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Supplementary Materials & Methods
Establishment of androgen-dependent (AD) and androgen-independent (AI) PCa xenograft
tumors
Basic procedures were previously described (1, 2). In brief, AD xenograft tumors were routinely
maintained in intact NOD/SCID mice (1, 2). To establish AI tumor lines, parental tumor cells were
purified from AD tumors, mixed with Matrigel and injected subcutaneously (s.c) in castrated
NOD/SCID mice, and then serially passaged in castrated mice.
Immunohistochemistry (IHC) and immunofluorescence (IF) staining in formalin-fixed
paraffin-embedded (FFPE) sample
Basic IHC and IF procedures have been detailed (3). For IHC, FFPE sections (including a tissue
microarray/TMA containing twenty CRPC patient sample), ref. 1) were cut (4 μm), deparaffinized in
xylene and hydrated in gradient alcohols to water, followed by antigen retrieval in 10 mM citrate
buffer (pH 6.0). For IF, PCa xenograft sections (4 μm) were deparaffinized, dehydrated via graded
alcohols followed by antigen retrieval in a pre-warmed target retrieval solution (S1699, Dako) in
boiling for 40 min. For both IHC and IF, slides were incubated with primary antibodies
(Supplementary Table S1) followed by secondary antibodies and DAPI counterstaining (when
applicable). Images were captured on an Olympus inverted epifluorescence microscope.
Purification of human PCa cells from xenograft tumors and primary human PCa (HPCa)
sample processing
Xenograft tumor processing was previously described (1-3). Xenograft tumors were harvested and
minced into ~1 mm3 pieces, rinsed with PBS and digested with 1x Accumax (1200-2000 U/ml
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proteolytic activity containing collagenase and DNase; Innovative Cell Technologies) for 30 min at
room temperature (RT) under rotating conditions. Cell suspension was then filtered through a prewetted 40-μm strainer, and single cells were loaded onto a layer of Histopaque-1077 (sigma)
gradient to deplete dead cells and debris.
Primary HPCa samples (Supplementary Table S2) with >75% tumor involvement were
obtained with the written informed consent from patients in accordance with federal and institutional
guidelines and under the coverage of IRB (Institutional Review Board) Protocol number LAB040498. Basic processing procedures for HPCa samples were processed as previously described (4).
Tumors were cut into small chunks, washed by PBS, and digested with collagenase/dispase digestion
solution (1 mg/ml each) at 10 ml per gram in a 37°C incubator overnight with continuous rotation.
After 8-12 h, cell mixture was washed by PBS, incubated with 0.05% Trypsin/EDTA (Gibco) for 510 min at 37°C followed by a 5-min treatment in DNase I digestion solution (0.02 mg/ml). The
resultant cell/medium mixture was triturated with an 18-G and/or 20-G needle for 5-10 times,
filtered through a 100-μm strainer, subjected to a 5-min treatment of a 1x RBC lysis buffer
(BioLegend) and passed through a 40-μm strainer to obtain single-cell suspension.
