Tissue Specimens Research Articles

Neurprotective Role of Amaranthus Dubius on Penicillin Induced Experimental Epileptic Rat Model by Combating the Multineurotransmitter Deficiency

Objective: The study aims to clarify the protective effects of Amaranthus dubius (AD) in penicillin (PCN)-induced experimental epilepsy models in rats. Method: In this study, twenty-four adult male Wistar albino rats, each weighing between 200-250 grams, were utilized. The rats were allocated into four groups: a control group, an Amaranthus dubius (AD) treated control group, a PCN-induced experimental epileptic rat model group, and an AD pretreated PCN-induced experimental epileptic rat model group. All groups were administered a daily oral dosage of 400 mg/kg body weight of AD aqueous leaf extract for fourteen days via an orogastric cannula. For the experimental epileptic model, PCN was injected into designated areas of the somatosensory cortex. On the concluding fifteenth day, subsequently, the rats were humanely euthanized. Tissue specimens from the cerebral cortex (CC), cerebellum (CB), caudate nucleus (CN), pons and medulla (PM), and midbrain (MB) were extracted, measured, and homogenized to facilitate subsequent biochemical analyses. Result: Pretreatment with AD has demonstrated significant changes in the levels of antioxidants and neurotransmitters in the brain. Conclusion: Amaranthus Dubius aids in combating multi-neurotransmitter deficiencies and enhances the body's antioxidant capabilities.

Selecting Targets for Molecular Imaging of Gastric Cancer: An Immunohistochemical Evaluation.

Tumor-targeted positron emission tomography (PET) and fluorescence-guided surgery (FGS) could address current challenges in pre- and intraoperative imaging of gastric cancer. Adequate selection of molecular imaging targets remains crucial for successful tumor visualization. This study evaluated the potential of integrin αvβ6, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor-2 (HER2) for molecular imaging of primary gastric cancer, as well as lymph node and distant metastases. Expression of αvβ6, CEACAM5, EGFR, EpCAM and HER2 was determined using immunohistochemistry in human tissue specimens of primary gastric adenocarcinoma, healthy surrounding stomach, esophageal and duodenal tissue, tumor-positive and tumor-negative lymph nodes, and distant metastases, followed by quantification using the total immunostaining score (TIS). Positive biomarker expression in primary gastric tumors was observed in 86% for αvβ6, 72% for CEACAM5, 77% for EGFR, 93% for EpCAM and 71% for HER2. Tumor expression of CEACAM5, EGFR and EpCAM was higher compared to healthy stomach tissue expression, while this was not the case for αvβ6 and HER2. Tumor-positive lymph nodes could be distinguished from tumor-negative lymph nodes, with accuracy ranging from 82 to 93% between biomarkers. CEACAM5, EGFR and EpCAM were abundantly expressed on distant metastases, with expression in 88-95% of tissue specimens. Our findings show that CEACAM5, EGFR and EpCAM are promising targets for molecular imaging of primary gastric cancer, as well as visualization of both lymph node and distant metastases. Further clinical evaluation of PET and FGS tracers targeting these antigens is warranted.

Strategic insights into the cultivation of pancreatic cancer organoids from endoscopic ultrasonography-guided biopsy tissue

BACKGROUND The frequent suboptimal efficacy of endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) to culture pancreatic cancer (PC) organoids (PCOs) poses a major challenge in the advancement of personalized medicine for advanced PC. AIM To explore how to obtain appropriate puncture tissues from EUS-FNB and optimize the strategy for efficiently constructing PCOs, providing an efficient tool for the advancement of personalized medicine. METHODS Patients who underwent EUS-FNB for the diagnosis of PC tissue were prospectively enrolled. We refined the endoscopic biopsy procedures and organoid cultivation techniques. All tissue specimens verified by on-site pathological assessment were cultured in a semi-suspended medium in a microfluidic environment. We assessed differences in PCOs cultured beyond and below five generations examining patient demographics, specimen and organoid attributes, and the sensitivity of organoids to a panel of clinical drugs through cell viability assays. RESULTS In this study, 16 patients with PC were recruited, one sample was excluded because onsite cytopathology showed no tumor cells. Successful organoid generation occurred in 93.3% (14 of 15) of the EUS-FNB specimens, with 60% (9 of 15) sustaining over five generations. Among these patients, those with a history of diabetes, familial cancer, or larger tumors exhibited enhanced PCO expandability. The key factors influencing long-term PCOs expansion included initial needle sample quality (P = 0.005), rapid initiation of organoid culture post-isolation (P ≤ 0.001), and high organoid activity (P = 0.031). Drug sensitivity analysis revealed a partial response in two patients following therapeutic intervention and surgery and stable disease in four patients, indicating a moderate correlation between organoid response and clinical outcomes. CONCLUSION Optimal initial needle sampling, rapid and precise biopsy sample processing, process isolated samples as soon as possible, and sufficient cellular material are crucial for successful cultivating PCOs. High organoid activity is an important factor in maintaining their long-term expansion, which is essential for shortening the time of drug sensitivity analysis and is the basis of PC research.

