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.

The tumor immune microenvironment (TME) of colorectal cancer contains key biomarkers for host anti-tumor immune responses and tumor immune escape mechanisms. The density of T cell subsets such as CD3⁺ and CD8⁺ T cells has been proven to more accurately predict disease recurrence risk than traditional histopathological staging. Additionally, tissue-resident memory T cells expressing CD103 play important roles in anti-tumor immunity for colorectal cancer and other tumors, with their density correlating with long-term oncological outcomes. Therefore, precise characterization of the phenotype, function, and spatial distribution of immune cells in the TME is crucial for discovering novel prognostic markers and guiding immunotherapeutic strategies.

In the field of glioma treatment, immunotherapy development has long been limited by unclear understanding of the tumor immune microenvironment. As the most aggressive malignant tumor in the central nervous system, gliomas, particularly glioblastoma (GBM), have long been labeled as "immune-cold" tumors. Barriers such as the blood-brain barrier hindering immune cell infiltration, high expression of immune checkpoint molecules like PD-L1 by tumor cells, and enrichment of immunosuppressive cells like M2 macrophages contribute to a clinical response rate of less than 15% for therapies like immune checkpoint inhibitors.

Tertiary Lymphoid Structures (TLSs) are ectopic lymphoid aggregates that can develop within or adjacent to tumors, activating anti-tumor immunity. Their maturity status is closely related to patients' immunotherapeutic efficacy and survival, making them highly promising prognostic markers. Opal-TSA multiplex immunofluorescence (mIF) technology, with its high sensitivity and staining flexibility, has become an ideal choice to break through these bottlenecks.

Multiplex immunofluorescence combined with tyramide signal amplification (TSA) technology, based on the principle of enzymatic signal cascade amplification, has broken through the limitations of traditional methods in sensitivity and multi-target detection, and its application research in the field of tumor pathological diagnosis has shown explosive growth in recent years.