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Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments

Nature Cell Biology volume 19, pages 120–132 (2017)Cite this article

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Abstract

Hypoxia is a poor-prognosis microenvironmental hallmark of solid tumours, but it is unclear how it influences the fate of disseminated tumour cells (DTCs) in target organs. Here we report that hypoxic HNSCC and breast primary tumour microenvironments displayed upregulation of key dormancy (NR2F1, DEC2, p27) and hypoxia (GLUT1, HIF1α) genes. Analysis of solitary DTCs in PDX and transgenic mice revealed that post-hypoxic DTCs were frequently NR2F1hi/DEC2hi/p27hi/TGFβ2hi and dormant. NR2F1 and HIF1α were required for p27 induction in post-hypoxic dormant DTCs, but these DTCs did not display GLUT1hi expression. Post-hypoxic DTCs evaded chemotherapy and, unlike ER− breast cancer cells, post-hypoxic ER+ breast cancer cells were more prone to enter NR2F1-dependent dormancy. We propose that primary tumour hypoxic microenvironments give rise to a subpopulation of dormant DTCs that evade therapy. These post-hypoxic dormant DTCs may be the source of disease relapse and poor prognosis associated with hypoxia.

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Figure 1: Hypoxia induces the expression of dormancy genes in mouse, PDX and human tumours.
Figure 2: Hypoxia induction using iNANIVID.
Figure 3: Primary tumour hypoxia induces the expression of quiescence and dormancy genes.
Figure 4: Hypoxia does not significantly influence intravasation and extravasation.
Figure 5: Post-hypoxic DTCs become quiescent in metastatic sites.
Figure 6: Post-hypoxic breast cancer cells become quiescent in vitro.
Figure 7: Post-hypoxic DTCs upregulate a dormancy signature.
Figure 8: Evasion of chemotherapy by post-hypoxic DTCs and NR2F1 induction in spontaneously seeded PyMT DTCs.

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Acknowledgements

We thank the Aguirre-Ghiso, Condeelis and Castracane laboratories for useful discussions. We thank N. Linde and M. S. Sosa for help and advice during initial phases of this study on the detection of TGFβ2 and NR2F1. This study was supported by: the Samuel Waxman Cancer Research Foundation Tumor Dormancy Program to J.A.A.-G.; the NIH/NCI TMEN U54CA163131 to J.Condeelis, P.J.K., J.Castracane and J.A.A.-G.; NIH/NCI grants CA109182 and CA191430 to J.A.A.-G.; DoD-BCRP Breakthrough Award (BC132674) to J.A.A.-G. and J.Condeelis; NCI Cancer Center P30 grant CA196521 to J.A.A.-G.; the TCI Young Scientist Cancer Research Award JJR Fund and NCI K22CA196750 grants to J.J.B.-C.; and the German Research Foundation (DFG) Fellowship (FL 865/1-1) and University Hospital Duesseldorf, Department of General and Visceral Surgery to G.F. Special optical devices were constructed and validated in the Gruss Lippoer Biophotonic Center and Integrated Imaging Program at Einstein. All imaging was performed in the Microscopy CORE at the Icahn School of Medicine at Mount Sinai. We thank N. Tzavaras (Microscopy CORE) for his technical help.

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Author notes

  1. Alvaro Avivar-Valderas

    Present address: Present address: AstraZeneca, iMED Oncology, Hodgkin Building, Chesterford Research Campus, Saffron Walden, Cambridge CB10 1XL, UK.,

  2. Georg Fluegen and Alvaro Avivar-Valderas: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine and Department of Otolaryngology, Tisch Cancer Institute, Black Family Stem Cell Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029, USA

    Georg Fluegen, Alvaro Avivar-Valderas, Ana Rita Nobre, Veronica Calvo, Julie F. Cheung, Jose Javier Bravo-Cordero & Julio A. Aguirre-Ghiso

  2. Department of General, Visceral and Pediatric Surgery, Medical Faculty, University Hospital of the Heinrich-Heine-University Düsseldorf, Düsseldorf 40225, Germany

    Georg Fluegen

  3. Department of Anatomy and Structural Biology, Gruss Lipper Biophotonics Center, Integrated Imaging Program, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA

    Yarong Wang, David Entenberg, Vladislav Verkhusha & John Condeelis

  4. Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, New York 12203, USA

    Michael R. Padgen, James K. Williams & James Castracane

  5. Department of Cell and Regenerative Biology, Laboratory of Molecular Biology, University of Wisconsin-Madison, 1525 Linden Drive, Madison, Wisconsin 53706, USA

