2,116
Views
22
CrossRef citations to date
0
Altmetric
Research Paper

Direct visualization of the phenotype of hypoxic tumor cells at single cell resolution in vivo using a new hypoxia probe

, , , , , & show all
Article: e1187803 | Received 22 Dec 2015, Accepted 05 May 2016, Published online: 16 May 2016

References

  • Talmadge JE, Fidler IJ. AACR centennial series: the biology of cancer metastasis: historical perspective. Cancer Res 2010; 70:5649-69; PMID:20610625; http://dx.doi.org/10.1158/0008-5472.CAN-10-1040
  • Patsialou A, Wang Y, Lin J, Whitney K, Goswami S, Kenny PA, Condeelis JS. Selective gene-expression profiling of migratory tumor cells in vivo predicts clinical outcome in breast cancer patients. Breast Cancer Res 2012; 14:R139; PMID:23113900; http://dx.doi.org/10.1186/bcr3344
  • Gligorijevic B, Bergman A, Condeelis J. Multiparametric classification links tumor microenvironments with tumor cell phenotype. PLoS Biol 2014; 12:e1001995; PMID:25386698; http://dx.doi.org/10.1371/journal.pbio.1001995
  • Bertout JA, Patel SA, Simon MC. The impact of O2 availability on human cancer. Nat Rev Cancer 2008; 8:967-75; PMID:18987634; http://dx.doi.org/10.1038/nrc2540
  • Chan DA, Giaccia AJ. Hypoxia, gene expression, and metastasis. Cancer Metastasis Rev 2007; 26:333-9; PMID:17458506; http://dx.doi.org/10.1007/s10555-007-9063-1
  • Gort EH, Groot AJ, van der Wall E, van Diest PJ, Vooijs MA. Hypoxic regulation of metastasis via hypoxia-inducible factors. Curr Mol Med 2008; 8:60-7; PMID:18289014; http://dx.doi.org/10.2174/156652408783565568
  • Nagelkerke A, Bussink J, Mujcic H, Wouters BG, Lehmann S, Sweep FC, Span PN. Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response. Breast Cancer Res 2013; 15:R2; PMID:23294542; http://dx.doi.org/10.1186/bcr3373
  • Staller P, Sulitkova J, Lisztwan J, Moch H, Oakeley EJ, Krek W. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. Nature 2003; 425:307-11; PMID:13679920; http://dx.doi.org/10.1038/nature01874
  • Zundel W, Schindler C, Haas-Kogan D, Koong A, Kaper F, Chen E, Gottschalk AR, Ryan HE, Johnson RS, Jefferson AB, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev 2000; 14:391-6; PMID:10691731
  • Blagosklonny MV, An WG, Romanova LY, Trepel J, Fojo T, Neckers L. p53 inhibits hypoxia-inducible factor-stimulated transcription. J Biol Chem 1998; 273:11995-8; PMID:9575138; http://dx.doi.org/10.1074/jbc.273.20.11995
  • Zhou W, Dosey TL, Biechele T, Moon RT, Horwitz MS, Ruohola-Baker H. Assessment of hypoxia inducible factor levels in cancer cell lines upon hypoxic induction using a novel reporter construct. PLoS One 2011; 6:e27460; PMID:22132102; http://dx.doi.org/10.1371/journal.pone.0027460
  • Zhang H, Wong CC, Wei H, Gilkes DM, Korangath P, Chaturvedi P, Schito L, Chen J, Krishnamachary B, Winnard PT, Jr, et al. HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs. Oncogene 2012; 31:1757-70; PMID:21860410; http://dx.doi.org/10.1038/onc.2011.365
  • Liao D, Corle C, Seagroves TN, Johnson RS. Hypoxia-inducible factor-1alpha is a key regulator of metastasis in a transgenic model of cancer initiation and progression. Cancer Res 2007; 67:563-72; PMID:17234764; http://dx.doi.org/10.1158/0008-5472.CAN-06-2701
