Correction Interactions between cancer stem cells and their niche govern metastatic colonization Ilaria Malanchi, Albert Santamaria-Martínez, Evelyn Susanto, Hong Peng, Hans-Anton Lehr, Jean-Francois Delaloye & Joerg Huelsken Nature 481, 85 89 (2012). In the Supplementary Figures, one of the controls in Supplementary Fig. 27 inadvertently duplicated the control picture in Fig. 3h. The error has been corrected. Our results and conclusions are not altered by this change.
SUPPLEMENTARY INFORMATION doi:10.1038/nature10694 Fig. S1: At the primary site, existing niches that are involved in regulation of stem cell function may be preserved upon tumour formation. This may involve additional activation and/or expansion of the niche in parallel to the increase of cancer stem cell numbers. Upon invasion and seeding of tumour cells to secondary target sites, tumour cells will suddenly be exposed to a different, potentially non-supportive environment. Only the education or re-programming of the stroma by infiltrating tumour cells will allow formation of a secondary niche. This novel niche resembles at least partially the primary environment. One essential ECM molecule contributing to the metastatic niche is POSTN, which is induced by tumour-derived TGFβ3. It supports cancer stem cells by recruiting Wnt ligands that have been shown to directly regulate stem cell maintenance for a variety of stem and cancer stem cell populations. Obviously, not all secondary sites can be expected to facilitate niche generation, which may explain in part the non-random organ distribution of metastases. WWW.NATURE.COM/NATURE 1
RESEARCH SUPPLEMENTARY INFORMATION Fig. S2: CD24 + CD90 + identifies a subset of the CD24 + CD29 + population. GFP + MMTV-PyMT tumour cells were transplanted orthotopically and were analysed by FACS after 8 weeks when tumours had formed. Gating of CD24 + CD29 + GFP + 7AAD - Lin - tumour cells show that CD90 + CD24 + cells are a subset of the previously described CD24 + CD29 hi population of breast cancer stem cells. CD90 + CD24 + cells are not found in the CD24 + CD29 low population. 2 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Fig. S3: CSCs (red) and non-cscs (black) show similar extravasation efficiency. GFP + tumour cells were intravenously injected and the relative amount of the two populations was evaluated at 2h (before extravasation) and 48h (after extravasation) post-injection. For the 48h time point, lungs were perfused with Ringer solution to remove cells trapped inside capillaries. Note that the CSCs population is stably maintained at 1% of the total tumour cells at these early time points. (n=3 each, error bars show s.d.) WWW.NATURE.COM/NATURE 3
RESEARCH SUPPLEMENTARY INFORMATION Fig. S4: Dynamics of metastatic colonization in the lung after tail vein injection. 10 6 GFP + tumour cells were tail vein-injected at different time points. Whole mount pictures show a rapid decrease of tumour cell numbers in the lungs during the first week and progressive growth of colonies giving rise to full-blown metastases by 4 weeks. Bar: 1mm. The samples were analyzed by FACS to quantify total tumour cell and CSC content, as depicted in Fig. 1d. 4 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH BrdU DAPI 2 weeks 1 week GFP Fig. S5: CSC proliferation during metastasis establishment. GFP+ tumour cells were tail vein injected and cell proliferation was measured by BrdU incorporation one and two weeks later. A single dose of BrdU was intraperitoneally injected two hours previous to euthanasia, and cryosections were stained for BrdU to show the abundance of proliferating GFP+ tumour cells. Bar: 20µm. W W W. N A T U R E. C O M / N A T U R E 5
