Tumor biopsy and blood DNA were isolated from fresh samples using TGuide M24 (Tiangen, Beijing, China). The purity and quantity of the total DNA were estimated by measuring absorbance at 260 nm (A260) and 280 nm (A280) with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA); the extracted DNA was considered pure and suitable for future experiments if the A260/A280 ratio was within 1.6–1.9. FUSCC NGS 511-gene panel sequencing [16 (link)] was conducted to detect somatic and germline mutations in both biopsy (n = 23) and ctDNA (n = 23) samples.
FFPE blocks of samples from 30 patients were retrieved, and 4–5-μm-thick samples on slides were used for IHC staining. IHC for TIM-3 (CST, Cat#45208T, 1/400 dilution), c-Myc (Abcam, Cat#ab32072, 1/200 dilution), STING (CST, Cat#13647S, 1/100 dilution), PD-L1 (Abcam, Cat#ab228462, 1/500 dilution) and CD31 (DAKO, Cat#M0823, 1/100 dilution) was performed on FFPE sections from 30 patients, with the staining status being independently assessed by two experienced pathologists. If multiple tissue sections from one patient were available, the highest score was used for classification.
FFPE blocks of samples from 30 patients were retrieved, and 4–5-μm-thick samples on slides were used for IHC staining. IHC for TIM-3 (CST, Cat#45208T, 1/400 dilution), c-Myc (Abcam, Cat#ab32072, 1/200 dilution), STING (CST, Cat#13647S, 1/100 dilution), PD-L1 (Abcam, Cat#ab228462, 1/500 dilution) and CD31 (DAKO, Cat#M0823, 1/100 dilution) was performed on FFPE sections from 30 patients, with the staining status being independently assessed by two experienced pathologists. If multiple tissue sections from one patient were available, the highest score was used for classification.
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