Spatial Mapping of Myeloid Cells and Macrophages by Multiplexed Tissue Staining

Abstract

An array of phenotypically diverse myeloid cells and macrophages (MC&M) resides in the tumor microenvironment, requiring multiplexed detection systems for visualization. Here we report an automated, multiplexed staining approach, named PLEXODY, that consists of five MC&M-related fluorescently-tagged antibodies (anti - CD68, - CD163, - CD206, - CD11b, and - CD11c), and three chromogenic antibodies, reactive with high- and low-molecular weight cytokeratins and CD3, highlighting tumor regions, benign glands and T cells. The staining prototype and image analysis methods which include a pixel/area-based quantification were developed using tissues from inflamed colon and tonsil and revealed a unique tissue-specific composition of 14 MC&M-associated pixel classes. As a proof-of-principle, PLEXODY was applied to three cases of pancreatic, prostate and renal cancers. Across digital images from these cancer types we observed 10 MC&M-associated pixel classes at frequencies greater than 3%. Cases revealed higher frequencies of single positive compared to multi-color pixels and a high abundance of CD68+/CD163+ and CD68+/CD163+/CD206+ pixels. Significantly more CD68+ and CD163+ vs. CD11b+ and CD11c+ pixels were in direct contact with tumor cells and T cells. While the greatest percentage (~70%) of CD68+ and CD163+ pixels was 0–20 microns away from tumor and T cell borders, CD11b+ and CD11c+ pixels were detected up to 240 microns away from tumor/T cell masks. Together, these data demonstrate significant differences in densities and spatial organization of MC&M-associated pixel classes, but surprising similarities between the three cancer types.

Macrophage targeting in cancer

“…This physical barrier can be TAMs, as they can promote T cell trapping at the border of the tumors by forming long-lasting interactions with CD8 + T cells. 99,100 Tumors defined as altered-immunosuppressed are characterized by poor, although not absent, T cell infiltration due to the presence of an immunosuppressive environment (e.g., myeloid-derived suppressor cells (MDSCs), 101 TAMs, and/or regulatory T cells) within the tumor. Preclinical studies have demonstrated that resistance to immune checkpoint therapy can be circumvented by targeting macrophages with CSF1R inhibitors in colorectal cancer.…”

Overexpression of HNF4α enhances umbilical cord mesenchymal stem cell function to treat acute liver failure in mice through macrophage polarization

“…Compared to undifferentiated cells, expression of human macrophage markers CD11b and CD68 were significantly higher (Fig.5b). In summary, the cells met the criteria of experimental macrophages [21].…”

Immune contexture analysis in immuno‐oncology: applications and challenges of multiplex fluorescent immunohistochemistry

“…114,152,153 There are already a growing number of image acquisition platforms in the market that combine image analysis with automated slide scanning to support fluorescence multiplexed staining. These include the Akoya Biosciences Vectra â and Vectra â Polaris TM , 24 TissueGnostics/TissueFAXS, 154 and Leica Biosystems/Aperio FL, 155 all of which can scan slides affixed to whole tissue or tissue microarray slices and provide some level of image analysis. Higher level of multiplexing for multivariate biomarker analysis requires spectral unmixing to separate spectral overlap.…”