15th European Molecular Imaging Meeting
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Immuno-Oncology Imaging

Session chair: Gilbert Fruhwirth (London, UK); Beatriz Salinas (Madrid, Spain)
Shortcut: PW17
Date: Friday, 28 August, 2020, 12:00 p.m. - 1:30 p.m.
Session type: Poster


Abstract/Video opens by clicking at the talk title.


Fluorescent amphipathic peptides as markers for apoptotic cells enable efficient monitoring of anti-cancer therapies

Nicole D. Barth1, Ramon Subiros-Funosas1, Lorena Mendive-Tapia1, Rodger Duffin1, Adriano Rossi1, Mikala Egeblad2, Ian Dransfield1, Marc Vendrell1

1 University of Edinburgh, CIR, QMRI, Edinburgh, United Kingdom
2 Cold Spring Harbor Laboratory, Cold Spring Harbor, United States of America


The incidence of cancer is increasing worldwide  and is considered to be a leading cause of death with an estimated 9.6 million deaths in 20181. This highlights the need to develop new imaging approaches for early diagnosis and effective treatment. Cancer therapies aim to inhibit division and induce cell death of the tumour. Assessment of efficiency of therapy is hampered due to the lack of adequate markers of cell death in vivo. We developed and characterised a library of fluorescent peptides to bind to apoptotic cells and tested their applicability to monitor response to anti-cancer therapy.


Amphipathic peptides were synthesised using SPPS containing Trp-BODIPY as fluorogenic amino acid for detection of binding under was-free conditions. To identify the optimal sequence for usage in in vivo, diverse peptides were screened for their optimal binding concentration and kinetics to apoptotic cells. Neutrophils were isolated from human blood and cultured 24 h in vitro to have a mixed population of apoptotic and viable cells, providing an optimal platform to test the binding of the peptides to apoptotic cells using flow cytometry. We tested the most promising peptide in an LPS-induced acute lung injury model in mouse as well as chemotherapy of mammary gland tumours to investigate its applicability in vivo.


Our selection process revealed that several peptides specifically label apoptotic cells by confocal microscopy and flow cytometry. We have undertaken further detailed molecular characterisation of the most promising amphipathic peptides (see Fig. 1B). Initial characterization of the peptides show little or no cytotoxicity, minimal effect on cell physiology, with a predicted biodistribution/clearance that is favourable for use in vivo. Furthermore, using multi-colour flow cytometry we demonstrated specific labelling of multiple apoptotic cell populations using Annexin V as a well described marker for apoptosis (see Fig. 1A). In further studies, we used the peptide for in vivo detection and imaging of apoptotic cells in a mouse model of tumour growth and metastasis. Our results demonstrate that the amphipathic peptides allow imaging of tumour cell death in situ following treatment with chemotherapeutic agents.


Our results demonstrate the specificity and in vivo applicability of small amphipathic fluorogenic peptides as effective and reliable markers of apoptotic cells. Using confocal imaging and intravital imaging we could demonstrate their utility for real-time imaging. Based on these findings, we propose that these peptides offer new opportunities to screen for the effective induction of apoptosis in cancer cells by therapeutic drugs.

AcknowledgmentN.D.B. acknowledges funding from OPTIMA (EP/L016559/1). R.S.F acknowledges an MSCA Individual Fellowship (659046). L.M.T. acknowledges the support of Fundacion Antonio Martin Escudero (FAME) in the form of a post-doctoral fellowship.  M. E. acknowledges funding from Cold Spring Harbor Laboratory, Northwell Health, and the Thompson Family Foundation. M.V. acknowledges funding from an ERC Consolidator Grant (771443), the Biotechnology and Biological Sciences Research Council (BB/M025160/1), and The Royal Society (RG160289). The authors thank the technical support from the QMRI Flow Cytometry and Confocal Advanced Light Microscopy facilities at the University of Edinburgh and Yaiza Varela (UPV), Luxembourg Bio Technologies Ltd. (Rehovot) for the kind supply of reagents for peptide synthesis, and Astex Therapeutics, who kindly provided AT7519 as a gift. 
[1] WHO. Cancer. (2019). Available at: https://www.who.int/news-room/fact-sheets/detail/cancer. (Accessed: 2nd February 2020)
[2] Hassan, M., Watari, H., Abualmaaty, A., Ohba, Y. & Sakuragi, N. Apoptosis and molecular targeting therapy in cancer. BioMed Research International 2014, (2014).
[3] Wong, R. S. Apoptosis in cancer: from pathogenesis to treatment. J. Exp. Clin. Cancer Res. 30, 87 (2011).
In vitro and in vivo association of newly developed Trp-BODIPY coupled peptide with apoptotic cells.

