Histochemistry and Cell Biology最新文献

Acyl-CoA synthetase long-chain family member 4 (ACSL4) is a lipid-metabolizing enzyme implicated in ferroptosis regulation and tumor aggressiveness. Although ACSL4 overexpression has been reported in various malignancies, its immunohistochemical profile in primary cutaneous melanoma has not been fully characterized. This study aimed to evaluate ACSL4 expression in melanoma compared with normal skin using quantitative digital image analysis. A total of 80 formalin-fixed paraffin-embedded samples were analyzed, including 50 primary cutaneous melanoma specimens and 30 control skin samples obtained from benign dermatologic excisions. Hematoxylin-eosin staining was used to assess histopathologic features, and ACSL4 immunostaining was performed using a standardized protocol. Quantitative evaluation was conducted with QuPath software by calculating the percentage of positive cells, mean intensity scores (0-3), and H-scores (0-300) in epidermal and dermal compartments. Group comparisons were performed using the independent t test, with p < 0.05 considered statistically significant. Control tissues exhibited minimal ACSL4 expression (epidermal H-score 12; dermal H-score 9), whereas melanoma specimens demonstrated markedly increased ACSL4 immunoreactivity. Dermal atypical melanocytic tumor cells showed the highest expression levels (mean intensity 2.10 ± 0.35; H-score 168; p < 0.001), while epidermal layers also exhibited moderately elevated staining (H-score 58; p < 0.001). Histopathologic evaluation revealed characteristic features of invasive melanoma, including atypical melanocytic nests, pagetoid spread, cytologic atypia, and architectural disorder. Overall, ACSL4 expression was significantly upregulated in primary cutaneous melanoma compared with normal skin, particularly within dermal atypical melanocytic tumor cells, suggesting that ACSL4 may contribute to melanoma biology through lipid metabolic pathways and may represent a potential biomarker of tumor aggressiveness, warranting further investigation into its diagnostic and prognostic relevance.

Peroxisomes are eukaryotic organelles primarily known for their conserved roles in fatty acid β-oxidation and hydrogen peroxide detoxification. These organelles are also involved in a diverse range of other metabolic and non-metabolic functions. We recently compared the transcriptome and proteome of Saccharomyces cerevisiae wild-type and peroxisome-deficient (pex3) cells. This study uncovered the major processes and metabolic pathways that are influenced by peroxisomes. Here we performed a mass spectrometry-based analysis of intracellular metabolites of the same two strains. This led to the identification of 160 compounds, of which seven exhibited significant differences between wild-type and pex3 cells (glycerol-3-phosphate, carnitine, pantothenate, acetyl-spermidine, propionyl-carnitine, and aminolevulinic acid). Notably, we observed elevated lysine levels in pex3 cells, consistent with previous findings, which confirms the reliability and accuracy of our analytical approach. In addition, changes in carnitine compounds were measured, aligning with the proposed occurrence of a carnitine shuttle across the peroxisomal membrane. By integration of the current metabolomic data with the previously obtained transcriptomic and proteomic data, we provide a broader view of the metabolic impact of peroxisome deficiency. We show that, in addition to the well-known function of yeast peroxisomes in lipid and fatty acid degradation, these organelles are also involved in lipid synthesis. Furthermore, our study revealed that peroxisome deficiency affects polyamine homeostasis.

Peroxidasin (PXDN) is a multi-domain heme peroxidase that catalyzes sulfilimine cross-links in type IV collagen, a reaction essential for basement membrane stability. In addition to its structural role, PXDN has been implicated in oxidative stress regulation, fibrosis, and tumorigenesis. Antibody-based detection is critical for defining PXDN expression and localization, yet few commercial reagents have been rigorously validated, limiting reproducibility. Here, we assessed the specificity and performance of a commercially available anti-PXDN antibody (Abbexa, abx101906) in immunohistochemistry and immunocytochemistry applications. Validation was performed in formalin-fixed, paraffin-embedded human kidney tissue and in a primary kidney fibroblast line under PXDN silencing and overexpression conditions. The antibody yielded reproducible labeling patterns and demonstrated specificity across cellular contexts. These findings establish abx101906 as a reliable tool for immunofluorescence-based detection of PXDN. This validation provides a foundation for future studies of PXDN biology in kidney development, disease, and potential therapeutic targeting.

