Although tumor immunotherapy has developed rapidly and achieved significant results, malignant tumors can escape immune surveillance by constructing an immunosuppressive microenvironment, which severely limits treatment efficacy. After the discovery of immune checkpoint molecules such as PD-1 and CTLA-4 and their roles in tumor immune evasion, clarifying the interaction between immune cells and cancer cells has become a core prerequisite for developing novel immunotherapies. Multiplex immunofluorescence (mIF) is a reliable high-throughput method that can directly observe multiple biomarkers expressed by individual cells and analyze the spatial relationships of these biomarkers in different cell populations, which is impossible with traditional immunohistochemistry (IHC) techniques. Therefore, the study "Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue" can identify multiple cell subpopulations by combining carefully selected antibodies. The tyramide signal amplification (TSA) manual protocol was used as a standard reference for validating multiplex staining. The automated stainer significantly reduced the original 4-5 day manual staining time to 14-17 hours while improving staining consistency. The literature demonstrates the optimization process and reproducibility of automated TSA staining, and validates its application value in tumor microenvironment research and cellular phenotype spatial distribution analysis in a small cohort of non-small cell lung cancer (NSCLC) samples.

Delayed graft function (DGF) after kidney transplantation is a common complication, defined as the need for dialysis within 7 days post-transplantation, and is an important risk factor for chronic kidney transplant injury. Interstitial fibrosis and tubular atrophy (IFTA) are typical manifestations of chronic kidney injury. However, not all DGF patients progress to IFTA, and the complex association between them remains unclear. The core bottleneck is that traditional assessment methods have difficulty accurately quantifying inflammatory infiltration characteristics and cannot early identify predictive markers for IFTA progression. Traditional histological assessment relies on the subjective judgment of pathologists, with accuracy greatly influenced by experience, and cannot simultaneously capture information on the phenotype, density, and spatial distribution of multiple types of immune cells, limiting in-depth exploration of the association between the inflammatory microenvironment and graft prognosis. "Quantitative assessment of inflammatory infiltrates in kidney transplant biopsies using multiplex tyramide signal amplification and deep learning" focuses on technological innovation in the quantitative assessment of inflammatory infiltration in kidney transplant biopsies, aiming to solve the clinical challenge of predicting interstitial fibrosis and tubular atrophy progression in patients with delayed graft function, and achieving precise quantitative analysis of inflammatory infiltration through technology integration.

In recent years, the analysis of the complex immune landscape of the tumor microenvironment (TME) has become the core of precision oncology research. However, routine clinical practice still relies on H&E staining and single-marker chromogenic IHC detection, which has limitations such as tissue waste, inability to simultaneously evaluate multiple markers, and unsuitability for quantitative and spatial analysis, making it difficult to meet the needs of tumor microenvironment research and immunotherapy response prediction. The cellular abundance and spatial arrangement of the tumor microenvironment affect tumor progression and treatment response. When using single IHC to evaluate PD-L1 expression to predict immunotherapy response, there are problems such as poor reproducibility, inability to clearly identify the cell type of expression, and difficulty in quantifying spatial arrangement. Multiplex staining, which can simultaneously target multiple proteins through fluorescent or chromogenic staining, can characterize the co-expression profile and spatial arrangement of markers, showing superior performance to multiple detection methods in predicting immunotherapy response. It has wide application value in tumor microenvironment research, spatial heterogeneity analysis of biomarkers, solving the problem of multi-marker detection in small samples, and patient stratification in clinical trials. With continuous optimization and decreasing implementation difficulty, it is more suitable for routine clinical use. "Multiplex Immunohistochemistry and Immunofluorescence: A Practical Update for Pathologists" is a review article for pathologists, focusing on multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) techniques. By simultaneously detecting multiple biomarkers on the same section, it greatly improves the information content and accuracy of pathological diagnosis. Currently, the technical approaches to achieve multiplex detection mainly include multiplex IHC/immunofluorescence based on TSA and other methods, mass spectrometry imaging and digital spatial profiling (DSP), and virtual multiplex staining achieved through consecutive section registration. Although these techniques still face challenges in standardized procedures and data analysis, with the maturity of digital pathology technology, multiplex staining has gradually gained feasibility for clinical translation and is expected to become a routine method for guiding tumor immunotherapy decisions in the near future.

