Synergistic effect of tumor chemo-immunotherapy induced by leukocyte-hitchhiking thermal-sensitive micelles

Some specific chemotherapeutic drugs are able to enhance tumor immunogenicity and facilitate antitumor immunity by inducing immunogenic cell death (ICD). However, tumor immunosuppression induced by the adenosine pathway hampers this effect. In this study, E-selectin-modified thermal-sensitive micelles are designed to co-deliver a chemotherapeutic drug (doxorubicin, DOX) and an A2A adenosine receptor antagonist (SCH 58261), which simultaneously exhibit chemo-immunotherapeutic effects when applied with microwave irradiation. After intravenous injection, the fabricated micelles effectively adhere to the surface of leukocytes in peripheral blood mediated by E-selectin, and thereby hitchhiking with leukocytes to achieve a higher accumulation at the tumor site. Further, local microwave irradiation is applied to induce hyperthermia and accelerates the release rate of drugs from micelles. Rapidly released DOX induces tumor ICD and elicits tumor-specific immunity, while SCH 58261 alleviates immunosuppression caused by the adenosine pathway, further enhancing DOX-induced antitumor immunity. In conclusion, this study presents a strategy to increase the tumor accumulation of drugs by hitchhiking with leukocytes, and the synergistic strategy of chemo-immunotherapy not only effectively arrested primary tumor growth, but also exhibited superior effects in terms of antimetastasis, antirecurrence and antirechallenge.


Content Page
Synthesis route of NTA-PEG-p-(AAm-co-AN) and the illustration of the introduction of E-selectin onto the surface of micelles. S4 Supplementary Fig. 2 The 1 H-NMR spectra of p-(AAm-co-AN), PEG, PEG-p-(AAm-co-AN) and NTA-PEG-p-(AAm-co-AN). S5 Supplementary Fig. 3 The particle size and potential of ES-DSM as E-selectin modifications increased. S6 Supplementary Fig. 4 Biocompatibility of blank micelles, DSM and ES-DSM to leukocytes. S7 Supplementary Fig. 5 Confocal microscopy images of T lymphocyte and neutrophil 24 hours after the intravenous injection of ES-DSM. S8 Supplementary Fig. 6 Flow cytometry detection of 4T1 cells exposed to free Nile red or Nile red-loaded micelles with (+) or without (-) hyperthermia. S9 Supplementary Fig. 7 4T1 cell viabilities after exposure to a series of concentrations of blank micelles for 48h. S10 Supplementary Fig. 8 Detection of MHC Ⅱ on DCs after co-incubation with tumor cells. S11 Supplementary Fig. 9 Detection of MHC Ⅱ on DCs after co-incubation with tumor cells in the presence of NECA. S12 Supplementary Fig. 10 Analysis of CD4 + Foxp3 + T cells in the ternary co-incubation system. S13 Supplementary Fig. 11 Analysis of CD4 + Foxp3 + T cells in the ternary co-incubation system containing NECA. S14 Supplementary Fig. 12 Average fluorescence intensity of ICG-loaded micelles and ES-modified ICG-loaded micelles in tumor and other major organs 24 h after intravenous injection S15 Supplementary Fig. 13 Curves showing the changes of tumor volume of individual mouse after various treatments. S16 Supplementary Fig. 14 Microscopic images of H&E-stained cross-sections of the tumors at the end of observation. S17 Supplementary Fig. 15 Evaluation of the mature DCs in tumors after different treatments in 4T1 tumor models. S18 Supplementary Fig. 16 Evaluation of the mature DCs in sentinel lymph nodes (SLNs) after different treatments in 4T1 tumor models. S19 Supplementary Fig. 17 Evaluation of T cells in PBMC and spleen after different treatments in 4T1 tumor models. S20 Supplementary Fig. 18 Positive percentage of T cells in tumor calculated based on Fig. 7a and b. S21 Supplementary Fig. 19 Immunohistochemistry was used to examine the levels of CD69 and perforin in 4T1 tumor sections. S22 Supplementary Fig. 20 The effect of CD39 and anti-CRT antibody on the tumor inhibition of ES-DSM+MW. S23 Supplementary Fig. 21 The effect of CD39 and anti-CRT antibody on the tumor infiltration of immune cells. Leukocyte viabilities after exposure to ES-DSM at different DOX concentrations (6.5, 12.5, 25, 31.5, 37.5 μg/mL) for various time (1, 2, 4, 8 h) at 37℃ (n=3 independent experiments). c Transwell assay was used to measure the chemotaxis and biological penetration ability of leukocytes (LEU). Images of leukocytes (LEU) transported in the lower chamber of the transwell system in the presence of CXCL2 and CXCL12 were presented. d The transwell percentage of LEU after incubation with DSM or ES-DSM in vitro was calculated based on c (n=3 independent experiments). e The transwell percentage of LEU isolated form mice after intravenous injection of DSM or ES-DSM in vivo was calculated based on c (n=3 mice). Data are presented as mean values ± SEM and unpaired two-tailed T test was performed in d, e. Source data are provided as a Source Data file.
Supplementary Fig. 5 Confocal microscopy images of T lymphocyte and neutrophil 24 hours after the intravenous injection of ES-DSM. Leukocytes were isolated from PBMC and incubated with APC-anti CD3 or CD16 antibody for 20 min at room temperature in dark to identify T lymphocyte and neutrophil, then observed by CLSM. The red fluorescence indicated CD3 on T lymphocyte or CD16 on neutrophil, and the green fluorescence indicated DOX of ES-DSM. The experiments were repeated independently for three times with similar results.