Fluorescence-activated cell sorting (FACS) and purification of single, double and/or triplemarker stained cell populations
Basic procedures for FACS were described in our previous publications (1, 2). In brief, xenograft
tumor-derived single cells were incubated with the FcR blocking reagent (Miltenyi Biotec) for 10
min at 4°C and then incubated with a primary antibody against α2β1 for 30 min at 4°C followed with
an APC-conjugated goat anti-mouse IgG antibody for 30 min at 4°C. Cells were washed with PBS
and incubated with a PE-conjugated anti-CD44 antibody and a biotinylated mouse H-2K[d] antibody
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for 30 min at 4°C. After PBS wash, cells were incubated with an Alexafluor 405-conjugated
streptavidin antibody for 15 min at 4°C. In all above staining steps, cells were incubated in a staining
buffer consisting of 1% BSA and 5 μg/ml insulin (Sigma). Subsequently, PCa cells were washed and
incubated with ALDEFLUOR assay buffer containing ALDH substrate (StemCell Technologies,
#01700) (1 μM/1 million cells) for 45 min at 37°C. As negative control, diethylaminobenzaldehyde
(DEAB) was added to cell suspension per protocol from the manufacturer. For primary patient tumor
(HPCa) samples (Supplementary Table S2), single HPCa epithelial cells were sorted into the CD45 Trop2+ cell population (5), which was then incubated with primary antibodies against integrin α2β1
and CD44 followed by incubation with the ALDEFLUORTM kit. Briefly, single HPCa cells were
incubated with primary antibodies against α2β1 for 30 min at 4°C followed with a Alexafluor 405conjugated goat anti-mouse IgG antibody for 30 min at 4°C. HPCa cells were washed with PBS and
incubated, sequentially, with a PE-conjugated anti-CD44 antibody, an APC-conjugated anti-Trop2
antibody and a APC-efluor 780-conjugated CD45 antibody for 30 min at 4°C. The following steps
for ALDH reaction were the same as in xenograft tumor cells. Single, double, and triple-marker
stained cell populations were sorted on FACSAria II or FACSAria Fusion (BD Biosciences), and
post-sort analysis was routinely carried out to check the purity of each population.
Clonal, clonogenic and sphere-formation assays in androgen-deprived conditions
For clonogenic assays (1, 2), we plated sorted cells at various numbers in Matrigel at 1:1 ratio. For
sphere-formation assays (1, 2), 1,000 -10,000 sorted cells were plated in ultra-low attachment
(ULA) plates. In both assays, cells were cultured in IMDM plus 15% charcoal-dextran stripped FBS
(i.e., CDSS, Gemini) and 10 μM enzalutamide (Selleck Chemicals) (LAPC9 or LAPC4), or in
CDSS-containing prostate epithelial basal medium (PrEBM) supplemented with 4 μg/ml insulin,
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B27 (Invitrogen), 20 ng/ml EGF. 20 ng/ml bFGF and 10 μM enzalutamide (PC3). Both clonogenic
and sphere-formation assays were repeated for ≥3 generations. For clonal assays in primary HPCa
cells (2), sorted cells were plated at a clonal density of 1,000-2,000 cells/well in a 6-well dish coated
by PureCol bovine collagen solution (Advanced BioMatrix). 10 μM enzalutamide was added into the
culture dishes. Clones, colonies and floating spheres were enumerated and imaged 1-2 weeks after
plating.
Quantitative RT-PCR (qRT-PCR) and Western blot
For qRT-PCR (2), total RNA was extracted via the RNeasy mini kit (Qiagen). iTaq Universal SYBR
Green Supermix (BIO-RAD) was used to quantify the mRNA levels of multiple genes
(Supplementary Table S3) by normalizing to GAPDH. qRT-PCR was carried out in ABI 7900HT
system. Western blot protocols were previously described (4).
Wafergen SmartChip human microRNA (miRNA) arrays
Total RNA (500 ng-1 μg) was extracted from FACS purified TM+ and TM-depleted LAPC9 AI cells
using mirVana miRNA isolation kit. MicroRNA array experiments were performed, according to the
manufacturer’s instructions, in quadruplicate in a real-time PCR based SmartChip Human microRNA
Panel (Wafergen Biosystems) that contains 1043 human microRNAs. The ∆Ct value of each
microRNA was calculated by averaging over all its quadruplicates and subtracting the mean Ct value
of internal controls. The panel contained 7 small RNAs as internal controls, and the most stable ones
identified by GeNorm algorithm (6) were used in the analysis. Differential expression was
statistically assessed by function “limmaCtData” in R/Bioconductor package HTqPCR (7). The
baseline difference between the two generations was adjusted in the model. MicroRNAs with P value
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≤ 0.05 were considered differentially expressed. The heatmap of log2 expression fold change (i.e., ∆∆Ct) was plotted by heatmap.2 function in R. Processed data was presented in Supplementary Table
S4.
