Mouse MC38 colon cancer cells were provided by Innovent Biologics, Inc. ICD effects and immune activation, which were hampered by immune system impairment caused by chemotherapy. Compared with traditional formulations, a low dose of nanomicelle-encapsulated PTX (nano-PTX) treatment induced immune-dependent tumor control, which increased the infiltration and function of both T cells and DCs within tumors. However, this antitumor immunity was hampered by highly expressed PD-1 on tumor-infiltrating CD8+ WZ4003 T cells and upregulated PD-L1 on both immune cells and tumor cells after nano-PTX treatment. Combination therapy with a low dose of nano-PTX and PD-1 antibodies elicited CD8+ T cell-dependent antitumor immunity and remarkably improved the therapeutic efficacy. Conclusions: Our results provide systemic insights into the immune-regulation ability of PTX to induce ICD, which acts as an inducer of endogenous vaccines through ICD effects, and also provides an experimental basis for clinical combination therapy with nano-PTX and PD-1 antibodies. and exert good tumor-control effect. We also provide evidence that PTX treatment increases programmed cell death-ligand 1 (PD-L1) expression within the tumor microenvironment; combination therapy with nano-PTX and PD-1 antibody effectively suppresses tumor growth and prolongs overall survival of tumor-bearing mice. The results of this study suggest a new immune regulation mechanism WZ4003 of PTX, which may be augmented by the nanomicelle package to facilitate immunotherapy. Materials and Methods Mice and cell lines Six-week-old female BALB/c-nude, BALB/c, and C57BL/6 mice were purchased from Beijing HFK Bioscience Co. Ltd., Beijing, China. Mouse cell lines including colon carcinoma (CT26), mammary carcinoma (4T1), lung carcinoma (LL/2, LLC1), and melanoma (B16-F10), as well as human cell lines including colon carcinoma (HCT116), mammary carcinoma (MDA-MB-231), and cervical cancer (HeLa) were purchased from American Type Ctgf Culture Collection (ATCC). CT26-RFP was constructed by lentiviral infection expressing red fluorescent protein (RFP). Mouse MC38 colon cancer cells were provided by Innovent Biologics, Inc. (Suzhou, Jiangsu, P.R. China). Mouse ID8 ovarian cancer cells were provided by Professor Xia Zhao (West China Second University Hospital, Sichuan University, Chengdu, China). Drugs and antibodies For chemotherapeutic drugs, CDDP was purchased from Hanson Pharma, Inc. (Lianyungang, Jiangsu, P.R. China); OXP was purchased from Hengrui Medicine, Inc. (Lianyungang, Jiangsu, P.R. China); and PTX was purchased from TAIJI Industry (Group), Inc. (Chengdu, Sichuan, P.R. China). PTX entrapped with methoxy-poly (ethylene glycol)-and supernatant was collected for detecting the release of ATP (D) and HMGB1 (E) , n = 3 replicates. F Immunofluorescence staining of HMGB1 secretion in CT26 cell after treatment (24 h), statistics was shown in right panel. G Immunohistochemistry staining of HMGB1 within CT26 tumor after PTX injection (scale bar, 100 m). H Flow-cytometry detection of CRT on CD45- cells within CT26 tumor after nano-PTX injection, n = 5 mice per group. I Western blot showed the expression of protein related to ER stress signaling pathway in CT26 and HCT116 cells after treatment for 4 h. Mean SEM was shown. * P 0.05, ** P 0.01, *** P 0.001, **** P 0.0001, ns (no statistical significance). Immunogenic release of ATP and HMGB1 from dying cells is another essential marker of ICD that can promote antitumor immune response 21, 23. We detected increased ATP in the supernatant of CT26 (Figure ?(Figure3D)3D) and MC38 cells (Figure S3E) after PTX and OXP treatment. Similar results were observed for HMGB1 in CT26 (Figure ?(Figure3E-F)3E-F) and MC38 cells (Figure S3F), and also observed a dose-dependent effect for PTX treatment. As ATP and HMGB1 release is a consequence of cell death, increased ATP and HMGB1 were observed after CDDP treatment in this study, consistent with the findings of other studies 24, 34. Moreover, HMGB1 was previously WZ4003 identified as an important marker for ICD after treatment in CT26 cells (Figure S4B), while the XBP1 protein and HSPA5 mRNA were attenuated (Figure ?(Figure3I3I and Figure S4A-B), which was consistent with previous report 38, 39. Similar findings were also observed in MC38 tumor cells.