Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging

Abstract

SummaryA highly multiplexed cytometric imaging approach, termed co-detection by indexing (CODEX), is used here to create multiplexed datasets of normal and lupus (MRL/lpr) murine spleens. CODEX iteratively visualizes antibody binding events using DNA barcodes, fluorescent dNTP analogs, and an in situ polymerization-based indexing procedure. An algorithmic pipeline for single-cell antigen quantification in tightly packed tissues was developed and used to overlay well-known morphological features with de novo characterization of lymphoid tissue architecture at a single-cell and cellular neighborhood levels. We observed an unexpected, profound impact of the cellular neighborhood on the expression of protein receptors on immune cells. By comparing normal murine spleen to spleens from animals with systemic autoimmune disease (MRL/lpr), extensive and previously uncharacterized splenic cell-interaction dynamics in the healthy versus diseased state was observed. The fidelity of multiplexed spatial cytometry demonstrated here allows for quantitative systemic characterization of tissue architecture in normal and clinically aberrant samples.

Novel Platforms of Multiplexed Immunofluorescence for Study of Paraffin Tumor Tissues

“…The antibodies to be revealed first generally have shorter overhangs than the antibodies to be visualized later ( Figure 1B). The platform can be performed on any three-color fluorescence microscope enabling conversion of regular fluorescence microscope into a tool for multidimensional tissue rendering and cell cytometry 14 , giving a good advantage to user this platform.…”

“…CO-Detection by indEXing (CODEX) is a new and recently imaging approach that use antibodies labeled with oligonucleotide duplexes that encode uniquely designed sequences with 5' overhangs 14 tagged antibodies at the same time. During iterative cycles of visualization of labeling the sequence of the 5'overhang determines the index (the combination of a polymerization cycle and a fluorescent channel) at which a given DNA tag incorporates one of two fluorescently labeled dNTP species.…”

Multiplex tissue imaging: An introduction to the scope and challenges

“…Such straightforward quantifiers of cell distress or anatomic distortion can find clinical utility as low‐plex diagnostic panels. Additionally, higher level in situ mapping of cell communities and functional interactions has revealed unmapped homeostasis and disease‐emergent properties of tissue: cell proximity analysis identified unsuspected cognate T‐follicular‐helper cell interaction networks driving inflammation within renal allografts; cell neighbor patterns revealed unsuspected linear threads of homotypically associated CD4 and CD8 T cells driving splenic tissue substructure . The possibility of unappreciated drug effects on this immune organization awaits exploration.…”

“…The possibility of unappreciated drug effects on this immune organization awaits exploration. Neighbor analysis also shows that local spatial relationships are a major contributor to gene expression variation, determining tissue function but unaccounted for in homogenate or cell isolate analysis. Such findings draw researchers toward in situ multiplexed analysis and away from spatially ignorant testing in dynamic settings, to dissect what pathologists intuit about the importance of local context …”

Single cell immune profiling in transplantation research

“…19,20 CO-Detection by indexing (CODEX) and cyclic immunofluorescence (CycIF) utilize staining with oligonucleotide-conjugated antibodies and an iterative process of sequential primer extension with fluorescently labeled nucleotides in order to image expression of two markers at a time,for as many iterations as necessary 21,22. Multiplexed immunohistochemistry (mIHC) involves staining one biopsy specimen with multiple antibodies, with either chemical or fluorescent detection.…”