The length and width of tumor tissues and body weights of mice were measured twice a week, and the tumor volume was calculated as (Length Width 2 )/2. efficacy on three AGC patients with different statuses of HER2. Materials and Methods Patients and Tumor Samples This study included three patients with AGC who received systematic treatment from 2017 to 2021 at Peking University Cancer Hospital, Beijing, China. Histopathology confirmation and HER2 detection were determined by two pathologists. This study was approved by the institutional review board at Peking University Cancer Hospital. The clinical response of treatment was evaluated by computed tomography (CT) and was categorized as a complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD), according to the RECIST 1.1 criteria. This study was approved by the Medical Ethics Committee of Peking University Cancer Hospital. All animal studies complied with the ARRIVE guidelines and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Experiments involving human were in accordance with the ethical standards of committees (institutional and national) and with The Code of Ethics of the World Medical Association (Declaration of Helsinki). All patients completed written informed consent prior to study entry. Reagents and Antibodies RC48-ADC was provided by RemeGenCo, Ltd., and dissolved in normal saline. Trastuzumab was purchased from Shanghai Roche Pharmaceutical Ltd. Antibodies specific for HER2, pHER2, AKT, pAKT, S6, pS6, ERK, pERK, pCDK1, CDK2, cyclin E1, p53, Bcl-2, and Bax were purchased from Cell BAIAP2 Signaling Technology (Boston, MA, USA). The antibody specific for weekly vein injection for 3 weeks. The length and width of tumor tissues and KT 5823 body weights of mice were measured twice a week, and the tumor volume was calculated as (Length Width 2 )/2. Mice were sacrificed after the administration cycle or when the tumor volume reached 2,000 mm3. Tumor growth inhibition (TGI) was determined as [1CT/C] 100% (T and C presented changes in tumor volume of the treatment group and vehicle group over the course of the KT 5823 treatment). Western Blotting Analysis Total protein was extracted from tumor tissues and the concentration was measured BCA Protein Assay Kit (Beyotime, Shanghai, China). Here, 50 g protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)and KT 5823 transferred onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ). After incubation with corresponding primary antibodies diluted in 5% bovine serum albumin (BSA)overnight at 4C and incubation with secondary antibodies for 1 h at room temperature, protein samples were visualized using ECL-plus Western Blotting Detection Reagents (GE Healthcare Life Sciences, Chalfont, UK). Protein bands were quantified and normalized with ImageJ software. Immunohistochemistry Staining Tumor tissues were isolated from euthanized mice, and then formalin-fixed paraffin-embedded (FFPE) tissue blocks were prepared. IHC staining for HER2 was performed according to the manufacturers instructions and interpreted by two independent pathologists. IHC scores for HER2 were interpreted as follows: 0, no staining; 1+, weak or focal staining; 2+, moderate staining; and 3+, strong staining. Statistical Analysis The differences between/among groups were analyzed using unpaired two-tailed t-tests, one-way ANOVAs, or factorial analysis by GraphPad Prism version 7.0 (GraphPad Software Inc., CA, USA). Results RC48-ADC Exerted Selective Antitumor Activity in Gastric Cancer Cells and Patient-Derived Xenograft Models Cell viability tests wereconducted to evaluate the antitumor activity of RC48-ADC on 4 GC cell lines, followed by protein expression analysis to clarify the profiling of those cells. Compared with trastuzumab, RC48-ADC had superior antiproliferative effects in a dose-dependent manner on 4 cell lines (Figure 1A). NCI-N87 and SNU-216 cells were more sensitive to RC48-ADC treatment, resulting from the superior expression of HER2 (Figures 1B, C). Open in a separate window FIGURE 1 RC48-ADC had superior antiproliferative effects than trastuzumab on 4 gastric cancer (GC) cell lines. (A) Cell viability of NCI-N87, SNU-216, NUGC-4, and HGC-27 was detected by CCK-8 assays after RC48-ADC (0C10,000 nM) treatment for 72 h. Data are presented as means SD of three independent experiments. (B) The expression of human epidermal growth factor receptor 2 (HER2) in 4 GC cells quantified by Western blotting. (C) The IC50 of RC48-ADC on GC cell lines evaluated by CCK-8 assay. Nine GC PDX models were exploited to evaluate the TGI of RC48-ADC and trastuzumab = 5 mice per group). TGI, tumor growth inhibition; i.v., intravenous injection. ** p 0.005, *** p 0.001.