IL-32/NFκB/miR-205 loop sustains the high expression of IL-32 and enhances the motility of cervical cancer cells.

Human papillomavirus (HPV) infection is a major contributor to cervical cancer. Persistent HPV infection can trigger the expression of IL-32, yet the precise role of IL-32 in the occurrence and development of cervical cancer remains elusive. To investigate this, qRT‒PCR and western blotting were utilized to measure the mRNA and protein expression levels; bioinformatics analysis was used to screen differentially expressed miRNAs; wound healing and transwell assays were conducted to evaluate cell migration and invasion capabilities. Comparative analysis revealed significantly elevated IL-32 expression in cervical cancer tissues and cell lines compared to control groups. In SiHa and/or HeLa, overexpression of IL-32 and IL-32 exposure markedly upregulated miR-205, whereas its knockdown resulted in a substantial downregulation of miR-205. Furthermore, miR-205 also could significantly regulate the expression of IL-32 in HeLa and SiHa cells. Upregulation and downregulation of IL-32 led to a significant increase or decrease in NFκB expression, respectively. Treatment with BAY11-7082 (an NFκB inhibitor) notably decreased miR-205 expression but had no effect on IL-32 levels. qRT‒PCR and western blotting analyses demonstrated that both overexpression and underexpression of IL-32 and miR-205 significantly enhanced or reduced MMP2 and MMP9 expression in cervical cancer cells, respectively. Knockdown of IL-32 significantly inhibited the migration and invasion of HeLa and SiHa; conversely, treatment with rIL-32α and rIL-32γ notably promoted their migration and invasion. In brief, IL-32 is highly expressed via the formation of a positive regulatory loop with NFκB/miR-205, contributing to the persistence of inflammation and promoting the progression of cervical cancer.

First case report of sunvozertinib for the treatment of HER2 exon 20 insertion in lung adenocarcinoma.

Human epidermal growth factor receptor 2 (HER2) is a transmembrane glycoprotein receptor with intracellular tyrosine kinase activity. It is generally considered as a poor prognostic marker. Targeted therapies, such as small molecule tyrosine kinase inhibitors (TKIs), showed limited efficacy in HER2-mutant advanced nonsmall cell lung cancer (NSCLC). In the 2023 National Comprehensive Cancer Network guidelines for NSCLC, antibody-drug conjugate trastuzumab emtansine is recommended for the treatment of HER2-mutant lung cancer. However, this medication is currently not approved in certain regions.So it is necessary to explore alternative treatment options for HER2-mutant NSCLC patients. In our study of a patient with HER2 exon 20 insertion lung adenocarcinoma who had previously failed multiple epidermal growth factor receptor (EGFR)-TKI treatments, we discovered that sunvozertinib could stabilize the patient's condition, achieving a progression-free survival of 87 days. This is a novel finding that may provide new treatment options for HER2 exon 20 insertion patients who have failed TKI therapy.

iTRAQ-based proteomic analysis of heteromorphic leaves reveals eco-adaptability of Populus euphratica Oliv.

BACKGROUND: Heterophylly is regard as adaptation to different environments in plant, and Populus euphratica is an important heterophyllous woody plant. However, information on its molecular mechanism in eco-adaptability remains obscure. RESULTS: In this research, proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ) technology in lanceolate, ovate, and dentate broad-ovate leaves from adult P. euphratica trees, respectively. Besides, chlorophyll content, net photosynthetic rate, stomatal conductance, transpiration rate and peroxidase activity in these heteromorphic leaves were investigated. A total number of 2,689 proteins were detected in the heteromorphic leaves, of which 56, 73, and 222 differential abundance proteins (DAPs) were determined in ovate/lanceolate, dentate broad-ovate/lanceolate, and dentate broad-ovate/ovate comparison groups. Bioinformatics analysis suggested these altered proteins related to photosynthesis, stress tolerance, respiration and primary metabolism accumulated in dentate broad-ovate and ovate leaves, which were consistent with the results of physiological parameters and Real-time Quantitative PCR experiments. CONCLUSION: This research demonstrated the mechanism of the differential abundance proteins in providing an optimal strategy of resource utilization and survival for P. euphratica, that could offer clues for further investigations into eco-adaptability of heterophyllous woody plants.

A nomogram-based immunoprofile predicts clinical outcomes for stage II and III human colorectal cancer.

