Skip to main content

Advertisement

SpringerLink
Log in

Evaluation of Two Optical Probes for Imaging the Integrin αvβ6− In Vitro and In Vivo in Tumor-Bearing Mice

  • Research Article
  • Published:
Molecular Imaging and Biology Aims and scope Submit manuscript

A Correction to this article was published on 06 April 2020

This article has been updated

Abstract

Purpose

The purpose of this study was to develop and evaluate two αvβ6-targeted fluorescent imaging agents. The integrin subtype αvβ6 is significantly upregulated in a wide range of epithelial derived cancers, plays a key role in invasion and metastasis, and expression is often located at the invasive edge of tumors. αvβ6-targeted fluorescent imaging agents have the potential to guide surgical resection leading to improved patient outcomes. Both imaging agents were based on the bi-PEGylated peptide NH2-PEG28-A20FMDV2-K16R-PEG28 (1), a peptide that has high affinity and selectivity for the integrin αvβ6: (a) 5-FAM-X-PEG28-A20FMDV2-K16R-PEG28 (2), and (b) IRDye800-PEG28-A20FMDV2-K16R-PEG28 (3).

Procedures

Peptides were synthesized using solid-phase peptide synthesis and standard Fmoc chemistry. Affinity for αvβ6 was evaluated by ELISA. In vitro binding, internalization, and localization of 2 was monitored using confocal microscopy in DX3puroβ6 (αvβ6+) and DX3puro (αvβ6−) cells. The in vivo imaging and ex vivo biodistribution of 3 was evaluated in three preclinical mouse models, DX3puroβ6/DX3puro and BxPC-3 (αvβ6+) tumor xenografts and a BxPC-3 orthotopic pancreatic tumor model.

Results

Peptides were obtained in > 99% purity. IC50 values were 28 nM (2) and 39 nM (3). Rapid αvβ6-selective binding and internalization of 2 was observed. Fluorescent intensity (FLI) measurements extracted from the in vivo images and ex vivo biodistribution confirmed uptake and retention of 3 in the αvβ6 positive subcutaneous and orthotopic tumors, with negligible uptake in the αvβ6-negative tumor. Blocking studies with a known αvβ6-targeting peptide demonstrated αvβ6-specific binding of 3.

Conclusion

Two fluorescence imaging agents were developed. The αvβ6-specific uptake, internalization, and endosomal localization of the fluorescence agent 2 demonstrates potential for targeted therapy. The selective uptake and retention of 3 in the αvβ6-positive tumors enabled clear delineation of the tumors and surgical resection indicating 3 has the potential to be utilized during image-guided surgery.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Change history

  • 06 April 2020

    This article was updated to correct the axes in Figures 4e and 5d.

References

  1. Lutje S, Rijpkema M, Frassen GM et al (2014) Dual-Modality Image-Guided Surgery of Prostate Cancer with a Radiolabeled Fluorescent Anti-PSMA Monoclonal Antibody. J Nucl Med 55(6):995–1001

    CAS  PubMed  Google Scholar 

  2. Haidong C, Chongwei C, Wenting S et al (2017) Precise integrin-targeting near-infrared imaging-guided surgical method increases surgical qualification of peritoneal carcinomatosis from gastric cancer in mice. Oncotarget 8:6258–6272

    Google Scholar 

  3. Handgraff HJM, Boonstra M, Preevoo H et al (2017) Real-time near-infrared fluorescence imaging using cRGD-ZW800-1 for intraoperative visualization of multiple cancer types. Oncotarget 8:21054–21066

    Google Scholar 

  4. Debie P, Hernot S (2019) Emerging Fluorescent Molecular Tracers to Guide Intra-Operative Surgical Decision-Making. Front Pharmacol 10:510

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Li C, Wang W, Wu Q et al (2006) Dual optical and nuclear imaging in human melanoma xenografts using a single targeted imaging probe. Nucl Med Biol 33(3):349–358

    CAS  PubMed  Google Scholar 

  6. Huang R, Vider J, Kovar JL et al (2012) Integrin αvβ3-Targeted IRDye 800CW Near-Infrared Imaging of Glioblastoma. Clin Cancer Res 18:5731–5740

