All Publications

  • Tissue schematics map the specialization of immune tissue motifs and their appropriation by tumors. Cell systems Bhate, S. S., Barlow, G. L., Schurch, C. M., Nolan, G. P. 2021


    A schematic of a biological system, i.e., a representation of its pieces, how they are combined, and what they do, would facilitate understanding its essential organization and alteration in pathogenesis or evolution. We present a computational approach for constructing tissue schematics (TSs) from high-parameter imaging data and a biological model for interpreting them. TSs map the spatial assembly of cellular neighborhoods into tissue motifs, whose modular composition, we propose, enables the generation of complex outputs. We developed our approach in human lymphoid tissue (HLT), identifying the follicular outer zone as a potential relay between neighboring zones and a core lymphoid assembly with modifications characteristic of each HLT type. Applying the TS approach to the tumor microenvironment in human colorectal cancer identified a higher-order motif, whose mutated assembly was negatively associated with patient survival. TSs may therefore elucidate how immune architectures can be specialized and become vulnerable to reprogramming by tumors.

    View details for DOI 10.1016/j.cels.2021.09.012

    View details for PubMedID 34653369

  • Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front. Cell Schürch, C. M., Bhate, S. S., Barlow, G. L., Phillips, D. J., Noti, L. n., Zlobec, I. n., Chu, P. n., Black, S. n., Demeter, J. n., McIlwain, D. R., Samusik, N. n., Goltsev, Y. n., Nolan, G. P. 2020


    Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains.

    View details for DOI 10.1016/j.cell.2020.07.005

    View details for PubMedID 32763154

  • CellSeg: a robust, pre-trained nucleus segmentation and pixel quantification software for highly multiplexed fluorescence images. BMC bioinformatics Lee, M. Y., Bedia, J. S., Bhate, S. S., Barlow, G. L., Phillips, D., Fantl, W. J., Nolan, G. P., Schürch, C. M. 2022; 23 (1): 46


    Algorithmic cellular segmentation is an essential step for the quantitative analysis of highly multiplexed tissue images. Current segmentation pipelines often require manual dataset annotation and additional training, significant parameter tuning, or a sophisticated understanding of programming to adapt the software to the researcher's need. Here, we present CellSeg, an open-source, pre-trained nucleus segmentation and signal quantification software based on the Mask region-convolutional neural network (R-CNN) architecture. CellSeg is accessible to users with a wide range of programming skills.CellSeg performs at the level of top segmentation algorithms in the 2018 Kaggle Data Challenge both qualitatively and quantitatively and generalizes well to a diverse set of multiplexed imaged cancer tissues compared to established state-of-the-art segmentation algorithms. Automated segmentation post-processing steps in the CellSeg pipeline improve the resolution of immune cell populations for downstream single-cell analysis. Finally, an application of CellSeg to a highly multiplexed colorectal cancer dataset acquired on the CO-Detection by indEXing (CODEX) platform demonstrates that CellSeg can be integrated into a multiplexed tissue imaging pipeline and lead to accurate identification of validated cell populations.CellSeg is a robust cell segmentation software for analyzing highly multiplexed tissue images, accessible to biology researchers of any programming skill level.

    View details for DOI 10.1186/s12859-022-04570-9

    View details for PubMedID 35042474

  • Subcellular localization of biomolecules and drug distribution by high-definition ion beam imaging. Nature communications Rovira-Clave, X., Jiang, S., Bai, Y., Zhu, B., Barlow, G., Bhate, S., Coskun, A. F., Han, G., Ho, C. K., Hitzman, C., Chen, S., Bava, F., Nolan, G. P. 2021; 12 (1): 4628


    Simultaneous visualization of the relationship between multiple biomolecules and their ligands or small molecules at the nanometer scale in cells will enable greater understanding of how biological processes operate. We present here high-definition multiplex ion beam imaging (HD-MIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added structurally-unmodified small molecules. With this technology, the atomic constituents of the biomolecules themselves can be used in our system as the "tag" and we demonstrate measurements down to ~30nm lateral resolution. We correlated the subcellular localization of the chemotherapy drug cisplatin simultaneously with five subnuclear structures. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. Unexpectedly, cells surviving multi-drug treatment with cisplatin and the BET inhibitor JQ1 demonstrated near total cisplatin exclusion from the nucleus, suggesting that selective subcellular drug relocalization may modulate resistance to this important chemotherapeutic treatment. Multiplexed high-resolution imaging techniques, such as HD-MIBI, will enable studies of biomolecules and drug distributions in biologically relevant subcellular microenvironments by visualizing the processes themselves in concert, rather than inferring mechanism through surrogate analyses.