  • Eisinger-Mathason TS, Zhang M, Qiu Q, Skuli N, Nakazawa MS, Karakasheva T, Mucaj V, Shay JE, Stangenberg L, Sadri N, et al. Hypoxia-dependent modification of collagen networks promotes sarcoma metastasis. Cancer Discov 2013; 3:1190-205; PMID:23906982; http://dx.doi.org/10.1158/2159-8290.CD-13-0118
  • Gilkes DM, Bajpai S, Wong CC, Chaturvedi P, Hubbi ME, Wirtz D, Semenza GL. Procollagen lysyl hydroxylase 2 is essential for hypoxia-induced breast cancer metastasis. Mol Cancer Res 2013; 11:456-66; PMID:23378577; http://dx.doi.org/10.1158/1541-7786.MCR-12-0629
  • Aro E, Khatri R, Gerard-O'Riley R, Mangiavini L, Myllyharju J, Schipani E. Hypoxia-inducible factor-1 (HIF-1) but not HIF-2 is essential for hypoxic induction of collagen prolyl 4-hydroxylases in primary newborn mouse epiphyseal growth plate chondrocytes. J Biol Chem 2012; 287:37134-44; PMID:22930750; http://dx.doi.org/10.1074/jbc.M112.352872
  • Bentovim L, Amarilio R, Zelzer E. HIF1alpha is a central regulator of collagen hydroxylation and secretion under hypoxia during bone development. Development 2012; 139:4473-83; PMID:23095889; http://dx.doi.org/10.1242/dev.083881
  • Koh MY, Powis G. HAF : the new player in oxygen-independent HIF-1alpha degradation. Cell Cycle 2009; 8:1359-66; PMID:19377289; http://dx.doi.org/10.4161/cc.8.9.8303
  • Shibata T, Giaccia AJ, Brown JM. Development of a hypoxia-responsive vector for tumor-specific gene therapy. Gene Ther 2000; 7:493-8; PMID:10757022; http://dx.doi.org/10.1038/sj.gt.3301124
  • He F, Deng X, Wen B, Liu Y, Sun X, Xing L, Minami A, Huang Y, Chen Q, Zanzonico PB, et al. Noninvasive molecular imaging of hypoxia in human xenografts: comparing hypoxia-induced gene expression with endogenous and exogenous hypoxia markers. Cancer Res 2008; 68:8597-606; PMID:18922936; http://dx.doi.org/10.1158/0008-5472.CAN-08-0677
  • Liu J, Qu R, Ogura M, Shibata T, Harada H, Hiraoka M. Real-time imaging of hypoxia-inducible factor-1 activity in tumor xenografts. J Radiat Res 2005; 46:93-102; PMID:15802864; http://dx.doi.org/10.1269/jrr.46.93
  • Indovina P, Collini M, Chirico G, Santini MT. Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy. FEBS Lett 2007; 581:719-26; PMID:17270179; http://dx.doi.org/10.1016/j.febslet.2007.01.040
  • Harney AS, Arwert EN, Entenberg D, Wang Y, Guo P, Qian BZ, Oktay MH, Pollard JW, Jones JG, Condeelis JS. Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA. Cancer Discov 2015; 5:932-43; PMID:26269515; http://dx.doi.org/10.1158/2159-8290.CD-15-0012
  • Doublier S, Belisario DC, Polimeni M, Annaratone L, Riganti C, Allia E, Ghigo D, Bosia A, Sapino A. HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast. BMC Cancer 2012; 12:4; PMID:22217342; http://dx.doi.org/10.1186/1471-2407-12-4
  • Lacroix M, Leclercq G. Relevance of breast cancer cell lines as models for breast tumours: an update. Breast Cancer Res Treat 2004; 83:249-89; PMID:14758095; http://dx.doi.org/10.1023/B:BREA.0000014042.54925.cc