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Rosenthal EL, Warram JM, de Boer E et al (2015) Safety and Tumor Specificity of Cetuximab-IRDye800 for Surgical Navigation in Head and Neck Cancer. Clin Cancer Res 21(16):3658–3666

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Sipos B, Hahn D, Carceller A et al (2004) Immunohistochemical screening for b6-integrin subunit expression in adenocarcinomas using a novel monoclonal anti- body reveals strong up-regulation in pancreatic ductal adenocarcinomas in vivo and in vitro. Histopathology 45:226–236

    CAS  PubMed  Google Scholar 

  9. Elayadi AN, Samli KN, Prudkin L et al (2007) A peptide selected by biopanning identifies the integrin alphavbeta6 as a prognostic biomarker for nonsmall cell lung cancer. Cancer Res 67:5889–5895

    CAS  PubMed  Google Scholar 

  10. Zhuang Z, Zhou R, Xu X et al (2013) Clinical significance of integrin avb6 expression effects on gastric carcinoma invasiveness and progression via cancer-associated fibroblasts. Med Oncol 30:580

    PubMed  Google Scholar 

  11. Ahmed N, Pansino F, Clyde R et al (2002) Overexpression of avb6 integrin in serous epithelial ovarian cancer regulates extracellular matrix degradation via the plas- minogen activation cascade. Carcinogenesis 23:237–244

    CAS  PubMed  Google Scholar 

  12. Hecht JL, Dolinski BM, Gardner HA et al (2008) Overexpression of the avb6 integrin in endometrial cancer. Appl Immunohistochem Mol Morphol 16:543–547

    CAS  PubMed  Google Scholar 

  13. Yang GY, Xu KS, Pan ZQ et al (2008) Integrin alpha v beta 6 mediates the potential for colon cancer cells to colonize in and metastasize to the liver. Cancer Sci 99:879–887

    CAS  PubMed  Google Scholar 

  14. Xue H, Atakilit A, Zhu W et al (2001) Role of the avb6 integrin in human oral squamous cell carcinoma growth in vivo and in vitro. Biochem Biophys Res Commun 288:610–618

    CAS  PubMed  Google Scholar 

  15. Janes SM, Watt FM (2004) Switch from avb5 to avb6 integrin expression protects squamous cell carcinomas from anoikis. J Cell Biol 166:419–431

    CAS  PubMed  PubMed Central  Google Scholar 

  16. John AE, Luckett JC, Tatler AL et al (2013) Preclinical SPECT/CT imaging of alphavbeta6 integrins for molecular stratification of idiopathic pulmonary fibrosis. J Nucl Med 54:2146–2152

    CAS  PubMed  Google Scholar 

  17. Thomas GJ, NytrÖm ML, Marshall JF (2006) αvβ6 integrin in wound healing and cancer of the oral cavity. J Oral Pathol Med 35:1–10

    CAS  PubMed  Google Scholar 

  18. Prudkin L, Liu DD, Ozburn NC et al (2009) Epithelial-to-mesenchymal transition in the development and progression of adenocarcinoma and squamous cell carcinoma of the lung. Mod Pathol 22:668–678

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Annes JP, Chen Y, Munger JS et al (2004) Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1. J Cell Biol 165:723–734

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Berghoff AS, Rajky O, Winkler F et al (2013) Invasion patterns in brain metastases of solid cancers. Neuro-Oncology 15:1664–1672

    PubMed  PubMed Central  Google Scholar 

  21. Liu Z, Liu H, Ma T et al (2014) Integrin αvβ6–Targeted SPECT Imaging for Pancreatic Cancer Detection. J Nucl Med 55:989–994

    CAS  PubMed  Google Scholar 

  22. Singh AN, McGuire M, Li S et al (2014) Dimerization of a Phage-Display Selected Peptide for Imaging of αvβ6- Integrin: Two Approaches to the Multivalent Effect. Theranostics 4:745–760

