15th European Molecular Imaging Meeting
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Understanding Tumour Biology

Session chair: Federica Pisaneschi (Houston, USA); Eric Kajzel (Leiden, Netherlands)
 
Shortcut: PW02
Date: Wednesday, 26 August, 2020, 5:30 p.m. - 7:00 p.m.
Session type: Poster

Contents

Abstract/Video opens by clicking at the talk title.

125

The role of endothelial adhesion in tumour progression and metastasis

Christina Arapatzi1, Pinelopi Nikolopoulou1, Vasso Kostourou1

1 BSRC Alexander Fleming, Intitute of Bioinovation, Athens, Greece

Introduction

Blood vessel formation and function are prerequisite for tumour development, malignant progression and metastasis. Endothelial cells that constitute blood vessels, form dynamic adhesions with each other and the extracellular matrix, capable to integrate signals by the tumour microenvironment that drive angiogenesis and tumour malignancy.

Methods

To explore the role of endothelial adhesion proteins in tumour biology, we have developed mouse genetic models with inducible and endothelial-specific deletion of key adhesion regulators, Talin and ILK. We examined tumour blood vessel formation and functionality in subcutaneously grown syngeneic tumours exploiting various in vivo animal imaging techniques, including μPET, ultrasound and whole animal fluorescent and luminescence imaging. We combined these studies with in vitro histopathological analysis and immunofluorescent staining for specific markers of blood vessels and tumour microenvironment.

Results/Discussion

We showed that endothelial Talin deletion at initial stages of tumour development blocks tumour angiogenesis and disrupts blood vessel morphology, resulting in strikingly reduced tumour growth. Using μPET imaging we demonstrated that endothelial Talin deletion, at established tumours, reduces significantly tumour glycolysis and causes tumour regression. We verified these findings by immunofluorescent analysis on tumour sections which showed reduced proliferation, increased hypoxia, apoptosis and necrosis. We also demonstrated that endothelial Talin deletion increases capillary leakage and results in higher number of small metastatic loci that are unable to develop into big malignant lesions. Similarly, we observed diminished tumour growth upon endothelial ILK deletion, caused by impaired tumour angiogenesis. We performed ultrasound experiments using microbubbles as contrast agent for small tumour blood vessels and showed that endothelial ILK deletion diminishes vessel functionality.

Conclusions

Taken together, different in vivo imaging modalities enabled the longitudinal studies of tumour development and gave us insight into dynamic changes in tumour pathology elicited by endothelial specific deletion of adhesion proteins. Specifically, we demonstrated that Talin and ILK are essential for blood vessel function during tumour development.

AcknowledgmentWe acknowledge support of this work by the project “A Greek Research Infrastructure for Visualizing and Monitoring Fundamental Biological Processes (BIOIMAGING-GR)” (MIS 5002755) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund) and the grant from World Wide Cancer Research
Keywords: tumour, animal, vessels
126

Optical Imaging as a Tool for Pre-Qualification of Tumour Vascularity in Rodent Cancer Models: Initial Experience

Cathryn Driver1, 2, Rose Hayeshi3, Jillene Visser2, 1, Ambrose Okem3, Jan Rijn Zeevaart1, 2, 3, Thomas Ebenhan2, 4

1 Necsa, Radiochemistry, Pretoria, South Africa
2 NuMeRI, Pre-clinical Imaging Facility, Pretoria, South Africa
3 North West University, DST/NWU Pre-Clinical Drug Development Platform, Potchefstroom, South Africa
4 University of Pretoria, Nuclear Medicine, Pretoria, South Africa

Introduction

Tumours are hypervascularised with vasculature that is hyperpermeable and with defective lymphatic drainage.1 This leads to the enhanced permeability and retention (EPR) effect.2 The EPR effect and tumour hypervascularity is a factor in the development of new tumour targeting macromolecules with improved tumour drug delivery. The aim of this study is to determine if fluorescent optical imaging using the macromolecular vascular imaging probe, Superhance 6803, can be used to determine the degree of tumour vascularity and compound uptake in tumours with different volumes in a rodent cancer model.

