Image-guided drug delivery (IGDD) study group meeting: Wellcome and brief introduction from the chairs (#750)
Rafael T.M de Rosales1, Roel Deckers2
1 King's College London, London, United Kingdom
2 UMC Utrech, Utrech, Netherlands
Molecular imaging in cancer patients: real-time information on drug delivery. (#628)
Willemien Menke-van der Houven van Oordt1
1 AmsterdamUMC location VUMC, Medical Oncology, Amsterdam, Netherlands
About half of the world population will be confronted with having cancer somewhere during their lifetime and despite significant advances in the treatment still about a third will be dying of it. Therefore, there is an urgent need to improve the current therapeutic options. Molecular imaging using labeled drugs as a PET tracer can help understand drug delivery and drug-target interactions in tumor lesions as well as healthy tissues1.
Many antibody-based drugs, such as monoclonal antibodies (mAbs) inhibiting specific receptor-signaling, bispecific antibodies f.i. targeting tumor antigens and attracting immune cells, antibody drug conjugates (ADCs) delivering cytotoxic agents to the tumor, and many other targeted therapeutics are being developed or already in use in the clinic. The delivery is assumed to depend on receptor/antigen-antibody interaction, however no direct measure of this interaction in the patient is available. Optimal drug efficacy has traditionally been achieved by selecting the maximum tolerated dose (MTD), however this approach may not be optimal for antibody-based drugs. Molecular imaging using zirconium-89 (89Zr)-desferal (Df)-labeled antibody as tracer can give insight in their biodistribution and target-mediated antibody uptake. Indeed, dose‐dependent inhibition of target-mediated tumor uptake of a labeled anti-HER3 mAb (89Zr‐GSK2849330) by unlabeled mAb confirmed target engagement of mAb to the HER3 receptor. Moreover, identification of saturating mass dose of the antibody potentially supports dose selection2.
Other options to increase therapeutic efficiency of f.i. standard chemotherapeutic agents is the use of nanoparticles. CPC634 is a nanoparticle entrapping docetaxel designed to improve the tumor accumulation and tolerability compared to conventionally administered docetaxel by taking advantage of the presumed enhanced permeability and retention (EPR) effect. In vivo imaging with 89Zr-Df-CPC634 showed retention in 16/37 evaluable tumor lesions compatible with the assumed EPR effect of a non-targeted nanoparticle which has been shown here for the first time in patients3.
Examples of clinical trials in drug development will be discussed.
1 Jauw YW, Menke-van der Houven van Oordt CW, Hoekstra OS, Hendrikse NH, Vugts DJ, Zijlstra JM, Huisman MC, van Dongen GA. Immuno-Positron Emission Tomography with Zirconium-89-Labeled Monoclonal Antibodies in Oncology: What Can We Learn from Initial Clinical Trials? Front Pharmacol. 2016;7:131.
2 Menke-van der Houven van Oordt CW, McGeoch A, Bergstrom M, McSherry I, Smith DA, Cleveland M, Al-Azzam W, Chen L, Verheul H, Hoekstra OS, Vugts DJ, Freedman I, Huisman M, Matheny C, van Dongen G, Zhang S. Immuno-PET Imaging to Assess Target Engagement: Experience from 89Zr-Anti-HER3 mAb (GSK2849330) in Patients with Solid Tumors. J Nucl Med. 2019 Jul;60(7):902-909.
3 Miedema IHC, Zwezerijnen GJC, Oprea-Lager DE, Verheul HMW, Vugts DJ, Huisman MC, Matthijssen RHJ, Rijken C, Hu Q, van Dongen GAMS, Menke-van der Houven van Oordt CW. First in human imaging of nanoparticle-entrapped docetaxel (CPC634) in patients with advanced solid tumors using 89Zr-Df-CPC634 PET/CT. J Clin Oncol 2019; 37 suppl; abstr 3093.
Keywords: immunoPET, drug development, nanoparticle, zirconium-89
Monitoring dynamics of the EPR effect using theranostic polymeric micelles (#159)
Ilaria Biancacci1, Benjamin Theek1, Yang Shi2, Felix Gremse1, Federica De Lorenzi1, Maike Baues1, Jan-Niklas May1, Wim Hennink2, Fabian Kiessling1, Twan Lammers1, 2
1 Institute for Experimental Molecular Imaging, University Clinic, Aachen, Germany
2 Department of Pharmaceutics, Utrecht University, Utrecht, Netherlands
Conventional chemotherapy has multiple drawbacks. Hence, plenty of nanomedicines have been evaluated over the years, aiming to enhance therapeutic outcomes via improving pharmacokinetics and biodistribution1. Nanomedicine rely on the Enhanced Permeability and Retention (EPR) effect for efficient tumor accumulation. However, the EPR effect shows a very high degree of inter- and intra-patient variability2 and it is subject to unknown changes during therapy. We here used theranostic polymeric micelles to monitor the EPR dynamics and tumor microenvironment changes during the course of nanotherapy.
