Optimized multiplex immunofluorescence for the characterization of tumor immune microenvironment in neoplastic paraffin-preserved tissues

Author(s)

Roversi, Fernanda Marconi ; Pereira Bueno, Maura Lima ; Francisco da Silva, Juliete Aparecida ; Assis-Mendonca, Guilherme Rossi ; Olalla Saad, Sara Teresinha

• Document type: Journal article
• Source: JOURNAL OF CELL COMMUNICATION AND SIGNALING; v. 17, n. 3, p. 11-pg., 2023-05-16.

Abstract

The study of neoplastic cells enabled the discovery of important tumor-related biomarkers which resulted in new forms of early diagnosis, therapeutic options, and prognostic markers. Thus, immunofluorescence (IF), a high throughput imaging technology, represents a valuable method that enables the virtual characterization and localization of diverse cell types and targets, preserving tissue architecture and spatial surroundings. IF staining and analysis of formalin-fixed paraffin-embedded (FFPE) tissues are considered a challenge due to several difficulties, such as tissue autofluorescence, non-specific antibody binding, and image acquisition and quality. This study aimed to develop a multiplex-fluorescence staining technique with high-contrast and high-quality multiple-color images to enrich the investigation of important biomarkers. We present a robust optimized multiple-immunofluorescence procedure that reduced sample autofluorescence, enabled the use of simultaneous antibodies on the same sample, and showed super-resolution imaging through precise antigen localization. We illustrated the utility of this powerful method in FFPE neoplastic appendix, lymph node and bone marrow biopsies, and a 3D-coculture system, in which cells are enabled to grow and interact with their surroundings in all 3D dimensions. Our optimized multiple-immunofluorescence method represents a powerful tool for better understanding the complexity of tumor cells, characterizing cell populations and spatial localization, revealing predictive and prognostic biomarkers, and identifying immunologic phenotypes in a single and limited sample. This valuable IF protocol successfully enables tumor microenvironment profiling that could contribute to the study of cellular crosstalk and the niche, and to the identification of predictive biomarkers for neoplasms. (AU)

• FAPESP's process: 17/21801-2 - Predictors of severity and new treatments for bone marrow neoplasias
• Grantee: Sara Teresinha Olalla Saad
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 21/05320-0 - Profile of Brazilian undergraduates who use electronic cigarettes: an epidemiological study on forbidden use
• Grantee: Gabriella dos Santos Maximino
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 19/25247-5 - Study of inhibitory and antitumor activity of natural substance-associated MAPK and WNT inhibitors in in vitro models
• Grantee: Maura Lima Pereira Bueno
• Support Opportunities: Scholarships in Brazil - Technical Training Program - Technical Training