Oligonucleotide transfection
We used Lipofectamine RNAiMAX (Invitrogen) to transfect PCa cells with 30 nmol/L miR-499-5P
mirVana mimic or non-targeting negative control (miR-NC) oligonucleotides (Life Technologies) per
the manufacturer's instructions. These are double-stranded oligonucleotides that mimic mature
miRNAs and have novel chemical modifications for high potency and specificity according to the
manufacturer’s specifications (Life Technologies). mirVana miR-499-5P inhibitor (anti-miR-499-5P)
or nontargeting negative control miRNA inhibitor (anti-NC) was used to inhibit miR-499-5P
expression under the same transfection conditions.
qRT-PCR for miRNAs
Total RNA was extracted with the mirVana miRNA isolation kit (Life Technologies). Levels of
mature miR-499-5P were measured using TaqMan MicroRNA Assay (Applied Biosystems) by
normalizing to the RNU48 levels. qRT-PCR was carried out in ABI 7900HT system.
RT-PCR and DNA sequencing for TMPRSS-ERG fusion
Total RNA was extracted by the RNeasy mini kit (Qiagen), and reverse transcribed to cDNA. Primers
to detect TMPRESS-ERG fusion were selected from (8). PCR was carried out in a standard protocol
with annealing temperature of 63°C, and PCR products were run on a ~ 1.5% agarose gel. GAPDH
was used as an internal control.
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For sequencing, the PCR products were cloned into pGEM-T Easy Vector System (Promega),
which were then transformed in Top10 competent cells. ~30 clones were randomly picked, and
plasmids were prepared (Qiagen) and sequenced to verify the fusion. T7 and SP6 primers were used
for sequencing (pGEM-T Easy Vector System; Promega), and sequencing results were analyzed in
the BLAST software.
Clone identification numbers for genes in the knockdown experiments
GIPZ Non-silencing (NS) shRNA lentiviral vector was used as control, and CD44 was used as
previously described (2). Other GPZ-based shRNA lentiviral vectors included integrin α2 (#1:
V2LHS_133421; #2: V2LHS_133424; #3: V3LHS_376867; #4: V3LHS_376868), ALDH1A1 (#1:
V2LHS_112035; #2: V2LHS_265598), ALDH7A1 (#1: V3LHS_331282; # V3LHS_331283),
RegIV (#1: V2LHS_117727; #2: V2LHS_225663), and SOX9 (#1: V2LHS_11387; #2:
V3LHS_396211).
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Supplementary References
1. Qin J, Liu X, Laffin B, Chen X, Choy G, Jeter CR, et al. The PSA-/lo prostate cancer cell
population harbors self-renewing long-term tumor-propagating cells that resist castration. Cell
Stem Cell 2012;10:556-69.
2. Liu X, Chen X, Rycaj K, Chao HP, Deng Q, Jeter CR, et al. Systematic dissection of
phenotypic, functional, and tumorigenic heterogeneity of human prostate cancer cells.
Oncotarget 2015; 6:23959-86.
3. Patrawala L, Calhoun T, Schneider-Broussard R, Li H, Bhatia B, Tang S, et al. Highly purified
CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and
metastatic progenitor cells. Oncogene 2006;25:1696-708.
4. Chen X, Liu B, Li Q, Honorio S, Liu X, Liu C, et al. Dissociated primary human prostate
cancer cells coinjected with the immortalized Hs5 bone marrow stromal cells generate
undifferentiated tumors in NOD/SCID-γ mice. PLoS One 2013;8:e56903.
5. Goldstein AS, Drake JM, Burnes DL, Finley DS, Zhang H, Reiter RE, et al. Purification and
direct transformation of epithelial progenitor cells from primary human prostate. Nat Protoc
2011;6:656-67.
6. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate
normalization of real-time quantitative RT-PCR data by geometric averaging of multiple
internal control genes. Genome Biol 2002;3:RESEARCH0034.
