Supplementary Information for

Maintaining manganese in tumor to activate cGAS-STING pathway evokes a robust abscopal anti-tumor effect

Chao Wanga, Zhaoyi Suna, Chenxuan Zhaoa, Zhewei Zhanga, Haoran Wanga, Yang Liua, Yunfei Guoa, Bingtao Zhanga, Lihong Gua,d, Yue Yua,d, Yiqiao Hua,c, Jinhui Wua,b,c*

a State Key Laboratory of Pharmaceutical Biotechnology, Medical School of Nanjing University, School of Life Sciences, Nanjing University, Nanjing 210093, P. R. Chinab Chemistry and Biomedicine Innovation Center, Nanjing University, Nanjing 210023, P. R. Chinac Jiangsu Key Laboratory for Nano Technology, Nanjing University, Nanjing 210093, P. R. Chinad Affiliated Drum Tower Hospital, Medical School of Nanjing University, Nanjing University, Nanjing 210008, P. R. China

* Corresponding author: Prof. Jinhui Wu

Email: wuj@nju.edu.cn (J. Wu)

This PDF file includes:

Figures S1 to S14

Tables S1 to S3

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Fig. S1: The cytotoxicity of Mn2+ for tumor cells at different concentrations. A, CT26 tumor cells were treated with MnCl2 as indicated concentrations for 24 h, and cell viability was detected by CCK-8. B, B16F10 tumor cells were treated with MnCl2 as indicated concentrations for 24 h, and cell viability was detected by CCK-8. The concentration of MnCl2 below 1 mM didn’t show cytotoxicity on CT26 tumor cells, whereas B16F10 tumor cells were sensitive to MnCl2 when its concentration is higher than 500 μM. (n=6, compared to control by one-way ANOVA). ns, represents no significance.

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Fig. S2: Prolonging the co-incubation time of Mn2+ with the mixed cells can upregulate the transcription of ifnb1 and the expression of IFN-β. A, B and C, qRT-PCR analyzes the transcription level of Ifnb1, Isg15 and Ifit1 in total RNA extracted in the mixed cells treated as specified, respectively. Irradiated or unirradiated CT26 tumor cells were mixed with Raw264.7 cells and treated with 200 μM MnCl2 for 2 h or 24 h (in A, B and C, n=3, one-way ANOVA). D, ELISA for determination of the concentration of IFN-β in the supernatants of the mixed cells . Irradiated or unirradiated CT26 tumor cells were mixed with Raw264.7 cells and treated with 200 μM MnCl2 for 24 h. (in D, n=6, one-way ANOVA). Data represent mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ns, represents no significance.

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Fig. S3: Flow cytometry for analyzing the activation of BMDCs and its ability of antigen presentation. A, Images represent the results of Flow cytometry for analyzing the activation of BMDCs and its ability of antigen presentation. B, Statistically analyzing the results of Flow cytometry in A. Flow cytometry analysis of CD86 expression on C57BL/6 BMDCs (CD11c+) mixed with irradiated (or unirradiated) B16F10 tumor cells after 24 hours of stimulation with 200 μM MnCl2 for 2h or 24 h. And flow cytometry analysis of the SIINFEKL-H2Kb+ presentation on C57BL/6 BMDCs (CD11c+) mixed with irradiated (or unirradiated) B16F10 tumor cells after 24 hours of stimulation with 200 M MnCl2 for 2h or 24 h. Prolonging the co-incubation time of Mn2+ with the mixed cells (BMDCs and B16F10 tumor cells) can promote the activation of BMDCs and its ability of antigen presentation. (n=5, two-way ANOVA). Data represent mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ns, represents no significance.

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Fig. S4: Growth curves of individual mice treated with MnCl2 at different times after RT. A, Growth curves of irradiated (primary) and unirradiated (abscopal) tumors in individual mice treated with intratumoral injection of MnCl2 immediately after RT (Saline, n = 9; Mn, n = 9; Saline+RT, n = 10; Mn+RT, n = 8). B, Growth curves of irradiated (primary) and unirradiated (abscopal) tumors in individual mice treated with intratumoral injection of MnCl2 at 24 h after RT (Saline, n = 5; Mn, n = 5; Saline+RT, n = 6; Mn+RT, n = 6).

