Supplementary Materials for - Science · 2013-07-10 · 3 Fig. S2. Sympathetic nerve infiltration...
Transcript of Supplementary Materials for - Science · 2013-07-10 · 3 Fig. S2. Sympathetic nerve infiltration...
www.sciencemag.org/content/341/6142/1236361/suppl/DC1
Supplementary Materials for
Autonomic Nerve Development Contributes to Prostate Cancer Progression
Claire Magnon,* Simon J. Hall, Juan Lin, Xiaonan Xue, Leah Gerber, Stephen J. Freedland,
Paul S. Frenette*
*Corresponding author. E-mail: [email protected] (C.M.); [email protected] (P.S.F.)
Published 12 July 2013, Science 341, 1236361 (2013)
DOI: 10.1126/science.1236361
This PDF file includes:
Figs. S1 to S12
Tables S1 to S5
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Fig. S1. Normal prostate epithelium is densely innervated by adrenergic and cholinergic nerve fibers. (A) Adrenergic varicose fibers staining for tyrosine hydroxylase (TH) activity are lined beneath the epithelium of healthy prostate acini within muscle bundles of the stroma. (B) In contrast, cholinergic fibers expressing the vesicular acetylcholine transporter (VAChT) surround epithelial cells, forming close bonds with smooth muscles and epithelial cells. Neural identity was confirmed by staining with the neuron-specific cytoskeletal subunits of neurofilament-L (NF-L, C, D, F) and neurofilament-H (NF-H, E, F). Scale bars, 50 µm.
TH DAPI
VAChT DAPI
NF-‐L DAPI
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NF-‐L NF-‐H DAPI
NF-‐H DAPI
NF-‐L DAPI
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Fig. S2. Sympathetic nerve infiltration in orthotopic xenogeneic PC3-luc tumors. Three representative views of peripheral tumor prostate tissues (t) surrounded by healthy prostate tissues (h), delineated by white dotted lines (right panels). Nearly consecutive frozen sections stained with H&E (A-C) or TH in red (Dapi, blue; D-F) showing discrete TH+ nerve fibers (white arrows) arising from normal areas and infiltrating cancer, 11 weeks after tumor cell injection. Note that large TH+ sympathetic fibers are mainly localized in the healthy compartment, around remaining benign prostate glands, while areas towards the core (c) of the tumor display sparse short fibers suggesting nerve sprouting from the periphery. Scale bars 50µm.
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TH DAPI
th
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Fig. S3. NF-L+ and NF-H+ nerve densities in PC-3luc tumors and in healthy adult prostate glands. Quantification of NF-L+ or NF-H+ neural areas per tumor field within sections of untreated tumor prostates at 11 weeks after xenografting (left, n=11). Quantification of periacinus nerve areas positive for NF-L or NF-H (right, n=3). Note that tumor tissues are mainly infiltrated by NF-L+ fibers. By contrast, healthy (tumor-free) prostate acini are preferentially innervated by NF-H+ mature fibers. LUMPlanFI 60x NA 0.90 ∞ objective, five random fields per animal, field surface=0.01 mm2, ***, P < 0.001. Results are shown as mean ± SEM.
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Fig. S4. Chemical sympathetic denervation induces epithelial cell death in vivo. (A) Immunofluorescence images for TH (left) and VachT (right) and (B) quantification of nerve fiber densities showing that 6OHDA damages specifically TH+ (red, DAPI, blue) four days after the last injection, without any detectable changes on cholinergic fibers (VAChT+). Data were obtained from 5 fields per section from field surface= 0.038 mm2, n=2-3 mice. (C) 6OHDA did not induce any direct cytotoxicity in vitro on cultured PC-3luc cells. (D) Quantification of apoptotic epithelial cells in vivo. Note that 6OHDA triggered apoptosis in healthy prostate. Data were obtained from 5 fields per section from field surface= 0.038 mm2, n=2-3 mice. (E) Representative images of TUNEL staining in prostate from 6OHDA- or PBS-treated mice. DAPI, blue ***, P < 0.001. Error bars indicate standard error. Scale bars 50µm.
