LDOC1 as Negative Prognostic Marker for Vulvar Cancer Patients

So far, studies about targeted therapies and predictive biomarkers for vulva carcinomas are rare. The leucine zipper downregulated in cancer 1 gene (LDOC1) has been identified in various carcinomas as a tumor-relevant protein influencing patients’ survival and prognosis. Due to the lack of information about LDOC1 and its exact functionality, this study focuses on the expression of LDOC1 in vulvar carcinoma cells and its surrounding immune cells as well as its correlation to clinicopathological characteristics and prognosis. Additionally, a possible regulation of LDOC1 in vulvar cancer cell lines via the NF-κB signaling pathway was analyzed. Vulvar carcinoma sections of 157 patients were immunohistochemically stained and examined regarding LDOC1 expression by using the immunoreactive score (IRS). To characterize LDOC1-positively stained immune cell subpopulations, immunofluorescence double staining was performed. The effect of the NF-κB inhibitor C-DIM 12 (3,3′-[(4-chlorophenyl)methylene]bis[1 H-indole]) on vulvar cancer cell lines A431 and SW 954 was measured according to MTT and BrdU assays. Baseline expression levels of LDOC1 in the vulvar cancer cell lines A431 and SW 954 was analyzed by real-time PCR. LDOC1 was expressed by about 90% of the cancer cells in the cytoplasm and about half of the cells in the nucleus. Cytoplasmatic expression of LDOC1 was associated with decreased ten-year overall survival of the patient, whereas nuclear staining showed a negative association with disease-free survival. Infiltrating immune cells were mainly macrophages followed by regulatory T cells. Incubation with C-DIM 12 decreased the cell viability and proliferation of vulvar cancer cell line A431, but not of cell line SW 954. LDOC1 expression on mRNA level was twice as high in the cell line A431 compared to the cell line SW 954. Overexpression of LDOC1 was associated with unfavorable overall and disease-free survival. Tumor growth could be inhibited by C-DIM 12 in vitro if the expressed LDOC1 level was high enough.     

Nanoparticle-Aided Detection of Colorectal Cancer-Associated Glycoconjugates of Extracellular Vesicles in Human Serum

Extracellular vesicles (EVs) are found in all biological fluids, providing potential for the identification of disease biomarkers such as colorectal cancer (CRC). EVs are heavily glycosylated with specific glycoconjugates such as tetraspanins, integrins, and mucins, reflecting the characteristics of the original cell offering valuable targets for detection of CRC. We report here on europium-nanoparticle (EuNP)-based assay to detect and characterize different surface glycoconjugates of EVs without extensive purification steps from five different CRC and the HEK 293 cell lines. The promising EVs candidates from cell culture were clinically evaluated on small panel of serum samples including early-stage (n = 11) and late-stage (n = 11) CRC patients, benign condition (n = 11), and healthy control (n = 10). The majority of CRC cell lines expressed tetraspanin sub-population and glycovariants of integrins and conventional tumor markers. The subpopulation of CD151 having CD63 expression (CD151^CD63) was significantly (p = 0.001) elevated in early-stage CRC (8 out of 11) without detecting any benign and late-stage samples, while conventional CEA detected mostly late-stage CRC (p = 0.045) and with only four early-stage cases. The other glycovariant assays such as CEA^Con-A, CA125^WGA, CA 19.9^Ma696, and CA 19.9^Con-A further provided some complementation to the CD151^CD63 assay. These results indicate the potential application of CD151^CD63 assay for early detection of CRC patients in human serum.     

Prognostic Value of Association of Copy Number Alterations and Cell-Surface Expression Markers in Newly Diagnosed Multiple Myeloma Patients

Multiple myeloma results from the clonal proliferation of abnormal plasma cells (PCs) in the bone marrow (BM). In this study, the cell surface expression markers (CD) on atypical PCs (detected by multiparametric flow cytometry (MFC)) were correlated with copy number alterations (CNAs) in the genome (detected by multiplex ligation-dependent probe amplification (MLPA)) to assess their impact on prognosis in newly diagnosed MM patients. Statistically significant results were obtained when different stages of PC maturation (classified based on CD19 and CD81 expression) were associated with CD117 expression and identified CNAs. In the intermediately differentiated PC group (CD19(−) CD81(+)), patients who didn’t express CD117 had a lower median progression free survival (PFS) (p = 0.024). Moreover, within this group, patients with less than three adverse CNAs, which harbor CD117, had a better outcome with a PFS of more than 48 months compared with 19 months (p = 0.008). Considering all the results, our study suggested the need to integrate both the CD markers and copy number alterations to evaluate the prognosis of newly diagnosed multiple myeloma patients.     

