SUPPLEMENTARY INFORMATION
LAMC2 enhances the metastatic potential of lung adenocarcinoma
Yong Wha Moon1,8, Guanhua Rao2, John J. Kim3, Hyo-Sup Shim4, Kang-Seo
Park1,2, Steven S. An5, Bouk Kim6**, Patricia S. Steeg7, Sami Sarfaraz1, Liam Changwoo
Lee1, Donna Voeller1, Eric Y. Choi5, Ji Luo1, Diane Palmieri7, Hyun Cheol Chung8, JooHang Kim8, Yisong Wang1,2 and Giuseppe Giaccone1,2
1
Medical Oncology Branch, National Cancer Institute, National Institutes of Health,
Bethesda MD, 20892;
2
Lombardi Comprehensive Cancer Center, Georgetown
University, Washington DC; 3Department of Bioengineering, Johns Hopkins University,
Baltimore, MD; 4Department of Pathology, Yonsei University College of Medicine,
Seoul, Korea; 5Department of Environmental Health Sciences, Johns Hopkins University,
Bloomberg School of Public Health, Baltimore, MD; 6Pathology Branch, National
Cancer Institute, National Institutes of Health, MD;
7
Women’s Cancers Section,
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of
Health, Bethesda, MD; 8Division of Medical Oncology, Yonsei Cancer Center, Yonsei
University College of Medicine, Seoul, Korea
1. SUPPLEMENTARY FIGURE LEGENDS
Figure S1
Figure S2
Figure S3
Figure S4
Figure S5
2. SUPPLEMENTARY METHODS
3. SUPPLEMENTARY REFERENCES
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Fig. S1. Identification of LAMC2 in metastatic lung cancer cell lines by
mRNA microarray profiling.
1
Expression profiles of A549 (our data) and PC9 metastatic cells (public data, GSE14107),
which were independently derived using the intracardiac injection mouse metastasis
model. Forty-eight differentially expressed genes between round 0 and round 3 were
identified in common in both systems, with criteria of false discovery rate (FDR) < 0.1
and log2 fold change > 1.5 or < -1.5.
Supplementary Fig. S2. Effect of LAMC2 on cell doubling time, traction and kinetics of
attachment.
A, doubling time of LAMC2-transfected or -knockdown cells. NS, non-significant p
values. Data are presented as mean±SEM of quadruplicate experiments. B, representative
images of phase contrast and traction map (left panel) and quantitative analyses of
projected cell area (right upper panel) and net contractile moments (right lower panel) in
A549R0, R3, and R4 cells. In traction maps, the white lines show the cell boundary,
colors show the magnitude of the tractions in Pascal (Pa), and arrows show the direction
and relative magnitude of the tractions. Scale bar: 50μm. P values were calculated by
student t-test. n, the number of cells analyzed. Data are presented as mean±SEM. C,
representative images of phase contrast and traction maps (left panel) and quantitative
analysis of projected cell area (right upper panel) and net contractile moments (right
lower panel) in PC9-shLAMC2 and control cells. Scale bars: 50μm. P value was
calculated by student t-test. n, the number of cells analyzed. Data are presented as
mean±SEM. (d and e) Analysis of cell attachment kinetics in (D) H358-shLAMC2 and
(E) PC9-shLAMC2 and their corresponding control cells. Time-lapse images of cell
attachment showed the duration (hours) after cells were plated in cell culture dishes.
Small, round, whitish, and glittering cells are considered as “not attached cells”. Spindlelike (PC9) or widely spreading (H358) cells are considered as “attached cells”. Student ttest was performed. Data are presented as mean±SEM of triplicate experiments.
Supplementary Fig. S3. Effect of conditioned media on cell migration and invasion.
A, schematic of collection and concentration of conditioned medium. When cells were
confluent in the culture plate, medium was replaced with fresh medium without additives,
serum or other growth factors for 24 h. The conditioned medium was then collected,
2
followed by Amicon Ultra-Centrifugal filter concentration with molecular weight cut-off
of 10 kDa. B, migration and invasion assay by adding conditioned medium from H358shLAMC2 and H358-shMock cells to H358-shLAMC2 cells. Student t-test was
performed. Data are presented as mean±SEM of triplicate experiments. C, migration
assay by adding serially diluted, concentrated conditioned medium from PC9-shMock
cells to PC9-shLAMC2 cells in the Boyden chamber inserts. Student t-test was performed.
