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.
Highlights
Diverse subsets of myeloid cells and macrophages (MC&M) are observed in tissues, including both resident and recruited MC&M populations [1,2,3]
Monocytes recruited to inflamed tissues can for instance become inflammatory macrophages or monocytederived dendritic cells [9], and myeloid cells produced in the bone marrow of tumor-bearing mice and humans can colonize the tumor microenvironment (TME) and develop immunosuppressive activities [10]
To analyze the spatial organization of tumor-associated myeloid cells and macrophages (MC&M) through visualization by antibodies, we developed an automated, multiplexed immunofluorescent assay
Summary
Diverse subsets of myeloid cells and macrophages (MC&M) are observed in tissues, including both resident and recruited MC&M populations [1,2,3]. While the functional programming of MC&M subpopulations is influenced by their origins, they can further differentiate or become activated in tissues in response to organ-specific microenvironmental factors [8]. Monocytes recruited to inflamed tissues can for instance become inflammatory macrophages or monocytederived dendritic cells [9], and myeloid cells produced in the bone marrow of tumor-bearing mice and humans can colonize the tumor microenvironment (TME) and develop immunosuppressive activities [10]. The immune infiltration of cancers consists of a complicated admixture of tissue and organ resident MC&M populations as well as MC populations from the bone marrow whose production, attraction and phenotypes in the TME are influenced by tumor-derived cytokines
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