Isolation and Enhancement of a Homogenous in Vitro Human Hertwig’s Epithelial Root Sheath Cell Population

Hertwig’s epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.     

A Comprehensive Prognostic Analysis of Tumor-Related Blood Group Antigens in Pan-Cancers Suggests That SEMA7A as a Novel Biomarker in Kidney Renal Clear Cell Carcinoma

Blood group antigen is a class of heritable antigenic substances present on the erythrocyte membrane. However, the role of blood group antigens in cancer prognosis is still largely unclear. In this study, we investigated the expression of 33 blood group antigen genes and their association with the prognosis of 30 types of cancers in 31,870 tumor tissue samples. Our results revealed that blood group antigens are abnormally expressed in a variety of cancers. The high expression of these antigen genes was mainly related to the activation of the epithelial-mesenchymal transition (EMT) pathway. High expression of seven antigen genes, i.e., FUT7, AQP1, P1, C4A, AQP3, KEL and DARC, were significantly associated with good OS (Overall Survival) in six types of cancers, while ten genes, i.e., AQP1, P1, C4A, AQP3, BSG, CD44, CD151, LU, FUT2, and SEMA7A, were associated with poor OS in three types of cancers. Kidney renal clear cell carcinoma (KIRC) is associated with the largest number (14 genes) of prognostic antigen genes, i.e., CD44, CD151, SEMA7A, FUT7, CR1, AQP1, GYPA, FUT3, FUT6, FUT1, SLC14A1, ERMAP, C4A, and B3GALT3. High expression of SEMA7A gene was significantly correlated with a poor prognosis of KIRC in this analysis but has not been reported previously. SEMA7A might be a putative biomarker for poor prognosis in KIRC. In conclusion, our analysis indicates that blood group antigens may play functional important roles in tumorigenesis, progression, and especially prognosis. These results provide data to support prognostic marker development and future clinical management.     

Inhibition of α(1,6)fucosyltransferase: Effects on Cell Proliferation,Migration, and Adhesion in an SW480/SW620 Syngeneic Colorectal Cancer Model

The present study explored the impact of inhibiting α(1,6)fucosylation (core fucosylation) on the functional phenotype of a cellular model of colorectal cancer (CRC) malignization formed by the syngeneic SW480 and SW620 CRC lines. Expression of the FUT8 gene encoding α(1,6)fucosyltransferase was inhibited in tumor line SW480 by a combination of shRNA-based antisense knockdown and Lens culinaris agglutinin (LCA) selection. LCA-resistant clones were subsequently assayed in vitro for proliferation, migration, and adhesion. The α(1,6)FT-inhibited SW480 cells showed enhanced proliferation in adherent conditions, unlike their α(1,6)FT-depleted SW620 counterparts, which displayed reduced proliferation. Under non-adherent conditions, α(1,6)FT-inhibited SW480 cells also showed greater growth capacity than their respective non-targeted control (NTC) cells. However, cell migration decreased in SW480 after FUT8 knockdown, while adhesion to EA.hy926 cells was significantly enhanced. The reported results indicate that the FUT8 knockdown strategy with subsequent selection for LCA-resistant clones was effective in greatly reducing α(1,6)FT expression in SW480 and SW620 CRC lines. In addition, α(1,6)FT impairment affected the proliferation, migration, and adhesion of α(1,6)FT-deficient clones SW480 and SW620 in a tumor stage-dependent manner, suggesting that core fucosylation has a dynamic role in the evolution of CRC.     

