IMAGING IMMUNITY – from Nanoscale to Macroscale | Insights from Biophysics
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POSTER SESSION I

Session chair: Frauke Alves (Göttingen, Germany); Chrit Moonen (Utrecht, Netherlands); Eric Ahrens (La Jolla, USA); Franck Debarbieux (Marseille, France)
 
Date: Wednesday, 15 January, 2020, 5:00 PM - 6:30 PM

Posters will be evaluated by two chairs, the "best" presentation will be awarded and the author invited to present her/his work as a lecture at Friday. Be prepared to present your work! These posters will be presented as poster presentations "only".

Contents

Click on an contribution to preview the abstract content.

P-01

CD69-ImmunoPET enables early response evaluation of cancer immunotherapies (#34)

Bredi Tako1, Philipp Knopf1, Andreas Maurer1, 2, Manfred Kneilling1, 2, 3, Bernd Pichler1, 2, Dominik Sonanini1, 4

1 University of Tuebingen, Werner Siemens Imaging Center, Tuebingen, Germany
2 University of Tuebingen, Cluster of Excellence iFIT (EXC 2180) "Image-Guided and Functionally Instructed Tumor Therapies", Tuebingen, Germany
3 University of Tuebingen, Department of Dermatology, Tuebingen, Germany
4 University of Tuebingen, Deparment of Internal Medicine VIII, Tuebingen, Germany

Introduction

T cell infiltration and activation is essential for successful cancer immunotherapies. Checkpoint inhibitor therapy (CIT) has become standard of care treatment for various cancers, but response is inconsistent and still remains unpredictable. As T cells highly upregulate the early activation antigen CD69 upon stimulation, CD69 might be an excellent candidate for monitoring immune responses by molecular imaging. Here, we developed an antibody based CD69 targeting PET approach and evaluated its potential as a novel imaging tool for monitoring T cell activation by CIT.

Methods

CD69 monoclonal antibodies (mAb) were radiolabeled with copper-64 (64Cu) by the radiochelator NODAGA. Immunoreactivity assays were performed on in vitro activated and non-activated mouse CD4+ and CD8+ T cells and macrophages. C57BL/6N mice with subcutaneously injected MC38 colon adenocarcinomas or OVA-B16 melanomas underwent combined intraperitoneal (i.p.) programmed death ligand 1 (PD-L1) and lymphocyte activation gene 3 (LAG-3) therapy once weekly. Another cohort of OVA-B16 tumor bearing mice were treated i.p. with cyclophosphamide, adoptively transferred T helper 1 (Th1) T cells and PD-L1/Lag-3. Control groups of both tumor models received no therapy. Simultaneous PET/MRI was acquired 48 hours after i.v. tracer injection on different time points after therapy start.

Results/Discussion

Simultaneous PET/MRI of MC38 tumor bearing mice showed a significant increase in 64Cu-NODAGA-CD69-mAb tumor uptake on day 3 (13.6 %ID/cc ± 1.1 SEM) and day 5 (13.4 %ID/cc ± 0.6) after onset of CIT compared to the untreated control group (10.3 %ID/cc ± 0.5 SEM). No differences to the control group were observed on day 8 (10.6 %ID/cc ± 0.3 SEM). In contrast, αPD-L1/αLAG-3 treatment of OVA-B16 melanoma bearing mice did not show therapy response and yielded similar tumor uptake values compared to non-treated mice (11.6 %ID/cc ± 0.3 vs. 10.9 %ID/cc ± 0.4). After induction of a tumor response by additional adoptive transfer of OVA-Th1 cells OVA-B16 tumors exhibited a significantly increased uptake (13.8 %ID/cc ± 0.5). Interestingly, beside T cells tumor infiltrating macrophages were identified as main CD69 source. Subsequent in vitro and ex vivo analyzes revealed that CD69 was highly expressed on antitumoral (M1) macrophages but not on resting (M0) or M2-polarized protumoral macrophages.

Conclusions

CD69-ImmunoPET enables monitoring of immune cell activation and to identify CIT responders as early as 3 days after therapy initiation. The simultaneous identification of T cells and macrophages may overcome limitations in the imaging sensitivity of rare immune cell populations. Immediate identification of CIT resistance by this technique facilitates early treatment modification and will probably better guide clinicians as current biomarkers do.

References

Acknowledgement

CD69-ImmunoPET enables early response evaluation of cancer immunotherapies

A. MC38 tumor bearing mice, which respond to a αPD-L1/αLAG-3 mAb therapy showed a significantly higher tumor uptake of 64Cu-NODAGA-CD69-mAb 3 and 5 days after therapy initiation compared to the non-treated control group. Tracer uptake was similar to the control groups 8 days after therapy start. B. OVA-B16 tumor revealed neither a therapy response nor any higher tracer uptake 3 days after αPD-L1/αLAG-3 therapy. Through adoptive transfer of OVA-Th1 cells and cyclophosphamide however the tumor growth slowed down and a significant higher 64Cu-NODAGA-CD69-mAb uptake in the tumor was observed.

Keywords: PET/MRI, Molecular Biology, ImmunoImaging, Cancer Immuntherapies
P-02

Effect of metformin as add-on therapy in a high-grade glioma mouse model (#10)

Silvia Valtorta1, 2, 3, Valentina Pieri4, Stefania Battaglia2, 3, Sara Giordano1, Elisa Toninelli5, Cristina Monterisi3, Sergio Todde1, Andrea Cappelli6, Rossella Galli4, Rosa Maria Moresco1, 2, 3

1 University of Milano-Bicocca, Medicine and Surgery Department and Tecnomed Foundation, Monza, Italy
2 CNR, Institute of Molecular Bioimaging and Physiology (IBFM), Segrate, Italy
3 IRCCS San Raffaele Scientific Institute, Experimental Imaging Center- Nuclear Medine Division, Milano, Italy
4 IRCCS San Raffaele Scientific Institute, Neural Stem Cell Biology Unit, Neuroscience Division, Milano, Italy
5 Vita-Salute San Raffaele University, Milano, Italy
6 University of Siena, Biotechnology Department, Siena, Italy

Introduction

Cancer-related inflammation influences the clinical outcome of patients with high-grade glioma(1). We showed that Metformin (MET) add-on to Temozolomide (TMZ) increases the overall survival of mice inoculated with EGFR-mutated GBM cells. Moreover, TMZ plus MET reduced brain inflammation in extratumoral brain regions as revealed by PET and the TSPO ligand [18F]VC701 (Valtorta et al., under submission). Here, we evaluated the effect of MET plus TMZ on animal survival, brain inflammation and cell proliferation in an orthotopic GBM model based on the inoculation of patient-derived GSC EGFR+ cells.

Methods

Athymic nude mice intracranially injected with L0627 GSCs or normal animals were included in the study. For efficacy studies, GBM mice were randomly divided into 4 different groups and treated with 28-days cycle of vehicle (10% DMSO daily), MET (250mg/kg/day, i.p.), TMZ (70mg/kg/day, 5 days, p.o., 28 days cycles) or TMZ plus MET starting at tumor comparison (2). Lesion growth and treatment effect were monitored by T2-weighed MRI and PET using [18F]FLT and [18F]VC701 (to measure thymidine salvage pathway activation or inflammation respectively) before and during treatment administration (7 and 28 days). Mice were sacrificed based on clinical signs or at 150 days post cell inoculation and brains processed for immunohistochemistry.

Results/Discussion

MET plus TMZ increased mice survival in comparison with TMZ alone. No effect was observed in the group treated with MET alone whose survival time was similar to that of vehicle-treated animals. In agreement with these data, all mice showed a progressive increase in tumor growth at MRI except those treated with MET plus TMZ. Surprisingly, only a slightly positive signal at [18F]FLT PET imaging was observed, independently from time or treatment schedule. [18F]VC701 uptake was increased in tumor and extra-tumor regions in comparison with normal mice in particular after one cycle of TMZ treatment.

