Download Template Activity Report_Postdoctoral Fellowship

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Postdoctoral Fellowship
Activity Report
 PERSONAL DETAILS
Name
Lentacker
First name
Fellowship start and end
date
Sabine
1/10/2013-25/02/2017 (prolonged due to parental leave)
Title of the research
project
Development and evaluation of theranostic microbubbles
and nanoparticles for cancer immunotherapy.
UGent
Prof. Stefaan De Smedt
Med1
Host Institution
Supervisor(s)
FWO Expertpanel
 ADMINISTRATIVE DATA
Diplomas or distinctions
obtained during the past
academic year
/
International mobility
Give an overview of
research visits abroad
(subject, duration,
outcome, …). Mention
other funding sources
where applicable
Percentage of
employment at FWO
Mention percentage of
employment elsewhere if
applicable, and provide
details on employer and
starting date of the
contract
Career breaks
If you have interrupted
your fellowship for
whatever reason during
the past year, you may
provide details about this
(optional)
/
100% till 30/9/2016
60% till 1/10/2016 combined with 40% ATP-function at UGent
Parental leave – 20% (from 1/10/2013 till july 2016)
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Activity Report
 SCIENTIFIC RESULTS
Description of the research performed (ca. 1500 words)
 Describe the research that you have conducted in the framework of your research project, and
the results obtained
 Include details about stays abroad (where applicable)
As I am mainly involved in project writing, reporting and guiding PhD students and early post-docs, I
refer to the specific projects that I supervise and that are dealing with the evaluation of theranostic
microbubbles and nanoparticles for cancer immunotherapy.
mRNA loaded microbubbles for ultrasound triggered cancer immunotherapy (project of dr. Heleen
Dewitte)
Earlier we developed mRNA loaded microbubbles and demonstrated that they can be used as an
efficient ultrasound-triggered transfection tool for DCs, without compromising DC viability or
maturation capacities. Moreover we proved that DCs that were sonoporated with antigen mRNA via
mRNA loaded microbubbles and ultrasound can induce potent antigen-specific immune responses in
vivo. Sonoporation with TriMix, to further modulate the DC’s antigen-presenting functionality, could
be used to further augment immunity. Especially in a therapeutic setting, vaccination with antigen
and TriMix mRNA sonoporated DCs resulted in a significant reduction in tumor growth, leading to a
marked increase in overall survival and long-lasting antigen-specific protection against tumor
recurrence . These findings are especially important in the context of a possible in vivo use of this
technique. Needless to say that direct in vivo transfection of DCs would not only eliminate the need
for costly ex vivo DC handling and transfection, but it could also allow transfection of several DC
subsets which would broaden the generated immune response.
Although the results described above were very promising, they still made use of ex vivo transfected
DCs which were injected in mice. A second milestone is to evaluate the in vivo transfection of
mRNA-lipoplex loaded microbubbles in the lymphatics. We therefore already evaluated, in
collaboration with Prof. Vanderperren (Faculty of Veterinary Sciences, Ghent University) the
lymphatic drainage of mRNA loaded microbubbles in dogs. These experiments revealed that it is
indeed possible to use the microbubbles for the delivery of antigen encoding mRNA in the lymph
nodes of these animals. A second question we tried to answer this year is whether we are able to
induce mRNA transfection in the lymph nodes. As acoustic settings are a crucial factor to induce
sonoprinting (De Cock et al. Biomaterials 2016) we evaluated transfection efficiencies with different
acoustic settings from a clinical imaging system (Philips iU22) or therapeutic ultrasound probe
(Sonitron 2000). In these experiments freshly euthanized pigs were injected intranodally with mRNA
loaded microbubbles and subsequently exposed to ultrasound. Lymph nodes were excised and
luciferase expression was measured using the IVIS in vivo imaging system after intranodal injection
of luciferin substrate.
Our results clearly indicated that higher pressures (>1MPa) and longer cycles are required to induce
sonoprinting in vivo. While the sonitron 2000 (2000 cycles at 1,5MPa) gave a really high intranodal
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expression efficiency, surprisingly the burst setting and power Doppler setting of the clinical
ultrasound imaging system were not able to induce any intranodal transfection. It is also evident
that mRNA delivery is indeed inducible by ultrasound as the injection of mRNA loaded microbubbles
without ultrasound exposure did not result in any transfection efficiency (Figure 1). Follow-up
experiments are planned where we will evaluate in which intranodal cells the mRNA becomes
expressed and where we will evaluate transfection efficiency after intradermal injection in the pigs.
