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. 2013 Jul;15(7):807-17.
doi: 10.1038/ncb2767. Epub 2013 Jun 2.

The perivascular niche regulates breast tumour dormancy

Cyrus M Ghajar  1 , Héctor Peinado, Hidetoshi Mori, Irina R Matei, Kimberley J Evason, Hélène Brazier, Dena Almeida, Antonius Koller, Katherine A Hajjar, Didier Y R Stainier, Emily I Chen, David Lyden, Mina J Bissell
Affiliations

The perivascular niche regulates breast tumour dormancy

Cyrus M Ghajar et al. Nat Cell Biol. 2013 Jul.

Abstract

In a significant fraction of breast cancer patients, distant metastases emerge after years or even decades of latency. How disseminated tumour cells (DTCs) are kept dormant, and what wakes them up, are fundamental problems in tumour biology. To address these questions, we used metastasis assays in mice and showed that dormant DTCs reside on microvasculature of lung, bone marrow and brain. We then engineered organotypic microvascular niches to determine whether endothelial cells directly influence breast cancer cell (BCC) growth. These models demonstrated that endothelial-derived thrombospondin-1 induces sustained BCC quiescence. This suppressive cue was lost in sprouting neovasculature; time-lapse analysis showed that sprouting vessels not only permit, but accelerate BCC outgrowth. We confirmed this surprising result in dormancy models and in zebrafish, and identified active TGF-β1 and periostin as tumour-promoting factors derived from endothelial tip cells. Our work reveals that stable microvasculature constitutes a dormant niche, whereas sprouting neovasculature sparks micrometastatic outgrowth.

