Multiplex Immunofluorescence and the Digital Image Analysis Workflow for Evaluation of the Tumor Immune Environment in Translational Research

A robust understanding of the tumor immune environment has important implications for cancer diagnosis, prognosis, research, and immunotherapy. Traditionally, immunohistochemistry (IHC) has been regarded as the standard method for detecting proteins in situ, but this technique allows for the evaluation of only one cell marker per tissue sample at a time. However, multiplexed imaging technologies enable the multiparametric analysis of a tissue section at the same time. Also, through the curation of specific antibody panels, these technologies enable researchers to study the cell subpopulations within a single immunological cell group. Thus, multiplexed imaging gives investigators the opportunity to better understand tumor cells, immune cells, and the interactions between them. In the multiplexed imaging technology workflow, once the protocol for a tumor immune micro environment study has been defined, histological slides are digitized to produce high-resolution images in which regions of interest are selected for the interrogation of simultaneously expressed immunomarkers (including those co-expressed by the same cell) by using an image analysis software and algorithm. Most currently available image analysis software packages use similar machine learning approaches in which tissue segmentation first defines the different components that make up the regions of interest and cell segmentation, then defines the different parameters, such as the nucleus and cytoplasm, that the software must utilize to segment single cells. Image analysis tools have driven dramatic evolution in the field of digital pathology over the past several decades and provided the data necessary for translational research and the discovery of new therapeutic targets. The next step in the growth of digital pathology is optimization and standardization of the different tasks in cancer research, including image analysis algorithm creation, to increase the amount of data generated and their accuracy in a short time as described herein. The aim of this review is to describe this process, including an image analysis algorithm creation for multiplex immunofluorescence analysis, as an essential part of the optimization and standardization of the different processes in cancer research, to increase the amount of data generated and their accuracy in a short time.