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SRX19507328: GSM7065543: DSP-1001660008744-D-A10; Homo sapiens; OTHER
2 ILLUMINA (Illumina NovaSeq 6000) runs: 17.5M spots, 981.5M bases, 300.3Mb downloads

External Id: GSM7065543_r1
Submitted by: Yale University School of Medicine
Study: Spatially defined gene signatures uncover the association of extracellular matrix genes with immunotherapy resistance in head and neck squamous cell carcinoma
show Abstracthide Abstract
Background: Immune-checkpoint inhibition (ICI) only benefits a subgroup of patients with head and neck squamous cell carcinoma (HNSCC). Several molecular and cellular components of the tumor microenvironment (TME) have been hypothesized to drive either response or resistance. Here, spatially defined whole transcriptome data were analysed in search of associations of compartment-specific gene-signatures with HNSCC immunotherapy outcomes. Methods: Pre-treatment biopsy samples from 50 immunotherapy-treated recurrent or metastatic HNSCC patients as well as 12 matched post-treatment biopsies obtained after 4 weeks of treatment, constructed in tissue microarray format (YTMA496), were included in the study. The GeoMx Human Whole Transcriptome Atlas (NanoString Technologies) assay was performed on samples to allow RNA quantification of 18,677 protein encoding genes, using in situ hybridization, in three molecularly defined tissue compartments; tumor (CK), leukocyte (CD45), macrophage (CD68). Differentially expressed genes (DEGs) (P<0.05) between pre- and post-treatment biopsies in each of the tissue compartments were identified. Next, these DEGs were used as a “biological” filter for the initial 18,677 gene set and analysed using LASSO logistic regression models with the aim to obtain pre-treatment gene expression signatures for best overall response (RECIST 1.1.). The performance of each compartment signature was evaluated using the receiver operating characteristic (ROC) curve and AUCs were calculated for Best Overall Response (BOR) to ICI. Results: A six-gene signature (DDX4, COL17A1, HBA1, MMP1, GPNMB, TTN) in the CD45 compartment presented the highest AUC (0.83), followed by signatures in the CK and CD68 compartments (AUC: 0.72 and 0.68, respectively). Cross-testing of the CD45 signature in the other two compartments, as well as in a third, artificially generated “pseudo-bulk” compartment (all compartments combined), showed poor performance, indicating spatial specificity. Interestingly, the CD45 signature included three extracellular-matrix protein-encoding genes (MMP1, COL17A1, TTN), all associated with resistance (negative coefficients in the BOR model) to ICI. Fibroblast and dendritic cell populations, characterized using the CIBERSORTx gene matrix, were the immune phenotypes most closely associated with the CD45 signature. Conclusions: Our results indicate that CD45 molecular tissue compartment gene expression demonstrates increased association with ICI resistance in HNSCC. Extracellular matrix genes rather than immune-cell related genes dominated the CD45 compartment signature, highlighting the importance of non-immune stromal components within the TME and the importance of the use of spatial information in the understanding of ICI resistance. Overall design: This dataset comprises gene expression data from 50 pre-treatment tissue samples of anti-PD-1 treated head and neck squamous cell carcinoma patients in three molecularly defined compartments; tumor (defined by CK), leukocyte (defined by CD45) and macrophage (defined by CD68). Samples were constructed in tissue microarray format and experiments were performed on slides from two separate paraffin blocks (2-fold redundancy), each containing a non-adjacent tissue core from each patient-biopsy. Nanostring GeoMx Digital Spatial Profiling (DSP) Whole Transcriptome Atlas (WTA) Panel (>18,677 target genes) was used per manufacturer's protocol. 240 ROIs corresponding to 240 AOIs were collected and analysed.
Sample: DSP-1001660008744-D-A10
SAMN33449608 • SRS16895278 • All experiments • All runs
Organism: Homo sapiens
Library:
Name: GSM7065543
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: day1:TMA slides were processed using the Nanostring GeoMx DSP manual slide preparation GeoMx®-DSP protocol (MAN-10150-01).Antigen retrieval of the FFPE tissue was performed for 20 minutes followed by deparaffinization and rehydration.Then, the slides were incubated with proteinase K for 20 minutes and RNA probes were placed on slides for overnight in situ hybridization. day2:slides were washed to remove off-target probes for 90 minutes. Morphology markers: CD68 for macrophage compartment (Cy5/666 nm, excitation 645/19 nm, emission 683/30 nm), CD45 for lymphocyte compartment (Texas Red/615 nm, excitation 588/19 nm, emission 623/30 nm), S100B (Cy3/568 nm, excitation 538/19 nm, emission 564/15nm) for tumor compartment to identify the compartments and SYTO 13 for nuclear stain, were added for 1-hour incubation. Slides were loaded onto the GeoMx DSP instrument and after scanning, ROI (region of interest: i.e., CD45, CD68 and S100B) selection and probe collection were performed according to the GeoMx®-DSP user manual protocol (MAN-10088-03). Samples were collected in a 96-well probe aspirate collection plate and each ROI representing a compartment from a patient tumor core was collected in a well. GeoMx®-DSP user manual (MAN-10117-01); Each GeoMx DSP aspirate in the plate contains photocleaved DNA oligos comprised of an RNA analyte identifier, a unique molecular identifier (UMI) barcode, and primer binding sites. Prior to purification, PCR reactions were pooled into three mixtures, separating CD68, CD45 and S100B ROIs to generate three pooled libraries. The libraries were pooled in a biased manner designed to target the smallest ROI pool with 6 parts of reads for CD68, 3 parts of reads for CD45 and 1 part of reads for S100B. Illumina i5 x i7 system adapter sequences and unique dual sample indices are added when PCR is performed on the probe aspirates. Each photocleaved oligo in the GeoMx DSP collection plate contains a readout tag sequence identifier (RTS ID) that identifies the target. It also includes a unique molecular identifier (UMI), to remove PCR duplicates when converting reads to digital counts. Read 1 (SPR1) and Read 2 (SPR2) are binding sites for Illumina sequencing primers. The GeoMx Seq code primers that hybridize to SPR1 and SPR2 contain i5 or i7 indexing sequences as well as P5 or P7 sequences for binding to Illumina flow cells. Sequencing was performed in both lanes of an Illumina Nova-Seq 6000 S2 flow cell resulting of 3 billion reads
Runs: 2 runs, 17.5M spots, 981.5M bases, 300.3Mb
Run# of Spots# of BasesSizePublished
SRR236230448,735,770489.2M149.8Mb2023-04-07
SRR236230458,791,500492.3M150.6Mb2023-04-07

ID:
26791655

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