In the field of histochemistry and cytochemistry (histocytochemistry), fixation is a critical process for preserving biological structures and enabling accurate analysis. Fixation methods, broadly categorized into precipitating and cross-linking techniques, stabilize biomolecules such as proteins, sugars (carbohydrates) and nucleic acids, although lipids often require specific handling due to the loss during a routine procedure. Traditional staining methods have evolved into advanced techniques like immunohistochemistry (IHC) and in situ hybridization (ISH), which allow for precise analysis of the expression of specific molecules. IHC employs antibodies to visualize specific antigens, with fixation playing a vital role in maintaining antigen integrity. However, excessive fixation can mask epitopes, requiring antigen retrieval techniques to restore antigenicity. Microwave-induced retrieval, for instance, enhances staining efficacy while introducing further fixation by promoting molecular interactions. ISH, which targets nucleic acids with specific base sequences, is also sensitive to fixation conditions. Formaldehyde-based fixatives react variably with purines and pyrimidines, affecting hybridization efficiency with a probe. Positive controls like 28S rRNA help to standardize ISH across facilities, ensuring reproducible and reliable results. Variability in fixation protocols among institutions brings fatal defects in achieving consistent results. Shared standards or the use of robust controls can alleviate these issues, enhancing the accuracy and reliability of histocytochemical analyses for both research and clinical applications.

Squamous cell carcinoma (SCC), a common malignancy affecting the skin, vagina, uterine cervix, anus, larynx, and upper digestive tract, is characterized by significant disruption of cell-cell adhesion in stratified squamous epithelium during tumorigenesis, progression, and metastasis. CALML5, a stratified epithelial-specific protein linked to desmosomal junctions, plays a key role in cell adhesion and is notably downregulated in human papillomavirus (HPV)-associated cervical SCC. Esophageal and pharyngeal cancers, commonly with a squamous cell phenotype, have distinct etiologies: oropharyngeal carcinoma is strongly associated with HPV, whereas esophageal carcinoma is linked to environmental factors such as smoking, alcohol, and diet. To investigate the role of CALML5 in these cancers, we performed immunohistochemical analyses on clinical samples and explored its regulatory mechanisms using in vitro studies with human esophageal SCC cell lines. Our findings revealed that CALML5 expression is suppressed in early-stage esophageal SCC but reactivated at invasive sites in well to moderately differentiated SCC undergoing keratinization. In specialized SCC with sarcomatoid component, CALML5 reactivation occurred alongside aberrant KLF4 expression, highlighting its context-dependent role in tumor progression. Conversely, while HPV-unrelated oropharyngeal SCC exhibited patterns similar to esophageal SCC, HPV-related oropharyngeal SCC consistently showed suppressed CALML5 expression due to impaired KLF4 nuclear translocation. These results suggest that CALML5 functions as a tumor suppressor in HPV-associated cervical SCC but may be reactivated in non-HPV-associated invasive SCC, emphasizing its complex role in SCC pathogenesis and the need for careful interpretation of its expression in clinical contexts.

Endoscopic ultrasound-guided fine-needle aspiration/biopsy (EUS-FNA/B) is critical for determining treatment strategies for patients with pancreatic cancer. However, conventional pathological examination using hematoxylin and eosin (H&E) staining is time-consuming. Microscopy with ultraviolet surface excitation (MUSE) enables rapid pathological diagnosis without requiring slide preparation. This study explores the potential of combining MUSE imaging with a cycle-consistent generative adversarial network (CycleGAN), an image generation algorithm capable of learning translations without paired images, to enhance diagnostic workflows for pancreatic EUS-FNA/B. Thirty-five pancreatic specimens were stained with Terbium/Hoechst 33342, and deep ultraviolet (DUV) fluorescence images were captured by exciting the tissue surface. These fluorescence images, along with H&E-stained formalin-fixed, paraffin-embedded (FFPE) sections from the same specimens, were divided into 256 × 256-pixel segments for CycleGAN training. The algorithm was employed to translate pseudo-H&E images from MUSE test images. The pseudo-H&E images generated by the CycleGAN showed improved inter-pathologist agreement among three pathologists compared with the original MUSE images. We established a technique to perform MUSE imaging on small pancreatic samples obtained through EUS-FNA/B and confirmed that H&E-style translation using CycleGAN simplified interpretation for pathologists. Integrating MUSE imaging with CycleGAN has the potential to offer a rapid, cost-effective, and accurate diagnostic tool.