Figure 1. Luciferase expression in excised pig lymph nodes after intranodal injection of mRNA loaded
microbubbles (25µg mRNA) and exposure to “burst function” of Philips IU22 (ultrasound settings
unknown) (A), exposure to sonitron transducer (1.5MPa, 2000 cycles, 1 minute exposure) (B) or
unexposed to ultrasound (C).
HIFU (high intensity focused ultrasound) induced immunotherapy (research project of dr. Heleen
Dewitte).
In collaboration with Prof. Moonen (Utrecht university) we want to explore HIFU, a non-invasive
cancer ablation technique, to induce immunogenic cell death in tumors with the aim to develop a
generalized immune response. HIFU is a non-invasive technique that has been used in the clinic to
ablate tumors (breast, prostate, bone). In these experiments B16F10 cells were exposed to
increasing acoustic pressures and subsequently analyzed at different time points after HIFU
exposure for ICD markers (ATP release, calreticulin expression). We were able to detect a CRT
exposure in a small fraction of cells (Figure 2). Unfortunately the Utrecht set-up only allows to treat
suspension cells in plastic tubes, which makes it impossible to get a good control over ultrasound
settings and cell exposure time. As explained further on (incoming research visit of dr. Guillaume
Lajoinie), we have installed a new set-up where we can treat adherent and suspension cells in
custom-made acoustically transparent cell culture chambers that are currently being optimized.
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Figure 2. Calreticulin expression in B16F0 cells 6 hours after HIFU exposure.
Design of mRNA and adjuvant nanoparticles for in vivo immunotherapy (research project of Rein
Verbeke, dr. Heleen Dewitte)
In this project we designed mRNA encapsulating, lipid-based nanoparticles which are able to
migrate to the lymph nodes upon intravenous injection, this with the final aim to reach dendritic
cells residing in the lymph nodes and induce an in situ immune response. This strategy would allow
to induce immunotherapy without the need for an expensive and time consuming ex vivo procedure
where monocytes are taken from the patient’s blood and cultured ex vitro to obtain dendritic cells.
In this project and optimized optimized lipid nanoparticle was developed containing DOTAP (cationic
lipid) and cholesterol. Within these nanoparticles we are able to co-encapsulate antigen encoding
mRNA and adjuvants which can assure that mature dendritic cells are able to initiate an effective
immune response. In earlier experiments we showed that nanoparticles containing antigen
encoding mRNA and MPLA as an adjuvant remain stable in serum containing medium and are able
to transfect and mature dendritic cells in vitro.
Recent experiments were performed to study distribution and transfection efficiency of the
nanoparticles in BL57/6 mice. In these experiments mice were injected with three different type of
luciferase mRNA containing and DIR labeled nanoparticles (non-modified mRNA, pseudo-uridine
modified mRNA and pseudo-uridine modified mRNA and MPLA). Although the biodistribution of the
different nanoparticles was very similar (lungs, spleen, liver and bone marrow), we noticed clear
differences in the expression level of the complexed mRNA. Lipoplexes containing the unmodified
mRNA showed relatively weak transfection efficiencies, while lipoplexes containing pseudouridine
modified mRNA were able to induce very high expression efficiencies, mainly in lungs, spleen and
bone marrow and were taken up by dendritic cells and macrophages in the respective tissues. It was
suggested earlier in literature that innate viral immune responses (type I IFN), provoked by
unmodified mRNA, can prevent mRNA expression (Pollard et al. Biomaterials 2014). It remains
however crucial to activate the immune system as this stimulation is required to acquire effective
immune responses that are able to eliminate the antigen expression cells (e.g. tumor cells). When
we added MPLA adjuvant to the nanoparticles we were indeed able to restore transfection
efficiency without hampering its transfection (Figure 3). Manuscript in preparation. Follow up
experiments are planned to evaluate T-cell activation and functionality in vivo, followed by a
therapeutic vaccination study.
