Targeting breast cancer cells and their microenvironment
Nienhuis, Hilje Harmina
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Publication date: 2019
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Nienhuis, H. H. (2019). Targeting breast cancer cells and their microenvironment: Pre-clinical models and translational studies. Rijksuniversiteit Groningen.
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Near infrared fluorescent antibody
imaging of tumors on ex ovo
chorioallantoic membrane assay
H.H. Nienhuis, S.J.H. Waaijer, H. Timmer-Bosscha, L. Pot, E.G.E. de Vries, C.P. Schröder Department of Medical Oncology, University Medical Center Groningen, the Netherlands
Revised and resubmitted
ABSTRACT
Antibodies are increasingly used as anticancer drugs. Molecular imaging with labeled antibodies can provide non-invasive information about tumor targeting and distribution. Preclinical antibody imaging in cancer research is predominantly performed in mouse models. We aimed to develop an alternative evaluation model by using the chicken chorioallantoic membrane (CAM). We used the fertilized egg in an ex ovo manner to be able to inject antibodies into the vasculature. In this study trastuzumab and cetuximab, antibodies targeting the human epidermal growth factor receptor (HER) 2 and epidermal growth factor receptor (EGFR), were labeled with the near infrared dye IRDye800CW for fluorescent imaging in the near infrared spectrum. These labeled antibodies were injected into the vasculature of CAMs, engrafted with human tumor cells, using a glass microcapillary. Negative controls were obtained by a non-target expressing cell line or 680RD-labeled nonspecific IgG. By means of IVIS imaging, both fluorescent antibodies showed excellent tumor specific uptake already 24 hours after injection. Our study demonstrates the feasibility of the CAM assay to study tumor targeting with fluorescently labeled antibodies.
INTRODUCTION
Cancer is a leading cause of mortality with over 8 million deaths worldwide in 2012 (1). Currently, systemic treatment increasingly incorporates specific targeted therapies directed towards one target or pathway. Monoclonal antibodies comprise an important part of these targeted therapies. By labeling these antibodies and perform molecular imaging, whole-body information can be obtained about biological processes at the molecular and cellular level (2). In the past years, molecular imaging has gained an important role for identifying potential targets, studying antibody behavior in vivo and evaluating treatment effects (3).
In early tracer development, preclinical imaging models are essential for the assessment of tracer engagement. Fluorescent tracers are a novel development in this field. In this preclinical setting, human tumor bearing mouse models are most widely used for in vivo imaging studies. An alternative in vivo model for using lab rodents is the chicken chorioallantoic membrane (CAM) assay (4). This highly vascularized membrane of fertilized chicken eggs, forms an immunodeficient host which allows the development of xenografts including growth of human tumors (4). Previous studies have successfully employed this model for studying angiogenesis, evaluating treatment effects on tumor growth and metastatic potential (5-7). This model can be used with the developing embryo inside the eggshell (in ovo) or without the eggshell in a dish (ex ovo) (8). The ex ovo system is particularly suitable for imaging purposes because it allows the direct visualization of the tumor including surrounding vasculature. For fluorescent tracers the direct visualization is interesting, as the signal has limited penetration depth.
The aim of this study was to evaluate whether the CAM assay can be used as model system for molecular tumor imaging of tumor cell characteristics using fluorescent-labeled antibodies.
MATERIALS AND METHODS
Cell linesHuman epidermal growth factor receptor (HER) 2 and hormone receptor negative breast cancer cell line MDA-MB-231 (obtained from American Type Culture Collection (ATCC)) and the stably transfected cell line MDA-MB-231-H2N that overexpresses HER2 (9) (9) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) with 10% fetal calf serum (FCS) and 1% glutamine. The epidermal growth factor receptor (EGFR) overexpressing cell line A431 (obtained from ATCC) was cultured in DMEM with 10% FCS, the EGFR negative breast cancer cell line T47D (obtained from ATCC) in Roswell Memorial Park Medium (RPMI) with 10% FCS. Cell lines were cultured at
37 °C in a humidified atmosphere containing 5% CO2 and were routinely tested for Mycoplasma.