Nude mice orthotopically inoculated with murine 4T1 breast cancer cells were randomly assigned to different treatment groups: control, free paclitaxel (Taxol®; 15 mg/kg), and an equi-dose (15 mg/kg) and double-dose (30 mg/kg) of paclitaxel-loaded and Cy7-labeled polymeric micelles. Hybrid Computed Tomography - Fluorescence Molecular Tomography (CT-FMT) scanning was longitudinally performed to assess micelle target site accumulation during nanotherapy (Fig. 1A). Ex vivo fluorescence reflectance (FRI) scans and histopathological examinations of tumor sections were conducted to investigate the macro-distribution of the micelles and to study the microenvironmental changes resulting from nanomedicine-mediated anticancer therapy.
Paclitaxel-loaded micelles outperformed free paclitaxel when administered at double dose (Fig. 1B). A correlation between EPR-mediated tumor accumulation and antitumor efficacy was only observed when comparing the single dose vs. the double dose group (Fig. 1C-J). For the double-dose-treated group, EPR-mediated tumor accumulation (in %ID/200 mm3) increased during the course of therapy, whereas it dropped for the single-dose group, indicating that efficient treatment promotes the EPR effect. Histological analyses confirmed the improved therapeutic effect of the micelles as compared to the free drug (Fig. 2A-B). We observed that nanotherapy impacted the tumor microenvironment, reducing the number of blood vessels but promoting their maturity and functionality (Fig. 2C-D). Nanotherapy furthermore resulted in a dose-dependent decrease in macrophage and collagen content (Fig. 2E-G).
Theranostic micelles enable non-invasive imaging of tumor accumulation. Within the relatively small dosing groups (n=5), no correlation was found between tumor accumulation and antitumor efficacy, but this was observed between the different dosing level groups. High dose micelle therapy affected the tumor microenvironment and promoted EPR-mediated accumulation. These insights are important for understanding and improving anticancer nanotherapy.
AcknowledgmentThe authors gratefully acknowledge financial support by the DFG (Research Training Group 2375 “Tumor-targeted Drug Delivery”; grant 331065168) and by the European Research Council (ERC-PoC-813086: PIcelles).
 Dasgupta A. et al. 2020, ‘Imaging-assisted anticancer nanotherapy’, Theranostics, 10(3), 956-967.
 Van Der Meel, R. et al. 2019, ‘Smart cancer nanomedicine’, Nat. Nanotechnol., 14(11), 1007-1017.
Figure 1: Monitoring EPR effect upon treatment.
Study design is represented in (A). The higher accumulation of high dose DDS results in a slower growth rate, with respect to the free drug and control groups (B-D). Both groups show similar cell uptake (E). Representative CT-FMT images showing the accumulation of NIR-micelles in the tumor over time (F). Exponential tumor growth and stable micelle accumulation over the time is observed in the low dose group, opposite trend is shown for high dose group (G-I). The combination of both groups shows a negative correlation between relative tumor growth and ptx-micelle accumulation (J). ** p<0.01.
Figure 2: Tumor microenvironment is subject to modification upon treatment with paclitaxel.
Exemplary images of cell proliferation, blood vessel perfusion, macrophages infiltration and collagen I in tumors, based on immunostaining (A). The employment of paclitaxel results in an improved therapeutic effect, as compared to free ptx (B). Furthermore, it highlights a modification of the tumor microenvironment, which exhibits a mild increase of vessel maturity and functionality and a decreased macrophage (pro- and anti-inflammatory phenotypes) and collagen I content (C-G). Scale bars size corresponds to 20 µm. * p<0.05, ** p<0.01, *** p<0.001.
Keywords: image-guided drug delivery, nanomedicine, tumor microenvironment, enhanced permeability and retention (EPR) effect
Mesenchymal Stem Cell-Derived Exosomes for Drug Delivery and PET Imaging (#512)
Arunkumar Pitchaimani1, Miguel Moreira1, Annalisa Palange1, Beatriz P. Samper2, Roberto Moratta1, Felipe P. Cardoso2, Paolo Decuzzi1
1 Istituto Italiano di Tecnologia, Nanotechnology for Precision Medicine, GE, Italy
2 University of Navarra, CIMA, Navarra, Spain
Extracellular vesicles like the nano-sized EXOsomes (EXO) from mammalian cells play a key role in various transcellular communication processes including genetic exchange. Particularly Mesenchymal Stem cells (MSCs) exosomes have functions similar to that of its parent cell in repairing tissue damage, the immune system modulation, etc.1,2 Interestingly, it has the ability to cross the BBB upon systemic injection and accumulates in the brain parenchyma.3 Herein, we investigate the role of MSC EXO in delivering therapeutic doxorubicin and PET imaging agents (64Cu) non-invasively via the nose.
