{
  "abstract": "Background Comprehensive spatial characterization of the tumor microenvironment requires integrated measurement of transcripts, proteins, and morphology at single-cell resolution in clinically relevant specimens. We present a high-throughput in situ profiling platform, the G4X Spatial Sequencer, that enables simultaneous detection of RNA transcripts, proteins, and study of morphological features from the same formalin-fixed, paraffin-embedded (FFPE) tissue section. The system supports up to 40 cm 2 tissue area per run, enabling clinical-scale studies across diverse tissue types.Methods We developed ~300-gene targeted transcript panels for kidney, lung, colon, and breast tissues, incorporating 150 shared immuno-oncology genes, 50 stromal genes, and ~100 tissue-specific transcripts relevant to cell typing, oncogenic signaling, and tumor microenvironment. An immunooncology-focused antibody panel was included for targeted protein detection. All analytes were analyzed from single 5 μm thick FFPE sections, enabling direct spatial co-registration of transcript, protein, and morphological features.Sections of different tissue types were transferred to custom slides in large format (10 x 10 mm2; 10 sections/slide) or small format (4.5 x 4.5 mm2; 32 sections/slide) arrays via a proprietary tissue transfer method. Each tissue was processed through antibody staining, hybridization of probes, amplification, and sequencing-by-synthesis (SBS). Tissue morphology was visualized using fluorescent stains that recapitulate hematoxylin and eosin contrast, generating an fH&ETM image co-registered with RNA and protein data.Results We detected 50-200 transcripts and 20-50 unique genes per cell, with dynamic ranges exceeding 500 transcripts in high-expressing cells. False discovery rates were maintained at or below 0.5%, with most samples below 0.1%, ensuring high-confidence signal detection. Cross-modality validation demonstrated a strong correlation between transcript and protein abundance at the single-cell level. Technical replicates across serial sections exhibited high reproducibility in both RNA and protein signal, and integrated analysis via multi-modal graph-based clustering leveraging RNA, protein, and spatial proximity revealed distinct spatial neighborhoods and tumor-associated cellular phenotypes.Serial 5 μm sections from a renal cell carcinoma sample were profiled and computationally registered using fH&E images to reconstruct the tissue in 3D. Integrated analysis revealed spatially organized cellular niches, including healthy structures, tumor regions, and tertiary lymphoid structure-rich tumor-normal interfaces, consistent across transcriptomic, proteomic, and morphological modalities. The ability to process all sections within a single flow cell highlights the scalability of the G4X platform for high-throughput 3D spatial profiling of tissue architecture.Conclusions G4X provides a unified workflow for multiplexed spatial transcriptomics, proteomics, and histopathology on the same FFPE section, enabling integrated molecular and morphological analysis of clinical specimens at scale.",
  "authors": [
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Kenneth H Gouin"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Sabrina Shore"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Yuji Ishitsuka"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Michael Lawson"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Daan Witters"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Eli Glezer"
    }
  ],
  "title": "159 High-throughput multimodal spatial profiling of RNA, protein, and morphology on the same FFPE section using the G4XTM Spatial Sequencer",
  "uid": "c9fd1c36-6851-5d6e-9f14-5ed991c3fd1a"
}