Search Results (162)

Scaffolds are widely used in bioengineering, both as 3D native tissue-mimicking models for investigating mechanisms under physiological and pathological conditions and also as implantable agents in regenerative medicine. Histological approaches, mainly formalin-fixed paraffin-embedded (FFPE) and frozen sample sectioning, are commonly applied to evaluate cell distribution and tissue-like properties of scaffolds. However, standard histological processing is not always compatible with the materials that scaffolds are made of. Thus, some adaptations to protocols are required to obtain intact sections. In this review we discuss challenges related to the histological processing of scaffolds and solutions to overcome them. We sequentially cover processing steps of the three main histological techniques for sample preparation—cryomicrotomy, FFPE samples microtomy and vibrating microtomy. Furthermore, we highlight the critical considerations in choosing the most appropriate method based on scaffold composition, mechanical properties and the specific research question. The goal of this review is to provide practical guidance on choosing reliable histological evaluation of complex scaffold-based systems in tissue engineering research. Full article

Synthetic biology has fundamentally advanced cell engineering and helped to develop effective therapeutics such as chimeric antigen receptor (CAR)-T cells. For these applications, the detection, localization, and quantification of heterologous fusion proteins assembled from interchangeable building blocks is of high importance. The V5 tag, a 14-residue epitope tag, offers promising characteristics for these applications but has only rarely been used in this context. Thus, we have systematically evaluated the murine anti-V5 tag antibody mu_SV5-Pk1 as well as its humanized version, hu_SV5-Pk1, to analyze cells expressing V5-tagged receptors in samples from various in vitro and in vivo experiments. We found that the V5 tag signal on cells is affected by certain fixation and detachment reagents. Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) mouse tissue samples was performed to sensitively detect cells in tissue. We improved IHC by applying the hu_SV5-Pk1 monoclonal antibody (mAb) to avoid cross-reactivity within and unspecific background signals arising on fixed mouse tissue. Conversely, the absence of unspecific binding by the mu_SV5-Pk1 mAb was evaluated on 46 human normal or cancer tissues. Our findings present a robust toolbox for utilizing the V5 tag and cognate antibodies in synthetic biology applications. Full article

Persistent high-risk human papillomavirus (HR-HPV) infection is closely associated with the development of cervical intraepithelial neoplasia (CIN) and cervical cancer. In recent years, the potential impact of viral co-infections on this process has also been investigated. This study investigated the presence of HR-HPV, HSV-1/2, and HHV-8 DNA in formalin-fixed paraffin-embedded (FFPE) cervical biopsy samples, as well as their association with lesion severity. A total of 276 FFPE cervical tissue samples were evaluated. Viral DNA was detected by real-time PCR. The samples were histopathologically classified as normal/non-dysplastic, low-grade (LSIL), and high-grade (HSIL) lesions. HR-HPV DNA was detected in 112 samples (40.6%), with the highest prevalence observed in the 30–39 age group (51.2%). Among the HPV-positive cases, 46.5% (52/112) had single-type infections, 32.1% (36/112) had multiple-type infections, and 21.4% (24/112) were untypable. Together, these categories accounted for all HPV-positive samples. The most common genotype was HPV-16 (16.7%). HHV-8 and HSV-2 DNA were not detected. HSV-1 DNA was detected in only three non-dysplastic, HPV-negative cervical samples. In conclusion, HR-HPV DNA was detected in 40.6% of cervical biopsy samples and showed a significant association with increasing histological severity, highlighting its critical role in the progression of cervical lesions. Although the absence of HHV-8 and HSV-2 suggests a limited contribution of these viruses to cervical disease, the use of a single real-time PCR assay limits the ability to draw generalized conclusions regarding their clinical relevance. Further large-scale, multicenter studies employing both tissue-based and serological approaches are needed to validate these findings and to better understand the dynamics of viral co-infections in cervical disease. Full article