Aa TH DAPI VAChT DAPI
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PBS6OHDA
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Fig. S5. Surgical sympathectomy of the prostate gland through selective micro-section of the hypogastric nerve. Immunofluorescence analyses showing the loss of sympathetic fibers (TH+, red) in the prostate of surgically sympathectomized mice (right, compared to a sham-operated mouse, left). DAPI, blue.
TH DAPI
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Fig. S6. The sympathetic control of prostate tumor development does not depend on cancer cell line. (A) In vivo bioluminescence of LNCaP-luc cells orthotopically implanted in the ventral prostate of nu/nu Adrβ2-/-Adrβ3-/- mice (n=8) or nu/nu Adrβ2+/+Adrβ3+/+ control littermates (n=8). (B) Illustrative examples of bioluminescence signal at week 7.
Intensity
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tons/sec X 105)
Adrβ2-‐
/-‐Ad
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Adrβ2-‐/-‐Adrβ3-‐/-‐
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A BOrthotopic LNCaPluc xenografts
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Fig. S7. Sympathetic innervation is predominantly localized in normal prostate area in cancer-bearing Hi-Myc transgenic mouse. Consecutive frozen sections stained with H&E or TH showing malignant (invasive cancer area, top row) or normal (ventral periurethral region, bottom row) areas from a 12-month-old Hi-Myc mouse (TH+, red, DAPI, blue). Scale bars, 50 µm.
TH DAPI
Cancer area
Normal area
Cancer area
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Fig. S8. Expression of muscarinic receptors in the normal prostate and cancer cell lines. Real-time quantitative PCR analyses of mRNA extracts from prostate cancer cell lines and healthy prostate tissues. The prostate gland predominantly expresses Chrm1 whereas PC-3 tumor cells mostly express Chrm3. Data are triplicates with error bars indicating standard error.
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Fig. S9. Cholinergic signals induce the invasion of lymph nodes surrounding the prostate. Representative images of bioluminescent signal in orthotopic prostate tumors (top row) and pelvic lymph nodes (bottom row) from mice described in Fig. 3.
Prostate tu
mor
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node
PZP + Carb Chrm1+/+ + Carb Chrm1-‐/-‐ + CarbCarb
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Fig. S10. Cholinergic stimulation induces prostate tumor cell proliferation in vivo but not in vitro. (A) Incubation of PC-3luc cells in vitro with carbachol does not induce tumor cell proliferation. (B) By contrast, carbachol (Carb) injections induced the proliferation of PC-3luc cells orthotopically implanted into the prostate, as determined by Ki-67 expression. Administration of pirenzepine (PZP), a selective antagonist of the type 1 muscarinic receptor, specifically blocked carbachol-induced tumor cell proliferation. Data obtained from five random fields/section, field surface= 0.01 mm2. n=3 mice per group; **, P < 0.01. Error bars indicate standard error.
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Fig. S11. Overall nerve fiber densities in human prostate tumor specimens. Quantification of nerve fibers by neurofilament-L (NF-L) and neurofilament-H (NF-H) staining in low-risk (n=30) and high-risk (n=13) human prostate adenocarcinomas (same patients shown in Fig. 7). (A) Paired bars represent the average areas for NF-L and NF-H, respectively, for each patient. Each bar represents average nerve densities of a patient obtained from 10 fields per Gleason grade or per normal area, field surface = 0.15 mm2. (B) Double-staining for TH and NF-H (left), VAChT and NF-H (middle) or NF-L and NF-H (right) confirms the neural origin of the fibers. (C) Average of NF-L+ / NF-H+ fiber densities in both normal and cancer tissues of low-risk (Lo, n=30) and high-risk (Hi, n=13) patients. Scale bar, 50 µm. ****p<0.0001.
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Fig. S12. Proliferative indexes of prostate epithelial cells in healthy tissues or prostate tumor cells in human prostatectomy specimens. Quantification of Ki-67+ nuclei in normal or tumor prostate tissue from low-risk (n=30) or high-risk (n=13) patients described in Fig. 7 and 8; table S3 to S5. While proliferative indexes of normal prostate epithelial cells do not change significantly among patients, cancer areas (Gleason grade from 3 to 5) display more proliferative cells in high-risk specimens compared to low-risk prostate tumors.