(A) Representative confocal image of in vitro association of Trp-BODIPY coupled peptide with PyMT MMTV cells undergoing apoptosis (programmed cell death).

(B) Scanning electron microscope images of peptide positive and peptide negative myeloid cells.

 (C) In vivo association of Trp-BODIPY coupled peptide with apoptotic neutrophils in the mouse lungs.

Keywords: Apoptosis, cancer imaging, fluorogenic peptide, amphipathic peptide, fluorescent probes for in vivo in real-time imaging

Confocal and 2-photon intravital microscopy analysis of Tumor-Associated Macrophage targeting by anti-Macrophage Mannose Receptor (MMR) Nanobodies

Marco Erreni1, Evangelia Bolli2, 3, Roberta Avigni4, Francesca D'Autilia1, Pieterjan Debie5, Fijs Van Leeuwen6, Andrea Doni1, Paola Allavena4, 7, Cecilia Garlanda4, 7, Alberto Mantovani4, 7, 8, Sophie Hernot5, Jo Van Ginderachter2, 3

1 Humanitas Clinical and Research Center – IRCCS, Unit of Advanced Optical Microscopy, Pieve Emanuele, Italy
2 Vrije Universiteit Brussel, Lab of Cellular and Molecular Immunology, Brussels, Belgium
3 VIB Center for Inflammation Research, Lab of Myeloid Cell Immunology, Gent, Belgium
4 Humanitas Clinical and Research Center – IRCCS, Dpt of Immunology and Inflammation, Pieve Emanuele, Italy
5 Vrije Universiteit Brussel, Lab for in vivo Cellular and Molecular Imaging, ICMI-BEFY/MIMA, Brussels, Belgium
6 Leiden University Medical Center, Interventional Molecular Imaging laboratory, Leiden, Netherlands
7 Humanitas University, Pieve Emanuele, Italy
8 Queen Mary University of London, William Harvey Research Institute, London, United Kingdom


Tumor-associated macrophages (TAMs) are the major host cell type infiltrating tumor tissue, promoting angiogenesis, metastasis and tumor-immune suppression. Targeting TAMs represents an opportunity for prognostic and therapeutic purpose.
Nanobodies (Nbs), the smallest natural available antigen-binding fragments derived from Camelid heavy-chain-only antibodies, are suitable for tumor targeting, due to their fast kinetic, rapid clearance and deep tissue penetration. Anti-mannose receptor (MMR)-Nbs have been successfully used to target TAMs in vivo for imaging and radioimmunotherapy approaches.


Cy5-conjugated α-MMR Nbs (Cy5-MMR-Nbs) were used to microscopically image MMR+-TAMs in a mouse model of transplanted fibrosarcoma. MN-MCA cells were injected i.m. in the mouse hind leg. 3 weeks after tumor cell injection, monovalent or bivalent α-Cy5-MMR-Nbs were injected i.v.: 1h post-injection, mice were sacrifice and organs collected for whole mount confocal analysis. Intraperitoneal pre-injection of a 16X molar excess of unconjugated-bivalent α-MMR Nbs was used to reduce the binding of α-Cy5-MMR-Nbs in off-tumor sites. For 2-photon intravital microscopy (IVM) approach, mice were injected with 1mg of FITC-conjugated dextran to visualize vasculature: 2 min after IVM acquisition started, bivalent or monovalent α-Cy5-MMR-Nbs were i.v injected and their diffusion kinetic analyzed


Whole mount confocal analysis of tumor tissue revealed a high specificity of both monovalent and bivalent α-Cy5-MMR-Nbs in targeting TAMs at cellular level (Fig.1A). Of note, only the MMR+-TAM subpopulation was identified by α-Cy5-MMR-Nbs. In the liver, monovalent and bivalent α-Cy5-MMR-Nbs preferentially accumulated in MMR+-liver sinusoidal vessels, with a minimal accumulation in liver macrophages and no retention within the portal spaces (Fig1A). Pre-treatment with a 16X molar excess of unconjugated-bivalent α-MMR-Nbs significantly reduced the accumulation of monovalent α-Cy5-MMR-Nbs in the liver. MN-MCA-derived tumors develop lung metastasis at late tumor stages: accordingly, α-Cy5-MMR-Nbs accumulate in the MMR+ macrophages at lung metastatic site. 2-photon IVM showed a rapid diffusion of α-Cy5-MMR-Nbs from the circulation into tumor tissues, with a specific cellular accumulation in MMR+-TAMs already 5-6 minutes after i.v injection (Fig.1B).