MET exon 14 skipping mutations have emerged as significant driver alterations in non-small-cell lung cancer (NSCLC), contributing to tumor progression. This study examines the immune microenvironment in NSCLC patients with these mutations and its prognostic implications. We performed multiplex immunofluorescence (mIF) staining on formalin-fixed paraffin-embedded (FFPE) tissue samples from nine NSCLC patients, including four recurrent/metastatic and five non-recurrent/non-metastatic patients. Two panels assessed immune cell markers (CD8, CD4, CD20, CD68, and FoxP3) and immune checkpoints (PD-L1, LAG3, and TIM3). Immune cell infiltration and checkpoint expression were analyzed using HALO^TM software (version 3.6.4134.464). Nearest neighbor analysis was conducted to assess the proximity of immune cells to tumor cells. Univariate Cox regression analysis assessed factors associated with disease-free survival (DFS). CD8+TIM3+ and CD8+LAG3+ cells were predominantly located in the tumor parenchyma of recurrent/metastatic patients but localized to the stroma in non-recurrent/non-metastatic patients. Non-recurrent/non-metastatic patients exhibited a higher density of tertiary lymphoid structures and closer proximity of CD20+ B cells, CD8+TIM3+, and CD8+LAG3+ cells to tumor cells compared to recurrent/metastatic patients, though the differences were not statistically significant. Cox regression analysis suggested a potential association between higher densities of CD8+TIM3+ cells and improved DFS (HR = 0.89), though these findings did not reach statistical significance. Our findings suggest that differences in immune microenvironmental factors, particularly those related to immune checkpoint expression (TIM3 and LAG3), may influence clinical outcomes in NSCLC patients with MET exon 14 skipping mutations. Further studies are needed to validate these observations and explore potential therapeutic implications. Full article

In the era of precision oncology, comprehensive molecular profiling is critical for guiding targeted and immunotherapy strategies. This study presents the analytical and clinical validation of a 1021-gene next-generation sequencing (NGS) panel, designed for use with both formalin-fixed paraffin-embedded (FFPE) tissue- and liquid-biopsy specimens. Analytical validation confirmed the assay’s high sensitivity and specificity across variant types—including SNVs (Single Nucleotide Variations), indels, CNVs (Copy Number Variations), and fusions—down to a 0.5% variant allele frequency. The assay also accurately identified microsatellite instability (MSI) and tumor mutational burden (TMB), essential biomarkers for immunotherapy. Clinical validation was performed on over 1300 solid tumor samples from diverse histologies, revealing actionable alterations in over 50% of cases. The panel detected on-label treatment biomarkers in 12.57% of patients, increasing to 20.15% when immunotherapy markers were included. Additionally, the assay demonstrated strong concordance with orthogonal methods and was effective in detecting variants in plasma-derived circulating tumor DNA in 70% of evaluable cases. These findings support the robust performance and broad clinical applicability of the 1021-gene panel for comprehensive genomic profiling in both tissue and liquid biopsies, offering a valuable tool for personalized cancer treatment. Full article

The upcoming wave of personalized medicine, driven by genomic diagnostics and artificial intelligence, demands clearly defined pre-laboratory and laboratory procedures to ensure the acquisition of DNA and RNA of sufficient quantity and quality. In prostate cancer oncogenetics, diagnostic and prognostic assessments increasingly rely on personalized approaches, including Comprehensive Genomic Profiling (CGP). In this pilot study, we aimed to establish optimal pre-analytical and analytical conditions for selected genetic diagnostic methods using tissue samples acquired through multiparametric MRI-guided biopsy. Tissue specimens from thirteen patients were processed for DNA isolation, fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS). Comparative analyses were performed on DNA derived from both fresh and formalin-fixed, paraffin-embedded (FFPE) samples. Sequencing quality metrics demonstrated markedly superior performance in fresh tissue compared to FFPE. These results highlight the importance of standardized tissue collection and processing protocols to enable reliable molecular diagnostics in prostate cancer. Our findings support the feasibility of integrating high-quality genomic testing into routine biopsy workflows and emphasize the need for further large-scale validation. Full article