Methods

C57/BL6 (n=5) and athymic nu/nu mice (n=5) were inoculated over 9 days with E0771 murine breast cancer cells (1 x 106 cells; mammary fat pad allograft) and MCF-7 breast cancer cells (1 x 106 cells; hind flank xenograft) respectively. 14 days after inoculation of the first mouse, the mice were injected intravenously with Superhance 680 (0.15 ml; 4 nmol) and imaged with the IVIS Lumina (ex: 640/660 nm; em: 710 nm) at an early (3h) and late (24h) time point.  The process was repeated 72 h later. The mice were euthanized after the final 24h image and the tumours excised for ex vivo imaging and gross pathology analysis. The fluorescence data was analysed using Living Image® 4.5 system software (PerkinElmer) with respect to tumour volume and Total Radiant Efficiency ([p/s]/[µW/cm²])(RE).

Results/Discussion

At 3h and 24h p.i, both E0771 (tumour volume = 30-1000 mm3) and MCF-7 (tumour volume = 20-600 mm3) animal models exhibited a RE that increased with tumour volume.  The correlation between the volume and RE at 3h is R2 = 0.94 (log) and 0.76 (log) respectively while at 24h the correlation was R2 = 0.88 (linear) and 0.76 (log).
When expressing the tumour fluorescence signal per mm3, a tentative correlation was found: the smaller tumours exhibited a higher RE and therefore compound uptake per mm3 than the larger tumours. The data acquisition for tumour analysis ex vivo was more robust achieving a comparable correlation (E0771: R2 = 0.88 (linear) and MCF-7: R2 = 0.69 (linear)) to the 24h in vivo analysis. Both in vivo and ex vivo analysis showed a clear increase in uptake (≈ 5 fold) of the Superhance 680 by the E0771 tumours over the MCF-7 tumours.

Conclusions

As the tumour volume increases, so does the RE by virtue of the amount of compound that has been accumulated in relation to the tumour size. However, per unit area, the smaller tumours have a higher RE. At this preliminary stage, the only conclusion is that the E0771 tumours are more highly vascularised than the MCF-7 tumours. Further immunohistochemical investigations would better correlate the results to the degree of tumour vascularisation.

Acknowledgment

The authors thank Perkin-Elmer SA for assistance with funding. Thanks also to NWU PCDDP staff  for assistance with the development of the animal models .

References
[1] Bertrand, N.; Wu, J.; Xu, X.; Kamaly, N.; Farokhzad O.C. Adv. Drug Deliv. Rev. 2014, 66, 2-25.
[2] Fang, J.; Nakamura, H.; Maeda, H. Adv. Drug Deliv. Rev. 2011, 63, 136-151.
[3] McMannus, C.; Vasquez, K.; Peterson, J.D.; Perkin-Elmer Inc, Hopkinton, MA, 2018, Application note 014081_01
Keywords: Fluorescent Imaging, Tumour vascularisation, EPR effect
127

Breast Cancer Imaging by way of targeting Glutamate Carboxypeptidase II ?

Thomas Ebenhan1, 7, Malvin T. Tshabalala2, Johncy Mahapane3, 4, Jillene Visser5, 7, Janke Kleynhans1, 7, Cathryn Driver5, 7, June Serem6, Rose Hayeshi3, Anne Grober3, Mike M. Sathekge4, 7, Duncan Cromaty2, Jan Rijn Zeevaart3, 5, 7

1 University of Pretoria, Nuclear Medicine, Pretoria, South Africa
2 University of Pretoria, Pharmacology, Pretoria, South Africa
3 North West University, Preclinical Drug Development Platform, Potcheftroom, South Africa
4 University of Pretoria, Nuclear Medicine and Steve Biko Academic Hospital, Pretoria, South Africa
5 The South African Nuclear Energy Corporation, Radiochemistry, Pelindaba, South Africa
6 University of Pretoria, Anatomy, Pretoria, South Africa
7 Nuclear Medicine Research Infrastructure, Preclinical Imaging Facility, Pretoria, South Africa

Introduction

Breast and prostate cancer mutually represent the most commonly occurring malignancies worldwide in women and men, respectively 1. The mutative states, recurrence capacity, resistance to conventional chemotherapy, low surgery success rate and risks associated with radiotherapy confound the management of both these malignancies. Glutamate Carboxypeptidase II (GCPII) is an emerging target for nuclear medicine, hence this study investigates the relationship between breast cancer related GCPII expression in vitro and in vivo to better understand the accumulation of [68Ga]Ga-PSMA11 in humans 2.