The add-on of metformin increased the effect of TMZ. However, differently to what observed with Gli36ΔEGFR, L0627 GSCs cells failed to display an evident uptake of [18F]FLT within tumor masses. In agreement with previous results, tumor increased [18F]VC701 and the signal was differently modulated by TMZ+MET treatment compared to TMZ alone.

Conclusions

Our data indicate that add-on metformin increases TMZ efficacy in GBM EGFR+ models possibly acting on the inflammatory microenvironment, but further studies are needed.

References

  1. Roesch S. et al., Int J Mol Sci. 2018; 19: 436.
  2. Valtorta S., et al., Oncotarget. 2017 ; 68:113090-113104.

Acknowledgement

This work was supported by AIRC, IG2018 n. 21635

Keywords: PET, MRI, Molecular Biology, Glioma, inflammation
P-03

In vitro characterisation of nanobodies targeting a pan-T-cell marker as potential universal immunotherapy T-cell trackers (#20)

Dario Gosmann1, Theresa Käsbauer1, Lisa Russelli2, Nick Devoogdt5, Florian Bassermann1, 3, 4, Wolfgang Weber2, 3, 4, Calogero D'Alessandria2, Angela Krackhardt1, 3, 4

1 Technische Universität München, IIIrd Medical Department, Munich, Bavaria, Germany
2 Technische Universität München, Department of Nuclear Medicine, Munich, Bavaria, Germany
3 Technische Universität München, Center for Translational Cancer Research (TranslaTUM), Munich, Bavaria, Germany
4 German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), partner-site Munich, Heidelberg, Bavaria, Germany
5 Vrije Universiteit Brussel, In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Brussels, Belgium

Introduction

Immunotherapeutic approaches like immune checkpoint modulation and adoptive T cell transfer have shown great potential for the treatment of various tumor entities. However, reliable and clinically implemented imaging strategies to monitor the therapeutic effect are still lacking. To directly depict both the endogenously stimulated as well as genetically modified T cells, we are using single domain antibodies (nanobodies) to develop an immuno-PET based imaging approach targeting the pan T cell marker CD7.

Methods

Nanobodies targeting CD7 were generated by the VIB Nanobody Core in Brussels. The nanobodies were then produced in E.coli, and their size and purity was validated by SDS page, HPLC, MALDI-TOF and nLC MS/MS. Binding to CD7 was validated by FACS analysis of PMBC-derived central memory T cells (Tcm) with and without previous blocking of CD7. The dissociation constant (Kd) was determined via flow cytometry analysis of Tcm in the presence of different nanbody concentrations. The effects of nanobody binding on functional properties of T cells was tested after coincubation of ML2-B7 tumor cells, TCR transduced Tcm and a wide range of nanobody concentrations by investigation of tumor cell killing and IFNy secretion. Dynamic cytotoxic effects were tested using an xCELLigence system.

Results/Discussion

We confirmed high purity of nanobodies at the expected size of around 15kDa. Specific binding of CD7 to PBMC-derived Tcm was confirmed using flow cytometry analysis and most tested nanobodies showed high affinities for their target. Assessment of T-cell functionality after coincubation with nanobodies showed promising results, as cytotoxic efficiency of T cell was preserved and there was no sign of functional T-cell impairment or modulation. The cytotoxic potential was tested using two different systems, and in both cases T cells coincubated with nanobodies retained their functionality and were able to kill off the respective tumor cells. Based on these experiments, nanobodies that showed a strong specific binding, no impairment of the cytotoxic effect and IFNy secretion of bound T cells were selected for further studies.

Conclusions

These initial in vitro assays show promising results regarding CD7 specific nanobodies as possible inert pan T-cell tracer. To validate these results and head closer to clinical application, in vivo studies monitoring the T-cell function in the presence of nanobodies and subsequent radioactive labelling will be performed.

References

Mall, S. et al. (2016). "Immuno-PET Imaging of Engineered Human T Cells in Tumors." Cancer Res June 28 2016 DOI: 10.1158/0008-5472.CAN-15-2784

Mayer & Mall et al. (2018). "T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target." Theranostics 2018 Nov 28;8(21):6070-6087. doi: 10.7150/thno.27275

Keywords: PET, PET/CT, Nanobody, Immunology, Oncology
P-04

High total metabolic tumor volume on 18F-FDG-PET/CT correlates with lack of immune response in advanced melanoma patients treated with pembrolizumab.  (#24)

Odrade Gondry1, 3, Gil Awada2, Ibrahim Özdemir1, Julia K. Schwarze2, Emma Daeninck2, Yanina Jansen2, Teofila Seremet2, Lode Goethals1, Hendrik Everaert1, Bart Neyns2, Marleen Keyaerts1, 3

1 Nuclear Medicine, Universitair Ziekenhuis Brussel, Jette, Belgium
2 Medical Oncology, Universitair Ziekenhuis Brussel, Jette, Belgium
3 ICMI, Vrije Universiteit Brussel, Jette, Belgium

Introduction

Advanced Melanoma is a known for its poor long-term prognosis and although the last few years an enormous improvement is seen due to the Immune Checkpoint Inhibitors (ICI), only a subset of patients (pts) benefit. Biomarkers to predict and guide treatment decisions are being investigated. The idea is that in tumorleasions with an immunosuppressive tumor micro-environment (TME) the ICI may not work, and that the lesions will become much larger. In this study we aimed to investigate the role of pre-treatment 18F-FDG PET/CT as a predictor of survival after pembrolizumab treatment.

Methods

Outcome data of 69 pts with advanced melanoma treated with pembrolizumab at the university hospital in Brussels were collected as part of a prospective therapeutically non-interventional trial. Objective responses were evaluated using the immune-related response criteria. Total Metabolic Tumor Volume  (TMTV) and the Total Lesional Glycolysis (TLG) were measured, using MIM Encore Software®. A log rank was performed to correlate TMTV, TLG, LDH and presence of brain metastasis to overall survival (OS) and progression free survival (PFS) after pembrolizumab administration. Multivariate analysis was conducted for selected parameters. For each parameter, treshold values were determined to dichotomize the patients, prior to analysis.

Results/Discussion

For all 69 pts, median PFS was 19 w and median OS was 130 w. A threshold of 90 mL of TMTV was identified as most optimal to distinguish a patient subpopulation with significantly worse PFS (median 7 w [95% CI 4-9] vs 56 w [95% CI 0-118]; HR 19.10, p<0.001) and OS (median 21 w [95% CI 2-41] vs not reached; HR 46.14, p<0.001). Also in a multivariate analysis (Cox multivariate logistic regression), the TMTV above 90 mL showed the strongest correlation with shorter PFS and shorter OS. Besides TMTV, the presence of brain metastasis was an independent factor associated with shorter PFS and OS. For TLG, similar patterns but with weaker correlations were detected.

Conclusions

TMTV above 90 mL was the best predictive parameter to identify patients who did not respond to pembrolizumab monotherapy. This threshold could reflect a tumor volume, above which the patient’s immune system is unable to become activated. In such patients, other cancer treatments or combination immunotherapy might be better options. Confirmation of our findings is currently ongoing in an additional patient cohort.

Acknowledgement

Marleen Keyaerts is a senior clinical investigator of the Research Foundation Flanders.

