-targeting activity depends on vessel-density within the tumor. VEGFR2 might be a relevant target that could be exploited for a novel CAR T cell-based immunotherapeutic approach against glioblastoma.

SIRT4 is an independent prognostic factor in bladder cancer and inhibits bladder cancer growth by suppressing autophagy

BackgroundNucleosome-localized sirtuin 4 (SIRT4) was found to function as an oncogene and tumor suppressor gene in different tumors. However, the clinical significance of SIRT4 in bladder urothelial carcinoma (BLCA) has not been assessed, nor has the function of SIRT4 in BLCA been analyzed.MethodsIn this study, we assessed the levels of SIRT4 protein in BLCA tissues and its association with clinicopathological parameters and overall survival time of BLCA patients by immunohistochemical staining of tissue microarrays containing 59 BLCA patients. Then, we constructed BLCA cell lines (T24) with overexpression or interference of SIRT4 by lentiviral infection. The effects of SIRT4 on the proliferation, migration and invasive ability of T24 cells were investigated using cell counting kit-8 (CCK-8) assays, wound healing assays, and migration and invasion assays. Moreover, we also investigated the effect of SIRT4 on the cell cycle and apoptosis of T24 cells. Mechanistically, we explored the relationship between SIRT4 and autophagy and its role in the inhibition of BLCA.ResultsWe found by immunohistochemistry that SIRT4 protein levels were reduced in BLCA and that lower SIRT4 levels were associated with larger tumor volumes, later T-staging and later AJCC staging in BLCA patients and were an independent prognostic factor in BLCA patients. Overexpression of SIRT4 significantly inhibited the proliferative viability, scratch healing capacity, migratory capacity, and invasive capacity of T24 cells, while interference with SIRT4 had the opposite effect. Moreover, overexpression of SIRT4 significantly inhibited the cell cycle and increased the apoptosis rate of T24 cells. Mechanistically, SIRT4 inhibits BLCA growth by suppressing autophagic flow.ConclusionsOur study suggests that SIRT4 is an independent prognostic factor for BLCA and that SIRT4 plays a tumor suppressor role in BLCA. This suggests a potential target for SIRT4 in the diagnosis and treatment of BLCA.

Ubiquitin-Specific Protease 43 Impacts Pancreatic Ductal Adenocarcinoma Prognosis by Altering Its Proliferation and Infiltration of Surrounding Immune Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer, and the therapy options for PDAC remain restricted. The distinctive tumor immunological microenvironment (TIME) of PDAC, comprising a high number of stromal cells and a limited infiltration of cytotoxic T lymphocytes (CTLs), rendered immunotherapy ineffective. The protein level of ubiquitin-specific protease 43 (USP43) was a prognostic predictor in numerous cancers; however, its function in PDAC is limited. This article focuses on the influence of USP43 expression on PDAC prognosis and TIME alteration. Based on TCGA database and tissue microarray staining, the expression of USP43 in PDAC was evaluated. The association between USP43 and prognosis was then investigated using tissue samples and online databases. In PDAC tumor tissues, the correlation between USP43 expression and clinicopathological characteristics, immune cell infiltration, and prognosis was investigated. The expression of USP43 in PDAC cell lines was evaluated using quantitative polymerase chain reaction. Using a cell counting kit-8 (CCK-8) and a cell colony formation test, the viability of the cells was determined. On the basis of online databases and tissue samples, the link between USP43 and immune cell infiltration around PDAC was also examined. For statistical analyses, the software GraphPad, R, and SPSS 26.0 were utilized. The expression of USP43 was considerably higher in PDAC compared to normal pancreatic tissue in both the TCGA database and the tissue microarrays of PDAC patients (P < 0.001). High USP43 expression was associated with poor overall survival in both the TCGA database and the tissue microarray of PDAC patients (P = 0.046 and 0.021, respectively). USP43 overexpression promoted PANC-1 cell proliferation (P = 0.0018), but USP43 knockdown decreased PANC02 cell proliferation (P < 0.001). According to the TCGA database, USP43 is associated with T cell activation and inhibits CD8+ T cell activation in PDAC, as proven by a study of cell lines. Moreover, in both TCGA and PDAC cell lines, USP43 expression was negatively linked with the chemokine signaling pathway. Overexpression of USP43 is a potential prognostic indicator for PDAC patients. USP43 is a potential biomarker associated with T cell activation, suppression of CD8+ T cell enrichment, and the cytokine signal pathway. Future multicenter studies are needed to confirm our findings and their potential application in the treatment of PDAC patients.

Integrated clinical analysis and data mining assessed the impact of NOX4 on the immune microenvironment and prognosis of pancreatic cancer.

The tumor microenvironment (TME) of pancreatic cancer is complex. which forms forms a microenvironment with high immunosuppression, ischemia and hypoxia, which promotes tumor proliferation and migration, inhibit the anti-tumor immune response. NOX4 plays an important role in tumor microenvironment and has a significant relationship with the occurrence, development and drug resistance of tumor. Firstly, NOX4 expression in pancreatic cancer tissues under different pathological conditions was detected by applying immunohistochemical staining of tissue microarray (TMA). Transcriptome RNA sequencing data and clinical data of 182 pancreatic cancer samples were downloaded and collated from the UCSC xena database. 986 NOX4-related lncRNAs were filtered by Spearman correlation analysis. prognosis-related NOX4-related lncRNAs and NRlncSig Score were finally obtained by univariate and multivariate Cox regression with Least Absolute Shrinkage and Selection Operator (Lasso) analysis in pancreatic cancer patients. we plotted Kaplan -Meier and time-dependent ROC curves (ROC) to assess the validity in predicting the prognosis of pancreatic cancer. The ssGSEA analysis was applied to explore the immune microenvironment of pancreatic cancer patients as well as to discuss the immune cells and immune status separately. We found that a mature tumor marker, NOX4, play different roles in different clinical subgroups by immunohistochemical analysis and clinical data. Finally, 2 NOX4-related lncRNAs were determined by least absolute shrinkage and selection operator (LASSO) analysis, univariate Cox analysis and multivariate COX analysis. The ROC curve and DCA curve showed that NRS Score had better predictive ability than independent prognosis-related lncRNA and other clinicopathologic indicators. We obtained the relative abundance of 28 infiltrating immune cells by ssGSEA analysis and found a significant positive correlation between the abundance of anti-tumor immune cells and tumor-promoting immune cells in the risk-classified microenvironment. No matter NRS Score or AC092667.2, RP11-349A8.3 was significantly correlated with immune infiltrating cells. Meanwhile, the IC50 of conventional chemotherapeutic agents in high-score group were significantly lower than those in low-score group. As a mature tumor marker, NOX4-related lncRNAs provide new research strategies for prognostic evaluation, molecular mechanism and clinical treatment of pancreatic cancer.