7. Dvinge H, Bertone P. HTqPCR: high-throughput analysis and visualization of quantitative
real-time PCR data in R. Bioinformatics 2009;25:3325-6.
8. Wang J, Cai Y, Ren C, Ittmann M. Expression of variant TMPRSS/ERG fusion messenger
RNAs is associated with aggressive prostate cancer. Cancer Res 2006;66:8347-51
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Supplementary Figure Legends
Figure S1. FACS gating strategies and enrichment of TM+ cells in LAPC9 AI tumors.
Shown in (A-C) are the representative histograms illustrating the FACS strategy to analyze TM+
PCa cells in LAPC9 AD (A), AI 3° (B) and AI 6° (C) tumors. Bulk cells were sorted from
respective tumors (a), gated into live cells (b) using Propidium Iodide (PI), and then into single
cells (c). After gating out Lin+ mouse cells (d), ALDHhi human PCa cells were selected (e) based
on its negative control (DEAB) (not shown), which were then sorted for CD44+ and α2β1+ cells (f)
according to respective isotype controls (not shown). The percentage of TM+ cells in these tumors
was then calculated (shown on top of each row).
Figure S2. Phenotypic marker-positive cells are enriched in AI xenograft tumors.
A. IHC analysis of CD44 expression in LAPC9 AD and AI FFPE tumor samples reveals that
CD44 levels are higher in AI tumors than AD tumors.
B-C. ALDH1 IHC (B) and ALDH7A1 IF(C) staining in LAPC9 AD and AI FFPE tumor samples
indicates that AI tumors contain more abundant ALDH1+ and ALDH7A1+ cells than AD
tumors.
D. IF staining of integrin α2 in LAPC4 AD and AI FFPE tumor samples shows that integrin α2+
cells were enriched in AI tumors.
(Original magnifications: x400 for IHC and x200 for IF).
Figure S3. TM+ cells can self-renew in vivo.
Left column (AD condition): During serial passages, the percentages of TM+ cells were
measured by FACS and compared among parental AD and 1°, 2° and 3° AD TM+ cellderived tumors. FACS gating strategies were similar to Figure S1. The percentage of TM+ in
these tumors was then calculated (top). Note that the % of TM+ cells gradually increased
during serial passaging. Importantly, however, in each generation, the TM+ cells represented
a relatively constant small population, suggesting that the TM+ subpopulation self-renewed in
AD tumors.
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Right column (AI condition): the percentages of TM+ cells were compared among parental AI
and 1° and 2° AI TM+ cell-derived tumors. Note increased % of TM+ cells in AI tumors than
in AD tumors. Importantly, the TM+ cells represented a relatively constant percentage
suggesting that they self-renewed in AI tumors during serial transplantation.
Figure S4. TM+ cell-derived tumors are less differentiated than TM- cell-derived tumors.
In two independent experiments (A & B), Western blot analysis was used to check several PCa
differentiation markers (i.e., AR, PSA, CK18, and/or PAP) in TM+ and TM- cell-derived LAPC9
tumors (serially) passaged in intact male NOD/SCID mice. In (A), TM+ cell-derived LAPC9
tumors had lower expression levels of AR and PSA, as compared to TM- cell-derived tumors
even after two consecutive passages in intact male mice (2°). In (B), TM+ cell-derived LAPC9
tumors had decreased levels of AR, PSA, and PAP compared to TM- cell-derived tumors, both of
which were maintained in intact male mice. LNCaP and Du145 cells were used as controls.
Figure S5. Characterizations of primary HPCa samples.
A. Biopsy schematic illustrating high-volume tumor involvement (~100%) in the two HPCa
samples (i.e., HPCa222 and HPCa223) used in our studies.
B. IHC images showing positive staining for AR, PSA and Racemase, and negative for CK5 in
the two HPCa samples. Original magnifications are indicated.