Figure 1
PFS in function of baseline TMTV
Figure 2
Overall Survival in function of baseline TMTV.
Keywords: PET, CT, FDG, Total Metabolic Tumor Volume
P-05

Imaging Immunity with [18F]DPA-714 PET-MRI in auto-immune limbic encephalitis (#12)

Wolfgang Roll1, Christine Strippel2, Bastian Zinnhardt1, 3, Philipp Backhaus1, 3, Robert Seifert1, 3, Andreas Jacobs3, Nico Melzer2, Michael Schäfers1, 3

1 University Hospital Münster, Department of Nuclear Medicine, Münster, North Rhine-Westphalia, Germany
2 University Hospital Münster, Department of Neurology and Institute of translational Neurology, Münster, North Rhine-Westphalia, Germany
3 University of Münster (WWU), European Institute for Molecular Imaging, Münster, North Rhine-Westphalia, Germany

Introduction

Auto-immune limbic encephalitis (LE) usually presents with mesial temporal lobe epilepsy and cognitive changes and is thought to be mediated by adaptive neural immunity. Targeting the translocator protein (TSPO) with a second generation PET-Tracer (F-18-DPA-714) permits visualisation of TSPO overexpression in activated microglia and other immune cells. The aim of this study is to investigate the feasibility of a combined F-18-DPA-714 PET-MRI protocol for visualisation of inflammation in seropositive and seronegative LE patients.

Methods

Seven LE patients underwent combined F-18-DPA-714-PET-MRI between 9/2017 and 03/2019. Dynamic images were acquired 0-60 min p.i. on a Siemens PET-MRI system (mMR). T1w, T2w and Flair MR-images were performed for correlation purposes. Inclusion criteria were recent diagnosis of limbic encephalitis (3 x seronegative; 4 x seropositive (anti-GAD65, anti-LGI1) and naivety to immunotherapies. Inflammation to background ratios (SUVmax tumor/SUVmean reference region) measured in 30-60 min p.i. summation images were standardized to a cortical reference region including grey and white matter on the less-affected brain hemisphere.

Results/Discussion

This study provides first insights into microglia activation in LE as assessed by DPA-714-PET-MRI. All LE patients showed increased uptake in the mesial temporal lobe (amygdala, hippocampus) on the affected side compared to the contralateral hemisphere and the reference region. TSPO expression correlated with signal alterations in T2 and FLAIR images.

Conclusions

We demonstrate the feasibility of [18F]DPA -714-PET-MRI to visualize individual inflammation patterns in seropositive and seronegative limbic encephalitis patients.

Keywords: PET-MRI, 18F-DPA-714, limbic encephalitis
P-06

Non-invasive imaging of immunotherapy response using camelid-derived single domain antibodies: a feasibility study (#16)

Isabel Remory1, 2, Christoph M. Griessinger3, Meher Majety4, Damya Laoui5, Tony Lahoutte1, 2, Nick Devoogdt1

1 Vrije Universiteit Brussel (VUB), In vivo cellular and molecular imaging laboratory, Faculty of Medicine and Pharmacy, Jette, Belgium
2 Universitair Ziekenhuis Brussel, Jette, Belgium
3 Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland
4 Roche Pharmaceutical Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany
5 Vrije Universiteit Brussel & Vlaams Instituut voor Biotechnologie, Cellular and Molecular Immunology lab/Lab of Myeloid Cell Immunology, Brussels, Belgium

Introduction

Non-invasive imaging of cancer immunotherapy (CIT) allows reduction of sample size in preclinical studies and enable personalized medicine in the clinic. Single domain antibodies (sdAbs) can be generated to be specific for any target on immune cells or tumor cells. sdAbs are a platform which has the potential to enable repeated imaging during CIT. As a first proof, we evaluated the decrease of tumor-associated macrophages in the tumor microenvironment (TME) after therapy with an anti-CSF-1R blocking antibody using an anti-CD206 sdAb as tracer for Single Photon Emission Computed Tomography.

Methods

Syngeneic C57BL/6J mice were inoculated with 1x106 MC38 colon carcinoma cells and treated twice with either the CSF-1R blocking or a control antibody at 9- and 15-days post-implantation. 99mTc-CD206-sdAb uptake in the tumor microenvironment (TME) was evaluated before and after anti-CSF-1R therapy by multipinhole SPECT/CT. In vivo quantification of the SPECT scans was performed after placing 3D ellipsoid Region of Interest on the anatomical CT image. After in vivo imaging, all major organs and tumors were dissected and radioactivity in each tissue was analyzed in a γ-counter.

Results/Discussion

99mTc-anti-CD206 sdAb SPECT imaging showed tracer signal in liver and spleen, cervical lymph nodes, kidney and bladder (fig.1A). Animals treated with the anti-CSF-1R antibody showed lower tracer uptake throughout the body except for liver and the clearance organs, kidney and bladder (fig.1B). The aCD206-sdAb tumor uptake was 1.00±0.28 %ID/cc in animals treated with the control antibody, while the tumor uptake of 0.624±0.05 %ID/cc in the therapy group was significantly lower (fig.2). Ex vivo gamma counting confirmed the in vivo observations, showing lower aCD206-sdAb uptake in tumor, heart, lung, pancreas, stomach, small and large intestine, muscle, cervical lymph nodes, brown and white fat.

Conclusions

After anti-CSF-1R therapy, animals clearly showed a lower CD206-sdAb uptake in macrophage-containing organs and the TME. Radiolabeled sdAb are able to detect changes elicited by CIT.

figure 1
Representative in vivo SPECT/CT whole-body maximum intensity projections (MIP) of MC38- tumor bearing mice 1 h after i.v. injection of 99mTc-anti-CD206-sdAb. (A) 9 days post inoculation; before therapy (B) 16 days post inoculation; after therapy; red box indicates tumor
figure 2
In vivo quantification of SPECT/CT with CD206-sdAb after anti-CSF-1R therapy. A 3D ellipsoid ROI was placed on the tumor as detected on the CT image.  Data expressed as %IA/cc. (Each replicate is represented, mean±SD; Student’s t-test  ** p<0.01)
Keywords: SPECT, CT, sdAb, cancer immunotherapy
P-07

Intra-peritoneal administered oncolytic viruses promote and catalyze immune checkpoint inhibitor based immune therapies of pancreatic cancer (#23)

Dimitri Stowbur1, Barbara F. Schörg1, Susanne Berchthold2, Julia Beil2, Dominik Sonanini1, Ulrich Lauer2, 4, Bernd Pichler1, 4, Manfred Kneilling1, 3, 4

1 University of Tuebingen, Department of Preclinical Imaging and Radiopharmacy, Tuebingen, Baden-Württemberg, Germany
2 University of Tuebingen, Abteilung Innere Medizin VIII, Tuebingen, Germany
3 University of Tuebingen, Department of Dermatology, Tuebingen, Baden-Württemberg, Germany
4 University of Tuebingen, Cluster of Excellnence iFIT (EXC 2180) , Tuebingen, Germany

Introduction

Cancer research is engaged to understand how tumor cells evade the immune system resulting in different approaches such as immune checkpoint blocking antibodies (mAbs) or oncolytic viruses (OVs). We have established an effective immunotherapy in RIP1-Tag2 (RT2) mice using tumor-antigen specific IFN-y producing CD4+ T cells (TA-Th1) combined with anti-PD-L1 and anti-LAG-3 mAbs (ICB)1. We aim to combine our approach with T-VEC, an OV applicable to selectively destroy cancer cells and to boost the immune response. In a second step we aim to establish 18F-[FHBG] imaging to monitor OV therapies.

Methods

RT2 mice endogenously develop insulin secreting pancreatic insular cell carcinomas resulting in low blood glucose levels (BGL) and death at 14 weeks of age. We initiated our combined immunotherapy with a 2 Gy whole body radiation followed by a weekly TA-Th1 administration and twice weekly ICB injections. T-VEC was administered twice at the onset of the combined treatment approach. Tumor progression was monitored twice weekly by BGL measurements. At the end of the experiment, at 14 weeks of age, we sacrificed experimental mice and performed histopathological (H&E staining) and CD3+ immunohistochemistry (IHC) of the pancreas and the spleen. In addition, we collected blood and analyzed organs of interest (pancreas, spleen, lymph nodes, liver, thymus) to check for viral infection.

