Click on an contribution preview the abstract content.
Click on an contribution preview the abstract content.
Introductory Talk by Jan Grimm - New York, USA
This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.
In vivo clinical imaging of breast cancer by means of multispectral optoacoustic tomography (#177)
K. Paul-Yuan1, G. Diot2, S. Metz3, A. Karlas2, E. Liapis1, E. Rummeny3, V. Ntziachristos1, 2
1 Helmholtz Zentrum Muenchen, Institute for biological and medical imaging (IBMI), Neuherberg, Bavaria, Germany
Multispectral optoacoustic tomography (MSOT) combines near-infrared light excitation along with ultrasonic detection to achieve high-resolution tissue imaging in vivo. MSOT provides tissue visualizations at unprecedented depths (~3-4cm) by mapping the distributions of intrinsic or extrinsic chromophores with optical contrast and acoustic resolution . Our current clinical applications of MSOT rely on a handheld device which achieves label-free imaging [2,3]. For breast cancer, clinical MSOT shows great potential as a novel tool for enhanced diagnostic and therapeutic monitoring.
A tunable short-pulsed laser, an array of ultrasound detectors and a DAQ connected to a PC were the main components of the measurement setup (Fig.1b). A fiber bundle connected the laser to the MSOT probe (Fig.1b) to deliver light at 50 Hz repetition rate (8ns pulse, wavelength ranges from 700 nm to 980 nm) to the examined region [2,3]. Ultrasound waves generated in tissue were recorded by an arch-shaped 256 element array of transducers and images were reconstructed in real-time [3,4]. The distribution of the endogenous chromophores (oxygenated-HbO2 and deoxygenated-Hb hemoglobin, lipids and water, Fig.1c), is determined by linear regression. High resolution MSOT image of malignant tumors in 10 female patients with previously diagnosed breast cancer were acquired and analyzed .
Acquired MSOT images of tumors showed recurring features such as high marginal and peripheral vascularization, increased heterogeneity of the total hemoglobin signal over normal tissue, low intratumoral vascularity and disruption of tissue layers as validated by histopathology analysis . Moreover, the spatial distribution of hemoglobin showed marked differences between the healthy and the malignant tissue, with increased homogeneity in healthy tissue areas compared to tumoral tissue (Fig.2 a,b).
MSOT is a versatile technology for characterizing human breast tumors in vivo by resolving oxygenated and deoxygenated hemoglobin and visualizing vascularization patterns with high resolution, outperforming other techniques such as ultrasonography and diffuse optical imaging (DOI). This allows investigating two main features of tumor pathophysiology: angiogenesis and hypoxia. MSOT-derived parameters may provide novel imaging biomarkers to enrich objective tumor classification tools, such as the BIRADS[WU1] classification .
[WU1] Breast Imaging-Reporting and Data System.
 Taruttis A. and V. Ntziachristos. 2015. Advances in real-time multispectral optoacoustic imaging and its applications. Nature Photonics Vol 9:219-227
 Diot G., S. Metz, A. Noske, E. Liapis, B. Schroeder, S.V. Ovsepian, R. Meier, E.J. Rummeny and V. Ntziachristos . (2017). Multi-Spectral Optoacoustic Tomography (MSOT) of human breast cancer. Clinical Cancer Research 10.1158/1078-0432.CCR-16-3200.
 Buehler A., M. Kacprowicz, A. Taruttis and V. Ntziachristos (2013). Real-time handheld multispectral optoacoustic imaging. Opt. Lett. 38(9), 1404—1406
 Rosenthal A., V. Ntziachristos, D. Razansky (2013). Acoustic Inversion in Optoacoustic Tomography: A Review. Current Medical Imaging Reviews 9(4), 318-336.
 Buehler A., G. Diot, T. Volz, J. Kohlmeyer, V. Ntziachristos. (2017). Imaging of fatty tumors: appearance of subcutaneous lipomas in optoacoustic images. Journal of Biophotonics 10(8), 983-989.
 Karlas A., J. Reber, G. Diot, D. Bozhko, M. Anastasopoulou, T. Ibrahim, M. Schwaiger, F. Hyafil, and V. Ntziachristos. (2017). Flow-mediated dilatation test using optoacoustic imaging: a proof-of-concept. Biomedical Optics Express 8(7), 3395-3403.
