Supp. Table 1: Summary of RT4 cell recombinant data. Supp. Figure 1: Controls for adenoviral infection of bladder epithelium

Transcription

1 Supplementary Materials for: Inactivation of p53 and Pten promotes invasive bladder cancer Puzio-Kuter et al. Materials & Methods Tables Supp. Table 1: Summary of RT4 cell recombinant data Figures Supp. Figure 1: Controls for adenoviral infection of bladder epithelium Supp. Figure 2: Additional examples of histology of mouse bladder tumors Supp. Figure 3: Immunohistochemical analyses of mouse bladder tumors Supp. Figure 4: Gene deletion in mouse bladder tumors Supp. Figure 5: Histology of bladder epithelium in the single and compound heterozygous p53 flox/flox and Pten flox/flox mutant mice Supp. Figure 6: Bladder phenotype of p53 flox/flox ; Rb flox/flox and Pten flox/flox ; Rb flox/flox mutant mice Supp. Figure 7: RT4 cell recombinants following knock-down of p53 or PTEN Supp. Figure 8: Pre-clinical treatment study 1

2 Supplementary Materials and Methods Mouse model The R26R reporter allele (GT(ROSA)26Sor tm1sor ) (C57Bl6;129SVJ) (Soriano 1999) was obtained from the Jackson Laboratory Induced Mutant Resource (Bar Harbor, Maine). Conditional alleles for Pten (Pten flox/flox, C57Bl6;129SVJ) (Lesche et al. 2002) and p53 (p53 flox/flox, FVB;129SJv) (Jonkers et al. 2001) were obtained from the NCI Mouse Models of Human Cancer Consortium repository ( An adenovirus expressing Cre-recombinase (Adeno-Cre) and control vector (Adeno-empty) were obtained from the University of Iowa Vector Core Facility (Ad5CMVCre). Concentrated virus (25 µl; 4 X10 11 PFU/ml) was mixed with 20 µl of Dulbeccoʼs Modified Eagle Medium (D-MEM) and 5 µl of hexadimethrine bromide (polybrene, 80 µg/ml), and 5 µl of diluted virus was injected into the bladder lumen of adult mice (2-3 months of age) under anesthesia; unless otherwise indicated, male mice were used. At the time of sacrifice, the bladder and relevant tissues were collected and fixed in 10% formalin or snap-frozen in liquid nitrogen. Real-time PCR was done using RNA extracted with Trizol (Invitrogen Carlsbad, California), SuperScript III Reverse Transcriptase (Invitrogen Carlsbad, California ) for 1 st strand cdna synthesis and real-time one step RT-PCR with QuantiTect SYBR Green RT-PCR kit (Qiagen Valencia California ) on a Eppendorf Mastercycle ep realplex 2 S (Eppendorf, Westbury New York). Analyses of bladder tissues Hematoxylin and Eosin (H&E) and immunohistochemical staining were performed on 5-micron paraffin sections. Immunohistochemistry was performed as described (Kinkade et al. 2008) following antigen retrieval in Antigen Unmasking Solution (Vector Laboratories H-3300; Burlingame, California). Slides were blocked and 2

3 incubated overnight at 4 C with primary antibody (see below) followed by secondary antibody (Vector Laboratories, Biotinylated Horse Anti-Mouse IgG (H+L) #BA_2000 or Biotinylated Goat Anti-Rabbit IgG (H+L) #BA_1000) diluted 1:250 or 1:500. Signal enhancement was performed using the Vectastain ABC System, followed by counterstaining with the NovaRed kit (Vector Laboratories). Quantification of proliferating cells with Ki67 was performed as previously (Kinkade et al. 2008); results represent a minimum of 10 sections from 3 mice and are expressed as the percentage of Ki67- labeled epithelial cells relative to total epithelial cells. Western blot analyses were performed using tissues prepared in RIPA buffer as described (Kinkade et al. 2008). Antibodies for immunohistochemistry were as follows: Beta-galactosidase (Rockland rabbit polyclonal, ; 1:20000); Cytokeratin 7 (Abcam mouse monoclonal, ab9021; 1:100 for IHC and 1:50 for IF); Broad Spectrum Cytokeratin (Dako Cytomation rabbit polyclonal, Z0622 1:10); Pten (Cell Signaling rabbit polyclonal, 9552; 1:200), p-akt (Cell Signaling Ser473 rabbit monoclonal, 3787; 1:50); p-s6 Ribosomal Protein (Cell Signaling Ser235/236 rabbit polyclonal, 2211; 1:250); Ki-67 (NovoCastra rabbit polyclonal, NCL-Ki67-p; 1:2000). Antibodies for immunoblotting were as follows: p53 (Santa Cruz rabbit polyclonal, sc6243; 1:500); Pten (Cell Signaling rabbit polyclonal, 9552; 1:500); p-akt (Cell Signaling Ser473 polyclonal 9271, 1:500); p-mtor (Cell Signaling Ser2448 rabbit polyclonal, 2971; 1:100); mtor (Cell Signaling rabbit polyclonal, 2983; 1:100); p-s6 Ribosomal Protein (Cell Signaling Ser235/236 rabbit polyclonal, 2211; 1:500); Raptor (Bethyl Laboratories Inc. rabbit polyclonal, A A; 1:100). Immunofluorescence was done using 5-micron paraffin sections or 12 micron cryosections, which were subjected to antigen retrieval by boiling in Antigen Unmasking Solution (H-3300, Vector Laboratories Burlingame, California). Slides were blocked in 3

