Advertisement

Breast Cancer Research and Treatment

, Volume 140, Issue 2, pp 263–272 | Cite as

Molecular imaging with a fluorescent antibody targeting carbonic anhydrase IX can successfully detect hypoxic ductal carcinoma in situ of the breast

  • Aram S. A. van Brussel
  • Arthur Adams
  • Jeroen F. Vermeulen
  • Sabrina Oliveira
  • Elsken van der Wall
  • Willem P. Th. M. Mali
  • Paul. J. van Diest
  • Paul M. P. van Bergen en HenegouwenEmail author
Preclinical Study

Abstract

Ductal carcinoma in situ (DCIS) of the breast is difficult to remove completely during surgery as it is not palpable and can therefore require re-excision. Real-time visualization of DCIS using near-infrared fluorescent probes could help the surgeon during surgery as well as the pathologist post-operatively to distinguish the tumor from healthy tissue. As hypoxia-induced necrosis is a common phenomenon in DCIS, we investigated the molecular imaging of DCIS using a fluorescent antibody targeting a hypoxia marker, carbonic anhydrase IX (CAIX), in a preclinical mouse model. A monoclonal antibody against human CAIX was fluorescently labeled with the near-infrared dye IRDye800CW and characterized in vitro. An in vivo study was performed in SCID/Beige mice that were orthotopically transplanted with human breast cancer cells mimicking human DCIS (MCF10DCIS) and MCF10DCIS stably expressing CAIX. A clinically approved fluorescence imaging system was used to monitor probe uptake and to determine tumor-to-normal tissue ratios (TNR). Mean in vivo TNR of CAIX-transduced (CAIX+) tumors was 7.5 ± 0.5. Mean in vivo TNR of DCIS tumors with hypoxic areas reached a plateau level at 48 h after injection of 2.1 ± 0.1 (mean ± SEM) compared to 1.7 ± 0.1 in DCIS without hypoxic areas. Mean intra-operative TNR of DCIS tumors with necrotic regions was higher than that of DCIS tumors without necrotic regions 96 h after injection—2.9 ± 0.1 and 1.5 ± 0.1, respectively—while the TNR of CAIX+ tumors was 11.2 ± 1.0. Specific tumor uptake of MabCAIX-IRDye800CW was confirmed by a biodistribution assay, and immunofluorescence imaging on tumor sections showed specific uptake in hypoxic tumor regions, with higher contrast than conventional chromagen-based immunohistochemistry. Molecular fluorescence imaging with MabCAIX-IRDye800CW can be successfully used to detect hypoxic DCIS before and during surgery to facilitate radical resection. Furthermore, it allows for sensitive CAIX-specific immunofluorescence microscopy of tumor sections, thereby introducing the concept of molecular fluorescence pathology.

Keywords

Carbonic anhydrase IX Hypoxia Monoclonal antibody DCIS Molecular imaging Near-infrared 

Notes

Acknowledgments

We would like to thank A. Martens for the pLV-CMV-LUC2-IRES-GFP vector and P.C. Pearlman for writing the algorithm in Matlab. We are indebted to P.W.B. Derksen and S.G. Elias for their suggestions concerning the mouse model. Also, we would like to thank the animal facility of the University Utrecht and the biobank of the University Medical Center Utrecht for their support. This research was supported by the Center for Translational Molecular Medicine—Mammary Carcinoma Molecular Imaging for Diagnosis and Therapeutics (CTMM—MAMMOTH, Project 203)—and by an unrestricted educational grant from Aegon to P.J.vD.