Data are presented as mean±SEM of duplicate experiments (bottom graph).
Supplementary Fig. S4. Confirmation of bioluminescence-labeled metastatic cells by
histological examination. A, baseline bioluminescent activity of luciferase-transfected
A549R4-shMock and -shLAMC2 cells. The same number of cells (2x105 cells) per well
were seeded and cultured for 24 h and then luciferase activities were measured by in vitro
bioluminescent imaging with an IVIS Imaging System. B, selected bioluminescent
signals (dotted circle) acquired by in vivo bioluminescent imaging were confirmed to be
metastatic tumors by histologic examination. Upper panel indicates bone metastasis and
lower panel indicates soft tissue metastasis.
Supplementary Fig. S5. Effect of Snail, Vimentin and ZEB1 on cell migration and
invasion, and influence of Vimentin expression on survival of stage I ADC.
A, ectopic expression of Snail restores migration and invasion of H322-shLAMC2 cells.
Student t-test was performed. Data are presented as means±SEM of triplicate experiments.
B, ectopic expression of Vimentin restores miƒgration and invasion of H322-shLAMC2
cells. Student t-test was performed. Data are presented as means±SEM of triplicate. C,
recurrence-free survival according to Vimentin expression in patients with completely
resected stage I ADC of lung from Yonsei Cancer Center.
Supplementary Fig. S6. A. Ectopic expression of LAMC2 induces migration and
invasion of H1703 lung squamous cell carcinoma cells. B. Lung adenocarcinomas with
fibrous stroma were associated with worse recurrence-free survival. Sixty-six evaluable
ADCs out of 250 pStage I NSCLC TMA samples were stratified into ADCs intermingled
3
with fibrous and thin stroma respectively by a pathologist (H-S S) according to the
previously described stratification criteria1
SUPPLEMENTARY METHODS
In Vivo Selection of Metastatic Derivatives
Metastatic derivatives were obtained by repeated intracardiac injection of A549 cells
and subsequent cultivation of metastatic tissues. Cell suspensions of parental A549,
designated round 0 (A549R0) in 100 μl RPMI medium without phenol red, were injected
into the left ventricle.
Four to 6 weeks later, mice were sacrificed, and the whole brains
were minced and placed in RPMI medium with 10% FBS for culture. Outgrowth of A549
cells from the cultivated brain tissue indicated that injected A549 cells metastasized to
the brain. A549R1, A549R2, and A549R3 cells were serially established by performing
intracardiac injection of A549R0 (5 × 105 cells), R1 (2.5 × 105 cells), and R2 (2.5 × 105
cells), respectively. For each round, 10 mice were injected. To monitor end-organ
metastasis of the metastatic derivatives, we generated a stable cell line expressing
luciferase activity using A549R3 cells (A549R3-LUC). In brief, firefly luciferaseexpressing retrovirus was produced by transfecting 293T cells with the luciferaseexpressing retroviral plasmids (Addgene, Cambridge, MA), VSV-G and gal/pol cDNA.
A549R3 cells were infected with luciferase-expressing retrovirus in the presence of 4
g/ml polybrene and selected with 5 g/ml of puromycin. Bioluminescent imaging was
performed in vivo after intracardiac injection of A549R3-LUC cells, as described in the
respective section.
Finally, round 4 A549 (A549R4)-LUC-Brain, -Femur, and -Spine
cells were established from the corresponding organ metastases.
Gene Expression Profiling
RNA was extracted in triplicate samples from subconfluent A549R0, A549R1,
A549R2, and A549R3 cells using the RNeasy mini kit (Qiagen, Venlo, Netherlands).