  • Harada H, Kizaka-Kondoh S, Itasaka S, Shibuya K, Morinibu A, Shinomiya K, Hiraoka M. The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts. Biochem Biophys Res Commun 2007; 360:791-6; PMID:17624305; http://dx.doi.org/10.1016/j.bbrc.2007.06.149
  • Krishnamachary B, Penet MF, Nimmagadda S, Mironchik Y, Raman V, Solaiyappan M, Semenza GL, Pomper MG, Bhujwalla ZM. Hypoxia regulates CD44 and its variant isoforms through HIF-1alpha in triple negative breast cancer. PLoS One 2012; 7:e44078; PMID:22937154; http://dx.doi.org/10.1371/journal.pone.0044078
  • Potzkei J, Kunze M, Drepper T, Gensch T, Jaeger KE, Buchs J. Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor. BMC Biol 2012; 10:28; PMID:22439625; http://dx.doi.org/10.1186/1741-7007-10-28
  • Jiang L, Greenwood TR, Artemov D, Raman V, Winnard PT, Jr, Heeren RM, Bhujwalla ZM, Glunde K. Localized hypoxia results in spatially heterogeneous metabolic signatures in breast tumor models. Neoplasia 2012; 14:732-41; PMID:22952426; http://dx.doi.org/10.1593/neo.12858
  • Raman V, Artemov D, Pathak AP, Winnard PT, Jr, McNutt S, Yudina A, Bogdanov A, Jr, Bhujwalla ZM. Characterizing vascular parameters in hypoxic regions: a combined magnetic resonance and optical imaging study of a human prostate cancer model. Cancer Res 2006; 66:9929-36; PMID:17047055; http://dx.doi.org/10.1158/0008-5472.CAN-06-0886
  • Takahashi E, Sato M. Imaging of oxygen gradients in monolayer cultured cells using green fluorescent protein. Am J Physiol Cell Physiol 2010; 299:C1318-23; PMID:20844249; http://dx.doi.org/10.1152/ajpcell.00254.2010
  • Cao Y, Li CY, Moeller BJ, Yu D, Zhao Y, Dreher MR, Shan S, Dewhirst MW. Observation of incipient tumor angiogenesis that is independent of hypoxia and hypoxia inducible factor-1 activation. Cancer Res 2005; 65:5498-505; PMID:15994919; http://dx.doi.org/10.1158/0008-5472.CAN-04-4553
  • Moeller BJ, Cao Y, Li CY, Dewhirst MW. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer Cell 2004; 5:429-41; PMID:15144951; http://dx.doi.org/10.1016/S1535-6108(04)00115-1
  • Vordermark D, Shibata T, Brown JM. Green fluorescent protein is a suitable reporter of tumor hypoxia despite an oxygen requirement for chromophore formation. Neoplasia 2001; 3:527-34; PMID:11774035; http://dx.doi.org/10.1038/sj.neo.7900192
  • Reid BG, Flynn GC. Chromophore formation in green fluorescent protein. Biochemistry 1997; 36:6786-91; PMID:9184161; http://dx.doi.org/10.1021/bi970281w
  • McKeown SR. Defining normoxia, physoxia and hypoxia in tumours-implications for treatment response. Br J Radiol 2014; 87:20130676; PMID:24588669; http://dx.doi.org/10.1259/bjr.20130676
  • Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003; 3:721-32; PMID:13130303; http://dx.doi.org/10.1038/nrc1187
  • Gorin F, Harley W, Schnier J, Lyeth B, Jue T. Perinecrotic glioma proliferation and metabolic profile within an intracerebral tumor xenograft. Acta Neuropathol 2004; 107:235-44; PMID:14712400; http://dx.doi.org/10.1007/s00401-003-0803-1
  • Drepper T, Huber R, Heck A, Circolone F, Hillmer AK, Buchs J, Jaeger KE. Flavin mononucleotide-based fluorescent reporter proteins outperform green fluorescent protein-like proteins as quantitative in vivo real-time reporters. Appl Environ Microbiol 2010; 76:5990-4; PMID:20601504; http://dx.doi.org/; http://dx.doi.org/10.1128/AEM.00701-10