The Infected Diabetic Foot: Does Negative Pressure Wound Therapy with Irrigation Reduce Bioburden and Improve Wound Healing?

The aim of this study was to compare the microbial loads of patients with diabetic foot infections treated with negative pressure wound therapy (NPWT) with and without irrigation with polyhexamethylene biguanide (NPWTi-P). This is a post hoc analysis of combined data of two randomized clinical trials. We evaluated people with diabetes treated with moderate and severe diabetic foot infections that required surgery. Tissue specimens were obtained after the initial surgery and following a second planned return to the operating room after 48-72 h of NPWT or NPWTi-P, prior to the second surgery. We used quantitative polymerase chain reaction (qPCR) to determine the total microbial loads (Log10 16S copies per gram of tissue). There was no difference in mean quantitative bacterial cultures among patients that received NPWT and NPWTi-P (before first surgery Log10: NPWT = 6.4 ± 1.8, NPWTi-P = 7.5 ± 1.7 vs before second surgery Log10: NPWT = 6.7 ± 1.8, NPWTi-P = 7.6 ± 1.9 p = .12). There was no difference in wound healing (59.5% vs 50.0%, p = .51) or time to heal (127 ± 109.3 vs 143 ± 95.9), p = .71). There were fewer re-infections in people that received traditional NPWT (28.6% vs 56.3%, p = .05). Level of Clinical Evidence: Level 1.

Abstract B069: Spatial and single-cell proteomic landscaping of the hypoxic microenvironment in glioblastoma

Abstract Glioblastoma (GBM) is a fatal adult solid tumour with median overall survival of 18-20 months post-diagnosis; contributing factors to therapeutic inefficacy include acquisition of genomic alterations post-therapy, immune evasion, deregulated hypervascularization, and tumor microenvironmental factors such as hypoxia. Combined with extensive inter- and intra-tumoral heterogeneity at bulk and single-cell level, hypoxia contributes to a gradient of molecular alterations that are specific to different cell populations that make up tumour bulk and reside in specific niches. Hitherto, high-dimensional histopathologic analyses of hypoxic regions within GBM tissue have not been performed. We took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study wherein the exogenous hypoxia marker, pimonidazole (PIMO), was administered to GBM-patients preoperatively. Tissue specimens were subjected to imaging mass cytometry, high-resolution imaging, and serial immunohistochemistry using a panel of markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. Our findings showed that PIMO staining is associated with histopathologic features of hypoxia and correlates with specific metabolic, immune, and stemness markers in GBM; specific lymphocyte populations were depleted from hypoxic regions alongside alterations in macrophagic and microglial landscape in a niche-specific manner; hypoxia reduced the proportion of proliferating glioma initiating cells and altered the proliferative, transcriptional, and translational capacity of different cell populations; microvessel density was reduced in hypoxic microenvironment. Cytometry by time-of-flight further validated the altered proportions of specific immune cell types enriched in hypoxic populations of GBM microenvironment. Our study is the first to report use of PIMO to interrogate spatial, single-cell, and phenotypic architecture associated with tissue hypoxia and altered expression of biomarkers associated with hypoxia/glycolysis, immune infiltration, proliferation, and stemness. Identification of targetable biomarkers and mediators of hypoxia-driven habitats in GBM may provide direction for future immunotherapeutic research. Citation Format: Shreya Gandhi, Sheila Mansouri, Olivia Singh, Mark Zaidi, Ronald Wu, Andrew Gao, Bradley Wouters, Gelareh Zadeh. Spatial and single-cell proteomic landscaping of the hypoxic microenvironment in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B069.

Sharing of data archive of radiation exposure animal experiments in QST/NIRS and IES.

Institute for Radiological Science (NIRS), National Institutes for Quantum Science and Technology (QST), and Institute for Environmental Sciences (IES) have conducted large-scale animal experiments for radiation risk analyses in terms of life shortening and cancer prevalence. It is important to store data and biological samples from these large-scale experiments for sharing and future use since the economic and practical limitations, as well as the ethical considerations, make it difficult. QST/NIRS established an archive called the Japan Storehouse of Animal Radiobiology Experiments (J-SHARE) for the purpose of storing and sharing these historic collections. We plan to integrate the data and tissue specimen images obtained at the IES into J-SHARE by standardizing the archive format, with the aim of maximizing the results of radiation biology research. This integration is expected to contribute to the generation of new knowledge for radiation risk assessment and the provision of scientifically based information on radiation protection.