Short tandem repeat profiling (BaseClear) was used to authenticate the cell lines.
Tracer development
Monoclonal antibodies trastuzumab (Roche) targeting HER2 and cetuximab (Merck) targeting EGFR were labeled with IRDye800CW-NHS (LI-COR Biosciences) (10, 11), according to the manufacturer’s protocol. In addition, human IgG (Sanquin) labeled with IRDye680RD-NHS (LI-COR Biosciences) served as a negative control in the experiment with cetuximab. Antibodies were reacted with IRDye 800CW at a molar ratio of 1:4 (trastuzumab) or 1:2 (cetuximab, IgG) in phosphate-buffered saline (pH 8.5). The product was purified by ultracentrifugation with Vivaspin sample concentrators (GE Healthcare) for trastuzumab and PD-10 columns (GE Healthcare) for cetuximab. Labeling efficiency and purity were determined by size-exclusion high-performance liquid chromatography, with ultraviolet detector wavelengths at 220, 280, and 790 nm. The labeling efficiency obtained was 72%, giving a labeling ratio of an average of 2.9 dyes per antibody. After purification, a purity of greater than 95% was accomplished. Ex ovo culture and tumor inoculation
For the experimental set up (Figure 1), fertilized chicken eggs (Gallus Gallus domesticus; DeKalb White; het Anker BV) were incubated (Savimat) at 38°C and after 3 days the embryos were transferred into plastic dishes to obtain ex ovo cultures (8). Eggs were removed from the incubator and placed horizontally on egg boxes to allow the embryo to move to the lateral side. Eggshells were broken on a metal strip and total egg content, including CAM and embryo, were transferred into a plastic dish (Makro). To ensure oxygenation holes were punctured in the lid of the dishes. Ex ovo egg contents were returned to the incubator. On day 6 of embryonic development, the CAM was damaged using a cotton tissue (Celltork) and each CAM was inoculated with a total of
5-10·106 cells suspended in a mixture of 25 μL culture media and 25 μL phenol red free Matrigel
(BD Biosciences). For HER2 imaging, the MDA-MB-231-H2N cell line was used, while MDA-MB-231 served as HER2 negative control. For EGFR imaging, cell line A341 was used with T47D as negative control. For the EGFR imaging also 680RD-labeled nonspecific IgG was used as negative control.
Click # HHN2014 0126 13 2803 _0 01 zon da g 2 6 j an 20 14 13 :28 :08 Le vel =Hi gh , Em =80 0, Ex= 74 5 Bin:M (8), F OV6.5 , f2, 4s Cam era: IS 0741 N437 9, Spe ctr al Instr um en ts TE Us er: Hi lde Grou p: Me dical on col og y Ex perim ent: e x ov o t ras cw80 0 iv Com men t1: H2N Com men t2: inj 1 ug + Tim e P oint: 24 hrs Ani mal Num be r: 7 0 5 6 7 8 9 10 -5 ImageMin = 0 M ax = 1.804e-05 efficienc y Col or Ba r M in = 4.1942e-06 Max = 1 .601e-05 bk g s ub flat-f ielde d co sm ic BKG 1 Click # HHN2014 0126 13 2803 _0 01 zon da g 2 6 j an 20 14 13 :28 :08 Le vel =Hi gh , Em =80 0, Ex= 74 5 Bin:M (8), F OV6.5 , f2, 4s Cam era: IS 0741 N437 9, Spe ctr al Instr um en ts TE Us er: Hi lde Grou p: Me dical on col og y Ex perim ent: e x ov o t ras cw80 0 iv Com men t1: H2N Com men t2: inj 1 ug + Tim e P oint: 24 hrs Ani mal Num be r: 7 0 5 6 7 8 9 10 -5 ImageMin = 0 M ax = 1.804e-05 efficienc y Col or Ba r M in = 4.1942e-06 Max = 1.6 01e-05 bk