CD44 Gene Research Articles

Abstract 7252: Characterization of targeting peptide sequences for peptide-mediated delivery of siRNA in HER2+ breast cancer

Abstract Introduction: This work presents the characterization of two novel HER2 targeting peptides to measure which has the most compatible properties to be used as a siRNA carrier to knock down the CD44 gene in HER2+ breast cancer. Procedures: Targeting peptides were electrostatically complexed with non-targeting siRNAs at different N:P ratios to determine the minimum binding ratios needed to form nanoparticles. Different ratios of peptide-siRNA complexes were tested to determine the minimum molar ratio at which the peptides would protect siRNA from degradation. Cytotoxicity assays were performed to determine cytotoxicity levels at different peptide concentrations. Dynamic light scattering assay was performed to determine peptide-siRNA nanoparticle size and zeta potential. Immunofluorescent microscopy was used to determine the targeting of the peptide to HER2+ cells compared to cells not expressing HER2. Summary of Results: Peptide-siRNA complexes for the P51 and P25 peptides formed at a minimum 20:1 N:P ratio. P25 and P51 peptides both successfully protects the siRNA at N:P ratios from 20:1 to 60:1 for both degradation agents. Cytotoxicity assays show that in the SKBR3 cell line, the P25 and P51 peptides both showed above 80% viability until the 70:1 ratio; however, in the BT474 cell line, the P25 showed above 80% viability to the 50:1 ratio, but the P51 was only viable in the 20:1 ratio. The results of these studies illustrate how both peptides protect siRNA but that the P51 peptide is toxic to some HER2+ breast cancer cell lines. Conclusions: Results show that both peptides target HER2+ breast cancer and are compatible carriers of siRNA, protecting the siRNA well within a cell-like environment. However, from MTS studies, P25 appears less cytotoxic to the cell lines than P51, which shows higher cytotoxicity at high molar ratios. We observed that the P25 protects siRNA from degradation, is cytocompatibility, and promotes efficient internalization into HER2+ cell lines. Future work will include the development of a tandem peptide, including a targeting peptide fused with a fusogenic peptide to increase cancer targeting and efficacy of this targeted treatment. Acknowledgments: This research was supported in part by the Dabo Swinney All-In Foundation as well as Materials Assembly and Design Excellence in South Carolina (MADE in SC) under the National Science Foundation EPSCoR Program under NSF Award #OIA-1655740. Data presented in this material was partially collected using SC BioCRAFT facilities supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number P30 GM131959. Citation Format: Audreanna Miserendino, Sevrina Tekle, James Kalogeros, Angela Alexander-Bryant. Characterization of targeting peptide sequences for peptide-mediated delivery of siRNA in HER2+ breast cancer [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 7252.

Abstract 2706: Identification and characterization of cancer-associated polymorphonuclear cell subsets in mice

Abstract Background: Neutrophils are the main type of polymorphonuclear cells (PMN) and comprise most immune cells in the body. PMN cells are upregulated in cancer and accumulate in the tumors. Tumor-associated PMNs comprise a heterogeneous population consisting of immunologically active mature neutrophils and immature polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). The phenotype of these cells evolves with disease progression and is implicated in modulating immune response. However, there is yet to be a consensus to define PMN populations in tumors and blood. Methods: Here, we used single-cell RNA-seq, bulk RNA sequencing, flow cytometry, and immunohistochemistry (IHC) analysis to characterize cancer-associated PMN-MDSCs in tumor-bearing mice. Results: Single-cell RNA-seq data from lung tumors and healthy individuals revealed five metaclusters (MC): MCA, MCB, MCC, MCD, and MCE. MCA and MCB made up 34.4 and 65.1% of PMNs in healthy tissues, respectively. MCA, MCB, MCC, MCD, and MCE comprised 3.8%, 23.5% 3.3% 14.6%, and 54.7% of PMNs in tumors, respectively. Genes enriched in MCA and MCE were associated with MDSCs and myeloid progenitor cells. MCB and MCC were associated with inflammatory responses mediated by TNF-alpha signaling and neutrophil degranulation. MCD was associated with a CD33 myeloid cell phenotype and had high expression levels of CD274, Pdcd1lg2, Havcr2, and Tigit genes. Cells from MCE were associated with protein translation signatures and had elevated levels of Nt5e. Gene expression signatures from single-cell RNA-seq data were compared with bulk transcriptomic data from healthy mouse prostates and Pten-null prostate tumors characterized by high PMN cell infiltration. Gene set enrichment analysis showed enrichments after androgen deprivation therapy and Cluster MCE was associated with castration-resistant tumors. Flow cytometric and IHC analysis confirmed the enrichment of PMN cells expressing elevated levels of proteins identified from gene expression analysis including CD73, PD-L1, and PD-L2. Conclusion: This study offers further insights into the distinct subsets of PMN-MDSCs present in tumors. Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Takashi Kikuchi, Yasunori Mori, Takafumi Minami, Kazuhiro Yoshimura, Kazutoshi Fujita, Kazuto Nishio, Hirotsugu Uemura. Identification and characterization of cancer-associated polymorphonuclear cell subsets in mice [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 2706.