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Table S1. Primer sequences used for PCR
Human genes Mouse genes Genes Primer sequences Genes Primer sequences CHRM1 For TGACCGCTACTTCTCCGTGACT Chrm1 For CAGAAGTGGTGATCAAGATGCCTAT
CHRM1 Rev CCAGAGCACAAAGGAAACCA Chrm1 Rev GAGCTTTTGGGAGGCTGCTT
CHRM2 For TCACAAAACCTCTGACCTACCC Chrm2 For TGGAGCACAACAAGATCCAGAAT
CHRM2 Rev TCCACAGTTCTCCACCCTACAA Chrm2 Rev CCCCTGAACGCAGTTTTCA
CHRM3 For ACCATCCCTCAACTCCACCAAGT Chrm3 For CCGCTCTACCTCTGTCCTTCA
CHRM3 Rev GGAAAACTGCCTCCATCGTC Chrm3 Rev GGTGATCTGACTTCTGGTCTTGAG
CHRM4 For TCGCTATGAGACGGTGGAAA Chrm4 For GTGACTGCCATCGAGATCGTAC
CHRM4 Rev AGCACAACCAATAGCCCAAG Chrm4 Rev CAAACTTTCGGGCCACATTG
CHRM5 For GAAAGCAGCCCAGACACTGA Chrm5 For GGCCCAGAGAGAACGGAAC
CHRM5 Rev AGCACAACCAACAGCCCAAG Chrm5 Rev TTCCCGTTGTTGAGGTGCTT
ACTB For TGTGATGGTGGGAATGGGTCAG Gapdh For GCATGGCCTTCCGTGTTC
ACTB Rev TTTGATGTCACGCACGATTTCC Gapdh Rev CCTGCTTCACCACCTTCTTGA
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Table S2. Hemodynamic measurements after carbachol administration Control Carbachol P value Intravital microscopy n = 10 vessels Venules Diameter (μm) 35.5 ± 1.2 33.7 ± 1.3 0.45 Blood flow rate (nl/sec)
Vmean x π x d2/4 821 ± 93 863 ± 128 0.25
Wall shear rate (g) 2.12 (8Vmean)/d 397 ± 38 469 ± 59 0.15 RBC velocity (mm/sec) 1.30 ± 0.10 1.48 ± 0.19 0.19 Arterioles Diameter (μm) 38.3 ± 1.2 38.1 ± 0.8 0.45 Blood flow rate (nl/sec)
Vmean x π x d2/4 1518 ± 110 1737 ± 148 0.08
Wall shear rate (g) 2.12 (8Vmean)/d 579 ± 27 698 ± 69 0.05 RBC velocity (mm/sec) 2.06 ± 0.11 2.47 ± 0.22 0.09 Echocardiography n = 4 mice Ejection fraction (%) (Endocardial SV/Endocardial
Vol;d) x 100 63 ± 8 65 ± 4 0.85
Endocardial volume, diastole (μl)
(4π/3) x (End Major;d/2) x {End Area;d/(π(End Major;d/2))}2
35.7 ± 7.9 34.4 ± 7.9 0.87
Endocardial volume, systole (μl)
(4π/3) x (End Major;s/2) x {End Area;s/(π(End Major;s/2))}2
14.8 ± 5.3 11.7 ± 3.0 0.51
Vmean, Mean blood flow velocity; d, diameter; SV, Stroke Volume which is calculated by subtraction of the left ventricle end-‐systolic volume from the left ventricle end-‐diastolic volume; Vol, volume; End Major, endocardial major represent the maximal ventricular length; End Area, endocardial area ; s, systole; d, diastole.