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Sakorafas GH, Farley DR, Peros G (2008) Recent advances and current controversies in the management of DCIS of the breast. Cancer Treat Rev 34(6):483–497. doi: 10.1016/j.ctrv.2008.03.001 PubMedCrossRefGoogle Scholar
  2. 2.
    Cutuli B, Lemanski C, Le Blanc-Onfroy M, de Lafontan B, Cohen-Solal-Le-Nir C, Fondrinier E, Mignotte H, Giard S, Charra-Brunaud C, Auvray H, Gonzague-Casabianca L, Quetin P, Fay R (2013) Local recurrence after ductal carcinoma in situ breast conserving treatment. Analysis of 195 cases. Cancer Radiother 17(3):196–201. doi: 10.1016/j.canrad.2013.01.011 PubMedCrossRefGoogle Scholar
  3. 3.
    Lee LA, Silverstein MJ, Chung CT, Macdonald H, Sanghavi P, Epstein M, Holmes DR, Silberman H, Ye W, Lagios MD (2006) Breast cancer-specific mortality after invasive local recurrence in patients with ductal carcinoma-in situ of the breast. Am J Surg 192(4):416–419. doi: 10.1016/j.amjsurg.2006.06.005 PubMedCrossRefGoogle Scholar
  4. 4.
    Shamliyan T, Wang SY, Virnig BA, Tuttle TM, Kane RL (2010) Association between patient and tumor characteristics with clinical outcomes in women with ductal carcinoma in situ. J Natl Cancer Inst Monogr 41:121–129. doi: 10.1093/jncimonographs/lgq034 CrossRefGoogle Scholar
  5. 5.
    Stillebroer AB, Zegers CM, Boerman OC, Oosterwijk E, Mulders PF, O’Donoghue JA, Visser EP, Oyen WJ (2012) Dosimetric analysis of 177Lu-cG250 radioimmunotherapy in renal cell carcinoma patients: correlation with myelotoxicity and pretherapeutic absorbed dose predictions based on 111In-cG250 imaging. J Nucl Med 53(1):82–89. doi: 10.2967/jnumed.111.094896 PubMedCrossRefGoogle Scholar
  6. 6.
    Vermeulen JF, van Brussel AS, Adams A, Mali WP, van der Wall E, van Diest PJ, Derksen PW (2013) Near-infrared fluorescence molecular imaging of ductal carcinoma in situ with CD44v6-specific antibodies in mice: a preclinical study. Mol Imaging Biol 15(3):290–298. doi: 10.1007/s11307-012-0605-8 PubMedCrossRefGoogle Scholar
  7. 7.
    Terwisscha van Scheltinga AG, van Dam GM, Nagengast WB, Ntziachristos V, Hollema H, Herek JL, Schroder CP, Kosterink JG, Lub-de Hoog MN, de Vries EG (2011) Intraoperative near-infrared fluorescence tumor imaging with vascular endothelial growth factor and human epidermal growth factor receptor 2 targeting antibodies. J Nucl Med 52(11):1778–1785. doi: 10.2967/jnumed.111.092833 PubMedCrossRefGoogle Scholar
  8. 8.
    Day KE, Beck LN, Heath CH, Huang CC, Zinn KR, Rosenthal EL (2013) Identification of the optimal therapeutic antibody for fluorescent imaging of cutaneous squamous cell carcinoma. Cancer Biol Ther 14(3):271–277. doi: 10.4161/cbt.23300 PubMedCrossRefGoogle Scholar
  9. 9.
    Zhang Y, Hong H, Engle JW, Yang Y, Barnhart TE, Cai W (2012) Positron emission tomography and near-infrared fluorescence imaging of vascular endothelial growth factor with dual-labeled bevacizumab. Am J Nucl Med Mol Imaging 2(1):1–13PubMedGoogle Scholar
  10. 10.
    Bos R, Zhong H, Hanrahan CF, Mommers EC, Semenza GL, Pinedo HM, Abeloff MD, Simons JW, van Diest PJ, van der Wall E (2001) Levels of hypoxia-inducible factor-1 alpha during breast carcinogenesis. J Natl Cancer Inst 93(4):309–314PubMedCrossRefGoogle Scholar
  11. 11.