The development of immunohistochemical and immunohistofluorescence assays is essential for investigating the tissue and cellular distribution of target proteins. In this study, we identify specific anti-ESR1 antibodies against rodent ESR1 proteins and evaluate their applicability for immunohistochemistry and dual immunohistofluorescence with the specific anti-ESR2 antibody PPZ0506. We assessed the specificity and cross-reactivity of six commercially available anti-ESR1 antibodies (Clones MC-20, C1355, E115, H4624, SP1, and F-10) against mouse and rat ESR1 proteins using immunoblotting and immunocytofluorescence assays. Among them, MC-20, C1355, E115, and H4624 exhibited specific immunoreactivity to mouse and rat ESR1 proteins. These four antibodies were subsequently applied to paraffin-embedded ovarian and uterine sections from mice and rats. Heat-induced antigen retrieval and an appropriate antibody dilution were required to obtain specific and adequate signals. MC-20 and E115 were suitable for immunohistochemical detection of ESR1 proteins, while C1355 was effective for uterine tissue staining. H4624 showed utility only in mouse tissues. Furthermore, rabbit-derived MC-20 and E115 antibodies were successfully employed in dual immunohistofluorescence assays with the mouse monoclonal PPZ0506 antibody, enabling simultaneous visualization of ESR1 and ESR2 proteins in paraffin-embedded ovarian sections. Notably, little cellular co-localization of ESR1 and ESR2 proteins was observed in mouse and rat ovarian sections. These findings provide a validated set of antibodies for ESR1 immunohistochemical detection and demonstrate their compatibility with ESR2 co-labeling, facilitating detailed analysis of estrogen receptor distribution in rodent tissues.

Fanconi anemia (FA) is primarily an autosomal recessive genetic disorder that leads to bone marrow failure, increased risk of developing cancer, and a plethora of developmental abnormalities. Patients are prone to recurrent infections and increased risk of hemorrhage, as well as delayed wound healing with poor results. FA is caused by a genetic mutation in the proteins needed for FA pathway activation; FA group D2 protein (FANCD2) is an indispensable part of this pathway and plays essential roles in some aspects of cellular life, especially in the cellular responses to DNA damage. Here, we found that depletion of FANCD2 induced reduction of proliferation and migration of NIH3T3 cells. Moreover, FANCD2 knockout decreased production of extracellular matrix (ECM) protein collagen III and cytoskeleton protein alpha-smooth muscle actin (α-SMA). In this process, FANCD2 knockout decreased the expression of DNA methyltransferase 1 (DNMT1), and DNMT1 inhibitor 5-aza-2'-deoxycytidine (5-AZA-CdR) also induced the decline of proliferation and migration ability of NIH3T3 cells, and reduced the expression of collagen III and α-SMA. These findings suggest that FANCD2 affects wound healing through DNMT1. These findings may provide novel therapeutic ideas for clinical treatment of patients with FA with poor wound healing.

The adrenal glands are essential endocrine organs whose cortex and medulla maintain systemic homeostasis and mediate stress responses via steroid hormone and catecholamine secretion. Despite anatomical and functional similarities between human and mouse adrenal glands, notable species-specific differences exist. Here, we leveraged spatial transcriptomics (10× Genomics Visium) to comprehensively map gene expression in adult human and mouse adrenal glands, aiming to identify canonical marker genes conserved across species. The analysis was based on a 31-year-old female human sample (GEO dataset) and four 10-week-old male CD-1 mice. Human adrenal sections were processed using optimal cutting temperature (OCT) embedding, whereas mouse adrenal sections were processed as formalin-fixed paraffin-embedded (FFPE) samples, highlighting differences in sample preparation. Using unsupervised clustering of spatial gene expression data, we delineated distinct adrenal cortex and medulla zones in both species, confirming known zonation patterns. Our cross-species analysis revealed highly conserved spatial expression of key known marker genes characteristic of the adrenal cortex (e.g., CYP11B2 for ZG, CYP11B1 for ZF) and medullary chromaffin cells (e.g., TH), as well as a core set of additional marker genes previously less characterized in adrenal biology. By integrating transcriptional profiles, we generated a catalogue of conserved canonical marker genes that define adrenal zonation and function in both humans and mice. These results highlight the fundamental molecular conservation of adrenal gland organization and support the translational value of mouse models in adrenal research. Our findings provide new insights into the evolutionary preservation of adrenal function and a valuable resource for studies on adrenal physiology and disease.