Results/Discussion

Addition of T-VEC therapy to our established combined treatment approach (ICB & TA-Th1 cells) yielded a superior treatment efficacy reflected by an impressive increase in BGL of 31.67 ± 7.63 mg/dl; n=6; p=0.002), whereas mice treated with ICB & TA-Th1 cells alone yielded only a slight increase in BGL, one week after onset of treatment. Interestingly, T-VEC treatment exhibited the strongest effect one week after administration. Combining single ICB, as in metastatic melanoma patients, with T-VEC treatment, revealed significantly increased BGL of 21.33 ± 6.88 mg/dl (n=4-6; p=0.0145), whereas treatment with ICB alone was not efficient. Histopathological analysis and IHC of the pancreatic tissue confirmed a T-VEC therapy related pronounced inflammation and treatment efficacy. Moreover, T-VEC treatment caused an impressive splenomegaly. PCR analysis confirmed a viral infection. Next, we will detect viral thymidine kinase expression to monitor the viral infection with 18F-[FHBG]-PET/MRI.

Conclusions

This is the first report clearly demonstrating an OV induced increased anti-tumoral treatment efficiency when combining with TA-Th1 cell & ICB or OV with ICB alone in an experimental model of pancreatic cancer. Thus, i.p. administration of OV in patients with adenocarcinomas in addition to ICB could represent a novel very promising treatment approach which could be monitored by 18F-[FHBG]-PET/MRI.

References

Paper excepted in Theranostics, Schwenk, Schoerg et. al., 2019

Acknowledgement

Keywords: MRI, PET, Immunotherapy
P-08

Towards precise PET and MRI arterial input functions through a novel extracorporeal circulation approach in mice (#53)

Philipp Backhaus1, 2, 3, Florian Büther1, 3, Lydia Wachsmuth2, Sven Hermann1, Klaus Schäfers1, Cornelius Faber2, Michael Schäfers1, 3

1 University of Münster, European Institute for Molecular Imaging (EIMI), Münster, North Rhine-Westphalia, Germany
2 University of Münster, Translational Research Imaging Center (TRIC), Münster, North Rhine-Westphalia, Germany
3 University Hospital Münster, Department of Nuclear Medicine, Münster, North Rhine-Westphalia, Germany

Introduction

Inflammation leads to drastic changes of perfusion and permeability setting the basis for functional imaging modalities as dynamic contrast-enhanced MRI (DCE-MRI) to characterize inflammatory processes. However, these changes can lead to unspecific uptake in molecular imaging modalities such as PET. Compartmental modeling of dynamic data is the key to resolve the specific component of PET radiotracer uptake and to quantify the vascular response to inflammation in MRI. The required arterial input function (AIF) however, is highly challenging to acquire, especially in small animals.

Methods

We developed an experimentally feasible setup that allows precise measurements of DCE-MRI contrast agend and PET radiotracer AIFs simultaneously in mice based on an extracorporeal circulation. Mice receive a shunt from the femoral artery to the tail vein. The extracorporeal line features reservoirs which reside in the MRI field of view (9.4 T Bruker). A MRI-compatible measuring chamber for a β-Microprobe (biospace lab) is included in the circulation. Clinical standard contrast agent Gadobutrol was used for DCE-MRI, PET radiotracers 18F-PSMA and 18F-FDG were co-injected. Different methods of dispersion correction were used and compared to undispersed AIF measurements using an intra-aortic β-Microprobe.

Results/Discussion

AIFs measured inside the extracorporeal circulation showed little noise, small interindividual variance and typical curve shapes. Validation with an intra-aortic probe for PET radiotracers and validation with blood withdrawals and mass spectrometry analysis for MR input functions demonstrated good quantitative accuracy and precision. Moreover, PET and MR input functions shared a similar shape and close accordance of peak concentrations. The shunt based AIFs were used to model quantitative plausible parameter maps in tumors, muscle and brain and outperformed image and population based input functions. Comparison of different methods of AIF measurements and dispersion correction demonstrated varying and model-dependend effects on PET and MRI derived parameters.

Conclusions

We present a novel approach for simultaneous PET and DCE-MRI AIF measurements in mice with conceivable potential compared to so far published methods. The method supports higher precision of MR-based quantification of the vascular response and PET-based quantification of molecular biomarkers of inflammation in preclinical studies increasing their significance for translation.

References

Acknowledgement

Figure 1
(A) Overview of the experimental setup Blood is shunted from the femoral artery (FA) to the tail vein (TV) through silicone tubings and passes two reservoirs, that reside inside the field-of-view (FOV) of the MRI (Rprox and Rdist). A tube containing a fixed gadolinium concentration of 1 mM resides between the two reservoirs (Rfix). The measuring chamber contains an outlet for blood withdrawals and a β-Microprobe for radiotracer measurments. (B) Dispersion and delay corrected MR-AIF is displayed in green, Rprox and Rdist in red and blue, co-injected raciotracer in black (18F-PSMA)
Keywords: MRI, PET, Arterial Input Function
P-10

Multimodal imaging of modified neuroinflammation in a mouse stroke model (#19)

Marina Dobrivojevic Radmilovic1, Dunja Gorup1, Sinisa Skokic1, Anton Glasnovic1, Helena Justic1, Paula Josic1, Anja Baric1, Srecko Gajovic1

1 University of Zagreb School of Medicine, Croatian Institute for Brain Research, ZAGREB, Croatia

Introduction

Stroke is characterized by complex interactions of protective mechanisms against damage and subsequent repair. The influence of inflammation on those mechanisms remains controversial. Toll-like receptors (TLR) located on microglia evoke an in situ inflammatory response after ischemia, which is suggested to have both detrimental and beneficial consequences depending of the timing and cellular context. The aim of this study was to visualize the influence of altered innate immunity on brain damage and repair after ischemic injury by performing multimodal bioluminescence and MRI in vivo imaging.

Methods

Tlr2-deficient mice (2-4 months old) were compared to their wild type counterparts. The ischemic lesion was induced by transient (60 minutes) middle cerebral artery occlusion (MCAO) followed by filament removal and reperfusion. The animals were monitored over 28 days by multiple magnetic resonance imaging (MRI, Bruker 7T Biospec 70/20 USR), bioluminescence imaging (BLI, Perkin Elmer IVIS Spectrum) and behavioral assessment sessions. At day 28 after MCAO the animals were sacrificed and the brains were processed for immunohistochemistry and Western blot protein analysis.

Results/Discussion

The ischemic lesion, edema and loss of tissue were evaluated by MRI. BLI included evaluation of neuroinflammation by Tlr2 gene activity, neurorepair by Gap43 gene and apoptosis by caged luciferin (DEVD-luciferin) activity. The neuroinflammation-modified group (Tlr2-deficient mice) and the wild type control differed in the various elements characterizing the post-ischemic brain status. The survival was better in Tlr2-deficient animals, although they showed increased neurological deficit, lesion size and edema in the acute phase. Tlr2-deficient mice showed enhanced repair GAP43 elements, although simultaneously demonstrating extensive apoptosis (caspases 3 and 7), which was visible as a marked reduction of the ipsilateral cortical and striatal tissue at 14 days after MCAO, progressing over time.

Conclusions

Multimodal in vivo imaging allowed the evaluation of both morphological and molecular changes following ischemia. Tlr2-deficient mice with modified inflammation showed enhanced neuronal repair accompanied by pronounced apoptosis.

References

Gorup D, Škokić S, Kriz J, Gajović S. Tlr2-deficiency is associated with enhanced elements of neuronal repair and caspase 3 activation following brain ischemia. Sci Reports. 2019;9(1):2821.

Gorup D, Bohaček I, Miličević T, Pochet R, Mitrečić D, Križ J, Gajović S. Increased expression and colocalization of GAP43 and CASP3 after brain ischemic lesion in mouse. Neurosci Lett. 2015;597:176-82.

Bohacek I et al. Toll-like receptor 2 deficiency leads to delayed exacerbation of ischemic injury. J Neuroinflammation. 2012;9:19.