The research leading to these results has received funding by the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich-824 (SFB-824), subproject A1 and Gottfried Wilhelm Leibniz Prize 2013 (NT 3/10-1).
Clinical handheld MSOT imaging
a) Illustration of imaging of a lesion . b) Hardware employed in image acquisition of a multi-spectral dataset. HS – probe (photography below); DAQ – data acquisition; OPO – laser source. c) Mixture of absorbers and coloring scheme of: oxygenated blood (HbO2), deoxygenated blood (Hb), lipids and water used in Fig.2 . d) Optical absorption of the four absorbers in the near infrared regime.
MSOT images of healthy and malignant breast tissue.
Composite MSOT image of a) healthy breast tissue and b) a breast cancer tumor showing all four key absorbers after multispectral analysis . Two characteristic features for tumors are indicated: feeding vessels and the core of the tumor which lacks blood signal presumably due to necrotic tissue. Scale bars are 5mm. The color bars are chosen as described by Fig.1,c).
Keywords: optoacoustic, photoacoustic, breast cancer
Caveolin-1 inhibition increases and accelerates 89Zr-trastuzumab uptake in HER2-positive tumours: preclinical studies in tumour xenografts and human tumours (#191)
P. M. R. Pereira1, K. J. Edwards1, L. M. Carter1, J. - P. Meyer1, J. Pourat1, J. C. Durack1, J. S. Lewis1
1 Memorial Sloan-Kettering Cancer Center, Radiology, NY, New York, United States of America
HER2 overexpression in tumours is necessary to initiate trastuzumab therapy. Although HER2 is a membrane receptor, the surface pool of HER2 is governed by a balance between endocytosis and recycling. HER2 dynamics present a challenge for cancer treatment agents and molecular imaging probes targeting HER2 receptor. Since trastuzumab needs to bind to the extracellular domain of HER2, if the surface pool of HER2 is low, less binding will occur. This study uses HER2-PET imaging to evaluate the effects of CAV1 inhibition (a protein involved in HER2 endocytosis) on HER2 stability at the membrane.
For in vitro experiments, siRNA or lovastatin were used to inhibit CAV1. HER2 membrane stability was studied with cell surface biotinylation assays on human cancer cells. Trastuzumab binding was analyzed by internalization and cellular-binding of [89Zr]Zr-DFO-trastuzumab. LigandTracer technology was used for real-time binding-kinetic studies. For in vivo evaluation, lovastatin was orally administrated twice (8.3 mg/kg of mice), starting 1 day prior to [89Zr]Zr-DFO-trastuzumab administration. A pretargeted in vivo strategy, using TCO-conjugated trastuzumab and 18F-labeled tetrazine, was also performed to image HER2 at the cell membrane after CAV1 inhibition. In addition to conventional in vitro and in vivo biological models, cultures of fresh human bladder tumours were used in our study.
CAV1 inhibition for HER2-positive cancer cells increased the HER2 half-life at the cell membrane, which also increased the levels of membrane bound 89Zr-labeled trastuzumab and reduced its internalization. The number of HER2s per cell was higher in CAV1 depleted cells when compared with control (CT) cells. Real-time binding experiments resulted in an association rate constant lower for CT cells when compared with CAV1 depleted cells. Lovastatin inhibits CAV1, resulting in reduced HER2 internalization and increased membrane HER2. Lovastatin treatment increased the accumulation of 89Zr-labeled trastuzumab in HER2-positive tumours. The accelerated tumour accumulation of 89Zr-labeled trastuzumab in mice treated with lovastatin, prompted us to perform a pretargeted strategy where 18F-labeled tetrazine was administrated after trastuzumab. 18F-PET images clearly delineated HER2-positive tumours with activity concentrations up to 4.2 %ID/g at 4 h after injection of the radioligand.
