Potential Anti-Metastatic Role of the Novel miR-CT3 in Tumor Angiogenesis and Osteosarcoma Invasion

Osteosarcoma (OS) is the most common primary bone tumor mainly occurring in young adults and derived from primitive bone-forming mesenchyme. OS develops in an intricate tumor microenvironment (TME) where cellular function regulated by microRNAs (miRNAs) may affect communication between OS cells and the surrounding TME. Therefore, miRNAs are considered potential therapeutic targets in cancer and one of the goals of research is to accurately define a specific signature of a miRNAs, which could reflect the phenotype of a particular tumor, such as OS. Through NGS approach, we previously found a specific molecular profile of miRNAs in OS and discovered 8 novel miRNAs. Among these, we deepen our knowledge on the fifth candidate renamed now miR-CT3. MiR-CT3 expression was low in OS cells when compared with human primary osteoblasts and healthy bone. Through TargetScan, VEGF-A was predicted as a potential biological target of miR-CT3 and luciferase assay confirmed it. We showed that enforced expression of miR-CT3 in two OS cell lines, SAOS-2 and MG-63, reduced expression of VEGF-A mRNA and protein, inhibiting tumor angiogenesis. Enforced expression of miR-CT3 also reduced OS cell migration and invasion as confirmed by soft agar colony formation assay. Interestingly, we found that miR-CT3 behaves inducing the activation of p38 MAP kinase pathway and modulating the epithelial-mesenchymal transition (EMT) proteins, in particular reducing Vimentin expression. Overall, our study highlights the novel role of miR-CT3 in regulating tumor angiogenesis and progression in OS cells, linking also to the modulation of EMT proteins.

Effects of host vimentin on Eimeria tenella sporozoite invasion

Background Chicken coccidiosis is a parasitic disease caused by Eimeria of Apicomplexa, which has caused great economic loss to the poultry breeding industry. Host vimentin is a key protein in the process of infection of many pathogens. In an earlier phosphorylation proteomics study, we found that the phosphorylation level of host vimentin was significantly regulated after Eimeria tenella sporozoite infection. Therefore, we explored the role of host vimentin in the invasion of host cells by sporozoites. Methods Chicken vimentin protein was cloned and expressed. We used qPCR, western blotting, and indirect immunofluorescence to detect levels of mRNA transcription, translation, and phosphorylation, and changes in the distribution of vimentin after E. tenella sporozoite infection. The sporozoite invasion rate in DF-1 cells treated with vimentin polyclonal antibody or with small interfering RNA (siRNA), which downregulated vimentin expression, was assessed by an in vitro invasion test. Results The results showed that vimentin transcription and translation levels increased continually at 6–72 h after E. tenella sporozoite infection, and the total phosphorylation levels of vimentin also changed. About 24 h after sporozoite infection, vimentin accumulated around sporozoites in DF-1 cells. Treating DF-1 cells with vimentin polyclonal antibody or downregulating vimentin expression by siRNA significantly improved the invasion efficiency of sporozoites. Conclusion In this study, we showed that vimentin played an inhibitory role during the invasion of sporozoites. These data provided a foundation for clarifying the relationship between Eimeria and the host. Graphical Abstract

THE ROLE OF IMMUNOHISTOCHEMICAL MARKERS IN PREDICTING RECURRENCE OR DEATH IN PATIENTS WITH MEDULAR THYROID CANCER

Introduction. Medullary thyroid cancer (MTC) belongs to a class of rare neuroendocrine aggressive tumors and arises from parafollicular cells (C-cells). An important modern problem is the development of ways to predict the recurrence of this disease. The aim of the study is to determine the role of immunohistochemical tumor markers of medullary thyroid cancer in predicting recurrence or death. Materials and methods. The analysis of the prospective study included 22 patients with MTC, 5 of whom have developed a recurrence and 4 have died at the end of the 10-year (120 months) follow-up period. Immunohistochemical examinations were performed using monoclonal antibodies of tumor markers calcitonin, chromogranin A, vimentin and Ki-67. Results. The discrepancy between the data of histological and immunohistochemical examinations in MTC is 12.0%, which indicates the hyperdiagnosis of this nosology and argues the importance of performing immunohistochemical examinations to verify the diagnosis. Patients who had a recurrence of MTC had significantly (p <0.05) lower levels of calcitonin expression (5.00 [5.00; 5.00] points) compared with patients who did not relapse, where this figure was 6.00 [6.00; 7.00] points. In patients with MTC, an increase in calcitonin expression was significantly associated with an increase in chromogranin A expression (r = + 0.49, p = 0.02); a similar relationship was found for the proportions of immunopositive cells of these tumor markers: r = + 0.68, p = 0.001. At the same time, it was found that the increase in the level of calcitonin expression was apparently combined with the decrease in the level of Ki-67 expression (r = -0.52, p = 0.02). It was also found that the increase in the level of vimentin expression is combined with an increase in the expression (r = + 0.64, p = 0.001) and the proportion of immunopositive cells of chromogranin A (r = + 0.45, p = 0.038). Conclusions. Low levels of calcitonin expression are prognostically unfavorable markers for the recurrence of MTC. Specific tumor markers are important in the treatment process and for the dynamic monitoring of patients with MTC.