    PubMed  PubMed Central  Google Scholar 

  23. Hung K, Harris PWR, Ami D et al (2017) Structure-activity relationship study of the tumour-targeting peptide A20FMDV2 via modification of Lys16, Leu13, and N- and/or C-terminal functionality. Eu J of Med Chem 136:154–164

    CAS  Google Scholar 

  24. Hausner SH, DiCara D, Marik J et al (2007) Use of a peptide derived from foot-and-mouth disease virus for the noninvasive imaging of human cancer: generation and evaluation of 4-[18F]fluorobenzoyl A20FMDV2 for in vivo imaging of integrin alphavbeta6 expression with positron emission tomography. Cancer Res 67:7833–7840

    CAS  PubMed  Google Scholar 

  25. Gagnon MK, Hausner SH, Marik J et al (2009) High-throughput in vivo screening of targeted molecular imaging agents. Proc Natl Acad Sci U S A 106:17904–17909

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Hausner SH, Bauer N, Sutcliffe JL (2014) In vitro and in vivo evaluation of the effects of aluminum [(1)(8)F]fluoride radiolabeling on an integrin alphavbeta(6)-specific peptide. Nucl Med Biol 41:43–50

    CAS  PubMed  Google Scholar 

  27. Hausner SH, Abbey C, Bold RJ et al (2009) Targeted in vivo imaging of integrin alphavbeta6 with an improved radiotracer and its relevance in a pancreatic tumor model. Cancer Res 69(14):5843–5850

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Berryman S, Clark S, Monaghan P et al (2005) Early Events in Integrin v6-Mediated Cell Entry of Foot-and-Mouth Disease Virus. J Virol 79:8519–8534

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Kimura RH, Teed R, Hackel BJ et al (2012) Pharmacokinetically Stabilized Cystine Knot Peptides that Bind Alpha-v-Beta-6 Integrin with Single-Digit Nanomolar Affinities for detection of Pancreatic Cancer. Clin Cancer Res 18:839–849

    CAS  PubMed  Google Scholar 

  30. Hausner SH, Bauer N, Hu LY et al (2015) The Effect of Bi-Terminal PEGylation of an Integrin alphavbeta(6)-Targeted (1)(8)F Peptide on Pharmacokinetics and Tumor Uptake. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 56:784–790

    CAS  Google Scholar 

  31. Tummers WS, Kimura RH, Abou-Elkacem L et al (2018) Development and Preclinical Validation of a Cysteine Knottin Peptide Targeting Integrin alphavbeta6 for Near-infrared Fluorescent-guided Surgery in Pancreatic Cancer. Clin Cancer Res 24:1667–1676

    CAS  PubMed  Google Scholar 

  32. Hausner SH, Bold RJ, Cheuy LY, et al. (2019) Preclinical Development and First-in-Human Imaging of the Integrin alphavbeta6 with [(18)F]alphavbeta6-Binding Peptide in Metastatic Carcinoma. 25:1206-1215

  33. Hausner SH, DiCara D, Marik J et al (2007) Use of a peptide derived from foot-and-mouth disease virus for the noninvasive imaging of human cancer: generation and evaluation of 4-[18F]fluorobenzoyl A20FMDV2 for in vivo imaging of integrin alphavbeta6 expression with positron emission tomography. Cancer Res 67(16):7833–7840

    CAS  PubMed  Google Scholar 

  34. Mondal SB, Gao S, Zhu N et al (2014) Real-time fluorescence image-guided oncologic surgery. Adv Cancer Res 124:171–211

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Nagaya T, Nakamura YA, Choyke PL et al (2017) Fluorescence-Guided Surgery. Front Oncol 7:314

    PubMed  PubMed Central  Google Scholar 

  36. Lanzardoa S, C L, Brioschi C et al (2011) A new optical imaging probe targeting αVβ3 integrin in glioblastoma xenografts. Contrast Media Mol Imaging 6(6):449–458

    Google Scholar 

  37. Chen X, Conti PS, Moats RA (2004) In vivo Near-Infrared Fluorescence Imaging of Integrin v 3 in Brain Tumor Xenografts. Cancer Res 64(21):8009–8014