    Patricia J. Keely

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Contributions

Conceptualization: J.A.A.-G., J.Condeelis, P.J.K., A.A.-V., G.F.; methodology: J.Condeelis, J.Castracane, D.E., A.R.N., J.F.C., V.C.; investigation: G.F., A.A.-V., A.R.N., J.F.C., V.C.; resources: Y.W., M.R.P., J.K.W., V.V., J.F.C., J.J.B.-C.; writing: G.F., J.A.A.-G.; visualization: G.F., A.A.-V.; funding acquisition: J.Condeelis, J.Castracane, P.J.K., J.A.A.-G., J.J.B.-C., G.F.; supervision: J.A.A.-G., J.Condeelis.

Corresponding authors

Correspondence to John Condeelis or Julio A. Aguirre-Ghiso.

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Competing interests

J.A.A.-G. receives funding from E. Lilly and co. J.Condeelis is a consultant for, and has equity in MetaStat, Inc.; J.Condeelis is also a consultant for Deciphera Pharmaceuticals.

Integrated supplementary information

Supplementary Figure 1 Quantification of the hypoxia-biosensor response, hypoxic adducts and influence of different oxygen tensions in vitro.

(A–C) Representative IF images and fluorescence histograms of MDA-231-HIF reporter cell xenografts in nude mice. Cells are GFP-tagged (green) and express mCherry (red) when hypoxic (5HR-ODD-mCherry). Sections were stained for (A) Glut1, (B) DEC2 and (C) NR2F1. Histograms show the percent fluorescence intensity normalized to maximum fluorescence for each channel over the boxed area (x-axis: distance, in arbitrary units). Scale bar: 50 μm; n = 3 tumors, see Fig 1A–C. (D) IF staining and quantification for pimonidazole adducts in T-HEp3 cell cytospins. Cells were in vitro cultured 72 h in hypoxia (1% O2) or normoxia (21% O2) and then treated with 50 μM pimonidazole for 3 h. Pimonidazole adducts are exclusively detectable in hypoxic cells. Bars show mean + SD, n = 4 independent experiments. Scale bar 25 μm. (E) Fold change of DEC2, GLUT1 and NR2F1 mRNA in T-HEp3, MDA-MB-231 and ZR-75-1 cells grown in different oxygen tensions (10%, 5%, 1% O2) versus 21% O2 for 72 hours. Following reverse transcription of whole RNA, quantitative qPCR for GLUT1, DEC2, NR2F1 and GAPDH was performed. GAPDH was used as a housekeeping gene. For the specific primer sequences see Table S3. Data points represent mean + SEM of n = 3 independent experiments, qPCR in triplicate; two tailed Student’s t test. (F) Images showing a 3-day-old Tet-On H2B-GFP T-HEp3 tumor on CAM with implanted PBS-iNANIVID (control), see Fig 2C. Top panel: stereoscopic image of the tumor in situ (4x). Panel a: merged image of a representative tumor area showing no GFP activation. Box indicates area in panel b. b: Detail of GFP negative tumor area. Scale bar: 250 μm; n = 3 tumors.

Supplementary Figure 2 iNANIVID microenvironments and hypoxia responses in vitro and in vivo.

