tissue: head and neck carcinoma gender: Male anatomical location: Left parotid age: 81 tumor level: B18 treatment: Vehicle mil113_grade: negative mil113_percent: 0
Extracted molecule
total RNA
Extraction protocol
Tissue sections were baked, deparaffinized, washed in PBS, and targets were retrieved for 20 minutes in 1x Tris-EDTA pH 9.0 buffer (Invitrogen #00-4956-58) at 100 °C. Next, tissues were washed in PBS, incubated in 0.1 μg/ml proteinase K (Thermo Fisher Scientific, #AM2546) in PBS for 15 minutes at 37 °C and washed again in PBS. Protein targets were post-fixed in 10% neutral-buffered formalin for 5 minutes, followed by two 5-minute incubations in 12.25 mg/ml Tris base, 7.5 mg/ml Glycine stop buffer diluted in DEPC-treated water. Tissues were incubated overnight at 37 °C with hybridization solution (Human Cancer Transcriptome Atlas probe mix (NanoString #121400101), Buffer R (NanoString #121300313), and DEPC-treated water). During incubation, slides were covered with HybriSlip hybridization covers (Grace BioLabs #714022). After incubation, HybriSlip covers were gently removed by soaking in 2x SSC buffer. Two 25 minute stringent washes were performed using 50% formamide in 2x SSC at 37 °C. Tissues were washed for 5 minutes in 2× SSC and incubated in blocking buffer W (NanoString #121300313) for 30 minutes at room temperature in a humidity chamber. Primary antibody targeted against MIL113 was diluted in buffer W and incubated with the tissues for 1 hour followed by a 1 hour incubation with fluorescently-labeled secondary antibody diluted in buffer W. After the slides were washed twice for 5 minutes in 5x SSC buffer they were incubated for 1 hour in SYTO13 (50 nM) and fluorescently conjugated antibody targeting PanCK in blocking 8 buffer. Following staining the sections were washed for 5 minutes in fresh 2x SSC. Following whole slide imaging, multiple geometric ROIs of 400 μm2 were placed in regions of subasumstat drug exposure according to MIL113 signal intensity and regions of vehicle or background. Indexing oligonucleotides were then cleaved and quantified via next gen sequencing on a NovaSeq 6000 Illumina sequencer as previously described(25)
Label
n/a
Label protocol
Nanostring
Hybridization protocol
n/a
Scan protocol
Nanostring GeoMx Digital Spatial Profiling (DSP) Platform
Description
TKA7_B18_006_20221217_Full
Data processing
Raw probe level counts from the NanoString DSP machine were subjected to QC and normalization as follows. ROIs with deduplicated read counts of <10,000 were excluded. Count data was then normalized using TMM normalization(26) prior to performing limma analysis
Trackable Intratumor Microdosing and Spatial Profiling Provide Early Insights into Activity of Investigational Agents in the Intact Tumor Microenvironment