We previously demonstrated that α-MMR Nbs can be used to monitor tumor progression by the in vivo imaging of TAMs. Here we showed, for the first time, MMR+-TAMs targeting with α-MMR Nbs at microscopic levels, showing their high cellular specificity and very rapid kinetic diffusion within tissues. These findings increase our knowledge on α-MMR Nb behavior in tissues, enforcing the potentiality of using α-MMR Nbs for tumor imaging and treatment.


This work was supported by the Italian Association for Cancer Research (AIRC) 5x1000, Kom op tegen Kanker, Stichting tegen Kanker, FWO and EU-COST action Mye-EUNITER.

[1] Tumour-associated macrophages as treatment targets in oncology. Mantovani A, Marchesi F, Malesci A, Laghi L, Allavena P. Nat Rev Clin Oncol. 2017 Jul;14(7):399-416. doi: 10.1038/nrclinonc.2016.217
[2] Nanobody-based targeting of the macrophage mannose receptor for effective in vivo imaging of tumor-associated macrophages. Movahedi K, Schoonooghe S, Laoui D, Houbracken I, Waelput W, Breckpot K, Bouwens L, Lahoutte T, De Baetselier P, Raes G, Devoogdt N, Van Ginderachter JA.
Cancer Res. 2012 Aug 15;72(16):4165-77. doi: 10.1158/0008-5472.CAN-11-2994
[3] Macrophage polarization in pathology. Sica A, Erreni M, Allavena P, Porta C. Cell Mol Life Sci. 2015 Nov;72(21):4111-26. doi: 10.1007/s00018-015-1995-y.
[4] Immuno-imaging using nanobodies. Vaneycken I, D'huyvetter M, Hernot S, De Vos J, Xavier C, Devoogdt N, Caveliers V, Lahoutte T. Curr Opin Biotechnol. 2011 Dec;22(6):877-81. doi: 10.1016/j.copbio.2011.06.009
[5] Stromal-targeting radioimmunotherapy mitigates the progression of therapy-resistant tumors.
Bolli E, D'Huyvetter M, Murgaski A, Berus D, Stangé G, Clappaert EJ, Arnouk S, Pombo Antunes AR, Krasniqi A, Lahoutte T, Gonçalves A, Vuylsteke M, Raes G, Devoogdt N, Movahedi K, Van Ginderachter JA. J Control Release. 2019 Oct 15;314:1-11. doi: 10.1016/j.jconrel.2019.10.024.

A) Whole mount confocal analysis of bivalent and monovalent Cy5-MMR-Nb (Red) staining in liver (upper panel) and tumor (lower panel). CD31 (white) shows blood vasculature. Original magnification: 20X

B) 2-photon Intravital microscopy analysis of monovalent Cy5-MMR-Nb (Red) diffusion kinetic within tumor tissues. White arrows indicated the identification of MMR+ TAMs after Cy5-MMR-Nb extravasation and tissue penetration. Diffusion time (t) indicate minutes after IVM acquisition started. Green: FITC-Dextran. Original Magnification: 25X.

