Dormant but migratory tumour cells in desmoplastic stroma of invasive ductal carcinomas

Vanisri Raviraj1, Hui Zhang1, Hsin-ya Chien1, Louise Cole2, Erik W. Thompson3, Lilian Soon1

1Australian Centre for Microscopy and Microanalysis (ACMM), AMMRF, The University of Sydney, NSW 2006, Australia

2Advanced Microscopy Facility, Bosch Institute, The University of Sydney, NSW 2006, Australia

3Invasion and Metastasis Unit, St. Vincent's Institute of Medical Research and University of Melbourne, Department of Surgery, St Vincent’s Hospital, Melbourne, Australia

1Corresponding author: Lilian Soon, ACMM, Madsen Building F09, Room 243, The University of Sydney, NSW 2006, Australia, PH +61 2 9351 5322, MOB +61 0423 371 272, FAX +61 2 9351 7682, Email:

Journal- Clinical & Experimental Metastasis

Supplementary Information

Supplementary Table 1. Primer list for MT-PCR and second round quantitative PCR

Gene / Outer primers sequence / Inner primers sequence
PAX2 / 5’-gagcacatcaaatcagaacagg-3’ / 5’-agtctatctgcatccaccaacc-3’
5’-acgaccagtcacaactgggtat-3’ / 5’-cgaccagtcacaactgggtat-3’
COLIA2 / 5'-agatagaggaccacgtggagaa-3' / 5'-atggtgaagatggtcccaca-3'
5'-aagtccaactccttttccatca-3' / 5'-agcaaagttcccaccgagac-3'
E-cadherin / 5'-ccaactggaccattcagtacaa-3' / 5’-ggtgggtgactacaaaatcaatc-3’
R- 5'-agtcacacacgctgacctctaa-3' / 5'-cctctaaggtggtcacttggtc-3’
CTGF / 5'-agctgacctggaagagaacatt-3' / 5'-gaagctgacctggaagagaaca-3'
5'-cgtcggtacatactccacagaa-3' / 5'-ttgataggcttggagattttgg-3'
N-Wasp / 5'-ggttccaactactgcaggaaac-3' / 5'-agagagggtgctcagctaaaaa-3'
5'-ggataccctgtcgtatctggtc-3' / 5'-gtctaacagtgcatctcgtcca-3'
WAVE2 / 5'-acacgtaaggaagagtgggaga-3' / 5'-acttctgggtatccacccactt-3'
5'-tacttgcatccacgttttcaac-3' / 5'-tacttgcatccacgttttcaac-3'
fascin / 5'-ctgctactttgacatcgagtgg-3' / 5'-caatggcaagtttgtgacctc-3'
5'-tgatgagcttcatgaggaagag-3' / 5'-ctctgagtcccctgctgtct-3'
ROCK1 / 5'-ttgagaaacagtgttccatgct-3' / 5'-tgaagcaatctcagcagaaac-3'
5'-tgtaacaacagccgcttatttg-3' / 5'-gctccagttgcagggttaga-3'
Cyclin-D1 / 5’-tctacaccgacaactccatcc-3’ / 5’-ctcctggtgaacaagctcaag-3’
5’-cggatgatctgtttgttctcct-3’ / 5’-gagaggaagtgttcaatgaaatcg-3’
p21 / 5'-tcactgtcttgtacccttgtgc-3' / 5'-ggagactctcagggtcgaaaac-3'
5'-gcttcctcttggagaagatcag-3' / 5'-ggcgtttggagtggtagaaat-3'
p27 / 5’-cctcagaagacgtcaaacgtaa-3’ / 5’-gcttgatgaagcaaggaagata-3’
5’-tcagtgcttatacaggatgtcca-3’ / 5’-gatgtccattccatgaagtcag-3’
TXNIP / 5'-gtatgcccctgagttcaagttc-3' / 5'-catgccaccaccgacttatact-3'
5'-tgagtgtccaggaagagagaca-3' / 5'-cttttcttccacatgctcactg-3'
GAPDH / 5'-acaactttggtatcgtggaagg-3' / 5'-acaactttggtatcgtggaagg-3'
5'-gtagaggcagggatgatgttct-3' / 5'-acagtcttctgggtggcagt-3'
NONO / 5'-cacatttcctcgtcctgtgac-3' / 5'-cccatggaccagttagatgatg-3'
5'-cgcatggcatattcatactcaa-3' / 5'-gttccttgtgaaattgctggtt-3'
GUSB / 5'-aacctttctatttccacggtgt-3’ / 5'-ggtgtcaacaagcatgaggat-3'
5'-cttcctctgcataggggtagtg-3’ / 5'-gttgaagtccttcaccagcag-3'

Supplementary Table 2. SPCs and pathology. H&E stained IDC sections were screened for SPCs based on nuclear morphology and cell shape. A total of 26 cancers were examined in a blinded study and segregated into two categories - presence or absence of SPCs. The percentage of samples in each category for ER, PR and Her-2 status, lymph node involvement, recurrence and metastasis were determined. Those with undetermined status were eliminated from analysis. Chi-squared analyses were performed to test the hypothesis that SPC status is not correlated with disease traits and patient outcomes. They null hypothesis was accepted for all categories except for breast cancer recurrence. There was a 0.03% chance that the correlation between SPC+ve tumours and recurrence is due to chance alone. *P<0.05.

Sample / ER Status / PR Status / Her-2/neu Status / Lymph Node Involvement / Recurrence / Metastasis
(+ve) SPCs / 73%; n=11 / 55%; n=11 / 30%; n=9 / 86%; n=7 / *27%; n=11 / 27%; n=11
(-ve) SPCs / 47%; n=14 / 36%; n=14 / 33%; n=15 / 67%; n=15 / *0%; n=14 / 29%; n=14

Supplementary Fig.1 Sorting of PMC42-ET/spc. Human breast cancer cells, PMC42-ET, a heterogeneous cell line with stem cell characteristics, were seeded into Boyden chambers containing high-density collagen matrices (20 mg/cm3). Cells were allowed to migrate through the matrix for one week. Those that migrated through the membrane pores were collected from the underside of the upper chamber by trypsinisation. The collected cells are called “PMC42-ET/spc. Both PMC42-ET/spc and PMC42-ET were grown onto high-density collagen matrices (20 mg/cm3) in complete RPMI supplemented with 10% FCS medium for 72 hours. The cells were then fixed and stained with Alexa Fluor 488 phalloidin. Images were taken on Olympus FV1000. (a) PMC42-ET/spc cells occur as solitary cells in high-density matrix and have a rounded morphology with protrusions. (b) PMC42-ET cells divide and form clusters. Scale bar = 10 μm.

Supplementary Fig.2 PMC42-ET/spc ceased proliferation in high-density matrix. (a), Both PMC43-ET/spc and PMC42-ET cells were grown on HD matrix for 72 hours and cells were counted 0, 24, 48 and 72 hours after seeding under a transmission light microscope (20x objective). PMC42-ET/spc cell number remained the same whereas PMC42-ET cells proliferated over time. (b), Seventy-two hours after seeding, cells were fixed in 4% PFA and stained with Alexa Fluor 488 phalloidin and confocal and reflection microscopes were used to image the cells and collagen, respectively. The image stacks were reconstructed in the x-z plane to measure the depth of cell migration. PMC42-ET/spcs were more migratory compared with PMC42-ET cells. Bar indicate the standard deviation from three biological replicates and the experiment was repeated twice. Student’s t-test demonstrates significant difference at * p < 0.05, ** p <0.01.