STING exhibits multi-cell type expression characteristics in cancer tissues, including lymphocytes, natural killer cells, endothelial cells, and epithelial cells. Current studies on STING expression in the cancer field are mostly based on RNA levels, which makes it difficult to distinguish cell-specific expression differences, and the clinical significance of STING in tumor cells and tumor-associated inflammatory cells is not yet fully clear. Based on this, a research paper published in Pathology titled "High-level STING expression in tumour and inflammatory cells is linked to microsatellite instability and favourable tumour parameters in a cohort of over 1,900 colorectal cancer patients" used multiplex fluorescent immunohistochemistry (mIHC) technology to quantitatively analyze STING expression in different cell types in colorectal cancer and explore its association with clinicopathological characteristics. This provides important basis for the study of immune mechanisms and potential therapeutic target exploration in colorectal cancer.

The core principle of TSA technology is that horseradish peroxidase (HRP)-conjugated secondary antibodies, in the presence of hydrogen peroxide, catalyze the conversion of fluorescently labeled tyramides into active forms, which then covalently bind to tyrosine residues near target antigens, achieving cascade amplification of signals. The study selected Opal fluorescent dyes as labels, which, compared to traditional fluorescent secondary antibodies, significantly improve signal sensitivity and allow imaging at low laser power, reducing tissue photobleaching.

Previous studies have shown that cyclophosphamide-methotrexate (CM) maintenance therapy does not significantly reduce the recurrence risk in patients with triple-negative breast cancer (TNBC) in a statistically meaningful way. However, patients with high levels of stromal tumor-infiltrating lymphocytes (sTILs, >500) experienced a more pronounced reduction in breast cancer recurrence risk after receiving CM maintenance therapy. Based on this finding, Rusakiewicz S et al. characterized the immune cell infiltration features of TNBC patients using 6-plex immunofluorescence staining technology. They evaluated the prognostic value of specific immune cell subsets and their predictive role in the efficacy of CM maintenance therapy, while also analyzing the impact of the spatial distribution characteristics of immune cells on treatment efficacy and patient prognosis. This study provides a basis for formulating precise treatment strategies for TNBC patients.

In recent years, immune checkpoint inhibitors (ICIs) have shown potential value in breast cancer treatment, but treatment responses vary significantly among different patients. The characteristics of the tumor immune microenvironment (TIME) are key factors affecting the efficacy of ICIs. Currently, there is still a lack of systematic and precise definition of predictive biomarkers for PD-L1 inhibitor treatment response in breast cancer patients, especially the differences in treatment response mechanisms among different receptor subtypes of breast cancer have not been fully elucidated. New detection technologies developed in recent years can simultaneously evaluate multiple markers while completely preserving the spatial relationships of cells in situ, and are being widely applied to tumor immune microenvironment characterization and mining of immune-related biomarkers. Among these, multiplex immunofluorescence (mIF) technology has demonstrated application value in predicting ICI efficacy in various tumors, significantly outperforming gene expression signatures, tumor mutational burden, and standardized PD-L1 immunohistochemistry detection.

GATA proteins comprise a group of transcription factors that are related by the presence of conserved zinc finger DNA-binding domains, which bind directly to the nucleotide sequence core element GATA. There are six vertebrate GATA proteins, designated GATA-1 to GATA-6. Although they are commonly divided as hematopoietic (GATA-1-3) or cardiac (GATA-4-6) factors, GATA proteins are expressed in a wide variety of tissue and play critical roles in embryonic development and organ differentiation.

Based on this core bottleneck, the research "In situ staining with antibodies cross-reactive in pigs, cattle, and white-tailed deer facilitates understanding of biological tissue status and immunopathology" developed a multiplex chromogenic immunohistochemistry scheme for comparative study of immune cells in lymph nodes across species, and verified the ability of fluorescent multiplex staining to detect protein co-localization signals in porcine tissues. Given the scarcity and preparation difficulty of protein-reactive antibodies in animals, the research further proposed to design immunoreagents targeting in-situ targets through RNA level design and combine them with species antibody staining in multiplex combinations to break through the limitations of immunoreagents in in-situ detection. To confirm the role of combined RNA and protein detection in elucidating tissue status and immunopathology, the research achieved combined staining of cell-specific protein markers with Seneca virus A (SVA) RNA in porcine tonsil tissue.

Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma that primarily affects older adults. The incidence is higher and occurs earlier in immunocompromised individuals, with a high rate of recurrence, metastasis, and mortality, necessitating effective prognostic evaluation and treatment approaches. MCC develops through two distinct pathways: most tumors are driven by clonal integration of the Merkel cell polyomavirus (MCPyV) and expression of oncogenic viral T antigens; MCPyV-negative tumors arise due to ultraviolet-induced high tumor mutation burden. Traditional predictors of immunotherapy response (such as PD-L1 expression and tumor mutation burden) are ineffective for MCC, while detailed analysis of the tumor microenvironment (TME) may more effectively predict treatment response. In-depth understanding of MCC-associated TME may provide new alternative treatment strategies for patients who are ineffective or intolerant to immunotherapy.