Human cutaneous malignant melanoma is a skin cancer that develops from melanocytes, the cells specialised in the production of eu- and pheomelanin. A growing body of evidence suggests that pheomelanin in particular is involved in melanoma development. The aim of this study was to develop a new method enabling the determination of the pheomelanin in formalin-fixed paraffin-embedded (FFPE) tissue specimens of human nodular (NM) and superficial spreading (SSM) melanomas. The pheomelanin level was evaluated in a small amount of material obtained from FFPE melanoma samples (less than 1 mg), using a multi-step procedure of paraffin removal, tissue rehydration, and homogenisation, omitting the melanin isolation step. The obtained product was studied for pheomelanin content using the Py-GC/MS/MS method operating in a multiple reaction monitoring (MRM) mode. The results of our research confirmed the presence of all the pheomelanin markers in the FFPE human melanoma specimens and showed that the tissues analysed contained different amounts of pheomelanin isomers (5-S-cysteinylDOPA and 2-S-cysteinylDOPA). The developed Py-GC/MS/MS procedure enables sensitive quantification of pheomelanin in FFPE human melanoma samples, facilitating broader studies on its role in melanoma development and progression. This method opens new avenues for investigating pheomelanin’s involvement in melanoma malignancy. Full article

Background/Objectives: Primary sclerosing cholangitis (PSC) is a rare, incurable liver disease characterized by chronic biliary inflammation and fibrosis. PSC is a significant risk factor for biliary tract cancer (BTC). This study aims to evaluate STAT3 expression in BTC and its prognostic significance as well as explore the potential of organoids derived from PSC and liver tumor patients as an in vitro model for testing novel therapeutic strategies in both PSC and BTC. Methods: Fresh tissue samples obtained from 10 PSC patients through targeted endoscopic retrograde cholangiography (ERC) and biopsy samples from liver tumor patients were used to establish organoid cultures. Organoids were treated with different agents and the therapeutic effect was measured by CellTiterGlo. Treatment with the JAK inhibitor baricitinib was followed by the measurement of cytokine concentrations in the supernatant. Archived formalin-fixed paraffin-embedded (FFPE) samples from 55 surgically resected BTC tumors were analyzed for STAT3 expression using immunohistochemistry. Results: We successfully established organoid cultures from all ERC samples. STAT3 protein expression was detected in 56% of tumor samples and 69% of the immune microenvironment. STAT3 positivity in the immune cell compartment was associated with longer disease-free survival, although the multivariate analysis could not confirm its value as an independent prognostic factor. Chemotherapy testing on liver tumor organoids showed various degrees of decreases in viability after treatment with gemcitabine, cisplatin, and cabozantinib. Baricitinib treatment significantly reduced IL-6 and MCP-1 secretion in cholangiocarcinoma Conclusions: The patient-derived organoid model of PSC and liver tumors is a valuable tool for testing novel and established therapeutic strategies, including JAK inhibitors and chemotherapy regimens. STAT3 expression in the immune microenvironment of BTC may serve as a prognostic marker. Further studies are needed to explore the integration of co-cultured organoid systems with stromal and immune components to improve physiological relevance. Full article

We developed a measurement setup and protocol reliably relating complex permittivity measurements with tissue characterization and specific histological features. We measured 148 fresh human tissue samples across 14 tissue types at 51 frequencies ranging from 200 MHz to 20 GHz, using an open-ended coaxial slim probe. Tissue samples were collected using a punch biopsy, ensuring that the sampled area encompassed the region where complex permittivity measurements were performed. This approach minimized experimental uncertainty related to potential position-dependent variations in permittivity. Once measured, the samples were then formalin-fixed and paraffin-embedded (FFPE) to obtain histological slides for microscopic analysis of tissue features. We observed that complex permittivity values are strongly associated with key histological features, including fat content, necrosis, and fibrosis. Most tissue samples exhibiting these features could be differentiated from nominal values for that tissue type, even accounting for statistical variability and instrumental uncertainties. These findings demonstrate the potential of incorporating fast in situ complex permittivity for fresh tissue characterization in pathology workflows. Furthermore, our work lays the groundwork for enhancing databases where complex permittivity values are measured under histological control, enabling precise correlations between permittivity values, tissue characterization, and histological features. Full article

