Tumor microenvironment, defined by surrounding stromal/immune cells as well as blood vessels, plays an important role in disease progression and therapy resistance. Increasing understanding of the heterogeneity in both tumor and its microenvironment will be crucial to development more effective therapies. Recently, several studies employing state-of-the-art single-cell sequencing methods reveal enormous complexity in tumor microenvironment. However, the spatial information and cell-to-cell interaction could not be preserved in these dissociated cells. Immunofluorescence has been widely used in different fields of biological and medical research for decades. The ability to obtain in situ and single-cell information makes this technique particularly important in tumor biology. However, biochemical and optical constraints limit the number of signals that could be captured simultaneously within the same sample. We have developed the CycIF (Cyclic Immunofluorescence), an easy and low-cost method to increase the multiplexity of conventional immunofluorescence. The CycIF method has been first applied in pre-clinical drug discovery, cancer and stem cell biology with adherent cell cultures. In here, we modified the original CycIF method for IHC/IF on FFPE samples, and used that to probe tumor heterogeneity, microenvironment and immune infiltration in various types of tumors. Up to 30 different antigens/markers could be simultaneously detected in different tumor samples, and these makers represent a wide range of biological processes, including the key molecules for lymphocyte surface makers (CD45, CD4, CD8, CD20, CD11c), immune checkpoints (PD-1, PD-L1), stromal/EMT proteins (E-Cadherin, Vimentin), cell cycle regulators (CycD1, PCNA, Ki67, pRB, p21/CIP), signaling proteins (EGFR, pERK, pS6) and apoptosis mediators (p53, Bax, Bcl-2, Survivin). Our study not only provides the first detailed map of tumor and its immune microenvironment, but also illustrates a robust multiplexed imaging platform for probing tumor heterogeneity.

Citation Format: Jia-Ren Lin, Benjamin Izar, Sabrina Hawthorne, Josh Nordberg, Eric Kaldjian, Peter Sorger. Probing tumor heterogeneity and immune infiltration with Cyclic Immunofluorescence (CycIF), a robust, multiplexed imaging method. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B21.