Endometrial Metaplastic/Reactive Changes Coexistent with Endometrial Hyperplasia and Carcinoma: A Morphological and Immunohistochemical Study

The aim of this study was to assess the relationship between endometrial metaplastic/reactive changes (EMRCs) and endometrial neoplastic lesions. Twenty cases of “simple” (without architecture complexity) EMRCs coexistent with endometrial malignant/premalignant lesions, twenty cases of neoplasia-unassociated EMRCs, and eight cases of complex metaplastic lesions were assessed by immunohistochemistry. EMRCs coexisted with endometrioid carcinoma (n = 12), atypical endometrial hyperplasia (n = 3), serous carcinoma (n = 2), and clear cell carcinoma (n = 3). Neoplasia-associated EMRCs showed a mean Ki67 labeling index of 12.6% (range 0–30%); with nuclear atypia in 16/20 (80%) cases; diffuse p16 expression in 15/20 (75%) cases; and heterogeneous ER, PR, and vimentin expression. Compared to the associated neoplasia, EMRCs showed a lower Ki67 expression (p < 0.001) and higher p16 expression (p < 0.001). No EMRC case showed mitotic activity, PTEN loss, MMR deficiency, nuclear β-catenin, p53-mutant pattern, Napsin A, or AMACR expression. No significant differences were found between neoplasia-associated and neoplasia-unassociated EMRCs. Complex metaplastic lesions showed a lower Ki67 expression than EMRCs (p = 0.044) and PTEN loss in 5/8 cases, even in the absence of nuclear atypia. In conclusion, neoplasia-associated simple EMRCs may show evident atypia and a worrisome immunophenotype, but no data support their involvement in endometrial carcinogenesis. Architectural complexity appears as a crucial factor to identify precancerous lesions.

PD-L1, Vimentin and Ki-67 Acting as Immunotherapy Predictive Biomarkers in Pulmonary Carcinomas Transthoracic and Bronchial Biopsies

Introduction: Programmed death-ligand 1 (PD-L1) expression became a routine biomarker to preview response to programmed death-1 (PD-1)/PD-L1 inhibitors, with diverging parameters concerning PD-L1 scoring and variable response to immunotherapy agents. The aim of this study was to evaluate association between PD-L1 expression and immunohistochemistry panel applied in Pathology practice, defining any of those antibodies as biomarkers concurrent in patients selection for PD-1/PD-L1 blockade therapy. Methods A total of 97 cTNM IIIb/IV staged pulmonary carcinoma biopsies were randomly selected between 2018/2020, after adequate representativeness and PD-L1 expression scored through Dako 22C3 antibody. The panel with cytokeratin 7, thyroid transcription factor 1 (TTF1), cytokeratin 5.6, cluster of differentiation 56 (CD56), periodic acid-Schiff (PAS-D), vimentin expression, and ki-67 labeling index (LI) was considered for retrieving reports and respective archival slides. Results PD-L1 expression in tumor cells (TCs) was identified in 56 samples and significantly associated with male gender (p=0.028), vimentin expression (p=0.018) and ki-67 LI>30% (p=0.029). A tendency to PD-L1 positivity came up in lymphocytic-predominant/immune-inflamed stroma (9/10), adenocarcinoma solid subtype (21/23) and CK7-negative squamous cell carcinomas (8/13). When more than 50% TCs expressed PD-L1, the risk of vimentin expression was 3.85 times higher (OR=3.85; p=0.013), and for ki-67 LI>30% the risk was 9.90 times higher (OR=9.90; p=0.033), compared with PD-L1-negative samples. Conclusion High proliferation status defined by ki-67 LI>30% and epithelial-mesenchymal transition phenotype verified by vimentin staining analysis might complement PD-L1-positive TCs percentage determination for immunotherapy prescription. These patients will more likely benefit from PD-1/PD-L1 blockade therapy, overcoming the limitations of selection based on PD-L1 immunohistochemistry status.

The Expression of Programmed Death Ligand 1 and Vimentin in Resected Non-Metastatic Non-Small-Cell Lung Cancer: Interplay and Prognostic Effects

Programmed death ligand 1 (PD-L1) is an immune checkpoint with a role in cancer-related immune evasion. It is a target for cancer immunotherapy and its expression is detected for the use of some immune checkpoint inhibitors in advanced non-small cell lung cancer patients (NSCLC). Vimentin is a key component of the epithelial-to-mesenchymal transition phenotype. Its expression has negative prognostic effects in NSCLC. In this study, we retrospectively evaluated PD-L1 and vimentin expression in tumor cells, immune infiltrate and PD-L1 positive immune infiltrate via immunohistochemistry in tissue samples from resected non-metastatic NSCLC patients. We explored the interplay between PD-L1 and vimentin expression through Spearman’s correlation test. We performed univariate analysis through the Cox models for demographic and clinico-pathological variables, and also for dichotomized biomarkers, i.e., PD-L1 and vimentin in tumor cells, both with 1 and 50% cutoffs. We used Kaplan-Meier method to estimate the overall survival, comparing both vimentin and PD-L1 positive patients with all the others. We found a weak positive correlation between PD-L1 and vimentin expressions in tumor cells (r = 0.25; p = 0.001). We also observed a statistically not significant trend towards a shorter overall survival in patients with both PD-L1 and vimentin expression &gt;1% (HR = 1.36; 95% CI: 0.96–1.93, p = 0.087). In conclusion, these findings suggest that interplay between PD-L1 and vimentin may exist in non-metastatic NSCLC patients and the positivity of both markers in tumor tissue is associated with a trend towards a worse prognosis.