Trastuzumab uptake increases in HER2-positive tumours after CAV1 inhibition. CAV1 signaling might explain the reasons by which trastuzumab does not always reach HER2-positive tumours. The accelerated accumulation of trastuzumab after CAV1 inhibition allowed us to perform a pretargeted methodology and this strategy deserves further investigation in HER2-positive tumours where CAV1 expression is low. Knowing that the therapeutic efficacy of trastuzumab depends on its ability to bind the extracellular domain of HER2, we are now exploring CAV1 inhibition with trastuzumab radioimmunotherapy.
(1) Network, T.C.G.A.R. (2014) Nature. 507, 315-322. (2) Martinez-Outschoorn, U.E., et al. (2015) Nat. Rev. Cancer. 15, 225-237. (3) Holland, J.P., et al. (2010) PLoS One. 5. (4) Janjigian, Y.Y., et al. (2013) J. Nucl. Med. 54, 936-943. (5) Meyer J.-P., et al. (2016) Bioconj. Chem. 27, 298-301.
PMRP acknowledges the Tow Foundation Postdoctoral Fellowship from the MSKCC Center for Molecular Imaging and Nanotechnology.
Figure 2. (A) PET images of [89Zr]Zr-DFO-trastuzumab and [18F]AlF-NOTA-PEG11-Tz/Trastuzumab-TCO pretargeting strategy. (B) Western blotting analysis of HER2 and CAV1 protein in cancer cells treated with lovastatin. (C) Quantification and autoradiographic images of [89Zr]Zr-DFO-trastuzumab in organotypic cultures of non-tumoural and bladder tumours treated with lovastatin.
Keywords: HER2, Caveolin-1, Endocytosis, PET imaging, Pretargeting
Prediction of chemotherapy resistance in preclinical models of ovarian cancer with [¹⁸F]FSPG positron emission tomography. (#4)
H. E. Greenwood1, P. N. McCormick1, M. Glaser2, T. Gendron2, K. Sander2, N. Koglin3, M. Zaw-Thin1, S. Patrick1, M. F. Lythgoe1, E. Årstad2, T. H. Witney1
1 University College London, 1Centre for Advanced Biomedical Imaging, London, United Kingdom
High-grade serous ovarian cancer (HGSOC) is a devastating disease, with a 5-year survival rate <42% (1). Following treatment, patients often relapse with a diminished response to subsequent therapy due to acquired drug resistance (2). Currently, there is no satisfactory way to identify patients that are refractive to the standard of care. Here, we propose (4S)-4-(3-[18F]fluoropropyl)-L-glutamate ([¹⁸F]FSPG) positron emission tomography (PET) as a non-invasive method for the prediction of drug resistance in HGSOC, enabling more appropriate treatment strategies to be instigated.
Drug sensitivity was characterised in wild-type (WT), cisplatin-resistant (CisR) and doxorubicin-resistant (DoxR) A2780 human ovarian cancer cells using an MTT assay (72h, 0.01-400 µM drug). Basal reactive oxygen species (ROS) were assessed by flow cytometry (CellROX orange), and levels of oxidised (GSSG) and reduced glutathione (GSH) were measured (GSH-Glo). Finally, [18F]FSPG cell retention was measured (1h uptake; 0.185MBq; 37°C).
Mice bearing s/c WT or DoxR xenograft tumours (~100 mm3) were imaged by PET following i.v. injection of ~3.7MBq [18F]FSPG (40-60 min p.i.). A second cohort of mice bearing s/c DoxR tumours were imaged with [18F]FSPG following liposomal doxorubicin treatment (doxil; 10 mg/kg, i.p on d0, d2 & d5). All tumours were excised and snap frozen for ex vivo analysis.
CisR cells displayed moderate sensitivity to cisplatin in comparison to WT cells (EC50 = 9µM vs. 0.9µM), with DoxR cells highly resistant to doxorubicin (EC50 = 50.5µM and 0.5µM for DoxR and WT, respectively; Fig 1A). Basal levels of intracellular ROS were 24% and 86% lower in CisR and DoxR cells, respectively versus WT (Fig 1B). [¹⁸F]FSPG is a substrate for the glutamate/cystine antiporter xCT. We have previously shown that activation of the cell’s antioxidant machinery reduces [18F]FSPG retention through changes in xCT activity (4). Here, [18F]FSPG cell retention (Fig 1C, insert) was related to the magnitude of drug resistance (n=3; P<0. 0001; Fig 1C). In vivo, [18F]FSPG uptake in DoxR tumours was ~80% lower than WT (7.9±0.7 %ID/g vs. 1.7±0.4 %ID/g; n=5-6; P<0.0001; Fig 1D, E), corresponding to a 21-fold increase in GSSG:GSH. In DoxR tumours, doxil treatment (Fig 2A) had no significant effect on tumour size and [18F]FSPG uptake compared to untreated mice (n=5-6; P>0.5; Fig 2B-D).
