Supporting Information

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1 Supporting Information of Laser Immunotherapy in Combination with Perdurable PD-1 Blocking for Treatment of Metastatic Tumor Lihua Luo, Chunqi Zhu, Hang Yin, Mengshi Jiang, Junlei Zhang, Bing Qin, Zhenyu Luo, Xiaoling Yuan, Jie Yang, Wei Li, Yongzhong Du, and Jian You. College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang , P. R. China *Corresponding Author: JianYou, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou , Zhejiang, China. Office: Fax:

2 Experimental Sections PD-1, PD-L1 expression in mice bearing tumor and PD-1 receptor antagonizing with APP Mice bearing 4T1 or CT26 tumors were obtained by subcutaneous injection of 4T1 or CT26 cells ( cells) and were used for subsequent study when the tumors grew up to about 500 mm 3. Firstly, PD-1 expression on PBMCs or splenic lymphocytes from mice bearing tumor was measured, and the expression in healthy mice was as control. Briefly, PBMCs and splenic lymphocytes from healthy and tumor bearing mice were isolated from blood and the spleen using the Ficoll-paque PREMIUM respectively and were cultured in complete RPMI 1640 medium for 24 h. The cells were collected, stained with FITC-anti-PD-1 antibody, and then tested by flow cytometry analysis. PD-L1 expression in the tumor tissues (CT26 and 4T1) was also determined using the immunohistochemistry. PBMCs from the mice bearing CT-26 tumors were incubated with PBS, free APP (6.8 nm), AA@PN (containing APP at 6.8 nm), HAuNS@PN (as blank carrier). The cells, incubated with AA@PN or HAuNS@PN were further irradiated with NIR laser (1.5 W/cm 2, 3 min). After 24 hours, PBMCs were incubated with FITC-anti-PD-1 antibody, followed by flow cytometric annalysis. In vitro antigen uptake into DC Immature BMDC were seeded in 35 mm 2 dishes (cover glass-bottom dish) at a density of per well and pre-cultured overnight at 37 C. OVA-FITC (2.5 µg/ml) were placed in the wells and incubated as above for 4 h. And then, the suspensions were removed by washing three times with PBS, and then cells were cultured in a medium containing LysoTracker Red for 2 h. DC were fixed with 4% paraformaldehyde for 15 min and treated with DAPI (0.8 ng/ml) to stain the nucleus. The cells were imaged with confocal laser scanning microscopy (CLSM, TCS SP8, Leica). Preparation of tumor cell lysate with PTA 4T1 tumor cells were planted into the 24-well plate at a density of / ml in a serum-free medium and were incubated with PBS, free APP, free CpG, HAuNS@PN, HAuNS@PN plus CpG and AA@PN plus CpG. The cells in the last three groups were then subjected to NIR laser (2 W/cm 2, 3 min). 24 hours later, the cell lysate was centrifuged for 10 min at 300 g to remove debris and the supernatant was collected. T-cell enrichment The PBMCs were isolated using lymphocyte density gradient centrifugation with Ficoll-paque PREMIUM. And after collection and processing of PBMCs, cells were stained for surface marker with a

