Probes for KIT

The CD169+ macrophages in lymph nodes are implicated in cytotoxic T lymphocyte (CTL) activation and are associated with improved prognosis in several malignancies. Here, we investigated the significance of CD169+ macrophages in oral squamous cell carcinoma (OSCC). Further, we tested the anti-tumor effects of naringenin, which has been previously shown to activate CD169+ macrophages, in a murine OSCC model. Immunohistochemical analysis for CD169 and CD8 was performed on lymph node and primary tumor specimens from 89 patients with OSCC. We also evaluated the effects of naringenin on two murine OSCC models. Increased CD169+ macrophage counts in the regional lymph nodes correlated with favorable prognosis and CD8+ cell counts within tumor sites. Additionally, naringenin suppressed tumor growth in two murine OSCC models. The mRNA levels of CD169, interleukin (IL)-12, and C-X-C motif chemokine ligand 10 (CXCL10) in lymph nodes and CTL infiltration in tumors significantly increased following naringenin administration in tumor-bearing mice. These results suggest that CD169+ macrophages in lymph nodes are involved in T cell-mediated anti-tumor immunity and could be a prognostic marker for patients with OSCC. Moreover, naringenin is a new potential agent for CD169+ macrophage activation in OSCC treatment.

Complex tissues such as tumors are comprised of multiple cells types and extracellular matrix. These cells include heterogenous populations of immune cells that infiltrate the tumors. Understanding the composition of these immune infiltrates in the tumor microenvironment (TME) can provide key insights to guide therapeutic intervention and predict treatment response. Thorough understanding of complex tissue dynamics and immune cell characterization requires a multi-omics approach. Simultaneous detection of RNA and protein using in situ hybridization (ISH) and immunohistochemistry/immunofluorescence (IHC/IF) can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. However, a sequential workflow of ISH followed by IHC/IF frequently yields suboptimal protein detection because the protease digestion step in the ISH protocol resulting in poor antibody signal. Here we demonstrate a newly developed integrated ISH/IHC workflow that can substantially improve RNA-protein co-detection, enabling the visualization and characterization of tumor immune infiltrates at single-cell resolution with spatial and morphological context. To characterize tumor-infiltrating immune cells in a tumor TMA (tumor microarray), we utilized the RNAscope Multiplex Fluorescence assay in combination with the RNA-Protein Co-detection Kit to detect multiple immune cell populations. Immune cells such as macrophages, T cells and NK cells were detected using specific antibodies against CD68, CD8, CD4 and CD56, respectively. Precise characterization of these immune cells was achieved by using probes against targets such as CCL5, IFNG, GNZB, IL-12, NCR1 etc. that not only help in identifying specific immune cells but also assist in determining their activation states. We identified subsets of T cells such as CD4+ regulatory T cells and CD8+ cytotoxic T lymphocytes. Additionally, we were able to determine the activation states of CD8+ T cells by visualizing the expression of IFNG and GZMB. Furthermore, infiltrating macrophages were identified by detecting the CD68 protein expression while the M1 and M2 subsets were differentiated by detecting the M2-specific target RNA for CD163. Similarly, NK cells were identified by detecting CD56 protein in combination with CCL5 and NCR1 RNA expression. Interestingly, the degree of infiltration of the different immune cell populations varied based on the tumor type. In conclusion, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies - including many previously incompatible antibodies - with RNAscope. This new workflow provides a powerful new approach to identifying and characterizing tumor infiltrating populations of immune cells.