SUPPLEMENTARY INFORMATION

Luis-Ravelo et al.

Metastasis Models of Lung Cancer Reveal the Impact of Tumor-Stromal Activities

SUPPLEMENTARY MATERIAL AND METHODS

Gene expression analysis

Total RNA was extracted from 70-80% confluent cultures using Trizol (Invitrogen) according manufacturer instructions. Only samples with 260/280 ratio between 1.9-2.1 were subsequently used. Two micrograms were treated with 2 units of DNase (Invitrogen) for 15 minutes at RT and EDTA (2mM) at 65ºC. Superscript II retrotranscriptase (Invitrogen) was added in presence of hexamers (200 ng per tube), dNTPs (0.4 mM), DTT (6 mM) and RNase Out (40 units).

For qualitative PCR, a mix containing MgCl2 (1.5 mM), dNTPs (200µM), DMSO (5%) and Taq polymerase (1 unit) were added to 1 µl cDNA. Reactions were carried as follows: 25ºC, 10´; 50ºC, 50´; 70ºC, 15´. Primer sequence and cycling conditions for each PCR are shown in Table 1.

GENE / Tm (ºC) / CYCLES / SENSE PRIMER / ANTISENSE PRIMER
MIP-1-α / 60 / 41 / ctcttcctgttcacgcttcc / atgctggtgatgacaccaaa
Osteopontin / 57 / 31 / cctgtgccataccagttaaaca / ggctaggagattctgcttctga
PTHrP / 56 / 36 / tccatccaagatttacggcg / cctgaatatgtccttggaagg
MMP-2 / 60 / 41 / cactttcctgggcaacaaat / tgatgtcatcctgggacaga
Galectin-3 / 60 / 31 / ggccactgattgtgccttat / ctgaccacttcaaggttgca
CTGF / 60 / 41 / ccgtactcccaaaatctcca / gtaatggcaggcacaggtct
IL8 / 59 / 36 / agctggccgtggctctct / ggtggaaaggtttggagtatgtctt
β-actin / 55 / 27 / agcctcgcctttgccga / ctggtgcctggggcg

Table 1: Primer sequence, annealing temperatures and cycling times for each amplicon.

Table 2 shows primer characteristics for quantitative PCR.

GENE / SENSE PRIMER / ANTISENSE PRIMER / LENGHT / EXON BOUNDARY
MMP2
(human) / cgaccgcgacaagaagtatggc / aagtgaaggggaagacacagggg / 95 / 6-7
mmp2
(mouse) / ctatgaccgggataagaagtatgga / gcaccttctgaatttccaccc / 75 / 6-7
MMP3
(human) / atggacctggaaatgttttggcc / gcagcaacgagaaataaattggtccc / 127 / 4-5
mmp3
(mouse) / gcctggaacagtcttggct / cagcaaccaggaataggttggtac / 122 / 4-5
MMP9
(human) / aaggatacagtttgttcctcgtg / gcccctcagtgaagcggtacat / 114 / 7-8
mmp9
(mouse) / aagggtacagcctgttcctggtg / aagccctcgaggtagctatacagcg / 115 / 7-8
MMP10
(human) / aagttaacagcagggacacc / cttggataacctgcttgtacc / 88 / 7-8
mmp10
(mouse) / gaggctcacaacacggacagt / ctttgggtagcctgcttggact / 90 / 7-8
GAPDH
(human) / ctgctcctcctgttcgacagt / ccatggtgtctgagcgatgt / 75 / 1-2
gapdh
(mouse) / tacccccaatgtgtccgtc / ggagttgctgttgaagtcgc / 163 / 5-6

Table 2: Sequence of primers for real time PCR. Reactions were performed using SYBR Green (Applied Biosystems) using human or murine- specific primers at 500 nM.

Histological analysis

Hind limbs were excised, soft tissues carefully removed from femur and tibia, fixed for 24 h in 10% neutral-buffered formalin, dehydrated and decalcified 72 h in Osteosoft® solution (Merck). Following complete alcohol dehydration, bones were embedded in paraffin and 5 mm sections were stained with H&E. For TRAP staining, sections were rehydrated and exposed to TRAP solution (Sigma) for 3 hours according to manufacturer instructions.

SUPPLEMENTARY FIGURES

Supplementary Figure 1: Screening by semi-quantitative RT-PCR of several human non-small cell lung cancer (NSCLC), H460, H727 and A549 as compared to normal human epithelial cells (NHBE) and human bronchoepithelial immortalized cells (BEAS). Genes including MIP1a, PTHrP, MMP-2, Galectin-3, and CTGF were compared to a housekeeping gene, b-actin.

Supplementary Figure 2: H-E images of periosteal new bone formation induced by A549. Left panel: Metastasis at periostium (20X magnification). Right panel: New bone formation between two tumor foci in the cortical bone (20X magnification). * denotes the presence of tumor cells and # indicates the formation of new bone.

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