An immunoscore for colorectal cancer (CRC) has higher prognostic significance than the TNM staging system. However, the tumor immune microenvironment contains various components that affect clinical prognosis. Therefore, a broader range of immune markers is required to establish an accurate immunoprofile to assess the prognosis of patients with CRC. Using immunohistochemistry combined with multispectral immunohistochemistry and objective assessments, the infiltration of four immune cell types (CD4+/CD8+/forkhead box p3+/CD33+ cells), as well as the expression of six co-signaling molecules [programmed cell death 1 (PD1) ligand 1/PD1/T-cell immunoglobulin mucin family member 3/lymphocyte-activating 3/tumor necrosis factor receptor superfamily, member 4/inducible T-cell costimulator] and indoleamine 2,3-dioxygenase 1 were investigated in two independent cohorts of CRC. The patients' overall survival (OS) was evaluated using the Kaplan-Meier method. Using the Cox proportional hazards model, independent prognostic factors of patients were assessed and a nomogram-based immunoprofile system was developed. The predictive ability of the nomogram was determined using a concordance index (C-index) and calibration curve. To facilitate clinical application, a simplified nomogram-based immunoprofile was constructed. Using receiver operating characteristic (ROC) analysis, the predictive accuracy for OS was compared between the immunoprofile and the TNM staging system for patients with stage II/III CRC. According to multivariate analysis for the primary cohort, independent prognostic factors for OS were CD8+ tumor-infiltrating lymphocytes, CD33+ myeloid-derived suppressor cells and TNM stage, which were included in the nomogram. The C-index of the nomogram for predicting OS was 0.861 (95% CI: 0.796-0.925) for the internal validation and 0.759 (95% CI: 0.714-0.804) for the external validation cohort. The simplified nomogram-based immunoprofile system was able to separate same-stage patients into different risk subgroups, particularly for TNM stage II (P<0.0001) and III (P=0.0002) patients. Pairwise comparison of ROC curves for the immunoprofile and TNM stage systems for patients with stage II/III CRC revealed statistically significant differences (P=0.046) and the Z-statistic value was 1.995. In conclusion, the nomogram-based immunoprofile system provides prognostic accuracy regarding clinical outcomes and is a useful supplement to the TNM staging system for patients with stage II/III CRC.

A Modified Method to Isolate Circulating Tumor Cells and Identify by a Panel of Gene Mutations in Lung Cancer.

The CellSearch system is the only FDA approved and successful used detection technology for circulating tumor cells(CTCs). However, the process for identification of CTCs by CellSearch appear to damage the cells, which may adversely affects subsequent molecular biology assays. We aimed to explore and establish a membrane-preserving method for immunofluorescence identification of CTCs that keeping the isolated cells intact. 98 patients with lung cancer were enrolled, and the efficacy of clinical detection of CTCs was examined. Based on the CellSearch principle, we optimized an anti-EpCAM antibody and improved cell membrane rupture. A 5 ml peripheral blood sample was used to enrich CTCs with EpCAM immunomagnetic beads. Fluorescence signals were amplified with secondary antibodies against anti-EpCAM antibody attached on immunomagnetic beads. After identifying CTCs, single CTCs were isolated by micromanipulation. To confirm CTCs, genomic DNA was extracted and amplified at the single cell level to sequence 72 target genes of lung cancer and analyze the mutation copy number variations (CNVs) and gene mutations. A goat anti-mouse polyclonal antibody conjugated with Dylight 488 was selected to stain tumor cells. We identified CTCs based on EpCAM+ and CD45+ cells to exclude white blood cells. In the 98 lung cancer patients, the detection rate of CTCs (≥1 CTC) per 5 ml blood was 87.76%, the number of detections was 1-36, and the median was 2. By sequencing 72 lung cancer-associated genes, we found a high level of CNVs and gene mutations characteristic of tumor cells. We established a new CTCs staining scheme that significantly improves the detection rate and allows further analysis of CTCs characteristics at the genetic level.

Upregulation of miR-205 induces CHN1 expression, which is associated with the aggressive behaviour of cervical cancer cells and correlated with lymph node metastasis.