Keywords: Tumor-associated Macrophages, Nanobodies, macrophage mannose receptor

Macrophage imaging in Optoacoustics

Ina Weidenfeld1, Lena Peters2, Christian Zakian1, Peter Duewell3, Andriy Chmyrov1, 4, Uwe Klemm1, Anita Loeschcke2, Robin Weihmann2, Juan Aguirre1, 4, Karl-Erich Jäger2, Thomas Drepper2, Vasilis Ntziachristos1, 4, Andre C. Stiel1

1 Helmholtz Zentrum München, Institute of Biological and Medical Imaging, Munich, Germany
2 Forschungszentrum Jülich, Institute of Molecular Enzyme Technology, Jülich, Germany
3 University of Bonn, Institute of Innate Immunity, Bonn, Germany
4 Technische Universität München, Chair of Biological Imaging and Center for Translational Cancer Research, Munich, Germany


Macrophages are one of the most functionally-diverse cell types with roles in innate immunity, homeostasis and disease making them attractive targets for diagnostics and therapy. Photo- or Optoacoustics could provide non-invasive, deep tissue imaging with high resolution and allow to visualize the spatiotemporal distribution and activity of macrophages in vivo. Here we present two pilot studies that explore innovative strategies for OA Macrophage imaging.


Methods as described in Peters et al., 2019 1 and Weidenfeld et al., 2019 2.


We introduce Rhodobacter as bacterial reporter for multispectral optoacoustic (photoacoustic) tomography (MSOT). Endogenous bacteriochlorophyll a in gives rise to strong optoacoustic signals.Importantly, our results suggest that changes in the spectral signature of Rhodobacter depend on macrophage activity inside the tumor. (Fig 1) 1.
Secondly, we present a homogentisic acid-derived pigment (HDP) for biocompatible intracellular labeling of macrophages with strong optoacoustic contrast and utilize this labeling method to track migration of proinflammatory macrophages in vivo with whole-body imaging (Fig 2) 2.


We expand the sparse palette of macrophage labels for in vivo optoacoustic imaging and facilitate research on macrophage functionality and behavior.


Funding statements as in the respective manuscripts.

[1] Peters, L. et al. Phototrophic purple bacteria as optoacoustic in vivo reporters of macrophage activity. Nat. Commun. 10, 1–9 (2019).
[2] Weidenfeld, I. et al. Homogentisic acid-derived pigment as a biocompatible label for optoacoustic imaging of macrophages. Nat. Commun. 10, 5056 (2019).
Spatiotemporal change of the bacterial spectral signature in the tumor environment.
Color coded regions indicate a mixture of 800 nm/ 860 nm peaks or predominance of the 860-nm peak as visualized in the images. 
Homogentisic acid derived pigment labeled Macrophages in vivo

The identity of macrophages is confirmed on immunofluorescence (middle) and brightfield (bottom) cross-sections. Note, the inset for OA is recorded using raster scanning optoacoustic mesoscopy (RSOM) the main image using multi spectral optoacoustic tomography (MSOT).

Keywords: Macrophages, Photo/Optoacoustic, Labeling, Rhodobacter, Tumor

Mannosylated PLGA-nanoparticles for tumor-associated macrophage detection by 19F MRI

Giorgia Zambito1, 2, 3, Siyuan Deng4, Natasa Gaspar2, 3, 7, Roberta Censi4, Clemens W. G. M. Lowik5, 2, Piera Di Martino4, Laura Mezzanotte2

1 Medres Medical research, Cologne, Germany
2 Erasmus MC, Radiology and Nuclear Medicine, Rotterdam, Netherlands
3 Erasmus MC, Molecular genetics, Rotterdam, Netherlands
4 University of Camerino, School of Pharmacy, Camerino, Italy
5 Institute of Chemical Sciences and Engineering (ISIC), Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland
6 Percuros B.V., Leiden, Netherlands


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). The PLGA NPs are mannosylated to significantly enhance the cellular uptake by targeting the mannose receptor (CD206/MRC1) expressing by TAMs, both in vitro and in vivo.


The surface of PFCE loaded PLGA NPs was modified to present polyethylene glycol (PEG) and Mannose ligand. 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). Toxicity test of nanoparticles for Raw264.7 cells was performed by LDH assay. PLGA NPs containing FITC were con-incubated with M2 polarized Raw264.7 to perform the uptake assay. Nanoparticle uptake by Raw264.7 was confirmed by FITC fluorescence absorbance and confocal microscopy. 19F-NMR and magnetic resonance spectroscopy (MRS) were applied to measure 19F spins and PFCE encapsulation yield , respectively.


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 (Fig.1C). PLGA-PEG-MANNOSE-PFCE showed increased uptake (1,49%) for M2-like macrophage compared to control macrophages (M0) (Fig.1D). Confocal microscopy confirmed the targeting of M2-like macrophages after 1h. In Fig.1E PLGA-nanoparticles (green), membrane (magenta), lysosomes (red) and nuclei (blue) are imaged. MRS was performed to demonstrate that targeted nanoparticles contain sufficient PFCE (9,50E20 19Fspins) for further in vivo imaging  for 19FMRI (Fig.1F).