Acknowledgement

The study was supported by EU European Regional Development Fund, Operational Programme Competitiveness and Cohesion, grant agreement No.KK.01.1.1.01.0007, CoRE – Neuro; Croatian Science Foundation projects RepairStroke (IP-06-2016-1892), and BRADISCHEMIA (UIP-2017-05-8082). The work of doctoral students Anja Barić and Paula Josić has been fully supported by the “Young researchers' career development project – training of doctoral students” of the Croatian Science Foundation funded by the European Union from the European Social Fund. Multimodal imaging was done at Laboratory for Regenerative Neuroscience - GlowLab, University of Zagreb School of Medicine.

Keywords: Tlr2, stroke, multimodal imaging, MRI, BLI
P-11

In vivo characterization of the hepatic lipid composition and the inflammatory response in a murine model of acute and chronic carbon tetrachloride induced hepatitis  (#32)

Bianca Blöchl1, Andreas Schmid1, Manuela Martella2, Natalie Mucha1, Bernd Pichler1, 3, Leticia Quintanilla-Martinez2, 3, Johannes Schwenck1, 3, 5, Manfred Kneilling1, 3, 4

1 Eberhard Karls University, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Germany
2 Eberhard Karls University, Institute of Pathology and Neuropathology and Comprehensive Cancer Center Tübingen, Tübingen, Germany
3 Eberhard Karls University, Cluster of Excellence iFIT (EXC 2180) "Image-Guided and Functionally Instructed Tumor Therapies, Tübingen, Germany
4 Eberhard Karls University, Department of Dermatology, Tübingen, Germany
5 Eberhard Karls University, Department of Nuclear Medicine and Clinical Molecular Imaging, Tübingen, Germany

Introduction

Steatosis in the liver tissue induced by high caloric diet causes non-alcoholic steatohepatitis (NASH) in a significant number of patients, which is a major cause of hepatocellular carcinomas (HCC). Currently, invasive biopsies and subsequent histopathological analysis are the gold standard in the diagnostic procedures of NASH.

Here, we studied the feasibility of non-invasive in vivo 1H magnetic resonance spectroscopy (MRS) in a murine model of acute and chronic carbon tetrachloride (CCl4)-induced hepatitis which exhibits similar histomorphological features, like steatohepatitis.

Methods

Mice received a single intraperitoneal (i.p.) CCl4 injection to induce acute hepatitis  (AH) and repetitive CCl4 injections (3x weekly over five weeks) to induce chronic hepatitis (CH). The hepatic lipid composition in AH and CH was determined by MRS. For ex vivo validation after the experiment, livers underwent histopathological (H&E) and RT-PCR analysis focusing on expression patterns of proinflammatory cytokines (TNF, IL-1b, IL-6), hypoxia inducible factor 1α (HIF-1α) and reactive oxygen species-associated superoxide dismutase 1 (SOD-1). In addition, we analyzed the liver enzymes in the blood serum of mice with AH and CH. Finally, we conducted flow cytometry analysis focusing on immune cells in livers and spleens of mice with CH.

Results/Discussion

MRS revealed in AH a tendency towards an increase in hepatic fatty acids and a higher fractional lipid mass compared to sham-treated healthy mice. Interestingly, we could not identify alterations in the hepatic lipid composition upon CH. Histopathological analysis of livers with AH uncovered necrosis, regeneration processes and microsteatosis, whereas livers with CH exhibited necrosis and steatosis, but only weak signs of inflammation. Elevated alanine transaminase values were exclusively determined in the serum of mice with AH. RT-PCR analysis of the liver revealed a drastically enhanced TNF expression in CH when compared to AH, whereas SOD-1 was decreased upon AH and CH when compared to healthy livers.  Flow cytometry analysis of livers with CH revealed a decreased expression of CD4+ T cells and an increase in regulatory T cells and PD-1+ CD4+ T cell, while splenic effector memory CD4+ T cells and PD-1+ CD4+ T cells were increased.

Conclusions

Our results support the feasibility of non-invasive MRS to assess molecular changes in chemically-induced AH and AH. Detailed analyses of the underlying inflammatory and molecular events revealed distinct changes in AH and CH. Nevertheless, further studies are needed for detailed characterization of the basic underlying inflammatory and molecular mechanisms as well as to investigate the diagnostic capabilities of MRS in NASH patients.

References

Acknowledgement

Keywords: 1H magnetic resonance spectroscopy (MRS), Steatosis, toxic inflammation, acute and chronic carbon tetrachloride (CCl4)-induced hepatitis, NASH
P-12

Mannosylated PLGA nanoparticles for tumor macrophage detection by 19F MRI (#29)

Giorgia Zambito1, 4, Siyuan Deng2, Natasa Gaspar1, Clemes Lowik3, 1, Piera Di Martino2, Laura Mezzanotte1

1 Dept of Radiology and Nuclear Medicine, Erasmus Medical Center, Rotterdam, Netherlands
2 School of Pharmacy, University of Camerino, Camerino, Italy
3 Institute of Chemical Sciences and Engineering (ISIC) , Swiss Federal Institute of Technology Lausanne , Lausanne, Switzerland
4 Medres Medical research , Cologne, Germany

Introduction

Tumor-associated macrophages (TAMs) promote tumor progression and angiogenesis [1,2]. In vivo 19FMRI is a non-invasive imaging technique for monitoring the trafficking of immune cells with a highly specific detection and quantification of cells [3-5]. Here, we encapsulated perfluoro‐15‐crown‐5‐ether (PFCE) into a series of poly lactic-co-glycolic acid nanoparticles (PLGA NPS), which are stable and non-toxic [6]. The PLGA NPs are mannosylated to significantly enhance the uptake by targeting the mannose receptor (CD206/MRC1) expressing TAMs, both in vitro and in vivo..

Methods

Thesurface of PFCE loaded PLGA NPs was modified to present polyethylene glycol (PEG) and Mannose. The nanoparticles were prepared by solvent-extraction evaporation method [3]. The particle size and zeta potential were characterized by dynamic light scattering (DLS). The morphology was characterized by scanning electron microscopy (SEM). The toxicity of the nanoparticles for Raw264.7 cells was performed by LDH assay. PLGA NPs containing FITC were con-incubated with polarized Raw264.7  to perform the uptake assay. The nanoparticle uptake by Raw264.7 was analysed by FITC fluorescence absorbance and confocal microscopy, quantitatively and qualitatively. 19F-NMR and magnetic resonance spectroscopy (MRS) were applied to measure PFCE encapsulation yield and 19F content, respectively.

Results/Discussion

All the NPs showed a diameter ranged from 200 to 390 nm with low PDI. Zeta potential were between -30 and -13 mV(Table1). SEM picture revealed that nanoparticles have spherical shape with a smooth surface (Fig.1 A). The PFCE encapsulation yield was measured by 19F NMR resulting in 1%(w/v) PFCE/PLGA NPs were dissolved in CDCl3 with 0.1M Trifluoroacetic acid as reference(Fig1B).For in vitro tests, the LDH toxicity assay indicated that cell viability was not significantly altered  after 24h of treatment with nanoparticles. PLGA-PEG-MANNOSE-PFCE showed higher uptake (1,49%) for M2-like macrophage compared to control macrophages (M0)(Fig.1C). Confocal microscopy confirmed internalization of nanoparticles after 1h (Fig.1D).MRS was performed to demonstrate that targeted nanoparticles contain sufficient 19F for detection by MRI (Fig.1E).

Conclusions

Based on these preliminary results, we suggest that Mannosylated PLGA-PEG nanoparticles containing PFCE can be a suitable tool to efficiently image TAMs in vivo by 19FMRI.

References

 

  1. Yang Li, Journal of Hematology and Oncology, 2017

  2. Quo G.et al. Journal of Immunol Res, 2016

  3. Srinivas M. et al. Biomaterials, 2010

  4. Bersen M.R. et al. BJR, 2015

  5. Chapelin F et al. Journal for Immunotherapy Of Cancer, 2018

  6. Makadia H.K. et al. Polymers, 2011

Acknowledgement

We acknowledge the funding for this work provided by the European Commission under a MSCA-ITN award, grant number 675743 (ISPIC).