BACKGROUND: Cervical cancer is the leading cause of cancer-related death in women worldwide. However, the mechanisms mediating the development and progression of cervical cancer are unclear. In this study, we aimed to elucidate the roles of microRNAs and a1-chimaerin (CHN1) protein in cervical cancer progression. METHODS: The expression of miR-205 and CHN1 protein was investigated by in situ hybridisation and immunohistochemistry. We predicted the target genes of miR-205 using software prediction and dual luciferase assays. The expression of mRNAs and proteins was tested by qRT-PCR and western blotting respectively. The ability of cell growth, migration and invasion was evaluated by CCK-8 and transwell. Cell apoptosis was analysed by flow cytometry analysis. RESULTS: We found that miR-205 and CHN1 were highly expressed in human cervical cancer tissue compared with paired normal cervical tissues. The CHN1 gene was shown to be targeted by miR-205 in HeLa cells. Interestingly, transfection with miR-205 mimic upregulated CHN1 mRNA and protein, while miR-205 inhibitor downregulated CHN1 in high-risk and human papilloma virus (HPV)-negative human cervical cancer cells in vitro,. These data suggested that miR-205 positively regulated the expression of CHN1. Furthermore, the miR-205 mimic promoted cell growth, apoptosis, migration, and invasion in high-risk and HPV-negative cervical cancer cells, while the miR-205 inhibitor blocked these biological processes. Knockdown of CHN1 obviously reduced the aggressive cellular behaviours induced by upregulation of miR-205, suggesting that miR-205 positively regulated CHN1 to mediate these cell behaviours during the development of cervical cancer. Furthermore, CHN1 was correlated with lymph node metastasis in clinical specimens. CONCLUSIONS: Our findings showed that miR-205 positively regulated CHN1 to mediate cell growth, apoptosis, migration, and invasion during cervical cancer development, particularly for high-risk HPV-type cervical cancer. These findings suggested that dysregulation of miR-205 and subsequent abnormalities in CHN1 expression promoted the oncogenic potential of human cervical cancer.

Alveolar macrophages in patients with non-small cell lung cancer.

OBJECTIVE: To observe alveolar macrophages (AMs) in the microenvironment of patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: 20 NSCLC patients received bronchoalveolar lavage, and the bronchial alveolar lavage fluid (BALF) was collected. The phenotypes of AMs were detected by the opal multiplex immunofluorescence assay (mIF), flow cytometry, and western blot. RESULTS: AMs could easily be made into paraffin sections after agar pre-embedding. The mIF results showed that AMs highly expressed M1-type marker CD86, and M2-type marker CD163 under PerkinElmer Vectra microscope, while there was a significant difference between the expression of CD86 and CD163 (**P<0.01), consistent with the flow cytometry results. Western blot revealed that the other markers of M1-type (CD16 and iNOS) expression in the AMs were compared with M2-type markers CD206 and ARG (*P<0.05). CONCLUSIONS: Our results showed that AMs simultaneously expressed M1-type markers and M2-type markers, while the M2 markers still dominated. This suggests agar pre-embedding is a very convenient method to embed cells to paraffin tissue, so that cell membrane or nuclear antigens are very easily detected by mIF.

Infiltration of CD8+ FOXP3+ T cells, CD8+ T cells, and FOXP3+ T cells in non-small cell lung cancer microenvironment.

BACKGROUND: Studies about CD8+ FOXP3+ T cells as a subtype of regulatory T cells (Treg cells) in non-small cell lung cancer (NSCLC) are few. Associations among the clinicopathologic factors of NSCLC and tumor-infiltrating lymphocytes (TILs) such as CD8+ FOXP3+ T cells, CD8+ T cells, FOXP3+ T cells and tumor PD-L1 expression are unclear. METHODS: We retrospectively enrolled 192 patients who underwent resections for NSCLC. We used tissue microarrays (TMA) with multiplex immunofluorescence and immunohistochemistry staining to evaluate the expression of CD8, FOXP3, cytokeratin, DAPI and PD-L1. We then used Wilcoxon test, Kaplan-Meier method, and Cox hazard proportion model to analyze their relationships with clinicopathologic factors and prognosis. RESULTS: Density of tumor CD8+ FOXP3+ T cells was significant by univariate analysis, and positively associated with tumor CD8+ T cells and FOXP3+ T cells. Density of tumor CD8+ T cells was higher in lung adenocarcinoma (LUAD) than squamous cell carcinoma (LUSC), and was an independent prognostic factor for NSCLC. The density of tumor FOXP3+ T cells decreased with tumor size. Tumor PD-L1 expression was higher in LUSC than LUAD. Cox hazard proportion model analysis correlated being younger than 65 years, early TNM stage, early T stage, high tumor CD8+ T cell density, and adjuvant chemotherapy with longer overall survival. CONCLUSION: Infiltration of CD8+ FOXP3+ T cells, CD8+ T cells, and FOXP3+ T cells is important in non-small cell lung cancer microenvironment, and needs to be investigated more.