(A) Representative intra-vital images of mCherry and GFP signal of MDA-231 HIF reporter cells grown on the CAM for 3 days in indicated distances of PBS- or Hi-NANIVIDs. Scale bar 10 μm; n = 3 tumors. (B) Fluorescence was measured: D = distal, M = middle distance, P = proximal to NANIVID. (C) Quantification of overall (all distances) mCherry signal in PBS- and Hi-NANIVID influenced MDA-231 HIF tumors. Bars show mean + SEM of n = 3 tumors per group, >180 cell cluster; one tailed Student’s t test. (D) Red mCherry signal at indicated distances after 3 days in vivo. D = distal, M = middle distance, P = proximal to iNANIVID (see Supplemental Figure 2B). Bars show mean + SD of n = 3 tumors, >60 cell clusters each distance. (E) T-HEp3 tumors in CAMs implanted with DFOM or PBS iNANAVIDs harvested at day 3 (D3) and day 6 (D6) and stained for HIF1α; scale bar 10 μm. Graph: average HIF1α fluorescence intensity (red) was quantified using MetaMorph. Each dot one cell. Bars show mean + SD; n = 400 cells, 2 tumors per treatment; Mann-Whitney test. (F) Representative images of IHC staining for DEC2, NR2F1 and Glut1 in 6 days PBS- or Hi-NANIVID treated T-HEp3 CAM tumors. White arrows: negative, black arrows positive cells; n = 2. (G) Fold change of expression in positive cells from PBS- to Hi-NANIVID treated CAM tumors; bars indicate mean + SEM. See Fig 2E + F for 3 day treated tumors. Each point one HPF. Scale bar 10 μm, n = 11 HPF of 2 tumors; Mann-Whitney test. (H) T-HEp3 tumors 6 days on Hi-iNANAVID CAMs were harvested and cultured for 24 hours with either PBS (n = 4) or 90 μM DFOM (n = 3), 120 cells per condition. Cells were stained for GLUT1. Representative images shown, scale bar 50 μm and 10 μm. Average fluorescence intensity per cell (green, GLUT1) was quantified using MetaMorph. Each dot one cell. Bars show mean + SD. Note re-induced GLUT1 expression in cells that had downregulated the response to DFOM in vivo (see Fig S2F + G). Mann-Whitney test. (I) Representative images of IF staining for phospho-Rb (pRb) and cleaved Caspase3 (Cl-C3) of 3 days PBS- or Hi-NANIVID treated T-HEp3 CAM tumors. Scale bar 50 μm; n = 3 tumors. For quantification see Fig 2G + H.

Supplementary Figure 3 Morphology of NANIVID treated tumors, correlation of HIF1α and p27 in NANIVID tumors, H3K4me3 staining, knock-down controls and plating efficiencies in hypoxia.

(A) Representative low-magnification images of H&E stained T-HEp3 CAM tumors treated for 6 days with PBS- or Hi-NANIVID or no iNANIVID at all. Scale bar 50 μm; n = 3 tumors. (B) Images and quantification of 3 and 6 days PBS- or Hi-NANIVID treated T-HEp3 CAM tumors stained for Glut1. Glut1 expression was measured and quantifying by red channel intensity (ImageJ). Scale bar 50 μm. Mean + SD; n = 5 low power images (10×) per tumor, 2 tumors each; Mann-Whitney test. (C) Representative images of nuclear HIF1α and p27 levels in PBS- and Hi-NANIVID T-HEp3 CAM tumor sections (day 3). Scale bar 10 μm; n = 3 tumors. (D) Quantification (integrated intensity) of nuclear levels of p27 and HIF1α in CAM sections of 3 days PBS- (blue) and Hi-NANIVID treated (red) tumors; negative control (background signal) in black. Note the higher levels of both antigens in DFOM exposed tumor cells. Bar graph: percentage of p27high/HIF1ahigh cells in CAM tumors, (high signal = 1.5x higher than background); bars show mean + SEM; n = 3 tumors, each dot one cell; Mann-Whitney test. (E) Pearson’s correlation between nuclear p27 and nuclear HIF1α in Hi-NANIVID tumors. Note the strong correlation between HIF1α induction and the growth arrest marker p27. Integrated intensities, see Fig S3D; n = 3 tumors, each dot one cell. (F) Quantification of H3K4me3 staining in normoxic (GLUT1 negative) and hypoxic (GLUT1 positive) areas of T-HEp3 CAM tumors (see Fig 3E, F). Bars show mean + SEM; n = 1500 cells scored of 3 tumors; two tailed Student’s t test. (G) Relative NR2F1 mRNA expression of T-HEp3 cells transfected 24 h with 50 nM NR2F1 siRNA compared to siControl (siCtr) transfected cells. Cells were cultured 24 h in normoxic (21% O2) or hypoxic (1% O2) tissue culture conditions. Cells from this pool were seeded on CAMs, see Fig 3H. PCR in triplicate, bars show mean + SD of n = 4 independent experiments; two tailed Student’s t test. (H) Relative HIF1α mRNA expression of T-HEp3 cells transfected 24 h with 20 nM NR2F1 siRNA compared to siControl (siCtr) transfected cells. Cells were cultured 24 h in normoxic (21% O2) or hypoxic (1% O2) tissue culture conditions. Cells from this pool were seeded on CAMs, see Fig 3I. PCR in triplicate, bars show mean + SD of n = 3 independent experiments; two tailed Student’s t test. (I) Graphs show plating efficiency (PE) for 24 h normoxic (N), 24 h hypoxic (24 h), 48 h hypoxic (48 h) and 72 h hypoxic (72 h) pre-treated cells. Bars show mean + SEM of n = 4 independent experiments; two tailed Student’s t test. (J) Relative NR2F1 mRNA expression of ZR-75-1 H2B-Dendra2 cells transfected 24 h with 50 nM NR2F1 or control siRNA and cultured in normoxia (21% O2) or hypoxia (1% O2) for 72 h. Cells were then photo converted and seeded in 3D Matrigel (see Fig 6C). Bars show mean NR2F1 level + SD of n = 4 independent experiments, PCR in triplicate; one tailed Student’s-t test.

