Identification of Different Classes of Luminal Progenitor Cells within Prostate Tumors

Transcription

1 Cell Reports Supplemental Information Identification of Different Classes of Luminal Progenitor Cells within Prostate Tumors Supreet Agarwal, Paul G. Hynes, Heather S. Tillman, Ross Lake, Wassim G. Abou-Kheir, Lei Fang, Orla M. Casey, Amir H. Ameri, Philip L. Martin, Juan Juan Yin, Phillip J. Iaquinta, Wouter R. Karthaus, Hans C. Clevers, Charles L. Sawyers, and Kathleen Kelly

2 Figure S1, related to Figure 1 WT Pten -/-,TP53 -/- Sca-1 - PE EpCAM - APC WT Pten -/-,TP53 -/- CD24 - PE EpCAM - APC

3 Figure S2, related to Figure 1 FACS Gating Strategy Viability Doublet Discrimination Lineage neg Selection Post-Sort Purity Scans Pre-Sort Sample CD49f hi CD49f - PE CD49f - PE PROM1 + PROM1 - APC EpCAM pos Selection CD49f lo PROM1 - APC

4 Figure S3, related to Figures 2, CD49f hi Sphere Formation PROM1 + Ductal Structure Formation PHASE PHASE

5 Figure S4, related to Figure 4 KRT8 PROM1 + Adenosquamous Tumor KRT5 DAPI KRT5/KRT8/DAPI

6 Figure S5, related to Figure 6 A B % ClC3 Positive Cell mrna Levels Clu Intact Castrated Post castration (days) 0.0 CD49f hi PROM1 + CD49f lo Cell Fraction TP63 C %OFU NS 40 Intact Castrated CD49f hi mrna Levels CD49f hi PROM1 + CD49f lo Cell Fraction Intact Castrated

7 SUPPLEMENTAL FIGURE LEGENDS Figure S1. FACS separation profiles for SCA-1 and CD24. Representative FACS plots of Sca-1 and EpCAM (top panel) and CD24 and EpCAM (bottom panel) co-staining of primary WT and Pten -/-,TP53 -/- prostate cells. Figure S2. FACS gating strategy. FACS gating strategy for isolation of primary Pten -/-,TP53 -/- prostate cells based on CD49f and PROM1 expression. Post-purity scans demonstrate purity of cell fractions. Figure S3. Time lapse images of organoid development. (A) Time-lapse images of Pten -/-,TP53 -/ - CD49f hi organoid formation. (B) Time-lapse images of Pten -/-,TP53 -/ - PROM1 + organoid formation leading to a twined structure. Organoid formation was imaged hourly over 4 days. Representative images are shown at 6 hour increments over 4 days. Figure S4. Individual keratin staining profiles in an adenosquamous tumor. KRT5, KRT8, and DAPI labeling of a representative adenosquamous tumor section. Note double-labeled cells within several areas of adenocarcinoma and squamous transitions (arrowheads). Figure S5. Histopathological and gene expression consequences of castration in Pten - /- Tp53 -/- tumors. (A) Quantification of cleaved CASP3 (ClC3) positive cells in Pten -/-,Tp53 -/- prostate tissue between 0-42 days post-castration. (B) qrt-pcr analysis of Clu and TP63 expression in CD49f hi, PROM1 + and CD49f lo cell fractions obtained from intact and 14 day castrated Pten -/-,Tp53 -/- prostate tissue. (C) G1 organoid formation from intact and castrated Pten -/-,Tp53 -/- CD49f hi cells. Data is reported as mean % OFU ± SEM. Movie S1 Time-lapse movie of CD49f hi organoid growth. Growth was captured hourly over 105 hours. Movie S2 Time-lapse movie of Prom-1 + organoid growth. Growth was captured hourly over 105 hours.

8 Table S1 Primer Itga6 (CD49f) For Itga6 (CD49f) Rev PROM1 For PROM1 Rev KRT 5 For KRT 5 Rev KRT 14 For KRT 14 Rev KRT 18 For KRT 18 Rev TP63 For TP63 Rev GAPDH For GAPDH Rev Clu For Clu Rev FKBP5 For FKBP5 Rev Sequence ACGGTGTTTCCCTCAAAGAC GAAGAAGCCACACTTCCACA CACCAACACCAAGAACAAGG TTGGTCTGTTTGATGGCTGT AACGTCAAGAAGCAGTGTGC TCCAGCTCTGTCAGCTTGTT GATGACTTCCGGACCAAGTT TGAGGCTCTCAATCTGCATC TTGCGAATTCTGTGGACAAT ATCTACCACCTTGCGGAGTC CAGTCAAGCACTGCCAAGTC CATCACCTTGATCTGGATGG CAGAACATCATCCCTGCATC CTGCTTCACCACCTTCTTGA GCCAGCAGCTAGAGGAGTTT CATCCAGGACTTGGCTCTG CTGTGGTGGAAGGACATTTG AAACCATAGCGTGGTCCAA

9 Table S2 Antibody Target Supplier & Cat # KRT5 Covance (PRB-160P) ; (SIG-3475) KRT8 Covance (MMS-162P) PTEN Cell Signaling (9188) TP63 Santa Cruz (sc-8431) ; (sc-8343) AR Abcam (ab133273) KI67 Cell Signaling (9449) Cleaved CASPASE 3 Cell Signaling (9661) S6 240 Cell Signaling (4838)

10 SUPPLEMENTAL EXPERIMENTAL PROCEDURES RNA Extraction, cdna Amplification and Quantitative Real-Time PCR RNA extraction, cdna synthesis and qrt-pcr were performed as previously described (Casey et al., 2012). Primers used are listed in Supplementary Table 1. Subcutaneous Tumor Initiation Assays FACS sorted cells were counted and re-suspended in PrEGM medium (Lonza) with 50% MatrigelTM (BD Biosciences). All subcutaneous injections were performed in four to six week old NOD/SCID mice. Mice that showed no evidence of tumor formation after 9 months were considered negative for tumor initiation. Histology, Immunohistochemistry and Immunofluoresence. Cells were prepared for cytospin staining as previously described (Abou-Kheir et al.). Preparation of prostate tissue and subcutaneous tumors for histological analysis was performed as previously described (Abou-Kheir et al., 2010). Immunohistochemical and immunofluorescence staining was performed as previously described (Abou-Kheir et al., 2010). Antibodies used for staining are listed in Supplementary Table 2. Confocal microscopy was performed using a Carl Zeiss LSM 510 Meta Mk4 confocal microscope with a Plan-Apochromat 63x/, NA 1.40 DIC oil objective. Images were acquired and analysed using Carl Zeiss LSM image software. Time-lapse imaging was performed using an Olympus Viva View FL Incubator Microscope with a 40X UPLSAPO40X, NA 0.95 objective. Images were acquired using Olympus metamorph software. Brightfield and multi-channel fluorescence imaging was performed using either a Carl Zeiss Axio Observer.Z71 inverted microscope with EC Plan Neofluar 40x,

11 NA 1.3 oil and N-Achroplan 10x, NA 0.25 objective and Axiovision software; a Carl Zeiss Axio Imager.1 microscope with Plan Neofluar 40X, NA 0.7 objective and Axiovision software; or a Carl Zeiss Axio Scan.Z1 slide scanner with a Plan-Apochromat 20X, NA 0.8 objective and Zen software. 2