g s ub flat-f ielde d co sm ic BKG 1 Click # HHN2014 0126 13 2803 _0 01 zon da g 2 6 j an 20 14 13 :28 :08 Le vel =Hi gh , Em =80 0, Ex= 74 5 Bin:M (8), F OV6.5 , f2, 4s Cam era: IS 0741 N437 9, Spe ctr al Instr um en ts TE Us er: Hi lde Grou p: Me dical on col og y Ex perim ent: e x ov o t ras cw80 0 iv Com men t1: H2N Com men t2: inj 1 ug + Tim e P oint: 24 hrs Ani mal Num be r: 7 0 5 6 7 8 9 10 -5 ImageMin = 0 M ax = 1.804e-05 efficienc y Col or Ba r M in = 4.1942e-06 Max = 1.6 01e-05 bk g s ub flat-f ielde d co sm ic BKG 1 Click # HHN2014 01 26 1328 03 _001 zon da g 2 6 j an 20 14 13 :28 :08 Le vel=Hi gh, E m =80 0, Ex= 745 Bin:M (8), F OV6.5 , f2, 4s Cam era: IS 0741 N437 9, Spe ctr al Instr um en ts TE Us er: Hi lde Grou p: Me dical on col og y Ex perim en t: e x ov o t ras cw80 0 iv Com men t1: H2N Com men t2: inj 1 ug + Time P oint: 24 hrs Ani mal Num be r: 7 0 5 6 7 8 9 10 -5 ImageMin = 0 Max = 1.804e-05 efficienc y Col or Ba r Min = 4.1942e-06 Max = 1.6 01e-05 bkg s ub flat-f ielde d cosm ic BKG 1
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Figur e 1 | O ver vie w of exper imental set up At d ay 3 o f e m br yo ni c d eve lo pm en t, e gg s w er e m ad e e x-ov o a nd t ra ns fe rre d t o p la st ic d ish es . O n d ay 6 t um or c el ls w er e i no cu la te d o n t he c ho rio al la nt oi c m em br an e ( CA M ). T w o d ay s l at er , fl u or es ce nt ly l ab el ed a nt ib od y w as i nj ec te d f ol lo w ed b y fl u or es ce nc e i m ag in g u p t o d ay 1 2 o f e m br yo ni c d eve lo pm en t.5
Tracer injection
At day 8 of embryonic development, trastuzumab-800CW or cetuximab-800CW/IgG-680RD mixture were intravenously injected into the CAM using a stereo microscope (Olympus). For this we designed an injection system based on a previous study (12). For the injection system, a 20 gauge needle (BD Medical Technology) was placed on 1 mL syringe (BD Medical Technology). Subsequently, the needle was inserted into Tygon plastic tubing (VWR) to connect the 25 µm microcapillar (Origio) to the syringe (Figure 2). Each CAM was injected with 1 µg tracer in a total volume of 10 µL.
Figure 2 | Microcapillary connected to syringe
The injection system consists of a 1 mL syringe with a 20 gauge needle. Subsequently, the needle was inserted into Tygon plastic tubing to connect the 25 µm microcapillair to the syringe
Fluorescence Imaging and microscopy
In vivo fl uorescence images were obtained with the IVIS spectrum (Caliper Life Sciences) in vivo imaging system. Images were acquired 24, 48, 72 and 96 hours or 1, 12, 24 and 36 hours after tracer injection for trastuzumab-800CW and cetuximab-800CW, respectively. Diff erent excitation wavelengths were used to measure 800CW and 680RD as well as background and auto-fl uorescence signal. Analysis of the images was performed by using Living Image Software 4.1 (Caliper Life Sciences). A region of interest (ROI) was drawn around the tumor as well as around adjacent background to quantify the tumor and background signal. White light images and/or auto-fl uorescent signal images (540 nm) were used to localize the tumor. Results are
presented in radiant effi ciency (p/sec/cm2/sr/µW/cm2) and tumor-to-background ratios (TBR).