3 monoclonal antibody FITC-anti-CD3. After 20 min on ice and protected from light, cells were washed once and sorted with Fluorescence Activated Cell Sorting (FACS). Furthermore the sorted cells were determined by flow cytometer. Long-term immune effect of on post-therapy recurrent and rechallenged tumor To investigate the long-term immune effect after the therapy with our strategy, recurrent and rechallenged tumor models were established. Briefly, T1 tumor cells were transplanted into the breast pad of mice which were weighted and randomly divided into 5 different groups (n = 5 to 10) 7 days later. Then the mice were tumorally injected with different formulations, including Saline, Free APP, AA@PN, HAuNS@PN plus CpG & Laser and AA@PN plus CpG & Laser. (APP, 3mg/kg; CpG, 20 ug per mouse). The tumors were measured with a digital caliper. The tumor volume (mm 3 ) was calculated as (long diameter short diameter depth) / 2. After 3 weeks treatment, when the tumors in the HAuNS@PN plus CpG & Laser and AA@PN plus CpG & Laser treated mice were almost eradicated, tumors in other three groups of mice were subjected to 99% resection. The about 1% residual tumor in the surgical bed was leaving for recurrence. As a parallel experiment, after 3 weeks treatment, a rechallenged tumor with of 4T1 tumor cells was transplanted in the other breast pad of the mice, and the rechallenged tumor volume was measured with a digital caliper. Parallel experiments were conducted to investigate the survival rate of the mice. Ex vivo analysis of different groups of central memory T cells To study the central memory T cells, spleen tissues were harvested from mice in different groups and stained with anti-cd8a-apc (Biolegend, Clone: , Catalog: ), anti-cd4-pe (Biolegend, Clone: GK1.5, Catalog: ), anti-cd122-fitc (Biolegend, Clone: 17A2, Catalog: ) and anti-cd44-percp (Biolegend, Clone: 17A2, Catalog: ) antibodies according to the manufacturer s protocols. Briefly, spleen tissues were cut into small pieces and put into a glass homogenizer containing PBS (ph7.4) with 2% heat-inactivated fetal bovine serum. Then, the single-cell suspension was prepared by gentle pressure with the homogenizer without addition of digestive enzyme. Finally, cells were stained with fluorescence-labelled antibodies after the removal of red blood cells (RBC) using the RBC lysis buffer.

4 Supplementary Figures Figure S1. (A) Cytotoxicity of CT26 and 4T1 cells at 24 h, 48h after treatment with or free, APxiP with various concentration. (B) Photothermal ablation of 4T cells with various treatments. NIR laser was delivered at an output power of 1.5 W/cm 2. (C) Cells were stained with calcein AM (green) and PI (red) to visualize of live and dead cells, respectively. The error represents the standard deviation from the mean (n = 4).

5 Figure S2. Expression of Heat shock protein (Hsp70) in 4T1 and CT26 cell lines after PTA immunotherapy through (A) immunofluorescence staining (green: Hsp70; blue: cell nucleus; laser: 1.5 W/cm 2 ). (B-C) The expression of Hsp70 in 4T1 and CT26 after treatment with PTA through western blotting (B) and its gray degree values (C).

6 Figure S3. PD-1 and PD-L1 expression and the effects of APP on antagonizing PD-1 receptor. (A) PD-1 expression on splenic lymphocytes and PBMCs isolated from Balb/c healthy and CT26, 4T1 tumor bearing mice. (B) PD-1 positive expressed lymphocytes gated on 4T1 tumor infiltrated CD3 + T cells. (C) PD-1 expression rate on PBMCs isolated from CT26 tumor bearing mice and stained with FITC-anti-PD-1 antibody after various treatments. (D) PD-L1 expression on CT26, 4T1 tumor tissues by immunohistochemistry staining; brown signal indicates PD-L1-positive expression. (Laser condition: 1.5 W/cm 2, 3 min).

7 Figure S4. The phagocytosis ability of DCs on OVA antigen. (A) The bright filed of DCs in control and (L) plus CpG treated groups. (B) The uptake of FITC-OVA antigen by DCs after being treated with PBS or (L) plus CpG. (C) The expression of MHC-II on DCs. (D) The matched signal of FITC and PE.

8 Figure S5. The antigen presenting ability of DCs. (A-B) The expression of MHC-I and MHC-II on DCs after being treated with tumor cell lysate. (C) The IL-12p70 secreted in suspensions of co-culture system after 48 h treatment. Data are expressed as mean ± sd, (n=4).

