Supplementary Information Recruitment of Mesenchymal Stem Cells Into Prostate Tumours Promotes Metastasis Younghun Jung 1, Jin Koo Kim 1, Yusuke Shiozawa 1, Jingcheng Wang 1, Anjali Mishra 1, Jeena Joseph 1, Janice E. Berry 1, Samantha McGee 1, Eunsohl Lee 1, Hongli Sun 2, Jianhua Wang 3, Taocong Jin 4, Honglai Zhang 5, Jinlu Dai 5, Paul H. Krebsbach 2, Evan T. Keller 5, Kenneth J. Pienta 5, and Russell S. Taichman 1, 1 Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI 4819, USA. 2 Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 4819, USA. 3 Institute of Medical Sciences, Shanghai Jiao-Tong University School of Medicine, Shanghai 225, P.R. China. 4 Department of Cariology, Restorative Sciences and Endodontics, University of Michigan School of Dentistry, Ann Arbor, MI 4819, USA. 5 Departments of Urology and Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 4819, USA. Supplementary Figures S1-S5 Supplementary Table S1 Supplementary Methods
a Undifferentiated MSC Alizarin Red S/Oil Red O/ Alcian Blue Alcian Blue c Prostate Tissue d CXCL16/DAPI CXCL16/DAPI CXCL16/DAPI CXCL16/DAPI.2.15.1.5 f P <.1.5 g P <.1.3.2.1 MC3T3-E1 s.c. PCa implantation () Tumour growth MSC cell recruitment to tumours (CXCR6 +/+ or CXCR6 -/- mice) k NMPE % MSC cells in marrow MCF-7 i Tramp MCF-1A MCF-7 2 RWPE-1 PC3 C4-2B CXCR16 CXCR6 (RWPE-1, PC3, C4-2B/ NMPE,, Tramp/ MCF-1A, MCF-7) MSC cell recruitment to tumours.4 CXCR6 +/+ CXCR6 -/- (SCID or CXCR6 +/+ mice) m P =.22 P =.1 1.5 1.8 1.5 3.1 Orthotopic cancer cell implantation.12 l P <.1 P <.1 P <.1.2 j n.s..16 % MSC cells in tumour MCF-1A % MSC cells in tumour.5 C4-2B LNCaP DU145 PC3 mrna of cells 1 4 HOB RWPE-1.4 1.5 mcxcl16 mrna hcxcl16 (ng ml -1 ).1 Gleason 8+9 Gleason 6+7 h.15.25 hcxcl16 mrna Gleason 4+5 hcxcl16 mrna Normal prostate P <.1 P <.1.3 e Chondrogenic conditions Adipogenic conditions Oil Red O NMPE Tramp % MSC cells in tumour b Osteoblastic conditions Alizarin Red S P =.7.3.2.1 MCF-1A MCF-7
n s.c.tumour CXCR6 +/+ CXCR6 -/ p s.c.tumour CXCR6 +/+ Control SCID SCID SCID C4-2B PC3 CXCR6 +/+ shcxcl16 q Orthotopic tumour (prostate) RWPE-1 r o Orthotopic tumour (prostate) NMPE Tramp Orthotopic tumour (fat pad) SCID MCF-1A SCID MCF-7 Supplementary Figure S1: Expression of CXCL16 by prostate cancers and breast cancers and MSC cell recruitment into tumours in orthotopic animal models. (a) Differentiation of murine MSCs into osteoblasts, adipocytes, and chondrocytes was confirmed by staining of Alizarin Red S, Oil Red O, and Alcian Blue, respectively. Scale bars, 5µm. (b) Immunohistochemistry (IHC) staining for the expression of CXCL16 (red, white arrows) in human prostate cancer tissue microarray is correlated with aggressive phenotype. Blue, DAPI nuclear stain. Scale bars, 1 µm. (c) CXCL16 mrna by human prostate cancer cell lines was determined by qrt-pcr. (d) CXCL16 mrna, and (e) CXCL16 secretion by human breast cancer cell lines were determined by qrt-pcr or ELISA. (f) CXCL16 mrna by murine prostate cancer cell lines was determined by qrt -PCR. (g) mrna levels for CXCR16 and CXCR6 by cells were determined by qrt-pcr. Data in (c-g) are representative of mean with s.d. for triplicates in each of three independent experiments (Student s t -test). (h) Experimental scheme of cell implantation to CXCR6 +/+ or CXCR6 -/- mice for examining tumour growth and MSC cell recruitment to tumours. (i) % MSC ( Lin - Sca-1+CD45 -) present in the bone marrow of CXCR6 +/+ or CXCR6 -/- mice (mean±s.d., n = 3 independent experiments, Student s t-test). n.s., not significant. (j) Experimental scheme of human and murine prostate cancer and human breast cancer cell orthotopic implantation to in the prostate or fat pad of SCID or C57BL/6 (CXCR6 +/+ ) mice for examining tumour growth and MSC cell recruitment to tumours. (k-m) % MSCs present in human and murine prostate tumours, and human breast tumours were grown in the prostate or fat pad of SCID or C57BL/6 (CXCR6 +/+) mice (error bars represent mean±s.d. for 3-5 animals/group, n = 1independent experiment, Student's t-test). (n-r) Tumours from various animal experiments (Supplementary Table S1) were stained by H&E. Scale bars, 1 µm.