Human adipose derived MSCs were isolated following the protocols developed at CIMA, University of Navarra (Spain). Further, EXO were isolated from the MSCs via a differential ultracentrifugation. The protein profile was analyzed using western blots and LC-MS (Proteinase-K method). Rhodamine-EXO were used for studying differential cellular uptake in macrophages (RAW 264.7), neuronal cells (Neuro-2A) and brain cancer cells (U87-MG) via confocal and flow cytometry. Doxorubicin was loaded into EXO and tested its encapsulation efficiencies and release rates using HPLC . Finally, exosomes were also labeled with lipid-DOTA molecules reacted with 64Cu, using fusogenic liposomes. Administration of EXO to mice was realized non-invasively through the nasal cavity in Nu/Nu mice.
The exosomes isolated from the hADSCs were spherical in shape with an average diameter of 119 ±7 nm under cryo-EM. The dynamic light scattering analysis showed exosomes with a hydrodynamic diameter of ~ 130 ± 18 nm and the surface zeta potential around – 30 ± 2mV. The characteristic functional protein markers exposed of the exosome membrane, CD-9 and CD-81, were clearly identified through western blot and mass spectroscopy analyses. Further, confocal microscopy and flow cytometry analyses showed that significant levels of naïve exosome were taken up by RAW 264.7, Neuro-2A and U87-MG cells, as compared to the PK treated EXO. The radiolabeling efficacy of EXO was found to be ~70% and showed a high stability for over 24h. Upon intra-nasal administration, EXO were slowly released from the nasal cavity at the systemic level with peak accumulations in the stomach, intestine, liver and tumor. The cytotoxicity of DOX-EXO was comparable to that of the free drug.
This preliminary work demonstrates that human adipose stem cell-derived exosomes can be efficiently loaded with a chemotherapeutic molecule and a radioactive agent. The biodistribution of the exosomes can be monitored over time using Nuclear Imaging and the therapeutic activity of doxorubicin loaded exosomes is comparable with the free drug molecule.
AcknowledgmentThis work was partially supported by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 754490.
 Bose, R. J.; Kim, B. J.; Arai, Y.; Han, I.-B.; Moon, J. J.; Paulmurugan, R.; Park, H.; Lee, S.-H. Bioengineered Stem Cell Membrane Functionalized Nanocarriers for Therapeutic Targeting of Severe Hindlimb Ischemia. Biomaterials 2018, 185, 360–370
 Gao, C.; Lin, Z.; Jurado-Sánchez, B.; Lin, X.; Wu, Z.; He, Q. Stem Cell Membrane-Coated Nanogels for Highly Efficient In Vivo Tumor Targeted Drug Delivery. Small 2016, 12 (30), 4056–4062.
 Betzer, O.; Perets, N.; Angel, A.; Motiei, M.; Sadan, T.; Yadid, G.; Offen, D.; Popovtzer, R. In Vivo Neuroimaging of Exosomes Using Gold Nanoparticles. ACS Nano 2017, 11 (11), 10883–10893.
Mesenchymal Stem Cell-Derived Exosomes for Drug Delivery and PET Imaging
Role of Mesenchymal stem cell exosome in delivering therapeutic doxorubicin and PET imaging agents (64Cu) non-invasively via nose to brain.
Keywords: Mesenchymal stem cells, Exosomes, PET, Doxorubicin
The impact of multidrug resistance on tumor microenvironment remodeling in murine 4T1 breast cancer models (#275)
Elena Rama1, Okan Tezcan1, Fabian Kiessling1, Twan Lammers1
1 University Clinic RWTH Aachen, Experimental Molecular Imaging, Aachen, Germany
Multidrug resistance (MDR) is a major limiting event in chemotherapy. MDR results from changes in several cellular mechanisms such as altered gene expression profiles and signaling networks. These cellular MDR events were evaluated in the direction of tumor microenvironment remodeling. To examine this, we developed sensitive and resistant breast tumor-bearing mouse models. We evaluated the deposition of the extracellular matrix (ECM) components and the density of microvascularization in tumors. Lastly, we studied the accumulation and distribution of nanoparticles in both tumors.