C. RT-PCR analysis of TMPRSS-ERG fusion. Total RNA from VCaP (positive control) and
LNCaP (negative control) cells or from the HPCa222 TM+ cell derived spheres was used in
RT-PCR analysis of TMPRSS-ERG fusion product (591 bp; left) or GAPDH (right). The
molecular ladder was shown on the left lane of the gels.
D. DNA sequencing of the PCR products validates TMPRSS-ERG fusion in HPCa222 TM+
spheres. VCaP cells were used as positive control (top). Shown are BLAST alignments.
Figure S6. Triple-marker or double-marker positive cells from primary HPCa samples possess
CSC activities in castrated conditions.
A. TM+ HPCa223 cells posses higher clonal and clonogenic activities than TM- cells. In clonal
assays (a), purified cells were plated (2k/well) in PureCol-coated 6-well plates, and images
were taken after 8 days. In clonogenic assays (b), sorted cells were mixed with Matrigel and
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plated in 12-well plates at the indicated cell doses. Colonies were counted after 10 days.
Colony numbers (mean± S.D; n=3) and size and representative images were shown.
B. ALDHhiCD44+ HPCa198 cells are more clonal and clonogenic than ALDHloCD44- cells. In
clonal assays (a), purified cells were plated (2k/well) in PureCol-coated 6-well plates, and
images taken after 10 days. In clonogenic assays (b), sorted cells were mixed with Matrigel
and plated in 12-well plates at the indicated cell doses. Colonies were enumerated after two
weeks. Colony size and representative images were shown. Data represent the mean ± S.D
from triplicate in each condition.
C. ALDHhiCD44+ HPCa206 cells show enhanced clonal and clonogenic capacities than
ALDHloCD44- cells. Experiments were performed using similar strategies described in (B).
In all above experiments, cells were cultured in a CDSS-containing medium plus 10 μM
enzalutamide.
Figure S7. Functional importance of phenotypic markers in CRPC development.
A-B. Integrin α2 knockdown reduces tumor initiation in LAPC9 AI (A) tumors and lowers tumor
burden in LAPC4 AI (B) tumors. Bulk LAPC9 (A) and LAPC4 (B) AI cells were infected
with the control or α2 shRNA-encoding lentiviral vectors for ~72 h at an MOI of 10-20, and
s.c injected in castrated NOD/SCID male mice. Tumor incidence, weight and P values were
indicated. Shown on the right were representative phase and GFP images of the endpoint
tumors.
C. CD44 knockdown inhibits LAPC9 tumor regeneration in castrated male hosts.
D. ALDH1A1 knockdown partially inhibits the growth of LAPC9 AI tumors.
Figure S8. qRT-PCR validation and functional characterizations of gene knockdown in AI
tumor cells.
A. Efficiency of integrin α2 knockdown in PC3 cells with 4 different shRNA vectors revealed by
qRT-PCR. Bar graphs represent the mean ± S.D. ***P<0.001.
B. qRT-PCR analysis to evaluate efficiency of ALDH7A1 knockdown in LAPC9 AI cells with 2
different shRNA vectors. Error bars represent the mean ± S.D (n=2). *P<0.05.
C. ALDH1A1 and ALDH7A1 knockdown in LAPC9 AI bulk cells impairs their sphere-forming
capacities under androgen-deprived conditions. Bulk LAPC9 AI were infected with
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ALDH1A1 and ALDH7A1-shRNA lentiviral vectors, plated (5k/well) in 6-well ULA plates
and cultured in CDSS-containing IMDM medium plus 10 μM enzalutamide. Spheres were
counted in 2 weeks. Data represent the mean ± S.D (n=3 for each condition). *P<0.05.
D-E. Knockdown efficiency of RegIV (D) and SOX9 (E) in LAPC9 AI cells with 2 different
shRNAs as assessed by qRT-PCR. Shown are the bar graphs (mean ± S.D; n=2; *P<0.05;
**P<0.01).
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