  • Coralli C, Cemazar M, Kanthou C, Tozer GM, Dachs GU. Limitations of the reporter green fluorescent protein under simulated tumor conditions. Cancer Res 2001; 61:4784-90; PMID:11406553
  • Shaner NC, Steinbach PA, Tsien RY. A guide to choosing fluorescent proteins. Nat Methods 2005; 2:905-9; PMID:16299475; http://dx.doi.org/10.1038/nmeth819
  • Carroll P, Schreuder LJ, Muwanguzi-Karugaba J, Wiles S, Robertson BD, Ripoll J, Ward TH, Bancroft GJ, Schaible UE, Parish T. Sensitive detection of gene expression in mycobacteria under replicating and non-replicating conditions using optimized far-red reporters. PLoS One 2010; 5:e9823; PMID:20352111; http://dx.doi.org/; http://dx.doi.org/10.1371/journal.pone.0009823
  • Brahimi-Horn MC, Chiche J, Pouyssegur J. Hypoxia signalling controls metabolic demand. Curr Opin Cell Biol 2007; 19:223-9; PMID:17303407; http://dx.doi.org/10.1016/j.ceb.2007.02.003
  • Behrooz A, Ismail-Beigi F. Stimulation of Glucose Transport by Hypoxia: Signals and Mechanisms. News Physiol Sci 1999; 14:105-10; PMID:11390832
  • Ameri K, Luong R, Zhang H, Powell AA, Montgomery KD, Espinosa I, Bouley DM, Harris AL, Jeffrey SS. Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype. Br J Cancer 2010; 102:561-9; PMID:20051957; http://dx.doi.org/10.1038/sj.bjc.6605491
  • Rademakers SE, Lok J, van der Kogel AJ, Bussink J, Kaanders JH. Metabolic markers in relation to hypoxia; staining patterns and colocalization of pimonidazole, HIF-1alpha, CAIX, LDH-5, GLUT-1, MCT1 and MCT4. BMC Cancer 2011; 11:167; PMID:21569415; http://dx.doi.org/; http://dx.doi.org/10.1186/1471-2407-11-167
  • Heilig C, Brosius F, Siu B, Concepcion L, Mortensen R, Heilig K, Zhu M, Weldon R, Wu G, Conner D. Implications of glucose transporter protein type 1 (GLUT1)-haplodeficiency in embryonic stem cells for their survival in response to hypoxic stress. Am J Pathol 2003; 163:1873-85; PMID:14578187; http://dx.doi.org/10.1016/S0002-9440(10)63546-8
  • Kaluz S, Kaluzova M, Chrastina A, Olive PL, Pastorekova S, Pastorek J, Lerman MI, Stanbridge EJ. Lowered oxygen tension induces expression of the hypoxia marker MN/carbonic anhydrase IX in the absence of hypoxia-inducible factor 1 α stabilization: a role for phosphatidylinositol 3'-kinase. Cancer Res 2002; 62:4469-77; PMID:12154057
  • Helmlinger G, Yuan F, Dellian M, Jain RK. Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat Med 1997; 3:177-82; PMID:9018236; http://dx.doi.org/10.1038/nm0297-177
  • Rundqvist H, Johnson RS. Hypoxia and metastasis in breast cancer. Curr Top Microbiol Immunol 2010; 345:121-39; PMID:20549469
  • Entenberg D, Wyckoff J, Gligorijevic B, Roussos ET, Verkhusha VV, Pollard JW, Condeelis J. Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging. Nat Protoc 2011; 6:1500-20; PMID:21959234; http://dx.doi.org/10.1038/nprot.2011.376
  • Paz H, Pathak N, Yang J. Invading one step at a time: the role of invadopodia in tumor metastasis. Oncogene 2013; 33(33):4193-202; PMID:24077283; http://dx.doi.org/; http://dx.doi.org/10.1038/onc.2013.393