Table1.Nanoparticle characterization
The diameter range is from 239 nm to 386 nm. All the particles show low poly dispersity index (PDI) and a relatively constant zeta potential. Measurements were made in triplicate.
Figure 1. Characterization of Nanoparticles and in vitro tests.
A) Scanning electron microscopy (SEM) analysis. The scale bar represents 200nm. B) 19FNMR analysis shows  PFCE encapsulation yiled.C) In vitro citotoxicity assay by LDH. Raw 264.7 M2-like were treated with different concentration of nanoparticles ranged from 0 to 2.5 mg/ml, and incubated for 24h. D) Uptake assay for M2-like macrophages treated with FITC entrapped nanoparticles (1mg/ml)  after 6h. E) MRS acquisition of 19F encapsulated by the PLGA-PEG-Mannose-PFCE nanoparticles by 7T MRI.
Keywords: 19F MRI, multimodality imaging, nanoparticles, magnetic resonance., macrophage
P-13

Reactive oxygen and nitrogen species (ROS/RNS) differentially impact acute and chronic cutaneous delayed-type hypersensitivity reaction (DTHR) (#25)

Roman Mehling1, Johannes Schwenck1, 2, Irene Gonzalez-Menendez3, Leticia Quintanilla-Martinez3, Birgit Fehrenbacher4, Martin Schaller4, Martin Röcken4, Bernd Pichler1, Manfred Kneilling1, 4

1 Eberhard Karls University, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Germany
2 Eberhard Karls University, Department of Nuclear Medicine, Tübingen, Germany
3 Eberhard Karls University, Institute of Pathology and Neuropathology, Tübingen, Germany
4 Eberhard Karls University, Department of Dermatology, Tübingen, Germany

Introduction

ROS and RNS are important modulators of inflammatory immune responses applicable to suppress or to promote them. Nevertheless, the role of ROS/RNS mainly provoked by neutrophils and macrophages during DTHR of the skin is still not well understood. Aim of our study was to dissect the dominant ROS/RNS sources non-invasively in vivo in mice with differently impaired ROS/RNS production, namely Myeloperoxidase deficient; MPO-/-, NOX2 deficient; gp91phox-/-, inducible nitric oxide synthase deficient; iNOS-/- mice, employing the ROS/RNS-sensitive chemiluminescence optical imaging probe L-012.

Methods

Mice were sensitized with 5% TNCB at the abdomen and challenged with 1% trinitrochlorobenzene (TNCB) at the right ear 7 days later to elicit acute DTHR. To induce chronic cutaneous DTHR mice were challenged every 48h up to 5 times. We determined ear swelling responses and conducted non-invasive in vivo optical imaging measurements 0h, 4h, 12h and 24h after the 1st, 3rd and 5th TNCB ear challenge (Ch) (n = 5-8 per experimental group). Images were analyzed by drawing regions of interest and determining the average radiance of the chemiluminescence. Additionally, we obtained the ear tissues for H&E histopathology (n=4), CD3 and MPO immunohistochemistry (IHC; n=4), dihydrorhodamin 123 (DHR) flow cytometry analysis (n=5) and 8-Oxoguanine glycosylase (OGG1) fluorescence microscopy (n=3).

Results/Discussion

In vivo L-012 optical imaging revealed in acute and chronic DTHR a completely abrogated ROS/RNS production in ears of gp91phox-/- mice and an up to 70% decreased ROS/RNS production in ears of MPO-/- when compared to wild-type mice. DHR flow cytometry analysis of leucocytes derived from ears with acute DTHR confirmed our in vivo L-012 optical imaging results. Interestingly, we determined no significant differences in ear swelling response between all experimental groups in acute and chronic DTHR. Nevertheless, H&E and IHC staining of ears with acute DTHR from gp91phox-/- mice exhibited an enhanced inflammation with prominent neutrophilic infiltrate. Contrary, we determined a reduced inflammatory immune response in gp91phox-/- mice with chronic DTHR, while inflamed ears from MPO-/- mice exhibited the strongest inflammation. Interestingly, OGG1 staining showed neutrophil extracellular trap formation in ears with acute DTHR in all experimental groups, but not in ears with chronic DTHR.

Conclusions

MPO and NOX2 are the dominant ROS/RNS sources in acute and chronic DTHR. Nevertheless, depletion of predominant ROS/RNS sources exhibited only moderate but conflicting clinical consequences. Thus ROS/RNS is not a single entity and each species has different properties at certain stages of the disease resulting in different outcomes. Consequently, proper therapeutic targeting of ROS/RNS is essential for an efficient treatment approach.

Keywords: Optical Imaging, Inflammation, ROS/RNS, Neutrophils
P-14

In vivo live imaging of human T/B cell lymphoma cross-linking mediated by bispecific CD20-TCB antibody (#44)

Elena Menietti1, Dario Speziale1, Johannes Sam1, Stefano Sammicheli1, Stanford Chen1, Marine RIchard1, Christian Klein1, Pablo Umana1, Marina Bacac1, Mario Perro1, Sara Colombetti1

1 Roche, Zürich, Switzerland

Introduction

Cancer Immune Therapies have shown unprecedented results in improving tumor control [1-3]. However, many patients are still refractory to treatment. A deeper understanding of the mode of action of the different CITs sub-classes may help improving therapeutic approaches to reach better anti-tumor response. For this reason, we developed a multi-photon intra-vital microscopy (MP-IVM) approach to study in vivo, at single cell level, the tumor microenvironment upon treatment with CD20-targeting T-cell bispecific antibodies (TCB) [4] in a preclinical model of diffuse large B cell lymphomas (DLBCL).

Methods

To selectively monitor clinical lead molecules in the context of human T cell responses, we developed a skinfold chamber model [5] in last generation humanized mice [6] that allows visualization, by MP-IVM, of labelled human T cells co-injected intra-dermally with WSU-DLCL2, a human DLBCL. We have used this model to investigate T cells recruitment to tumors upon CD20-TCB therapy: by intra-venously injecting labeled T cells in mice treated with selected blocking antibodies, we were able to identify dedicated pathways induced by CD20-TCB and regulating T cell influx into the tumor bed. Furthermore, we developed a user-independent quantification platform to assess changes in the dynamics of T cell motility and time of interaction with tumor cells.

Results/Discussion

We have developed an experimental preclinical model that aims to reduce xenoreaction (human T cell reaction against mouse tissue) by utilizing T cells derived from humanized mice, educated within murine thymus. We demonstrate that such model is optimal to quantify human T cell dynamics in vivo. We show that CD20-TCB localizes in the tumor and acts on tumor-resident T cell motility within 1 hour post i.v. injection (defined as functional PK), causing a sharp reduction in their speed (from 4 to 2 µm/min) and an increase in tumor/T cell interaction time; those changes last up to 72h post-treatment. In addition, we prove how the initial tumor/T cell interaction mediated by CD20-TCB lead to peripheral T cells recruitment into the tumor. This mechanism is dependent on the presence of tumor-resident T cells and on IFNg-CXCL10 pathway. Inhibiting any of these two parameters resulted in reduced T cells infiltration from the periphery and reduced anti-tumor efficacy.

Conclusions

We developed a reliable imaging and analysis pipeline to investigate in vivo T cell dynamics and recruitment and applied it to the study of CD20-TCB treatment of DLBCL model. Our approach has shed new lights into the MoA of this new class of immune-therapeutics, demonstrating that the IFNɣ-CXCL10 pathway is involved in T cell recruitment upon CD20-TCB treatment

References

  1. Yousefi, H., et al., Expert Rev Clin Immunol, 2017. 13(10): p. 1001-1015.
  2. Pishko, A. and S.D. Nasta, T Transl Cancer Res, 2017. 6(1): p. 93-103.
  3. Emens, L.A., et al., Eur J Cancer, 2017. 81: p. 116-129.
  4. Bacac, M., et al., Clin Cancer Res, 2018. 24(19): p. 4785-4797.
  5. Koehl, G.E., A. Gaumann, and E.K. Clin Exp Metastasis, 2009. 26(4): p. 329-44.
  6. Shultz, L.D., F. Ishikawa, and D.L. Greiner, Nat Rev Immunol, 2007. 7(2): p. 118-30.

