2.2 Differential expression and prognosis of MUC18 in
GEPIA2
The internet database GEPIA2 (Gene Expression Profiling Interactive
Analysis 2) was used to assess MUC18 gene expression in several types of
tumors and normal tissues by matching TCGA normal and GTEx data.
Survival maps, survival curves for overall survival (OS) and
disease-free survival (DFS) were generated based on the gene expression
patterns of 33 different cancers using GEPIA2 through log-rank and
Mantel–Cox tests, and the group cutoff was based on the median
(cutoff-high: 50%, cutoff-low: 50%). The hazard ratio (HR) was
calculated based on the Cox PH model, and the confidence interval (CI)
was 95%. Special survival graphs with log-rank p values were generated
using the “Survival Analysis” module in GEPIA2. The results were
considered to be statistically significant if p < 0.05.
The data were downloaded from
http://gepia2.cancer-pku.cn/\#analysis.
2.3 DFO conjugation and89Zr-labeling of IP150
The MUC18-specific mAb IP150 was conjugated to DFO-NCS (Innochem) via
lysine residues. DFO was prepared at 10 mg/mL (20 μL) in neat DMSO and
mixed with 0.1 M Na2CO3 (pH 9.0, 200
μL). Then, 500 μg of mAb was added and incubated at 37 °C for 60 min.
The nonconjugated chelate was removed by size exclusion chromatography
using metal-free PBS as the eluent and a PD-10 column (Cytiva). The
products were collected into five centrifuge tubes (500 µL). Then, the
concentration was determined by a NanoDrop, and the average molecular
weight of the mAb (20 µL) before and after the reaction was analyzed by
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS).
The 89Zr-IP150 probe was labeled by reacting89Zr-oxalate and DFO-IP150 at 37 ℃ for 60 min (Figure
1A), and 89Zr-oxalate (48.1 MBq; 125 µL in 1 M oxalic
acid) was mixed with HEPES buffer (1375 µL, pH 7.0). Then, 200 µL of
DFO-IP150 (200 µg) was added to the mixed liquor. After incubating at
room temperature for 60 minutes, the reaction mixture was purified on a
PD-10 column using metal-free PBS as the eluent. Similar to DFO
conjugation, 89Zr-IP150 was collected in five
centrifuge tubes (500 µL). Before and after purification, 3 μL of sample
was spotted on a TLC silica gel strip and developed in 0.05 M citric
acid (pH 5.0)/saturated EDTA as the eluent, after which the
radiolabeling rate and radiochemical purity were analyzed by BIOSCAN.
Two hundred microliters of 89Zr-IP150 in 5% HSA
(Baxter) was incubated at 4 ℃ to assess the in vitro stability by
analyzing the radiochemical purity every 24 h.
2.4 Binding properties of IP150 and DFO-IP150 to the