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Annotation of spatially resolved single-cell data with STELLAR

Nature Methods volume 19, pages 1411–1418 (2022)Cite this article

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Abstract

Accurate cell-type annotation from spatially resolved single cells is crucial to understand functional spatial biology that is the basis of tissue organization. However, current computational methods for annotating spatially resolved single-cell data are typically based on techniques established for dissociated single-cell technologies and thus do not take spatial organization into account. Here we present STELLAR, a geometric deep learning method for cell-type discovery and identification in spatially resolved single-cell datasets. STELLAR automatically assigns cells to cell types present in the annotated reference dataset and discovers novel cell types and cell states. STELLAR transfers annotations across different dissection regions, different tissues and different donors, and learns cell representations that capture higher-order tissue structures. We successfully applied STELLAR to CODEX multiplexed fluorescent microscopy data and multiplexed RNA imaging datasets. Within the Human BioMolecular Atlas Program, STELLAR has annotated 2.6 million spatially resolved single cells with dramatic time savings.

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Fig. 1: STELLAR is a geometric deep learning framework for annotating spatially resolved single-cell datasets.
Fig. 2: STELLAR accurately identifies cell types from the reference set and discovers novel cell types that have never been characterized in the reference set.
Fig. 3: STELLAR transfers granular cell-type labels across tissue regions and donors from HuBMAP data and identifies main structures of healthy human intestine tissue.
Fig. 4: STELLAR’s embeddings reveal higher-order tissue structures.

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Data availability

The CODEX datasets presented in this study can be found in the online repository Dryad at https://datadryad.org/stash/share/1OQtxew0Unh3iAdP-ELew-ctwuPTBz6Oy8uuyxqliZk. Specifically, the quantified single-cell data are provided (with cells in rows and protein expression, xy position and cell-type labels in columns). Additionally, we provide datasets used to transfer from the tonsil to BE tissue (BE_Tonsil_dryad.csv) and expert-annotated healthy human intestine (B004_training_dryad.csv), which was used to test the accuracy of STELLAR across the four regions of the colon regions of this dataset and also for training for transferring cell-type labels to unlabeled donors (B0056_unannotated_dryad.csv). MERFISH mouse cortex datasets are from Ref. 8.

Code availability

STELLAR was written in Python v.3.8 using the PyTorch library. The source code is available on Github at https://github.com/snap-stanford/stellar. The project website with links to data and code can be accessed at http://snap.stanford.edu/stellar/.

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Acknowledgements

This work was supported by the US National Institutes of Health (grant nos. 2U19AI057229-16, 5P01HL10879707, 5R01GM10983604, 5R33CA18365403, 5U01AI101984-07, 5UH2AR06767604, 5R01CA19665703, 5U54CA20997103, 5F99CA212231-02, 1F32CA233203-01, 5U01AI140498-02, 1U54HG010426-01, 5U19AI100627-07, 1R01HL120724-01A1, R33CA183692, R01HL128173-04, 5P01AI131374-02, 5UG3DK114937-02, 1U19AI135976-01, IDIQ17X149, 1U2CCA233238-01 and 1U2CCA233195-01); Cancer Research UK (grant no. C27165/A29073); and the Parker Institute for Cancer Immunotherapy. J.W.H. was supported by an NIH T32 Fellowship (grant no. T32CA196585) and an American Cancer Society: Roaring Fork Valley Postdoctoral Fellowship (grant no. PF-20-032-01-CSM). We also gratefully acknowledge the support of DARPA under grant nos. HR00112190039 (TAMI), N660011924033 (MCS); ARO under grant nos. W911NF-16-1-0342 (MURI), W911NF-16-1-0171 (DURIP); NSF under grant nos. OAC-1835598 (CINES), OAC-1934578 (HDR), CCF-1918940 (Expeditions), IIS-2030477 (RAPID), NIH under grant no. R56LM013365; Stanford Data Science Initiative, Wu Tsai Neurosciences Institute, Amazon, JPMorgan Chase, Docomo, Hitachi, Juniper Networks, Intel, KDDI and Toshiba.

Author information

Author notes

  1. These authors contributed equally: Maria Brbić, Kaidi Cao, John W. Hickey.

Authors and Affiliations

  1. Department of Computer Science, Stanford University, Stanford, CA, USA

    Maria Brbić, Kaidi Cao & Jure Leskovec

  2. School of Computer and Communication Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Maria Brbić

  3. Baxter Laboratories Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford University, Stanford, CA, USA

    John W. Hickey, Yuqi Tan & Garry P. Nolan

  4. Department of Genetics, Stanford University School of Medicine, Stanford University, Stanford, CA, USA

    Michael P. Snyder

  5. Department of Pathology, Stanford University School of Medicine, Stanford University, Stanford, CA, USA

    Garry P. Nolan

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Contributions

M.B., K.C., J.W.H. and J.L. conceived the research. M.B., K.C., J.W.H. and Y.T. performed research and analyzed results. M.B., K.C. and J.L. contributed new analytical tools and created the algorithmic framework. J.W.H., M.P.S. and G.P.N. generated and analyzed the data. J.L., G.P.N. and M.P.S. supervised the research. All authors participated in interpretation and wrote the manuscript.

Corresponding authors

Correspondence to Garry P. Nolan or Jure Leskovec.

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Competing interests

M.P.S. is cofounder and advisory board member of Personalis, Qbio, January AI, Mirvie, Filtricine, Fodsel, Protos. RTHM, Marble Therapeutics and Crosshair Therapeutics. G.P.N. has equity in and is a scientific advisory board member of Akoya Biosciences, Inc. The other authors declare no competing interests.

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Nature Methods thanks Ellis Patrick, Darren Tyson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 STELLAR overview.

