SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells

Roberta Santini1^, Silvia Pietrobono1^, Silvia Pandolfi^1, Valentina Montagnani1, Massimo D’Amico2, Junia Y. Penachioni1, Maria C. Vinci1§, Lorenzo Borgognoni3 and Barbara Stecca1*

SUPPLEMEMENTARY TABLES

Supplementary Table S1. Histo-pathological features of the melanomas used in this study.

Cell line / Type1 / Site / Location / Breslow2 / Stage
SSM2c / MM / Skin / Trunk / 8.5 / IIIB
M3 / MM / Lymph Node / Groin / 6.4 / IIIC
M5 / MM / Skin / Arm / 1.1 / IIIB
M6 / MM / Lymph Node / Arm / n.a. / IIIC
M9 / MM / Skin / Arm / n.a. / IV
M11 / MM / Skin / Trunk / 9.3 / IIIC
M14 / MM / Skin / Leg / 8.1 / IIIC
M15c / MM / Skin / Scalp / 11 / IIC
M16 / PM / Cheek / 10 / IIB
M17 / MM / Skin / Clavicular region / i.s. regr / IVB
M21 / MM / Skin / Trunk / 5 / IIIC
M25 / MM / Skin / Leg / 2.6 / IV
M26c / MM / Lymph Node / Groin / 0.47 / IV
M27 / MM / Lymph Node / Groin / 1.2 / IV
M28 / MM / Lymph Node / Armpit / 2.35 / IIIC
M30 / MM / Skin / Clavicular Region / n.a / n.a
M31 / MM / Skin / Trunk / 0.64 / IA
M33c / MM / Skin / Back / 4.95 / IIIC
M37 / MM / Skin / Leg / 9.80 / IIIC
A375 / Cell line

1PM: primary melanoma, MM: metastatic melanoma;2Thickness (mm) of the primary melanoma from which metastasis originated. i.s. regr: melanoma in situ with marker regression.n.a.: data not available. In bold are indicated melanomas used for MIC cultures.

Supplementary Table S2. List of primers used for quantitative real time PCR.

Primer / Sequence (5’ to 3’)
SOX2-F / GAGCTTTGCAGGAAGTTTGC
SOX2-R / GCAAGAAGCCTCTCCTTGAA
SOX10-F / CTGAAGGCAGGAAGGAGTTG
SOX10-R / GAGGGAGGCTCTGTGAATTG
NESTIN-F / AACAGCGACGGAGGTCTCTA
NESTIN-R / TTCTCTTGTCCCGCAGACTT
PAX3-F / GTTTCGCCTTCACCTGGATA
PAX3-R / GTTGATAAAAACACCGCCGA
ABCB5-F / TTCATCCTCCGTGGCTTATC
ABCB5-R / ATCCACACCATCAAACAGCA
ABCF2-F / TGGCCTGGAGACATCCTGGCTT
ABCF2-R / GCCAATGTCAGGGTGCACACGTT
JARID1B-R / GGGCTGGCCCCGTATTCAGC
JARID1B-F / TCGTGGCCTGGCCTTGAGGA
CD271-R / GCCTGGACAGCGTGACGTTCT
CD271-F / CACGGCGCCGACATGCTCTG
NANOG-F / ACCTTGGCTGCCGTCTCTGG
NANOG-R / AGCAAAGCCTCCCAATCCCAAACA
OCT4-F / TTTTGGTACCCCAGGCTATG
OCT4-R / GCAGGCACCTCAGTTTGAAT
KLF4-F / GCAGCCACCTGGCGAGTCTG
KLF4-R / CCGCCAGCGGTTATTCGGGG
GLI1-F / CCCAGTACATGCTGGTGGTT
GLI1-R / GCTTTACTGCAGCCCTCGT
TP73-F / GGGACGCAGCGAAACCG
TP73-R / CGAAGTAGGTGCTGTCTGGTT
GADD45A-F / GAGAGCAGAAGACCGAAAGG
GADD45A-R / CACAACACCACGTTATCGGG
NOXA-F / ACTGTTCGTGTTCAGCTCGC
NOXA-R / GAGTAGCACACTCGACTTCCA
BCL2-F / CTTTGAGTTCGGTGGGGTCA
BCL2-R / GGGCCGTACAGTTCCACAAA
BCLXL-F / GGTAAACTGGGGTCGCATTG
BCLXL-R / GCTGCTGCATTGTTCCCATAG
GAPDH-F / GACGCTGGGGCTGGCATTG
GAPDH-R / GCTGGTGGTCCAGGGGTC
-ACTIN-F / GAAAATCTGGCACCACACCT
-ACTIN-R / TAGCACAGCCTGGATAGCAA
hSOX2prom1-F / GCGTCCCATCCTCATTTAAG
hSOX2prom1-R / AGCAACAGGTCACACCACAC

SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure S1. Confocal images of patient-derived primary melanoma cells M26c, M33c, M5, M25 and of A375 melanoma cellsafter immunolabelling with anti-SOX2 antibody. Nuclei were counterstained with DAPI. Scale bar = 10m.