  • Desmarais V, Yamaguchi H, Oser M, Soon L, Mouneimne G, Sarmiento C, Eddy R, Condeelis J. N-WASP and cortactin are involved in invadopodium-dependent chemotaxis to EGF in breast tumor cells. Cell Motil Cytoskeleton 2009; 66:303-16; PMID:19373774; http://dx.doi.org/10.1002/cm.20361
  • Gligorijevic B, Wyckoff J, Yamaguchi H, Wang Y, Roussos ET, Condeelis J. N-WASP-mediated invadopodium formation is involved in intravasation and lung metastasis of mammary tumors. J Cell Sci 2012; 125:724-34; PMID:22389406; http://dx.doi.org/10.1242/jcs.092726
  • Tolde O, Folk P. Stress-induced expression of p53 target genes is insensitive to SNW1/SKIP downregulation. Cell Mol Biol Lett 2011; 16:373-84; PMID:21461980; http://dx.doi.org/10.2478/s11658-011-0012-1
  • Nagelkerke A, Bussink J, van der Kogel AJ, Sweep FC, Span PN. The PERK/ATF4/LAMP3-arm of the unfolded protein response affects radioresistance by interfering with the DNA damage response. Radiother Oncol 2013; 108:415-21; PMID:23891100; http://dx.doi.org/; http://dx.doi.org/10.1016/j.radonc.2013.06.037
  • Salnikov AV, Liu L, Platen M, Gladkich J, Salnikova O, Ryschich E, Mattern J, Moldenhauer G, Werner J, Schemmer P, et al. Hypoxia induces EMT in low and highly aggressive pancreatic tumor cells but only cells with cancer stem cell characteristics acquire pronounced migratory potential. PLoS One 2012; 7:e46391; PMID:23050024; http://dx.doi.org/10.1371/journal.pone.0046391
  • Raheja LF, Genetos DC, Wong A, Yellowley CE. Hypoxic regulation of mesenchymal stem cell migration: the role of RhoA and HIF-1alpha. Cell Biol Int 2011; 35:981-9; PMID:21574962; http://dx.doi.org/10.1042/CBI20100733
  • Park JE, Tan HS, Datta A, Lai RC, Zhang H, Meng W, Lim SK, Sze SK. Hypoxic tumor cell modulates its microenvironment to enhance angiogenic and metastatic potential by secretion of proteins and exosomes. Mol Cell Proteomics 2010; 9:1085-99; PMID:20124223; http://dx.doi.org/10.1074/mcp.M900381-MCP200
  • Wyckoff J, Wang W, Lin EY, Wang Y, Pixley F, Stanley ER, Graf T, Pollard JW, Segall J, Condeelis J. A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res 2004; 64:7022-9; PMID:15466195; http://dx.doi.org/10.1158/0008-5472.CAN-04-1449
  • Wyckoff JB, Wang Y, Lin EY, Li JF, Goswami S, Stanley ER, Segall JE, Pollard JW, Condeelis J. Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res 2007; 67:2649-56; PMID:17363585; http://dx.doi.org/10.1158/0008-5472.CAN-06-1823
  • Roussos ET, Balsamo M, Alford SK, Wyckoff JB, Gligorijevic B, Wang Y, Pozzuto M, Stobezki R, Goswami S, Segall JE, et al. Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer. J Cell Sci 2011; 124:2120-31; PMID:21670198; http://dx.doi.org/; http://dx.doi.org/10.1242/jcs.086231
  • Patsialou A, Wyckoff J, Wang Y, Goswami S, Stanley ER, Condeelis JS. Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor. Cancer Res 2009; 69:9498-506; PMID:19934330; http://dx.doi.org/10.1158/0008-5472.CAN-09-1868
  • Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med 2003; 9:677-84; PMID:12778166; http://dx.doi.org/10.1038/nm0603-677