These results confirm the sensitivity of [18F]FSPG uptake to upregulated antioxidant pathways present in drug-resistant A2780 tumours. In the drug-resistant lines, low [18F]FSPG uptake corresponded with reduced ROS and higher GSH utilisation in comparison to drug-sensitive lines. [18F]FSPG may therefore enable the identification of ovarian cancer patients that are refractory to the standard-of-care, as well as monitor their response post-treatment. Transferral of drug-resistant patients to alternative therapies has the potential to increase tumour response and patient survival.
[18F]FSPG uptake is reduced in drug-resistant ovarian cancer.
A. Cell viability in A2780 cells after cisplatin or doxil treatment. B. Intracellular ROS in A2780 cells. C. The effect of drug sensitivity on [18F]FSPG uptake (insert: [18F]FSPG chemical structure). D. PET/CT maximum intensity projection images of WT and DoxR tumour-bearing mice 40-60 min p.i. White arrows indicates the tumour. E. Quantification of [18F]FSPG tumour uptake (n=5, WT; n=6, DoxR).
[18F]FSPG analysis as a predictive marker for treatment response following doxil treatment regime.
A. The treatment and imaging protocol. B. PET/CT maximum intensity projection images representing chemotherapy resistant A2780 DoxR tumour bearing mice prior treatment, 24h and 6d following initial doxil treatment. C. Quantification of tumour uptake in all three treatment groups (UT, n=6; 24h, n=5; 6d, n=6). D. A2780 DoxR tumour growth curves in untreated and drug-treated mice (n=5-6 mice).
Keywords: drug resistance, predict response, ovarian cancer, positron emission tomography, preclinical, non-invasive
CXCR4-directed PET imaging for lymphoma of the central nervous system (#277)
P. Herhaus1, T. Vag2, S. Habringer1, 8, J. Slotta-Huspenina3, M. Deckert4, K. Steiger2, 8, T. Pukrop5, C. Lapa6, H. - J. Wester7, 8, M. Schwaiger2, 8, U. Keller1, 8
1 MRI, TU München, III. Medical Department, Munich, Germany
The chemokine receptor CXCR4 is a master regulator of migration, organogenesis and maintenance of the hematopoietic stem cell niche. In lymphoma, mutations and aberrant expression are involved in pathogenesis and CXCR4 over-expression is often associated with poor survival and treatment resistance (1). In vivo imaging of CXCR4 by means of the CXCR4-directed PET tracer [68Ga]Pentixafor provides a novel tool for investigating CXCR4 expression (2,3). We here describe the proof-of-concept analysis of CXCR4-directed PET imaging in patients with biopsy-proven central nervous system (CNS) lymphoma.
We hypothesized that CXCR4-directed PET with [68Ga]Pentixafor might add valuable information to the standard diagnostic procedure magnet resonance imaging (MRI) in patients with CNS lymphoma. 8 patients with lymphoma of the CNS with different disease subtypes and stages (6 with first diagnosis of primary CNS lymphoma (PCNSL), 2 with CNS relapse of diffuse large B-cell lymphoma (DLBCL)) were imaged with [68Ga]Pentixafor by PET-MRI or PET-CT after signing informed consent in this observational assessment (see Table 1 for patient characteristics). The administered dose of [68Ga]Pentixafor ranged from 96 to 275 MBq.