4 0.1% blocking solution (Invitrogen/Molecular Probes TSA Kit #2 T or TSA Kit #12 T-20922) followed by incubation in primary antibody diluted in 0.1% blocking solution in a humid chamber overnight at 4 C. Slides were then incubated with secondary antibody (Invitrogen/Molecular Probes Carlsbad, California AlexaFluor 555 goat anti-mouse IgG (H+L) #A or AlexaFluor 488 goat anti-rabbit IgG (H+L) #A ) for 1 hour in a dark humid chamber at room temperature and mounted with Vectashield+DAPI (Vector Laboratories # H-1200). If tyramide amplification was used, slides were incubated with secondary antibody HRP goat anti-mouse IgG or HRP goat anti-rabbit IgG (Invitrogen/Molecular Probes Carlsbad, California TSA Kit #2 with HRPgoat anti-mouse IgG and Alexa Fluor 488 tyramide #T or TSA Kit #12 with HRP-goat anti-rabbit IgG and Alexa Fluor 488 tyramide #T-20922) for 1 hour in a dark humid chamber at room temperature followed by incubation with Alexa 488 tyramide (Invitrogen/Molecular Probes Carlsbad, California TSA Kit #2 with HRP-goat antimouse IgG and Alexa Fluor 488 tyramide #T or TSA Kit #12 with HRP-goat anti-rabbit IgG and Alexa Fluor 488 tyramide #T-20922) for 10 minutes at room temperature at RT and mounted with Vectashield+DAPI (Vector Laboratories # H-1200). Immunofluorescence was visualized using a Zeiss LSM 510 META inverted confocal microscope equipped with argon and helium/neon lasers using excitation wavelengths 488 and 543nm. Cell recombination assays For cell recombination assays, RT-4 human bladder cells (ATCC) were grown in McCoyʼs 5a Medium with 1.5 mm L-glutamine and 2.2 g/l sodium bicarbonate supplemented with 10% fetal bovine serum. For knock-down studies, lentiviral particles expressing RNAi for p53 and/or Pten (or control) were obtained from the Mission shrna 4

5 library (Sigma-Aldrich St. Louis, Missouri). For each gene, four alternative RNAi constructs were evaluated, and the two that produced the most significant knock-down (>50%) were selected for analyses (Supp. Table 1). Cells were infected with lentiviral particles at 5 MOI at a low (~20%) density for 12 hours in growth media. Procedures for generating cell recombinants were adapted from (Oottamasathien et al. 2006). Briefly, following lentiviral infection, 1 X 10 5 of the bladder epithelial cells were recombined with rat mesenchyme from a 15.5 dpc embryo obtained from Sprague- Dawley pregnant rats. The recombinants were mixed with 40 µl collagen and plated on Falcon dishes (BD Biosciences/Falcon, San Jose, CA ). Following overnight incubation at 37 C, cell recombinants were surgically implanted under the kidney capsule of NCr nude mice (Taconic) and grown for 2 to 3 months. Following sacrifice, the kidneys were removed and recombinants were fixed in 10% formalin and processed for histological and immunohistochemical analyses as above. Tissue recombinants were made as described for the cell recombinants, with the exception that bladder tumors from the mutant mice provided the source of epithelium. Pre-clinical studies Rapamycin (LC labs Catalog #R-5000, lots #ASW-105 and #ASW-109) was dissolved in 100% ethanol to make a working stock of 25 mg/ml, and diluted to 1.25 mg/ml in a solution of 5.2% Tween 80, 5.2% PEG400. Rapamycin was delivered once daily via i.p. injection at 10 mg/kg into cohorts of Adeno-Cre injected p53 flox/flox ; Pten flox/flox mice or nude mice harboring bladder tumor tissue recombinants as described (Kinkade et al. 2008). At the conclusion of the study, mice were sacrificed and processed for histological analyses as above. 5