STELLAR is a unique method in its ability to simultaneously recognize cell types seen in the reference set and discover novel cell types that have never been characterized in the reference set. This is made possible by an objective function that consists of two main components (Methods). First, STELLAR learns to gradually separate cell types from the reference set by controlling intra-class variance to allow the model to simultaneously learn to discover novel cell types. Simultaneously, STELLAR discovers novel classes by generating auxiliary labels (pseudo-labels) in the unannotated graph that are used to guide the training. The auxiliary labels are generated based on the nearest neighbors of each cell in the embedding space.

Extended Data Fig. 2 CODEX image of reference dataset from human tonsil.

Ground-truth labels of the tonsil CODEX multiplexed imaging dataset. Colors denote different cell types.

Extended Data Fig. 3 Cell-type distributions on tonsil and BE datasets.

Cell-type distributions of ground-truth labels on (a) tonsil reference dataset and (b) Barrett’s esophagus dataset. PDPN stands for Podoplanin (PDPN) positive stromal cells.

Extended Data Fig. 4 Neighborhoods found in tonsil and Barrett’s esophagus (BE) dataset.

(a) Neighborhood heatmap showing the neighborhoods found across both tissues and cell types enriched compared to tissue averages. (b) Neighborhood composition between BE and tonsil tissues. (c) Neighborhood types mapped back to tissue coordinates.

Extended Data Fig. 5 Comparison of STELLAR to baseline methods on the Barrett’s esophagus (BE) dataset.

(a) Accuracy of STELLAR and scANVI on the BE dataset. Performance was evaluated as a mean score across n=5 runs of each method. Error bars are from standard deviation. scANVI stands for the setting evaluated in the same manner as STELLAR in which we train the model on tonsil dataset and evaluate on BE dataset. scANVI_leaky stands for the approach in which we use fraction of labels from BE dataset as the training data and use the rest of the BE dataset as the test set. Although the setting in which scANVI_leaky is evaluated does not present a fair comparison to STELLAR and other baselines, it indicates that the performance of drop of scANVI is caused by differences between tonsil and BE datasets. (b-d) Performance of STELLAR and alternative baselines on the BE dataset evaluated as (b) mean macro F1-score, (c) macro precision score, and (d) macro recall score across n=5 runs of each method. Error bars are from standard deviation. XGB stands for XGBoost, SVM for Support Vector Machine, RF for Random Forest, ADA for ADABoost, and Seurat for Seurat V4.

Extended Data Fig. 6 Robustness of STELLAR evaluated on the Barrett’s esophagus (BE) dataset.

(a) Performance of STELLAR using different normalization strategies. ‘Unnorm’ stands for raw (unnormalized) data. Performance was evaluated as a mean accuracy score across n=5 runs of each normalization strategy. Error bars are from standard deviation. (b) Performance of STELLAR when misannotating proportion of randomly selected cells. In each run, cells were randomly selected and labels different than ground truth annotations were randomly assigned to cells in the annotated reference tonsil dataset. Performance was evaluated as an accuracy score across n=5 runs. Individual data points are shown. (c) Performance of STELLAR when removing different number of marker genes. In each run, different set of randomly selected marker genes was withheld from the reference tonsil dataset and BE datasets. Performance was evaluated as a mean accuracy score across n=5 runs. Error bars are from standard deviation.

Extended Data Fig. 7 Performance of STELLAR on the MERFISH dataset from mouse cortex.

We applied STELLAR to a large-scale mouse primary motor cortex MERFISH dataset consisting of 23 granular cell types from two mice [8]. (a) Annotation accuracy of STELLAR on the MERFISH mouse cortex dataset with different numbers of withheld cell types. Position of scatter plot points is computed as a mean accuracy score across n=5 runs. Error bars are from standard deviation. We randomly removed a number of cell types from the reference set and evaluated STELLAR’s performance by gradually increasing the number of removed cell types. We measured accuracy separately on classes seen in the reference set and classes withheld from the reference set. Performance is evaluated on the reference cell types, novel cell types withheld from the reference set during training, and jointly on all cell types. (b, c) UMAP visualization of MERFISH mouse cortex dataset from mouse used as the test set. Cells are colored according to (b) ground-truth annotations, and (c) STELLAR’s predictions without any withheld cell types.

Extended Data Fig. 8 STELLAR predictions on the dataset from healthy intestine.

CODEX-imaged regions with cell types colored by prediction from STELLAR using data from the healthy intestine of a different donor as the reference set. Data from both small intestine and colon are shown. Colors denote different cell types. DC stands for dendritic cell, ICC stands for interstitial cells of Cajal, TA stands for transit amplifying cell.

Extended Data Fig. 9 Multicellular structures discovered bt STELLAR on CODEX healthy intestine data.

Characterization of multicellular structures by clustering the embedding space from STELLAR on CODEX healthy intestine data. (a) Heatmap of average cell-type composition in clustered embeddings. (b) Representative tissue image colored by embedding structure. IEL stands for intraepithelial lymphocytes.

Extended Data Fig. 10 Multicellular structures discovered bt STELLAR on MERFISH mouse cortex data.

Clusters in STELLAR’s embedding space identify multicellular structures in tissues in MERFISH data from mouse cortex. (a) Heatmap of average cell-type composition in STELLAR clustered embeddings. (b) Representative tissue image colored by overall structure. L, lateral; OPC, oligodendrocyte precursor cell; PVM, perivascular macrophage; SMC, smooth muscle cell; VLMC, vascular leptomeningeal cell.

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Brbić, M., Cao, K., Hickey, J.W. et al. Annotation of spatially resolved single-cell data with STELLAR. Nat Methods 19, 1411–1418 (2022). https://doi.org/10.1038/s41592-022-01651-8

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