Supplementary Figure 4 Controls for Vimentin specific detection, pRb-detection in DTCs, Dendra2 photoconversion and NR2F1 expression in PyM tumors.

(A) Gray scale image of mock tail vein injected (no T-HEp3 cells, only PBS) Foxn1 nu/nu mouse lung stained for human vimentin and DAPI. Vimentin antibody showed no reaction with mouse lung tissue. Boxed area in 40x indicates higher magnification shown in 100x. Scale bar 25 μm (40x) and 10 μm (100x). (B) Representative images and quantification of T-HEp3 DTCs in mouse lungs at day 10 after injection and 5 days cis-platin treatment (see Fig 8A), stained for p-Rb (green), human vimentin (red) and DAPI (blue). Graph shows mean + SD, n = 3 mice, >500 cells per group; one tailed Student’s t test. (C) Frozen section of PyMT-Dendra2 PT photo converted with UV channel for 15sec (see dashed line). Scale bar 250 μm. (D) Representative images of a spontaneous MMTV-PyMT-Dendra2 (green) lung micro-metastasis stained for macrophage marker CD45 (red) and DAPI (blue). No significant green Dendra2 signal was detected in CD45 positive macrophages. Asterisks indicate cytoplasmic aggregates of Dendra2 protein observed in MMTV-PyMT-Dendra2 cells, as in Fig 8G-H. Scale bar 10 μm; n = 3 mice. (E) Increase in red pixel intensity of 34 label retaining (LRC) and 34 not label retaining (NLRC) PyMT-Dendra2 lung DTCs measured using MetaMorph. Graph LRC: fold increase normalized to red pixel intensity before photo conversion of slide. Graph NLRC: absolute red pixel intensity in arbitrary units, no red pixel intensity before PC. Cells marked with # are shown in Figure 8H. (F) Quantification of cells positive for NR2F1 in PyMT-Dednra2 primary tumor (PT) and spontaneous lung DTCs (see Fig 8E-I). Bars show mean + SD. n = 3 mice, 10 HPF per PT, 187 lung DTCs.

Supplementary Figure 5 Graphic illustrating the effect of PT hypoxic microenvironments on DTCs.

We hypothesize that hypoxic PT microenvironments (red area) give rise to a more heterogeneous population of DTCs consisting of proliferative but also larger amounts of dormant DTCs. The latter can be dormantn for varying time periods but sufficient to possibly escape cycles of chemotherapy. These dormant DTCs may evade via passive and active mechanisms antiproliferative therapies fueling subsequent relapse. Compared to DTCs originating from normoxic PT microenvironments (blue area), hypoxic DTCs (red) are more prone to enter a dormancy (green cells) program in secondary organs driven by NR2F1 +, DEC2 +, p27 + and TGFβ2 + signals. Still in secondary organs post-hypoxic DTCs do not appear to maintain hypoxic gene expression (GLUT1-). However, the induction of p27 is dependent of at elast HIF1α and NR2F1 in primary sites. In secondary organs post-hypoxic DTCs may home to TGFβ2 + niches, create their own TGFβ2 + niches and/or fail to disrupt niches that contain and induce TGFβ2 expression. The persistence of dormancy gene expression in secondary organs may be linked to histone-H3 PTMs that control expression of dormancy (up) and hypoxic (down) genes as post-extravasation events in DTCs. We propose that understanding how hypoxia induces dormancy may reveal novel therapies that target selectively dormant DTCs. These therapies could be combined with standard anti-proliferative treatments.

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Fluegen, G., Avivar-Valderas, A., Wang, Y. et al. Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments. Nat Cell Biol 19, 120–132 (2017). https://doi.org/10.1038/ncb3465

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