Supplementary Figure S2. Growth curves of A375 and M5 melanoma cells transduced with LV-c, LV-shSOX2-1 or LV-shSOX2-2. Three to five thousands transduced cells/well were plated in 12-well plates and cells were counted on days 3, 5 and 7.

Supplementary Figure S3. Flow cytometric analysisof cell cycle distribution in SSM2c (a) and M26c (b) melanoma cells transduced with LV-c, LV-shSOX2-1, LV-shSOX2-2 and SSM2c stably transfected with LV-SOX2 (c). For the cell cycle analysis cells were syncronized for 24hrs without serum, seeded for 48hrs in 10% FBS DMEM, harvested and resuspended in 50 µg/ml propidium iodide.Data were analyzed on a FACSCanto cytometer (BD Bioscience, San Diego, CA, USA) using FlowJo software (Treestar, Ashland, OR, USA).

Supplementary Figure S4. Representative images of Aldefluor assay in M26c cells transduced with LV-c, LV-shSOX2-1 and LV-shSOX2-2. Dissociated cells were incubated in Aldefluor assay buffer containing the ALDH protein substrate for 30 minutes at 37°C. Sorting gates for fluorescence-activated cell sorting (FACS) were drawn relative to cell baseline fluorescence, which was determined by the addition of the ALDH-specificinhibitor diethylaminobenzaldehyde (DEAB) during the incubation. DEAB-treated samples served as negative controls.

Supplementary Figure S5. Characterization of the melanoma spheres used in this study. (a) Representative phase-contrast images of the different melanoma spheres. Scale bar = 50m. (b) SOX2 protein expression in spheres from patient-derived primary melanoma cells SSM2c, M26c, M5 and in the established melanoma cell line A375. HSP90 was used as loading control. (c) Number of primary spheres obtained after limiting dilutions (5, 2.5, 1, 0.5, 0.1, 0.05 cells/l) in SSM2c, M26c, M5 and A375 cells. (d) qPCR analysis of SOX2 and of melanoma stem cell markers in melanoma spheres. The y-axis in each graph represents expression ratio of gene/(GAPDH+ACTIN average). Error bars indicate SEM of at least three independent experiments.

Supplementary Figure S6. (a)Proliferation index measured by CFSE staining in SSM2c and M26c spheres stably transduced with LV-c and LV-shSOX2-1. (b) Quantification of the fraction of early (Annexin V+/7-AAD-) and late (Annexin V+/7-AAD+) apoptotic cells in SSM2c and M26c spheres stably transduced with LV-c and LV-shSOX2-1. Error bars indicate SEM of at least three independent experiments.

Supplementary Figure S7. (a)Proliferation index measured by CFSE staining in SSM2c and M33c spheres stably transfected with LV-c and LV-SOX2. (b) Quantification of the fraction of early (Annexin V+/7-AAD-) and late (Annexin V+/7-AAD+) apoptotic cells in SSM2c and M33c spheres stably transfected with LV-c and LV-SOX2. Error bars indicate SEM of at least three independent experiments.

Supplementary Figure S8. qPCR analysisof GLI1(a) and SOX2(b) in M26c, M33c and M5 cells transduced with LV-c and LV-shPTCH1, showing the increase of GLI1 and SOX2mRNA upon HH pathway activation. qPCR values reflect Ct values after normalization with 2 housekeeping genes (GAPDH andACTIN), with the values in control equated to 1.

Supplementary Figure S9. (a)Quantification of sphere size in SSM2c and M26c spheres transduced with LV-c, LV-shSOX2-1, LV-shPTCH1 or LV-shPTCH1/LV-shSOX2-1.(b) Proliferation index measured by CellTraceTM Violet staining in M26c spheres stably transduced with LV-c, LV-shSOX2-1, LV-shPTCH1 orLV-shSOX2-1/LV-shPTCH1. (c) Quantification of the fraction of early (Annexin V+/7-AAD-) and late (Annexin V+/7-AAD+) apoptotic cells in M26c spheres stably transduced with LV-c, LV-shSOX2-1, LV-shPTCH1 orLV-shSOX2-1/LV-shPTCH1.Data shown are the mean  SEM of at least three independent experiments.

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