6 Analyses of human tissue microarrays Human tissues were obtained following guidelines of the Institutional Review Board. Two independent cohorts were used in this study. The first included 165 patients diagnosed with bladder cancer from whom paraffin embedded specimens had been previously obtained. These included 136 cases of transitional cell carcinoma and 16 cases of squamous cell carcinoma; the remaining 13 cases corresponded to anaplastic, small cell, sarcomatoid or signet ring carcinomas. Tumor stage was assigned according to the Tumor-Node-Metastasis staging system (Sobin 2002) on the basis of the depth of invasion in transurethral resection or cystectomy specimens. Tumors were classified as follows: 1 case of ptcis; 23 cases of pta; 28 cases of pt1; 25 cases of pt2; 75 cases of pt3; 8 cases of pt4, and 6 cases of ptx. 97 tumors showed a high grade (G3) histology. 85 patients were treated by radical cystectomy and pelvic lymphadenectomy with the following N staging: 31 pn0, 54 pn1-2. The second cohort included 86 patients with muscle-invasive bladder cancer (median age 69 years, range 25-86) undergoing radical cystectomy. For these patients, clinical follow-up data was available and entered into a prospective database. Median follow-up time after cystectomy was 6.6 years. Normal bladder tissue samples (N=10) were obtained from non-affected, distant urothelium from cystectomy specimens. For immunohistochemistry, TMA sections (5 µm) processed using the avidin-biotin immunoperoxidase method as above. Antibodies were as follows: p53 (Calbiochem mouse monoclonal, OP09L; 1:500); Pten (Cascade mouse monoclonal, ABM2052; 1:75); p-akt (Ser473 rabbit monoclonal, 3787; 1:50); p70 S6 Kinase (Rabbit polyclonal, 9202; 1:75) from Cell Signaling. Staining was scored by estimating the percentage of tumor cells with immunoreactivity as well as the intensity of the staining was determined using an ordinal 6

7 scale (undetectable = 0, weak = 1, strong = 2, very strong = 3). If different staining intensities were found in a given sample, the most intense (>25% of cells) was used for analysis. Statistical analyses were done using the Mann-Whitney U test, the chix 2 test, or Fisherʼs exact test, and the Spearman correlation, as appropriate. Survival analysis (Kaplan-Meier) was conducted by the log rank test and the Cox proportional hazard model. All calculations were performed using StatView 4.5 software (Abacus Concepts, Inc., Berkeley, CA). References Jonkers, J., Meuwissen, R., van der Gulden, H., Peterse, H., van der Valk, M., and Berns, A Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat Genet 29: Kinkade, C.W., Castillo-Martin, M., Puzio-Kuter, A., Yan, J., Foster, T.H., Gao, H., Sun, Y., Ouyang, X., Gerald, W.L., Cordon-Cardo, C., and Abate-Shen, C Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model. J Clin Invest 118: Lesche, R., Groszer, M., Gao, J., Wang, Y., Messing, A., Sun, H., Liu, X., and Wu, H Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene. Genesis 32: Oottamasathien, S., Williams, K., Franco, O.E., Thomas, J.C., Saba, K., Bhowmick, N.A., Staack, A., Demarco, R.T., Brock, J.W., 3rd, Hayward, S.W., and Pope, J.C.t Bladder tissue formation from cultured bladder urothelium. Dev Dyn 235: Sobin, L TNM Classification of Malignant Tumours. John Wiley & Sons, Hoboken, NJ. Soriano, P Generalized lacz expression with the ROSA26 Cre reporter strain. Nat Genet 21:

8 Supplementary Table 1: Summary of cell recombinants of RT4 human bladder cancer cells Experimental groups N Lentivirus RNAi Description Control 4 Control Moderate size grafts with well-organized morphology and histological appearance of of low grade transitional cell carcinoma p53 RNAi 7 Pten RNAi 8 p53 + Pten RNAi 9 p53(h)-clone 1 (N=4) p53(h)-clone 2 (N=3) Pten(h)-clone 1 (N=3) Pten(h)-clone 2 (N=5) p53(h)-clone 1 + Pten(h)-clone 2 (N=5) p53(h)-clone 2 + Pten(h)-clone 2 (N=4) Moderate size grafts with well-organized morphology and histological appearance of of low grade transitional cell carcinoma Moderate size grafts with well-organized morphology and histological appearance of of low grade transitional cell carcinoma Large size grafts with invasive features; histological appearance of high grade transitional cell carcinoma with invasion of the surrounding mesenchyme and host kidney Details of Lentiviral RNAi used in this study Gene Name Species Product number 1 Region targeted Abbreviation Control N/A Control-SHC002V Scrambled Control p53 PTEN Human Human TRCN Coding region p53(h)-clone 1 TRCN Coding region p53(h)-clone 2 TRCN Coding region Pten(h) clone 1 TRCN ʼ UTR Pten(h) clone 2 Notes: (1) Lentiviral particles were purchased from Sigma Aldrich MISSION RNAi.