Abstract B012: M-PACT: Methylation-based predictive algorithm for CNS tumor liquid biopsies

Abstract Background: DNA methylation-based classification has been transformative in the molecular diagnostics of pediatric central nervous system (CNS) tumors. Current diagnostic pipelines integrate histopathology and molecular tools in accordance with the WHO classification of CNS tumors which rely on the availability of tissue specimens. However, some tumors are not amenable to neurosurgical resection due to their high-risk locations and tissue collection is not routinely performed at relapse. In addition, molecular biomarkers are largely lacking for longitudinal disease monitoring in pediatric neuro-oncology. Liquid biopsies (LBs) have emerged as a minimally invasive, longitudinal source of tumor-derived cell-free DNA (cfDNA) ideally suited for detecting minimal residual disease (MRD). In addition, LBs provide a sensitive means of studying tumor evolution at high temporal resolution. Success rates of LB analyses in neuro-oncology have varied drastically between studies, warranting the deployment of more robust and reproducible assays. Methods: We applied a novel cfDNA methylation-based workflow to a large cohort of cerebrospinal fluid (CSF) samples collected from pediatric brain tumor patients (n>200 patients). Enzymatic methylation sequencing (EM-seq) was adapted for CSF-derived cfDNA inputs in the picogram range, enabling the acquisition of genome-wide cfDNA methylation profiles across the cohort. Utilizing non-oncological cfDNA profiles and CNS tumor DNA methylation datasets as a reference, we developed an innovative computational pipeline that incorporates DNA methylation imputation and deep learning of a neural network. In addition, deconvolution of cfDNA profiles facilitated classification of low tumor burden samples, including those with balanced genomes that would have been missed using previous generation assays. Results: Our CSF-based classifier, M-PACT (Methylation-based Predictive Algorithm for CNS Tumors), yielded accurate tumor detection and entity prediction (AUC=0.9) in our benchmarking cohort (n>100 CSF samples). Proof-of-concept studies showed the potential of this methodology to track tumor evolution in serial CSF samples at both the genetic and epigenetic level, illuminating potential mechanisms driving treatment resistance and recurrence. Conclusions: Collectively, this study provides the framework for robust methylation- based tumor detection and classification of CSF LBs. M-PACT can be applied to aid in tumor diagnostics and to detect MRD during follow-up, warranting prospective validation of its broad applicability and clinical utility in future trials. Citation Format: Tom T. Fischer, Kyle S. Smith, Katie Han, Anna Kostecka, Hong Lin, Daniel Senfter, Tatjana Wedig, Nathalie Schwarz, Natalia Stepien, Sibylle Madlener, Christine Haberler, Esther Hulleman, Sandeep K. Dhanda, Stefan M. Pfister, Santhosh Upadhyaya, Amar Gajjar, Giles W. Robinson, Joonas Haapasalo, Hannu Haapasalo, Kristiina Nordfors, Johannes Gojo, Kendra K. Maass, Kristian W. Pajtler, Paul A. Northcott. M-PACT: Methylation-based predictive algorithm for CNS tumor liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B012.

Abstract 4137845: A single-cell lung atlas of human pulmonary arterial hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a disaster disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Hypothesis: We hypothesis that single-cell RNA sequencing analysis can help understand the cellular and molecular mechanisms that drive the disease initiation and progression. Methods: 10 healthy donors, 10 idiopathic PAH, 10 drug and toxin induced PAH, 10 systemic and pulmonary shunting induced PAH patients from both male and female, and different ethical groups and races, and ages were obtained from PHBI. Lung tissue specimens were processed for fixed scRNA-seq. The cell proportion change, gene expression, and pathway analysis were evaluated between different disease subclasses, sex, age on different cell types. Results: Our analysis reveals 317,414 cells, 33 clusters and 9 major cell types, including endothelial cells (ECs), alveolar cells, fibroblast, smooth muscle cells (SMCs), pericytes, myeloid cells, lymphocytes, airway epithelial cells, platelet. Cell proportion analysis demonstrated an increase in the proportions of venous ECs, adventitial fibroblasts and myofibroblasts, alveolar macrophages, B cells, plasma cells and a reduction in capillary ECs 2 and pericytes in PAH lungs. KEGG Pathway analysis showed that upregulated pathways related Herpes simplex virus 1 infection, ECM-receptor interaction, Complement and coagulation cascades, Hedgehog signaling, TGF-beta signaling pathway, and downregulated pathways related to IL-17 signaling, TNF signaling, MAPK signaling pathway were enriched in the PAH patients. Female PAH patients exhibit an increase of myofibroblasts and platelets proportion compared to male PAH patients. MsigDB Hallmark Pathway analysis showed that p53 pathway was upregulated, whereas Inflammatory Response, Hypoxia, Epithelial Mesenchymal Transition were downregulated in female PAH patients. PAH patients older than 21 years exhibit an increase in the cell proportion of Platelet, SMCs, Arterial ECs and Venous ECs, and alveolar macrophages and a reduction of B cells and plasma cells. Conclusions: Our integrated analysis of human PAH lung atlas dataset provides a comprehensive understanding of lung cell populations and molecular signature in PAH patients with different sex, age and subclasses

