Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes

Abstract

39The architecture of normal and diseased tissues strongly influences the development and 40 progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based 41 cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of 42 formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used 43 specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex 44 images using an iterative process (a cycle) in which conventional low-plex fluorescence images are 45 repeatedly collected from the same sample and then assembled into a high dimensional representation. t- 46 CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; 47 we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune 48 markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective 49 method for pre-clinical and clinical research and a natural complement to single-cell genomics. 50 51 52Histopathology is among the most important and widely used methods for diagnosing human disease 53 and studying the development of multi-cellular organisms. As commonly performed, imaging of formalin-fixed, 54 paraffin-embedded (FFPE) tissue has relatively low dimensionality, primarily comprising Hematoxylin and 55 Eosin (H&E) staining supplemented by immunohistochemistry (IHC). The potential of IHC to aid in diagnosis 56 and prioritization of therapy is well established 1 but IHC is primarily a single channel method: imaging multiple 57 antigens usually involves the analysis of sequential tissue slices or harsh stripping protocols (although limited 58 multiplexing is possible using IHC and bright-field imaging 2,3 ). Antibody detection via formation of a brown 59 diamino-benzidine (DAB) or similar precipitate is also less quantitative than fluorescence 4 . The limitations of 60 IHC are particularly acute when it is necessary to quantify complex cellular states and multiple cell types, such 61 as tumor infiltrating regulatory and cytotoxic T cells, 5 in parallel with tissue and pharmaco-dynamic markers. 62 Advances in DNA and RNA profiling have dramatically improved our understanding of oncogenesis 63 and propelled the development of targeted anti-cancer drugs. 6 Sequence data are particularly useful when an 64 oncogenic driver is both a drug target and a biomarker of drug response, such as BRAF V600E in melanoma 7 or 65 BCR-ABL 8 in chronic myelogenous leukemia. However, in the case of drugs that act through cell non- 66 autonomous mechanisms, such as immune checkpoint inhibitors, tumor-drug interaction must be studied in the 67 context of multi-cellular environments that include both cancer and non-malignant stromal and infiltrating 68 immune cells. Multiple studies have established that these components of the tumor microenvironment strongly 69 influence the initiatio...

A Cancer Cell Program Promotes T Cell Exclusion and Resistance to Checkpoint Blockade

“…To test if the resistance program in malignant cells is associated with T cell exclusion in situ, we used multiplexed immunofluorescence (t-CyCIF) (Lin et al, 2018). We stained histological sections of 19 tumors (472,771 cells per image on average) from our single-cell cohort for 14 proteins: six cell type markers (CD3, CD8, MHC-II, FOXP3, S100, and MITF) and seven resistance program members (induced: p53, Myc, and DLL3; repressed: HLA-A, c-Jun, SQSTM1, and LAMP2).…”

“…Imager5 software (RareCyte) was used to sequentially scan the region of interest in 4 fluorescence channels. Image processing and single-cell quantification was performed as previously described (Lin et al, 2018). Briefly, background subtraction was performed using the established rolling ball algorithm (with a 50-pixel radius) followed by registration in ImageJ.…”

Multidimensional profiling of drug‐treated cells by Imaging Mass Cytometry

“…In the field of fluorescent cellular imaging microscopy, biomarker analysis in single cells is limited to 4–6 due to the overlapping spectra of fluorescent dyes . Repetitive rounds of staining using the same biological sample are required to achieve multiplexing . New imaging technologies are needed to significantly increase multiplicity in a single sample preparation experiment and perform replicate analyses .…”

Modelling cell-cell interactions from spatial molecular data with spatial variance component analysis

“…There is a growing appreciation of the role of spatial distribution of proteins, transcripts and other molecules in determining tissues functioning and its deregulation in disease, with potential value as predictors of clinical outcomes (Bodenmiller, 2016) . This is largely driven by vigorous development of novel technologies that enable us to capture such data (Aichler & Walch, 2015;Bodenmiller, 2016;Goltsev et al, 2018;Lin et al, 2017;Schulz et al, 2018) .…”