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Abstract/Video opens by clicking at the talk title.
EpCAM-targeting DARPins for photodynamic therapy of ovarian cancer (#288)
Sanne A. M. van Lith1, Dirk van den Brand2, 3, Jelske M. de Jong2, Mark A. J. Gorris4, Valentina Palacio-Castañeda2, Stijn Couwenberg2, Mark Goldman2, Inge Ebisch5, Leon F. Massuger3, William P. J. Leenders2, Roland Brock2, Wouter P. R. Verdurmen2
1 Radboudumc, Radiology and Nuclear Medicine, Nijmegen, Netherlands
Ovarian cancer is the most lethal gynecological malignancy. Current treatment of advanced stage of disease is cytoreductive surgery with chemotherapy, however relapse often occurs due to remaining (micro)metastases in the abdominal cavity. Since over 90% of the metastatic lesions express epithelial cell adhesion molecule (EpCAM), treatment with EpCAM-targeted photodynamic therapy (PDT) is an interesting treatment option. Here, we evaluated the potency of EpCAM-targeting designed ankyrin repeat proteins (DARPins) conjugated to a photosensitizer for eradication of EpCAM positive cancer cells.
DARPins with either picomolar (Ec1) or nanomolar (Ac2) affinity for EpCAM, and a negative control DARPin (Off7) were functionalized site-specifically with 1) fluorophores for microscopy and flow cytometry 2) the photosensitizer IRDye700DX for PDT. Binding of DARPins to the EpCAM-expressing ovarian carcinoma cell line OV90 was determined with flow cytometry. Furthermore, penetration into OV90 spheroids and binding to primary patient samples of ovarian cancer metastases was assessed with confocal microscopy. The cell death induced by DARPins-IRDye700DX upon illumination was determined in OV90 adherent cell cultures and spheroids, and in co-cultures of OV90 cells with C5120 fibroblasts. Finally, tumor homing of the DARPins-IRDye700DX was assessed in mice bearing subcutaneous OV90 xenografts.
Ec1 and Ac2 showed concentration dependent binding to EpCAM-expressing OV90 cells, with a stronger binding for Ec1 compared to Ac2, and no binding of negative control Off7. Furthermore, EpCAM-specific binding to cells in primary patient samples of ovarian cancer metastases was found (Figure 1A). Both Ec1 and Ac1 penetrated into OV90 spheroids within 2.5 hours, while no penetration of Off7 into the spheroid was observed (Figure 1B). Efficient cell death of OV90 adherent cultures was induced by Ec1 (EC50: 23.06 nM) and Ac2 (EC50: 14.73 nM), and not by Off7, upon illumination with 60 J/cm2 690 nm light (Figure 2A). In OV90 spheroids, Ec1 and Ac2 decreased cell viability to 20.1% and 20.4%, respectively, while Off7 decreased cell viability to 65.8% (Figure 2B). Furthermore, cell death of only tumor cells was induced by Ec1 in a 3D co-culture of tumor cells and fibroblasts upon illumination. At last, both Ec1 and Ac2, and not Off7, effectively homed to OV90 subcutaneous xenografts in mice.
From this work we conclude that DARPin-IRDye700DX conjugates efficiently induce EpCAM-specific cell death upon illumination in vitro. Furthermore, they are able to penetrate into tissue and bind to target expressing cells both in vitro and in vivo, indicating the potential use of these molecules for treatment of metastasized ovarian cancer.
Figure 1. EpCAM-specific binding of DARPin-AlexaFluor680 conjugates
A) 300 µM tissue slices from ovarian cancer metastases were incubated for 4h with 200 nM DARPin-AlexaFluor680 conjugates in culture medium. Subsequently cytokeratin and EpCAM were visualized with immunohistochemistry. Composite and separate channels are shown. All signal derived from DARPins colocalized with EpCAM. Ec1 showed the most effective binding, while for Ac2 less and for Off7 no binding was observed. B) OV90 spheroids were incubated for 2.5 h with 500 nM DARPin-AlexaFluor680 conjugates and imaged with confocal miscroscopy.