    CAS  PubMed  Google Scholar 

  38. Ding J, Feng M, Wang F et al (2015) Targeting effect of PEGylated liposomes modified with the Arg-Gly-Asp sequence on gastric cancer. Oncol Rep 34(4):1825–1834

    CAS  PubMed  Google Scholar 

  39. Tang YSC, Davis RA, Ganguly T, et al. (2019) Identification, Characterization, and Optimization of Integrin alphavbeta(6)-Targeting Peptides from a One-Bead One-Compound (OBOC) Library: Towards the Development of Positron Emission Tomography (PET) Imaging Agents. 24

  40. Zhang C, Zhang Y, Hong K, et al. (2017) Photoacoustic and Fluorescence Imaging of Cutaneous Squamous Cell Carcinoma in Living Subjects Using a Probe Targeting Integrin alphavbeta6. Molecules (Basel, Switzerland) 7:42442

  41. Achilefu S, Jimenez HN, Dorshow RB et al (2002) Synthesis, in vitro receptor binding, and in vivo evaluation of fluorescein and carbocyanine peptide-based optical contrast agents. J Med Chem 45:2003–2015

    CAS  PubMed  Google Scholar 

  42. Hu LY, Bauer N, Knight LM et al (2014) Characterization and evaluation of (64)Cu-labeled A20FMDV2 conjugates for imaging the integrin αvβ6. Mol Imaging Biol 16(4):567–577

    PubMed  PubMed Central  Google Scholar 

  43. Ghannad F, Nica D, Fulle MI et al (2008) Absence of alphavbeta6 integrin is linked to initiation and progression of periodontal disease. Am J Pathol 172:1271–1286

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Saha A, Ellison D, Thomas GJ et al (2010) High-resolution in vivo imaging of breast cancer by targeting the pro-invasive integrin alphavbeta6. J Pathol 222:52–63

    CAS  PubMed  Google Scholar 

  45. Rolleman EJ, Valkema R, de Jong M et al (2003) Safe and effective inhibition of renal uptake of radiolabelled octreotide by a combination of lysine and arginine. Eur J Nucl Med Mol Imaging 30:9–15

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported in part by NIH (RO1 CA199725). We thank the staff of CMGI at UC Davis for their technical support, and Dr. Ryan Davis for assistance with MALDI. We would also like to thank Dr. Michael Paddy for providing training and technical support for confocal microscopy, at The Molecular and Cellular Biology Light Microscopy Imaging Facility at UC Davis. Special thanks to Dr. Rhonda Oates from the Campus Veterinary Service at UC Davis, for performing the orthotopic pancreatic surgeries and assisting with the post-operative care.

Author information

Authors and Affiliations

  1. Department of Internal Medicine, Division of Hematology & Oncology, University of California, Davis, CA, USA

    Tanushree Ganguly, Sarah Y. Tang, Nadine Bauer & Julie L. Sutcliffe

  2. Department of Biomedical Engineering, University of California, Davis, CA, USA

    Julie L. Sutcliffe

  3. Center for Molecular and Genomic Imaging, University of California, Davis, CA, USA

    Julie L. Sutcliffe

Authors
  1. Tanushree Ganguly
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah Y. Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nadine Bauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Julie L. Sutcliffe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Julie L. Sutcliffe.

Ethics declarations

Conflict of Interest

Julie Sutcliffe is a named inventor of intellectual property related to the αvβ6-BP. Julie Sutcliffe is the founder and stock holder of Luminance Biosciences, Inc.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This original version of this article was updated to correct the axes in Figures 4e and 5d.

Electronic supplementary material

ESM 1

(PDF 2428 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ganguly, T., Tang, S.Y., Bauer, N. et al. Evaluation of Two Optical Probes for Imaging the Integrin αvβ6− In Vitro and In Vivo in Tumor-Bearing Mice. Mol Imaging Biol 22, 1170–1181 (2020). https://doi.org/10.1007/s11307-019-01469-5

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11307-019-01469-5

Key Words

Search

Navigation