Cutting-Edge Platforms for Analysis of Immune Cells in the Hepatic Microenvironment—Focus on Tumor-Associated Macrophages in Hepatocellular Carcinoma

SIMPLE SUMMARY: Hepatocellular carcinoma is the most common primary liver malignancy in the United States. Macrophages are immune cells that play a critical role in the promotion of cancer growth and configuration of the hepatic microenvironment. Studying intrahepatic macrophages is challenging because they are difficult to isolate, they transform their phenotype upon manipulation, and in vivo animal models poorly replicate the liver microenvironment. Understanding the complexity of intrahepatic macrophage populations is crucial because they coordinate antitumoral immunity. Application of novel methods that can detect immune cell phenotypes, along with their spatial co-localization in situ is critical and timely. ABSTRACT: The role of tumor-associated macrophages (TAMs) in the pathogenesis of hepatocellular carcinoma (HCC) is poorly understood. Most studies rely on platforms that remove intrahepatic macrophages from the microenvironment prior to evaluation. Cell isolation causes activation and phenotypic changes that may not represent their actual biology and function in situ. State-of-the-art methods provides new strategies to study TAMs without losing the context of tissue architecture and spatial relationship with neighboring cells. These technologies, such as multispectral imaging (e.g., Vectra Polaris), mass cytometry by time-of-flight (e.g., Fluidigm CyTOF), cycling of fluorochromes (e.g., Akoya Biosciences CODEX/PhenoCycler-Fusion, Bruker Canopy, Lunaphore Comet, and CyCIF) and digital spatial profiling or transcriptomics (e.g., GeoMx or Visium, Vizgen Merscope) are being utilized to accurately assess the complex cellular network within the tissue microenvironment. In cancer research, these platforms enable characterization of immune cell phenotypes and expression of potential therapeutic targets, such as PDL-1 and CTLA-4. Newer spatial profiling platforms allow for detection of numerous protein targets, in combination with whole transcriptome analysis, in a single liver biopsy tissue section. Macrophages can also be specifically targeted and analyzed, enabling quantification of both protein and gene expression within specific cell phenotypes, including TAMs. This review describes the workflow of each platform, summarizes recent research using these approaches, and explains the advantages and limitations of each.