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Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Dormant breast tumor cells reside on microvascular endothelium in distant tissues in vivo. (a) GFP-Luc MDA-MB-231 cells were injected into the inguinal mammary gland of NOD-SCID mice. Tumors were resected at 3 wks (Vavg = 0.5 cm3; representative bioluminescence shown). Mice that were relapse-free after 6 wks (4/20 mice) were sacrificed and visceral organs were dissected. (b) Representative image of a primary tumor section fixed and stained for endothelial-specific marker CD31 (red), and cell cycle marker Ki67 (white). DNA was labeled with Hoechst 33342 (blue). Dormant (Ki67-negative) DTCs (white asterisks) were found residing on microvascular endothelium in (c) lung and (d) BoMa tissues isolated from mice sacrificed 6 wks after primary tumor resection. (e) mCherry T4-2 cells (false-colored green here for consistency) were introduced via intra-cardiac injection, and mice that did not show any evidence of metastatic burden were sacrificed 8 wks later. In this second model, dormant (Ki-67 negative) T4-2 BCCs (white asterisks) also were found residing perivascularly in (f) lung, (g) bone marrow, and (h) brain. Scale bars = 20 μm.
Figure 2
Figure 2
Microvascular endothelium induces sustained quiescence of breast tumor cells in engineered cultures. (a) Lung and BoMa stroma (LFs and MSCs, respectively) were seeded alone or with mCherry-E4-ECs. In co-culture, mCherry-E4-ECs self-assembled into 3D microvascular networks over 7d. YFP-expressing BCCs (T4-2) were then seeded sparsely (240/cm2) in SFM onto stroma or microvascular niche cultures and overlaid with a drip of laminin-rich ECM (LrECM) diluted in media to provide BCCs with a 3D microenvironment64. Entire wells were imaged 10 days later. (b) Representative images of T4-2 cell growth within lung-like or BoMa-like niches containing stroma only, or stroma + ECs, 10 days post-seeding. Scale bars = 500 μm. (c, d) Tumor cell area fraction of YFP T4-2 at day 10 (normalized by value measured immediately post-seeding to correct for any minor variations in initial seeding density) in lung-like or BoMa-like niches, respectively (n=5 sets of co-cultures analyzed per condition). Error bars denote s.e.m. **p=0.001 and ***p<0.0001 by two-tailed t test. Day 10 co-cultures were fixed and stained for CD31 to label ECs and Ki67 to identify actively cycling tumor cells (b, inset; Scale bar = 50 μm). The percentage of Ki67-negative clusters (white asterisk in b, inset) was quantified for T4-2 cells seeded on (e) lung- and (f) BoMa-like niches (n=5 sets of co-cultures analyzed per condition). Error bars denote s.e.m. *** p<0.0001 by two-tailed t test. Tumor cell growth was measured over an additional 7 days (day 17 normalized by day 10) in (g) lung- and (h) BoMa-like niches to determine whether quiescent tumor clusters at day 10 remained quiescent (n=5 sets of co-cultures analyzed per condition). Error bars denote s.e.m. *** p<0.0001 by two-tailed t test. Live images of representative T4-2 cells on (i) lung-like stroma and microvascular niche are shown for day 10 and day 17, with IF staining to confirm Ki67 status. (j) The same is shown for representative T4-2 cells on BoMa-like stroma and microvascular niche cultures. Note that stroma culture scale bars = 100 μm and microvascular niche culture scale bars = 50 μm.
Figure 3
Figure 3
Thrombospondin-1 is an angiocrine tumor suppressor. Lung- and BoMa-like stroma and microvascular niche cultures were decellularized and residual proteins were acid extracted and subjected to LC-MS/MS analysis. (a) Heatmap of ECM proteins (spectral counts) from lung-like microvascular niche (LF + EC) normalized by lung stroma (LF; sorted high to low) and BoMa-like microvascular niche (MSC + EC) normalized by BoMa stroma (MSC). Log2 intensity scale shown at lower left. Localized expression of TSP-1 at the interface between dormant DTCs and lung microvasculature was confirmed in (b) spontaneous and (c) experimental metastasis models (white arrowheads). Scale bars = 10 μm. TSP-1 localization to the vascular BM was also confirmed in non-tumor bearing mice in (d) lung, (e) bone and (f) brain. Scale bars: 10 μm for lung, 20 μm for others. (g) Endothelial source of TSP-1 was confirmed by utilizing a 3D model of capillary morphogenesis where sprouting ECs are separated from inductive LFs by several millimeters. TSP-1 colocalized with type IV collagen in the BM of established microvessel stalks (white arrowheads), but TSP-1 appeared to be downregulated at neovascular tips (white asterisks). Scale bar = 50 μm. This was confirmed by (h) quantification of TSP-1 intensity at stalks vs. tips (n=16 microvessels pooled from 3 different experiments). Error bars denote s.e.m. ***p=0.0005 by two-tailed paired t-test. (i) Add-back of TSP-1 to T4-2 cells plated on lung stroma effectively substituted for the presence of ECs by causing a significant reduction in tumor cell growth (normalized to vehicle condition; n=3 sets of co-cultures analyzed per condition). Error bars denote s.e.m. * p<0.05 when compared to Vehicle by one-way ANOVA and Dunnett’s multiple comparisons test. (j) Treating lung-like microvascular niches with a TSP-1 blocking antibody resulted in significantly enhanced tumor cell growth (vs. IgG control; n=5 sets of co-cultures analyzed per condition). Error bars denote s.e.m. **p=0.0054 by two-tailed unpaired t-test.
Figure 4
Figure 4