Tumor-Stroma Ratio and Programmed Cell Death Ligand 1 Expression in Preoperative Biopsy and Matched Laryngeal Carcinoma Surgical Specimen

Programmed cell death ligand 1 (PD-L1) seems to rely on close relations between neoplastic and immune cells in the tumor microenvironment. Tumor to stroma ratio (TSR) has been associated with prognosis in different malignancies. The aims of this exploratory investigation were to analyze for the first time the: (i) association between TSR, PD-L1 expression and other clinical–pathological features in laryngeal squamous cell carcinoma (LSCC) biopsies and paired surgical specimens; (ii) prognostic and predictive role of TSR and PD-L1. TSR, PD-L1 expression (in terms of combined positive score [CPS]), and other clinical–pathological features were analyzed in biopsies and surgical specimens of 43 consecutive LSCC cases. A CPS < 1 evaluated on surgical specimens was associated with a low TSR (stroma rich) on both biopsies and surgical specimens (p = 0.0143 and p = 0.0063). Low TSR showed a significant negative prognostic value when evaluated on both biopsies and surgical specimens (HR = 8.808, p = 0.0003 and HR = 11.207, p = 0.0002). CPS ≥ 1 appeared to be a favorable prognostic factor (HR = 0.100, p = 0.0265). The association between bioptic and surgical specimen TSR and PD-L1 expression should be further investigated for a potential impact on targeted treatments, also with regard to immunotherapeutic protocols.     

PD-L1 Expression in High-Risk Early-Stage Colorectal Cancer—Its Clinical and Biological Significance in Immune Microenvironment

Programmed death-ligand 1 (PD-L1) is an immune checkpoint molecule that can regulate immune responses in the tumor microenvironment (TME); however, the clinical applications of PD-L1 in early-stage colorectal cancer (CRC) remain unclear. In this study, we aimed to investigate the relationship between PD-L1 expression and survival outcome and explore its relevant immune responses in CRC. PD-L1 expression was evaluated by immunohistochemical staining to determine the tumor proportion score and combined positive score (CPS) in a Taiwanese CRC cohort. The oncomine immune response research assay was conducted for immune gene expression analyses. CRC datasets from the TCGA database were reappraised for PD-L1-associated gene enrichment analyses using GSEA. The high expression of PD-L1 (CPS ≥ 5) was associated with longer recurrence-free survival (p = 0.031) and was an independent prognostic factor as revealed by multivariate analysis. High PD-L1 expression was related to six immune-related gene signatures, and CXCL9 is the most significant overexpressed gene in differential analyses. High CXCL9 expression correlated with increased infiltration levels of immune cells in the TME, including CD8+ T lymphocytes and M1 macrophages. These findings suggest that high PD-L1 expression is a prognostic factor of early-stage CRC, and CXCL9 may play a key role in regulating PD-L1 expression.     

Application of Immunoprofiling Using Multiplexed Immunofluorescence Staining Identifies the Prognosis of Patients with High-Grade Serous Ovarian Cancer

Immunoprofiling has an established impact on the prognosis of several cancers; however, its role and definition in high-grade serous ovarian cancer (HGSOC) are mostly unknown. This study is to investigate immunoprofiling which could be a prognostic factor in HGSOC. We produced tumor microarrays of 187 patients diagnosed with HGSOC. We performed a multiplexed immunofluorescence staining using Opal Multiplex IHC kit and quantitative analysis with Vectra-Inform system. The expression intensities of programmed death-ligand 1 (PD-L1), CD4, CD8, CD20, FoxP3, and CK in whole tumor tissues were evaluated. The enrolled patients showed general characteristics, mostly FIGO stage III/IV and responsive to chemotherapy. Each immune marker showed diverse positive densities, and each tumor sample represented its immune characteristics as an inflamed tumor or noninflamed tumor. No marker was associated with survival as a single one. Interestingly, high ratios of CD8 to FoxP3 and CD8 to PD-L1 were related to the favorable overall survival (77 vs. 39 months, 84 vs. 47 months, respectively), and CD8 to PD-L1 ratio was also a significant prognostic factor (HR 0.621, 95% CI 0.420–0.917, p = 0.017) along with well-known clinical prognostic factors. Additionally, CD8 to PD-L1 ratio was found to be higher in the chemosensitive group (p = 0.034). In conclusion, the relative expression levels of CD8, FoxP3, and PD-L1 were significantly related to the clinical outcome of patients with HGSOC, which could be a kind of significant immunoprofiling of ovarian cancer patients to apply for treatment.     