Abstract 3405: CT-assessed body composition and tumor immunologic characteristics in patients with non-small-cell lung cancer

Abstract Objectives: Obesity is associated with higher immunotherapy efficacy, but body mass index is a convoluted marker of adipose and muscle tissue. We conducted a discovery scale study to assess the association of skeletal muscle and adipose tissue areas annotated on computed tomography (CT) scan images with selected tumor immunologic characteristics among patients with non-small-cell lung cancer (NSCLC). Methods: We analyzed publicly available data from 102 patients (mean age = 67.2, 47.1% female, and 70.6% stage I-II) who had diagnostic CT images in The Cancer Image Archive. All patients had the 12th thoracic vertebra (T12) image, and a subset (62 patients) had the 3rd lumber vertebra (L3) image. Paravertebral muscle areas on T12 and skeletal muscle and adipose tissue areas on L3 were annotated using Slice-O-Matic. RNA-seq data were provided by The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium; percentages of CD8, regulatory T cells, and activated NK cells were identified using the CIBERSORT. ESTIMATE algorithm was used to derive the stromal score and the immune score. Differences in the immune markers were examined using ANOVA adjusting for stage, histology, and study. Results and Conclusion: Patients with higher paravertebral muscle area (3rd tertile) had the lowest stromal scores, compared to the 2nd and 1st tertile (P<0.05). Patients in the 2nd tertile of paravertebral muscle areas had the highest immune score and CD274 (PD-L1) gene expression. Among the body composition types classified using L3 images, low muscle/high adiposity was associated with lower stromal scores, and low muscle was associated with lower immune scores, compared to high adiposity and high muscle/low adiposity types. Other immunological markers did not differ across the body composition types. Significance: These findings suggest associations between deconvoluted skeletal and adipose tissue components and tumor markers relevant to immune checkpoint inhibitor efficacies in NSCLC. Further validation using immune phenotyping in a large patient sample is warranted. Funding: The study is in part supported by NIH/NCI R37CA248371 and OSU Comprehensive Cancer Center - James. Citation Format: Ting-Yuan David Cheng, Cuthbert Mario Mahenge, Rand T. Akasheh, Ayse Selen Yilmaz, Ben Kinder, Xuan Nguyen, Maciej Pietrzak, Carolyn J. Presley, Peter G. Shields. CT-assessed body composition and tumor immunologic characteristics in patients with non-small-cell lung cancer [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 3405.

Abstract 2520: CD133 expression is associated with less DNA repair, better response to chemotherapy, and survival in ER-positive/ER-negative breast cancer

