Molecular Pixelation: Single cell spatial proteomics by sequencing

The spatial distribution of cell surface proteins govern vital processes of the immune system such as inter-cell communication and mobility. However, tools for studying these at high multiplexing scale, resolution, and throughput needed to drive novel discoveries are lacking. We present Molecular Pixelation, a DNA-sequencing based method for single cell analysis to quantify protein abundance, spatial distribution, and colocalization of targeted proteins using Antibody Oligonucleotide Conjugates (AOCs). Relative locations of AOCs are inferred by sequentially associating these into local neighborhoods using DNA-pixels containing unique pixel identifier (UPI) sequences, forming >1,000 connected spatial zones per single cell in three dimensions. DNA-sequencing reads are computationally arranged into spatial single cell maps for 76 proteins without cell compartmentalization. By studying immune cell dynamics and using spatial statistics on graph representations of the data, previously known and novel patterns of protein spatial polarization and co-localization were found in chemokine-stimulated T-cells.

[1]  S. Gottschalk,et al.  Synapse-tuned CARs enhance immune cell anti-tumor activity , 2023, Nature Biotechnology.

[2]  Michelle M. Li,et al.  Graph representation learning in biomedicine and healthcare , 2022, Nature Biomedical Engineering.

[3]  D. Raulet,et al.  Induced CD45 Proximity Potentiates Natural Killer Cell Receptor Antagonism , 2022, bioRxiv.

[4]  A. Jainarayanan,et al.  The interplay between membrane topology and mechanical forces in regulating T cell receptor activity , 2022, Communications biology.

[5]  H. Nijman,et al.  Tumor-infiltrating lymphocytes in the immunotherapy era , 2020, Cellular & Molecular Immunology.

[6]  Lestat R. Ali,et al.  Immune receptor inhibition through enforced phosphatase recruitment , 2020, Nature.

[7]  Weian Zhao,et al.  CAR-T design: Elements and their synergistic function , 2020, EBioMedicine.

[8]  Edward M Marcotte,et al.  Bringing Microscopy-By-Sequencing into View. , 2020, Trends in biotechnology.

[9]  Pekka Orponen,et al.  A computational framework for DNA sequencing microscopy , 2019, Proceedings of the National Academy of Sciences.

[10]  Mats Nilsson,et al.  Profiling surface proteins on individual exosomes using a proximity barcoding assay , 2019, Nature Communications.

[11]  Allon M Klein,et al.  Scrublet: Computational Identification of Cell Doublets in Single-Cell Transcriptomic Data. , 2019, Cell systems.

[12]  Aviv Regev,et al.  DNA Microscopy: Optics-free Spatio-genetic Imaging by a Stand-Alone Chemical Reaction , 2018, Cell.

[13]  Leland McInnes,et al.  UMAP: Uniform Manifold Approximation and Projection , 2018, J. Open Source Softw..

[14]  M. Wright,et al.  The Many and Varied Roles of Tetraspanins in Immune Cell Recruitment and Migration , 2018, Front. Immunol..

[15]  Samuel L. Wolock,et al.  Scrublet: computational identification of cell doublets in single-cell transcriptomic data , 2018, bioRxiv.

[16]  H. Swerdlow,et al.  Large-scale simultaneous measurement of epitopes and transcriptomes in single cells , 2017, Nature Methods.

[17]  Xiaohui Wang,et al.  Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells. , 2016, Bioconjugate chemistry.

[18]  Vito Mennella,et al.  Super-Resolution Microscopy: From Single Molecules to Supramolecular Assemblies. , 2015, Trends in cell biology.

[19]  D. Davis,et al.  Rituximab causes a polarization of B cells that augments its therapeutic function in NK-cell-mediated antibody-dependent cellular cytotoxicity. , 2013, Blood.

[20]  G. Pantaleo,et al.  Functional Avidity: A Measure to Predict the Efficacy of Effector T Cells? , 2012, Clinical & developmental immunology.

[21]  Natasha S. Barteneva,et al.  Imaging Flow Cytometry , 2016, Methods in Molecular Biology.

[22]  F. Sánchez‐Madrid,et al.  Bringing up the rear: defining the roles of the uropod , 2009, Nature Reviews Molecular Cell Biology.

[23]  Hanlee P. Ji,et al.  Multiplexed protein detection by proximity ligation for cancer biomarker validation , 2007, Nature Methods.

[24]  Doron Lancet,et al.  Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification , 2005, Bioinform..

[25]  C. Martínez-A,et al.  Mastering time and space: immune cell polarization and chemotaxis. , 2005, Seminars in immunology.

[26]  Wilhelm Friedrich,et al.  Lymphocyte microvilli are dynamic, actin-dependent structures that do not require Wiskott-Aldrich syndrome protein (WASp) for their morphology , 2004 .

[27]  Michael Loran Dustin,et al.  The immunological synapse , 2002, Arthritis research.

[28]  N. Liabakk,et al.  Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens. , 2001, Cytometry.