Multidrug resistant 4T1 cells were established by stepwise selection using doxorubicin dose increments. Sensitive and resistant 4T1 cells were orthotopically injected into mammary fat pad of mice. When tumors reached 10-12 mm, mice were injected with rhodamine lectin and sacrificed. Tumors were excised for the fluorescence-based immunohistochemistry (IHC) analyses. To characterize the extracellular matrix deposition in both tumors, antibodies against collagen-1 (col-1), hyaluronan (HA) and collagen-4 (col-4) were used. The IHC analysis was performed also for the characterization of vascularization by using antibodies against endothelial cells (CD31) and pericytes (αSMA). CT-FMT and fluorescence microscopy were employed to visualize the nanoparticle accumulation and distribution.
Histological analyses revealed that the deposition of ECM components, col-1 (Fig.1A), HA (Fig.1B) and col-4 (Fig.1C) was significantly higher in resistant 4T1 tumors than the sensitive ones. The IHC analysis also showed that the density of the microvessels was higher in resistant 4T1 tumors as compared to the sensitive 4T1 tumors (Fig. 1D). The results from CT-FMT imaging showed a higher polymer and liposome accumulation in resistant 4T1 tumors than in sensitive 4T1 tumors (Fig.2A). The fluorescence microscopy analyses demonstrated that, despite having a higher accumulation, the distribution of nanoparticles was poor in resistant 4T1 tumors (Fig.2B). These findings demonstrate that cellular multidrug resistance signaling affects tumor microenvironment remodeling, specifically the composition of the ECM and the microvascularization. These remodeling features of multidrug resistant cells might further limit the success of nanoparticle delivery to MDR tumors.
Our findings show a relative increase in ECM deposition in multidrug resistant 4T1 tumors. We also supply evidence that the amount of angiogenic vessels in MDR tumors is higher than in sensitive tumors. This indicates that when cancer cells are continuously exposed to doxorubicin via stepwise dose increases, they present with a different microenvironment, which may affect the drug delivery to and into tumors.
This work was supported by DAAD (57048249) and Deutsche Forschungsgemeinschaft (DFG) (403039938).
 Tezcan O, Ojha T, Storm G, Kiessling F, Lammers T. Targeting cellular and microenvironmental multidrug resistance.
 Chen Y, Tezcan O, Li D, Beztsinna N, Lou B, Etrych T, Ulbrich K, Metselaar JM, Lammers T, Hennink WE. Overcoming multidrug resistance using folate receptor-targeted and pH-responsive polymeric nanogels containing covalently entrapped doxorubicin. Nanoscale. 2017;9(29):10404-19.
Figure 1. Microenvironment characterization of 4T1 sensitive and resistant tumor models through IHC:
Representative images show the ECM components, collagen-1 (A), hyaluronan (B) and collagen-4 (C) in sensitive and resistant 4T1 tumors. These findings demonstrate that the deposition of ECM components is significantly higher in 4T1 resistant tumors. The analyses for microvessel density reveals a stronger angiogenic profile in in resistant 4T1 tumors as compared to the sensitive 4T1 tumors (D). All images were captured from periphery (P) and core (C) regions of each tumor section (*p<0.05, **p<0.001 and ***p<0.0001).
Figure 2. Accumulation and distribution of polymers and liposomes through CT-FMT and fluorescence mi
CT-FMT images show the accumulation of polymers and liposomes in sensitive and resistant 4T1 tumors in 48h and 72h (A). The accumulation of polymers and liposomes was higher in 4T1 resistant tumors than in sensitive 4T1 tumors. Fluorescence microscopy images demonstrate the distribution of polymers and liposomes (B). These results show that the distribution of polymer and liposomes were poor in resistant 4T1 tumor as compared to sensitive ones (*p<0.05, **p<0.001 and ***p<0.0001).
Keywords: Tumor microenvironment heterogeneity, Multidrug resistance, CT-FMT imaging, Fluorescence microscopy, Nanoparticle delivery
Towards imaging cisplatin resistance with 64Cu PET (#714)
Fahad Al-salemee1, Joanna Bartnicka1, Timothy H. Witney1, Philip Blower1
1 King's College London, Imaging Chemistry and Biology, London, United Kingdom
Cisplatin is the backbone of several treatment regimens for various cancer types, including ovarian cancer; however, its efficacy is often abolished by the early development of resistance, especially in ovarian cancer patients. Consequently, many patients suffer the debilitating side effects of cisplatin in vain. A correlation between cellular accumulation of cisplatin and copper transporters (CTR1, ATP7A, ATP7B) has been repeatedly reported in the literature.1–4 We investigated the novel possibility of using unchelated 64Cu(II) PET imaging to predict cisplatin accumulation in tumours in vivo.