RESEARCH SUPPLEMENTARY INFORMATION Fig. S6: Non-CSCs do not give rise to CSCs. CSCs and non-cscs population were FAC-sorted from GFP + tumour cells freshly isolated from primary tumours and separately iv injected into recipient mice. The CSC content was evaluated by FACS analysis 1 week after injection. Note that non-cscs do not give rise to CSCs in vivo. (n=3 each, error bars show s.d.) 6 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH a b c CD34 POSTN -catenin POSTN K14 POSTN Fig. S7: Niche expression of POSTN in the skin and the small intestine. (a) Expression of the established bulge stem cell marker CD34 and of POSTN in the associated niche as detected by immunofluorescence in an early anagen hair follicle of murine skin. (b) Expression of POSTN in the fibroblast stroma underlying the intestinal crypt and of β-catenin in all intestinal epithelial cells as detected by immunofluorescence. Note that POSTN is not expressed by the stroma within the intestinal villi (cf. Kikuchi, Y. et al., J. Histochem. Cytochem. 2008). (c) Breast duct of a pregnant mouse 10.5 days post coitum shows POSTN staining in fibroblasts along the ducts however not in Keratin14 (K14) positive myoepithelial cells. WWW.NATURE.COM/NATURE 7
RESEARCH SUPPLEMENTARY INFORMATION Lung Brain Liver Lymph node Bone KO PyMT WT Fig. S8: POSTN staining pattern in mouse tissues. IHC of different tissues of wild type, tumour harbouring but metastasis free MMTV-PyMT and of POSTN-/- mice. POSTN staining is present in the bone of wt and PyMT animals while there is no detectable staining in the lung parenchyme, the lymph node, the brain or the liver. 8 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH lymph nodes with stromal 17/23 reaction (75%) POSTN + stroma 17/23 a b Fig. S9: Axillary lymph node metastases of human breast cancer patients often contain a desmoplastic reaction positive for POSTN. Immunohistochemical analysis revealed that 75% of axillary lymph node metastases of human breast cancer patients show induction of POSTN expression in the desmoplastic stroma adjacent to nodal metastases (cf. Fig. 2i,j). The remaining 25% are characterized by a complete absence of a desmoplastic reaction, thus no POSTN is detected. The pictures show adjacent sections of a such a sample (lacking a desmoplastic reaction) stained for (a) POSTN and (b) pan-cytokeratin. WWW.NATURE.COM/NATURE 9
RESEARCH SUPPLEMENTARY INFORMATION POSTN wt allele POSTN targeted allele +/+ +/- +/+ -/- wt wt ES cell Southern Blot null mice genotyping PCR null Fig. S10: Generation of the POSTN gene ablation in mice. The top panel depicts the wt and targeted allele of the POSTN gene. A stop and a polya cassette was introduced in the first exon that contains the signal sequence required for secretion of the protein. Green lines symbolize exons. The lower panel shows (left) a Southern blot used to identify targeted embryonic stem (ES) cells and (b) the result of a PCR genotyping. The mutant allele was backcrossed into the FVB background for at least 8 generations. 10 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH 4 weeks old virgin Control POSTN-/- Pregnant 15.5 d Fig. S11: Analysis of mammary gland development in POSTN-/- mice. Wild type and POSTN-/- females were analysed at different developmental stages. Images show Carmine Alum stained whole mounts and (inserts) H&E stained sections of the mammary gland of 4 weeks old and 15.5 days pregnant wt and -/- females. No morphologic differences were observed in wt and mutant mammary glands. WWW.NATURE.COM/NATURE 11
RESEARCH SUPPLEMENTARY INFORMATION % mammary gland stem cells control POSTN -/- Fig. S12: Analysis of mammary gland stem cell content. Mammary glands of 6 to 7 week old Bl6 females were digested to isolate single cells and stained for FACS. No significant differences were observed in the abundance of the Lin - CD24 + CD29 hi population of mammary gland stem cells between wt and POSTN-/- mice. (n=4 each, errorbars show s.d.) 12 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH a 3.0 b 60 size primary tumour (g) 2.5 2.0 1.5 1.0 0.5 number of metastases 50 40 30 20 10 p<0.003 control POSTN -/- control POSTN -/- control POSTN-/- control POSTN-/- Fig. S13: Comparison of primary tumour size and number of metastases of wt vs. POSTN-/- MMTV-PyMT mice. Although mutant mice show no differences in terms of primary tumour size compared to wt animals, the number of metastases is dramatically reduced in the former. WWW.NATURE.COM/NATURE 13
RESEARCH SUPPLEMENTARY INFORMATION GATA-3 SMA K8 GATA-3 SMA K8 KO WT Fig. S14: Differentiation status of POSTN-/- tumours. Pictures show representative sections of wild type MMTV- PyMT and MMTV-PyMT;POSTN-/- tumours (n=8) stained for SMA, cytokeratin 8 and the previously defined marker of tumour cell differentiation in breast cancer, GATA-3. 14 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Fig. S15: Hematoxylin-eosin staining of (a,b) wt and (c,d) ko tumours. No major differences were observed in terms of histological features in tumour samples from wt and ko animals. Bars: a,c 200µm; b,d 500µm WWW.NATURE.COM/NATURE 15
