Tumor Ablation and Therapeutic Immunity Induction by an

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1 Tumor Ablation and Therapeutic Immunity Induction by an Injectable Peptide Hydrogel Honglin Jin, 1 Chao Wan, 1 Zhenwei Zou, 1 Guifang Zhao, LingLing Zhang, Yuanyuan Geng, Tong Chen, Ai Huang, Fagang Jiang, Jue-Ping Feng, Jonathan F. Lovell, & Jing Chen, * Gang Wu, * and Kunyu Yang * Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China Department of Oncology, PuAi Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China & Department of Biomedical Engineering, University at Buffalo, State University of New York. Buffalo, New York 14260, USA * Correspondence: Kunyu Yang, yangkunyu@medmail.com.cn; Gang Wu, xhzlwg@163.com; Jing Chen, chenjingunion@163.com Address: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China. Fax: ; Tel: ; Kunyu Yang (yangkunyu@medmail.com.cn) is the corresponding author to communicate with the Editorial and Production offices.

2 1 These authors contributed equally to this work. KEYWORDS. Chemoimmunotherapy, melanoma treatment, biomaterials, tumor microenvironment, hydrogel, macrophage depletion, cytotoxic T cells Supporting Figure 1: Effects of the salt and peptide concentration on gel formation. Supporting Figure 2: Effect of the ph on hydrogel formation. Supporting Figure 3: Thixotropic property of the MR hydrogel. Supporting Figure 4: Effect of the MR hydrogel on cell viability. Supporting Figure 5: Quantitative data of the average number of living cells. Supporting Figure 6: Measurement of cytokine levels in DCs. Supporting Figure 7. Effects of tumor lysate on the DC maturation. Supporting Figure 8: Evaluation of the in vivo mice weight changes. Supporting Figure 9: Hemanalysis analyses. Supporting Figure 10: Representative photograph of the mice footpad. Supporting Figure 11. Anti-tumor effects of MRD hydrogel. Supporting Figure 12: Measurement of in vivo immune activation effects. Supporting Figure 13: Flow cytometry measurement of the M1-like TAMs. Supporting Figure 14: In vivo analysis of generation of adaptive immunity. Supporting Figure 15: In vivo analysis of the generation of CD8 + IFN-γ + T cells. Supporting Figure 16: Measurement of the serum cytokine levels in mice. Supporting Figure 17. Effects of RADA and MR hydrogel on tumor growth.

3 Supporting Figure 18: Flow cytometry gating strategy for the measurement of T EM cells. Supporting Figure 19: Effects of the RADA hydrogel on DC maturation. Figure S1. Effect of the saline and peptide concentration on hydrogel formation. MR peptide can gelate at a concentration of 0.5-2% in the presence of 0.9% NaCl (w/w) (top panel), while hydrogel was no formed in the absence of 0.9% NaCl (w/w) (bottom panel). Figure S2. Effect of the ph on hydrogel formation. MR peptide can gelate at ph , while hydrogel was not formed at ph 8.5.

4 Figure S3. Thixotropic property of the MR hydrogel. Figure S4. Effect of the MR hydrogel on cell viability. (A) Fragmented MR hydrogel was made by pre-mixing MR hydrogel with culture medium, followed with repeat pipetting to disrupt the solid MR hydrogel. (B) Comparison of the in vitro anti-tumor effects of MR hydrogel and RADA hydrogel. The B16-F10 cells were incubated with various concentration of fragmented MR hydrogel or RADA hydrogel for 24 h, and cell viability was measured using the CCK-8 assay. (C-E) Measurement of cell viability in B16, DCs amd murine mouse fibroblasts L929 cells.

5 Figure S5. Quantitative data of the average living cells from confocal images of B16-GFP cells treated with MR hydrogel. Figure S6. Measurement of cytokine levels in MR hydrogel-treated DCs. ImDCs were incubated with solid MR hydrogel (10 μm) or LPS for 24 h and cell supernatants were measured by cytokine antibody array.

6 Figure S7. Effects of cell supernatants generated from MR hydrogel-treated cells on the DC maturation. B16-F10 cells were treated with MR hydrogel for various time (0-48 h) and cell supernatants were collected, followed with 24 h incubation with imdcs. Figure S8. Evaluation of the in vivo mice weight changes. The subcutaneous B16-F10 tumor-bearing mice were treated by a single peritumoral injection of 50 μl PBS, DOX, RD or MRD hydrogel, and their weight changes were recorded.

7 Figure S9. Hemanalysis analyses. Hemanalysis were performed for the blood withdrawn from the mice on the 8 th day post drug treatment. The number of the monocytes (A), lymphocytes (B) and neutrophils granulocytes (C) were measured. Data are presented as the mean ± SEM (n = 3). Figure S10. Representative photograph of the mice footpad at 18 days post footpad injection of B16-Luc cells. Data show obvious tumor growth in PBS and DOX groups.

8 Figure S11. Anti-tumor effects of MRD hydrogel. Tumor growth measurement for mice peritumorally injected with PBS, DOX, MR, RD, or MRD hydrogel. Data are presented as the mean ± SEM (n = 6-7).

9 Figure S12. Measurement of the in vivo immune activation effects. (A) Representative flow cytometry results of the proportions of activated DCs (CD11c + CD80 + CD86 + ) within PLNs. (B) Representative flow cytometry results of the proportions of activated NKs (Granzyme B + NK1.1 + CD3 - ) within tumors. Data are presented as the mean ± SEM (n = 6-7). Figure S13. Flow cytometry measurement of the proportion of leukocyte and M1-like TAMs at 8 days post drug administration. Data are presented as the mean ± SEM (n = 6-7).

10 Figure S14. Adaptive immunity analysis. Representative flow cytometry results of the proportions of CD8 + T cells (A), and CD4 + T cells (B). Data are presented as the mean ± SEM (n = 6).

11 Figure S15. Adaptive immunity analysis. Representative flow cytometry results of the proportions of cytotoxic CD8 + IFNγ + T cells. Data are presented as the mean ± SEM (n = 6). Figure S16. Measurement of the serum cytokine levels in mice. Measurement was performed at 14 days post drug treatment using flow cytometry. (A) IFN-γ level. (B) IL-2 level. Data are presented as the mean ± SEM (n = 5-6).

12 Figure S17. Effects of RADA and MR hydrogel on tumor growth. Tumor growth measurement for mice peritumorally injected (day 7/11/15) with PBS, RADA, or MR hydrogel. Data are presented as the mean ± SEM (n = 7). Figure S18. Flow cytometry gating strategy for the measurement of the proportions of splenic T EM (CD3 + CD8 + CD44 + ) cells.

13 Figure S19. Effects of RADA hydrogel on the DC maturation. In vitro evaluation of the portion of mature DCs (CD11c + CD80 + CD86 + ) upon RADA hydrogel treatment.