Figure 2. EpCAM-specific cell death induced by DARPin-IRDye700DX conjugates upon illumination
A) OV90 adherent cells were incubated for 2h with varying concentrations of DARPin-IRDye700DX conjugates. Subsequently they were washed and illuminated with 60 J/cm2 690 nm light, and cell viability was determined with a resazurin assay after overnight incubation. B) Spheroids from OV90 cells were incubated with 500 nM of the DARPin-IRDye700DX conjugates. They were washed and illuminated with 60 J/cm2 690 nm light. Cell viability was assessed with the APH assay after overnight incubation. Mean and s.e.m. are shown (n=3; *** = p < 0.001)
Keywords: photodynamic therapy, DARPins, EpCAM, Ovarian cancer
Riboflavin-targeted peptostars as potential theranostic cancer agents (#241)
Milita Darguzyte1, Regina Holm2, Natascha I. Drude1, Jasmin Baier1, Jennifer Schultze3, Kaloian Koynov3, Seyed M. Dadfar1, Twan Lammers1, Matthias Barz2, Fabian Kiessling1, 4
1 University Hospital RWTH Aachen, Institute for Experimental Molecular Imaging, Aachen, Germany
Particles in the size range of antibodies can provide long circulation times, passive tumor accumulation and efficient tumor penetration. Active targeting can further increase their retention and internalization. Peptostars are star-like polymers consisting of polylysine and polysarcosine. They can be synthesized with a precise size control. Peptostar core and arms can be modified with various ligands i.e. riboflavin (RF), whose transporters have been shown to be overexpressed in certain tumors1-4. Therefore, we investigate antibody sized RF-targeted peptostars as ideal drug delivery systems.
Peptostars were synthesized as described previously5. The end groups were modified with RF or acetylated and the core was labeled with Cy5.5. The polymers were characterized using UV-Vis spectroscopy and fluorescence correlation spectroscopy (FCS). The biocompatibility was checked using XTT assay on mouse fibroblasts (NIH). In vitro uptake experiments were done on squamous cell carcinoma (A431) and prostate cancer (PC3) cells using flow cytometry. In vivo, A431 tumor xenografts-bearing BALB/c nude mice were intravenously injected with peptostars and sacrificed after 30 min. The organs of interest and tumors were dissected and imaged using 2D Fluorescence Reflectance Imaging. Histological analysis of accumulated vs. internalized polymers in tumors was done using fluorescence microscopy.
Peptostars labeled with dye showed characteristic absorbance peaks at 633 and 685 nm, while RF functionalized ones had additional peaks at 260 and 460 nm (Fig. 1A). Control peptostars had a 3.5 nm hydrodynamic radius, while targeted had 4.0 nm according to FCS. No toxic effects were observed on NIH for polymer concentrations up to 50 nmol (Fig. 1B). Targeted peptostars had superior uptake in comparison to control ones in both cell lines (Fig. 1C) and competitive blocking experiments further confirmed that targeted peptostars are taken up via the transporter-mediated pathway. Ex vivo, the highest fluorescence signal was seen in the liver followed by the kidney (Fig. 1D). The polymers showed a moderate tumor accumulation, and no difference between targeted and control peptostars was seen (Fig. 1E). Histological analysis indicated that targeted peptostars were internalized significantly more than non-targeted ones (Fig. 1F). This points to a successful tumor cell targeting of our systems.
We conclude that passive accumulation dominates the biodistribution of peptostars, while tumor cell internalization is strongly promoted by RF-targeting. However, the tumor accumulation of our system is not yet competitive to other drug delivery systems with similar size. Thus, further optimization will focus on refinement of the passive targeting capabilities and incorporation of a therapeutic drug to have a theranostic system.
This research was supported by the Deutsche Forschungsgemeinschaft (DFG) in the framework of the Research Training Group 2375 “Tumor-targeted Drug Delivery” grant 331065168, DFG FOR 2591 to FK Grant No. 321137804 and CRC 106.
