Additional file 1 of Novel FLT3/AURK multikinase inhibitor is efficacious against sorafenib-refractory and sorafenib-resistant hepatocellular carcinoma

Additional file 1: Figure S1. Development of sorafenib-acquired resistant Huh7 tumors. Huh7 tumor-bearing animals were treated with 30 mg/kg sorafenib once a day, 5 days per week by oral route for 4 weeks. Tumor-bearing animals with a volume increase of ≥ 30% during treatment were removed at the end of study. The remaining animals with sorafenib treatment-sensitive tumors, that is, tumors with a volume increase of < 30% or regressive tumors, were harvested and reimplanted into recipient mice. These animals were randomized when the average tumor size reached 100 mm3 and received sorafenib treatment for 4 weeks. This process was repeated twice when tumors developed acquired resistance to sorafenib. Acquired resistance development is defined as responsive to sorafenib for at least 2 weeks but then exhibited a > 30% increase in tumor volume within 5 days. Figure S2. Histologic analysis of DBPR114 and sorafenib treatment in PLC/PRF/5 xenograft tumors. Tumor tissues were formalin-fixed and paraffin-embedded, with the paraffin sections used for immunohistochemical staining. Cell proliferation was measured using the proliferation marker Ki-67 (upper panel), and microvessel density was measured using the endothelial cell marker CD31 (bottom panel). Digital scans were performed with a 3DHITECH PANNORAMIC Midi slide scanner, and images were captured with PANNORAMIC Viewer software. Representative images were extracted from two separate animals in each group at × 40 magnification. Red arrows indicate mononucleated and multinucleated giant cells. Bar: 50 µm. Figure S3. Individual animal tumor growth curve for the control and treated sorafenib-acquired resistant Huh7 xenograft tumors. The mice bearing sorafenib-acquired resistant Huh7 tumors were randomized and treated with the vehicle control, sorafenib, DBPR114, or regorafenib when the average tumor size reached 100 mm3. DBPR114 (40 mg/kg) was administered once a week intravenously for 3 weeks. Sorafenib and regorafenib were administered at 30 mg/kg once a day, 5 days per week by oral gavage for 25 days. The treatment was then discontinued, and the animals were monitored for tumor growth (A) and body weight change (B). n = 8 mice per group.