RESEARCH SUPPLEMENTARY INFORMATION 1.0 10 6 GFP + -/- tumour cells orthotopic transplantation tumour size [g] 2.0 1.5 1.0 0.5 0.8 0.6 0.4 0.2 tumour cells in lung [%] control host -/- host control host -/- host Fig. S16: POSTN deficient tumour cells form more metastases in wt than in POSTN-/- recipients. POSTN-/- tumour cells were orthotopically transplanted into Rag2-/- or RAG2-/-;POSTN-/- mice. Tumour size in the mammary gland was determined and pulmonary metastasis formation (p<0.08) was scored by FACS analysis of GFP + tumour cells after 6 weeks (n=3 each; error bars show s.d.). 16 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH 15 10 5 GFP + -/- tumour cells tail vein injection 2 control host 1 -/- host 10 5 0 number of metastases Fig. S17: Tail vein injection of tumour cells demonstrates an important function of POSTN at the metastatic site. Tail vein injection of 10 5 GFP + POSTN-/- tumour cells into wt or POSTN-/- Nude mice reveals a striking >6.5 fold reduction of pulmonary metastasis formation in POSTN-/- compared to wt recipients after 6 weeks (n=5 each, p<0.035, error bars show s.d.). WWW.NATURE.COM/NATURE 17
RESEARCH SUPPLEMENTARY INFORMATION Fig. S18: Immunohistochemistry reveals similar levels of POSTN expression in wt tumours (left) and tumours generated by POSTN-/- tumour cell transplantation into wt recipients (right). A tumour from a POSTN-/- animal is shown as a negative control (middle panel). Bars: 25µm. 18 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Fig. S19: Evaluation of candidate factors for induction of POSTN expression in lung stromal cells. TGFβ2, Wnt3a, Wnt5a, BMP4 were added to lung stromal cells to test their ability to induce POSTN expression. RNA expression by qpcr and protein expression by Western blot analysis (inserts) were measured at the indicated concentration of factor (n=3 each). WWW.NATURE.COM/NATURE 19
RESEARCH SUPPLEMENTARY INFORMATION Vimentin plus tumour cells control POSTN GFP Fig. S20: Tumour cells induce POSTN secretion in a primary lung fibroblast co-culture system. Wild type lungs were mechanically and enzymatically digested and then seeded in DMEM/F12 supplemented with 2% FBS, 20ng/ml EGF, 10 μg/ml insulin for 3 days to establish lung fibroblast cultures. On day 3, medium was changed to alphamem supplemented with 0.3% FBS and 10 μg/ml insulin. For co-cultures, GFP+ tumour cells were added one day later. After 72 hours, cells were fixed with 4% PFA and stained for vimentin and POSTN. Immunostainings show a dramatic increase in POSTN secretion when tumour cells are added to the cultures. 2 0 W W W. N A T U R E. C O M / N A T U R E
SUPPLEMENTARY INFORMATION RESEARCH 20% Relative to GAPDH [%] 10% 0% CSC non-csc Fig. S21: TGFβ3 mrna levels in CSCs vs non-cscs. MMTV-PyMT tumour cells were FACS-sorted, collected in RLP lysis buffer and RNA was extracted and reverse-transcribed to cdna. Real-time PCR analysis shows that there are no significant differences in terms of TGFβ3 mrna expression in CSCs compared to non-cscs. (n=3, error bars show s.d.) WWW.NATURE.COM/NATURE 21
RESEARCH SUPPLEMENTARY INFORMATION Fig. S22: Block of TGFβ signaling prevents POSTN induction in lung metastases in vivo. POSTN+/+ GFP + tumour cells were transduced with lentiviral vectors expressing a secreted decoy receptor (TGFβR2ΔTM) and hcd2 or hcd2 alone (control) and iv injected in recipient mice. Expression of the decoy receptor prevents POSTN induction in the metastasis stroma (n=5). Bar: 25µm 22 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Fig. S23: Distribution of micrometastasis size and number in control and POSTN deficient animals (n=5 each). WWW.NATURE.COM/NATURE 23