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.


We acknowledge the funding for this work provided by the European Commission under a MSCA-ITN award (ISPIC) and  under the H2020-RISE- Cancer.
We acknowledge Applied Molecular Imaging Erasmus MC (AMIE) for acquisition, analysis and interpretation of data by MRI. We acknowledge prof. Timo ten Hegen and dr. Reza Amim from the Laboratory of Experimental Surgical Oncology (LECO) dept. of the Erasmus MC, for nanoparticle characterization.

[1] Yang, L., Zhang, Y. Tumor-associated macrophages: from basic research to clinical application. J Hematol Oncol 10, 58 (2017)
[2] Guo, Q., Jin, Z., Yuan, Y., et al. 'New Mechanisms of Tumor-Associated Macrophages on Promoting Tumor Progression: Recent Research Advances and Potential Targets for Tumor Immunotherapy'. Journal of immunology research, 9720912,(2016).
[3] Srinivas M,  Cruz Luis J.,  Bonetto F.,et al.'Customizable, multi-functional fluorocarbon nanoparticles for quantitative in vivo imaging using 19F MRI and optical imaging, Biomaterials, Volume 31, Issue 27,(2010).
[4] Bersen M., Guenoun J., Van Tiel S."Nanoparticles for diagnostic imaging and radiotherapy special feature: review article' Br J Radiol; 88: 20150375; (2015)
[5] Chapelin, F., Capitini, C.M. & Ahrens, E.T. 'Fluorine-19 MRI for detection and quantification of immune cell therapy for cancer'. j. immunotherapy cancer 6, 105 (2018)
PLGA-nanoparticles characteristics and in vitro test.

A) Scanning electron microscopy (SEM) analysis. The scale bar represents 200nm. B) 19FNMR analysis showing PFCE encapsulation yield.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-PFCE PLGA nanoparticles (1mg/ml) after 6h. E)Confocal imaging of uptake of PLGA-PEG-FITC-Mannose-PFCE nanoparticles by Raw 264.7 cells. F) MRS acquisition of PFCE encapsulated in mannosylated PLGA-PEG nanoparticles by 7T MRI.

Table1.Nanoparticle characterization

The diameter range is from 239 nm to 386 nm. All the nanoparticles show low poly dispersity index (PDI) and a relatively constant zeta potential ranged between -17 and -31 mV. Measurements were made in triplicate.

Keywords: 19F-MRI, tumor-associated-macrophage, Cancer, TAM

Imaging immune system activity in preclinical Glioblastoma with MRSI: a metabolome-based biomarker

Pilar Calero-Pérez1, 2, Shuang Wu1, Lucía Villamañan1, Martí Pumarola3, 2, Paola V. Casanova4, Mario Vázquez4, Carlos Barcia4, Carles Arús1, 2, 5, Ana Paula Candiota2, 1, 5

1 Universitat Autònoma de Barcelona, Department of Biochemistry and Molecular Biology, Biosciences Faculty, Cerdanyola del Vallès, Spain
2 CIBER Centro de Investigación Biomédica en Red, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
3 Universitat Autònoma de Barcelona, Department of Animal Medicine and Animal Surgery, Veterinary Faculty, Cerdanyola del Vallès, Spain
4 Universitat Autònoma de Barcelona, Department of Biochemistry and Molecular Biology & Institut de Neurociències, School of Medicine, Cerdanyola del Vallès, Spain
5 Universitat Autònoma de Barcelona, Institut de Biotecnologia i de Biomedicina (IBB), Cerdanyola del Vallès, Spain


Glioblastomas (GB) are malignant brain tumours with poor prognosis even after surgery and aggressive therapy. The participation of the immune system is key for sustained response. We have used MRSI-based nosological images (1, 2) for GB therapy response assessment through tumour responding index (TRI) calculation. An oscillatory TRI pattern (6-7 days) was shown in longitudinal studies. The purpose of our present work was to gain further insight into the contribution of immune cell populations to the MRSI spectral pattern changes recorded from Temozolomide (TMZ) - treated preclinical GL261 GB.


