advances.sciencemag.org/cgi/content/full/6/5/eaax4690/DC1

Supplementary Materials for

Intratumoral delivery of CCL25 enhances immunotherapy against triple-negative

breast cancer by recruiting CCR9+ T cells

Hongmei Chen, Xiuxiu Cong, Chenxi Wu, Xuan Wu, Jialiang Wang, Kuirong Mao, Jie Li, Ge Zhu, Feiqi Liu, Xiandi Meng, Jia Song, Xu Sun, Xin Wang, Shuhan Liu, Shi Zhang,

Xianzhu Yang, Yanqiu Song*, Yong-Guang Yang*, Tianmeng Sun*

*Corresponding author. Email: tsun41@jlu.edu.cn (T.S.); songyqmd@163.com (Y.S.);

yongg@jlu.edu.cn (Y.-G.Y.)

Published 29 January 2020, Sci. Adv. 6, eaax4690 (2020) DOI: 10.1126/sciadv.aax4690

This PDF file includes:

Fig. S1. CCR9 expression in human peripheral blood T cells and mouse CD11b+ cells. Fig. S2. CCL25 expression in thymus and TNBCs and CCR9 expression in human colon cancer and TNBC. Fig. S3. Distribution of CCR9+ cells in tumor tissue. Fig. S4. Expression of CCR9, CD47, and PD-L1 in 4T1 cells in vitro and in vivo. Fig. S5. Characterization of NP-siCD47/CCL25 under neutral and tumoral acidic pH environments. Fig. S6. Cellular uptake of CD47 siRNA and CCL25 by 4T1 cells in vitro and down-regulation of the surface CD47 expression in 4T1 cells after treated with NP-siCD47/CCL25. Fig. S7. Tissue distribution of NP-siCD47/CCL25 in vivo. Fig. S8. Body weights of 4T1 tumor–bearing mice. Fig. S9. NP-siCD47/CCL25 significantly inhibits tumor growth and metastasis in 4T1-luc tumor-bearing mice. Fig. S10. Flow cytometric analysis of T cell depletion in anti-CD8 or anti-CD4 antibody–treated mice and the antitumor effects of anti–PD-1 antibodies in the 4T1 tumor model.

Fig. S1. CCR9 expression in human peripheral blood T cells and mouse CD11b+ cells.

Surface CCR9 expression in lymphocytes was measured using the peripheral blood mononuclear

cells (PBMCs) harvested from 11 healthy female volunteers (HV) and 10 breast cancer patients

(TNBC). (A) Representative flow cytometry plots showing CCR9 expression on CD3+CD4

+,

CD3+CD8

+, CD3

+CD45RO

+, CD3

+CD45RA

+ and CD3

+CD71

+ T cells in PBMCs of HV and

TNBC. (B) Frequencies of CCR9+CD3

+CD4

+, CCR9

+CD3

+CD8

+, CCR9

+CD3

+CD45RO

+,

CCR9+CD3

+CD45RA

+ and CCR9

+CD3

+CD71

+ T cells in CD45

+ PBMCs of HV and TNBC. (C)

Frequencies of CCR9+ cells in CD3

+CD4

+, CD3

+CD8

+, CD3

+CD45RO

+, CD3

+CD45RA

+ and

CD3+CD71

+ T cells in PBMCs of HVs and TNBC. (D) Representative flow cytometry plots

demonstrating the CCR9+ cells in CD11b

+ cells in CD45

+ cells in spleens and tumors harvested

from normal and tumor-bearing mice. Data are presented as mean ± SEM. *, p < 0.05.

Fig. S2. CCL25 expression in thymus and TNBCs and CCR9 expression in human colon

cancer and TNBC. (A) Representative photomicrographs of CCL25 proteins in human thymus

harvested from a 24-week fetus (used as a positive staining control for CCL25) and tumors

harvested from TNBC patients. Red arrowheads, CCL25 positive cells. The scale bar is 50 µm.

(B) Representative photomicrographs of CCL25 proteins in BALB/c mouse thymus (used as a

positive staining control for CCL25) and 4T1 tumor. Red arrowheads, CCL25 positive cells. The

scale bar is 50 µm. (C) Representative photomicrographs of CCR9 proteins in tumors harvested

from colon cancer patients and TNBC patients. Red arrowheads, CCR9 positive cells. The scale

bar is 50 µm.

Fig. S3. Distribution of CCR9+ cells in tumor tissue. Immunofluorescence staining of CCR9 in

BALB/c mouse thymus and 4T1 tumors harvested from tumor-bearing BALB/c mice treated by

PBS, NP-siNC, NP-siNC/CCL25, NP-siCD47 and NP-siCD47/CCL25 as scheduled in Fig. 3D.

CCR9 was stained with goat anti-mouse monoclonal antibody and Alexa Flour 647 labeled

secondary antibody. The cell membrane and nuclei were stained with DAPI (blue). The scale bar

is 100 µm.

Fig. S4. Expression of CCR9, CD47, and PD-L1 in 4T1 cells in vitro and in vivo. Flow

cytometry analysis show the surface expression of CCR9, CD47 and PD-L1 on 4T1 cells

cultured in vitro or prepared from 4T1 tumor-bearing mice when the tumor volume was about

700 mm3. Open histograms, isotype control; filled histograms, specific antibody staining.

