For slide preparation, we followed the GeoMx DSP slide preparation user manual (MAN-10087-04). In brief, FFPE tissue section of 5-µm were mounted on positively charged histology slides by grouping 3 patients per slide. Sections were incubated at 65 °C for 1 hour. Slides were deparaffinized in 3 xylene baths of 5 min, then rehydrated in ethanol gradient: 2 baths of 5 min in 100% EtOH, followed by 5 min in 95% EtOH. Slides were then washed with PBS 1X. Antigen retrieval was performed in Tris-EDTA pH 9.0 at 100°C for 15 min at low pressure. Slides were first dived into hot water for 10 seconds, and then into Tris-EDTA buffer. Cooker vent stayed open during the procedure to ensure low pressure. Slides were then washed in PBS 1X, incubated in proteinase K in PBS (1ug/ml) for 15 min at 37°C and washed again with PBS 1X. Tissues were post-fixed in 10% neutral-buffered formalin (NBF) for 5 min, washed twice for 5 min in NBF stop buffer (0.1M Tris Base, 0.1M Glycine) and finally once in PBS 1X. The RNA probe mix (CTA (https://www.nanostring.com/products/geomx-digital-spatial-profiler/geomx-rna-assays/geomx-cancer-transcriptome-atlas/), a pool of in situ hybridization probes with UV photocleavable oligonucleotide barcodes) was placed on each section and covered with HybriSlip Hybridization Covers. Slides were then incubated for hybridization overnight at 37°C in a Hyb EZ II hybridization oven (Advanced Cell Diagnostics). The day after, HybriSlip covers were gently removed and 25-min stringent washes were performed twice in 50% formamide and 2X Saline Sodium Citrate (SSC) at 37 °C. Tissues were washed for 5 min in 2X SSC, then blocked in Buffer W (Nanostring Technologies) for 30 min at room temperature in a humidity chamber. Next, tissues were stained with panCK-532 (clone AE1+AE3) (Novus, NBP2-33200) at 1:20 and SYTO 13 at 1:10 (Thermo Scientific S7575) in Buffer W for 1 h at room temperature. Slides were washed twice in fresh 2X SSC then loaded on the GeoMx Digital Spatial Profiler (DSP).
Growth protocol
FFPE blocks
Extracted molecule
total RNA
Extraction protocol
For GeoMx DSP sample collections, entire slides were imaged at 20X magnification and morphologic markers were used to select ROI using organic shapes. Automatic segmentation of ROI based on panCKpos markers were used to define AOIs, allowing to separate tumor cells (panCKpos) from adjacent stromal cells (panCKneg). A total of 95 AOIs were exposed to 385 nm light (UV), releasing the indexing oligonucleotides which were collected with a microcapillary and deposited into a 96-well plate for subsequent processing. The indexing oligonucleotides were dried overnight and resuspended in 10 μl of DEPC-treated water.
Label
NanoString
Label protocol
The spatial transcriptomic panel included 1812 genes targeted cancer transcriptome atlas panel to encompass a comprehensive detection of tumor-host interactions.
Hybridization protocol
Immunoflourecent tissue profiling was performed using PanCK (488) cytokeratin, and Syto13 for DNA staining to enable selection of 48 regions of interest (ROIs), divided into 48 tumor (panCK-positive) and 47 stromal (panCK-negative) areas of interest (AOIs) for targeted spatial transcriptomics using the commercial Nanostring GeoMx.
Scan protocol
Nanostring GeoMx Digital Spatial Profiling (DSP) Platform
Data processing
Sequencing libraries were generated by PCR from the photo-released indexing oligos and AOI-specific Illumina adapter sequences. Unique i5 and i7 sample indexes were added. Each PCR reaction used 4 μl of indexing oligonucleotides, 4 μl of indexing PCR primers, 2 μl of Nanostring 5X PCR Master Mix. Thermocycling conditions were 37 °C for 30 min, 50 °C for 10 min, 95 °C for 3 min; 18 cycles of 95 °C for 15 s, 65 °C for 1 min, 68 °C for 30 s; and 68 °C for 5 min. PCR reactions were pooled and purified twice using AMPure XP beads (Beckman Coulter, A63881), according to the manufacturer’s protocol. Pooled libraries were paired-sequenced at 2 × 27 base pairs and with unique dual indexes workflow on an Illumina HiSeq 4000 instrument. HiSeq-derived FASTQ files for each sample were compiled for each compartment using the bcl2fastq program of Illumina, and then demultiplexed and converted to digital count conversion (DCC) files using the GeoMx DnD pipeline (v.1) of Nanostring according to manufacturer’s pipeline. DCC files were imported back into the GeoMx DSP instrument for QC and data analyses using GeoMx DSP analysis suite version 2.2.0.111 (Nanostring). A minimum of 10,000 reads were required for each sample. Probes were checked for outlier status by implementing a global Grubb’s outlier test with alpha set to 0.01. The counts for all remaining probes for a given target were then collapsed into a single metric by taking the geometric mean of probe counts. For each sample, an RNA-probe-pool-specific negative probe normalization factor was generated on the basis of the geometric mean of negative probes in each pool. To ensure good data quality, we calculated the 75th percentile of the gene counts (that is, geometric mean across all non-outlier probes for a given gene) for each AOI, and normalized to the geometric mean of the 75th percentile across all AOIs to give the upper quartile (Q3) normalization factors for each AOI. The distribution of these Q3 normalization factors were then checked for outliers.
