Filter plate

The tissues were lysed (1:10, w/v) in RIPA (Solarbio Life Sciences; Beijing, China) containing protease inhibitors to quantify the protein production of IFN-α, IFN-β, IL-6, and TNF-α from mouse organs. The supernatants from these tissue lysates were examined by ELISA, using a VeriKine Mouse IFN Alpha ELISA Kit (PBL Interferon Source), Legend Max Mouse IFN-β ELISA kit (BioLegend; CA, United States), Legend Max Mouse IL-6 ELISA kit (BioLegend), or Legend Max Mouse TNF-α ELISA kit (BioLegend). To further quantify the protein production of the major inflammatory cytokines, the supernatants from tissues lysates (brain, spleen, and liver) and serum were examined by flow cytometry, using a LEGENDplex™ Mouse Inflammation Panel (13-plex) with a filter plate (BioLegend), following the manufacturer’s instructions.

The subcutaneous pancreatic tumor-bearing C57BL/6 mice were randomly divided into six groups and systematically injected with 0.2 mL saline, PBS solution of SPIN₀, SPIN_D2 (1.2 mg/mL, on days 0, 3, 6, 9, 12, 15, and 18) or free-drug mixture (8 mg/kg body weight for NLG919 and aPD-L1, on day 0, 3, 6, 9, 12, 15, and 18) via tail vein. US irradiation of tumors was conducted on day 1, 4, 7, 10, 13, 16, and 19 (1.0 MHz, 1.2 W/cm², 50% duty cycle, 10 min). The body of mice was measured every 2 days for 18 days. After treatments for 30 days, the mice in each group (n = 4) were used to collect blood and spleen for flow cytometry analysis of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells after staining with antibodies as described above. The major organs (heart, liver, spleen, lung, and kidney) were collected for H&E and immunofluorescence staining. The cytokine levels in the serum of mice (n = 5) after different treatments were measured using LEGENDplex™ mouse inflammation panel (13-plex) with a filter plate (Biolegend, cat. no. 740150) according to the manufacturer’s protocol. The blood from mice in each group (n = 5) was collected for blood routine and biochemical analysis.