Background: Cocaine abuse represents a serious health issue. The cardiovascular system is one of the main sites on which cocaine elicits its toxicity, as indicated by deadly events mainly related to myocardial infarction. The main aim of this study was to characterize the histological and immunohistochemical alterations related to cocaine abuse in cardiac tissue. Methods: Cardiac tissue samples derived from cocaine-related (n = 30) and not-cocaine-related deaths (n = 30). Histomorphology evaluations and immunohistochemistry for inflammatory biomarkers (CD45 and CD3) have been performed on formalin-fixed, paraffin-embedded (FFPE) cardiac tissue samples. Results: A higher frequency of cardiac alterations, such as wavy fibers, interstitial edema, fibrosis and hemorrhagic extravasation, were found in the group of cocaine users compared to the control group. Moreover, immunohistochemical analysis showed higher levels of inflammatory cells infiltrate within the cocaine-related deaths group. Conclusions: These data could shed new light on the complex relationship between cocaine use and cardiac alterations. Specifically, our data support the evidence that cocaine abuse is related to cardiac inflammation. Therefore, the generation of an inflammatory state could promote functional and structural cardiac alterations and lead ultimately to myocardial infarction. This would explain the high frequency of acute myocardial infarction in cocaine users. Full article

Sensitive and specific detection of DNA methylation is crucial for the early diagnosis of various human diseases, particularly cancers. However, conventional methylation detection methods often face challenges in balancing both sensitivity and specificity. In this study, we present a novel approach that integrates the high specificity of methylation-dependent restriction endonuclease (GlaI) digestion with the amplification efficiency of specific terminal-mediated polymerase chain reaction (STEM-PCR). This combination enables selective amplification of methylated DNA, which is then detected through lateral flow detection (LFD), providing a simple, visual readout. As a proof of concept, a STEM-PCR-LFD assay was applied to detect methylated Septin 9, a biomarker for colorectal cancer. The assay demonstrated a sensitivity of approximately 0.1% (10 copies of methylated template per reaction), with no cross-reactivity observed when 10,000 copies of unmethylated DNA were included as background. Furthermore, the assay was validated with ten formalin-fixed paraffin-embedded (FFPE) tissue samples, achieving 100% consistency with standard real-time STEM-PCR. This method offers a highly sensitive, specific, and accessible platform for DNA methylation detection, with potential for early disease diagnosis. Full article

Background/Objectives: The oncofetal membrane protein Claudin 6 (CLDN6) is an attractive target for T cell-based therapies. There is a lack of detailed analyses on the age-dependent expression of CLDN6 in normal tissues is lacking, which limits the expansion of CLDN6 CAR-T cell clinical trials to pediatric populations. Methods: We analyzed CLDN6 expression in extracranial solid tumors and normal tissues of children using RNA-sequencing data from over 500 pediatric solid tumor samples, qRT-PCR and immunohistochemistry (IHC) in more than 100 fresh-frozen tumor samples and, approximately, 250 formalin-fixed paraffin-embedded (FFPE) samples. We examined normal tissue expression via qRT-PCR in 32 different infant tissues and via IHC in roughly 290 tissues from donors across four age groups, as well as in fetal autopsy samples. Results: In fetal tissues, we detected CLDN6 expression primarily in the epithelial cells of several organs, including the skin, lungs, kidneys, intestinal tract, and pancreas, but not in undifferentiated blastemal cells. Postnatally, we found CLDN6-positive epithelial progenitors only during the first few weeks of life. In older-age groups, isolated clusters of CLDN6-positive progenitors were present, but in scarce quantities. In tumor tissues, we found strong and homogeneous CLDN6 expression in desmoplastic small round cell tumors and germ cell tumors. Wilms tumors demonstrated heterogeneous CLDN6 expression, notably absent in the blastemal component. Conclusions: These findings highlight an organ-specific presence of CLDN6-positive epithelial precursors that largely disappear in terminally differentiated epithelia within weeks after birth. Therefore, our data support CLDN6 as a viable therapeutic target in pediatric patients and justify their inclusion in basket studies for anti-CLDN6-based therapies. Full article