CD86+/CD206+, Diametrically Polarized Tumor-Associated Macrophages,Predict Hepatocellular Carcinoma Patient Prognosis

Tumor-associated macrophages (TAMs), the most abundant infiltrating immune cells in tumor microenvironment, have distinct functions in hepatocellular carcinoma (HCC) progression. CD68⁺ TAMs represent multiple polarized immune cells mainly containing CD86⁺ antitumoral M1 macrophages and CD206⁺ protumoral M2 macrophages. TAMs expression and density were assessed by immunohistochemical staining of CD68, CD86, and CD206 in tissue microarrays from 253 HCC patients. Clinicopathologic features and prognostic value of these markers were evaluated. We found that CD68⁺ TAMs were not associated with clinicopathologic characteristics and prognosis in HCC. Low presence of CD86⁺ TAMs and high presence of CD206⁺ TAMs were markedly correlated with aggressive tumor phenotypes, such as multiple tumor number and advanced tumor-node-metastasis (TNM) stage; and were associated with poor overall survival (OS) (p = 0.027 and p = 0.024, respectively) and increased time to recurrence (TTR) (p = 0.037 and p = 0.031, respectively). In addition, combined analysis of CD86 and CD206 provided a better indicator for OS (p = 0.011) and TTR (p = 0.024) in HCC than individual analysis of CD86 and CD206. Moreover, CD86⁺/CD206⁺ TAMs predictive model also had significant prognosis value in α-fetoprotein (AFP)-negative patients (OS: p = 0.002, TTR: p = 0.005). Thus, these results suggest that combined analysis of immune biomarkers CD86 and CD206 could be a promising HCC prognostic biomarker.     

The Prognostic Significance of FKBP1A and Its Related Immune Infiltration in Liver Hepatocellular Carcinoma

Liver hepatocellular carcinoma (LIHC) remains a global health challenge with poor prognosis and high mortality. FKBP1A was first discovered as a receptor for the immunosuppressant drug FK506 in immune cells and is critical for various tumors and cancers. However, the relationships between FKBP1A expression, cellular distribution, tumor immunity, and prognosis in LIHC remain unclear. Here, we investigated the expression level of FKBP1A and its prognostic value in LIHC via multiple datasets including ONCOMINE, TIMER, GEPIA, UALCAN, HCCDB, Kaplan–Meier plotter, LinkedOmics, and STRING. Human liver tissue microarray was employed to analyze the characteristics of FKBP1A protein including the expression level and pathological alteration in cellular distribution. FKBP1A expression was significantly higher in LIHC and correlated with tumor stage, grade and metastasis. The expression level of the FKBP1A protein was also increased in LIHC patients along with its accumulation in endoplasmic reticulum (ER). High FKBP1A expression was correlated with a poor survival rate in LIHC patients. The analysis of gene co-expression and the regulatory pathway network suggested that FKBP1A is mainly involved in protein synthesis, metabolism and the immune-related pathway. FKBP1A expression had a significantly positive association with the infiltration of hematopoietic immune cells including B cells, CD8⁺ T cells, CD4⁺ T cells, macrophages, neutrophils, and dendritic cells. Moreover, M2 macrophage infiltration was especially associated with a poor survival prognosis in LIHC. Furthermore, FKBP1A expression was significantly positively correlated with the expression of markers of M2 macrophages and immune checkpoint proteins such as PD-L1, CTLA-4, LAG3 and HAVCR2. Our study demonstrated that FKBP1A could be a potential prognostic target involved in tumor immune cell infiltration in LIHC.     

Expression and Significance of CD44, CD47 and c-met in Ovarian Clear Cell Carcinoma

Aims: The aim of the present study is to investigate the differential expression of CD44, CD47 and c-met in ovarian clear cell carcinoma (OCCC), the correlation in their expression and their relationship with the biological behavior of OCCC. Methods: We used immunohistochemistry to examine the expression of CD44, CD47 and c-met in OCCC (86 cases) and investigated the effects of the expression and interaction of these molecules on the development of OCCC. Results: CD44, CD47 and c-met expression was significantly high in OCCC. Expression of CD44 and CD47 correlated with patient surgical stage, chemotherapy resistance and prognosis (all p < 0.05), and expression of c-met correlated with chemotherapy resistance and prognosis (all p < 0.05), but did not correlate with lymph node metastasis (all p > 0.05). The surgical stage, CD44, CD47 and c-met expression were independent risk factors for OCCC prognosis (all p < 0.05). Patients with low levels of CD44, CD47 and c-met showed better survival than those with high levels (all p < 0.05). There was a positive correlation between CD44 (or CD47) and c-met, as well as between CD44 and CD47 (the Spearman correlation coefficient r_s was 0.783, 0.776 and 0.835, respectively, all p < 0.01). Additionally, pairwise correlation analysis of these three markers shows that the high expression of CD44/CD47, CD44/c-met and CD47/c-met were correlated with patient surgical stage, chemotherapy resistance and prognosis (all p < 0.05), but did not correlate with lymph node metastasis (all p > 0.05). Conclusions: Expression of CD44, CD47 and c-met was upregulated in OCCC and pairwise correlation. CD44, CD47 and c-met may have synergistic effects on the development of OCCC and are prognostic factors for ovarian cancer.     