Abstract Introduction: Cancer stem cells (CSC) constitute only 0.1-1% of all the cells in a bulk tumor, yet they play a crucial role in treatment resistance and relapses. Its role in breast cancer (BC) has been studied but a consensus remains elusive. We focused on gene expression of a CSC marker CD133 and studied its clinical significance in ER-positive/HER2-negative (ER+/HER2-) BC, the most prevalent subtype. We utilized multiple independent large patient cohorts of gene expression and clinical variables for transcriptome analysis. Methods: A total of 2969 patients from The Cancer Genome Atlas (TCGA, n=1065) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC, n=1904) BC cohorts, as well as BC patients who underwent neoadjuvant chemotherapy (NAC; GSE25066, GSE20194, GSE32646 cohorts) were analyzed. Results: By analyzing SCP1039 and SCP1106 single-cell sequencing cohorts, we found that epithelial cells were the predominant source of CD133 gene expression in the tumor microenvironment. High expression of CD133 in ER+/HER2- BC correlated with elevated gene expressions of the other CSC markers; CD24, NOTCH1, DLL1, and ALDH1A1 (all p<0.001), and significantly enriched WNT/β-Catenin, Hedgehog, and Notch signaling (all FDR<0.25), which are all known pathways related with CSC in TCGA and validated in METABRIC cohort. Consistent with CSC phenotype, CD133-high BC was negatively enriched with Hallmark cell proliferation-related gene sets, such as E2F Targets, G2M Checkpoint, MYC Targets V1, Ki67 gene expression and proliferation score (FDR<0.25 and p<0.001, respectively) in both cohorts. CD133-high BC was negatively associated with DNA repair genes, such as BRCA1, E2F7 in both cohorts (all p<0.001). In agreement, CD133-high BC was associated with less homologous recombination deficiency, lower silent and non-silent mutation rates. Surprisingly, CD133-high BC exhibited a proinflammatory microenvironment, as represented by higher cytolytic activity in the METABRIC and higher lymphocyte infiltrations, TGF-beta response, and TCR richness in TCGA (both p<0.001), but less Th1 cells, M1 macrophages and dendritic cell infiltrations consistently in both cohorts. It also enriched signaling pathways related to inflammation and immune responses such as inflammatory response, allograft rejection, TNF-alpha, IL-6, TGF-beta signaling, and complement signaling (all FDR<0.25). CD133-high tumors had better pathological complete response after NAC in GSE25066 cohort and improved Disease-Free Survival and Overall Survival in both TCGA and METABRIC cohorts (all p<0.05). Conclusion: CD133-high ER+/HER2- BC is associated with CSC phenotype, reduced cell proliferation, and impaired DNA repair, but enhanced inflammation, potentially explaining the better response to NAC and favorable patient prognosis. Citation Format: Takumi Sato, Masanori Oshi, Jing Lee, Arya M. Roy, Kohei Chida, Itaru Endo, Masato Obayashi, Kazuaki Takabe. CD133 expression is associated with less DNA repair, better response to chemotherapy, and survival in ER-positive/ER-negative breast cancer [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 2520.

New Genetic Markers of Skin T-Cell Lymphoma Treatment.

Cutaneous T-cell lymphomas (CTCL) can be described as chronic skin inflammation lesions with the content of malignant T cells and they are considered to be T-cell-mediated skin diseases. CD147 is recognized as a 58-kDa cell surface glycoprotein of the immunoglobulin superfamily; it can induce the synthesis of MMPs (matrix metalloproteinases) on the surface of tumor cells where it was originally identified. It can also function in adjacent tumor fibroblasts using CD147-CD147 interactions. The polymorphism rs8259 T/A is situated in the untranslated region (3'UTR) of the CD147 gene. HLA DRB1*1501 takes part in the process of presentation and recognition of different antigens to T cells. It can be expressed by antigen-presenting cells-macrophages, dendritic cells, and B cells. The aim of the study is to test genotype-phenotype associations of both polymorphisms including therapy in a large cohort of CTCL patients. A final total of 104 CTCL patients were enrolled in the study. For the first remission at the clinic department, they were treated by means of local skin-directed therapy, phototherapy, and systemic therapy. Genomic DNA was isolated from peripheral blood leukocytes. A standard technique using proteinase K was applied. The polymorphisms rs8259 T/A (CD147 gene) and rs3135388 (HLA DRB1*1501) were detected through standard PCR-restriction fragment length polymorphism methods. The severity of the disease (patients with parapsoriasis, stages IA and IB, vs patients with stages IIB, IIIA, and IIIB) was associated with the CD147 genotype: the AA variant was 3.38 times more frequent in more severe cases, which reflects the decision on systemic therapy (p = 0.02, specificity 0.965). The AA genotype in the CD147 polymorphism was 12 times more frequent in patients who underwent systemic therapy of CTCL compared to those not treated with this therapy (p = 0.009, specificity 0.976). The same genotype was also associated with radiotherapy-it was observed 14 times more frequently in patients treated with radiotherapy (p = 0.009, specificity 0.959). In patients treated with interferon α therapy, the AA genotype was observed to be 5.85 times more frequent compared to the patients not treated with interferon therapy (p = 0.03, specificity 0.963). The HLA DRB1*1501 polymorphism was associated with local skin-directed therapy of CTCL. The CC genotype of the polymorphism was observed to be 3.57 times more frequent in patients treated with local therapy (p = 0.008, specificity 0.948). When both polymorphisms had been calculated together, even better results were obtained: the AACC double genotype was 11 times more frequent in patients with severe CTCL (p = 0.009, specificity 0.977). The TACT double genotype was associated with local skin-directed therapy (0.09 times lower frequency, p = 0.007, sensitivity 0.982). The AACC genotype was 8.9 times more frequent in patients treated by means of systemic therapy (p = 0.02, specificity 0.976) and as many as 18.8 times more frequent in patients treated with radiotherapy (p = 0.005, specificity 0.969). Thus, the AACC double genotype of CD147 and DRB1*1501 polymorphisms seems to be a clinically highly specific marker of severity, systemic therapy and radiotherapy of patients with T-cell lymphoma. Although genotyping results were not known during the treatment decision and could not modify it, the clinical decision on severity and therapy reflected some aspects of the genetic background of this complicated T-cell-associated disease very well.