MRI represents the standard diagnostic imaging procedure for secondary and primary lymphomas of the central nervous system (PCNSL). However, MRI does not provide high specificity with regard to differentiating PCNSL or secondary CNS involvement of lymphoma from primary brain cancers or metastasis of solid cancers. CXCR4-directed PET imaging with [68Ga]Pentixafor correlated in all patients well with the lymphoma lesions determined by the current standard, MRI. The maximum standard uptake values (SUVmax) ranged from 4.2 to 23.3. The SUVmax of the background (brain parenchyma on the contralateral site) ranged from 0.1 to 0.8. The tumor-surrounding edema did not show any uptake of tracer (see Figure 1 for representative images). Importantly, the single patient with negative CXCR4-directed PET imaging received an extended biopsy and did not show visible lymphoma lesions in MRI either.
CXCR4-directed PET imaging with [68Ga]Pentixafor is feasible in patients with lymphoma of the CNS. Due to the low uptake of this tracer in normal brain tissue and resulting excellent tumor-to-background ratio this imaging modality has high potential for differentiating lymphoma of the CNS from other cancers, and for theranostic purposes. Prospective studies of this novel imaging modality are urgently required.
(1) Chen J, Xu-Monette ZY, Deng L, Shen Q, Manyam GC, Martinez-Lopez A, et al. Dysregulated CXCR4 expression promotes lymphoma cell survival and independently predicts disease progression in germinal center B-cell-like diffuse large B-cell lymphoma. Oncotarget. 2015;6(8):5597-614.
(2) Gourni E, Demmer O, Schottelius M, D'Alessandria C, Schulz S, Dijkgraaf I, et al. PET of CXCR4 expression by a (68)Ga-labeled highly specific targeted contrast agent. J Nucl Med. 2011;52(11):1803-10.
(3) Wester HJ, Keller U, Schottelius M, Beer A, Philipp-Abbrederis K, Hoffmann F, et al. Disclosing the CXCR4 expression in lymphoproliferative diseases by targeted molecular imaging. Theranostics. 2015;5(6):618-30.
Keywords: PET, CXCR4, lymphoma, cns, diagnostic
Is there a rational for combining 177Lu-PSMA radioligand- with androgen deprivation-therapy for the treatment of prostate cancer? (#9)
K. Lückerath1, 2, A. D. Stuparu1, W. P. Fendler1, 3, L. Wei1, R. Slavik1, J. Czernin1, K. Herrmann3, M. Eiber4
1 University of California Los Angeles, Molecular & Medical Pharmacology, Los Angeles, California, United States of America
PSMA-directed radioligand therapy (PSMA RLT) is emerging as new therapeutic option, often applied in combination with androgen deprivation therapy (ADT). Upregulation of PSMA expression and radiosensitization, both previously described for ADT, may synergistically enhance RTL. This study aimed at testing the rationale for combining 177Lu-PSMA RLT and ADT in mouse models of prostate cancer.
Scid mice bearing C4-2, LnCap or 22Rv1 human prostate cancer tumors were treated with 50 mg/kg enzalutamide (ENZ), 10 mg/kg bicalutamide (BIC) or vehicle (control) for 21 days. Kinetics of PSMA expression were quantified by biopsy/flow cytometry as reference method and by 68Ga-PSMA11 PET/CT before treatment (day -1) and on days 23, 29, 34 and 38 post therapy indcution. For the combination approach, mice bearing C4-2 or 22Rv1 tumors were treated with 50 mg/kg enzalutamide or vehicle for 21 days before receiving either 15MBq (84GBq/µmol) 177Lu-PSMA617 RLT or vehicle. Efficacy of treatment was determined as reduction of tumor volume on serial CT and survival. For estimating the 177Lu tumor dose, activity retained in tumor and organs was quantified ex vivo in a subset of mice.
Tumor growth was significantly retarded by ADT (day 29, ~0.3 of control tumor volume). Absolute 68Ga-PET signal increased in all mice over time with ~1.2-fold higher %IA/gmax in ADT treated groups. Correcting 68Ga-PSMA PET for tumor volume revealed up to 3.6-fold higher PSMA levels at day 29. Flow cytometry confirmed up to 1.9-fold upregulation of PSMA expression in tumors compared to untreated controls.
