Unlocking the Archives: How Spatial Multiomics is Revolutionizing FFPE Tissue Analysis

Breakthroughs in high-throughput spatial sequencing are transforming decades-old tissue samples into dynamic molecular maps

The Time Capsules of Disease

Nestled in hospital archives worldwide lie over a billion formalin-fixed paraffin-embedded (FFPE) tissue samples—each a meticulously preserved snapshot of human health and disease 4 .

Traditional Limitations

For decades, these clinical "time capsules" offered only morphological insights through traditional microscopy. Their molecular secrets remained locked away due to formaldehyde-induced RNA crosslinking and fragmentation 1 3 .

The Breakthrough

Now, breakthroughs in high-throughput spatial sequencing are cracking open these archives, enabling simultaneous mapping of proteins and RNA directly in FFPE tissues 6 .

Spatial Context Matters

This revolution is transforming dusty blocks of paraffin into dynamic maps of cellular battles against cancer, inflammation, and degeneration—all while preserving the critical spatial context where these molecular dramas unfold 6 .

Decoding the Archives: Key Technological Advances

Formalin fixation creates methylene bridges that crosslink nucleic acids and proteins—a process preserving tissue architecture but shattering RNA into fragments. Traditional single-cell RNA sequencing methods relying on poly(A) tails fail with these degraded fragments.

"The irreversible modifications caused by formalin fixation on macromolecules always make it challenging for molecular biology applications" 1 .
Recent Innovations:
  • Random primer-based capture: Replacing oligo(dT) with random primers (e.g., snRandom-seq) to bind RNA fragments regardless of polyadenylation status 1
  • Padlock probes: Circular DNA probes that selectively amplify degraded transcripts in situ 7
  • RCA-enhanced detection: Rolling circle amplification generates detectable "rolonies" from single molecules 7
Table 1: Performance Comparison of Spatial Technologies on FFPE Tissues
Technology Targets Resolution Genes Detected Key Innovation
snRandom-seq 1 RNA Single-nucleus >3,000/nucleus Random-primer capture
DART-FISH 7 RNA Subcellular 121-300 genes Enzyme-free decoding
G4X 5 RNA + Protein Subcellular 128-plex protein Integrated multiomics
CosMx SMI 4 RNA + Protein Single-cell 1,000 RNA + 64 protein Cyclic hybridization

The true power emerges when spatial transcriptomics integrates with proteomics.

"Proteins are the basic functional unit of the cell and predicting expression based solely on mRNA levels has proven unreliable" 4 .
Pioneering Platforms:
G4X In Situ Multiomics

Maps RNA, proteins, and morphology simultaneously using combinatorial indexing.

128 FFPE samples per run at <50% cost of alternatives 5
DART-FISH

Features "RiboSoma" cytoplasmic staining to segment cells in autofluorescence-prone human tissues.

Critical for diseased kidneys and brains 7
snRandom-seq

Detects non-coding RNAs and nascent transcripts missed by poly(A)-based methods.

Reveals proliferative cancer subclones in liver tumors 1

Spotlight Experiment: snRandom-seq's FFPE Breakthrough

Methodology: Total RNA Capture in a Microdroplet

Nature Communications details this landmark approach 1 :

  1. Nucleus Extraction:
    • Deparaffinize/rehydrate FFPE sections → collagenase/pepsin treatment to reverse crosslinks
  2. Random-Primed RT:
    • Permeabilize nuclei → reverse transcription with barcoded random primers
    • Critical innovation: Pre-indexing reduces doublets to 0.3%
  3. Poly(dA) Tailing:
    • Add poly(dA) tails to cDNA 3' ends using terminal transferase (TdT)
  4. Droplet Barcoding:
    • Encapsulate nuclei + barcode beads in microfluidic droplets
    • Poly(dT) beads capture poly(dA)-tailed cDNAs
  5. Library Prep:
    • Amplify barcoded cDNA → sequence (fragment size: 300-800 bp)
Table 2: snRandom-seq Performance Metrics
Metric Result Significance
Doublet Rate 0.3% Enables complex tissue analysis
Median Genes/Nucleus >3,000 2× higher than oligo(dT) methods
Intronic Mapping 75% of reads Captures nascent transcription
Non-coding RNA snoRNA, miRNA, lncRNA Reveals regulatory networks
Transformative Results

Applied to a human liver cancer FFPE block, snRandom-seq revealed:

Proliferative subclones

A rare hepatocyte population with elevated MKI67, TOP2A, and PCNA expression

Stromal-immune niches

Spatial mapping of fibroblast-infiltrating lymphocyte crosstalk

Disease trajectories

Pseudotime analysis traced progression from normal → dysplastic → malignant states

"Our method unlocks archival FFPE specimens for single-nucleus analysis and promises enormous applications in biomedical research" 1 .

The Scientist's Toolkit: Essential Reagents

Table 3: Key Reagents for FFPE Spatial Multiomics
Reagent Function Example/Innovation
Padlock Probes Target-specific circularization DART-FISH's array-synthesized probes (cost: <25% commercial) 7
RiboSoma Stain Cytoplasmic segmentation Oligos binding cDNA 5' handles in DART-FISH 7
Barcoded Beads Single-cell tagging snRandom-seq's hydrogel beads (40 μm) with split-pool barcodes 1
Poly(dA) Tailing Enzyme cDNA tagging Terminal deoxynucleotidyl transferase (TdT) in snRandom-seq 1
Decoding Oligos Rolony identification DART-FISH's isothermal probes (no enzymes needed) 7

From Bench to Bedside: Clinical Impact

Cancer Heterogeneity Decoded

In melanoma/dysplastic nevi FFPE sections (The Journal of Pathology):

  • Poly(A)- vs probe-capture protocols were optimized for degraded RNA (DV200 >30%)
  • Integration with CODEX protein imaging revealed immune exclusion neighborhoods where PD-L1+ macrophages surrounded T cell-deficient tumor zones 3
Kidney Disease Niches

DART-FISH applied to a diseased human kidney:

  • Mapped 300 genes across 20 cell states
  • Identified fibrotic niches with altered epithelial cells and activated myofibroblasts 7
"This workflow requires no specialized equipment and is adaptable to clinical settings"
3D Tumor Atlases

G4X reconstructed kidney cancer using 10 serial FFPE sections:

  • 6.2 million cells mapped
  • 438 million transcripts analyzed
  • Revealed angiogenic gradients driving tumor invasion 5

The Future: Spatiotemporal Omics Horizons

Three Emerging Frontiers
AI-Driven Pathology

Integrating H&E stains with spatial omics to predict metastatic risk

Live-Cell Electroseq

Pairing electrophysiology with spatial transcriptomics in real-time 6

Multi-Omic Integration

Combining snRandom-seq (RNA), CODEX (protein), and spatial ATAC (chromatin) 4 6

As Nature Methods declared, spatial omics is the "Method of the Year" for good reason. With FFPE archives now accessible, we stand at the threshold of a new era—where every biopsy, preserved across decades, becomes a multidimensional map guiding precision medicine. The molecular ghosts in the paraffin machines are finally telling their stories.

"The development of direct digital counting of nucleic acids has unlocked valuable insights from archival FFPE tissue from health and diseased patients" 4 .

References