C57BL/6 mice bearing GL261 GB tumours (n=33) were treated with TMZ in an immune-enhanced metronomic schedule (IMS) every 6 days, at 60 mg/kg (n=17).  T2w MRI and consecutive 14ms volumetric TE MRSI were acquired every 2 days (1) and nosologic maps calculated (3). Fourteen mice (n=6 controls, n=8 treated) were euthanized at TRI-guided time points for in vitro evaluation. Quantitative polymerase chain reaction (qPCR) was performed with 6 genes associated to M1-M2 macrophage polarization. Immunostainings for CD3 and Iba-1 were performed in n=6 additional mice (1). Histo-cytometry for M1 and M2 markers is currently in progress. Cured mice (n=8) were followed-up by T2w MRI and in case of non-tumour mass within one month of cure, a “rechallenge” experiment with GL261 cells was carried out.


IMS-TMZ increased GL261 GB bearing mice survival, from 21±1 days in untreated mice up to 242±205 days, improving previous results [3]. TRI oscillations (6.2±1.5 days, Fig. 1A) were in agreement with immune cycle length (Fig 1B). MRSI spectral changes could reflect immune system action involving lymphocytes and especially macrophages, since they can represent up to 30% of GB mass (4). Namely, immunohistochemistry shows CD3 and Iba-1 content significantly higher in responding zones (Fig 2A-D). Furthermore, average expression levels of genes related with M1 profiling presented 4-fold change increase in comparison with the untreated tumour expression level. M2 profile gene content showed a lower increase (2.8-fold). This agrees with an increase in anti-tumour immune cells content at TRI-high peak times (Figs 1 and 2). Regarding the re-challenged mice, only 1/8 tumour grew after 10 days, which vanished again after being treated with only one additional IMS-TMZ dose.


Our results indicate that IMS-TMZ treated GL261 GB recruited significantly more macrophages than GB in untreated mice, with predominance of the M1 antitumor phenotype, which can be imaged non-invasively by MRSI. This can be of interest in therapies in which immune system participation is foreseen. IMS-TMZ induced immune memory in GB cured mice, as described in (5), although the ongoing mechanism needs further clarification.


This work was funded by the Ministerio de Economía y Competitividad (MINECO) grant MOLIMAGLIO (SAF2014-52332-R) to CA. Also funded by Centro de Investigación Biomédica en Red – Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN, [http://www.ciber-bbn.es/en], CB06/01/0010), an initiative of the Instituto de Salud Carlos III (Spain) co-funded by EU Fondo Europeo de Desarrollo Regional (FEDER). APC received funding from the ATTRACT project funded by the EC under Grant Agreement 777222. We acknowledge the UAB Predoctoral training programme (14ª Convocatoria PIF-19612 and 13ª Convocatoria PIF, predoctoral fellowships for Ms P. Calero and L. Villamañan), and also China Scholarship Council (predoctoral fellowship 201606990027 for Ms S. Wu). Work also supported by grants from the Spanish Ministry of Economy and Competitiveness, and the European Regional Development Fund (Fondo Europeo de Desarrollo Regional, FEDER) (Reference Grants: RYC-2010-06729, SAF2013- 45178-P and SAF2015-64123-P), Generalitat de Catalunya (Reference Grant: 2014 SGR-984), and Spanish Ministry of Science, Innovation and Universities (Reference Grant: PGC2018-096003-B-I00), to CB.

[1] Arias-Ramos, N, et al. Metabolites (2017) 7: pii: E20.
[2] Wu, S, et al. NMR Biomed (2019) in press
[3] Delgado-Goñi, T, et al. NMR Biomed (2016) 29: 732-43
[4] Glass, R and Synowitz, M. Acta Neuropathol (2014) 128: 347-62
[5] Wu, J and Waxman, DJ. Oncoimmunology (2015) 4: e1005521
Figure 1
A) Bottom: tumour volume (mm3, black line) and % of responding pixels  (% TRI, green line) for case C1264. Green columns indicate TMZ administration. Top: nosological images superimposed to MRI. Color-coding: Blue, normal; Red, non-responding tumour; Green, responding tumour. Black arrows indicate TMZ administration days. B) Scheme of cancer immunity cycle, starting with cellular damage during treatment, release of immunogenic signals, antigen presentation, lymphocyte amplification followed by tumour infiltration, interaction with macrophages and cell killing.
Figure 2
Boxplot of A) CD3+ positive cells (n=147) and B) % of Iba-1 positive areas (n=148) in fields of red (unresponsive) and green (responding) regions of all studied cases. C) CD3+ and D) Iba-1 immunostaining for case C971. Nosological images superimposed to MRI. Arrows point to positive cells. Magnification (40×). E) Fold increase in IMS-TMZ treated cases (n=8), compared to expression in control GL261 GB of untreated mice, of macrophage phenotype gene expression markers for M1 (NOS2, grey bar) and M2 (ARG1, black bar) subtypes.
Keywords: glioblastoma, macrophage profiling, immunocompetent models

