Molecular, Metabolic, and Subcellular Mapping of the Tumor Immune Microenvironment via 3D Targeted and Non-Targeted Multiplex Multi-Omics Analyses
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Patient Samples
2.2. Sample Preparation for 3D Multi-Omics Analyses
2.3. Sequential Immunofluorescence Staining (seqIF)
2.3.1. Sequential Immunofluorescence Data Analysis
2.3.2. 3D Reconstruction of seqIF Images
2.4. Stereo-Seq Analysis
2.4.1. Stereo-Seq Raw Data Processing
2.4.2. Stereo-Seq Data Processing
2.5. MSI Analysis
2.5.1. MSI Sample Preparation
2.5.2. Mass Spectrometry Imaging
2.6. Integration of Multi-Omics Platforms
3. Results
3.1. Sequential Immunofluorescence Analysis
3.2. Generation of Robust Spatially Resolved Transcriptomic Profiles Using the Stereo-Seq Chip
High Levels of Tumor Heterogeneity Identified by Stereo-Seq Analysis and Leiden Clustering Analysis
3.3. Mass Spectrometry Imaging Analysis
3.4. Multi-Modality Data Integration
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Metabolites | PNGaseF | Glycan Matrix | Trypsin | Peptide Matrix | |
---|---|---|---|---|---|
Matrix/Enzyme | DAN | PNGaseF | CHCA | Trypsin | CHCA |
Concentration (mg/mL) | 10 | 0.1 | 10 | 0.1 | 10 |
Solvent | 50% ACN | Water | 70% ACN, 0.1% TFA, 10 mM AmPhos | 9% ACN, 100 mM AmBic | 70% ACN, 0.1% TFA, 10 mM AmPhos |
Flow Rate (mL/min) | 0.1 | 0.025 | 0.12 | 0.01 | 0.12 |
Number of Passes | 4 | 15 | 3 | 12 | 4 |
Nozzle Temperature (°C) | 60 | 45 | 75 | 30 | 75 |
Track Speed (mm/min) | 1200 | 1200 | 1200 | 750 | 1200 |
Track Spacing (mm) | 3 | 3 | 3 | 3 | 3 |
Track Pattern | CC | CC | HH | HH | HH |
Nozzle Height (mm) | 40 | 40 | 40 | 40 | 40 |
Metabolites | Glycans | Peptides | |
---|---|---|---|
Polarity | Negative | Positive | Positive |
m/z range | 50–600 | 700–3500 | 600–4500 |
Number of laser shots | 200 | 300 | 300 |
Funnel 1 RF (Vpp) | 75 | 450 | 450 |
Funnel 2 RF (Vpp) | 100 | 500 | 500 |
Multipole RF (Vpp) | 150 | 500 | 600 |
Collision Energy (eV) | 10 | 10 | 10 |
Collision RF (Vpp) | 500 | 2700 | 3400 |
Quadrupole Ion Energy (eV) | 5 | 5 | 5 |
Transfer time (μs) | 35 | 140 | 180 |
Pre Pulse Storage (μs) | 2 | 14 | 18 |
Sample Type | Sample Name | MID under Tissue Area | Median Reads (per bin200) | Median MID (per bin200) | Median Gene types (per bin200) | Mitochondria Transcripts | Microbiome Transcripts |
---|---|---|---|---|---|---|---|
HGSOC | HGSOC_4 | 82.40% | 349,016 | 8442 | 4102 | <2% | 104,870 |
HGSOC_9 | 66.80% | 489,331 | 6193 | 3122 | <2% | 52,250 | |
HGSOC_14 | 77.70% | 504,198 | 9964 | 4396 | <2% | 42,360 | |
AEH | AEH_4 | 59.68% | 253,798 | 9401 | 4169 | <2% | 35,650 |
AEH_9 | 66.70% | 145,040 | 9055 | 4000 | <2% | 24,980 | |
AEH_14 | 74.18% | 159,337 | 10,224 | 4345 | <2% | 40,300 |
Mean Gene Type Number | Mean Gene Type Number | ||||||
---|---|---|---|---|---|---|---|
Bin | RNA Capture Area | HGSOC_4 | HGSOC_9 | HGSOC_14 | AEH_4 | AEH_9 | AEH_14 |
1 | 0.5 μm × 0.5 μm | 1.2 | 1.16 | 1.25 | 1.2 | 1.21 | 1.21 |
20 | 10 μm × 10 μm | 82.62 | 59.44 | 97.85 | 91.64 | 90.87 | 102 |
50 | 25 μm × 25 μm | 461 | 333 | 536 | 505 | 502 | 561 |
100 | 50 μm × 50 μm | 1501 | 1101 | 1706 | 1592 | 1586 | 1752 |
150 | 75 μm × 75 μm | 2741 | 2045 | 3050 | 2816 | 2810 | 3078 |
200 | 100 μm × 100 μm | 3985 | 3010 | 4362 | 3989 | 4000 | 4339 |
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Share and Cite
Ferri-Borgogno, S.; Burks, J.K.; Seeley, E.H.; McKee, T.D.; Stolley, D.L.; Basi, A.V.; Gomez, J.A.; Gamal, B.T.; Ayyadhury, S.; Lawson, B.C.; et al. Molecular, Metabolic, and Subcellular Mapping of the Tumor Immune Microenvironment via 3D Targeted and Non-Targeted Multiplex Multi-Omics Analyses. Cancers 2024, 16, 846. https://doi.org/10.3390/cancers16050846
Ferri-Borgogno S, Burks JK, Seeley EH, McKee TD, Stolley DL, Basi AV, Gomez JA, Gamal BT, Ayyadhury S, Lawson BC, et al. Molecular, Metabolic, and Subcellular Mapping of the Tumor Immune Microenvironment via 3D Targeted and Non-Targeted Multiplex Multi-Omics Analyses. Cancers. 2024; 16(5):846. https://doi.org/10.3390/cancers16050846
Chicago/Turabian StyleFerri-Borgogno, Sammy, Jared K. Burks, Erin H. Seeley, Trevor D. McKee, Danielle L. Stolley, Akshay V. Basi, Javier A. Gomez, Basant T. Gamal, Shamini Ayyadhury, Barrett C. Lawson, and et al. 2024. "Molecular, Metabolic, and Subcellular Mapping of the Tumor Immune Microenvironment via 3D Targeted and Non-Targeted Multiplex Multi-Omics Analyses" Cancers 16, no. 5: 846. https://doi.org/10.3390/cancers16050846