Genes associated with antigen presenting cells, such as ITGAX (CD11c) and MHC-II, and chemokines of CXCL9 and CXCL10, were all up-regulated

Genes associated with antigen presenting cells, such as ITGAX (CD11c) and MHC-II, and chemokines of CXCL9 and CXCL10, were all up-regulated. responses, which provided a strong rationale for combining CRA and PD-L1 blockade in the clinical treatment for HCC. Key words: Hepatocellular carcinoma, Immunotherapy, Cryoablation, Microwave ablation, CXCL9, NK cells, Antibody-dependent cell-mediated cytotoxicity, Immunosuppressive microenvironment Graphical abstract Anti-PD-L1 antibody plays a key role in improving curative effect of cryoablation of HCC blocking PD-L1. It can enhance antitumor activity of CTL/NK cells and eliminating PD-L1highCD11b+ cells by antibody-dependent cell-mediated cytotoxicity (ADCC) effect. Open in a separate window 1.?Introduction Cryoablation (CRA) and microwave ablation (MWA) are the most commonly used local hepatocellular carcinoma (HCC) treatments1,2. Many studies have verified that CRA activates T cells in tumor microenvironment in many types of malignant tumors. It acts by releasing tumor antigen peptides from frozen tumor cells3,4, which are easily degraded at high temperatures. Multiple studies have demonstrated that, compared to other forms of thermal ablation, CRA induces a more potent immune response and up-regulates the PD-L1/PD-1 pathway in HCC5, 6, 7. The combination of ablation and checkpoint block inhibitors (CBI), such as anti-PD-1 or anti-CTLA-4 antibodies, can efficiently induce the abscopal effect and increase the prognosis of colorectal cancer, breast cancer, and prostate cancer8,9. However, some studies have revealed that CRA does not significantly improve the overall survival of HCC patients compared to MWA and radio-frequency-ablation (RFA)10, 11, 12. Whether CRA or MWA combined with immunotherapy can improve therapeutic efficacy for HCC treatments still needs to be further studied. Importantly, thermal ablation induces immunosuppression in residual tumors by triggering infiltration of immunosuppressive CD11b+ myeloid cells up-regulating PD-L1, CCL2, TGF-intraperitoneal injection every 3 days after ablation therapy. Anti-mouse PD-L1 antibody were intraperitoneal injection every 3 days. For ADCC blocking assay, 10?mg/kg anti-mouse CD16 (BE0307, BioXcell, Lebanon, NH, USA) was administered intraperitoneal injection every 3 days. The tumor sizes (volume) were calculated every day using Eq. (1): Volume = 0.5 is the longer LCI-699 (Osilodrostat) diameter, is the shorter diameter, and is the perpendicular dimension. The tumor weight at end point was tested. All experiment protocols were approved by the Ethics Committee Board for Human and Animal Experiments in Zhongshan School of Medicine of Sun Yat-sen University (ethics approval ID: SYSU-IACUC-2022-000335). The procedure was performed in accordance to the National Commission for the Protection of Subjects of Biomedical and Behavioral Research guidelines for animal experiments. All efforts were made to minimize suffering. 2.4. LCI-699 (Osilodrostat) Flow cytometric analysis Single-cell suspensions were surface stained in fluorescence activated cell sorter (FACS) buffer (phosphate buffer solution (PBS) supplemented with 1% fetal bovine serum (FBS) with the following antibodies for cell membrane staining and flow cytometric analysis: Zombie NIR Fixable Viability Kit (Biolegend, 423105, San Diego, CA, USA) for live/died cells staining, PE/Cyanine7 anti-mouse CD45 (Biolegend, 157613, San Diego, CA, USA), Pacific Blue anti-mouse CD3 (Biolegend, 100213, San Diego, CA, USA), Alexa Fluor? 488 anti-mouse CD11b (ThermoFisher, 53-0112-82, Carlsbad, CA, USA), PerCP/Cy5.5 anti-mouse Gr-1 (Elabscience, E-AB-F1120J-50T, wuhan, China), APC anti-mouse F4/80 (Biolegend, 123116, San Diego, CA, USA), APC anti-mouse CD11c (Biolegend, 117309, San Diego, CA, USA), PE anti-mouse PD-L1 (Elabscience, E-AB-F1132D-200T, wuhan, China), PerCP/Cyanine5.5 anti-mouse CD3 (Biolegend, 100218, San Diego, CA, USA), PE anti-mouse CD8a (Biolegend, 100708, San Diego, CA, USA), APC anti-mouse CD39 (Biolegend, 143809, San Diego, CA, USA), and FITC anti-mouse CD49b (Biolegend, 103503, San Diego, CA, USA). For intracellular staining, collected cells were fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD, 554714, Franklin Lake, NJ, USA) as recommended by LCI-699 (Osilodrostat) the manufacturer. Pacific Blue anti-mouse IFN-(Biolegend, 505818, San Diego, CA, USA) and APC anti-mouse Granzyme B (Biolegend, 372203, Rabbit polyclonal to IL18R1 San Diego, CA, USA) were used for intracellular staining. For testing the affinity between PD-L1 antibodies and mouse CD16, atezolizumab (tecentriq), Avelumab (Bavencio), Mutant Anti-PD-L1-mIgG1e3 (InvivoGen, hpdl1-mab15, San Diego, CA, USA), Mouse IgG1 Isotype Control (R&D, MAB002, Emeryville, CA, USA), Human IgG1 (N298A) isotype control (InvivoGen, bgal-mab12, San Diego, CA, USA) were labeled with FITC fluorescence using the ZENON ALEXA FLUOR 488 MOUSE IG 1 KIT (ThermoFisher, “type”:”entrez-nucleotide”,”attrs”:”text”:”Z25002″,”term_id”:”395641″,”term_text”:”Z25002″Z25002, Carlsbad, CA, USA) as recommended by the manufacturer. FITC anti-mouse CD16 (101305, Biolegend, San Diego, CA, USA) was used as a positive control. Stained samples were visualized on a BD FACSAria and analyzed with FlowJo software (Becton, Dickinson and Company, Ashland, OR, USA). 2.5. Total RNA extraction and RNA-seq Total RNA was extracted from tumor tissues, using.