68GaNOTA-anti-MMR-Nb for PET/CT assessment of protumorigenic macrophages in patients with solid tumors: preliminary results of a phase I clinical trial

Odrade Gondry1, 2, Catarina Xavier1, 2, Johannes Heemskerk2, Nick Devoogdt1, Hendrik Everaert2, Karine Breckpot5, Lore De Coster3, Christel Fontaine3, Bart Neyns3, Denis Schallier3, Sofie Joris3, Sandrine Aspeslagh3, Ilse Vaneycken2, Kiavash Movahedi4, Geert Raes4, Jo Van Ginderachter4, Tony Lahoutte1, 2, Vicky Caveliers1, 2, Marleen Keyaerts1, 2

1 Vrije Universiteit Brussel, In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Brussels, Belgium
2 Universitair Ziekenhuis Brussel, Nuclear Medicine Department (NUCG), Brussels, Belgium
3 Universitair Ziekenhuis Brussel, Department of Medical Oncology, Brussels, Belgium
4 Vrije Universiteit Brussel, Laboratory of Cellular and Molecular Immunology (CMIM), Brussels, Belgium
5 Vrije Universiteit Brussel, Laboratory for Molecular and Cellular Therapy, Brussels, Belgium


Tumor Associated Macrophages (TAM) play a role in shaping the tumor micro-environment (TME). An imbalance between pro-inflammatory and immunosuppressive, protumorigenic TAMs can support tumor growth and induce resistance to therapy. The presence of protumorigenic TAMs can be assessed by immunohistochemistry (IHC), but due to tumor heterogeneity and locations unable to take biopsies, the use of whole-body PET/CT is wanted. That is why a new PET-tracer is developed, a 68Ga labeled nanobody (Nb) targeting macrophage mannose receptor (MMR), expressed by the protumorigenic TAMs.


Patients (pts) with a solid tumor of at least 10 mm, ECOG score of 2 or lower and a good renal and hepatic function are eligible for inclusion in this phase I study. Safety is assessed using clinical examination and blood sampling before and 3h post injection (p.i.). Biodistribution and dosimetry is assessed using multiple blood samples for blood activity assessment and using PET/CT scans at 10, 90 and 150 min p.i. Blood and urine is assessed for metabolites. Cytokines are measured before and up to 24h p.i. to exclude macrophage activation.


Up to today, two pts were injected with 174MBq and 132MBq of Nb. Both were lung cancer patients treated with immunotherapy without complete radiological response. No adverse events (AE) were recorded, confirming safety in the first two subjects. Biodistribution analysis showed rapid blood clearance with <10% in total blood volume at 3h p.i. (fig. 1). PET/CT showed high tracer uptake in liver, spleen, adrenals and kidneys, as was expected from preclinical data. Other organs showed a very low background activity, with good potential to assess tumor lesions in such parts of the body. No uptake in tumor lesions was seen, but correlation to IHC was not available for these patients. Full biodistribution and dosimetry analysis is ongoing and will be presented at the meeting. Four additional patients will be recruited in the coming weeks.


68Ga-MMR-Nb PET/CT was well tolerated, without AE reported in the first two patients. The biodistribution is favorable, with the highest uptake in the kidneys, liver, spleen and adrenals. Further assessment in 4 pts is needed to complete the phase I study. A phase II study to compare with IHC is subsequently planned in a seamless phase I/II design.


We would like to thank Yasmine De Maeyer, Sonja Van den Block and Gratienne Van Holsbeeck for their contribution to the study. Also Kom op tegen Kanker and FWO to make our project possible.