 Bartmann, L.; Schumacher, D.; Von Stillfried, S.; Sternkopf, M.; Alampour-Rajabi, S.; van Zandvoort, M.; Kiessling, F.; Wu, Z., 2019, ' Evaluation of Riboflavin Transporters as Targets for Drug Delivery and Theranostics.' Front. Pharmacol., 10, Place of publication: Frontiers.
 Long, L.; Pang, X.-X.; Lei, F.; Zhang, J.-S.; Wang, W.; Liao, L.-D.; Xu, X.-E.; He, J.-Z.; Wu, J.-Y.; Wu, Z.-Y.; et al., 2018, 'SLC52A3 Expression Is Activated by NF-ΚB P65/Rel-B and Serves as a Prognostic Biomarker in Esophageal Cancer.' Cellular and Molecular Life Sciences, 75 (14), 2643–2661, Place of publication: Springer.
 Fu, T.; Liu, Y.; Wang, Q.; Sun, Z.; Di, H.; Fan, W.; Liu, M.; Wang, J., 2016, 'Overexpression of Riboflavin Transporter 2 Contributes toward Progression and Invasion of glioma.' Neuroreport , 27 (15), 1167–1173, Place of publication: Lippincott Williams & Wilkins.
 Jiang, X.-R.; Yu, X.-Y.; Fan, J.-H.; Guo, L.; Zhu, C.; Jiang, W.; Lu, S.-H., 2014, 'RFT2 Is Overexpressed in Esophageal Squamous Cell Carcinoma and Promotes Tumorigenesis by Sustaining Cell Proliferation and Protecting against Cell Death.', Cancer Lett., 353 (1), 78–86, Place of publication: Elsevier.
 Holm, R.; Weber, B.; Heller, P.; Klinker, K.; Westmeier, D.; Docter, D.; Stauber, R. H.; Barz, M., 2017, 'Synthesis and Characterization of Stimuli-Responsive Star-Like Polypept(o)Ides: Introducing Biodegradable PeptoStars.' Macromol Biosci, 17 (6), Place of publication: Wiley.
Keywords: polypept(o)ide stars, Riboflavin, theranostics, nanomedicines, targeted drug delivery
Bleomycin plus ultrasound and microbubbles to treat feline oral squamous cell carcinoma, as a model for human head and neck cancer, preliminary results of the BUBBLEFISH Trial (#264)
Josanne S. de Maar1, Maurice M. J. M. Zandvliet2, Stefanie Veraa2, Mauricio Tobón Restrepo2, Chrit T. W. Moonen1, Roel Deckers1
1 University Medical Center Utrecht, Utrecht University, Imaging Division, Utrecht, Netherlands
Most Head and Neck Squamous Cell Carcinoma patients(~60%) present with locally advanced disease, when primary surgery is seldom an option. Despite combination treatment, around half develops (often incurable) local recurrences. Improved local therapy is needed. Sonopermeation with ultrasound and microbubbles (USMB) can increase local efficacy of several drugs, particularly bleomycin[2,3]. Studying Feline Oral Squamous Cell Carcinoma (FOSCC) can bridge between preclinical and clinical research and these cats (with a life expectancy of only ±1 month) could benefit from a low-burden treatment.
A single-arm prospective study in cats with spontaneously arisen FOSCC without treatment options except for palliative care is currently ongoing. During general anaesthesia, the cats receive intravenous bleomycin (10.000 IU/m2) combined with USMB treatment of the oral tumour using intravenous injections of SonoVue (Bracco, conc. 1-5x108 bubbles/mL). USMB treatment is performed with an EPIQ5 (Philips) in Pulsed Wave doppler mode and US parameters were optimized during the treatment of the first two cats. Each cat is treated three times, once per week. Feasibility, adverse events and quality of life are monitored. Tumour response is evaluated by clinical (caliper) and ultrasound measurement of the tumour.
So far two feline patients, respectively with sublingual and right maxillary SCC, have been treated.