Perfusable adipose decellularized extracellular matrix biological scaffold co-recellularized with adipose-derived stem cells and L6 promotes functional skeletal muscle regeneration following volumetric muscle loss

Muscle tissue engineering is a promising therapeutic strategy for volumetric muscle loss (VML). Among them, decellularized extracellular matrix (dECM) biological scaffolds have shown certain effects in restoring muscle function. However, researchers have inconsistent or even contradictory results on whether dECM biological scaffolds can efficiently regenerate muscle fibers and restore muscle function. This suggests that therapeutic strategies based on dECM biological scaffolds need to be further optimized and developed. In this study, we used a recellularization method of perfusing adipose-derived stem cells (ASCs) and L6 into adipose dECM (adECM) through vascular pedicles. On one hand, this strategy ensures sufficient quantity and uniform distribution of seeded cells inside scaffold. On the other hand, auxiliary L6 cells addresses the issue of low myogenic differentiation efficiency of ASCs. Subsequently, the treatment of VML animal experiments showed that the combined recellularization strategy can improve muscle regeneration and angiogenesis than the single ASCs recellularization strategy, and the TA of former had greater muscle contraction strength. Further single-nucleus RNA sequencing (snRNA-seq) analysis found that L6 cells induced ASCs transform into a new subpopulation of cells highly expressing Mki67, CD34 and CDK1 genes, which had stronger ability of oriented myogenic differentiation. This study demonstrates that co-seeding ASCs and L6 cells through vascular pedicles is a promising recellularization strategy for adECM biological scaffolds, and the engineered muscle tissue constructed based on this has significant therapeutic effects on VML. Overall, this study provides a new paradigm for optimizing and developing dECM-based therapeutic strategies.

Correlation analysis of cancer stem cell marker CD133 and human endogenous retrovirus (HERV)-K env in SKOV3 ovarian cancer cells.

Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells.

S100A9+CD14+ monocytes contribute to anti-PD-1 immunotherapy resistance in advanced hepatocellular carcinoma by attenuating T cell-mediated antitumor function

BackgroundThe paucity of reliable biomarkers for predicting immunotherapy efficacy in patients with advanced hepatocellular carcinoma (HCC) has emerged as a burgeoning concern with the expanding use of immunotherapy. This study endeavors to delve into the potential peripheral biomarkers capable of prognosticating efficacy in HCC patients who are poised to receive anti-PD-1 monotherapy within the phase III clinical trial, KEYNOTE394. Additionally, we sought to elucidate the underlying molecular mechanisms for resistance to immune checkpoint blockade (ICB) and propose innovative combination immunotherapy strategies for future clinical application.MethodsPatient blood samples were collected for single-cell RNA sequencing to evaluate the immune cell signature before receiving ICB therapy. Subsequently, in vitro assays and in vivo murine model experiments were conducted to validate the mechanism that S100A9+CD14+ monocytes play a role in ICB resistance.ResultsOur study demonstrates a notable enrichment of S100A9+CD14+ monocytes in the peripheral blood of patients exhibiting suboptimal responses to anti-PD-1 therapy. Moreover, we identified the Mono_S100A9 signature as a predictive biomarker, indicative of reduced efficacy in immunotherapy and decreased survival benefits across various tumor types. Mechanistically, S100A9 activates PD-L1 transcription by directly binding to the CD274 (PD-L1) gene promoter, thereby suppressing T-cell proliferation and cytotoxicity via the PD-1/PD-L1 axis, consequently diminishing the therapeutic effectiveness of subsequent anti-PD-1 treatments. Furthermore, our in vivo studies revealed that inhibiting S100A9 can synergistically enhance the efficacy of anti-PD-1 drugs in the eradication of hepatocellular carcinoma.ConclusionsOur study underscores the significance of S100A9+CD14+ monocytes in predicting inadequate response to ICB treatment and provides insights into the monocyte cell-intrinsic mechanisms of resistance to ICB therapy. We also propose a combined therapeutic approach to enhance ICB efficacy by targeting S100A9.

Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer.

Subversion of RUNX3 developmental gene targets to metastasis program indicates the oncogenic nature of inappropriate RUNX3 regulation in gastric cancer.

Abstract WP318: Increased Synapse Elimination by Inflammatory Cells Contribute to Lon-Lasting Post-Stroke Memory Dysfunction in Aged Mice

Background and Purpose: Memory impairments are common after stroke. Memory dysfunction lasted longer in aged than in young mice, but the mechanisms are still unclear. Microglia maintain normal memory via removing excessive synapses and increase turnover of synaptic connections after stroke. Long-term memory deficits in aged mice may be associated with increased microglia in the brain. Hypothesis: Increased synapse removal by inflammatory cells contribute to the long-lasting post-stroke memory dysfunction of aged mice. Methods: Permanent distal middle cerebral artery occlusion (pMCAO) was induced in young and aged mice. The memory functions were analyzed weekly for 8 weeks by Y-maze test and at 8 weeks post stroke by novel objective recognition (NOR) test. Brain samples were collected 8 weeks after pMCAO induction. RNAseq analysis was performed to study gene expression profiles in peri-infarct area and hippocampus. Nissl staining was used to measure infarct volume and immunostaining was done to detect CD68 + and GFAP + cells and synapse in peri-infarct region and hippocampus. Results: RNAseq data shown up-regulation of CD68, GFAP, pro-inflammatory cytokines, immune response, microglia chemotaxins and phagocytosis genes; and down-regulation of synaptic assembly and transmission, synaptic plasticity, and long-term potentiation genes in aged mice at peri-infarct region and ipsilateral hippocampi. Aged mice had larger infarct volumes, more CD68 + and GFAP + cells in the peri-infarct region and ipsilateral hippocampi than young mice and had more CD68 + cells in the ipsilateral than the contralateral hippocampi. In Y-maze test, aged stroke mice made fewer spontaneous alternations from 3 to 8 weeks after pMCAO than young stroke and sham operated aged mice. In the NOR test, aged stroke mice spent less time on the novel objects than young stroke and sham aged mice. Increased synaptophysin positive CD68 + cells were detected in the hippocampi of aged stroke mice. Conclusions: Increase of synaptic elements by inflammatory cells contribute to the long-lasting post-stroke memory deficit in the aged mice, and reduction of neuroinflammation may improve their memory function.

Increased CD44 expression in primary meningioma: its clinical significance and association with peritumoral brain edema.

CD44 is a major cell surface receptor involved in cell adhesion and migration. The overexpression of CD44 is a poor prognostic factor in many neoplasms, including meningiomas. The aim of this study was to investigate the association between CD44 gene expression and clinical signatures of primary meningiomas. CD44 gene expression was quantitatively evaluated by snap freezing tumor tissues obtained from 106 patients with primary meningioma. The relationships between CD44 expression and clinical signatures of meningiomas, including histological malignancy, tumor volume, and peritumoral brain edema (PTBE), were analyzed. PTBE was assessed using the Steinhoff classification (SC) system (from SC 0 to SC III). CD44 gene expression in WHO grade 2 and 3 meningiomas was significantly higher than that in grade 1 meningiomas. In addition, CD44 expression increased with the severity of PTBE. Particularly, among the grade 1 meningiomas or small-sized tumors (maximum tumor diameter < 43 mm), CD44 expression in tumors with severe PTBE (SC II or III) was significantly higher than that in tumors without or with mild PTBE (SC 0 or I). Multivariate logistic regression analysis also revealed that overexpression of CD44 was an independent significant factor of severe PTBE development in primary meningiomas. In addition to tumor cell aggressiveness, CD44 expression promotes the development of PTBE in meningioma. Since PTBE is a strong factor of tumor-related epilepsy or cognitive dysfunction in patients with meningioma, CD44 is thus a potential therapeutic target in meningioma with PTBE.

Genome-wide analyses reveal the contribution of somatic variants to the immune landscape of multiple cancer types.

