Quantitative Multiplex Immunohistochemistry Reveals Myeloid-Inflamed Tumor-Immune Complexity Associated with Poor Prognosis

Abstract

SUMMARY Here we describe a multiplexed immunohistochemical platform, with computational image processing workflows including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas, and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination, and revealed that response to therapy correlated with degree of mono-myelocytic cell density, and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification, and provide digital image processing pipelines (https://github.com/multiplexIHC/cppipe) to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to thus improve biomarker discovery and assessment.

Quantifying tumor-infiltrating immune cells from transcriptomics data

“…Currently, alternative technologies for the quantification of tumor-infiltrating immune cells like multiplexed IF or IHC [67] are within the reach only of specialized laboratories due to the high costs and complex experimental procedures they entail. Moreover, RNA-seq data generated from bulk tumor can be used to simultaneously extract, besides immune cell fractions, different immunological features relevant for cancer immunology like HLA types, T- and B-cell receptor repertoires, tumor neoantigens [68], and information about the cell functional orientation and state, including exhaustion or anergy.…”

Caspase-1 from Human Myeloid-Derived Suppressor Cells Can Promote T Cell–Independent Tumor Proliferation

“…What has not been clear is MDSCs' non-T cell-dependent role in the human tumor microenvironment (TME). Tumors that are refractory to immune checkpoint inhibitors tend to be T-cell poor (21)(22)(23), so understanding MDCSs' T cell-independent, pro-carcinogenic mechanisms are important for the future development of combinatorial immuno-oncologic trials.…”

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (C e 3D)

“…This imaging/analysis pipeline allows multiplex phenotypic identification and quantification of cells directly in tissues, akin to in situ flow cytometry. This technique has been used to study the composition, distribution, and function of densely packed cells with complex phenotypes and morphology in tissue sections (13)(14)(15)(16)(17)(18)(19)(20)(21). Even for cell types with relatively simple morphology, such as T and B lymphocytes, histo-cytometry requires a high degree of spatial signal resolution, achieved with high numerical aperture (N.A.)…”