After treating two feline patients, bleomycin combined with USMB on a clinical US system using EMA/FDA approved microbubbles seems a feasible palliative treatment for FOSCC patients. Preliminary results suggest that tumour perfusion increases after sonopermeation at MI levels corresponding to stable cavitation. Eventually the obtained safety and efficacy results should facilitate the translation of USMB treatment into the clinic.
AcknowledgmentWe thank the patient owners for participating in this study with their cat.
 Lammertink BHA, Bos C, Deckers R, Storm G, Moonen CTW, Escoffre J-M, 2015 ‘Sonochemotherapy: from bench to bedside’,Frontiers in Pharmacology, 6(138): 1-17.
 Iwanaga K, Tominaga K, Yamamoto K, Habu M, Maeda H, Akifusa S, Tsujisawa T, Okinaga T, Fukuda J, Nishihara T, 2007, ‘Local delivery system of cytotoxic agents to tumors by focused sonoporation’, Cancer Gene Therapy, 14: 354-363.
 Lamanauskas N, Novell A, Escoffre J-M, Venslauskas M, Šatkauskas S, Bouakaz A, 2013, ‘Bleomycin delivery into cancer cells in vitro with ultrasound and SonoVue® or BR14® microbubbles’, Journal of Drug Targeting, 21(4): 407-414.
Keywords: Sonopermeation, Contrast enhanced ultrasound, USMB, head and neck cancer, feline oral squamous cell carcinoma
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
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
Porphyrin Metalla-Assemblies in Cellulose Nanocrystals, an Innovative Theranostic Agent (#147)
João C. Simões1, 2, Sophia Sarpaki1, Panagiotis Papadimitroulas1, Bruno Therrien2, George Loudos1
1 BioEmission Technology Solutions, Research & Development, Athens, Greece
There is an urgent need of new drugs and practices to treat cancer. Photodynamic therapy (PDT) imposes itself as one of the most preponderant voices to answer this demand. With the combination of a photosensitizer, light and oxygen, PDT achieves a unique selectivity by the production of localized reactive oxygen species inside tumor cells, which leads to their destruction with limited side effects.1 Our study aims to use cellulose nanocrystals to transport and deliver radiolabeled photo-responsive molecules to biological targets and create a new generation of theranostic agents.
In order to achieve our goal of using these cellulose-metal-porphyrin assemblies and to trace them using SPECT imaging, several steps have been taken so far. First, we have prepared and characterized CNCs2 as well as photo-responsive molecules through stoichiometric quantities of aldehydes and pyrrole in propionic acid under reflux for 1h.3
The aniline and phenol derivatives of 5,10,15,20-Tetra(4-pyridyl)-21H,23H-porphine were chosen as photo-responsive molecules, due to their ability to simultaneously be linked to the cellulose nanocrystals (CNC) and coordinated with the Ru complexes to make a metalla-assembly. Synthesis and coordination have been carried out efficiently.
Our preliminary results are very promising regarding the synthesis of a multimodal agent that combines a photosensitizer for PDT treatment, and a radioactive agent for imaging (before, during and after the process), thus aiming to develop an innovative drug with exciting theranostic properties. Their conjugation with cellulose nanocrystals and coordination to inorganic entities intend to ameliorate their delivery, solubility and metabolism.
This project has received financial support from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement n°764837.
 Robertson, C. A.; Evans, D. H.; Abrahamse, H., Photodynamic therapy (PDT): a short review on cellular mechanisms and cancer research applications for PDT. Journal of Photochemistry and Photobiology B: Biology 2009, 96 (1), 1-8.
 Chauhan, P.; Hadad, C.; Sartorelli, A.; Zarattini, M.; Herreros-Lopez, A.; Mba, M.; Maggini, M.; Prato, M.; Carofiglio, T., Nanocrystalline cellulose-porphyrin hybrids: synthesis, supramolecular properties, and singlet-oxygen production. Chem Commun (Camb) 2013, 49 (76), 8525-8527.
 Adler, A. D.; Longo, F. R.; Finarelli, J. D.; Goldmacher, J.; Assour, J.; Korsakoff, L., A simplified synthesis for meso-tetraphenylporphine. The Journal of Organic Chemistry 1967, 32 (2), 476-476.