b 16 Tramp f MSC P2 CXCR6 -/- h RAPA 2 g -1 (pg ml ) 4 IKKI P <.1 6 SB CXCL16 (1 ng ml -1) 1 m 8 SP H&E 1 µm P <.1 P <.1 U126 CXCR6 +/+ Gleason 4+5 e Prostate Tissue Benign (CXCR6 +/+ or CXCR6 -/- mice) NMPE CM: Tramp IKKI d 4 RAPA CAF formation in tumours 8 SP.1 CM: NMPE MSCP2 CXCR6-/MSCP2 12.2 () SB.3 s.c. PCa implantation U126 MSCP2 CXCR6-/MSCP2-1 (pg ml ).4 c P =.19 P =.7 P =.18 P =.37 Vimentin mrna a -SMA mrna a CXCL16 (1ng ml -1) MSC P2 U126 6 4 2 U126 IKKI U126+IKKI 1nM 5nM 1nM IKKI 25nM 5nM U126+IKKI CXCL16 (1ng ml -1 ) Supplementary Figure S2: Conditioned media by prostate cancer cells promote the generation of CAF and by CAFs was regulated through Erk and NF-kB signaling. MSCs isolated from CXCR6 +/+ or CXCR6-/- mice (P2 ) were exposed to murine prostate cancer cell conditioned media for 7 days. The expression of a-sma (a), and vimentin (b) mrna were evaluated. Data in (a,b) are representative of mean with s.d. for triplicates in each of three independent experiments (Student's t-test). (c) Experimental scheme of cells into CXCR6 +/+ or CXCR6-/- mice for examining CAF formation in tumours. (d) H&E staining for human prostate cancer tissue microarray in Fig. 2h. Scale bars, 1 µm. (e) Expression of (green, white arrows) from MSC cells or MSC CXCR6-/- cells were observed following exogenous CXCL16 treatment by IHC. Blue, DAPI nuclrea stain. Scale bars, 1 µm. (f) Erk and NFkB signaling are both required for induction of. production was stimulated by treating MSC cells with the presence or absence of CXCL16 in U126, SB2358 (SB), SP6125 (SP), IKK-2 inhibitor VI (IKKI), or rapamycin (RAPA). production was quantified by ELISA. (g) ELISA confirmed signaling by the inhibitors, U126, IKK-2 inhibitor VI (IKKI), and combination of U126 and IKK-2 inhibitor VI (IKKI). Data in (f,g) are representative of mean with s.d. for triplicates in each of three independent experiments (Student's t-test). (h) IHC staining confirmed (green, white arrows) by the inhibitors, U126, IKK-2 inhibitor VI (IKKI), and combination of U126 and IKK-2 inhibitor VI (IKKI). Scale bars, 1µm. implantation to (c) +/+ CXCR6 or
a b P =.16 a -SMA mrna Vimentin mrna P =.23.5 16 12 8 4 Control CM: MSC.2.1 CM: shcxcl16.4.3 MSC Control MSC shcxcl16 MSC d c Tumour s.c. PCa implantation a-sma/dapi Vimentin CAF formation in tumours (CXCR6 +/+ mice) a -SMA a-sma/ / DAPI DAPI Control Vimentin/DAPI Tumour Vimentin shcxcl16 shcxcl16 Control a -SMA a -SMA a-sma/ a-sma/ / Vimentin/DAPI f Tumour a-sma/dapi a -SMA (Control or shcxcl16 ) e shcxcl16 Control Vimentin Vimentin Vimentin/ Vimentin/ / DAPI Vimentin/ / DAPI Supplementary Figure S3: knockdown of CXCL16 in prostate cancer cells reduces generation of CAF. MSCs isolated from CXCR6 +/+ mice (P 2) were exposed to Control or shcxcl16 cell conditioned media for 7 days. The a- SMA (a) and vimentin (b) mrna were evaluated. Data in (a,b) are representative of mean with s.d. for triplicates in each of three independent experiments (Student's t-test). (c) Experimental scheme of Control or shcxcl16 cell implantation to CXCR6 +/+ mice for examining CAF formation in tumors. (d) IHC of localization of a -SMA and vimentin positive cells within Control or shcxc L16 tumours grown in CXCR6 +/+ mice (red, a -SMA or vimentin, white arrows; blue, DAPI nuclear stain). Scale bars, 1µm. Colocalization of expression with α-sma (e) and vimentin (f) positive cells (white arrows) within Control or shcxcl16 tumours grown CXCR6 +/+ mice. DAPI nuclear stain. Scale bar, 1µm.