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Table S3. Clinical and pathological characteristics of men with prostate cancer Patient characteristics Tumor characteristics
# Age (years)
Race Risk PSA (ng/ml)
Date of the
surgery
Clinical stage
Pathological stage
Recurrence 0-‐/1+
1 60 Black Low 8.4 2007 T1c T2a 0 2 60 White Low 7.8 2007 T1c T2c 1 3 61 White Low 5.4 2006 T1c T2b 0 4 50 White Low 4.9 2007 T1c T2c 0 5 61 Black Low 6.1 2007 T1c T2c 0 6 58 White Low 3.2 2005 T1c T2c 0 7 51 White Low 5.17 2005 T1c T2a 0 8 59 White Low 6.4 1999 T1c T2c 0 9 66 White Low 4 2001 T2a T2a 0 10 69 White Low 4 2006 T2b T2c 0 11 60 White Low 5.9 2001 T1c T2c 0 12 60 White Low 6.1 1999 T1c T2a 1 13 63 Black Low 4.7 2010 T1c T2c 0 14 50 Black Low 4.4 2008 T1c T2c 0 15 64 White Low 8.24 2003 T1c T2c 0 16 51 Black Low 3.7 2006 T1c T2c 0 17 61 Black Low 5.26 2010 T1c T2c 0 18 63 White Low 3.6 2006 T1c T2c 0 19 60 White Low 7.9 2008 T1c T2c 0 20 48 White Low 6.18 2003 T2a T2c 0 21 66 Asian Low 5.9 2001 T1c T2c 0 22 59 White Low 4.4 2004 T1c T2c 0 23 72 White Low 5.64 2002 T1c T2c 0 24 58 Black Low 4.5 2006 T1c T2c 0 25 59 Black Low 1.6 2010 T1c T2c 0 26 61 White Low 3.77 2005 T2a T2c 0 27 54 White Low 5.1 2005 T1c T2c 0 28 55 Black Low 2.68 2002 T1c T2c 0 29 60 White Low 4.6 2006 T1c T2a 0 30 53 Black Low 2.04 2001 T1a T2c 0 31 61 Black High 23.2 2008 T1c T3b 1 32 59 Black High 13.1 2009 T2 T3b 1 33 65 White High 50.7 2001 T1c T3a 1 34 65 White High 5.8 2009 T2b T3b 1 35 62 Black High 45.1 1999 T2a T3b 1 36 65 White High 12.7 2000 T2a T3a 1 37 60 White High 75.35 2004 T1c T3a 0 38 69 Black High 13.85 2003 T1c T3c 1 39 53 White High 5.51 2009 T2a T3a 0 40 66 Black High 5.1 2008 T1c T3a 0 41 60 White High 20.8 2004 T1c T4a 0 42 69 White High 6.2 2006 T1c T3a 0 43 58 White High 20.3 2003 T2a T3c 0
Low-‐risk prostate cancer was defined as prostate-‐specific antigen (PSA) levels <10 ng/ml, Gleason score <7, and stage T1c or T2a disease. High-‐risk (shaded grey) was defined as PSA levels ≥10 ng/ml, Gleason score ≥7, or disease stage ≥T2b.
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Table S4. Cox proportional hazards model for association between nerve densities and biochemical tumor recurrence Nerve type Healthy prostate tissue
HR100 95% CI p value Tumor prostate tissue
HR100 95% CI p value TH+ 1.108 1.036-‐1.485 0.0029 0.916 0.674-‐1.243 0.5719 VAChT+ 0.907 0.160-‐ 5.139 0.9117 1.283 0.974-‐ 1.690 0.0768 NF-‐L+ 1.258 1.082-‐1.461 0.0028 1.311 1.057-‐1.627 0.0138 NF-‐H+ 1.327 1.093-‐1.610 0.0043 1.326 1.069-‐1.646 0.0103 Recurrence was defined as a biochemical recurrence with PSA >0.2ng/ml, or two values at 0.2 ng/ml, or a secondary treatment for a rising PSA. Abbreviations: HR100, Hazard Ratio per 100 units; CI, Confidence intervals. Regression model was adjusted for race. Table S5. Logistic regression model for association between nerve densities and extra-‐prostatic extension Nerve type Healthy prostate tissue
OR100 95% CI p value Tumor prostate tissue
OR100 95% CI p value TH+ 1.553 1.077-‐2.239 0.0183 1.147 0.878-‐1.498 0.3146 VAChT+ 2.131 0.406-‐11.176 0.3708 26.830 2.356-‐305.527 0.0080 NF-‐L+ 4.242 1.584-‐11.360 0.0040 8.724 1.782-‐42.716 0.0075 NF-‐H+ 5.610 1.433-‐21.970 0.0133 7.003 2.037-‐24.079 0.0020 Extra-‐prostatic extension was defined as disease involving one or more of extracapsular, bladder neck, or seminar vesicle extension. Abbreviations: OR100, Odds Ratio per 100 units; CI, Confidence intervals. Regression model was adjusted for race.