 Mannancherril, V.; Therrien, B., Strategies toward the Enhanced Permeability and Retention Effect by Increasing the Molecular Weight of Arene Ruthenium Metallaassemblies. Inorg Chem 2018, 57 (7), 3626-3633.
Keywords: photosensitizer, photodynamic therapy, theranostic, imaging
Fluorescence Molecular Endoscopy (FME) using bevacizumab-800CW to evaluate response to neoadjuvant chemoradiotherapy in esophageal cancer: preliminary results (#217)
Iris Schmidt1, Xiaojuan Zhao1, Gursah Kats-Ugurlu2, Anne M. van der Waaij1, Ruben Y. Gabriels1, Jouke J. H. van der Laan1, Jan Willem Haveman3, Boudewijn van Etten3, Frederieke A. Dijkstra3, Dominic Robinson4, Wouter B. Nagengast1
1 University Medical Center Groningen, Department of Gastroenterology and hepatology, Groningen, Netherlands
Currently, patients diagnosed with locally advanced esophageal adenocarcinoma receive neoadjuvant chemoradiotherapy (nCRT) followed by esophagectomy independent of the patients’ response to nCRT. Roughly 16-43% of esophageal cancer patients will achieve a pathological complete response (pCR) after nCRT1. Current imaging devices (MRI, CT, PET) cannot adequately distinguish pCR from pathological partial response (pPR) before esophagectomy2. There is a need for a reliable pre-operative method which can precisely distinguish pCR from pPR and prevent (possibly) unnecessary surgery.
This study aims to determine the safety and feasibility of fluorescence molecular endoscopy (FME) using bevacizumab-800CW for identification of pCR after nCRT in patients with diagnosed with esophageal adenocarcinoma. Patients were intravenously injected with either 4.5 (n=5), 10 (n=3) or 25 mg (n=3) bevacizumab-800CW 2-3 days prior to the endoscopy. FME was performed to monitor the effect of the nCRT. Multi-diameter single fiber spectroscopy / single fiber fluorescence (MDSFR/SFF) spectroscopy was used to quantify the intrinsic fluorescence intensity, both in-vivo and ex vivo. Afterwards, biopsies were taken from normal and (residual) tumor area. Subsequently, widefield back-table imaging was performed on the whole surgical specimen directly after excision.
In this ongoing trial, patients diagnosed with locally advanced esophageal adenocarcinoma (cT1b-4a N0-3 M0) scheduled for neoadjuvant chemoradiotherapy (CROSS) followed by surgery were enrolled between November 2018 and November 2019. No tracer related (serious) adverse events were observed. The preliminary results show uptake of fluorescence in both normal ant tumor tissue, resulting in a mean tumor-to-background ratio (TBR) with spectroscopy of 1.28 in the 4.5 mg group. Optimization of the tracer dosage could increase the TBR, as previous studies showed higher target-to-background ratios after increasing the dosage3,4. Moreover, considering the high percentage of complete responders in the 10 mg group, the two groups of 10 mg and 25 mg will be extended to 6 patients each. In the future, a reliable restaging modality can be used to create a method of active surveillance as a clinical treatment option.
First results show that Bevacizumab-800CW is safe for administration in patients with locally advanced esophageal adenocarcinoma. The current dose escalation study will be extended to find the most optimal dose for treatment monitoring.
 Duan X-F, Tang P, Yu Z-T. Neoadjuvant chemoradiotherapy for resectable esophageal cancer: an in-depth study of randomized controlled trials and literature review. Cancer Biol Med. 2014;11(3):191–20
 De Gouw D, Klarenbeek BR, Driessen M, Bouwense SAW, van Workum F, Fütterer JJ, et al. Detecting pathological complete response in esophageal cancer after neoadjuvant therapy based on imaging techniques: a diagnostic systematic review and metaanalysis. J Thorac Oncol. 2019
 Koller, M., Qiu, S. Q., Linssen, M. D., Jansen, L., Kelder, W., de Vries, J., ... & Jorritsma-Smit, A. (2018). Implementation and benchmarking of a novel analytical framework to clinically evaluate tumor-specific fluorescent tracers. Nature communications, 9(1), 3739.