Labeling and hybridization to the GeneChip Human Gene 1.0 ST array (Affymetrix,
Santa Clara, CA) were performed by the Affymetrix Core Facility of the National Cancer
Institute (Frederick, MD). To discover differentially-expressed genes, we used the criteria
of false discovery rate < 0.1 and log2 fold change > 1.5 or < -1.5. Comparing mRNA
4
expression profiles between round 0 and round 3 cells revealed 48 differentially
expressed genes, which were shared by both A549 and PC9 ADC cell lines.
mRNA
microarray data of parental and metastatic derivatives of PC9 with the repeated
intracardiac injection model are publicly available (HG-U133A 2.0; GSE14107)2. To
evaluate the relationship between LAMC2 mRNA expression level and prognosis in
NSCLC, we used three publicly available mRNA microarray data: the first cohort of 204
ADCs from Japanese National Cancer Center (JNCC set; HG-U133A Plus 2.0;
GSE31210)3, the second cohort of a mix of 63 ADCs and 75 SCCs from the Samsung
Medical Center (SMC set; HG-U133A Plus 2.0; GSE8894)4, and the third cohort of 59
ADCs and 52 SCCs from Duke University Medical Center (DUMC set; HG-U133A Plus
2.0; GSE3141)5.
All raw CEL data were normalized with RMA algorithm before
analysis. An optimal cut-off point for normalized intensity of LAMC2 mRNA was
determined using minimum P value approach in predicting recurrence-free or overall
survival6.
Immunohistochemistry (IHC) in Human NSCLC Specimens
Expression of LAMC2 and Vimentin was determined by IHC from formalin-fixed,
paraffin-embedded surgical specimens of 250 patients with NSCLC. All tumor specimens
were obtained from a pathological stage I cohort which underwent complete surgical
resection between 1998 and 2007, without neoadjuvant treatment at Yonsei Cancer
Center (Seoul, Korea).
Tumor staging was performed according to TNM staging
revised in 2002 by American Joint Cancer Committee7. Tissue microarray (TMA) blocks
were generated with punctures of areas with >80% tumor content in each tumor tissue.
For IHC, all paraffin sections were cut at 3-μm thickness, deparaffinized through xylene,
and dehydrated with graded ethanol. Heat-induced antigen retrieval pH6 solution for
LAMC2 and pH9 solution for Vimentin were used for antigen retrieval. Endogenous
peroxidase activity was blocked with 3% H2O2 in methanol, and primary incubations
were performed with mouse monoclonal LAMC2 antibody D4B5 (Millipore, Billerica,
MA) at 1:100 for 60 min and with rabbit monoclonal Vimentin antibody D21H3 (Cell
Signaling Technology, Danvers, MA ) at 1:500 for 60 min. Subsequently, sections were
incubated with DAKO Env+ secondary antibody for 30 min, visualized with 3,35
diaminobenzadine for 10 min for chromogenic development, washed and counterstained
with hematoxylin. Positivity for LAMC2 was assessed from 0 to 100% of stained cells by
cytoplasmic staining with any intensity. The cut-off of 30% was used for LAMC2
positivity as in previous reports with the same antibody8, 9, 10. Vimentin was considered to
be positive if ≥ 1% of cancer cells were stained in the cytoplasm as previously reported11,
12
.
Ectopic Expression Studies
To establish stable cell lines expressing LAMC2, Snail, and Vimentin, the constructed
expression vectors pBOS-CITE-Neo-LAMC213 (a kind gift from Dr. Kaoru Miyazaki,
Yokohama City University, Japan) that contained a cDNA for the full-length LAMC2
chain (amino acid no. 1–1193), pEGFP-C2-Snail (Addgene), and pPSmOrange-N1Vimentin (Addgene) were transfected, respectively, using GenJet Plus DNA transfection
reagent (SignaGen Laboratories, Rockville, MD) following the manufacturer’s
instruction. For stable expression, the transfected cells were selected with 500-1,000
g/ml of the antibiotic G418 (Invitrogen, Carlsbad, CA). The parental cells transfected
with empty vectors were generated as controls.
shRNA-mediated Knockdown
LAMC2 shRNA and ZEB1 shRNA (Open Biosystems shRNA Library) were cloned
into PMSCV-PM retroviral vector and viral supernatants were generated by cotransfecting 293T cells with VSV-G and gag/pol expression vectors.