It has been well established that cancer cells can evade immune surveillance by mutating themselves. Understanding genetic alterations in cancer cells that contribute to immune regulation could lead to better immunotherapy patient stratification and identification of novel immune-oncology (IO) targets. In this report, we describe our effort of genome-wide association analyses across 22 TCGA cancer types to explore the associations between genetic alterations in cancer cells and 74 immune traits. Results showed that the tumor microenvironment (TME) is shaped by different gene mutations in different cancer types. Out of the key genes that drive multiple immune traits, top hit KEAP1 in lung adenocarcinoma (LUAD) was selected for validation. It was found that KEAP1 mutations can explain more than 10% of the variance for multiple immune traits in LUAD. Using public scRNA-seq data, further analysis confirmed that KEAP1 mutations activate the NRF2 pathway and promote a suppressive TME. The activation of the NRF2 pathway is negatively correlated with lower T cell infiltration and higher T cell exhaustion. Meanwhile, several immune check point genes, such as CD274 (PD-L1), are highly expressed in NRF2-activated cancer cells. By integrating multiple RNA-seq data, a NRF2 gene signature was curated, which predicts anti-PD1 therapy response better than CD274 gene alone in a mixed cohort of different subtypes of non-small cell lung cancer (NSCLC) including LUAD, highlighting the important role of KEAP1-NRF2 axis in shaping the TME in NSCLC. Finally, a list of overexpressed ligands in NRF2 pathway activated cancer cells were identified and could potentially be targeted for TME remodeling in LUAD.

NASOPHARYNGEAL ACE2, CD147, AND TMPRSS2 EXPRESSIONS IN MIS-C PATIENTS

Objective: Multisystem Inflammatory Syndrome in Children (MIS-C) is a disease developed after about eight weeks of the new coronavirus disease (COVID-19) infection in children. The underlying reason for MIS-C is still unknown. This study aims to assess the nasopharyngeal expressions of Angiotensin-converting enzyme 2 (ACE2), the differentiation 147 receptor cluster (CD147), and the transmembrane serine protease 2 (TMPRSS2) receptors in the MIS-C population. Since these receptors are related to COVID-19 infection, children with previous COVID-19 history were included as a control group. This study is important for future studies to see if there is any significant expression difference between these receptors in control and MIS-C populations. Methods: The prospective cohort study took place at XXXXX University. The participants of this study consisted of patients aged 0-18 years who were diagnosed with MIS-C due to COVID-19 infection or patients with only previous COVID-19 pneumonia but without MIS-C development. A total of 79 cases were registered in this study. Nasopharyngeal samples of children were collected. Total RNA was isolated from all samples. ACE2, CD147, and TMPRSS2 genes’ expression was accessed by real-time quantitative PCR (RT-qPCR). Results: Nasopharyngeal expression of CD147 and TMPRSS2 were not changed in COVID-19 (n=42) and MIS-C (n=37) cases. Expression of ACE2 was increased in the under 10-year-old MIS-C group (n=23) (p=0.004381). Conclusion: Future studies may exclude the nasopharyngeal expression of CD147 and TMPRSS2 to develop MIS-C. Further investigations are required to learn how ACE2 expression contributes to MIS-C development.

Relationship between CD274 gene polymorphism and systemic lupus erythematosus risk in a Chinese Han population.

Relationship between surface antigen differentiation cluster 274 (CD274) gene polymorphism and systemic lupus erythematosus (SLE) risk is limited. This study aims to discuss whether in a Chinese Han population, CD274 gene polymorphisms may relate to SLE susceptibility. Three hundred and ten SLE patients and 390 healthy controls were included in this case-control study. Using the Kompetitive Allele-Specific PCR (KASP) approach, five single nucleotide polymorphisms (SNPs), including rs2890658, rs4143815, rs822339, rs2282055, and rs2297137, were genotyped for CD274 gene polymorphisms. Correlation between the polymorphisms and clinical, laboratory features in SLE patients were discussed. Frequency of C allele was substantially lower in SLE patients than in healthy controls (p = .015), and CC genotype was significantly negatively related to developing SLE at locus rs4143815 (p = .013). At locus rs822339, frequency of GA genotype was higher than that of the healthy controls (p = .006). At locus rs2282055, frequency of GG genotype was lower than that of healthy controls (p = .024). According to subgroup analysis, the CD274 gene polymorphisms rs2890658, rs4143815, rs822339, rs2282055, and rs2297137 were partly linked to some clinical symptoms of SLE patients, such as Complement 4 (C4), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). CD274 gene polymorphisms may be susceptible to SLE in the Chinese Han people.

Polymorphisms of CD44 rs187115 as a Predictive Biomarker in Early Colorectal Cancer Diagnostic.