RESEARCH SUPPLEMENTARY INFORMATION MTT activity days in culture Fig. S24: Unchanged growth potential on plastic of POSTN-/- cells. Wild type and POSTN-/- tumour cells were plated in DMEM/F12 supplemented with 2% FBS, 20ng/ml EGF, 10 μg/ml insulin and assayed for MTT activity at different time points. Both show similar proliferation potential.(n=3) 24 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH POSTN SMA POSTN vimentin POSTN K14 Fig. S25: POSTN is found close to SMA + and vimentin + cells in tumour mammospheres. Whole mount fluorescent stainings of tumour mammospheres show the presence of POSTN close to vimentin-positive and αsma-positive cells but not to Keratin14 (K14) positive cells. This indicates that POSTN is produced by myofibroblasts in these cultures which are directly derived from cancer associated fibroblasts of the primary tumour. WWW.NATURE.COM/NATURE 25
RESEARCH SUPPLEMENTARY INFORMATION MTT activity control mab1 mab2 mab3 mab4 plated cell number Fig. S26: POSTN blocking antibodies do not interfere with short-term tumour cell growth under adherent culture conditions. When tumour cells from tumuor mammosphere cultures are seeded on collagen-treated plastic, they can continue to proliferate for up to 14 days and start to differentiate. Under these conditions, the POSTN blocking antibodies do not interfere with cell growth as determined by a MTT assay. Four different blocking hybridoma supernatants (mab1-4) and one control migg1 were tested. Either 500 or 3000 tumour cells were seeded and incubated with antibodies for 7 days until the MTT assay was performed. (n=6 each) 26 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH previously incubated with anti-postn control 1 week 2 weeks Fig. S27: Transient exposure to a POSTN blocking antibody does not prevent tumour mammosphere formation. While the continued exposure of a POSTN blocking antibody can block sphere formation, a transient exposure can be overcome demonstrating that POSTN function does not directly influence cell survival. Wild type tumour mammospheres were incubated with either control mab (top) or anti-postn hybridoma supernatant for 10 days. The cells were then washed to remove antibodies, trypsinized and again assayed under sphere forming conditions for 1 and 2 weeks. (n=3) WWW.NATURE.COM/NATURE 27
RESEARCH SUPPLEMENTARY INFORMATION signal sequence FSC FSC FSC FSC SBP His FSC: fasciclin domain Fig. S28: Schematic structure of TAP-tagged POSTN. POSTN encodes for a secreted protein with four fasciclin domains. For tandem affinity purification (TAP)-tag enrichment, the cdna was engineered to contain a C-terminal streptavidin-binding-peptide domain (SBP; MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) and a 8x poly-histidine tag for streptavidin and Ni-NTA enrichment, respectively. 28 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH LTR cppt TATA GFP PGK hcd2 WPRE LTR 20x LEF sites anti-postn control Fig. S29: Wnt signalling is abrogated in the absence of POSTN. The scheme depicts the design of the lentiviral Wnt reporter construct that contains a Wnt-responsive minimal promoter with 20 copies of a LEF/TCF responsive element driving GFP expression and a second, ubiquitous promoter (PGK) that drives expression of a hcd2 fragment. hcd2 expression was used to detect and enrich for reporter containing cells. Infected tumour cells were purified by MACS sorting and plated under ultra low attachment condition in presence or absence of POSTN blocking antibody. Spheres were analyzed after 10 days using an inverted fluorescent microscope to detect GFP reporter activity (n=5). Note the absence of Wnt signalling in presence of POSTN blocking antibody. Bar: 25µm. WWW.NATURE.COM/NATURE 29
RESEARCH SUPPLEMENTARY INFORMATION Fig. S30: Inhibition of tumour mammosphere formation (from POSTN wt tumour cells) by a blocking POSTN mab is overcome by Wnt3a addition. (n=6, error bars show s.d.) sphere numbers Fig. S31: Wnt3a addition can rescue sphere formation of POSTN deficient tumour cells. Wnt3a ligand was added to cultures of primary POSTN -/- tumour cells at the indicated concentrations. (n=3, error bars show s.d.) 30 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH % primary tumour stem cells control POSTN-/- Fig. S32: Cancer stem cell content in primary wt tumours vs. ko tumours. The frequency of Lin - CD24 + CD90 + tumour cells in wt MMTV-PyMT primary tumours is not statistically different from CSCs isolated from MMTV-PyMT;POSTN-/- primary tumours (n=10, error bars show s.d.). WWW.NATURE.COM/NATURE 31