CD137 ligand feedback upregulates PD-L1 expression on lung cancer via T cell production of IFN-γ.

BACKGROUND: The expression of PD-L1 and its regulation in tumors remains unclear. The importance of IFN-γ in upregulating the PD-L1 expression in various tumors, and the effects of other essential cytokines in the tumor microenvironment (TME), need to be further elucidated. METHODS: Constitutive expression of PD-L1 and CD137L in all 13 lung cancer cell lines were tested by flow cytometry. CD137L mRNA of lung cancer cell lines was detected by RT-PCR. PD-L1 expression rates following stimulation with these cytokines (IFN-γ, TNFα and IL2) were measured. After coculture of cells expressing CD137L (lung cancer cells or 293FT cells transfected with CD137L plasmid) with T cells, the PDL1 expression of lung cancer cells and IFN-γ in supernatant was detected. RESULTS: Our data revealed that adenocarcinoma and squamous cell carcinoma cells had the highest positive expression rate. IFN-γ was the core-inducing factor for enhancing the PD-L1 expression. CD137L was also widely expressed in the lung cancer cell lines at the mRNA level, whereas its expression was generally low at the protein level. However, the low expression of CD137L protein was still enough to induce T cells to produce IFN-γ, which subsequently increased the PD-L1 expression by lung cancer cells. The CD137 signal induces IFN-γ secretion by T cells, which stimulates high-level of PD-L1 expression in cancer cells; this negative immune regulation may represent a mechanism of immune escape regulation. CONCLUSIONS: CD137L mRNA was widely expressed in lung cancer cell lines whereas levels of protein expression were generally low. The low level of CD137L protein was still enough to induce T cells to produce IFN-γ that subsequently increased PD-L1 expression. The CD137L-induced negative immune regulation may represent a mechanism of immune escape.

Human-specific LAIR2 contributes to the high invasiveness of human extravillous trophoblast cells.

The placenta is a temporary vital organ for intra-uterine development and growth. The anatomical structure of the placenta has evolved substantially, resulting in broad inter-species diversity. In particular, human placental extravillous trophoblast cells (EVTs) have evolved aggressive features, although the mechanism underlying this aggressiveness remains elusive. In the present study, we compared the human and mouse homologous gene databases and obtained 2272 human-specific genes, 807 of which are expressed in the placenta according to the UniGene database. Using the human trophoblast cell line HTR8/SVneo, we further verified the expression and function of one of these genes, the leukocyte-associated immunoglobulin-like receptor 2 (LAIR2). This gene shows increased expression during pregnancy and its reduced expression is associated with pregnancy complications. Although LAIR2 was expressed in the human placenta villus and decidua in the first trimester of pregnancy, it was not expressed in mouse tissues. Knockdown of LAIR2 markedly improved cell viability and inhibited the invasive ability of HTR8/SVneo cells. These data suggest that species-specific genes are pivotal to the evolution of a more aggressive human placenta to match the physiological demands of human development. Further investigation is required to obtain evidence on the function of LAIR2 and other specific genes in the placenta, providing insight on the mechanism, properties, and possible applications of this in humans.

Preparation of Astragalus membranaceus lectin and evaluation of its biological function.

Astragalus membranaceus lectin (AML) was abstracted as a supposedly novel agglutinin of 67 kDa from the seeds of Astragalus membranaceus. The seeds of Astragalus membranaceus were treated with acetate, ammonium sulfate precipitation, and purified by HiTrap SP XL ion column and Superdex G25 gel filtration chromatography to obtain the AML. AML contained 16.4% sugar, ~70% polar amino acids and ~30% hydrophobic amino acids. The AML exhibited agglutination activity toward human and animal erythrocytes, particularly human blood type O and rabbit erythrocytes. It also exhibited acid/alkali resistance and thermal denaturation above 64°C. Compared with human normal liver HL-7702 cells, different concentrations of AML (6.25, 12.50, 25.00 and 50.00 µg/ml) exhibited superior inhibitory effects on the growth of SGC-7901, HepG2 and H22 carcinoma cell lines, and displayed marked antibacterial effects on bacteria; the half maximal inhibitory concentration for B. dysenteriae, S. aureus and E. coli were 85.4, 80.2 and 65.3 µg/ml, respectively.