 Hartmans, E., Tjalma, J. J., Linssen, M. D., Allende, P. B. G., Koller, M., & Jorritsma-Smit, A. (2018). Potential red-flag identification of colorectal adenomas with wide-field fluorescence molecular endoscopy. Theranostics, 8(6), 1458.
Keywords: Fluorescence, endoscopy, esophageal cancer
Study of the effects of a combination therapy in a subcutaneous mouse models of colorectal cancer (CRC) with ultrasound and photoacoustic imaging (#460)
Tiziano Lottini1, Matteo Stefanini1, Jessica Iorio1, Annarosa Arcangeli1
1 University of Florence, Department of Experimental and Clinical Medicine, Florence, Italy
Neoangiogenesis promotes progression and influence the distribution of drugs. The combination of Bevacizumab (Bv) with drugs that modulate the activity of hERG1 potassium channel, enhances the effect of Bv in gastric cancer1. Moreover, the expression of hERG1 is correlated with an angiogenetic pathway CRC. Blocking hERG1 switches this pathway off also in vivo2. Our objective is to study the effects of Ril and Bv co-treatment in a CRC mouse models by using Ultrasound (US) and Photoacoustic (PA) imaging.
Two CRC xenograft models were produced on athymic mice: a) generated by a subcutaneous (s.c.) inoculation of 3x106 of CRC cells (Hct-116 wt); b) produced by inoculation of Hct-116-Bev/A cells (resistant to Bv effects)4. Mice were divided into the following groups of treatment: i): control saline; ii): BV; iii): Ril; iv): co-treatment Ril and BV. We used US and PA imaging techniques to monitor the changes of tumor size and oxygenation (% sO2) following the different treatments.
In the CRC model derived from Hct-116 WT injection, the combination of Ril and BV produced a slight reduction of tumor volume and a reduction of average % sO2 , which was also triggered, although at lower levels, by Bv alone. In the CRC model that is resistant to Bv no significant difference in tumor growth emerged among the 4 groups of treatment. On the contrary, we observed a significant reduction of oxygen saturation in the masses of the group treated with a combination of Bv and Ril compared to the other three groups of treatment.
Overall, the use of US and PA imaging can allow to study the development of disease and the evaluation of the effect of treatment. In particular is possible to the determine the antiangiogenic activity of Ril in vivo, which can overcome BV resistance.
AcknowledgmentThanks to Visualsonics team group fot the training of VevoLAZR-X imaging System
 Crociani O et al. hERG1 channels modulate integrin signaling to trigger angiogenesis and tumor progression in colorectal cancer. 2013, Sci Rep 3: 3308
 Crociani O et al. hERG1 channels regulate VEGF-A secretion in human gastric cancer: clinicopathological correlations and therapeutical implications. 2014, Clin Cancer Res 20(6): 1502-12
 Fan F et al. Chronic exposure of colorectal cancer cells to bevacizumab promotes compensatory pathways that mediate tumour cell migration. 2011, British Journal of Cancer; 104, 1270 – 1277.
Keywords: VevoLazr-X, Colorectal cancer, Orthotopic mouse model
Light Sheet Fluorescence Microscopy to visualize new treatment outcome in patient-derived Glioblastoma spheroids (#596)
Mariam-Eleni Oraiopoulou1, 2, Stylianos Psycharakis2, Eleftheria Tzamali1, Eleftheria Parasiraki3, 4, Giorgos Tzedakis1, Antonis F. Vakis5, 6, Vangelis Sakkalis1, Joseph Papamatheakis3, 4, Giannis Zacharakis2
1 Foundation for Research and Technology - Hellas, Institute of Computer Science, Heraklion, Greece
Glioblastoma (GB) adjuvant chemotherapy includes Temozolomide (TMZ); yet, not all GB patients are responsive . The latest trends in GB (pre-)clinical trials [2-3] usually refer to Doxorubicin (DOX) with a low effective dose. Much effort is given towards DOX to adequately overpass the blood brain barrier  with no adverse reaction. Here, a range of TMZ and DOX doses was used to treat patient-derived GB spheroids, as either monotherapy or in combination. We present a drug-screening tool based on Light Sheet Fluorescence Microscopy (LSFM) in order to test a new combination GB treatment.