The identification of fungal pathogens in formalin-fixed paraffin-embedded (FFPE) tissues is an unmet need in human and animal medicine, and sequence-agnostic approaches are needed to identify emerging pathogens. Eleven FFPE biopsy specimens with etiologic diagnoses of fungal disease based on standard testing of paired fresh tissue samples were utilized here to evaluate metabarcoding approaches. The cases included tissues from three dogs, three cats, one box turtle, one goat, one common loon, and one gray tree frog. The diagnoses from the fresh tissues in these cases were Microsporum canis, Penicillium sp., Exophiala sp. (likely E. jeanselmei), Verticillium sp., Rhizopus sp., atypical Cryptococcus neoformans, Conidiobolus spp., Aspergillus fumigatus, Cryptococcus neoformans var grubii, Batrachochytrium dendrobatidis, Fusarium solani, Blastomyces dermatitidis, Coccidiodes immitis, and Histoplasma capsulatum. We compared the ITS1 and 28S D1 rRNA gene genetic markers in combination with several bioinformatic strategies to identify fungal pathogens in the FFPE tissue samples, with a success rate of 9/11. These methods could allow diagnosticians who receive only FFPE tissues and see fungal pathogens to speciate the pathogens and could be of value in retrospective studies wherein FFPE tissue is the only archived tissue. Furthermore, these techniques could be of use to researchers investigating polymicrobial communities where DNA preservation is suboptimal. Full article

The purpose of this study was to evaluate the suitability of formalin-fixed and paraffin-embedded (FFPE) samples and fixed fresh (FF) samples for single-cell RNA sequencing (scRNAseq). To this end, we compared single-cell profiles from FFPE and matched FF tissue samples of one invasive carcinoma of no special type carcinoma (invasive ductal carcinoma–IDC) and one invasive lobular carcinoma (ILC) to assess consistency in cell type distribution and molecular profiles. The results were validated using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and electron microscopy. Additionally, immune cell proportions identified by IHC were quantified using QuPath and compared to the scRNAseq results. FFPE- and FF-derived libraries demonstrated high-quality sequencing metrics, and cellular heterogeneity was similar. No exclusive cell populations were identified by either approach. The four samples analysis identified six types of epithelial cells, as well as tumoral microenvironment populations. The scRNAseq results from epithelial neoplastic cells were concordant with common IHC markers. The proportion of immune cells identified by IHC in FFPE sections were similar to those obtained by scRNAseq. We identified and validated a previously poorly recognized subpopulation of neoplastic multi-ciliated cells (MCCs) (FOXJ1, ROPN1L). Analysis of FOXJ1 in 214 ER-positive invasive carcinomas demonstrated protein expression in one third of tumors, suggesting frequent focal MCC differentiation. Our results support the suitability of scRNAseq analysis using FFPE tissue, and identified a subpopulation of neoplastic MCC in breast cancer. Full article

Background: The mammalian NAD-dependent deacetylase sirtuin-1 family (named also silent information regulator or SIRT family, where NAD stands for “nicotinamide adenine dinucleotide” (NAD)) appears to have a dual role in several human cancers by modulating cell proliferation and death. This study examines how SIRT1 protein levels correlate with clinicopathological characteristics and survival outcomes in patients with breast cancer. Methods: A total of 407 BC formalin-fixed paraffin-embedded (FFPE) samples were collected from King Abdulaziz University Hospital, Saudi Arabia. SIRT1 was stained on tissue microarray slides using automated immunohistochemistry. Results: All BC subtypes expressed more nuclear SIRT1 proteins than their cytoplasm counterparts. In luminal A, luminal B, and TNBC, nuclear and cytoplasmic SIRT1 were highly associated (p < 0.001). Kaplan–Meier analysis showed reduced disease-specific survival (DSS) in H2BC with high SIRT1 nuclear expression (p = 0.001, log-rank). Moreover, the cytoplasmic expression of SIRT1 in HER2-positive BC was associated with a larger tumor size (p = 0.036) and lymph node metastasis (p = 0.045). Nuclear SIRT1 expression was also positively associated with lymph node metastasis (LNM) (p = 0.048). As low-grade tumors had a higher frequency of SIRT1 protein expression than other groups, SIRT1 expression was associated with a favorable prognosis in patients with luminal A BC (p < 0.001). Conclusions: SIRT1 expression seems to be involved in different molecular pathways either suppressing or promoting tumor growth depending on the subtype of BC. These molecular functions require further investigations and validation on larger BC cohorts. Full article