Fig. S5. Characterization of NP-siCD47/CCL25 under neutral and tumoral acidic pH

environments. (A) Optimum N/P ratio of the anionic CD47 siRNA with protamine/R10 was

determined by gel retardation assay. The CD47 siRNA was labeled with Cy5. Fluorescent image

shows the binding affinities at various N/P ratios. (B) Representative of cryo-transmission

electronic microscopic image of NP-siCD47/CCL25. Scale bar = 100 nm. (C and D) Intensity

distribution of particle size (C) and zeta potential (D) of NP/siCD47, NP-siCD47 and

NP-siCD47/CCL25 determined by dynamic light scattering (n = 3). (E) Particle size of

NP-siCD47/CCL25 after incubation in PB (pH = 7.4 or 6.8) for 90 min at 37 oC detected by

dynamic light scattering. (F) In vitro release kinetics of Cy3-CCL25 from the

NP-siCD47/Cy-3CCL25 incubated in PBS with pH 7.4 or 6.8. Data are presented as mean ±

SEM.

Fig. S6. Cellular uptake of CD47 siRNA and CCL25 by 4T1 cells in vitro and

down-regulation of the surface CD47 expression in 4T1 cells after treated with

NP-siCD47/CCL25. (A) Representative flow cytometry histograms of cellular uptake of CD47

siRNA and CCL25 by 4T1 cells. 4T1 cells were incubated in DMEM with or without

NP-siCD47/CCL25 at pH 7.4 or 6.8 for 30 min. The CD47 siRNA and CCL25 were labeled with

FAM and Cy3, respectively. (B) Representative flow cytometry histograms of CD47 on

cell-surface of 4T1 cells continuously incubated in DMEM containing lipofectamine 2000-siNC

complexes (LiposiNC), lipofectamine 2000-siCD47 complexes (LiposiCD47), NP-siNC,

NP-siCD47/CCL25 at pH 7.4 or 6.8 for 4 days. (C) Representative flow cytometry histograms

showing CD47 expression on 4T1 cells that were incubated for 4 days in DMEM medium with

or without lipofectamine 2000-siCD47 complexes (LiposiCD47) at pH 7.4 or 6.8.

Fig. S7. Tissue distribution of NP-siCD47/CCL25 in vivo. (A) 4T1 tumor-bearing mice were

intravenously administrated with PBS, free Cy5-siCD47 + Cy3-CCL25, NP-Cy5-siCD47,

NP-Cy5-siCD47/Cy3-CCL25. Brains, hearts, lungs, tumors, livers, spleens, kidneys and

intestines were harvested at 24 h after the treatment. The fluorescent images were conducted

using a Xenogen IVIS system. The CD47 siRNA and CCL25 were labeled with Cy5 and Cy3,

respectively. The dose of Cy5-siRNA and Cy3-CCL25 were 4.7 mg/kg and 294 µg/kg,

respectively. (B) Corresponding quantification of Cy5 (left) and Cy3 (tight) signals in brain, lung,

heart, liver, spleen and kidney in (A). (C) CLSM images showed each color channel of the

merged images in Fig. 2C. The CD47 siRNA and CCL25 were labeled with Cy5 (red) and Cy3

(yellow), respectively. The cell membrane and nuclei were stained with FITC-phalloidin (green)

and DAPI (blue), respectively. The scale bar is 10 µm.

Fig. S8. Body weights of 4T1 tumor–bearing mice. The body weights of 4T1 tumor-bearing

mice in the tumor inhibition experiment described in Fig. 4A were measured every other day.

Fig. S9. NP-siCD47/CCL25 significantly inhibits tumor growth and metastasis in 4T1-luc

tumor-bearing mice. (A) Tumor growth of the 4T1-luc tumors in the experiment described in

Fig. 3C was measured every other day. Data are presented as mean ± SEM. *, p < 0.05; ****, p

< 0.0001. (B) Photographs of the whole lungs with metastatic nodules harvested from the

4T1-luc tumor-bearing mice at the end the experiment described in Fig. 4C. Photo credit:

Hongmei Chen, Jilin University.

Fig. S10. Flow cytometric analysis of T cell depletion in anti-CD8 or anti-CD4 antibody–

treated mice and the antitumor effects of anti–PD-1 antibodies in the 4T1 tumor model. (A)

Representative flow cytometry plots demonstrating the CD8+ and CD4

+ T cells in spleens and

blood in BALB/c mice received two i.p. injections with a two-day interval of anti-mouse CD8α

(53-6.7, 12.5 mg/kg) or anti-mouse CD4 (GK1.5, 12.5 mg/kg) antibodies. (B) Tumor volumes

were measured every other day after 4T1 tumor implantation (3 105 cells per mouse). PD-1

antibody or PBS was given by i.p. injection every three days for 8 times starting from day 2 (n =

3 per group). The dose of PD-1 antibody for each injection was 12.5 mg/kg. Data are presented

as mean ± SEM.