Associations between dynamic-contrast enhanced MRI and tumor infiltrating lymphocytes and tumor-stroma ratio in head and neck squamous cell cancer

Objectives The present study used dynamic-contrast enhanced MRI (DCE-MRI) to elucidate possible associations with tumor-infiltrating lymphocytes (TIL), stroma ratio and vimentin expression in head and neck squamous cell cancer (HNSCC). Methods Overall, 26 patients with primary HNSCC of different localizations were involved in the study. DCE-MRI was obtained on a 3 T MRI and analyzed with a whole lesion measurement using a histogram approach. TIL- and vimentin-expression was calculated on bioptic samples before any form of treatment. P16 staining was used to define HPV-status. Results Tumor-stroma ratio correlated with entropy derived from Ktrans (r = − 0.52, p = 0.0071) and with kurtosis derived from Ve (r = − 0.53, p = 0.0058). Several Ve derived parameters correlated with expression of TIL within the stroma compartment. TIL within the tumor compartment correlated with entropy derived from Ktrans (r = 0.39, p = 0.047), p90 derived from Ve (r = 0.41, p = 0.036) and skewness derived from Ve (r = 0.41, p = 0.037). Furthermore, these associations were different between HPV positive and negative tumors. Conclusions DCE-MRI might be able to reflect tumor compartments and TIL expression in HNSCC. The most promising parameters were values derived from Ktrans and Ve.

C-EBPβ mediates in cigarette/IL-17A-induced bronchial epithelial–mesenchymal transition in COPD mice

Background Cigarettes smoking and IL-17A contribute to chronic obstructive pulmonary disease (COPD), and have synergistical effect on bronchial epithelial cell proliferation. CCAAT/enhancer-binding protein β (C-EBPβ) could be induced by IL-17A and is up-regulated in COPD. We explored the effect of cigarettes and IL-17 on bronchial epithelial–mesenchymal transition (EMT) in COPD mice and potential mechanism involved with C-EBPβ in this study. Methods COPD model was established with mice by exposing to cigarettes. E-Cadherin, Vimentin, IL-17A and C-EBPβ distributions were detected in lung tissues. Primary bronchial epithelial cells were separated from health mice and cocultured with cigarette smoke extract (CSE) or/and IL-17A. E-Cadherin, Vimentin and IL-17 receptor (IL-17R) expressions in vitro were assessed. When C-EBPβ were silenced by siRNA in cells, E-Cadherin, Vimentin and C-EBPβ expressions were detected. Results E-Cadherin distribution was less and Vimentin distribution was more in bronchus of COPD mice than controls. IL-17A and C-EBPβ expressions were higher in lung tissues of COPD mice than controls. In vitro, C-EBPβ protein expression was highest in CSE + IL-17A group, followed by CSE and IL-17A groups. E-cadherin expression in vitro was lowest and Vimentin expression was highest in CSE + IL-17A group, followed by CSE or IL-17A group. Those could be inhibited by C-EBPβ silenced. Conclusions C-EBPβ mediates in cigarette/IL-17A-induced bronchial EMT in COPD mice. Our findings contribute to a better understanding on the progress from COPD to lung cancers, which will provide novel avenues in preventing tumorigenesis of airway in the context of cigarette smoking.

Detection of cancer stem cells by EMT-specific biomarker-based peptide ligands

The occurrence of epithelial-mesenchymal transition (EMT) within tumors, which enables invasion and metastasis, is linked to cancer stem cells (CSCs) with drug and radiation resistance. We used two specific peptides, F7 and SP peptides, to detect EMT derived cells or CSCs. Human tongue squamous carcinoma cell line-SAS transfected with reporter genes was generated and followed by spheroid culture. A small molecule inhibitor-Unc0642 and low-dose ionizing radiation (IR) were used for induction of EMT. Confocal microscopic imaging and fluorescence-activated cell sorting analysis were performed to evaluate the binding ability and specificity of peptides. A SAS xenograft mouse model with EMT induction was established for assessing the binding affinity of peptides. The results showed that F7 and SP peptides not only specifically penetrated into cytoplasm of SAS cells but also bound to EMT derived cells and CSCs with high nucleolin and vimentin expression. In addition, the expression of CSC marker and the binding of peptides were increased in tumors isolated from Unc0642/IR-treated groups. Our study demonstrates the potential of these peptides for detecting EMT derived cells or CSCs and might provide an alternative isolation method for these subpopulations within the tumor in the future.