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Fig. S5: Therapeutic effect of combining RT with intratumoral injection of MnCl2 at 24 h after RT in unilateral CT26 xenografts. A, Growth curves of irradiated tumors in individual mice treated with intratumoral injection of MnCl2 at 24 h after RT. B, Average tumour-growth curves of irradiated tumors in the mice treated as specified (Saline, n = 6; Mn2+ 50 μM, n = 6; Mn2+ 200 μM, n = 6; RT, n = 10; Mn2+ 50 μM+RT, n = 6; Mn2+ 200 μM+RT, n = 6). C, Average body weight curves of treated mice in (A). D, Survival curves of treated mice in (B). Tumour growth over time was compared by two-way ANOVA with Bonferroni correction. Differences in survival were determined for each group by the Kaplan-Meier method and the overall P value was calculated by the log-rank test. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. represents no significance.

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Fig. S6: Immunofluorescence characterized the co-localization of micronuclei with cGAS. B16F10 tumor cells were received irradiation (8Gy) or unirradiated, followed by stained with PicoGreen and anti-cGAS antibody for characterizing the co-localization of cytosolic micronuclei induced by radiation with cGAS. For 60 × magnification, scale bar = 30 μm; For 100 × magnification, scale bar = 10 μm.

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Fig. S7: Characterization of the elastic modulus of Alg-Mn at different time after peritumoral injection. The Alg-Mn was explanted and cut into cubic pieces at 0h, 6h, 12h, 48h and 72h after peritumoral injection to determine the elastic modulus.

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Fig. S8: Alg-Mn can improve RT and the abscopal effect in CT26 xenografts and B16-F10 xenografts. A, Growth curves of irradiated (primary) and unirradiated (abscopal) tumors in individual mice of CT26 xenografts treated as specified at 24 h after RT (Saline, n = 9; MnCl2, n = 10; Alg, n = 10; Alg-Mn, n = 10; RT+Saline, n = 9; RT+Alg, n = 10, RT+MnCl 2, n = 12, RT+Alg-Mn, n = 12). B, Growth curves of irradiated (primary) and unirradiated (abscopal) tumors in B16F10 xenograft individual mice of B16F10 xenografts treated as specified at 24 h after RT (Saline, n = 7; MnCl2, n = 7; Alg, n = 7; Alg-Mn, n = 7; RT+Saline, n = 8; RT+Alg, n = 7, RT+MnCl2, n = 8,RT+Alg-Mn, n = 8).

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Supplementary Fig. 9: Real shot of bilateral B16F10 tumor-bearing mice. Representative images of the B16F10 tumor-bearing mice 8 d after initiation of treatment as specified. (Alg-Mn and MnCl2 were peritumorally or intratumorally injected into the right-flank tumor, respectively.).

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Fig. S10: RNA-seq for tumor tissue treatment as specified. Heatmap of RNA-seq for analyzing the transcription level of interferon-stimulated genes (ISGs, genes listed on left of the heatmap) in irradiated B6F10 tumor at 2 days following peritumoral injection (n = 3 biologically independent samples).

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Fig. S11: Immunofluorescence for characterization of CD3+CD8+ T lymphocytes in tumor. CT26 tumor tissue was explanted 8 d after initiation of treatment as specified. Subsequently, tumor tissue section was collected and stained with APC-conjugated CD3+ antibody, PE-conjugated CD8+ antibody. Finally, the stained tumor sections were shot by using laser confocal microscope. Scale bar, 50 μm.

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Fig. S12: The addition of MnCl2 to the cell culture medium had no significant effect on its osmotic pressure. The osmotic pressure of cell culture contained different concentrations (50 μM, 100 μM, 250 μM, 500 μM, 1 mM, 2.5 mM, 5 mM, 10 mM and 20 mM) of MnCl2.