  • Patsialou A, Bravo-Cordero JJ, Wang Y, Entenberg D, Liu H, Clarke M, Condeelis JS. Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors. Intravital 2013; 2:e25294; PMID:25013744; http://dx.doi.org/10.4161/intv.25294
  • Nehls V, Herrmann R, Huhnken M. Guided migration as a novel mechanism of capillary network remodeling is regulated by basic fibroblast growth factor. Histochem Cell Biol 1998; 109:319-29; PMID:9562381; http://dx.doi.org/10.1007/s004180050232
  • Secomb TW, Alberding JP, Hsu R, Dewhirst MW, Pries AR. Angiogenesis: an adaptive dynamic biological patterning problem. PLoS Comput Biol 2013; 9:e1002983; PMID:23555218; http://dx.doi.org/10.1371/journal.pcbi.1002983
  • Diaz B, Yuen A, Iizuka S, Higashiyama S, Courtneidge SA. Notch increases the shedding of HB-EGF by ADAM12 to potentiate invadopodia formation in hypoxia. J Cell Biol 2013; 201:279-92; PMID:23589494; http://dx.doi.org/10.1083/jcb.201209151
  • Lucien F, Brochu-Gaudreau K, Arsenault D, Harper K, Dubois CM. Hypoxia-induced invadopodia formation involves activation of NHE-1 by the p90 ribosomal S6 kinase (p90RSK). PLoS One 2011; 6:e28851; PMID:22216126; http://dx.doi.org/10.1371/journal.pone.0028851
  • Md Hashim NF, Nicholas NS, Dart AE, Kiriakidis S, Paleolog E, Wells CM. Hypoxia-induced invadopodia formation: a role for β-PIX. Open Biol 2013; 3:120159; PMID:23740575; http://dx.doi.org/10.1098/rsob.120159
  • Bravo-Cordero JJ, Marrero-Diaz R, Megias D, Genis L, Garcia-Grande A, Garcia MA, Arroyo AG, Montoya MC. MT1-MMP proinvasive activity is regulated by a novel Rab8-dependent exocytic pathway. EMBO J 2007; 26:1499-510; PMID:17332756; http://dx.doi.org/10.1038/sj.emboj.7601606
  • Goswami S, Philippar U, Sun D, Patsialou A, Avraham J, Wang W, Di Modugno F, Nistico P, Gertler FB, Condeelis JS. Identification of invasion specific splice variants of the cytoskeletal protein Mena present in mammary tumor cells during invasion in vivo. Clin Exp Metastasis 2009; 26:153-9; PMID:18985426; http://dx.doi.org/10.1007/s10585-008-9225-8
  • Mader CC, Oser M, Magalhaes MA, Bravo-Cordero JJ, Condeelis J, Koleske AJ, Gil-Henn H. An EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion. Cancer Res 2011; 71:1730-41; PMID:21257711; http://dx.doi.org/10.1158/0008-5472.CAN-10-1432
  • Sharma VP, Eddy R, Entenberg D, Kai M, Gertler F, Condeelis J. Tks5 and SHIP2 regulate invadopodium maturation, but not initiation, in breast carcinoma cells. Curr Biol 2013; 23(21):2079-89; PMID:24206842; http://dx.doi.org/10.1016/j.cub.2013.08.044
  • Wyckoff JB, Segall JE, Condeelis JS. The collection of the motile population of cells from a living tumor. Cancer Res 2000; 60:5401-4; PMID:11034079

Reprints and Corporate Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

To request a reprint or corporate permissions for this article, please click on the relevant link below:

Academic Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

Obtain permissions instantly via Rightslink by clicking on the button below:

If you are unable to obtain permissions via Rightslink, please complete and submit this Permissions form. For more information, please visit our Permissions help page.