Introduction: Colorectal cancer (CRC) has exhibited an increasing incidence worldwide in recent years, underscoring the importance of early diagnosis methods. This study aimed to assess the influence of CD44 gene polymorphism rs187115 on CRC susceptibility. Material and Methods: The study encompassed 470 CRC patients and 165 healthy controls. Genotyping of all biological blood samples was conducted using the TaqMan assay on the ABI 7500 Real Time PCR System (Applied Biosystems, USA). Results: The genotyping revealed that carriers of the variant G allele, including the genotypes AG and GG, exhibited a heightened risk of CRC occurrence, with an odds ratio (OR) of 1.89 (95% confidence interval [CI] = 1.57-1.97; p = 0.047) compared to those carrying the AA genotype. Conclusions: The findings underscore the potential utility of CD44 rs187115 polymorphisms as a novel predictive biomarker for CRC prognosis.

Histological and immunohistochemical characteristic of the gingival stroma in the portion of the third molars in children of various age.

Aim: To evaluate the state of the gingival stromal elements in the portion of the third molars requiring extraction of these teeth due to orthodontic indications considering the stage of tooth germ formation. Materials and Methods: The surgery to extract third molars due to orthodontic indications was performed on 95 children aged 11 to 18 years. The three groups of observation were isolated according to clinical-radiological signs: І (n=30) - children aged 11-13 years; ІІ (n=35) - children aged 13-16 years, and ІІІ (n=30) - children aged 16-18 years. During surgery, the samples of gums were taken from the adjacent areas for examination. The samples were fixed, dehydrated, paraffinized for further histological processing. Immunohistochemical methods were used according to the protocols supplied by a producer. In particular, by means of immunohistochemical method, Ki-67, CD-34 antigens and vimentin with primary antibodies against them were determined. The primary antibodies were visualized by the polymeric visualization system with diaminobenzidine giving a brown color to the places of location of the antigens examined. The data obtained were statistically processed. Results: The results of the study showed that specific gravity of the vascular bed in the gingival papillary layer of children was the most variable. It ranges from (12,7±0,09) % at the stage of "D" root formation to (54,8±0,17) % at the "H" stage. Lower concentrations of CD-34 antigens and vimentin are found in the endotheliocytes of children aged 13-16 and 16-18 years, compared to the children aged 11-13 years (p<0,05). No changes were found in the specific volume of the blood vessels, CD-34 antigens and vimentin in the reticular gingival layer of children from the groups of observation. Conclusions: Therefore, the conducted histological and immunohistochemical study of the connective gingival tissues in the portion of the third molars in children enables to draw a conclusion that in the process of formation of the root of this tooth a number of changes occur in the gingival stroma. They include an increase of the blood flow volume in the papillary gingival layer on the background of a decreased concentration of CD-34 genes and vimentin, a longer stage of development of the third molar root. The specific volume of the islets of neoangiogenesis of the papillary gingival layer is the largest in children aged 13-16 years.

Transcriptional analysis of landmark molecular pathways in lung adenocarcinoma results in a clinically relevant classification with potential therapeutic implications.

Lung adenocarcinoma (LUAD) is a molecularly heterogeneous disease. In addition to genomic alterations, cancer transcriptional profiling can be helpful to tailor cancer treatment and to estimate each patient's outcome. Transcriptional activity levels of 50 molecular pathways were inferred in 4573 LUAD patients using Gene Set Variation Analysis (GSVA) method. Seven LUAD subtypes were defined and independently validated based on the combined behavior of the studied pathways: AD (adenocarcinoma subtype) 1-7. AD1, AD4, and AD5 subtypes were associated with better overall survival. AD1 and AD4 subtypes were enriched in epidermal growth factor receptor (EGFR) mutations, whereas AD2 and AD6 showed higher tumor protein p53 (TP53) alteration frequencies. AD2 and AD6 subtypes correlated with higher genome instability, proliferation-related pathway expression, and specific sensitivity to chemotherapy, based on data from LUAD cell lines. LUAD subtypes were able to predict immunotherapy response in addition to CD274 (PD-L1) gene expression and tumor mutational burden (TMB). AD2 and AD4 subtypes were associated with potential resistance and response to immunotherapy, respectively. Thus, analysis of transcriptomic data could improve patient stratification beyond genomics and single biomarkers (i.e., PD-L1 and TMB) and may lay the foundation for more personalized treatment avenues, especially in driver-negative LUAD.