Two variants of the human ovarian carcinoma cell line A2780, wild type (WT) and cisplatin-resistant (CisR),5 were used. Cisplatin resistance was compared between the two cell lines using a Sulforhodamine B assay allowing the determination of the half-maximal inhibitory concentration (IC50). Cellular accumulation and retention of [64Cu]CuCl2 and non-radioactive cisplatin (10 μM) were compared between the two cell lines at 10, 30, 60 and 120 minutes via gamma counting and ICP-MS, respectively. PET/CT imaging of xenograft-bearing (WT or CisR) female Balb/c nu/nu mice intravenously injected with acetate-buffered [64Cu]CuCl2 (1.5-4 MBq) was used to compare 64Cu accumulation between the two types of xenograft in vivo, dynamically for 2 hours post-injection (p.i.) and again at 24 hours p.i.
The IC50 value for WT cells was 2.2(±0.55) μM, compared to 10.3(±1.44) μM for the CisR cell line (n=3, p<0.001). 64Cu accumulation was significantly lower in CisR cells in vitro compared to WT (1.2±0.4 and 4.1±0.3 Bq/10^3 cells at 120 minutes, respectively)(n=3, p<0.001). The same was true for cisplatin accumulation (0.105±0.022 vs. 0.299±0.067 ng Pt/10^3 cells at 120 minutes)(n=3, p=0.009). 64Cu retention and that of platinum were comparable between the 2 cell lines at the time points tested. PET/CT images showed that the difference in 64Cu accumulation between CisR and WT xenografts was not statistically significant over the first 2 hours p.i. (3.3±1.3 and 1.6±0.5 %ID/g respectively at 120 minutes p.i.)(n=3, p=0.107). At 24 hours p.i., 64Cu accumulation was similar between WT and CisR xenografts (2.9±1.6 and 2.9±0.5 %ID/g, respectively). Ex vivo biodistribution at 24 hours confirmed imaging results (3±1.2 vs. 2.9±0.9 %ID/g for WT and CisR, respectively).
64Cu accumulation correlates well with cisplatin accumulation and sensitivity/resistance in vitro in A2780 cells. However, A2780 WT and CisR xenografts in mice did not differ in their 64Cu uptake. This highlights the limitations of in vitro experimentation as a predictor of in vivo behaviour of molecules, due to an inherent inability to take into account key factors influencing such behaviour, particularly pharmacokinetics and metabolism.
 Ishida S, Lee J, Thiele DJ, Herskowitz I. Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals. Proc Natl Acad Sci. 2002. doi:10.1073/pnas.162491399
 Holzer AK. The Copper Influx Transporter Human Copper Transport Protein 1 Regulates the Uptake of Cisplatin in Human Ovarian Carcinoma Cells. Mol Pharmacol. 2004. doi:10.1124/mol.104.001198
 Larson CA, Blair BG, Safaei R, Howell SB. The Role of the Mammalian Copper Transporter 1 in the Cellular Accumulation of Platinum-Based Drugs. Mol Pharmacol. 2008. doi:10.1124/mol.108.052381
 Kim ES, Tang XM, Peterson DR, et al. Copper transporter CTR1 expression and tissue platinum concentration in non-small cell lung cancer. Lung Cancer. 2014. doi:10.1016/j.lungcan.2014.04.005
 Ozols RF, Masuda H, Grotzinger KR, et al. Characterization of a cis-Diamminedichloroplatinum(II)-resistant Human Ovarian Cancer Cell Line and its use in Evaluation of Platinum Analogues. Cancer Res. 1987.
In vitro Results
A) Normalised survival % of WT and CisR cells as a function of log(cisplatin concentration) used. These curves were used to calculate IC50 values for the 2 cell lines B) Cu64 accumulation in WT and CisR cells over 120 minutes, represented as Bq/cell (n=3) C) Platinum accumulation in WT and CisR cells over 120 minutes, shown as ng platinum/10^3 cells (n=3) D) Cu64 retention % in WT and CisR cells over 120 minutes E) Platinum retention % in WT and CisR cells over 120 minutes.
In vivo results
A) Represantative PET/CT images showing 64Cu accumulation in WT and CisR xenografts at 10, 30, 60, 90 and 120 minutes. B) Imaging data represented as %ID/g of 64Cu in WT and CisR xenografts over 2 hours and at 24 hours p.i. (n=3). C) Ex vivo biodistribution at 24 hours showing the %ID/g in the two types of xenograft (n=5).
Keywords: Cu-64, cisplatin, PET, resistance, ovarian cancer