Cells were
infected with LAMC2 shRNA or ZEB1 shRNA retrovirus in the presence of 4 g/ml
polybrene and selected with 0.5-5 g/ml puromycin.
siRNA Transfection
A549R0 and R4 cells were seeded in 6-well plates overnight and transfected with
integrin 1 or control-siRNA (Santa Cruz Biotechnology Inc, Dallas, TX) and 3μl
PepMuteTM siRNA transfection reagent (SignaGen Laboatories) for 6 hours, and then
6
cultured in complete medium for additional 24~48 hours. After that, cells were harvested
for western blotting or functional assays.
Migration and Invasion Assay
Modified Boyden chamber method with 24-Well Millicell Cell Culture Insert
(Millipore, Billerica, MA) were used for cell migration and invasion assay. Inserts were
coated with BD matrigel (BD Biosciences, Franklin Lakes, NJ) with concentration of 200
g/ml for invasion assay according to the manufacturer’s instructions. Cells were serumstarved overnight and seeded in the upper chamber of transwell plates in 200 l medium
without FBS. The lower chamber was filled with 700 l of medium supplemented with
10% FBS. Cells in the transwell plates were incubated at 37°C in humidified air
containing 5% CO2. The number of seeded cells and incubation time were adjusted for
each cell line. To quantify the migrated and invaded cells, crystal violet-stained cells
were counted in five different microscopic fields under 200x magnification.
Fourier Transform Traction Microscopy (FTTM)
The contractile stress arising at the interface between each adherent cell and its
substrate was measured with traction microscopy14, 15. In brief, cells were plated sparsely
on elastic gel blocks coated with collagen type I, and allowed to adhere and stabilize for
48 h. A549R0 cells showed delayed adhesion and spreading dynamics on soft (1kPa) gels
and, as such, we used stiffer (8kPa) gel. On 8kPa gel, A549 cells spread to similar size to
that of H358 and PC9 cells by 48 h. For both H358 and PC9 cells, we used 1kPa gels. For
each adherent cell, images of fluorescent microbeads (0.2 μm in diameter, Molecular
Probes, Eugene, OR) embedded near the gel apical surface was taken at different times;
the fluorescent image of the same region of the gel after cell detachment with trypsin was
used as the reference (traction-free) image. The displacement field between a pair of
images was then obtained by identifying the coordinates of the peak of the crosscorrelation function15. From the displacement field and known elastic properties of the
gel (Young’s modulus of 1-8 kPa with a Poisson’s ratio of 0.48), the traction field was
computed using both constrained and unconstrained Fourier transform traction
cytometry15. The computed traction field was used to obtain net contractile moment,
7
which is a scalar measure of the cell’s contractile strength15. Net contractile moment is
expressed in units of pico-Newton meters (pNm).
Conditioned Medium and Antibody Blocking Assay
When cells became confluent, RPMI-1640 medium containing 10% FBS was replaced
with fresh serum-free medium. After 24 h the conditioned medium was collected and
concentrated using Amicon Ultra-Centrifugal filter (Millipore, Billerica, MA) with
molecular weight cut-off of 10 kDa at 3800g at 4°C until 500 μl left on the top of filter
(~20 min). Concentrated conditioned medium was used for immunoblotting with antiLAMC2 antibody (Santa Cruz Biotechnology Inc) after quantification of secreted total
protein. Migration and invasion assay was performed by adding concentrated conditioned
medium collected from shLAMC2- or shMock-transfected cells to the upper chamber of
the transwell plate containing shLAMC2-transfected. For LAMC2 blocking assay,
A549R0 cells (8 X 105 and 4 X 105 cells for migration and invasion, respectively) were
suspended in conditioned medium supplemented with 40 μg/ml mouse monoclonal
LAMC2 antibody D4B5 (Millipore) as a blocking antibody or mouse IgG antibody as
control and were plated in the upper chamber of the transwell plate. Subsequent steps of
migration assay were the same as described in the respective section.
Quantitative RT-PCR
Quantitative RT-PCR was performed on total RNA isolated from metastatic series of
A549 cells, using RNeasy mini kit (Qiagen, Valencia, CA) according to the
manufacturer’s instructions. cDNA was synthesized with High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Foster City, CA). RT-PCR was performed with
Taqman gene expression assay (Applied Biosystems, Foster City, CA) using 7900HT
Fast Real-Time PCR system (Applied Biosystems). GAPDH expression was used as an
internal reference to normalize input cDNA. Taqman gene expression assay IDs were
Hs00165042_m1
(LAMA3),
Hs00165078_m1
(LAMC2).