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Figure 3: Fluorescence and bioluminescence images of C57BL/6 mice after tail vein injection of
nanoparticles containing 10µg pseudo-uridine modified mRNA and MPLA.
Incoming research visit of dr. ir. Guillaume Lajoinie (Physics of Fluids, University of Twente).
During the research stay of dr. Lajoinie ( April-July 2016) we installed two different ultrasound setups. The first set-up contains a focused ultrasound transducer and enables us to visualize direct
microbubble cell interactions under very precise acoustic conditions (set-up on spinning disk
confocal microscope) (research project Silke Roovers). The second set-up was developed to enable
HIFU treatment of cancer cells (see project dr. Dewitte). Moreover a new ultrasound transparent
acoustic chamber was developed for both set-ups that minimizes acoustic reflections and allows to
treat adherent and suspension cells with very high precision. These windows can replace the very
expensive ClinicellTM units (35 euro/piece) that were used previously.
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Evaluation of DOX-loaded microbubbles in a spheroid model (research project of Silke Roovers).
Previous in vitro work on cell monolayers has demonstrated that our DOX-liposome loaded
microbubbles are very efficient to locally delivery DOX-liposomes to cells under ultrasound guidance
In collaboration with prof. Versluis (UTwente) we found out with high speed imaging (50.10^3 and
28. 10^6 fps) that liposomes become released from the microbubbles within the first few cycles of
microbubble cavitation and are subsequently dragged (manuscript in preparation) onto the cell
membrane by the translating microbubble gas core. Since these experiments were performed on
single cell layers, we would like to evaluate how deep the released liposomes can be deposited into
tissue. We recently developed an optimized protocol to grow reproducible spheroids containing 4T1
and fibroblasts (Figure 4) and are currently performing toxicity assays (Cell-Titer GLO 3D, Promega).
To enhance interaction between spheroids and DOX-liposome loaded microbubbles we have
designed nanobody coupled DOX-liposome loaded microbubbles (SBO project in collaboration with
Prof. Serge Muyldermans, VUB)to be able to study microbubble-spheroid interactions in real-time.
Figure 4. Confocal Fluorescence images of spheroids containing 4T1 cells (green) only (left) and
4T1 cells and fibroblasts (red).
Interdisciplinarity and ongoing Research Collaborations
- Collaboration with the Biomedical engineering group (Prof. De Jong, Erasmus MC, Rotterdam, The
Netherland) and the Physics of fluids group (Prof. versluis, Twente, The Netherlands). Ongoing
research collaboration on the high-speed imaging of drug release from drug loaded microbubbles at
high frame rates.
- Collaboration with prof. Jai Prakash and dr. Séverine Le Gac (University of Twente) on sferoid
sonoporation in microfluidic devices.
- Collaboration with the Biomedical Nuclear Magnetic Resonance group (Prof. Himmelreich,
KULeuven). Evaluation of PFC nanoparticles for cell tracking of T-cells in diabetes. (FWO research
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project B0B2814N, promotor Prof. himmelreich) and the use of microbubbles as theranostic agents
(IWT SBO project , Promotor Prof. Himmelreich).
- Collaboration with Laboratory of Molecular and Cellular Therapy (Prof. Thielemans and Prof.
Breckpot, VUB) on the use of mRNA nanoparticles and mRNA lipoplex loaded microbubbles for
cancer immunotherapy (FWO research project G016513N, promotor Prof. De Smedt). FWO postdoctoral research project Heleen Dewitte.
- Collaboration with Imaging Department of the faculty of Veterinary Sciences (Prof. Vanderperren,
UGent). Ultrasound contrast imaging with drug-loaded microbubbles.
- Collaboration with Imaging Department of Utrecht University Hospital (Prof. Moonen).
Collaboration on the use of HIFU (High Intensity Focused Ultrasound) as a method to induce
immune responses against tumors.
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List of scientific publications resulting from the fellowship
A1. Papers since start fellowship.
1. Geers B., De Wever O., Demeester J., De Smedt S.C., Lentacker I. Targeted liposome-loaded
microbubbles for cell-specific ultrasound-triggered drug delivery. Small 2013. 9(23); 4027-4035.
Own contribution:, involved in design of experiments and writing of the manuscript (30%).
IF 2014: 7,51. Citations: 13.