A comprehensive genome-based analysis identifies the anti-cancerous role of the anoikis-related gene ADH1A in modulating the pathogenesis of breast cancer.

Breast cancer (BC), a widespread and lethal neoplasm, is irrespective of the subtype of BC. Metastasis remains a crucial determinant for unfavorable outcome. The identification of novel diagnostic markers is instrumental in optimizing the treatment regime for BC. The direct correlation between anoikis and the progression/outcome of BC is well established. Nevertheless, the contribution of anoikis-related genes (ARGs) in BC remains obscure at present. We implemented the METABRIC dataset to scrutinize and assess differentially expressed ARGs in BC versus healthy breast tissues. An unsupervised consensus clustering approach for ARGs was employed to classify patients into diverse subtypes. ESTIMATE algorithms were utilized to assess immune infiltrative patterns. Prognostic gene expression patterns were derived from LASSO regression and univariate COX regression analysis. Subsequently, these signatures underwent examination via use of the Kaplan-Meier survival curve. 6 pairs of fresh tissue specimens (tumor and adjacent non-tumor) were employed to assess the expression of 7 ARGs genes via qPCR. Notably, DCN and FOS were not expressed in BC tissue, which had been excluded in our subsequent experiments. Also, among remaining 5 ARGs, solely the expression of ADH1A demonstrated a statistically remarkable disparity between freshly collected cancer tissues and the adjacent ones. ADH1A-overexpressed and ADH1A-sh vectors were transfected into MCF-7 and MCF-7-AR cell lines, respectively. The expression status of FABP4, CALML5, ADH1A, C1orf106, CIDEC, β-catenin, N-cadherin, and Vimentin in the clinical samples were scrutinized using RT-qPCR and western blotting techniques. Migration and invasion through transwell chambers were employed to assess the migratory and invasive potential of the cells. Detailed evaluation of cell proliferation was conducted utilizing a Cell Counting Kit-8 (CCK-8) assay. The apoptotic index of the cells was determined by flow cytometry analysis. An innovative anoikis-associated signature consisting of seven genes, namely ADH1A, DCN, CIEDC, FABP4, FOS, CALML5, and C1orf106, was devised to stratify BC patients into high- and low-risk cohorts. This unique risk assessment model, formulated via the distinctive signature approach, has been validated as an independent prognostic indicator. Additional analysis demonstrated that distinct risk subtypes manifested variances in the tumor microenvironment and drug sensitivities. Suppression of ADH1A enhanced the migratory and invasive capacities and reduced these tumorigenesis-related protein levels, underscoring the prognostic role of ADH1A in the progression of BC. Through our meticulous study, we have elucidated the possible molecular markers and clinical implications of ARGs in BC. Our model, which incorporate seven ARGs, has proven to accurately forecast the survival outcomes of BC patients. Moreover, the thorough molecular study of ADH1A has augmented our comprehension of ARGs in BC and opened a novel avenue for guiding personalized and precise therapeutic interventions for BC patients.