Supplementary Fig. 6 Flow cytometry detection of 4T1 cells exposed to free Nile red or Nile red-loaded micelles with (+) or without (-) hyperthermia. 4T1 cells were exposed to test agents and the hyperthermia treated groups were placed in the cell incubator (43℃ and 5% CO2, 30 min) immediately, followed by incubation at 37℃ for 6 h and detected by flow cytometry. Nile red emits strong fluorescence only if it is released from the micelles and binds to cellular lipids. n=2 independent experiments. Supplementary Fig. 15 Evaluation of the mature DCs in tumors after different treatments in 4T1 tumor models. At the end of the observation, mice were sacrificed, tumors were isolated and ground through a 200-mesh cell sieve to generate single-cell suspensions. The tumor cells were incubated with FITC-CD11c, PE-CD80 and APC-CD86 antibodies for 20 min at room temperature in dark, analyzed by flow cytometry. The ratios of a CD11c + CD80 + , b CD11c + CD86 + and c CD11c + CD80 + CD86 + DCs in tumors of different groups were presented. The percentages of d CD11c + CD80 + , e CD11c + CD80 + and f CD11c + CD80 + CD86 + DCs were calculated based on a, b and c, respectively (n=3 mice). Data are presented as mean values ± SEM and unpaired twotailed T test was performed in d, e, f. Source data are provided as a Source Data file.
Supplementary Fig. 16 Evaluation of the mature DCs in sentinel lymph nodes (SLNs) after different treatments in 4T1 tumor models. At the end of the observation, mice were sacrificed, SLNs were isolated and ground through a 200-mesh cell sieve to generate single-cell suspensions. The cells of lymph nodes were incubated with FITC-CD11c, PE-CD80 and APC-CD86 antibodies for 20 min at room temperature in dark, analyzed by flow cytometry. The ratios of a CD11c + CD80 + , b CD11c + CD86 + and c CD11c + CD80 + CD86 + DCs in SLNs of different groups were presented. The percentages of d CD80 + , e CD80 + and f CD80 + CD86 + DCs were calculated based on a, b and c, respectively (n=3 mice). Data are presented as mean values ± SEM and unpaired two-tailed T test was performed in d, e, f. Source data are provided as a Source Data file.
Supplementary Fig. 17 Evaluation of T cells in PBMC and spleen after different treatments in 4T1 tumor models. At the end of the observation, peripheral blood was collected in anticoagulant tubes by enucleation of the eyeball, and PBMC was isolated by density gradient centrifugation.
The spleen was isolated after the mice were sacrificed and was ground through a 200-mesh cell sieve to generate single-cell suspensions, the lymphocytes in spleen were isolated by density gradient centrifugation. The PBMC and spleen lymphocytes were incubated with FITC-CD3, PE-CD4 and APC-CD8 antibodies for 20 min at room temperature in dark, analyzed by flow cytometry. a Flow cytometry analysis of the ratios of CD3 + CD4 + and CD3 + CD8 + T cells in PBMC and spleen lymphocytes. The percentages of b CD3 + CD4 + and c CD3 + CD8 + T cells in PBMC were calculated based on a (n=3 mice). The percentages of d CD3 + CD4 + and e CD3 + CD8 + T cells in spleen were calculated based on a (n=3 mice). Data are presented as mean values ± SEM and unpaired two-tailed T test was performed in b-e. Source data are provided as a Source Data file.
Supplementary Fig. 18 Positive percentage of T cells in tumor calculated based on Fig. 7a and b. The percentages of a CD3 + CD4 + , b CD3 + CD8 + and c CD3 + CD4 + Foxp3 + T cells in tumor (n=3 mice). Data are presented as mean values ± SEM and unpaired two-tailed T test was performed in a-c. Source data are provided as a Source Data file.
Supplementary Fig. 19 Immunohistochemistry was used to examine the levels of CD69 and perforin in 4T1 tumor sections. The experiments in a, b showed similar results in three independent mice.
Supplementary Fig. 20 The effect of CD39 and anti-CRT antibody on the tumor inhibition of ES-DSM+MW. a Schematic of the treatment regimen. The ES-DSM was i.v. injected on day 1, 4, 7, 10, and the microwave radiation was applied 24 hour later. The CD39 (1 μg/mice per injection) and anti-CRT antibody (10 μg/mice per injection) were injected i.p. every 3 days to deplete ATP and CRT generated during the ICD process. b Curves showing tumor volumes of mice after various treatments (n=6 mice). Data are presented as mean values ± SEM and unpaired two-tailed T test was performed in b. Source data are provided as a Source Data file.
Supplementary Fig. 23 Curves showing volumes of rechallenged CT26 tumor of mice after different treatments (n=6). After orthotopic 4T1 breast tumor-bearing mice were treated with ES-DSM+MW, CT26 cells were inoculated subcutaneously in the left hind limb. The rechallenged CT26 tumor was also monitored every 2 days and the growth curves were presented. Data are presented as mean values ± SEM. Source data are provided as a Source Data file.
Supplementary Fig. 24 Hemolysis assay to evaluate the biocompatibility. a The hemolysis ratios of RBCs after treated with different agents (n=3 independent experiments). b The images of the mixture of RBCs and different agents after centrifugation. Source data are provided as a Source Data file.