    Greijer AE, de Jong MC, Scheffer GL, Shvarts A, van Diest PJ, van der Wall E (2005) Hypoxia-induced acidification causes mitoxantrone resistance not mediated by drug transporters in human breast cancer cells. Cell Oncol 27(1):43–49PubMedGoogle Scholar
  12. 12.
    Gort EH, Groot AJ, van der Wall E, van Diest PJ, Vooijs MA (2008) Hypoxic regulation of metastasis via hypoxia-inducible factors. Curr Mol Med 8(1):60–67PubMedCrossRefGoogle Scholar
  13. 13.
    Seeber LM, Horree N, Vooijs MA, Heintz AP, van der Wall E, Verheijen RH, van Diest PJ (2011) The role of hypoxia inducible factor-1alpha in gynecological cancer. Crit Rev Oncol Hematol 78(3):173–184. doi: 10.1016/j.critrevonc.2010.05.003 PubMedCrossRefGoogle Scholar
  14. 14.
    Jans J, van Dijk JH, van Schelven S, van der Groep P, Willems SH, Jonges TN, van Diest PJ, Bosch JL (2010) Expression and localization of hypoxia proteins in prostate cancer: prognostic implications after radical prostatectomy. Urology 75(4):786–792. doi: 10.1016/j.urology.2009.08.024 PubMedCrossRefGoogle Scholar
  15. 15.
    Kornegoor R, Verschuur-Maes AH, Buerger H, Hogenes MC, de Bruin PC, Oudejans JJ, Hinrichs B, van Diest PJ (2012) Fibrotic focus and hypoxia in male breast cancer. Mod Pathol 25(10):1397–1404. doi: 10.1038/modpathol.2012.101 PubMedCrossRefGoogle Scholar
  16. 16.
    Wykoff CC, Beasley N, Watson PH, Campo L, Chia SK, English R, Pastorek J, Sly WS, Ratcliffe P, Harris AL (2001) Expression of the hypoxia-inducible and tumor-associated carbonic anhydrases in ductal carcinoma in situ of the breast. Am J Pathol 158(3):1011–1019. doi: 10.1016/S0002-9440(10)64048-5 PubMedCrossRefGoogle Scholar
  17. 17.
    Li XF, O’Donoghue JA (2008) Hypoxia in microscopic tumors. Cancer Lett 264(2):172–180. doi: 10.1016/j.canlet.2008.02.037 PubMedCrossRefGoogle Scholar
  18. 18.
    Bos R, van der Groep P, Greijer AE, Shvarts A, Meijer S, Pinedo HM, Semenza GL, van Diest PJ, van der Wall E (2003) Levels of hypoxia-inducible factor-1alpha independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer 97(6):1573–1581. doi: 10.1002/cncr.11246 PubMedCrossRefGoogle Scholar
  19. 19.
    Wang Z, Shi Q, Gu Y, Shen Y, Sun M, Deng M, Zhang H, Fang J, Zhang S, Xie F (2011) Clinicopathologic correlation of cancer stem cell markers CD44, CD24, VEGF and HIF-1alpha in ductal carcinoma in situ and invasive ductal carcinoma of breast: an immunohistochemistry-based pilot study. Pathol Res Pract 207(8):505–513. doi: 10.1016/j.prp.2011.06.009 PubMedCrossRefGoogle Scholar
  20. 20.
    van der Groep P, van Diest PJ, Smolders YH, Ausems MG, van der Luijt RB, Menko FH, Bart J, de Vries EG, van der Wall E (2013) HIF-1alpha overexpression in ductal carcinoma in situ of the breast in BRCA1 and BRCA2 mutation carriers. PLoS ONE 8(2):e56055. doi: 10.1371/journal.pone.0056055 PubMedCrossRefGoogle Scholar
  21. 21.
    Chen CL, Chu JS, Su WC, Huang SC, Lee WY (2010) Hypoxia and metabolic phenotypes during breast carcinogenesis: expression of HIF-1alpha, GLUT1, and CAIX. Virchows Arch 457(1):53–61. doi: 10.1007/s00428-010-0938-0 PubMedCrossRefGoogle Scholar
  22. 22.
    De Simone G, Supuran CT (1804) Carbonic anhydrase IX: biochemical and crystallographic characterization of a novel antitumor target. Biochim Biophys Acta 2:404–409. doi: 10.1016/j.bbapap.2009.07.027 Google Scholar