a b Prostate Tissue Benign Gleason 4+5 s.c. PCa implantation () H&E EMT formation in tumours (CXCR6 +/+ or CXCR6 -/- mice) c P <.1 P =.2.4.3 P =.3.12 P =.1.8.2.1 P <.1.16 E-Cadherin mrna CXCR4 mrna.5 CXCR4Ab AMD31 CXCR4Ab AMD31 WT EMT e P =.7.4 CXCR4Ab AMD31 CXCR4Ab AMD31 WT EMT P =.1 P =.6 2. N-Cadherin mrna d P <.1 P =.7. 1.6 1.2.8.4 CXCR4Ab AMD31 WT CXCR4Ab AMD31 EMT Supplementary Figure S4: EMT formation in prostate tumours and CXCR4 inhibitors reduce the induction of an EMT phenotype in vitro. (a) Experimental scheme of cell implantation to CXCR6 +/+ or CXCR6 -/- mice for examining EMT formation in tumours. (b) H&E staining for human prostate cancer tissue microarray in Fig. 4d. Scale bars, 1 µm. (c-e) mrna expression of CXCR4, E- cadherin, and N-cadherin in the WT and EMT cells following treatment with CXCR4 inhibitors in vitro. Data in (c-e) are representative of mean with s.d. for triplicates in each of three independent experiments (Student's t-test).
(pg ml ) -1 a IgG/DAPI Control RFP IgG/DAPI b Incubation of WT cells or EMT cells with, AMD31 in vitro i.c. injection WT cells or EMT cells RFP/DAPI RFP/DAPI Tumour metastasis 1 Days (CXCR6 +/+ or CXCR6 -/- mice) c 8 6 4 2 Calvaria Mandible Spine Pelvis Humerus Femur Tibia Blood Supplementary Figure S5: Experimental scheme of prostate tumour metastasis and the levels of secretion in murine tissues. (a) Verification that RFP expression following lentiviral transduction. Expression of RFP protein was confirmed in cells. Scale bars, 1µm. (b) Experimental scheme of WT or EMT cell implantation to CXCR6 +/+ or CXCR6 -/- mice for examining tumour metastasis. (c) levels in murine tissues were identified by ELISA (n = 3 independent experiments).