Acknowledgement

We would like to acknowledge the oDTA of Roche pRED for the support of the project. We would like to thanks our colleagues of Large Molecule Research for providing the therapeutics, their  fluorescent conjugation as well as the genetically modified fluorescent cells. We would like to thanks the informatic teams for the help with analysis of the data and maintenance of the informatic infrastructure.

Keywords: In vivo Imaging, immunotherapy, T cell bi-specific, cancer immunotherapy, humanized mouse model
P-15

Molecular imaging of T-cell infliltrates after irradiation using radiolabelled anti-CD8CDb-diabodies. (#35)

Gemma M. Dias1, Edward S. O'Neill1, Felix B. Salazar2, Richard Tavare2, Anna Wu2, Bart Cornelissen1

1 University of Oxford, Department of Oncology, Radiobiology Research Institute, Oxford, United Kingdom
2 UCLA, Dept. of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America

Introduction

The combination of irradiation (IR) and immunotherapy (IT) has been shown to be an effective regime for cancer therapy. The combination approach however requires optimized IR and IT regime. In order to measure the effects of anti-CTLA-4 on T-cell infliltrates in multiple lesions in the same individual during radiotherapy, we used SPECT imaging with a radiolabelled anti-CD8-cys-diabody (α-CD8-CDb).

Methods

Female Balb/c mice were inoculated with CT-26 cells on the left and right lower flank. Fourteen days after, only the right-hand side tumours were irradiated (single dose, 4 Gy). On day 15, ¾ of the mice were administered anti-CTLA4 antibody (250 μg ). αCD8-CDb was conjugated with malemide-DTPA and radiolabelled with 111InCl3. [111In]In-mDTPA-αCD8-CDb (1.9 MBq, 10 μg) was administered intravenously on day 21, and SPECT/CT images were acquired day 22 (MILabs VECTor4CT). Images were analysed with PMOD. Tumours and organs were excised, weighed and measured on a gamma counter. Once flash frozen and stored at -80 °C tumours were sectioned and mounted on microscope slides. Slides were exposed to a storage phosphor screen for 72 h to obtain autoradiographs. Adjacent sections were obtained for CD8.

Results/Discussion

CT-26 tumours were slow to grow over the first 10 days post inoculation, however rapidly grew after 12 days post inoculation, no significant differences were observed in tumour sizes between the irradiated (right) tumour and the non-irradiated (left) tumour. SPECT/CT images showed uptake of the [111In]In-anti-CD8-CDB in spleen, kidneys, tumours and lymph nodes. The axillary and brachial nodes were clearly visible with the [111In]In-anti-CD8-CDB, while any signal from inguinal nodes was overshadowed by the location of the tumours (Figure 1).

Uptake of [111In]In-anti-CD8-CDB in the left tumours (LT)  was marginally higher than the right tumours (RT) regardless of α-CTLA treatment; LT 3.21 %ID/g, RT 2.36 %ID/g in α-CTLA-4 and LT 3.22 %ID/g, RT 2.68 %ID/g in non-treated. Tumour to muscle ratios did not differ between the tumours or between groups. Heterogeneity within tumours was evident from autoradiographs (Figure 2).

Conclusions

In vivo CD8 expression was assessed post irradiation and αCTLA4 treatment using a radiolabelled anti-CD8-cys-diabody. High tumour uptake was evident from the images and biodistribution data, and uptake to background ratio demonstrates the use of αCD8-CDb as a promising imaging probe. Further in vivo studies are underway to assess the relationship between T-cell response to combinations of irradiation and αCTLA4 treatment

References

Tavaré R, Escuin-Ordinas H, Mok S, et al. An Effective Immuno-PET Imaging Method to Monitor CD8-Dependent Responses to Immunotherapy. Cancer Res. 2016;76(1):73–82.

Acknowledgement

Figure.1 SPECT/CT images of [111In]In-mDTPA-αCD8-CDb uptake.

SPECT/CT images of the combined treated mouse (left) and the IR only treated mouse (right). Axillary (a) and brachial (b) lymph nodes as well as the kidney (k), spleen (s) demonstrate high uptake of [111In]In-mDTPA-αCD8-CDb at 24 hr post injection.

Figure. 2 Autoradiography images of [111In]In-mDTPA-αCD8-CDb uptake in tumour sections

Autoradiography images of tumour sections in a combined treated mouse (above) and an IR only treated mouse (below). Heterogeneity of [111In]In-mDTPA-αCD8-CDb distribution is evident in all tumours.

Keywords: SPECT, Diabodies, CD8
P-16

Imaging inflammation in amyotrophic lateral sclerosis (#31)

Pavle Andjus1, Stefan Stamenković1, Dunja Bijelić1, Tanja Dučić2

1 University of Belgrade Faculty of Biology, Center for laser microsopy, Belgrade, Serbia
2 CELLS - ALBA Synchrotron Light Source, Cerdanyola del VallèsBarcelona, Spain

Introduction

Amyotrophic lateral sclerosis (ALS) is an adult onset fatal neurodegenerative disease The immune mechanisms in the disease are prevalent. In vivo MRI studies on the brain of the transgenic hSOD1 G93A ALS rat model reveal a compromised blood-brain barrier and magnetically labelled anti- CD4 or CD8 antibodies show infiltration of lymphocytes in lesioned brain regions also in presymptomatic animals.  IgGs from ALS patients (ALS IgGs) increase intracellular Ca2+ in motor neurons and induce excitotoxic synaptic release [1-3], also affecting non-motor neurons [4], and glial cells as well (5-7).

Methods

The cell cultures of rat astrocytes and microglia treated with ALS and control IgGs, and ATP (as physiological control) were grown on CaF2 slides. After fixing cells were and scanned for high resolution synchrotron radiation-based Fourier Transform Infrared (FTIR) micro-spectroscopy with the aperture 10x10μm to achieve single cell resolution. Prinicipal Componenat Analysis (PCA) was used to investigate macromolecular expression and biochemical characteristics of glial cells treated with ALS IgGs from 2 patients (ALS 1 & 2) compared to healthy and disease (polydiscopathy) control IgGs and 0.2 mM ATP action. All the treatments were performed for 20 min or 24 h.

Results/Discussion

The PCA revealed the following prominent observations: 1) in astrocytes only the 20 min treatment gave apparent differences for the lipids (3100 - 2800 cm-1) and protein and esters (1800-1480 cm-1) bands between the two ALS patients, and between ALS1 and healthy control and disease control as well; 2) the difference between the two ALS patients was also retained for the carbohydrate and nucleic acids band (1480-900 cm-1); 3) in microglia after 20 min treatment again more apparent effects were observed as compared to 24 h, thus, most apparent differences were seen in the lipids band with ALS1 IgG compared to the healthy control while 4) microglia from ALS1 and 2 patients differed in the protein and ester band.

Conclusions

Basic changes in glial cell metabolism were revealed as induced by immune humoral factors, that may also serve for better stratification of patients for personalized medicine.

References

1) Appel et al. 1991. Proc. Natl. Acad. Sci. 88: 647; 2) Uchitel et al.1992. Neurology 42: 2175; 3) Mosier et al. 1995. Ann. Neurol. 37, 102; 4) Andjus et al. 1997. J Physiol. 504 ( Pt 1):103; 5) Milošević et al. 2013. Cell Calcium. 54:17; 6) Stenovec et al. 2011. Acta Physiol (Oxf). 203:457; 7) Milošević et al. 2017. Front Immunol. 8:1619.