  23. 23.
    Ahlskog JK, Schliemann C, Marlind J, Qureshi U, Ammar A, Pedley RB, Neri D (2009) Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours. Br J Cancer 101(4):645–657. doi: 10.1038/sj.bjc.6605200 PubMedCrossRefGoogle Scholar
  24. 24.
    Divgi CR, Pandit-Taskar N, Jungbluth AA, Reuter VE, Gonen M, Ruan S, Pierre C, Nagel A, Pryma DA, Humm J, Larson SM, Old LJ, Russo P (2007) Preoperative characterisation of clear-cell renal carcinoma using iodine-124-labelled antibody chimeric G250 (124I-cG250) and PET in patients with renal masses: a phase I trial. Lancet Oncol 8(4):304–310. doi: 10.1016/S1470-2045(07)70044-X PubMedCrossRefGoogle Scholar
  25. 25.
    Oliveira S, van Dongen GA, Stigter-van Walsum M, Roovers RC, Stam JC, Mali W, van Diest PJ, van Bergen en Henegouwen PM (2012) Rapid visualization of human tumor xenografts through optical imaging with a near-infrared fluorescent anti-epidermal growth factor receptor nanobody. Mol Imaging 11(1):33–46PubMedGoogle Scholar
  26. 26.
    Marshall MV, Draney D, Sevick-Muraca EM, Olive DM (2010) Single-dose intravenous toxicity study of IRDye 800CW in Sprague–Dawley rats. Mol Imaging Biol 12(6):583–594. doi: 10.1007/s11307-010-0317-x PubMedCrossRefGoogle Scholar
  27. 27.
    Siebels M, Rohrmann K, Oberneder R, Stahler M, Haseke N, Beck J, Hofmann R, Kindler M, Kloepfer P, Stief C (2011) A clinical phase I/II trial with the monoclonal antibody cG250 (RENCAREX(R)) and interferon-alpha-2a in metastatic renal cell carcinoma patients. World J Urol 29(1):121–126. doi: 10.1007/s00345-010-0570-2 PubMedCrossRefGoogle Scholar
  28. 28.
    Schackmann RC, van Amersfoort M, Haarhuis JH, Vlug EJ, Halim VA, Roodhart JM, Vermaat JS, Voest EE, van der Groep P, van Diest PJ, Jonkers J, Derksen PW (2011) Cytosolic p120-catenin regulates growth of metastatic lobular carcinoma through Rock1-mediated anoikis resistance. J Clin Invest 121(8):3176–3188. doi: 10.1172/JCI41695 PubMedCrossRefGoogle Scholar
  29. 29.
    Behbod F, Kittrell FS, LaMarca H, Edwards D, Kerbawy S, Heestand JC, Young E, Mukhopadhyay P, Yeh HW, Allred DC, Hu M, Polyak K, Rosen JM, Medina D (2009) An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ. Breast Cancer Res 11(5):R66. doi: 10.1186/bcr2358 PubMedCrossRefGoogle Scholar
  30. 30.
    MacAusland SG, Hepel JT, Chong FK, Galper SL, Gass JS, Ruthazer R, Wazer DE (2007) An attempt to independently verify the utility of the Van Nuys Prognostic Index for ductal carcinoma in situ. Cancer 110(12):2648–2653. doi: 10.1002/cncr.23089 PubMedCrossRefGoogle Scholar
  31. 31.
    Eccles SA (2011) The epidermal growth factor receptor/Erb-B/HER family in normal and malignant breast biology. Int J Dev Biol 55(7–9):685–696. doi: 10.1387/ijdb.113396se PubMedCrossRefGoogle Scholar
  32. 32.
    Leone F, Perissinotto E, Cavalloni G, Fonsato V, Bruno S, Surrenti N, Hong D, Capaldi A, Geuna M, Piacibello W, Aglietta M (2003) Expression of the c-ErbB-2/HER2 proto-oncogene in normal hematopoietic cells. J Leukoc Biol 74(4):593–601. doi: 10.1189/jlb.0203068 PubMedCrossRefGoogle Scholar
  33. 33.