PD-L1 Expression in 65 Conjunctival Melanomas and Its Association with Clinical Outcome

Conjunctival melanoma (CM) iss a rare and aggressive tumour that is increasing in frequency. The prognostic value of PD-L1 expression, alone or in combination with CD8 and PD-1 expression and the BRAF and NRAS status, has not been determined in CM to date. We evaluated the expression of PD-L1, CD8, PD-1 in CM and investigated whether there was an association between the expression of these markers and the BRAF and NRAS molecular profile as well as some clinico-pathological criteria. A total of sixty-five CM were assessed for PD-L1, PD-1, and CD8 expression by immunohistochemistry (IHC) and for BRAF and NRAS genomic alterations using molecular biology techniques and anti-BRAF and anti-NRAS antibodies. PD-L1 expression in tumour cells (TC) was very low or absent but detected in tumour-infiltrating immune cells (IC). A correlation was observed between the expression of PD-L1, CD8, and PD-1 in IC. No correlation between PD-L1 expression (in tumour and/or immune cells) and BRAF or NRAS mutations was observed. PD-L1 expression in IC correlated with a higher pTNM stage and PD-L1 expression in TC with worse disease-specific survival. PD-L1 expression is a potential prognostic biomarker that correlates with poor prognosis in CM patients.     

CD44V3, an Alternatively Spliced Form of CD44, Promotes Pancreatic Cancer Progression

Pancreatic cancer is one of the most lethal malignant tumors. However, the molecular mechanisms responsible for its progression are little known. This study aimed to understand the regulatory role of CD44V3 in pancreatic cancer. A Kaplan–Meier analysis was performed to reveal the correlation between CD44/CD44V3 expression and the prognosis of pancreatic cancer patients. CD44V3 and U2AF1 were knocked down using shRNAs. The proliferation, migration, invasion, and stemness of two pancreatic cell lines, BxPC-3 and AsPC-1, were examined. The expression of CD44V3, cancer-associated markers, and the activation of AKT signaling were detected by qRT-PCR and Western blot. Both CD44 and CD44V3 expression levels were associated with a poor prognosis in pancreatic cancer patients. Interestingly, the expression of CD44V3, instead of CD44, was greatly increased in tumor tissues. CD44V3 knockdown inhibited the proliferation, migration, invasion, and stemness of cancer cells. CD44V3 splicing was regulated by U2AF1 and downregulation of U2AF1 enhanced CD44V3 expression, which promoted pancreatic cancer progression. CD44V3 is an important cancer-promoting factor, which may serve as a potential candidate for pancreatic cancer intervention.     

CD82 and Gangliosides Tune CD81 Membrane Behavior

Tetraspanins are a family of transmembrane proteins that form a network of protein–protein interactions within the plasma membrane. Within this network, tetraspanin are thought to control the lateral segregation of their partners at the plasma membrane through mechanisms involving specific lipids. Here, we used a single molecule tracking approach to study the membrane behavior of tetraspanins in mammary epithelial cells and demonstrate that despite a common overall behavior, each tetraspanin (CD9, CD81 and CD82) has a specific signature in terms of dynamics. Furthermore, we demonstrated that tetraspanin dynamics on the cell surface are dependent on gangliosides. More specifically, we found that CD82 expression increases the dynamics of CD81 and alters its localization at the plasma membrane, this has no effect on the behavior of CD9. Our results provide new information on the ability of CD82 and gangliosides to differentially modulate the dynamics and organization of tetraspanins at the plasma membrane and highlight that its lipid and protein composition is involved in the dynamical architecture of the tetraspanin web. We predict that CD82 may act as a regulator of the lateral segregation of specific tetraspanins at the plasma membrane while gangliosides could play a crucial role in establishing tetraspanin-enriched areas.