After the fi nal fl uorescent scan, chicken embryos were placed on ice for euthanasia. Tumors were harvested, paraffi n embedded and stained with Hoechst 33342 (Life Technologies). Fluorescent images were obtained with inverted Leica DMI600B fl uorescence microscope equipped with a
RESULTS
Trastuzumab-800CW was injected in ex ovo CAMs with a HER2 positive MDA-MB-231-H2N (n=4) or a negative MDA-MB-231 (n=5) xenograft. Already at 24 hours after injection, distinct tracer tumor uptake was shown for MDA-MB-231-H2N compared to vasculature (TBR of 4.8 and 1.7 respectively; Figure 3). In addition, no tracer tumor uptake was observed in the MDA-MB-231 wildtype xenografts (TBR of 1.1 and 1.0; Figure 3). Over time, uptake in MDA-MB-231-H2N decreased to a TBR of 2.3 and 1.2, 72 and 96 hours after injection, respectively. This indicates HER2 specific uptake of trastuzumab-800CW at 24 hours after injection in the ex ovo xenograft model.
Figure 3 | HER2 imaging
MDA-MB-231 (n = 5) or MDA-MB-231-H2N (n = 4) xenograft bearing CAMs were injected with trastuzumab-800CW. CAMs were scanned after 24, 48, 72 and 96 hours after injection. Tumor-to-background ratios were calculated for each xenograft. White dotted circle indicates tumor location.
Moreover cetuximab-800CW was coinjected with IgG-680RD in ex ovo CAMs with an A431 (n=4) or a T47D (n=4) xenograft with a high and low EGFR expression, respectively, as previously described (13). Maximal TBR uptake of cetuximab-800CW in A431 xenograft was reached 24 hours after injection (TBR of 1.7; Figure 4). For the IgG-680RD control no increase in TBR was observed over time (TBR range 1.0 – 1.2). Altogether, this indicates specific tumor uptake of cetuximab-800CW in A431 xenografts. In an EGFR negative T47D xenograft, no specific uptake for cetuximab-800CW was demonstrated (TBR range 1.1 – 1.3). With fluorescent microscopy of A431 xenografts, cetuximab-CW800 could be visualized, while uptake was barely seen in the T47D xenografts (Figure 5). These results show EGFR specific tumor uptake of cetuximab-800CW in the ex ovo xenograft model.
Figure 4 | EGFR imaging
A431 (n = 4) or T47D (n = 4) xenograft bearing CAMs were coinjected with cetuximab-800CW and IgG-680RD. CAMs were scanned after 1, 12, 24 and 36 hours after injection. Tumor-to-background ratios were calculated for both tracers. Equal symbol correspond to tumor-to-background ratio for each tracer in one xenograft. White dotted circle indicates tumor location.
Figure 5 | Fluorescent microscopy
Fluorescent microscopy of A431 tumors, of which the CAM was injected with cetuxima-800CW (left panels). Visualization of CW800, nuclear staining with Hoechst and their combination show the presence of the tracer in the tumors. The right panels show fl uorescent microscopy of T47D tumors of which the CAM was injected with cetuximab-800CW. Visualization of CW800, nuclear staining with Hoechst and their combination shows barely tracer presence.
DISCUSSION
This study illustrates the feasibility of molecular fluorescent antibody imaging in the ex ovo CAM assay to visualize tumor characteristics and tracer distribution. For HER2 as well as EGFR imaging, tumor specific tracer uptake was seen in human tumor xenografts.
To our knowledge, this is the first study to use the ex ovo CAM assay for molecular fluorescent imaging. Previous studies have used imaging techniques for visualization of tumor or embryos
using the CAM assay. Tumor uptake with fluor-18-deoxyglucose (18F-FDG) was shown in a U87
glioblastoma xenograft (14). However, quantification of tumor uptake remains difficult due to unknown weight of the embryo (15). In our study, signal intensity could be quantified using IVIS scanning and thereby allowed us to calculate TBR. In addition, by including IgG control labeled with a different fluorophore, we were able to determine tumor specificity in the same xenograft. Others showed that the CAM assay can also be used to study new compounds to image xenografts using magnetic resonance imaging (16).
