Acta Histochemica Et Cytochemica最新文献

High-resolution spatial transcriptomics has emerged as a powerful approach for linking genome-wide gene expression with preserved tissue architecture, enabling new insights into cellular heterogeneity and microenvironmental organization in complex tissues. In neuropathology, where morphological context is central to disease interpretation, these technologies are particularly appealing. Using glioblastoma multiforme (GBM) as a representative and highly heterogeneous model, this review critically evaluates the strengths and limitations of high-resolution spatial transcriptomic platforms in relation to classical histochemistry and cytochemistry. Illustrative analyses of human GBM tissue demonstrate that spatial transcriptomics robustly captures disease-relevant gene expression patterns and enables comprehensive mapping of key pathological features, including pseudopalisading necrosis, aberrant tumor vasculature, and therapy-resistant tumor niches. When integrated with histopathological observations, spatially resolved transcriptomic data can generate biologically meaningful hypotheses, offering insight into immune suppression, and proliferative signaling networks. These examples underscore the potential of spatial transcriptomics to bridge morphology and molecular biology, thereby expanding the conceptual framework of histopathological research. At the same time, spatial transcriptomic data should not be regarded as a replacement for direct microscopic evaluation. Limitations in morphological fidelity, lack of subcellular resolution, indirect inference of functional states, and reliance on computational interpretation necessitate careful integration with established histochemical and immunohistochemical methods. Without a solid foundation in tissue and cellular morphology, spatial transcriptomic findings may be misinterpreted or overstated. Collectively, this review emphasizes that spatial transcriptomics and histochemistry are complementary approaches, whose effective integration depends critically on rigorous histochemical knowledge to ensure accurate pathological interpretation and translational relevance.

Ampullary carcinoma exhibits marked histological heterogeneity. Although genetic alterations partially account for this diversity, the contribution of epigenetic regulation remains largely unexplored. To elucidate the epigenetic alterations underlying this phenotypic heterogeneity, we investigated methyl-CpG-binding protein 2 (MeCP2) and its downstream target CDX2, and versican (VCAN), a major extracellular matrix proteoglycan implicated in tissue remodeling and tumor-stroma interactions. Seventeen surgically resected cases were analyzed using an integrative approach combining immunohistochemistry, spatial transcriptomics, methylation mapping, electrophoretic mobility shift assays (EMSA), and bioinformatic profiling. In non-neoplastic mucosa, MeCP2 and CDX2 showed reciprocal nuclear expression, a relationship partially preserved in differentiated adenocarcinomas. Spatial transcriptomics identified VCAN as a key MeCP2-associated target gene. Unexpectedly, VCAN, although detectable in MeCP2-negative carcinoma cells, was abundantly expressed in MeCP2-positive cancer-associated fibroblasts (CAFs). Notably, such transcriptional activation by MeCP2, rather than repression, has been reported in neural tissue by previous studies, indicating a conserved mechanism of context-dependent gene regulation. EMSA further demonstrated that hydroxymethylated CpG sites within the VCAN promoter specifically recruited MeCP2, which interacted with CREB to activate VCAN transcription. These findings reveal a dual role of MeCP2: its loss contributes to epithelial heterogeneity, whereas its retained function in CAFs promotes stromal remodeling through VCAN activation.

DNA methylation is a key epigenetic modification that regulates transcriptional activity and is frequently altered during carcinogenesis. To elucidate how cytosine methylation becomes selectively localized to CpG loci during tumor development, we analyzed sequential renal lesions induced by streptozotocin (STZ) in rats. Using microdissection combined with an agarose-bead-based bisulfite sequencing method, the methylation status of the p16^Ink4a promoter region was examined from early Armanni-Ebstein lesions through renal cell carcinomas. In early and advanced tubular lesions, cytosine methylation occurred non-selectively at both CpG and non-CpG (CH) sites, whereas in small renal tumors and carcinomas, methylation was progressively restricted to CpG loci. Immunohistochemistry demonstrated strong nuclear expression of p16^Ink4a in early lesions, suggesting that active transcription persisted at least until this early stage before the onset of epigenetic silencing. Concomitant expression of Dnmt3b and Dnmt1 was observed, supporting their cooperative involvement in de novo and maintenance methylation, respectively. Notably, the analyzed region overlapped with a CpG-rich segment within the gene body of the alternatively spliced p14^Arf gene, which completely fulfill the structural prerequisites for Dnmt3b-mediated methylation. In conclusion, our findings suggest that Dnmt3b-dependent de novo methylation is preferentially initiated within transcriptionally active, CpG-rich regions and subsequently refined to discrete CpG loci as neoplastic transformation advances. This stepwise process provides a mechanistic basis for the selective accumulation of DNA methylation during tumorigenesis and highlights its potential utility as an early epigenetic biomarker of cancer development.