9 Supplementary Figure Legends Supplementary Figure 1: Controls for Adeno-virus Cre injection Shown are examples of β-gal expression staining or H&E staining from sections from p53 flox/flox ; Pten flox/flox ; R26R/+ mice that were injected or not injected with Adeno-Cre. These are control experiments showing that the tumors in the p53 flox/flox ; Pten flox/flox mice are dependent on Adeno-Cre delivery (compare injected and non-injected) and that tumors in the mice having the R26R allele have B-Gal staining and are therefore derived from the bladder epithelium. Supplementary Figure 2: Histology of tumors arising from the p53 flox/flox ; Pten flox/flox mice injected with Adeno-Cre Examples of H&E staining from tumors showing additional examples of the histological phenotypes from bladder tumors from the p53 flox/flox ; Pten flox/flox mice at 4 months following delivery of Adeno-Cre. Supplementary Figure 3: Immunohistochemical analyses of mouse bladder tumors (A-D) Immunohistochemical staining with the indicated antibodies of sections from normal bladder (p53 +/+ ; Pten +/+ ; A, C) or bladder tumors (p53 flox/flox ; Pten flox/flox ; B,D). (E-G) Metastases from p53 flox/flox ; Pten flox/flox mice with large tumors showing gross phenotype (E), and sections from a liver metastases showing H&E staining (F), and immunohistochemical staining for broad cytokeratin (G). Scale bars represent 100 microns. 8

10 Supplementary Figure 4: Gene deletion of Pten and p53 in the mouse bladder tumors Data showing that Pten and p53 are effectively deleted in the mouse bladder tumors. Shown are Real-time PCR analyses (left) and western blot analyses (right) comparing expression in normal bladder versus in the mouse bladder tumors. Supplementary Figure 5: Histology of single and compound heterozygous mutant mice. Examples of H&E staining from mice of the indicated genotypes at 4 months following delivery of Adeno-Cre showing no obvious histological abnormalities. Supplementary Figure 6: Analyses of Analyses of bladder phenotype in Adeno-Cre injected p53 flox/flox ; Rb flox/flox and Pten flox/flox ; Rb flox/flox mutant mice Examples of the gross bladder tumors and H&E staining from mice of the indicated genotypes at 8-12 months following delivery of Adeno-Cre into the bladder lumen showing no gross bladder tumors or gross histological abnormalities. Supplementary Figure 7: RT4 cell recombinants from the single knock-down of p53 or Pten (Top) Diagram of the functional analyses of p53 and PTEN knock-down in human bladder cells. RT4 human bladder cancer cells were infected with lentiviral RNAi (MOI=5) for p53 and/or PTEN, recombined with rat embryonic bladder mesenchyme, 9

11 and grown under the renal capsule of a nude mouse for 3 months. Right, Western blot showing the efficacy of knock-down using two different RNAis. (Bottom) Renal grafts from cell recombinants made following knock-down in RT4 cells of p53 or Pten individually with lentiviral RNAi showing that the histology is similar to the control RT4 cells (see Fig. 3). Supplementary Figure 8: Pre-clinical analyses (A-D) Immunostaining for activated p-akt and p-s6 kinase in non-invasive or invasive human bladder cancer. (E-N) Treatment Study: Mouse bladder tumors were combined with rat embryonic bladder mesenchyme, and grown as renal grafts in nude mouse hosts; one week following grafting, the mice were randomly enrolled into vehicle and rapamycin groups and delivered rapamycin (or vehicle) via daily i.p. injection for up to 3 months. (E,F) Gross morphology. (G,H) H&E stained sections; (I-L) immunostained sections. (M) Summary of bladder weights. (N) Summary of proliferation rates. Scale bars represent 100 microns. 10

12 Supp. Figure 1 11

13 Supp. Figure 2 12

14 Supp. Figure 3 13

15 Supp. Figure 4 14

16 Supp. Figure 5 15

17 Supp. Figure 6 16

18 Supp. Figure 7 17

19 Supp. Figure 8 18