[1] Xavier, C et al. 2019 'Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages', Mol imaging Biol. Oct;21(5):898-906.
[2] Keyaerts, M et al. 2016 ‘Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma', J Nucl Med. Jan;57(1):27–33.
[3] Blykers, A et al. 2015 ‘PET Imaging of Macrophage Mannose Receptor-Expressing Macrophages in Tumor Stroma Using 18F-Radiolabeled Camelid Single-Domain Antibody Fragments', J Nucl Med. Aug;56(8):1265-71. 
[4] Movahedi, K et al. 2012 ‘Nanobody-based targeting of the macrophage mannose receptor for effective in vivo imaging of tumor-associated macrophages', Cancer Res. Aug 15;72(16):4165-77.
Figure 1: Blood Clearance Curve MMR

Several bloodsamples were taken to evaluate the biodistribution. The analysis showed rapid blood clearance with <10% in total blood volume at 3h p.i. The curve starts at >100%, due to the use of a standardized formula to calculate the total blood volume. Underestimating the total blood volume in this specific patient. 

Keywords: Macrophages, Nanobodies, Phase I, PET/CT, macrophage mannose receptor (MMR)

Imaging cancer immunology: Monitoring CD47 mAb Treatment in vivo by Magnetic Particle Imaging

Jeff Gaudet1, Gang Ren1, Marco Gerosa2, Yanrong Zhang2, James Mansfield1, Max Wintermark2, Patrick Goodwill1

1 Magnetic Insight, Alameda, California, United States of America
2 Stanford University, Stanford, United States of America


The rapid growth of research into immuno-oncology research has fueled a need to be able to evaluate the efficacy of immunotherapies in a timely and comprehensive fashion. However, established non-invasive multimodality imaging approaches have been insufficient to meet all the requirements. Magnetic Particle Imaging (MPI) is a novel tomographic molecular imaging technique that can be used to non-invasively track iron-oxide in 3D in vivo, with sensitivity and specificity similar to nuclear medicine but without the complex workflow, safety, and half-life limitations.


A murine breast tumour model was established by injecting 3 x 105 4T1 cells into the 4th mammary fat pad of 8-10 week female Blab/c mice. A CD47 monoclonal antibody (mAb) treatment was initiated by intraperitoneal injection of CD47 mAb (Bio-X Cell) at day 6 after tumour implantation. The treated and the control groups were then injected with an iron-oxide MPI tracer (6mg/kg) and then 3D MPI images were acquired 8, 11, 14 and 17 days after implantation. µCT and MRI were also acquired with a T2-weighted multi-slice, multi-echo (MSME) sequences at above time points. Both MPI and MRI images were co-registered and quantified. Tumours, liver, spleen and draining lymph nodes were then harvested, imaged, fixed, and stained with Perls Prussian blue for analysis of iron content.


Superparamagnetic iron oxide nanoparticles accumulation has been shown qualitatively to increase following CD47 mAb treatment [1]. To determine iron accumulation within the tumour, MPI was co-registered to both MRI and µCT for anatomical context. All mice showed an accumulation of nanoparticles in the tumour and liver following injection in both MRI and MPI. Quantitation of iron at the tumour was performed and compared between the CD47-treated and untreated groups at every imaging time point with MPI. CD47 mAb treated breast tumours demonstrated enhanced positive contrast on 3D MPI images while a shortened T2 relaxation on MRI images. Iron content within the tumor was confirmed by ex vivo scanning followed by staining.


By combining the sensitivity, specificity and quantitation potentials of MPI, information can be obtained on a preclinical model that monitors the efficacy of CD47 mAb cancer immunotherapy. With the high spatial resolution provided by MRI, coregistered MRI-MPI can be utilized to evaluate the response of tumor-associated macrophages to CD47 mAb and other cancer immunotherapies.

AcknowledgmentThis research was supported by NIDA and NIBIB of  the National Institutes of Health under award  numbers R43EB020463 and R43DA041814
[1] Mohanty S, Yerneni K, Theruvath JL, et al. Nanoparticle enhanced MRI can monitor macrophage response to CD47 mAb immunotherapy in osteosarcoma. Cell Death Dis. 2019;10(2):36. Published 2019 Jan 15. doi:10.1038/s41419-018-1285-3
Keywords: magnetic particle imaging, MPI, CD47, Immunotherapy