Clear cell foci (CCF) are frequently observed in metabolic dysfunction-associated steatohepatitis (MASH) and are considered potential precursor lesions of hepatocyte nuclear factor 1α-inactivated hepatocellular adenoma (H-HCA). To clarify their chronological development, we examined 55 male TSOD mice at 24, 32, 40, and 48 weeks of age using histology and immunohistochemistry for glutamine synthetase (GS), liver fatty acid-binding protein (L-FABP), β-Klotho, and fibroblast growth factor 21 (FGF21). CCF first appeared at 24 weeks and increased markedly with age (from 11% to 81%). All CCF were positive for β-Klotho, and a subset showed FGF21 expression, indicating that CCF represent a hepatocellular state associated with metabolic dysregulation. H-HCA, characterized by GS negativity and reduced L-FABP expression, emerged at 40 weeks and reached an incidence of 29% at 48 weeks. Notably, multiple H-HCA were partially or completely surrounded by β-Klotho-positive CCF, suggesting a morphologic continuum from CCF to H-HCA. Raman spectroscopic analysis demonstrated that CCF exhibit prominent autofluorescence and possess spectral characteristics distinct from both background hepatocytes and tumor tissue, supporting the concept that CCF represent a unique hepatocellular state. These findings indicate that metabolic abnormalities in TSOD mice promote the sequential formation of CCF and H-HCA, establishing this model as a useful platform for studying adenoma development in metabolic liver disease.

Facial nerve palsy profoundly affects facial expression and quality of life, with recovery frequently hindered by aberrant nerve regeneration leading to synkinesis. A comprehensive understanding of three-dimensional (3D) cytoarchitectural organization and adaptive changes within the facial motor nucleus remains constrained by conventional two-dimensional analyses. In this study, retrograde neural tracers (DiI, DiO, Dextran Alexa Fluor^TM 488/546) were integrated with tissue clearing techniques (CUBIC and LUCID) to reconstruct and analyze the 3D distribution of neurons innervating the orbicularis oris and orbicularis oculi muscles in guinea pigs. Cleared brainstem tissues were imaged using two-photon microscopy, revealing distinct region-specific neuronal clusters within the facial motor nucleus. In a facial nerve injury model, disrupted regional specificity and disorganized neuronal distribution were observed, suggesting misdirected central reinnervation. This novel 3D imaging method enables high-resolution spatial neuronal analysis and reorganization following nerve injury. This approach provides a valuable tool for elucidating facial nerve regeneration and synkinesis, and may facilitate the development of improved therapeutic strategies.

Cancer cells escape immune surveillance by suppressing immune responses through the binding of Programmed cell Death-Ligand 1 (PD-L1), which is abundantly expressed on the cell surface, to PD-1 on the surface of T cells. The regulation of cell surface PD-L1, one of these immune checkpoint molecules, is extremely important because it is a target for cancer immunotherapy; however, the intracellular trafficking pathway of PD-L1 has not been fully elucidated. Recently, we reported that Rab10, a small GTPase, localizes to a novel tubular endocytic pathway that evades the lysosomal degradation system. In this study, using live cells expressing GFP-PD-L1 and mScarlet-Rab10, we revealed that PD-L1 localizes in Rab10-positive endocytic tubules in some types of cancer cells. Typically, in HeLaM cells, Rab10-positive tubular structures of which membranes have PD-L1 extend from the plasma membrane toward the cell-central region. However, in Rab10-knockout HeLaM cells, no PD-L1-localized tubular structures were observed. We also found that PD-L1 dimerized by the PD-L1 inhibitor BMS-202 was removed from the cell surface and Rab10-positive tubular endosomes and transported to the lysosomal degradation system. Taken together, this study provides novel insights that the Rab10-dependent tubular endocytic pathway may play an important role in the intracellular reservoir and recycling of PD-L1 to the surface of cancer cells, possibly regulating the amount of PD-L1 on the cell surface.