Opposite regulation of tumor dormancy and growth by endothelial sub-niches: stable endothelium inhibits—whereas neovascular tips promote—breast tumor cell growth. (ai) Immunofluorescence of YFP T4-2 on BoMa-like microvascular niche after 10d. *: Ki67-negative tumor cluster; T: neovascular tips surrounding proliferative tumor. Scale bar = 100 μm. Insets ii–iv: additional examples of Ki67-negative BCCs residing on/near microvasculature from that same culture shown in (ai). Scale bar = 50 μm. (b) Stills from live tracking of histone H2B-GFP T4-2 cells on BoMa-like microvascular sub-niches (stable endothelium and neovascular tip) from 0–72h (see also Supplemental Movies 1–2). White arrowheads denote approaching tip and subsequent T4-2 division. Scale bars: left-most panel= 100 μm, right panels= 50 μm. (c) Scatter plot of T4-2 cell dwell time fraction (tdwell/tdiv) within stable (tdwell, stable), neovascular (tdwell, neo) or stromal (tdwell, stroma) sub-niches vs. division time (tdiv, n= 229 cells pooled from 3 separate time-lapse experiments). Pearson correlation analysis yielded significant correlation of tdwell, stable with tdiv and significant anti-correlation of tdwell, neo with tdiv, meaning that BCCs with longer division times tended to dwell more around stable endothelium and less around neovascular tips. Pearson coefficients (r) for stable endothelium, neovascular tips, and stroma are listed to right of plot. * p= 0.014 and ** p= 0.001 (two-tailed). (d) Dwell time (% of time to first division) as a function of sub-niche for fastest dividing tumor cells (t < tavg − SD; n=30 cells) and slowest dividing tumor cells (t > tavg + SD; n=59 cells).
Figure 5
Figure 5
Notch1-mediated reduction in neovascular tips suppresses breast tumor cell outgrowth. Microvascular niches were created with stromal cells mixed with shCtrl E4-EC and/or shNotch1 E4-EC. YFP T4-2 cells were then seeded in SFM and growth was analyzed 10 days later. (a) Microvascular niches composed of shCtrl E4-EC, shNotch1 E4-EC, or a 1:1 mix of the two (‘sh1:1’) were fixed and stained for CD31 at day 7. Scale bar = 200 μm. (b) Neovascular tip number/field (large white dots in a) and (c) branch point density (small yellow dots in a) were quantified (n=15 fields of microvascular networks pooled from 5 separate co-cultures). Error bars denote s.e.m ***p<0.001 when compared to shCtrl condition by one-way ANOVA and Dunnett’s post-test. (d) Representative images of YFP T4-2 seeded upon shCtrl EC, shNotch1 EC, or sh1:1 cultures, fixed and stained for CD31 and Ki67 10d post-seeding. Scale bar = 200 μm. (e) Quantification of normalized tumor cell area fraction and (f) %Ki67-negative clusters (each normalized by shCtrl condition; n=5 sets of co-cultures analyzed per condition). Error bars denote s.e.m. *p<0.05 when compared to shCtrl condition by one-way ANOVA and Dunnett’s post-test in (e).
Figure 6
Figure 6
Ectopic vascular sprouting promotes growth of injected breast tumor cells in zebrafish larvae. (a) ~1–10 mCherry-MDA-MB-231 BCCs were injected into the subintestinal space of 3.5 dpf mtp−/− mutant zebrafish and WT siblings (both containing the fli1:eGFP transgene) and imaged 4 days later. The injection time point (3.5 dpf) was chosen because (b) WT subintestinal vessels had few sprouts by this time point, while (c) the ectopic sprouting phenotype of the mtp−/− mutant was exaggerated (cyan asterisks denote neovascular sprouts). Scale bar = 50 μm. (d) Quantification of ectopic/neovascular sprouts in subintestinal space of WT and mtp−/− mutant siblings (n=25 WT zebrafish analyzed; n=23 mtp−/− zebrafish analyzed). Error bars denote s.e.m. ***p<0.0001 by two-tailed unpaired t-test. Representative images of (e) WT and (f) mtp−/− mutant zebrafish 4 days post-injection (i.e., 7.5 dpf) with mCherry-MDA-MB-231 cells. Scale bar = 100 μm. White arrow in (e) points to small cluster on abluminal surface of subintestinal vessel of WT, while white arrows in (f) point to larger clusters localized to neovascular tips in mtp−/− mutant. (g) Tumor cell area fraction in the subintestinal space was quantified at 7.5 dpf and normalized to the corresponding value post-injection for each surviving zebrafish with viable tumor cells in its subintestinal space to account for any variations in injection density (n=16 WT zebrafish analyzed; n=9 mtp−/− zebrafish analyzed). Error bars denote s.e.m. **p=0.005 by Mann-Whitney test.
Figure 7
Figure 7
Neovascular tips comprise ‘micrometastatic niches’ enriched for POSTN and TGF-β1. (a) Heatmap of ECM proteins (spectral counts) from 1) neovascular tiphigh cultures (LF + shCtrl EC) normalized by lung stroma (LF), 2) neovascular tiplow cultures (LF + shNotch1 EC) normalized by lung stroma (LF), and 3) tiphigh cultures normalized by tiplow cultures (sorted high to low with respect to this comparison, log2 scale). Representative images of microvessels stained for (b) POSTN, (c) active TGF-β1 and (d) latent TGF-β1. Scale bar = 20 μm. (e) Quantification of relative POSTN (left) and active TGF-β1 intensity (right) at the tip vs. stalk of microvessels (n=15 microvessels were pooled from 3 different experiments for analysis of POSTN intensity quantification; n=16 microvessels were pooled and analyzed for active TGF-β1 intensity quantification). Error bars represent s.e.m. ***p<0.0001 by paired two-tailed t-test. Representative images of microvascular niche cultures seeded with T4-2 cells and treated with (f) vehicle or (g) a combination of POSTN (50 ng/ml) and TGF-β1 (10 pg/ml) twice over the first 48h, and imaged at day 10. Scale bar = 500 μm. (h) Normalized tumor cell area fraction of YFP T4-2 at day 10 treated by vehicle or by said combination of POSTN and TGF-β1 (‘combo’; n=5 sets of co-cultures analyzed per condition). Error bars represent s.e.m. ***p<0.0001 by two-tailed t test. (i) A visual summary of our findings: In distant microenvironments, single or small clusters of DTCs reside in the perivascular niche and are maintained in a quiescent state by endothelial-derived factors. Here, we have identified TSP-1 as one such factor, while perlecan was identified by others as an EC-derived factor that suppresses tumor growth. Other ECM molecules such as laminins, type IV collagen and latent TGF-β binding proteins (LTBPs) may contribute directly or indirectly to the dormant niche. As vascular homeostasis is disrupted with induction of neovascular sprouting, endothelial architecture is perturbed. The result is not only loss of suppressive signals (e.g., TSP-1), but deposition of ECM molecules and growth factors that promote micrometastatic outgrowth. Thus, maintaining vascular homeostasis could be the key to sustaining DTC dormancy long-term.
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