Ab214488

Tumor cells were seeded at 3×10⁴ cells per well in glass slides and cultured for 12 hours. Then activated T cells were added to the culture in the absence or presence of F7AK3 (1 µg/mL) for 30 min. Afterwards, cells were washed twice with PBS and fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Cells were incubated with anti-TROP2 (Abcam, ab214488) and anti-CD3 (Servicebio, GB13440) overnight at 4°C. Secondary fluorescent antibodies conjugated with FITC and Cy3 (Servicebio, GB22303 and GB21301) were added for 1 hour and 4',6-diamidino-2-phenylindole (DAPI) (Servicebio, G1012) was used for nuclear staining. The percentages of CD3⁺ T cells in tumor tissues were quantified manually as the percentage of green labeled cells (CD3⁺ T cells) to all cells (DAPI). Images were obtained with a confocal microscope (Olympus) under a 60×oil objective.

Cells were lysed with RIPA lysis buffer containing phenylmethylsulfonyl fluoride (1 mM). Cell lysates were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore). The blots were probed with the following primary antibodies overnight at 4°C: anti-TROP2 (Abcam, ab214488) and anti-GAPDH (Servicebio, GB11002). Afterwards, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody for 1 hour, followed by detection using a chemiluminescence assay kit.

Paired tumor and adjacent non-tumor tissue samples were obtained with informed consent from Tongji Hospital of HUST (Wuhan, China). The demographic and clinical characteristics of the enrolled patients are presented in online supplemental table S1. The specimens were isolated at the time of surgery, formalin fixed and paraffin embedded, and stained with H&E. TROP2 expression was scored as follows: multiplication of the intensity of immunostaining ((1) mild; (2) moderate; (3) strongly positive) and the percentage of positive tumor cells, which resulted in a score of 0–300. A score <10 was considered as 0, a score of 10–40 was considered as 1, 41–140 as 2 and 141–300 as 3. Standard immunohistochemistry was performed with antibodies against TROP2 (Abcam, ab214488), CD3 (Servicebio, GB13440) then examined by two blinded pathologists.