Immunoblot Analysis
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(LAMB3),
and
Hs01043711_m1
Cell lysates were prepared using RIPA buffer (Sigma-Aldrich, St. Lous, MO)
according to the manufacturer’s instruction. Protein samples were applied to the wells of
NuPAGE 4-20% Tris-Gly gel, electrophoresed in SDS running buffer (Invitrogen), and
transferred to nitrocellulose membranes using the iBlot transfer apparatus (Invitrogen).
Membranes were blocked in Tris-buffered saline containing 0.5% Tween 20 (TBS-T) and
5% BSA for 1 h at room temperature followed by incubation with primary antibody
overnight at 4°C. After membranes were washed three times for 10 min each in TBS-T,
HRP-conjugated secondary antibody (Bio-Rad) in TBS-T containing 2% BSA was
applied for 1 h at room temperature. Proteins were visualized using G-box Chemi
Systems (SynGene, Cambridge, UK). Antibodies were purchased from Santa Cruz
Biotechnology Inc (LAMC2), Cell Signaling Technology (ZEB1, ZEB2, Snail, Slug,
Twist, E-cadherin, N-cadherin, Vimentin), BD Transduction LaboratoriesTM (Integrin
β1), and Sigma-Aldrich (actin).
Immunoprecipitation
A549R4, PC9 and H2122-LAMC2 cells were lysed with 1ml of lysis buffer plus
protease & phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA) for 30 min
on ice. After centrifugation for 15 min at 15,000 ×g, the supernatant were transferred to
new tubes and then immunoprecipitated with indicated antibodies and Protein A/G
magnetic beads (Pierce Biotechnology, Rockfort, IL) overnight at 4°C. Thereafter, the
beads were washed four times with lysis buffer, the precipitants were eluted with 1%
SDS sample buffer for 5 min at 95 °C and analyzed by immunoblotting.
In Vivo Metastasis Assay Using Intracardiac Injection and Bioluminescent Imaging
Baseline luciferase activity of A549R4-LUC-shLAMC2 and A549R4-LUC-shMock
cells was assessed by in vitro bioluminescent imaging with an IVIS Imaging System
(Xenogen, Alameda, CA) following addition of D-luciferin (Caliper Life Science,
Hopkinton, MA) at 150 μg/ml in cell culture medium. The same number (1 × 105 cells) of
A549R4-LUC-shLAMC2 cells (9 mice) and A549R4-LUC-shMock cells (10 mice) in
100 μl volume were injected into the left ventricle of the mouse. In vivo bioluminescent
imaging was performed with an IVIS Imaging as previously described16. In brief, D9
luciferin at 150 mg/kg in DPBS was injected intraperitoneally into mice 5 min prior to
imaging. Anesthetized mice were placed dorsally in the imaging box and imaged for 3
min and then imaged ventrally for another 3 min. Images and measurements of
bioluminescent signals were acquired and analyzed using Living Image software
(Xenogen). Serial bioluminescent imaging was performed weekly for four weeks and
then biweekly for the next two weeks. Metastasis was defined as the presence of
bioluminescent signals at the same anatomic locations on 3 consecutive images weekly or
biweekly.
Statistical Analysis
Statistical analysis was performed using SPSS version 17.0 (SPSS, Chicago, IL).
Recurrence-free survival (RFS) was defined as the time from curative surgery to NSCLC
recurrence or the last date at which the patient was known to be free of recurrence
(censoring time). Overall survival (OS) was defined as the time from curative surgery to
death or the date at which the patient was last confirmed to be alive (censoring time).
Kaplan-Meier plots were used to estimate survival. Comparisons of survival curves were
made by log-rank test. Multivariate analysis for prognostic factors was performed using
the Cox proportional hazards regression model. Student t-test was used to compare
migration, invasion and number of metastasis in mice between two groups. All P values
were two tailed, and P values of less than 0.05 were regarded as significant.
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