TMET-38. BIOCHEMICAL CHARACTERIZATION OF PSAMMOMATOUS MENINGIOMA USING MAGNETIC RESONANCE SPECTROSCOPY

Abstract Psammomatous meningioma is a histologic subtype of meningioma and is presented as a highly calcified intracranial or spinal lesion. Although computed tomography (CT) and short/zero echo-time MRI techniques can detect calcification in tumors, a complete biochemical profile of psammomatous meningiomas is not known. Here, we compare the metabolomic profiles of common meningiomas (WHO grade-I and grade-II) and a psammomatous meningioma using ex vivo proton magnetic resonance spectroscopy (1H MRS). The tumor specimens were collected from meningioma patients undergoing surgery for the resection of the tumor mass. Tumor samples were extracted in 5% perchloric acid, centrifuged, supernatants were vacuum dried and reconstituted in D2O containing 1.0 mM sodium 2,2-dimethyl- 2-silapentane-5-sulfonate-d₆ (DSS-d6, internal standard). Previously, we have identified the following metabolites in grade-I and grade-II meningioma tumors: leucine, isoleucine, valine, lactate, alanine, acetate, glutamate, succinate, glutamine, aspartate, creatine, phosphocreatine, phosphocholine, glycerophosphocholine, myo-inositol, scyllo-inositol, taurine, hypotaurine, glycine, phosphoethanolamine, and glucose. In the current study, psammomatous meningioma also showed the presence of all the above metabolites; however, the levels of these metabolites were lower concentrations compared to the grade-I and grade-II meningiomas, except for lactate. In addition to the above-mentioned metabolites, psammomatous meningioma showed the presence of citrate which was not detected in other meningioma subtypes. Citrate is a major component of bone and the presence of citrate (~614 µM/g, tissue) in psammomatous meningioma could be due to the calcification occurring in these tumors. Surgical pathology report of resected tissue specimen showed clusters and small whorls of meningothelial cells with scattered calcifications, consistent with psammomatous meningioma (WHO grade-I; Ki-67 = 3%). In conclusion, detection of citrate by 1H MRS in meningiomas can be helpful in the differential diagnosis of psammomatous meningiomas.

TMIC-22. MYELOID CELLS INDUCE IMMUNOSUPPRESSION AND HYPORESPONSIVENESS OF CHIMERIC ANTIGEN RECEPTOR T CELLS IN THE NEURONAL MICROENVIRONMENT OF GLIOBLASTOMA

Abstract Chimeric antigen receptor T (CART)-cell therapy has shown little success in glioblastoma and other solid tumors, unlike in hematologic malignancies. The immunosuppressive milieu and intratumoral heterogeneity are some obstacles limiting the effectiveness of CART-cell therapy in glioblastoma. Our goal in this study is to investigate how CART-cell transcriptional adaptations and cellular interactions in the glioblastoma microenvironment drive T-cell hyporesponsiveness. Using a human neocortical slice model, we performed physically interacting cell sequencing (PIC-seq) to investigate the transcriptional diversity of NKG2D CAR-T cells and control T-cells in the glioblastoma ecosystem. Through the integration of spatially resolved T-cell receptors sequencing and PIC-seq, we investigated the cellular and receptor-ligand interactions in CART-treated glioblastoma cells. Gene regulatory networks were reconstructed to identify key transcriptional regulators of CD8 CART-cell dysfunction. We used in-silico perturbation to understand transcriptional drivers that could potentially enhance the cytotoxic response of CAR-Ts. Primary patient-derived glioma cells were inoculated into human neocortical slices from surgical access tissue specimens. After 3 days of tumor growth, slices were treated with NKG2D CARTs or control T-cells. We observed that after 2 days of anti-tumor T-cell response in the CART group, tumor growth did not differ between conditions. After 7 days of treatment, PIC-seq was used to profile CARTs (tomato+) and tumor cells (GFP+) as well as physically connected cells (+/+). We identified a transcriptional shift towards T-cell exhaustion in CART cells compared to T-cells lacking the NKG2D construct. This transcriptional phenotype was driven by myeloid-CART and myeloid-tumor interaction. We identified that tumor-associated macrophages with enhanced phagocytic function in proximity to mesenchymal-like tumor cells located within hypoxic niches are the main drivers of T-cell dysfunction. Gene regulatory network analysis demonstrated that MAF and BACH2 are key transcriptional regulators of CART-cell function. Myeloid-tumor and myeloid-T-cell interactions promote an immunosuppressive microenvironment and hyporesponsive T-cells in glioblastoma. Our data has the potential to catalyze the re-engineering and optimization of CART-cell therapies with sustained cytotoxic effect against glioblastoma.