9 Figure S6. The immune responses mediated by (L) plus CpG in vitro against 4T cancer cells.

10 Figure S7. Immune responses mediated by PTA. (A) The amount of CD4 +, CD8 + T cells in the 4T1 primary tumor post 72 h-treatment with saline, AA@PN, laser alone, free CpG, AA@PN plus laser and AA@PN plus CpG & Laser. (B) The percentage of dendritic cells (DCs) in the 4T1 primary tumor post 72 h-treatment with various treatments. (C) Quantitation of CD4 +, CD8 + T cells based on the flow cytometric plot in (A). (D) Quantitation of DCs based on the flow cytometric plot gated on CD11c + in (B). (E) Cytokine levels in sera from mice isolated at 0, 24, 48 and 72 h post different treatments (NIR laser was conducted on 0 h and 48 h with an output power of 1.5 W/cm 2, 3 min).

11 Figure S8. Histology analyses of major organs and distant tumor tissues. H&E sections of the main organs from Balb/c mice bearing 4T1 tumors after different treatments (left). TUNEL, Ki 67-stained distant tumor slices collected from 4T1 tumor-bearing mice after different treatments (right).

12 Figure S9. Anti-tumor effects against CT26 and 4T1 tumor model. (A-B) Curves showing the body weight of mice after various treatments (A) for 4T1 tumor model, and (B) for CT26 tumor model (n=3-7). (C) Curves showing the survive time of mice in Luci-CT26 lung metastasis tumor model treated with various treatments (n=7).

13 Figure S10. (A) The CD86 + DCs in the primary tumor isolated from 4T1 tumor bearing mice at the end of treatment through immumohistochemical staining. (B-C) The amount of CD3 + CD4 + T cells in splenic lymphocytes (B) and distant tumors (C) isolated from 4T1 tumor-bearing mice and counted through flow cytometry after various treatments.

14 Figure S11. (A-B) Quantitation of CD8 + T cells and IFN-γ in the distant 4T1 tumor and 4T1 lung metastatic nodules based on flow cytometry using Image J software. (C-F) IL-2, IL-10, TNF-α, IFN-γ and IL-12p70 levels in the distant tumors tissues (C-E) and spleen tissues (D-F) of each group of mice isolated at the end of different treatments detected using ELISA assay. The error represents the standard deviation from the mean (n = 4). *p<0.05, **p<0.01.

15 Figure S12. Effects of (L) plus CpG against Luci-CT26 lung metastatic tumors. (A) Photographs, (B) H&E and (C) Ki67 staining of the metastatic foci of the CT26 tumors.

16 Figure S13. CD86 + DCs expression in the primary tumors by flow cytometry (A) and immunohistochemical staining (B) at the end of treatments in CT26 anti-tumor models. (C) The level of il-12p70 in the primary tumor isolated from the CT26 tumor bearing mice at the end of treatments.

17 Figure S14. (A) The expression of F4/80 in the primary tumor isolated from 4T1 tumor bearing mice at the end of treatment through immumohistochemical staining. (B) The amount of CD3 + T cells in splenic lymphocytes isolated from CT26 tumor-bearing mice and counted through flow cytometry after various treatments.

18 Figure S15. Individual tumor growth kinetics and the tumor recurrent rate after 3 weeks treatments in control and other treated groups. (n = 5 to 10, as indicated in the figure).

19 Figure S16. Individual rechallenged tumor growth kinetics after 3 weeks treatments in control and other treated groups. (n = 5 to 10, as indicated in the figure).

20 Body Weight (g) Saline 16 Free APP 14 (L) plus CpG 12 (L) plus CpG Time (Day) Figure S17. Measurement of body weight of control and treated mice.

21 Figure S18. In vivo anti - tumor treatment on CT26 bilateral subcutaneous tumors with Saline, Free APP (Single dose or multitude doses) and AA@PN (laser). (A) Schematic diagram of the model and administration method. (B) Curves showing the primary and distant tumor volumes of mice after various treatments (n=7). (C) Weights of the primary and distant tumors measured at the end of treatments (n=3-4).