Protein Co-detection Data Release Showcase

MERSCOPE FFPE Anti-body Co-staining Data Release:

Unraveling Immuno-Oncology Signatures in Human Uterine Cancer with MERSCOPE

 

Cancer immunotherapy has emerged as a promising avenue for treating various malignancies, including human uterine cancer. Research to understand the interplay between immune cell populations and tumor microenvironments is critical for developing effective therapies. Advancements in single-cell spatial transcriptomic imaging technologies, such as Multiplexed Error-robust Fluorescence in Situ Hybridization (MERFISH), have enabled the simultaneous profiling of hundreds to thousands of genes at single-molecule resolution and detection of protein in intact tissue. For this data showcase, we demonstrate the capability of spatial multi-omics profiling with the Vizgen MERSCOPE® Platform. Specifically, we combined protein staining for CD45RA and CD45RO, key immune cell markers involved in T-cell differentiation and activation, with MERFISH to provide a multi-dimensional view of the tumor microenvironment within FFPE human uterine cancer samples. Analyzing these multi-omics datasets not only resolved different cell types with distinct transcriptomic features and their spatial organization/interaction in the tissue, but also enabled the identification of specific immune cell type/gene expression patterns that correlate with the presence or absence of CD45RA and CD45RO, providing a comprehensive picture of the immuno-oncology landscape in uterine cancer. Taken together, simultaneous profiling of RNA and protein with the MERSCOPE Platform offers an unparalleled insight into the complex interplay between immune cells and tumor cells, holding significant potential for research uncovering novel biomarkers and therapeutic targets. 

Protein Co-detection Data Release Overview: 

Species Type Human
Tissue Type  FFPE uterine cancer tumor
Gene Panel Size 500 
Number of Samples  2 replicates
Total Transcripts Detected  826,725,264
Total Cells   1,468,971​

 

Three Ways to Explore the Data Release

1. MERSCOPE® Web Vizualizer

The FFPE human uterus section datasets displayed in the MERSCOPE® Web Vizualizer contain a MERFISH measurement of a gene panel containing 500 total genes. Use the MERSCOPE Web Vizualizer to interactively view the full output of the MERSCOPE experiment, including each detected transcript, the segmented cell boundaries, underlying high-resolution images of DAPI and cell boundary stains, and expression of  CD45RA and CD45RO proteins. Explore the dataset by:

  • Zooming from a whole-tissue view down to sub-cellular resolution
  • Visualizing the expression of all or a select subset of genes across single-cells
  • Coloring cells based on their cell type annotation
  • Switching between the 2 datasets by clicking the Datasets dropdown menu on the upper right
  • Note: Gene panel size is 500; the Web Vizualizer is capturing 50 blanks in the total number, scroll through list to turn off blanks. CTRL+ Click allows you to select genes of interest.

2. Download the Datasets

You can download the MERFISH data files directly to your computer to explore using your data analysis program of choice. Below is the list of downloadable file types:

  • Information on all transcripts detected in the sample (List of Detected Transcripts)
  • Single-cell information from cell segmentation (Cell by Gene Matrix)
  • Mosaic images of the full tissue

Sample Name 

Total Transcripts

Total Cell Number

Antibody

Individual Datasets (Includes transcripts, cells, and images)

Uterine Cancer CD45RA

379,924,231

723,666

CD45RA, marker for T-cell differentiation and activation

Access Data Set

Uterine Cancer CD45RO

446,801,033

745,305

CD45RO, marker for T-cell differentiation and activation

 Access Data Set

To cite this data, please use: Vizgen MERFISH Protein Co-detection Data Release,  September 2023.

Legal Notice:

By submitting the registration form and accessing or using any data from this dataset, you hereby agree on your own behalf and on behalf of any entity for which you are accessing or using such information or data, to defend, indemnify and hold harmless Vizgen, Inc. and each of its officers, directors, shareholders, employees, agents, and attorneys, from and against any and all claims, losses, damages, liabilities, and costs (including without limitation, attorney’s fees) arising from or related to your use of such information and/or data, except to the extent such claim, losses, damages or costs resulting from the gross negligence or willful misconduct of Vizgen.

3.  Jupyter Notebook on Google Colab

The spatial data analysis performed by our Juypter Notebook offers insights into the protein expression profiles and cellular composition within the human uterine cancer samples. It examines the protein expression patterns and classifies cells based on their protein expression status.

First, the Notebook identifies cell types across the tissue samples using the cell-by-gene matrix and cell_meta files and unsupervised cell clustering algorithms. Next, the Notebook uses protein expression to classify cells as protein expression-positive or protein expression-negative. This enables the comparison of the cell composition between protein expression-positive and protein expression-negative cells. 