BIOM-17. EVALUATION OF CARBONIC ANHYDRASE TYPE IX (CA IX) EXPRESSION AS TARGET FOR RADIOLIGAND THERAPY IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma represents an unmet need in neuro-oncology. Radioligand therapies are effective and approved in extracranial malignancies such as prostate cancer and neuroendocrine tumors, but data on their use is limited in brain tumors. In addition, rational target selection in gliomas is complicated by tumor heterogeneity. In extracranial tumors, clinical trials of radionuclide treatments targeting carbonic anhydrase IX (CA IX) are ongoing. METHODS Tumor samples of glioblastoma (isocitrate dehydrogenase [IDH]-wildtype) were retrieved from the Neuro-Biobank of the Medical University of Vienna. Formalin-fixed, paraffin-embedded tissue specimen were stained using rabbit anti-CA IX antibody (clone EP161, Cell Marque) on a Ventana Benchmark Ultra platform (Roche Diagnostics). CA IX expression was assessed semiquantitatively. RESULTS Overall, 29 patient samples were included in this study. Median age at surgery was 65 years (range: 55 – 74), and 22/29 (75.9%) were male. Median overall survival (OS) reached 11.6 months (95% confidence interval: 7.3 – 16.3). In total, 24/29 (82.8%) of specimen were positive for CA IX, of whom 20/29 (69.0%) exhibited moderate to high membranous CA IX expression and 4/29 (13.8%) showed weak immunoreactivity. CA IX staining displayed pronounced spatial heterogeneity, as positive areas were primarily seen in pseudo-palisading tumor cells around necrotic foci. Patients with stronger CA IX staining tended to have longer OS (median: 14.2 months) compared to those with absent or weak CA IX expression (median OS: 6.2 months; p = 0.075). Inclusion of further samples of different glioma subtypes is ongoing. CONCLUSIONS CA IX could represent a promising target for radioligand therapy in glioblastoma given the strong CA IX immunoreactivity in most samples. Intratumoral heterogeneity in CA IX expression should be considered in the design of appropriate compounds and the choice of optimal radionuclides to maximize efficacy.

Olaparib enhancing radiosensitization and anti-metastatic effect of oral cancer by targeting IL-17A signal.

We tested whether the PARP inhibitor, Olaparib, can effectively enhance radiosensitivity while inhibiting OSCC growth and metastasis in vitro and in vivo. Patient samples were used for survival validation. The present study investigated the effect of Olaparib and ionizing radiation (IR) on clonogenic, migratory, and invasive ability in human IR-sensitive (OML1) and IR-resistant (OML1-R) OSCC cell lines. We next explored the underlying mechanism with ELISA and a Western blotting assay. Two in vivo mouse models were established to investigate the efficacy of Olaparib combined with radiotherapy (RT) on local tumor growth and lung metastasis. IL-17A expression was confirmed in tissue specimens of OSCC patients by immunohistochemistry. We found that Olaparib, in combination with IR, substantially inhibited cell growth, migration, and invasion in vitro. Mechanistically, the Olaparib treatment significantly reduced the secretion of IL-17A in irradiated OSCC cells by attenuating NF-κB and p38 activity. Consistently, Olaparib enhanced the radiosensitivity and, with RT, synergistically reduced both tumor growth and lung metastasis in mice. In addition, OSCC patients with high IL-17A expression were substantially associated with an increased risk of lymph node involvement and worse survival. This study has highlighted that Olaparib displays radiosensitizing and antimetastatic effects by inhibiting the IL-17A-dependent signal. Remarkably, Olaparib could provide a remarkable anticancer efficacy to improve treatment response in OSCC patients with recurrent/metastatic disease after RT.

Establishment and Validation of Patient-Derived Non-Small Cell Lung Cancer Organoids as In Vitro Lung Cancer Models

Background: Recent advances in the personalized treatment of non-small cell lung cancer (NSCLC) require representative in vitro model systems that reflect tumor heterogeneity and maintain the characteristic genetic aberrations. We therefore aimed to establish patient-derived NSCLC organoids that offer a reliable platform for further investigations. Methods: NSCLC organoids were cultured between May 2020 and February 2022 from surgically resected NSCLC tissue specimens. After histological and immunohistochemical validation, genetic validation was performed by targeted next-generation sequencing of tissue and organoid specimens using the Oncomine Focus Assay (ThermoFisher Scientific). Results: From 37 resected NSCLC samples, 18 primary organoid cultures were successfully established and expanded during early passages. Upon histomorphological validation, organoids showed complementary characteristics when compared to the resected parental tumor, including adenocarcinoma, squamous cell carcinoma, mucoepidermoid carcinoma, and lung carcinoid differentiation. Among nine parental tumors, traceable genetic alterations were detected, and three corresponding organoids lines retained this mutational profile, including a KRAS p.Gly12Val mutation, KRAS p.Gly12Cys mutation, and RET-fusion. Conclusions: The establishment of primary NSCLC organoids from surgically resected tissue is feasible. Histological, immunohistochemical, and genetic validation is essential to identify representative NSCLC organoids that maintain the characteristics of the parental tumor. Overall, low establishment rates remain a challenge for broad clinical applications.