This proof of principle study supports use of the CAM model for future in vivo evaluation of treatment effects on tumor molecular characteristics without performing an animal experiment. In addition, this model could be used to study the tumor targeting properties of fluorescently labeled antibodies or antibody constructs. Also other applications are possible. Recent studies in oncology focus on the interaction between tumor and microenvironment including the immune system. Challenges in this field include the search for suitable models. The CAM assay allows growth of tumor cells in combination with stromal cells (17). For studying the interaction between immune cells and tumor cells, human immune cells (such as peripheral blood mononuclear cells) can be used instead of stromal cells. Another future use of the ex ovo CAM assay is the possibility for live cell imaging of fluorescently labeled antibodies. Thereby cellular dynamics can be studied in vivo while also gathering information regarding cellular receptor characteristics with fluorescent imaging. The CAM assay can potentially be further optimized for culturing patient derived xenografts existing of both human cancer cells and stromal cells. This would also preserve a part of the heterogeneity that is lost when using xenografts consisting of human cell lines.
Compared to the use of rodents for tumor imaging, the CAM assay has several advantages. As the lymphoid system develops late in the incubation period of the chicken eggs, the chicken embryo is naturally immunodeficient (18). Combined with its high vascularity, this makes the CAM an ideal host for human xenografts. Due to the fast tumor growth, experiments are less time consuming and of smaller expenses, which allows screening on relative large scale. Moreover, egg incubators are small compared to the space needed for rodent housing and few
embryos are used before the third trimester, making the CAM assay attractive as alternative for rodent experiments. Imaging experiments with fluorescently or radiolabeled antibodies in tumor bearing mice usually take up to 6 days after injection to reach maximal TBR (10, 11). Using the CAM assay, optimal TBR is already established at 24 hours after injection, allowing more rapid assessment of tumor targeting properties of fluorescently labeled antibodies compared to mouse experiments.
The CAM assay has also limitations compared to rodent experiments regarding molecular imaging of human xenografts. The time frame for the experimental procedures is relatively short, up to a maximum of 14 days. This is much shorter than the possible time frame for rodent experiments. As the pain sensation of the embryo develops somewhere between day 7 and day 13 of embryonic development, this should be taken into account after 10 days of development, by using anesthetics prior to euthanasia (21). Another issue with the CAM assay is the limited options for ex vivo analysis as many antibodies cross react with chicken cells (20).
In this study we have specifically chosen the ex ovo CAM assay as we are using fluorescently labeled antibodies. The in ovo experimental set up would give rise to imaging problems. Fluorescent imaging relies on light emitted to the tumor, which excites our fluorescent tracer and subsequently emits light of a different wavelength to our camera. If using the in ovo CAM assay, light would not be able to pass freely to and from the tumor because of interference of the eggshell. Another benefit for ex ovo fluorescent imaging is that no anesthesia is needed to fixate the embryo during the procedure. In contrast, anesthesia is indispensable for mouse models, when antibodies with a radioactive label are used for nuclear imaging. Although the ex ovo model can be used as a short-term assay, the in ovo model is less fragile in comparison, which would allow longer experiments such as studying effects of tumor growth and assessing pharmacodynamics. Indeed, earlier models such as catheter based injection system allowed assessment of bone metabolism over several days, using repeated injections of the bone seeker
agent 18F-labeled sodium fluoride (15).
In spite of the above, the CAM assay will not completely replace rodents for studying fluorescently labeled targeted agents. Whole body distribution and metabolism studies would still require fully developed organisms. However, the initial evaluation of new tracers could be performed with the, relatively high throughput, CAM assay. This set up supports experimentation in line with the three Rs (refinement, reduction, replacement).
Conflicts of interest
None of the authors has a conflict of interest to disclose regarding the publication of the present manuscript.
REFERENCES
1. Ferlay J, Soerjomataram I, Ervik M et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer; 2013: Available from: http:// globocan.iarc.fr, accessed 04/07/2018.