Our notebook serves as a simplified example, and we encourage you to adapt and customize the analysis pipeline according to your specific dataset and research goals.

 

Quality Spatial Multiomic Data from MERSCOPE

Accuracy and sensitivity are critical features in generating biologically meaningful spatial genomics data. The MERSCOPE FFPE Sample Prep Solution and Protein Stain Kits provide superior accuracy and sensitivity to ensure successful, impactful spatial multioimc data generation.

High reproducibility of RNA and protein co-detection with MERSCOPE​

Figure 1. High reproducibility of RNA and protein co-detection with MERSCOPE (A) Correlation of MERSCOPE data between two human uterine cancer FFPE tissue sections from the same patient, with CD45RA or CD45RO protein staining. The correlation coefficient is over 0.99, demonstrating high reproducibility. ​(B) UMAP visualization of the 17 cell clusters identified from the human uterus cancer samples. Datasets with CD45RA and CD45RO are combined for this analysis. ​(C) UMAP visualization of the cells from different datasets (CD45RA or CD45RO). ​(D) The number of cells from CD45RA/CD45RO samples are almost identical across different cell clusters. ​(E) Spatial distribution of different cell clusters in uterine cancer sections with CD45RA or CD45RO staining. 

Figure 1

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Gene expression and spatial distribution of cell types in uterine cancer sample​

Figure 2. Gene expression and spatial distribution of cell types in uterine cancer sample (A) Dotplot showing the expression of top marker genes across cell clusters. Cell clusters were assigned to different cell types based on their gene expression patterns. ​(B) UMAP visualization of cell types in human uterine cancer samples.  ​(C to F) Spatial distribution of different immune cell types in human uterine cancer section (upper panels). Selected marker of each immune cell type are shown in zoom-in pictures (lower panels). 

Figure 2

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Co-detection of RNA and protein in FFPE uterine cancer tissue​

Figure 3. Co-detection of RNA and protein in FFPE uterine cancer tissue (A) UMAP visualization of PRTRC gene (encoding both CD45RA and CD45RO protein) expression across cells from uterine cancer tissue. ​(B) Spatial distribution of PTPRC in uterine cancer sections with CD45RA (left) or CD45RO (right) staining. ​(C) UMAP visualization of CD45RA and CD45RO expression in cells of uterine cancer tissue. Each dot represents a cell, which is color-coded based on the protein staining signal intensity. CD45RA and CD45RO protein are enriched in different cell types. ​(D) Whole-slice view of CD45RA and CD45RO expression in uterine cancer sections. ​(E) Spatial distribution of CD45RA and CD45RO in uterine cancer sections. Each cell is color-coded based on the protein staining signal intensity. ​(F and G) Zoom-in pictures showing the distinct expression patterns of CD45RA (F) and CD45RO (G) in uterine cancer tissue. Although PTPRC is expressed in both T cells and B cells, CD45RA is highly enriched in B cells (F, blue cells), while CD45RO is highly enriched in T cells (G, yellow cells). ​

Figure 3

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Accurate protein detection in human uterine cancer tissue ​

Figure 4. Accurate protein detection in human uterine cancer tissue To validate the results of protein co-detection, the immune cell types of MERSCOPE datasets were extracted and integrated with published CITE-seq data of PBMC. ​(A) UMAP visualization of immune cell types in CITE-seq data. ​(B) UMAP visualization of immune cell types identified in uterine cancer MERSCOPE datasets. The cell type labels are predicted from integration with CITE-seq data, and cells are embedded in the same UMAP space as (A). ​(C and D) CD45RA expression in CITE-seq (C) and MERSCOPE (D) dataset. In both datasets, high CD45RA expression level is observed in B cells. Note that based on integration, the CD45RA+ NK cells and naive T cells (arrows) are largely depleted in the MERSCOPE uterine cancer dataset. The same UMAP embeddings are used as (A) and (B). ​(E and F) CD45RO expression in CITE-seq (C) and MERSCOPE (D) dataset. In both datasets, high CD45RA expression level is observed in T cells. The same UMAP embeddings are used as (A) and (B). ​

Figure 4

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MERSCOPE® Platform Data Images 

Figure 5.​ Spatial distribution of different cell types in human uterine cancer tissue

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Figure 6.Spatial distribution of different transcripts in human uterine cancer tissue

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Figure 7. Expression of PRTRC mRNA and CD45RO protein in human uterus cancer tissue 

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