Mitochondrial Mutations in Cardiovascular Diseases: Preliminary Findings

Background/Objectives: Mitochondria are the main organelles for ATP synthesis able to produce energy for several different cellular activities. Cardiac cells require high amounts of energy and, thus, they contain a high number of mitochondria. Consequently, mitochondrial dysfunction in these cells is a crucial factor for the development of cardiovascular diseases. Mitochondria constitute central regulators of cellular metabolism and energy production, producing approximately 90% of the cells’ energy needs in the form of ATP via oxidative phosphorylation. The mitochondria have their own circular, double-stranded DNA encoding 37 genes. Any mitochondrial DNA sequence anomaly may result in defective oxidative phosphorylation and lead to cardiac dysfunction. Methods: In this study, we investigated the potential association between mitochondrial DNA mutation and cardiovascular disease. Cardiac tissue and serum samples were collected from seven patients undergoing coronary artery bypass grafting. Total DNA was extracted from cardiac muscle tissue specimens and serum and each sample was subjected to polymerase chain reaction (PCR) to amplify the NADH dehydrogenase 1 (ND1) gene, which is part of the mitochondrial complex I enzyme complex and was screened for mutations. Results: We identified one patient with a homoplasmic A to G substitution mutation in cardiac tissue DNA and two patients with heteroplasmic A3397G mutation in serum DNA. Specifically, amplicon sequence analysis revealed a homoplasmic A3397G substitution in the ND1 gene in a tissue sample of the patient with ID number 1 and a heteroplasmic mutation in A3397G in serum samples of patients with ID numbers 3 and 6, respectively. The A to G substitution changes the amino acid from methionine (ATA) to valine (GTA) at position 31 of the ND1 gene. Conclusions: The detection of this novel mutation in patients with coronary artery disease may contribute to our understanding of the association between mitochondrial dysfunction and the disease, implying that mitochondria may be key players in the pathogenesis of cardiovascular diseases.

Higher detectability of amyloid with phenol Congo red compared with alkaline Congo red

IntroductionAmyloidosis is a heterogeneous group of conditions associated with tissue deposition of insoluble abnormal proteins that damage vital organs. Early diagnosis, when the deposits are minimal, determines the prognosis and requires histological confirmation. The commonly adopted gold standard technique is alkaline Congo red (ACR) staining, though its sensitivity is limited. There is a need for a simple and inexpensive screening method offering a better chance of detecting mini­mal amyloid deposits. The aim of this study was to compare amyloid detectability with ACR and phenol Congo red (PHCR) staining techniques for early detection of minimal deposits.Material and methodsWe assessed 452 tissue specimens (including adipose tissue, gastrointestinal mucosa, labial salivary gland, myocardium, and bone marrow) from 425 patients with clinically suspected systemic or local amyloidosis, which had been sent to the Pathology Laboratory of the National Institute of Geriatrics, Rheumatology and Rehabilitation in Warsaw. Adjacent sections from each specimen were stained with ACR and PHCR. If amyloid was detected, immunohistochemical typing was conducted. The consistency of the two staining methods was expressed as Cohen’s κ coefficient.ResultsA total of 169 tissue specimens (37%) yielded positive readings, with 93 cases ACR(+) and PHCR(+); 75 cases ACR(–) and PHCR(+), and 1 case ACR(+) and PHCR(–). The percentage agreement between the staining methods was 83%, with the Cohen’s κ coefficient value of 0.60 (95% CI: 0.52–0.69), which corresponds to moderate agreement according to Fleiss. Additional immunohistochemical amyloid typing, conducted in ACR(–) and PHCR(+) specimens, yielded conclusive results in 82% of cases.ConclusionsThe use of PHCR staining as a screening method in suspected amyloidosis improves amyloid (light chain, transthyretin, and amyloid A protein) detectability in various tissues. The PHCR staining specificity should be verified via electron microscopy and/or mass spectrometry.

Utilizing collagen-coated hydrogels with defined stiffness as calibration standards for AFM experiments on soft biological materials: the case of lung cells and tissue

Abstract Atomic Force Microscopy (AFM) is crucial in mechanobiology for high-resolution imaging and nanomechanical measurements of biological samples, providing insights into their mechanical properties. However, AFM faces challenges such as tip damage and cantilever selection errors, impacting measurement accuracy. This study proposes a methodology using collagen-coated hydrogels with predefined stiffness for calibrating AFM measurements on soft biological materials. By facilitating appropriate cantilever selection, assessing systematic errors, and evaluating tip damage, this approach ensures reliable Young’s modulus measurements. The proof of concept with human lung cells and tissue specimens demonstrates improved accuracy and reliability of AFM-based nanomechanical characterizations, essential for understanding cellular mechanics and disease progression.

Increased oscillation rate may improve morcellation efficiency in HoLEP.

