Gb111499

The mice with average tumor volume of about 100 mm³ were randomly allocated to 7 treatment groups (biological replicates n = 5 for each group), which were PBS, BPY, BPY@HSA, PBS + L, BPY + L, BPY-HSA + L, BPY@HSA + L. The tumor volume and body weight of each mouse was measured every two days to evaluate the tumor treatment efficacy. The dose of each formulation was 20 mg/kg (counted as BPY-Mal₂) for tail vein administration, and the laser irradiation (1 W/cm², 10 min) was performed at 24 h after administration. During the PTT processes, the mice were anesthetized by isoflurane and the temperatures of the irradiated sites were monitored by the thermal imaging camera (auto-capture mode with 30 s intervals). The tumors of represented mice were harvested for H&E staining, TUNEL and Ki67 (Anti-Ki67 Rabbit pAb, Servicebio, GB111499, dilution 1:500; Cy3-conjugated Goat Anti-Rabbit IgG, Servicebio, GB21303, dilution 1:300) analysis to evaluate the tumor inhibition effect 24 h after the first laser treatments (or 48 h after the first administration for groups without laser treatments). At the end of the treatments, the blood samples of each mouse were collected for blood routine analysis and blood biochemistry determination, and the major organs of represented mice were harvested for H&E staining to evaluate the safety of our formulations.

The tumor tissue was fixed using formalin, then embedded in paraffin, and sectioned into 4‐mm slides. Immunohistochemistry experiment was performed using the enhanced Polymer Assay kit (PV‐9001; ZSGB ‐ BIO). Then sections were incubated with anti‐SPP1 antibody (1:2000), anti‐STAT3 antibody (1:500), anti‐pSTAT3 antibody (1:500), and anti‐PI3K antibody (Catalog GB111499, Servicebio, 1:800). (Primary antibodies catalog is the same as that of WB).

After tumor tissues were formalin-fixed and paraffin-embedded, they were sliced into 4-μm thick sections for HE (hematoxylin-eosin) staining and Immunohistochemical staining (IHC) for Ki67 (GB111499,1:200, Servicebio, Wuhan, China), P16-INK4A (10883-1-AP, 1:100, Proteintech, Wuhan, China) and P21 (YT3794, 1:50, Immunoway, Jiangsu, China). HE staining was performed using HE staining kit (C0105S, Beyotime). Besides, sections were subjected to deparaffinization, rehydration and antigen retrieval. After blocking, these samples were incubated with primary antibody overnight at 4°C. Subsequently, the slides were covered with HRP-conjugated secondary antibody goat anti-rabbit IgG(H+L) HRP (GAR0072, 1:200, Liankebio, Huangzhou, China) or goat anti-mouse IgG(H+L) (bs-40296G-HRP, 1:200, Bioss, Beijing, China) and then stained with diaminobenzidine (DAB) chromogen kit. Images were captured using an inverted microscope (Eclipse Ci-L, Nikon, Japan). The staining results were analyzed using Image-pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) and quantified by integrated optical density (IOD).

After resection, the tumor tissues were fixed in 4% paraformaldehyde overnight. Then, the fixed tumor tissues were embedded with paraffin and sectioned at 5 μm thickness and then stained with anti-Ki67 (ServiceBio #GB111499). Pictures were taken with the Olympus VS120 imaging system, and representative pictures were presented in the manuscript. The percentage of proliferative tumor cells was quantified by the ratio of Ki67-positive tumor cells to total tumor cells in the tumor tissues.

Tumor tissues from different treatment groups were divided into sections. The tissue sections were placed into a citric acid (pH 6.0) antigen retrieval buffer (pH 6.0) (Servicebio, China) for antigen retrieval, and specimens were washed three times with PBS. The sections were sequentially soaked in a 3% hydrogen peroxide solution (Servicebio, China) and 3% BSA (Servicebio, China), each process lasting 25 minutes. Subsequently, specific primary antibodies (anti-Ki67, 1:400, GB111499, Servicebio, China) were added and membranes were incubated at 4°C for 12 h. The sections were immersed in a dilution of HRP-conjugated secondary antibody for 50 minutes. Finally, sections were re-stained with hematoxylin and sealed with sealing gel.