    Varia MA, Calkins-Adams DP, Rinker LH, Kennedy AS, Novotny DB, Fowler WC Jr, Raleigh JA (1998) Pimonidazole: a novel hypoxia marker for complementary study of tumor hypoxia and cell proliferation in cervical carcinoma. Gynecol Oncol 71(2):270–277. doi: 10.1006/gyno.1998.5163 PubMedCrossRefGoogle Scholar
  34. 34.
    Picchio M, Beck R, Haubner R, Seidl S, Machulla HJ, Johnson TD, Wester HJ, Reischl G, Schwaiger M, Piert M (2008) Intratumoral spatial distribution of hypoxia and angiogenesis assessed by 18F-FAZA and 125I-Gluco-RGD autoradiography. J Nucl Med 49(4):597–605. doi: 10.2967/jnumed.107.046870 PubMedCrossRefGoogle Scholar
  35. 35.
    Dolbier WR Jr, Li AR, Koch CJ, Shiue CY, Kachur AV (2001) [18F]-EF5, a marker for PET detection of hypoxia: synthesis of precursor and a new fluorination procedure. Appl Radiat Isot 54(1):73–80PubMedCrossRefGoogle Scholar
  36. 36.
    Okuda K, Okabe Y, Kadonosono T, Ueno T, Youssif BG, Kizaka-Kondoh S, Nagasawa H (2012) 2-Nitroimidazole-tricarbocyanine conjugate as a near-infrared fluorescent probe for in vivo imaging of tumor hypoxia. Bioconjug Chem 23(3):324–329. doi: 10.1021/bc2004704 PubMedCrossRefGoogle Scholar
  37. 37.
    Tafreshi NK, Bui MM, Bishop K, Lloyd MC, Enkemann SA, Lopez AS, Abrahams D, Carter BW, Vagner J, Grobmyer SR, Gillies RJ, Morse DL (2012) Noninvasive detection of breast cancer lymph node metastasis using carbonic anhydrases IX and XII targeted imaging probes. Clinical Cancer Res 18(1):207–219. doi: 10.1158/1078-0432.CCR-11-0238 CrossRefGoogle Scholar
  38. 38.
    Bao B, Groves K, Zhang J, Handy E, Kennedy P, Cuneo G, Supuran CT, Yared W, Rajopadhye M, Peterson JD (2012) In vivo imaging and quantification of carbonic anhydrase IX expression as an endogenous biomarker of tumor hypoxia. PLoS ONE 7(11):e50860. doi: 10.1371/journal.pone.0050860 PubMedCrossRefGoogle Scholar
  39. 39.
    Fillies T, Werkmeister R, van Diest PJ, Brandt B, Joos U, Buerger H (2005) HIF1-alpha overexpression indicates a good prognosis in early stage squamous cell carcinomas of the oral floor. BMC Cancer 5:84. doi: 10.1186/1471-2407-5-84 PubMedCrossRefGoogle Scholar
  40. 40.
    Horree N, van Diest PJ, van der Groep P, Sie-Go DM, Heintz AP (2007) Hypoxia and angiogenesis in endometrioid endometrial carcinogenesis. Cell Oncol 29(3):219–227PubMedGoogle Scholar
  41. 41.
    Seeber LM, Horree N, van der Groep P, van der Wall E, Verheijen RH, van Diest PJ (2010) Necrosis related HIF-1alpha expression predicts prognosis in patients with endometrioid endometrial carcinoma. BMC Cancer 10:307. doi: 10.1186/1471-2407-10-307 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Aram S. A. van Brussel
    • 1
    • 2
  • Arthur Adams
    • 3
  • Jeroen F. Vermeulen
    • 2
  • Sabrina Oliveira
    • 1
    • 2
  • Elsken van der Wall
    • 4
  • Willem P. Th. M. Mali
    • 3
  • Paul. J. van Diest
    • 2
  • Paul M. P. van Bergen en Henegouwen
    • 1
    Email author
  1. 1.Division of Cell Biology, Department of Biology, Science FacultyUtrecht UniversityUtrechtThe Netherlands
  2. 2.Department of PathologyUniversity Medical Center UtrechtUtrechtThe Netherlands
  3. 3.Department of RadiologyUniversity Medical Center UtrechtUtrechtThe Netherlands
  4. 4.Division of Internal Medicine and DermatologyUniversity Medical Center UtrechtUtrechtThe Netherlands

Personalised recommendations