The incidence of colorectal cancer (CRC) and the associated mortality in CRC patients have been rising in recent years. Quiescin sulfhydryl oxidase 1 (QSOX1), a secreted disulfide catalyst essential for extracellular matrix (ECM) assembly, is upregulated in several tumors (e.g. pancreatic, breast, and lung cancer), often correlating with aggressive tumor phenotypes and worse prognosis. In contrast, colorectal and hepatocellular carcinoma specimens show significant downregulation of QSOX1 compared to normal or adjacent tissue. Recognizing cancer as a heterocellular tissue where stromal cell types are crucial to tumor behavior, we evaluated by immunohistochemistry stromal and epithelial QSOX1 expression in 140 CRC cases (mean age: 64 years, 56% female, 83% without neoadjuvant therapy) and 10 normal colon samples. We found that stromal QSOX1 expression is significantly reduced in CRC compared to normal colon tissue (p < 0.0001). The stromal, rather than the epithelial, compartment determines total QSOX1 levels in tumor samples. Stromal QSOX1 demonstrates a negative relation with tumor size (ß = -0.04, p < 0.05), but not with other histopathological characteristics. This finding suggests that stromal QSOX1 loss may play a key role in early tumor cell proliferation rather than in clinical progression. Importantly, stromal QSOX1 expression shows excellent discriminatory power between tumor and non-tumor tissues, with an AUC of 0.98 in ROC analysis. Altogether, QSOX1 downregulation is a defining feature of tumor-associated stroma in CRC, likely affecting ECM integrity and modulating epithelial-stromal crosstalk. Thus, further characterization of stromal molecular signatures may identify novel biomarkers and therapeutic target strategies in CRC.

In the human placenta, trophoblast cells give rise to two unique cell layers that envelop the surface of chorionic villi: the outer syncytiotrophoblast (STB) layer and the inner cytotrophoblast (CTB) cell layer. The structural changes in CTB cells during gestation are still not fully understood. This study examined the ultrastructural integrity of the CTB layer within chorionic villi of the human term placenta using Fast Red immunohistochemistry in conjunction with proteinase K/Triton X-100 tissue-clearing and serial block-face scanning electron microscopy (SBF-SEM). Visualization of the CTB layer in whole-mount peripheral villous trees was facilitated by Fast Red immunohistochemistry of SPINT1, a marker specific to CTB cells, under both bright-field and fluorescence imaging modes in light microscopy. CTB cells displayed a thin, flattened morphology and extended multiple cellular projections, resulting in a spider-like transformation that envelops grape-like terminal villi. In contrast, in more proximal villi (e.g., stem villi), CTB cells showed a thick, cuboidal, or polygonal appearance, covering the villous subsurface. SBF-SEM imaging demonstrated the structure of a thin, mesh-like CTB layer, where the basal domain of the villous surface STB infiltrated through the small gaps of the CTB layer and contacted fetal capillaries via the basal lamina in terminal villi. Our data suggest that terminal villi undergo structural changes to facilitate fetomaternal exchange.

The expression of different sarco/endoplasmic reticulum calcium ATPase (SERCA) isoforms is controversial in various cancers and is not clear in the experimental cancer model. The present study attempts to evaluate the expression dynamics of SERCA isoforms in the azoxymethane/dextran sulphate sodium salt (AOM/DSS) model of colorectal carcinogenesis in mice. Inflammation-associated colorectal cancer was induced in the mice by administration of a single dose of AOM and three alternative cycles of DSS in drinking water. Body weights were recorded weekly. Mice were killed at weeks 0, 8, 12 and 16. At those times, the number of tumours was recorded, and colon tissues were processed for histopathological, immunohistochemical and gene expression analysis. The number of tumours and the formation of aberrant crypt foci were found to be significantly higher in the AOM/DSS group compared to the control. Histopathology of the colon revealed a higher percentage of dysplasia, adenoma and adenocarcinoma formation in the AOM/DSS group, further supported by high intensity of immunohistochemical staining for PCNA in the same. Gene expression analysis indicated higher expression of cyclin D1, β-catenin and low expression of E-cadherin, suggesting carcinogenic transformation of the colon. Immunohistochemical and gene expression analysis of SERCA isoforms indicated higher expression of SERCA1 and SERCA2 and low expression of SERCA3 in colon tissues of the AOM/DSS-exposed animals. The present study confirmed a similar expression pattern of SERCA isoforms in the AOM/DSS model of carcinogenesis as reported in clinical samples. Further, this study highlights the fact that altered SERCA patterns could be a contributing factor in the development of colorectal carcinogenesis.