Supplementary Table S1. Frequency of MSCs recruited into tumours. Exp. Cells Implantation Animal Tumour harvest % MSC cells Fold change P value (day) (mean + s.d.) 4 1 (1x1 ) s.c. CXCR6 +/+ (male) (n=7) 25 19.5 2.1 1 (1x14) s.c. CXCR6 -/- (male) (n=7) 25 12.7 2.3.7.17 2 Cotrol (1x1 4) s.c. CXCR6 +/+ (male) (n=5) 23.8 + -.3 1 shcxcl16 (1x14) s.c. CXCR6 +/+ (male) (n=5) 23.4.1.5.23 3 RWPE-1(2x1 5) orthotopic SCID (male) (n=3) 27.2 + -.1 1 PC3 (2x15) orthotopic SCID (male) (n=4) 27 34.7.9 28.4.1 C4-2B (2x15) orthotopic SCID (male) (n=3) 27.8.1 4.7.1 5 4 NMPE (5x1 ) orthotopic (male) (n=5) 13.3.2 1 (5x14) orthotopic (male) (n=3) 13 1. + -.3 3.1.1 Tramp (5x1 5) orthotopic (male) (n=3) 13.6.1 1.7.22 5 MCF-1A (1x16) orthotopic SCID (female) (n=5) 28. +.1 1 MCF-7 (1x16) orthotopic SCID (female) (n=5) 28.2 + -.1 7.3.7 Significance was determined using a Student's t-test. - -
Supplementary Methods RNA analysis and qrt-pcr. RNA was isolated using RNeasy Mini or Micro Kit (Qiagen, Valencia, CA). First-strand cdna synthesis and qrt-pcr were performed according to the directions of manufacturer (Applied Biosystems, Foster City, CA). Taqman predeveloped assay reagents were used for detection of CXCR6 (Mm472858_m1, Hs174843_m1), CXCL16 (Mm469712_m1, Hs222859_m1), CXCR4 (Mm1292123_m1), E-cadherin (Mm1247357_m1), N-cadherin (Mm483213_m1), α-sma (Mm1546133_m1), vimentin (Mm133343_m1), and β-actin (Mm67939_s1, Hs9999993_m1) (FAM/MGB probes, Applied Biosystems, Carlsbad, CA). Real-time detection of PCR products was performed using an ABI PRISM 77 sequence detector (Applied Biosystems). The qrt-pcr product and mrna expression were normalized to β-actin. RNA interference. Stable cell lines expressing pgipz lentiviral shrna constructs for scrambled and mouse CXCL16 were generated by the University of Michigan Vector Core, Ann Arbor, MI. Plasmids were co-transfected with pspax2 and pmd2.g into HEK-293T cells using CaPO 4. Supernatants were collected after 6 h and used to infect to the cells. Infected cells were selected for 7 days in RPMI medium containing supplemented with 1% FBS and 1 g ml -1 puromycin. qrt- PCR or ELISA analyzed silenced CXCL16. Fluorescence-activated cell sorting isolated the high levels of GFP expressing cells. ELISA. ELISAs were used to quantify and CXCL16 expression in conditioned media and isolated from the extracellular milieu from tumour masses (cat. DY35, DCX16, and DY54, R&D Systems, Minneapolis, MN). In some cases cells were serum-starved overnight, and 1 ng ml -1 CXCL16 or 2 ng ml -1 was added for 24 h either in the presence or absence of selective pathway inhibitors; MEK inhibitor U126 (cat. 6625, Calbiochem, La Jolla, CA), p38 inhibitor SB2358 (SB, cat. 559389, Calbiochem), JNK inhibitor SP6125 (SP, cat. 42119, Calbiochem), IKK-2 inhibitor VI (IKKI, cat. 41483, Calbiochem), and mtor inhibitor
rapamycin (RAPA, cat. 994, Cell Signaling, Danvers, MA). and CXCL16 levels were normalized to total protein (cat. 5-114, BioRad, Hercules, CA). Coculture of Prostate cancer cells with MSCs. cells were cocultured with mouse bone marrow derived-mscs (P 2 ) from CXCR6 +/+ or CXCR6 -/- mice. MSCs (2x1 4 ) from CXCR6 +/+ or CXCR6 -/- mice were placed into the top chambers of 24-well Transwell plates (.4 m, polycarbonate, Corning Life Sciences, Lowell, MA). prostate cancer cells were plated at a final density of 2x1 4 /well in serum-free RPMI into the bottom chambers of the plates. Coculture systems were maintained for 3-7 days. Transwell chemotaxis assays. MSCs (P or P 2 ) from CXCR6 +/+ or CXCR6 -/- mice were resuspended in serum-free α-mem or DMEM and equilibrated for 1 min at 37 C. Cells were loaded into the top chambers of 5-8 μm Transwell microporous membrane 24-well plates (Costar Corp, Cambridge, MA). Mouse CXCL16 protein (-1 ng ml -1, cat. 53-CX, R&D system), (2 ng ml -1, cat. 35- NS, R&D system) or 1% serum were added into the bottom chamber. At 3h the number of cells, which had migrated, was determined by trypan blue. In some cases the cells were labeled with 2.5 μg ml -1 of the lipophilic dye carboxyfluorescein diacetate (CFDA, cat. V12883, Molecular Probes, Eugene, OR) to facilitate enumeration. AMD31, a selective CXCR4 antagonist (4 ng ml -1, cat. A562, Sigma, St. Louis, MO), anti-cxcr4 antibody (25 μg ml -1, cat. MAB21651, R&D Systems) or an IgG control antibody was included.