2. Moonen C.T., lentacker I. Ultrasound assisted drug delivery: preface. Advanced drug Delivery
Reviews 2014. 72; 1-2.
Own contribution: writing of the preface (50%)
IF 2014: 15. Citations: 2
3. Lentacker I., De Cock I., Deckers R., De Smedt S.C., Moonen C.T. Understanding ultrasound
induced sonoporation: definitions and underlying mechanisms. Advanced drug Delivery Reviews
2014. 72; 49-64.
Own contribution: writing of the manuscript (80%)
IF 2014:15 . Pages: 15. Citations: 63 (highly cited paper web of science).
4. Dewitte H., Verbeke R., Breckpot K., Vandenbroucke R.E., Libert C., De Smedt S.C., Lentacker I.
Choose your models wisely; How different murine bone marrow-derived dendritic cell protocols
influence the success of nanoparticulate vaccines in vitro. Journal of Controlled Release 2014.
Own contribution: involved in the design of the experiments and the writing of the manuscript (30%)
IF 2014: 7.7 .Citations: 3.
5. Dewitte H., Van Lint S., Heirman C., Thielemans K., De Smedt S.C., Breckpot K., Lentacker I. The
potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasoundtriggered cancer immunotherapy. Journal of controlled Release 2014. 194. 28-36.
Own contribution: involved in the design of the experiments and the writing of the manuscript (30%)
IF 2014: 7.7 . Citations:8.
6. Van Lint S., Renmans D., Broos K., Dewitte H., Lentacker I., Heirman C., Breckpot K., Thielemans K.
The ReNAissanCe of mRNA-based cancer therapy. Expert Reviews of Vaccines 2015. 14(2), 235-251.
Own contribution: partially involved in writing of the manuscript (5%)
IF 2043: 4.22. Citations: 9.
7. Dewitte H., Vanderperren K., Haers H., Stock E., Duchateau L, Hesta M., Saunders J.H., De Smedt
S.C, Lentacker I. Theranostic mRNA-loaded microbubbles in the lymphatics of dogs: implications for
drug delivery. Theranostics 2015, 5(1), 97-109.
Own contribution: involved in the design of the experiments and the writing of the manuscript (30%)
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IF 2014: 8. Citations: 5.
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8. Xiong R., Raemdonck K., Peynshaert K., Lentacker I., De Cock I., Demeester J., De Smedt S.C.,
skirtach A.G., Braeckmans K. ACS Nano 2014, 8(6), 6288-6296.
Own contribution: involved in experimental design and writing of the manuscript (5%)
IF 2014: 12.88. Citations: 14.
9. Dewitte H., Verbeke R., Breckpot K., De Smedt S.C., Lentacker I. Nanoparticle design to induce
tumor immunity and ceallenge the suppressive tumor micro environment. Nano Today 2014.
9(6)743-785.
Own contribution: involved in writing of the manuscript (30%).
IF 2014: 13,2. Citations: 10.
10. De Cock I., Zagato E, Braeckmans K, Luan Y, de Jong N, De Smedt SC, Lentacker I. Ultrasound and
microbubble mediated drug delivery: acoustic pressure as determinant for uptake via membrane
pores or endocytosis. Journal of Controlled Release 2015. 197. 20-28.
Own contribution: involved in the design of the experiments and the writing of the manuscript (30%)
IF 2014: 7.7. Citations: 21 (Highly cited paper-web of sciences).
11. De Cock I., Lajoinie G., Versluis M., De Smedt S., Lentacker I. Sonoprinting and the importance of
microbubble loading for the ultrasound mediated delivery of nanoparticles. Biomaterials 2016.
83.294-307.
Own contribution: involved in the design of the experiments and the writing of the manuscript (30%)
IF 2014: 8,387. Citations: 2.
12. Lajoinie G., De Cock I., Coussios C.C., Lentacker I., Le Gac S., Stride E., Versluis M. In vitro
methods to study bubble-cell interactions for therapeutic applications. Invited review.
Biomicrofluidics 2016. 10(1).
Own contribution: involved in writing of the manuscript (15%)
IF 2014: 2,7. Citations: 0.