Acknowledgement

Supported by EC H2020 MSCA-RISE project No 778405. The experiments were performed at BL01-MIRAS beamline at ALBA AV-2018022751, CALIPSOplus Grant 730872.

Keywords: ALS, astrocytes, microglia, FTIR, synchrotron light source
P17

Developing activatbale probe for imaging T cell mediated cytotoxic response to immunotherapy by targeting granzyme B activity (#47)

Jianghong Rao1

1 Stanford Univeristy, Palo Alto, California, United States of America

Introduction

Immunotherapy is revolutionizing the clinical management of cancer. The lack of accurate and non-invasive diagnostics monitoring early immune response, however, remains an urgent need in advancing precision immunotherapy. Granzyme B (grzm B) is a serine proteinase uniquely governing the cytotoxic T cell mediated cancer killing and represents an ideal target for molecular imaging of early response to immunotherapies.

Methods

We tethered our highly translatable target-enabled in-situ ligand aggregation (TESLA) platform (Ref 1-3) and developed the first grzm B activated self-assembly small molecule probe for imaging T cell mediated cytotoxicity. This probe was characterized in a series of enzymatic studies and successfully imaged the grzm B activity in a B cell lymphoma (A20)/CAR T- and a melanoma (B16-mCD19)/CAR T-cells therapy model by fluorescence imaging and fluorescnece activated cell sorting (FACS).

Results/Discussion

The grzm B probe has been characterized in vitro with recombinant grzm B enzyme, showing the formation of the self-assembled products in reporting the activtiy of grzm B. The gramB probe has also been found not to be cleaved by caspase-3, indicating its specificty for grzm B. Treatment of a B cell lymphoma cell line (A20) with resting, untransduced or CAR T cells as an in vitro therapy model in the presence of fluorescent grzm B probes demonstrated T cell-mediated cytotoxicity dependent intracellular probe aggregation and signal retention with a confocal microscope. Flow cytometry analysis including a grzm B specific inhibitor further confirmed the grzm B dependent probe activation and signal retention. Weternblotting and mRNA analysis confirmed the elevation of grzm B level and acitivity in CAR T.

Conclusions

The novel design of this grzm B activated self-assembly imaging probe together with promising results revealed great potential for further development of the probe for PET imaging of immune response to CAR T and checkpoint blockade therapy in vivo.

References

1. Shen, B., J. Jeon, M. Palner, D. Ye, A. Shuhendler, F.T. Chin, and J. Rao, Positron emission tomography imaging of drug-induced tumor apoptosis with a caspase-triggered nanoaggregation probe.Angew Chem Int Ed Engl, 2013. 52(40): p. 10511-4.

2. Ye, D., A.J. Shuhendler, L. Cui, L. Tong, S.S. Tee, G. Tikhomirov, D.W. Felsher, and J. Rao, Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in vivo.Nat Chem, 2014. 6(6): p. 519-26.

3. Palner, M., B. Shen, J. Jeon, J. Lin, F.T. Chin, and J. Rao, Preclinical Kinetic Analysis of the Caspase-3 7 PET Tracer 18F-C-SNAT Quantifying the Changes in Blood Flow and Tumor Retention After Chemotherapy. J Nucl Med, 2015. (56): 1415-1421

Acknowledgement

Keywords: Fluorescence, Granzyme B, CAR-T
P-18

Development of a multimodal imaging agent for unstable atherosclerosis: PLGA-perfluorocarbon nanoparticles for imaging macrophage-like smooth muscle cells (#51)

Kim R. Cortenbach1, Massis Krekorian1, Alexander H. Staal1, Koen van Riessen1, Martin ter Beest1, Roland R. van Kimmenade1, I. Jolanda M. de Vries1, 2, Mangala Srinivas1

1 Radboud Institute for Molecular Life Sciences, Department of Tumor Immunology, Nijmegen, Netherlands
2 Radboud university medical centre, ) Department of Medical Oncology, Nijmegen, Netherlands

Introduction

Atherosclerosis, the inflammatory disease preceding potential fatal clinical events (myocardial infarction and stroke), is usually silent until onset. Smooth muscle cells (SMCs) are believed to contribute to inflammation by LDL uptake and forming foam cells (macrophage-like-SMCs or MAC-SMCs) and might be important in plaque vulnerability. Noninvasive imaging providing early information is not sufficient with no precise imaging agents present. The aim of this in vitro study is to investigate the uptake by macrophage(like) cells of nanoparticles (NP), suitable for different imaging modalities[1]

Methods

Our multimodal NPs consist of poly(lactic-co-glycolic acid) (PLGA) with perfluoro-15-crown-5-ether (PFCE) entrapped, produced by sonication. A fluorescent dye will also be entrapped. Monocyte-derived dendritic cells (moDCs) from a buffy, macrophages originating from THP-1 cells and MAC-SMCs will be incubated with these nanoparticles for several time points. SMCs will be harvested from murine thoracic aortas after enzymatic digestion; SMC origin will be confirmed by α-SMC actin reactivity. After 72 hours of incubation with cholesterol, SMCs will be differentiated into MAC-SMCs.[2-4] NP uptake will then be assessed by flow cytometry and confocal microscopy. Phagocytosis and efferocytosis will be studied using fluorescent latex beads and fluorescent labeled apoptotic cells respectively.

Results/Discussion

Our preliminary results show that after 90 minutes incubation 99% of the moDCs are positive for the fluorescent dye signal with FACS (atto647). Widefield microscopy shows signal from the dye at the same position as some cells, with confocal Z-stacks confirming the origin of the dye signal from the inside of the cells. The discrepancy between FACS and microscopy may be due to sticking of the particles to the cell surface which already can be measured by the sensitive FACS apparatus. Another possibility is the burst release of the hydrophilic dye, however nanoparticles containing a lipophilic dye showed a burst release too. Our NPs will further be optimized regarding dye concentration and coating or binding of targeting molecules for specific targeting of the MAC-SMCs. By the time of writing this abstract there is no data yet concerning the isolation of SMCs and differentiation into MAC-SMCs.

Conclusions

From these preliminary data we can conclude that with FACS 99% of the moDCs are positive for the dye signal. Confocal microscopy confirms intracellular dye signal in these cells, however the number of positive cells is much lower than measured with FACS. Further optimization of the nanoparticles and the protocol should elucidate this. Hopefully this results in a useful targeting strategy for MAC-SMC and thereby visualization of unstable plaques.

References

  1. Swider, E., et al., Clinically-Applicable Perfluorocarbon-Loaded Nanoparticles For In vivo Photoacoustic, (19)F Magnetic Resonance And Fluorescent Imaging. Nanotheranostics, 2018. 2(3): p. 258-268.
  2. Rong, J.X., et al., Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading. Proceedings of the National Academy of Sciences of the United States of America, 2003. 100(23): p. 13531-13536.
  3. Vengrenyuk, Y., et al., Cholesterol loading reprograms the microRNA-143/145-myocardin axis to convert aortic smooth muscle cells to a dysfunctional macrophage-like phenotype. Arterioscler Thromb Vasc Biol, 2015. 35(3): p. 535-46.
  4. Feil, S., et al., Transdifferentiation of vascular smooth muscle cells to macrophage-like cells during atherogenesis. Circ Res, 2014. 115(7): p. 662-7.

Acknowledgement

We thank ERA-CVD JTC2017-044 for funding the SCAN consortium.
Confocal microscopy was performed at Microscopic Imaging Centre (MIC).

Microscopy image of moDC after nanoparticle incubation

Image from a confocal Z-stack of moDCs after two hours incubation. Arrows indicating signal from dye. Blue is DAPI, red is the dye signal from the nanoparticles and green is wheat germ agglutinin staining for moDCs. (Zeiss LSM900).

Dye signal in moDCs after 90 minutes incubation

FACS data of moDCs incubated with nanoparticles. Percentages are calculated by dividing the cells which are positive in the dye channel (APC for atto647) by the total number of cells.

Keywords: Microscopy, Immunology, Cell biology