GB tissue was collected during the patient’s brain biopsy and subsequently used to establish a primary GB cell line. Spheroids were generated and treated with TMZ-only, DOX-only and cocktail (TMZ+DOX) from day 4 to day 7 (as in ), based on the IC50 values previously estimated in 2D. Optical Microscopy was used to monitor the growth pattern for up to 23 days and LSFM was used to visualize the drug penetration (ex488nm-em605/70nm, DOX autofluorescence) and cell death (Draq7, ex635nm-em650nmLP) in multiple time points. Multiple projections LSFM scans were performed to obtain for multispectral high-resolution 3D imaging and optical sectioning. Diametrical projections were stacked and registered, and finally, combined together in order to achieve the isotropic resolution in all directions.
Comparing the effective dose regime of the two antineoplastic agents in the primary GB spheroids, we observed that DOX appeared to be very effective in less concentrated doses; even in four orders of magnitude less than TMZ. On the other hand, in TMZ-treated spheroids growth-inhibiting effects were observed in a non-consistent dose-response relationship. According to the fluorescent images, DOX had a low elimination rate over time and was able to accumulatively cause necrosis. There was no differential pattern of the TMZ-induced cell death compared to the control spheroids. The differential growth inhibition pattern among the monotherapy-treated spheroids (as depicted in Figure 1), indicates either cell proliferation arrest and/or cell death. Driven by our observations together with the reported mechanisms of action [2, 5], we showed that the therapeutic effect for the cocktail-treated spheroids can be enhanced over time as compared to either monotherapy effect, even of a higher dose.
Our results demonstrate a time-efficient and reproducible preclinical drug-screening tool to assess the distribution and cytotoxic potency in primary 3D cell cultures treated with either monotherapy or combination therapeutic scheme, and the beneficial effect of the latter. By further integrating our results to GB-specific mathematical models, we expect to further predict the optimal TMZ-DOX combination for the purposes of precision medicine.
Authors would like to thank Evangelos Liapis and Maria Tampakaki for all the help they provided, Elias Drakos for his collaboration, and Katerina Manolitsi for her advisory comments, as well as Despina Tsoukatou and Venediktos Makatounakis for the expert technical assistance. This work was supported by the NSRF 2014-2020 projects “BIOIMAGING-GR” (MIS5002755) and KRIPIS II-VITAD (MIS 5002469). Mariam-Eleni Oraiopoulou wishes to acknowledge support from the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under the HFRI PhD Fellowship grant (GA. no. 130178/I2/31-7-2017).
 Lee, S.Y., Temozolomide resistance in glioblastoma multiforme. Genes & Diseases, 2016. 3(3): p. 198-210.
 Villodre, E.S., et al., Low Dose of Doxorubicin Potentiates the Effect of Temozolomide in Glioblastoma Cells. Mol Neurobiol, 2018. 55(5): p. 4185-4194.
 Jiang, P., et al., Novel anti-glioblastoma agents and therapeutic combinations identified from a collection of FDA approved drugs. J Transl Med, 2014. 12: p. 13.
 Tzedakis, G., et al. A hybrid discrete-continuous model of in vitro spheroid tumor growth and drug response. in Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS. 2016.
 Thomas, R.P., L. Recht, and S. Nagpal, Advances in the management of glioblastoma: the role of temozolomide and MGMT testing. Clinical Pharmacology : Advances and Applications, 2013. 5: p. 1-9.
Keywords: Glioblastoma, Light Sheet Fluorescence Microscopy, Precision Medicine, Doxorubicin, Preclinical trials