In our model, ADT with ENZ or BIC upregulates PSMA expression on three different tumors in vivo. Upregulation can be monitored using size corrected 68Ga-PSMA PET/CT. 177Lu-PSMA617 alone effectively decreases tumor size. Despite upregulation of the target, addition of ADT did not lead to higher 177Lu tumor doses or an additive effect in our xenograft mouse model.
Keywords: prostate cancer, androgen deprivation therapy, 177Lu-PSMA617 radioligand therapy, 68Ga-PSMA11 PET/CT, PSMA, combination treatment
Gelofusine improves the feasibility of insulinoma treatment in patients using radiolabeled exendin (#104)
T. Jansen1, M. Buitinga1, I. van der Kroon1, W. Woliner-van der Weg1, M. Boss1, M. Janssen1, M. Béhé2, D. Wild3, M. Brom1, M. Gotthardt1
1 Radboud university medical center, Radiology and Nuclear Medicine, Nijmegen, Netherlands
Endogenous hyperinsulinemic hypoglycemia is most commonly caused by insulinomas, with surgery as only curative treatment option. However, patients can have multiple lesions or metastatic disease (≈10%), hampering successful surgical intervention. In those patients, peptide receptor radionuclide therapy (PRRT) using radiolabeled exendin might be a valuable alternative. A major drawback of radiolabeled exendin is high renal uptake, Gelofusine might considerably reduce this uptake. We studied the effect of Gelofusine and explored the feasibility for insulinoma treatment using radiolabeled exendin
In a cross-over design (2 study arms) 10 healthy volunteers were included and 2 SPECT/CT scans per subject were performed, 24 h after the injection of 50 MBq 111In-exendin. First arm: 111In-exendin was injected with a co-injection of GELO, 3 weeks later the control scan was performed with co-injection of saline instead of GELO. Second arm: co-injections were done in reverse order. The effect of GELO was then used as input for a whole organ and pancreatic islet dosimetry model to calculate changes in therapeutic dose (177Lu-exendin) for insulinomas . Dosimetric data of 3 patients were used: 1 benign insulinoma (10 mm) and 2 malignant insulinomas (14 and 15 mm). Additionally, pancreatic islet and pancreas doses were calculated. The maximum tolerable absorbed kidney dose was set to 23 Gy.
GELO reduced renal uptake of 111In-exendin significantly with a mean reduction of 18.1 ± 4.2% (p<0.001). Accompanied by a significantly higher cumulative urinary excretion of 111In-exendin. No significant reduction in pancreatic uptake was observed. The allowable injected dose of 177Lu-exendin increased using GELO from 1.5 to 1.8 GBq before reaching the maximum tolerable kidney dose. The absorbed doses were (without/with GELO): islets (3.3/4.0 Gy), pancreas (0.3/0.3 Gy), insulinoma (10 mm: 105/127 Gy, 14 mm: 92/112 Gy, 15 mm: 64/78 Gy). Thus, GELO increased the allowable injected dose to the insulinomas with more than 20%. The islet dose remained very low and therefore also the risk of diabetes remains low , whereas the radiation dose to the pancreas did not change.
Gelofusine significantly reduces renal uptake of radiolabeled exendin and thereby the radiation dose to the kidneys. The estimated insulinoma dose increases by 20% when using Gelofusine, improving the feasibility of PRRT with radiolabeled exendin.
1. van der Kroon I, Woliner-van der Weg W, Brom M, Joosten L, Frielink C, Konijnenberg MW, Visser EP and Gotthardt M. Whole organ and islet of Langerhans dosimetry for calculation of absorbed doses resulting from imaging with radiolabeled exendin. Sci. Rep. Nature Publishing Group; 2017;7:39800
2. de Vathaire F, El-Fayech C, Ben Ayed FF, Haddy N, Guibout C, Winter D, et al. Radiation dose to the pancreas and risk of diabetes mellitus in childhood cancer survivors: A retrospective cohort study. Lancet Oncol. Elsevier Ltd; 2012;13:1002–10.
This work was supported by the project BetaCure, under grant agreement no. 602812.
