The slide preparation was performed as per GeoMx DSP Slide Preparation Protocol using the Semi-Automated BOND Rx protocol. In brief, 5 µm tissue sections were positioned in the GeoMx imaging area on a SuperFrost Plus slide. Sections were incubated at 60 ˚C for over 30 minutes prior to BOND Rx protocol. BOND Rx protocol consisted of Bake & Dewax, HIER for 20 minutes with ER1.
Growth protocol
FFPE blocks, tepotinib-treated surgical resection was obtained following 42 days of tepotinib treatment
Extracted molecule
protein
Extraction protocol
n.a.
Label
n.a.
Label protocol
Following BOND Rx protocol, slides were incubated overnight at 37 ˚C with the selected protein panels: Immune Cell Profiling, Immune Cell Typing, Immune Activation Status, IO Drug Targets, PI3K/AKT Signaling; as well as the morphology markers: TTF1, CD3 and CD33. The following day, post-fixation of section was performed, after washing the slides 3 times in TBS-T for 10 minutes, using 4% paraformaldehyde in a humidity chamber for 30 minutes. Slides were then washed twice for 2 minutes in TBS-T. Nuclear staining was then performed in a humidity chamber using SYTO13 DNA stain for 15 minutes. Slides were finally washed twice in TBS-T before being loaded into the GeoMx DSP instrument.
Hybridization protocol
n.a.
Scan protocol
Scanned using the GeoMx DSP (NanoString Technologies)
Description
20231006_P1001660003346A_P1001660003346A_10.RCC DNA barcode cleaved from RNA probe
Data processing
For an nCounter readout, the collected samples were first hybridized overnight to the GeoMx Hyb Code master mixes, consisting of probes U and R in hybridization buffer, as outlined in the GeoMx nCounter protein readout user guide. Hybridization products were then pooled for readout on the MAX/FLEX nCounter system. RCC files were loaded back onto the GeoMx instrument for downstream analysis in the GeoMx Analysis Suite.
Protein expression from paired biopsies from a patient with METex14 skiping non-small cell lung cancer before and after treatment with neoadjuvant tepotinib (42 days)
Data table header descriptions
ID_REF
VALUE
Results were obtained after processing of the nCounter RC files using the GeoMx Analysis Suite. First, quality check (QC) was performed with a binding QC density of 0.1-2.25, positive control normalization factor QC of 0.3-3.0, 75% of fields of view (FOV) detected, minimum surface area QC threshold of 1600 µm^2, and minimum nuclei count threshold of 20. This was followed by normalization using the metric mean of the Ms IgG1 and Rb IgG as negative controls, selected for normalization after comparing normalization methods using the Evaluate normalization custom script, available through NanoString.