Gastric cancer (GC), particularly the undifferentiated type, is frequently associated with peritoneal metastasis, which significantly worsens prognosis due to its resistance to conventional treatments. Photodynamic therapy (PDT) is localized treatment using a photosensitizer (PS) activated by light of a specific wavelength to generate cytotoxic reactive oxygen species that induce cell death. Severe adverse events were reported from clinical trials investigating PDT for peritoneal dissemination conducted until the early 2000s, leaving its safety and clinical effectiveness unestablished. The present study explored whether “non-cytotoxic” PDT using talaporfin sodium (TS) could enhance efficacy of chemotherapeutic agents in undifferentiated GC cell line HGC27. Cell viability was evaluated with MTT assay following TS-PDT, and the synergistic effect between non-cytotoxic TS-PDT and anticancer drug SN-38 was assessed. Changes in expression of drug resistance markers were analyzed through qRT-PCR, Western blotting, and immunocytochemistry. We found that non-cytotoxic TS-PDT enhanced the efficacy of chemotherapy in the undifferentiated GC cell line and reduced the expression of C-X-C chemokine receptor type 4, a key marker associated with GC stem-like properties. These findings highlight the potential of non-cytotoxic TS-PDT as a synergistic treatment approach. We conclude that non-cytotoxic TS-PDT could enhance drug sensitivity and offers a promising therapeutic strategy for GC.

Conventional histopathological techniques, such as hematoxylin and eosin staining, are limited to 4–5 μm-thick tissue sections, restricting visualization to two-dimensional planes. Moreover, acquisition of three-dimensional horizontal images from the skin surface remains challenging, hindering precise assessment of tumor margins in skin lesions. This challenge is particularly pronounced in extramammary Paget’s disease (EMPD), in which diffuse epidermal tumor cell spread complicates accurate evaluation of lesion extent. We hypothesized that combining horizontal sectioning with identification of individual tumor cells would enhance the determination of surgical margins. In this study, we developed a deep-imaging technique utilizing fluorescent solvatochromic dyes (LipiORDER^® and HistoBright^®) and two-photon microscopy to achieve high-resolution tumor margin visualization in EMPD. This technique enables identification of tumor cells in frozen and paraffin-embedded tissue blocks, as well as in live skin tissue under physiological conditions. Our novel approach holds substantial promise for improving the precision of surgical-margin assessment in EMPD and other cutaneous malignancies.

Ibrutinib is an oral irreversible Bruton’s tyrosine kinase (BTK) inhibitor that blocks BTK activity by forming covalent bonds with the thiol group of cysteine in the ATP-binding pocket via Michael addition. However, it also reacts with a variety of off-target nonspecific proteins. In this study, we attempted to generate a specific antibody against ibrutinib and develop an immunohistochemical method to detect the ibrutinib-protein conjugates. Ibrutinib has the same amino group as the nucleobase adenine. Paraformaldehyde fixation could not fix it to the tissue via this amino group. However, ibrutinib covalently binds to proteins such as BTKs to exert its action and is therefore immobilized in tissue as ibrutinib-protein conjugates. Thus, immunohistochemistry for ibrutinib detects the location of the ibrutinib-protein conjugates, that is, the sites of covalently bound to the tissue via Michael addition. Using this immunohistochemical method, we visualized the ibrutinib-protein conjugates in the rat gastrointestinal tract (gastric body, duodenum, jejunum, ileum, and colon). This study is the first to elucidate the location of the ibrutinib-protein conjugates in the rat gastrointestinal tract and helps to clarify the mechanism of ibrutinib-induced toxicity.

SNX25 is a member of the sorting nexin (SNX) superfamily, which plays crucial roles in membrane trafficking, cell signaling, and organelle dynamics. Our research has focused extensively on SNX25, demonstrating that SNX25-positive macrophages participate in inflammatory responses and pain perception through various signaling pathways. Atherosclerosis is now widely recognized as a chronic inflammatory disease of the vasculature, with macrophages serving as central contributors to its progression. These macrophages accumulate after internalizing oxidized low-density lipoproteins (oxLDL), transforming into foam cells that elicit inflammatory responses and promote atherosclerotic progression. To explore the impact of SNX25 on atherosclerosis, we induced the condition in apolipoprotein E-deficient (APOE^−/−) mice using a high-fat diet. As expected, SNX25 expression was observed in macrophages within atherosclerotic plaques. In SNX25^+/− mice on an APOE^−/− genetic background, plaque size was significantly smaller than in their SNX25^+/+ counterparts. Furthermore, bone marrow transplantation from SNX25^+/− mice into APOE^−/− recipients resulted in a marked reduction in foam cell formation and accumulation compared to transplants from SNX25^+/+ donors. These histopathological findings suggest that SNX25 may regulate macrophage activity under pathological conditions, identifying a novel role for SNX25 in the pathogenesis of atherosclerosis.