Keywords: insulinoma, PRRT, Radiolabeled exendin, Gelofusine, dosimetry, clinical study, radiation dose, exendin, GLP-1 receptor
HER3 PET Imaging as a Biomarker of Drug Resistance (#462)
C. D. Martins1, C. D. Pieve1, T. A. Burley1, D. M. Ciobota1, L. Allott1, R. Smith1, K. J. Harrington1, W. J. G. Oyen1, G. Smith1, G. Kramer-Marek1
1 The Institute of Cancer Research, Division of Radiotherapy and Imaging, London, United Kingdom
Intratumoural heterogeneity of breast cancer (BC) presents a clinical challenge, as histological techniques can fail to provide a representative indication of molecular variation, due to dependence on the section of tumour that is chosen for sampling. This underscores the need to introduce novel imaging biomarkers that allow for the examination of the whole tumour mass. Therefore, we developed a novel PET radiotracer that provides information on receptor expression changes due to downstream signalling inhibition, which is increasingly being recognised as a key player in therapeutic resistance.
ZHER3:8698 affibody molecules were conjugated to DFO-maleimide for 89Zr radiolabelling. The probe was characterised in vitro in a panel of human breast cancer cell lines. Pharmacokinetic studies in vivo were performed 3 and 24 h following 89Zr-DFO-ZHER3:8698 injection (1-3 μg, 2.7-8.1 MBq) in mice bearing MCF-7, BT474 and MDA-MB-231 xenografts. Additionally, the radioconjugate was tested as a tool to monitor treatment with AUY922, an Hsp90 inhibitor. AUY922 (50 mg/kg) was administered (i.p.) every second day for a total of 7 doses. Mice bearing MCF-7 (HER2+, HER3+++) or BT474 (HER2+++, HER3+++) xenografts were imaged before treatment initiation, and two weeks after the first dose. Following the last scan, the animals were euthanized and the tumours fixed and/or frozen for further analysis.
The Kd of the conjugate was determined to be 0.55+0.05 nM. Rapid clearance of the 89Zr-DFO-ZHER3:8698 in vivo enabled high-contrast imaging of HER3-positive MCF-7 xenografts (2.7 ± 0.32 %ID/g) as early as 1 h post injection. Additionally, in vitro data suggested that treatment with the Hsp90 inhibitor, AUY922, leads to an increase in HER3 expression in cells with non-amplified HER2, but with high-HER3 level e.g. MCF-7. Furthermore, AUY922 treatment triggered an alternative survival pathway promoting HER3/IGF-1R heterodimerization in these cells. In vivo, an increase in radioconjugate uptake, in MCF-7 xenografts, correlated with Hsp90-induced up-regulation of HER3 expression. An increase in IGF-1R levels in response to AUY922 treatment further confirmed a role for HER3/IGF-1R heterodimerization in acquired resistance to Hsp90 inhibition. Conversely, in BT-474 xenografts, AUY922 treatment led to a significant decrease in radioconjugate uptake due to HER2/HER3 downregulation.
Recent Hsp90 inhibitor-based clinical trials have shown that the use of such agents may not be an effective therapeutic strategy in BC. Therefore, combinatorial therapeutic approaches with agents targeting resistance-conferring receptors (e.g. HER3, IGF-1R) may be required for a more effective clinical use of Hsp90 inhibitors. 89Zr-DFO-ZHER3:8698-based imaging may prove to be an important measure in quantifying changes in HER3 expression resulting from acquired resistance to Hsp90 inhibition and thus serve as a valuable tool in facilitating treatment personalisation.
The authors gratefully thank AffibodyAB (Stockholm, Sweden) for supplying the affibody molecules. This work was supported by the funding from the Institute of Cancer Research and Cancer Research UK-Cancer Imaging Centre (C1060/A16464).
PET/CT imaging and ex vivo analysis of 89Zr-DFO-ZHER3:8698 in mice bearing subcutaneous breast cance
A. Ex vivo biodistribution (n=3) at 3 h after injection of the 89Zr-DFO-ZHER3:8698.
B. Histopathological analysis of HER3 expression in MCF-7, MDA-MB-468, and MDA-MB-231 xenografts.
D. PET/CT images of mice bearing MCF-7 and BT474 tumours after AUY922 treatment.
Keywords: HER3, affibody-based PET imaging, Hsp90 inhibition, breast cancer