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Fig. S13: H&E staining of organ sections to evaluate the safety of Alg-Mn. H&E-stained tissue sections of major organs including liver, heart, spleen, lung, and kidney of mice receiving i.t. injection of Alg-Mn (2% v/v, 100 μL). Those mice were examined at the 2 th (Day2) and 10th

(Day10) day post injection. Saline treated mice were used as the control. (For lung and liver, scale bar=100μm; for heart, spleen and kidney, scale bar =200μm).

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Fig. S14: Blood biochemistry and complete blood analysis of mice receiving i.t. injection of saline or Alg-Mn (200 μM, 100 μL) at the various time points (the 2th (2 d) and 10th (10 d) day post injection). The examined parameters included (A) alanine aminotransferase (ALT); (B) aspartate aminotransferase (AST); (C) alkaline phosphatase (ALP); (D) blood urea nitrogen (BUN); (E) creatinine (CR); (F) lymphocyte counts; (G) mean corpuscular volume (MCV); (H) mean corpuscular hemoglobin (MCH) and (I) hemoglobin (HGB). There were three mice per group. Error bars represent the mean ± s.d. (n = 3).

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Table S1: List of antibodies, cytokines and dyes used in flow cytometric analysis, western blotting and immunofluorescence staining.

Antibody Clone Fluorophore Manufacturer

CD11c N418 APC Biolegend

CD86 GL1 FITC Biolegend

SIINFEKL-H2Kb 25-D1.16 PE Biolegend

CD3 17A2 APC Biolegend

CD3 RM4-5 PE Biolegend

CD4 RM4-5 FITC Biolegend

CD8a 53-6.7 APC Biolegend

CD8a 53-6.7 PE Biolegend

STING D1V5L N/A CST

p-STING D8F4W N/ACST

TBK-1 D1B4 N/ACST

p-TBK-1 D52C2 N/ACST

IRF-3 D83B9 N/ACST

p-IRF-3 D6O1M N/ACST

GAPDH D16H11 N/ACST

GM-CSF N/A N/A Abcam

IL-4 N/A N/A Abcam

PicoGreen N/A N/A Yeasen

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Table S2. List of primers used in qRT-QPCR experiments.

Genes Forward primer (5’ to3’) Reverse primer (5’ to3’)

Ifnb1 CACAGCCCTCTCCATCAACT TCCCACGTCAATCTTTCCTC

Isg15 CTAGAGCTAGAGCCTGCAG AGTTAGTCACGGACACCAG

Ifit1 CTCTGAAAGTGGAGCCAGAAAAC AAATCTTGGCGATAGGCTACGA

Gapdh-1 TGATGGGTGTGAACCACGAG TAGGGCCTCTCTTGCTCAGT

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Table S3: Details for statistical analyses. F indicates F-values for ANOVA tests, T indicates t-values for t-tests, and df indicates degrees of freedom.

Figure Analysis One-tail or two-tailed F or T, df

Fig. 1b (24 h) Student T test Two-tailed T=20.45 df=4

Fig. 1b (72 h) Student T test Two-tailed T=5.486 df=4

Fig. 1c (24 h) Two-way ANOVA Two-tailed F (15, 90) = 447.1

Fig. 1c (48 h) Two-way ANOVA Two-tailed F (3, 90) = 733.8

Fig. 1c (72 h) Two-way ANOVA Two-tailed F (5, 90) = 1887

Fig. 1f (Mn) Two-way ANOVA Two-tailed F (9, 163) = 0.2921

Fig. 1f (RT) Two-way ANOVA Two-tailed F (9, 160) = 0.1412

Fig. 1g Two-way ANOVA Two-tailed F (21, 267) = 0.2508

Fig. 1i (Saline) Two-way ANOVA Two-tailed F (8, 72) = 0.1256

Fig. 1i (RT) Two-way ANOVA Two-tailed F (8, 90) = 1.435

Fig. 1i (Mn+RT) Two-way ANOVA Two-tailed F (16, 117) = 5.413

Fig. 1j (Saline) Two-way ANOVA Two-tailed F (16, 116) = 0.1153

Fig. 1j (Mn+RT) Two-way ANOVA Two-tailed F (8, 90) = 0.8417

Supplementary Fig. 1a One-way ANOVA Two-tailed F (2.372, 11.86) = 4.083

Supplementary Fig. 1b One-way ANOVA Two-tailed F (2.138, 10.16) = 1.484

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Table S3 continued:

Figure Analysis One-tail or two-tailed F or T, df

Supplementary Fig. 2a (RT+Mn-24h) Student T test Two-tailed T=5.144,

df=4

Supplementary Fig. 2a (RT+Mn-2h) One-way ANOVA Two-tailed

F (1.122, 2.244) = 10.54

Supplementary Fig. 2d One-way ANOVA Two-tailedF (1.192, 5.166) = 50.08

Supplementary Fig. 3b (CD86, RT+Mn-2h) Student T test Two-tailed t=0.1003,

df=5

Supplementary Fig. 3b (CD86, RT+Mn-24h) Student T test Two-tailed t=5.605,

df=5

Supplementary Fig. 3b (SIINFEKL-H2Kb, RT) Student T test Two-tailed t=4.014,

df=7

Supplementary Fig. 3b (SIINFEKL-H2Kb, RT+Mn-2h)

Student T test Two-tailed t=3.280, df=8

Supplementary Fig. 5b (Saline+RT) Two-way ANOVA Two-tailed F (14, 150)

= 5.114

Supplementary Fig. 5b(Mn2+ 50μM+RT) Two-way ANOVA Two-tailed F (14, 150)

= 0.7916

Supplementary Fig. 5d Log-rank (Mantel-Cox) test N/A N/A

Fig. 3e (RT6h) Student T test Two-tailedT=0.6868, df=4

Fig. 3e (RT48h) Student T test Two-tailedT=12.37, df=4

Fig. 3e (RT72h) Student T test Two-tailedT=18.44, df=4

Fig. 4b (MnCl2) Two-way ANOVA Two-tailed F (21, 271) = 0.6971

Fig. 4b (RT+Saline) Two-way ANOVA Two-tailed F (7, 136) = 1.906

Fig. 4b (RT+Alg) Two-way ANOVA Two-tailed F (7, 144) = 12.77

Fig. 4b (RT+Alg-Mn) Two-way ANOVA Two-tailed F (28, 343) = 37.40

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Figure Analysis One-tail or two-tailed F or T, df

Fig. 4c (Saline) Two-way ANOVA Two-tailed F (20, 294) = 0.1608

Fig. 4c (RT+MnCl2) Two-way ANOVA Two-tailed F (4, 90) = 0.8570

Fig. 4c (RT+Alg) Two-way ANOVA Two-tailed F (4, 90) = 9.396

Fig. 4c (RT+Alg-Mn) Two-way ANOVA Two-tailed F (4, 95) = 2.952

Fig. 4d Log-rank (Mantel-Cox) test N/A N/A

Fig. 4f (RT+Saline) Two-way ANOVA Two-tailed F (9, 140) = 1.280

Fig. 4f (RT+Alg-Mn) Two-way ANOVA Two-tailed F (9, 130) = 4.300

Fig. 4g (RT+Alg) Two-way ANOVA Two-tailed F (9, 130) = 0.5169

Fig. 4g (RT+MnCl2) Two-way ANOVA Two-tailed F (9, 140) = 3.068

Fig. 4g (RT+Alg-Mn) Two-way ANOVA Two-tailed F (9, 140) = 12.26

Fig. 4h Log-rank (Mantel-Cox) test N/A N/A

Fig. 5c Two-way ANOVA Two-tailed F (7, 80) = 50.33

Fig. 5d Two-way ANOVA Two-tailed F (7, 80) = 104.8

Fig. 5e Two-way ANOVA Two-tailed F (7, 79) = 10.25

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