Tissue morcellation has become increasingly efficient, yet remains a rate limiting step in holmium enucleation of the prostate (HoLEP). Limited data exists on how the rate of oscillation by the morcellator blades affects morcellation efficiency (ME). We undertook a retrospective review of HoLEP procedures performed by two surgeons from July 1, 2019 to August 25, 2022. All morcellation was performed with the Wolf Piranha device and enucleation was performed with Moses 2.0 technology. Surgeon 1 routinely uses 1500 oscillations/min (low rate [LR]) and Surgeon 2 uses a rate of 6000 oscillations/min (high rate [HR]). These rates were confirmed upon EMR review of each case. The primary endpoint was ME (g/minute). Secondary endpoints included enucleation efficiency (EE), mean tissue specimen weight, and preoperative prostate volume. A total of 894 HoLEPs were analyzed, 592 by Surgeon 1 and 302 by Surgeon 2. Surgeon 1 had larger preoperative prostate volumes (126 vs. 101, p<0.001) and specimen tissue weights (86.0 vs, 61.1, p<0.001). EE was higher in the LR group (1.67 vs. 1.33 g/min, p<0.001). Morcellation time was longer in the LR group (11.3 vs. 6.09 min, p<0.001) and ME was lower in the LR group (9.26 vs. 12.1 g/min, p<0.001). The difference in ME was inversely proportional to specimen weight. Increased oscillation rate during morcellation may lead to decrease in morcellation time and increased ME during prostate enucleation. The primary limitation of this paper is the inclusion of only two surgeons. Future studies will serve to evaluate this finding across a larger number of institutions, and evaluate ways to increase ME in large prostate cohorts.

The clinical management and efficacy of metagenomic next-generation sequencing in patients with pyogenic spinal infection: a single-center cohort study

ObjectiveThis study aims to evaluate the clinical management and effectiveness of metagenomic next-generation sequencing (mNGS) in patients with pyogenic spinal infections.MethodsWe conducted a retrospective review of 17 patients diagnosed with pyogenic spinal infections and treated at our institution between October 2022 and February 2024. The cohort included 8 males and 9 females, with a mean age of 63.59 ± 10.18 years (range: 41–71 years). The infections comprised 9 epidural abscesses, 6 intervertebral space infections, and 2 deep abscesses. All patients underwent open surgical procedures and mNGS-based bacterial identification using intraoperative pus or tissue specimens, in addition to conventional blood bacterial cultures. Clinical outcomes were assessed using CRP, PCT, WBC inflammatory markers, and VAS scores postoperatively.ResultsAll 17 patients with pyogenic spinal infections underwent open surgery and mNGS bacterial detection at our institution. Among the 17 patients, mNGS yielded positive results in 14 cases (82.4%), significantly higher than the 5.9% positivity rate of conventional bacterial cultures (p < 0.001). The mNGS test time was notably shorter than conventional cultures (1.0 vs. 5.88 days, p < 0.001). Postoperative antibiotic therapy was adjusted based on mNGS findings. There were significant reductions in postoperative VAS, WBC, PCT, and CRP values compared to preoperative levels (p < 0.01).ConclusionMetagenomic next-generation sequencing is effective in managing pyogenic spinal infections by facilitating rapid and sensitive detection of pathogens. This technique improves the timeliness and accuracy of diagnosis, highlighting its potential for broader clinical use.

Long-Term Infection With a Particular Human Papillomavirus (HPV) Genotype, HPV Subtype, or HPV Genomic Variant Does not Significantly Influence the Clinical Course of Recurrent Respiratory Papillomatosis.

Recurrent respiratory papillomatosis (RRP) is caused by human papillomaviruses (HPV) 6 and 11, but the role of their genomic variants in the disease's clinical course is unclear. This study investigated whether long-term persistence of a particular HPV genotype, subtype or genomic variant influences the RRP clinical course. HPV genotyping was performed in paired baseline and follow-up RRP laryngeal tissue specimens of 59 patients. HPV6 and HPV11 genomic variants were determined in paired tissue specimens taken at least 10 years apart in 20 selected patients. HPV was identified in 58/59 patients, most commonly HPV6 (40/58), followed by HPV11 (17/58). The most prevalent HPV genomic variant was HPV11 A2. HPV6 A and HPV6 B1 were most frequent in aggressive RRP. In all patients, identical HPV genomic variants were identified in both paired specimens. RRP results from a long-term infection with the same HPV genomic variant that can be identified decades after disease onset. We report the longest duration of genetically confirmed persistent HPV infection in peer-reviewed literature, during a 44-year interval in a patient with HPB6 B1. This study suggests that infection with a particular HPV genotype, subtype, or genomic variant does not significantly influence the clinical course of RRP.