State-of-the-Art of Profiling Immune Contexture in the Era of Multiplexed Staining and Digital Analysis to Study Paraffin Tumor Tissues

Abstract

Multiplexed platforms for multiple epitope detection have emerged in the last years as very powerful tools to study tumor tissues. These revolutionary technologies provide important visual techniques for tumor examination in formalin-fixed paraffin-embedded specimens to improve the understanding of the tumor microenvironment, promote new treatment discoveries, aid in cancer prevention, as well as allowing translational studies to be carried out. The aim of this review is to highlight the more recent methodologies that use multiplexed staining to study simultaneous protein identification in formalin-fixed paraffin-embedded tumor tissues for immune profiling, clinical research, and potential translational analysis. New multiplexed methodologies, which permit the identification of several proteins at the same time in one single tissue section, have been developed in recent years with the ability to study different cell populations, cells by cells, and their spatial distribution in different tumor specimens including whole sections, core needle biopsies, and tissue microarrays. Multiplexed technologies associated with image analysis software can be performed with a high-quality throughput assay to study cancer specimens and are important tools for new discoveries. The different multiplexed technologies described in this review have shown their utility in the study of cancer tissues and their advantages for translational research studies and application in cancer prevention and treatments.

Keywords: cancer tissues; image analysis; immune profiling; multiplexed methodologies; spatial analysis.

Figure 1

Technical comparison of fluorescent-based platforms and mass spectrometer-based platforms for digital image analysis. Digital image analysis for cancer research applications can be achieved with several methodologies. Some of them have advantages over others depending on the sample available, the specific antibodies against biological markers of interest, the detection system needed, and in some cases, the amplification of the signal for poorly expressed markers.

Figure 2

Byproducts and fundamentals of the imaging mass spectrometer. The application of mass spectrometry to biological research began in the last half century and it represents the conjunction of biological and deep physical and technological knowledge in biomedicine. Imaging Mass Spectrometry (IMS) came from the idea of building a 2D image with the elemental composition of a biological surface. The way that the surface is evaporated allowed the generation of two methods: one based on an ion beam and the second using a laser. The application of a tag-mass strategy to IMS is the most recent efficient and highly multiplexed platform for the digital image analysis of biological samples.

Figure 3

Basic fundamentals and similarities between multiplexed ion beam imaging and imaging mass cytometry. Characterization of multiplexed ion beam imaging and imaging mass cytometry.

Figure 4

Multiplex immunoflorescencce microphotography. Images representing the immunoprofiling of different tumor types using the multiplexed tyramine signal amplification system: (A) esophageal squamous cell carcinoma, (B) malignant melanoma, (C) lung squamous cell carcinoma, (D) lung adenocarcinoma, (E) colorectal adenocarcinoma, (F) Hodgkin’s lymphoma. Scale bar: 200× magnification.

Figure 5

Multiplex immunofluorescence microphotography. Images representing the immunoprofiling of different stages of progression in lung, pancreas and breast cancer using the multiplexed tyramine signal amplification system: (A) pre-neoplastic lung lesion, (B) lung adenocarcinoma, (C) pre-neoplastic pancreatic lesion, (D) invasive pancreatic carcinoma, (E) non-invasive breast carcinoma, (F) invasive breast carcinoma. Scale bar: 200× magnification.