Niosomal hesperidin attenuates the M1/M2-macrophage polarization-based hepatotoxicity followed chlorpyrifos -induced toxicities in mice

Chlorpyrifos(CPF) is a well-known hepatotoxic agent that has side effects on several organs. On the contrary, hepatic macrophages are crucial in maintaining liver tissue integrity. The main objective of this study was to evaluate the effects and possible mechanisms of niosomal hesperidin (Nio + Hesp), a flavanone glycoside found in citrus fruits, on M1-M2 liver macrophage polarization and inflammatory cells in the brain, liver, and ovarian tissues. Forty C57 mice were divided into CPF(3 mg/kg), Sham(Dimethyl sulfoxide 40 μL/kg), CPF + Hesp(100 mg/kg), and CPF + Nio + Hesp (100 mg/kg) groups. The activity of sera superoxide dismutase (SOD) and malondialdehyde (MDA), brain, liver, and ovary tissues changes, and M1-M2 liver macrophage polarization were evaluated by examining the expression of CD163 and CD68 genes. Hepatic lesions consisting of sporadic foci of coagulation necrosis, inflammatory cell reaction, and regenerative fibrosis were seen following CPF injection, reflected by significant overexpression of CD163 and CD68 genes. In comparison, Nio + Hesp declined the amount of cell apoptosis in the liver and downregulated CD163 and CD68 gene expression. Both Nio + Hesp and Hesp alleviated CPF-induced hepatotoxicity, however, Nio + Hesp was superior to hesperidin in the downregulation of the CD163 and CD68 gene expression. Even though a significant difference between hesperidin and Nio + Hesp was observed in the number of Graafian follicles, corpus luteum, and peri-antral follicles, no substantial difference was observed in primary follicles. The ameliorative effects of Hesp and Nio + Hesp may be at least in part due to their antioxidant and anti-inflammatory properties. These findings showed that both M1- and M2-macrophages contributed to the development of hepatic lesions induced by CPF and provided information about macrophage activation, indicating the importance of analysis of macrophage phenotypes for hepatotoxicity based on M1/M2-polarization which can be downregulated by niosomal nesperidin.

SHC4 orchestrates β-catenin pathway-mediated metastasis in triple-negative breast cancer by promoting Src kinase autophosphorylation

Triple-negative breast cancer (TNBC) is highly aggressive and metastatic, and has the poorest prognosis among all breast cancer subtypes. Activated β-catenin is enriched in TNBC and involved in Wnt signaling-independent metastasis. However, the underlying mechanisms of β-catenin activation in TNBC remain unknown. Here, we found that SHC4 was upregulated in TNBC and high SHC4 expression was significantly correlated with poor outcomes. Overexpression of SHC4 promoted TNBC aggressiveness in vitro and facilitated TNBC metastasis in vivo. Mechanistically, SHC4 interacted with Src and maintained its autophosphorylated activation, which activated β-catenin independent of Wnt signaling, and finally upregulated the transcription and expression of its downstream genes CD44 and MMP7. Furthermore, we determined that the PxPPxPxxxPxxP sequence on CH2 domain of SHC4 was critical for SHC4-Src binding and Src kinase activation. Overall, our results revealed the mechanism of β-catenin activation independent of Wnt signaling in TNBC, which was driven by SHC4-induced Src autophosphorylation, suggesting that SHC4 might be a potential prognostic marker and therapeutic target in TNBC.

Common Variants in Osteopontin and CD44 Genes as Predictors of Treatment Outcome in Radiotherapy and Chemoradiotherapy for Non-Small Cell Lung Cancer.

Osteopontin (OPN)-CD44 signaling plays an important role in promoting tumor progression and metastasis. In cancer, OPN and CD44 overexpression is a marker of aggressive disease and poor prognosis, and correlates with therapy resistance. In this study, we aimed to evaluate the association of single nucleotide polymorphisms (SNPs) in the OPN and CD44 genes with clinical outcomes in 307 non-small cell lung cancer (NSCLC) patients treated with radiotherapy or chemoradiotherapy. The potential impact of the variants on plasma OPN levels was also investigated. Multivariate analysis showed that OPN rs11730582 CC carriers had a significantly increased risk of death (p = 0.029), while the CD44 rs187116 A allele correlated with a reduced risk of locoregional recurrence (p = 0.016) in the curative treatment subset. The rs11730582/rs187116 combination was associated with an elevated risk of metastasis in these patients (p = 0.016). Furthermore, the OPN rs1126772 G variant alone (p = 0.018) and in combination with rs11730582 CC (p = 7 × 10-5) was associated with poor overall survival (OS) in the squamous cell carcinoma subgroup. The rs11730582 CC, rs187116 GG, and rs1126772 G, as well as their respective combinations, were independent risk factors for unfavorable treatment outcomes. The impact of rs11730582-rs1126772 haplotypes on OS was also observed. These data suggest that OPN and CD44 germline variants may predict treatment effects in NSCLC.