    View details for DOI 10.1038/s41467-021-24822-1

    View details for PubMedID 34330905

  • Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction. European journal of immunology Kennedy-Darling, J. n., Bhate, S. S., Hickey, J. W., Black, S. n., Barlow, G. L., Vazquez, G. n., Venkataraaman, V. G., Samusik, N. n., Goltsev, Y. n., Schürch, C. M., Nolan, G. P. 2021


    Multiparameter tissue imaging enables analysis of cell-cell interactions in situ, the cellular basis for tissue structure, and novel cell types that are spatially restricted, giving clues to biological mechanisms behind tissue homeostasis and disease. Here, we streamlined and simplified the multiplexed imaging method CO-Detection by indEXing (CODEX) by validating 58 unique oligonucleotide barcodes that can be conjugated to antibodies. We showed that barcoded antibodies retained their specificity for staining cognate targets in human tissue. Antibodies were visualized one at a time by adding a fluorescently labeled oligonucleotide complementary to oligonucleotide barcode, imaging, stripping, and repeating this cycle. With this we developed a panel of 46 antibodies that was used to stain five human lymphoid tissues: three tonsils, a spleen, and a lymph node. To analyze the data produced, an image processing and analysis pipeline was developed that enabled single-cell analysis on the data, including unsupervised clustering that revealed 31 cell types across all tissues. We compared cell-type compositions within and directly surrounding follicles from the different lymphoid organs and evaluated cell-cell density correlations. This sequential oligonucleotide exchange technique enables a facile imaging of tissues that leverages pre-existing imaging infrastructure to decrease the barriers to broad use of multiplexed imaging. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/eji.202048891

    View details for PubMedID 33548142

  • Immune cell topography predicts response to PD-1 blockade in cutaneous T cell lymphoma. Nature communications Phillips, D., Matusiak, M., Gutierrez, B. R., Bhate, S. S., Barlow, G. L., Jiang, S., Demeter, J., Smythe, K. S., Pierce, R. H., Fling, S. P., Ramchurren, N., Cheever, M. A., Goltsev, Y., West, R. B., Khodadoust, M. S., Kim, Y. H., Schürch, C. M., Nolan, G. P. 2021; 12 (1): 6726


    Cutaneous T cell lymphomas (CTCL) are rare but aggressive cancers without effective treatments. While a subset of patients derive benefit from PD-1 blockade, there is a critically unmet need for predictive biomarkers of response. Herein, we perform CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced CTCL patients enrolled in a pembrolizumab clinical trial (NCT02243579). We find no differences in the frequencies of immune or tumor cells between responders and non-responders. Instead, we identify topographical differences between effector PD-1+ CD4+ T cells, tumor cells, and immunosuppressive Tregs, from which we derive a spatial biomarker, termed the SpatialScore, that correlates strongly with pembrolizumab response in CTCL. The SpatialScore coincides with differences in the functional immune state of the tumor microenvironment, T cell function, and tumor cell-specific chemokine recruitment and is validated using a simplified, clinically accessible tissue imaging platform. Collectively, these results provide a paradigm for investigating the spatial balance of effector and suppressive T cell activity and broadly leveraging this biomarker approach to inform the clinical use of immunotherapies.

    View details for DOI 10.1038/s41467-021-26974-6

    View details for PubMedID 34795254

  • Cellular neighborhoods predict pembrolizumab response in cutaneous T cell lymphoma Schurch, C. M., Phillips, D. J., Gutierrez, B., Matusiak, M., Bhate, S. S., Barlow, G. L., Fling, S. P., Ramchurren, N., Pierce, R. H., Cheever, M. A., Khodadoust, M. S., West, R., Kim, Y. H., Nolan, G. P. AMER ASSOC CANCER RESEARCH. 2020
  • Combining RNA in situ hybridization and spectral flow cytometry to investigate the leukocyte glycocalyx in autoimmunity Marshall, P. L., Kaber, G., Linde, M. H., Barlow, G. L., Haddock, N. L., Wagar, L., Nagy, N., Bollyky, P. L. AMER ASSOC IMMUNOLOGISTS. 2020
  • Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection. Matrix biology : journal of the International Society for Matrix Biology Marshall, P. L., Nagy, N. n., Kaber, G. n., Barlow, G. L., Ramesh, A. n., Xie, B. J., Linde, M. H., Haddock, N. L., Lester, C. A., Tran, Q. L., de Vries, C. n., Hargil, A. n., Malkovskiy, A. n., Gurevich, I. n., Martinez, H. A., Kuipers, H. F., Yadava, K. n., Zhang, X. n., Evanko, S. P., Gebe, J. A., Wang, X. n., Vernon, R. B., de la Motte, C. n., Wight, T. N., Engleman, E. G., Krams, S. M., Meyer, E. n., Bollyky, P. L. 2020


    A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

    View details for DOI 10.1016/j.matbio.2020.12.001

    View details for PubMedID 33290836

  • Dynamics of the Cutaneous T Cell Lymphoma Microenvironment in Patients Treated with Pembrolizumab Revealed By Highly Multiplexed Tissue Imaging Schuerch, C. M., Phillips, D. J., Bhate, S. S., Barlow, G. L., Fling, S. P., Ramchurren, N., Pierce, R., Cheever, M. A., Khodadoust, M. S., Kim, Y. H., Nolan, G. P. AMER SOC HEMATOLOGY. 2019
  • Dynamics of the Bone Marrow Microenvironment during Leukemic Progression Revealed By Codex Hyper-Parameter Tissue Imaging Schuerch, C., Barlow, G. L., Bhate, S. S., Samusik, N., Nolan, G. P., Goltsev, Y. AMER SOC HEMATOLOGY. 2018
  • 50-dimensional microenvironment analysis of human and mouse bone marrow during malignant transformation Schuerch, C. M., Barlow, G. L., Bhate, S. S., Samusik, N., Nolan, G., Goltsev, Y. NATURE PUBLISHING GROUP. 2018: 550