The Hedgehog (Hh) signaling pathway is essential for organ development and tissue homeostasis; its dysregulation is associated with various congenital anomalies and human diseases. Hedgehog-interacting protein 1 (Hhip1) functions as a membrane-bound negative regulator of Hh signaling by directly binding to Hh ligands and limiting their activity in target tissues. While global knockout models have demonstrated the importance of Hhip1 during embryonic development, perinatal lethality has precluded investigations into its postnatal functions. To overcome this limitation, a novel conditional allele of Hhip1 (hereafter referred to as the Hhip1^flox or floxed allele, in which exon 2 is flanked by loxP sites) was generated using CRISPR/Cas9-based gene targeting. Cre-mediated excision of exon 2 produced a conditional null allele (Hhip1^ex2Δ ) with a frameshift mutation that annulled the Hedgehog ligand-binding domain. Germline recombination through CMV-Cre recapitulated the known pulmonary defects of global Hhip1 knockout mice, validating the model's functionality. Furthermore, using Prx1-Cre to generate limb-specific Hhip1 conditional knockout (cKO) mice, a previously unrecognized role for Hhip1 in maintaining postnatal growth plate architecture was detected. cKO mice exhibited progressive growth plate expansion and long bone overgrowth, together with sustained upregulation of Gli1 expression. These findings establish the Hhip1^flox model as a powerful tool for tissue- and stage-specific functional studies of Hhip1 and provide new insights into the spatiotemporal regulation of Hedgehog signaling in development and disease. Importantly, this model offers broad utility for dissecting Hedgehog signaling mechanisms across diverse biological contexts and disease models.

Eribulin, a microtubule inhibitor, is effective as later-line therapy for metastatic breast cancer (MBC) and has been reported to remodel the tumor microenvironment and inhibit epithelial-mesenchymal transition (EMT). However, the association between pretreatment EMT status and eribulin efficacy remains unclear. We retrospectively analyzed 41 patients with MBC (excluding invasive lobular carcinoma) treated with eribulin between 2013 and 2020. Formalin-fixed, paraffin-embedded biopsy specimens were examined by immunohistochemistry (IHC) using anti-E-cadherin (24E10) and anti-vimentin (V9) antibodies. Complete membranous E-cadherin expression (3+) was defined as normal; reduced expression (2+, 1+, 0) as altered. Negative vimentin was considered normal; positive expression, altered. Co-localization of E-cadherin and vimentin was assessed by multi-immunofluorescent staining. Of the 41 patients, 24 responded to eribulin and 17 did not. Progression-free survival (PFS) and overall survival (OS) were significantly longer in responders than in nonresponders (p < 0.001 and p = 0.0044). Altered E-cadherin and/or vimentin expression was more frequently observed in responders (p = 0.013) and associated with longer progression-free survival (p = 0.048). These results suggest that eribulin efficacy may be predicted by altered E-cadherin and vimentin expression before treatment.

Secondary hyperparathyroidism (SHPT) associated with chronic kidney disease (CKD) is characterized by parathyroid hyperplasia, which progresses from diffuse-type to nodular-type lesions. Nodular hyperplasia in SHPT is often considered to exhibit monoclonal proliferation, suggesting a shift toward neoplastic behavior, but the molecular mechanisms underlying this transformation remain poorly defined. In this study, we analyzed 340 surgically resected parathyroid glands from long-term dialysis patients who met clinical indications for parathyroidectomy. Based on histological architecture, lesions were classified into diffuse, nodular, or diffuse-nodular (mixed) hyperplasia. We conducted immunohistochemical analysis of Ki-67 and p16^INK4a (CDKN2A), and further assessed region-specific DNA methylation of the p16^INK4a promoter using a bisulfite padlock probe method combined with rolling circle amplification. Nodular-type lesions exhibited significantly higher Ki-67 indices and lower p16^INK4a expression compared to diffuse-type lesions. In situ methylation analysis revealed increased methylation of the p16^INK4a promoter specifically in nodular regions, suggesting epigenetic silencing. Our findings suggest that p16^INK4a silencing through promoter methylation may play a critical role in the clonal expansion and histopathological transformation of parathyroid tissue in SHPT. These results underscore the importance of epigenetic regulation in SHPT progression and suggest that p16^INK4a methylation could represent a potential biomarker for nodular transformation. The padlock probe-based detection system enabled high-resolution spatial analysis of methylation patterns and may serve as a valuable tool for dissecting epigenetic events in early phase of cellular alterations.

In our recent study, we identified Rab10-positive long tubular endosomes, which originate from macropinocytic cups, as a novel endocytic pathway in RAW264 cells. This pathway is unique because it bypasses the lysosomal degradation route and proceeds toward the Golgi region, distinguishing it from previously known endocytic routes. However, its function remains entirely unknown. Upon exploring the cargo transported by Rab10-positive tubular endosomes, we discovered that PD-L1, a cancer immune checkpoint molecule expressed on cancer cells and macrophage surfaces, was abundantly localized in Rab10-positive tubular structures in RAW264 cells. This suggests that PD-L1 may be a significant cargo for these endosomes in macrophages. These findings offer new insights into the role of Rab10-positive tubular endosomes in the intracellular transport of PD-L1, potentially influencing its expression on the cell surface.