13. Lajoinie G., Luan Y, Gelderblom E., Dollet B., Lentacker I., Dewitte H., De jong N., Versluis M.
Nonspherical oscillations drive the lipid shedding of microbubbles near a boundary. Submitted to
PNAS.
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Activity Report
 PROGRESS OF PHD DISSERTATIONS THAT YOU SUPERVISE
Describe the progress of the PhD candidates you supervise (if any), and mention the expected date of
the public defense when applicable.
Supervision PhD Rein Verbeke (Research Assistant, started Januari 2014).
Rational design of nanoparticles for in vivo cancer immunotherapy.
The PhD project of Rein aims at designing adjuvant containing lipid-based mRNA nanoparticles which
are able to migrate to the lymph nodes or tumor micro-environment with the aim to reach dendritic
cells residing in the lymph nodes or tumor micro-environment.
A first publication revealed differences in bone-marrow derived dendritic cell yields and
immunological properties as a consequence of in vitro cell culture protocols of dendritic cells. We
showed that the BM-DC model in which novel nanoparticles are tested, acts as an important
confounding factor in both transfection efficiency and T cell activation assays which makes it very
difficult to compare in vitro results of different research groups (Published in journal of controlled
release).
The second aim of his project as the development of lipid nanoparticles that are able to efficiently
delivery mRNA to dendritic cells and simultaneously induce their maturation. Earlier attempts where
TAA encoding mRNA and TLR-ligands were co-injected in the lymph nodes have failed in this task Up
till now there is no nanoparticle developed to initiate this process although the presentation of TAA
by mature DCs is a crucial step towards effective T-cell responses. In a recently written paper we
prove that mRNA transfection and DC maturation is feasible and possible at much lower adjuvant
doses when encapsulating them within the same nanoparticle. We also prove that this nanoparticle
remains stable (aggregation, mRNA complexation, transfection capacity) in biological media and that
an effective T-cell response is developed when DCs become transfected. These nanoparticles have
been evaluated in vivo (biodistribution, DC transfection and maturation) and very promising results
were obtained (see before). Paper submission is planned for december 2016.
Supervision PhD Silke Roovers (BOF-UGent personal scholarship, started october 2015).
Evaluation of chemotherapeutic delivery with DOX-loaded microbubbles in complex in-vitro tumor
models .
In the project of Silke we want to evaluate how deep DOX or DOX-liposomes can penetrate in more
complex in vitro tumor models like tumor spheroids and which microbubble-cell interactions are
involved and how we can optimize acoustic settings and microbubble design to enhance the
penetration depth. As mentioned above a new set-up has been installed allowing us to control
acoustic conditions and visualize microbubble-cell interactions in real time. Spheroid and
microbubble preparation have been optimized the last year and current cell vialibity studies are
performed to estimate the efficiency of DOX delivery in spheroids with microbubbles and ultrasound.
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 SCIENCE COMMUNICATION
FWO encourages its researchers to disseminate the results of their research widely, and valorize
them where possible. In this part you have to indicate which actions you have undertaken in the
context of science communication and science in society.
Results are published in high impact, peer-reviewed journals and are presented at
international conferences, organized by amongst others: the ESGCT (European Society of
Gene and Cell Therapy, CRS (Controlled Release Society) and ISTU (International society on
Therapeutic Ultrasound). Several poster and oral presentations have been awarded at these
conferences. Results are also presented at conferences, such as Knowledge for Growth to
trigger the interest of the industry. The interest of industry in our research is evidenced by the
fact that our abstract was selected in 2011 for an oral presentation at Knowledge for Growth.
In 2013 our poster was awarded with one of the two poster prizes of the conference.
Presentation at summer schools and during doctoral school meetings have resulted in the
dissemination of our research results to a wider and less specialized audience. Moreover,
selected lessons are also given within the course “bionanotechnology” (open to pharmacy
students, biotechnology students…) to trigger student’s interest in pharmaceutical research.
Dr. Heleen Dewitte received the second price in the first PhD Cup in flanders. She presented
our work on mRNA loaded microbubbles for ultrasound triggered immunotherapy” to a broad
audience during the final that was streamed live at canvas.be. Moreover her participation
resulted in several other presentations at science communication events (“dag van de
wetenschap” amongst others) and a few publications in dutch that aim to explain the concept
to society.