2. Mankoff DA. A definition of molecular imaging. J Nucl Med 2007; 48: 18N, 21N.
3. Lamberts LE, Williams SP, Terwisscha van Scheltinga AG et al. Antibody positron emission tomography imaging in anticancer drug development. J Clin Oncol 2015; 33: 1491-1504.
4. Nowak-Sliwinska P, Segura T, Iruela-Arispe ML. The chicken chorioallantoic membrane model in biology, medicine and bioengineering. Angiogenesis 2014; 17: 779-804.
5. Turtoi A, Blomme A, Bellahcene A et al. Myoferlin is a key regulator of EGFR activity in breast cancer. Cancer Res 2013; 73: 5438-5448.
6. Zijlstra A, Lewis J, Degryse B et al. The inhibition of tumor cell intravasation and subsequent metastasis via regulation of in vivo tumor cell motility by the tetraspanin CD151. Cancer Cell 2008; 13: 221-234.
7. Zhou F, Xue M, Qin D et al. HIV-1 Tat promotes Kaposi’s sarcoma-associated herpesvirus (KSHV) vIL-6-induced angiogenesis and tumorigenesis by regulating PI3K/PTEN/AKT/GSK-3beta signaling pathway. PLoS One 2013; 8: e53145. 8. Dohle DS, Pasa SD, Gustmann S et al. Chick ex ovo culture and ex ovo CAM assay: how it really works. J Vis Exp 2009; 33:
e1620.
9. du Manoir JM, Francia G, Man S et al. Strategies for delaying or treating in vivo acquired resistance to trastuzumab in human breast cancer xenografts. Clin Cancer Res 2006; 12: 904-916.
10. Terwisscha van Scheltinga AG, van Dam GM, Nagengast WB et al. Intraoperative near-infrared fluorescence tumor imaging with vascular endothelial growth factor and human epidermal growth factor receptor 2 targeting antibodies. J Nucl Med 2011; 52: 1778-1785.
11. Tjalma JJ, Garcia-Allende PB, Hartmans E et al. Molecular Fluorescence Endoscopy Targeting Vascular Endothelial Growth Factor A for Improved Colorectal Polyp Detection. J Nucl Med 2016; 57: 480-485.
12. Leong HS, Steinmetz NF, Ablack A et al. Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles. Nat Protoc 2010; 5: 1406-1417.
13. Aerts HJ, Dubois L, Hackeng TM et al. Development and evaluation of a cetuximab-based imaging probe to target EGFR and EGFRvIII. Radiother Oncol 2007; 83: 326-332.
14. Warnock G, Turtoi A, Blomme A et al. In vivo PET/CT in a human glioblastoma chicken chorioallantoic membrane model: a new tool for oncology and radiotracer development. J Nucl Med 2013; 54: 1782-1788.
15. Wurbach L, Heidrich A, Opfermann T et al. Insights into bone metabolism of avian embryos in ovo via 3D and 4D 18F-fluoride positron emission tomography. Mol Imaging Biol 2012; 14: 688-698.
16. Zuo Z, Syrovets T, Wu Y et al. The CAM cancer xenograft as a model for initial evaluation of MR labelled compounds. Sci Rep 2017; 7: 46690.
17. Nienhuis HH, Arjaans M, Timmer-Bosscha H et al. Human stromal cells are required for an anti-breast cancer effect of zoledronic acid. Oncotarget 2015; 6: 24436-24447.
18. Janse EM, Jeurissen SH. Ontogeny and function of two non-lymphoid cell populations in the chicken embryo. Immunobiology 1991; 182: 472-481.
19. Alcoser SY, Kimmel DJ, Borgel SD et al. Real-time PCR-based assay to quantify the relative amount of human and mouse tissue present in tumor xenografts. BMC Biotechnol 2011; 11: 124-6750-11-124.
20. Ribatti D. The chick embryo chorioallantoic membrane (CAM). A multifaceted experimental model. 2016; 141: 70-77. 21. Aleksandrowicz E, Herr I. Ethical euthanasia and short-term anesthesia of the chick embryo. ALTEX 2015; 32: 143-147.