Methods

Cultured breast- (MCF-7, MDA-MB231) and prostate cancer cells (LNCaP; positive control) were used in this study. Cells were fixed, permeabilised and exposed to a YPSMA-1 monoclonal mouse anti-human GCPII antibody and were analysed by confocal microscopy (CM) and flow cytometry (FC). Athymic nude mice breast cancer xenografts were developed by inoculating cells (106; matrigel) subcutaneously in the hind flank (MCF-7) or the mammary fat pad (MDA-MB231). The 68Ga-labeled GCPII ligand known as Glu-NH-CO-NH-Lys(Ahx)-HBED ([68Ga]Ga-PSMA11) was optimised for administration into mice; currently MCF-7 and MDA-MB231 xenografts were imaged with [18F]FDG-µPET/CT, and 24 h later  [68Ga]Ga-PSMA11-µPET/CT (up to 150 min) and ex vivo biodistribution analysis (%ID/g) were performed.

Results/Discussion

FC resulted in GCPII-positive (80%) cohort of LNCap and low enzyme expression in MCF-7 (24%) and MDA-MB231 (22%). CM exhibited faint expression of GCPII in breast cancer cells. [68Ga]Ga-PSMA11 prurification using a SepPakC18 cartridge (0.3 mL 25% ethanolic saline) yield sufficient radiochemical efficiency (~69%; n=5) and radiochemical yield (165±70 MBq; n=7) of [68Ga]Ga-PSMA11. The tumour volume (mm3) was similar for MCF-7 (136±99; n=4) and MDA-MB231 (150±31; n=5; p=0.365). Mice (20±3 g) were administered with 12±4 MBq and 16±4 for [18F]FDG and [68Ga]Ga-PSMA11, respectively. MCF-7 tumours showed faint uptake of [18F]FDG compared to good visualization of the MDA-MB231 tumours. Despite expected tracer uptake, both tumour xenografts did not accumulate [68Ga]Ga-PSMA11 in vivo. [68Ga]Ga-PSMA11 uptake (%ID/g) in MCF-7 tumours (2.8±1.3) was significant over MDA-MB231 tumours (0.25±0.12; p=0.026); 3-fold over muscle (p=0.123) 4-fold over liver (p=0.097) and 5-fold over heart uptake (p=0.082).

Conclusions

Investigation of MCF-7 and MDA-MB231 may suggest expression of GCPII in vitro; however uptake in the respective tumour xenografts imaged with [68Ga]Ga-PSMA11 μPET/CT was not adequate to visualize those tumours in vivo, which is possibly due to the suboptimal expression of GCPII. Further investigations are required to better understand the successful clinical imaging of breast cancer with [68Ga]Ga-PSMA11-PET/CT.

AcknowledgmentThe authors thank the NuMeRI for funding contribution to this project. The PCCDP staff and Mrs Delene van Wyk are thanked for support of the animal animal development and assistance with animal handling and PET/CT imaging.
References
[1] Siegel, RL, Miller, KD and Jemal, A. 2019, ‘Cancer statistics, 2019’, CA Cancer J Clin, 69, 7-34.
[2] Sathekge, M, Lengana, T, Modiselle, M, Vorster, M, Zeevaart, J, Maes, A, Ebenhan, T, Van de Wiele, C. 2017, ‘68Ga-PSMA-HBED-CC PET imaging in breast carcinoma patients.’, Eur J Nucl Med Mol Imaging, 44(4),689-694
Keywords: breast cancer, microPET/CT, PSMA, GCP II ligands
128

Effect of hypoxia and choline kinase inhibitor on choline metabolism of brain tumour cells

Claire L. Kelly1, 2, Martyna Wydrzynska2, Sofya Osharovich4, Marie Phelan3, Violaine See2, Edward J. Delikatny4, Harish Poptani1

1 University of Liverpool, Center for Pre Clinical Imaging, Liverpool, United Kingdom
2 University of Liverpool, Center for Cell Imaging, Liverpool, United Kingdom
3 University of Liverpool, NMR Center, Liverpool, United Kingdom
4 University of Pennsylvania, Department of Radiology, Philadelphia, United States of America

Introduction

Cellular dysregulation occurs during hypoxia1, which is commonly observed in Glioblastoma (GBM) and is responsible for its invasive phenotype1, as well as radiation resistance. Tumour cells adapt to hypoxia via the hypoxia inducible factor HIF-1α. 2, 3 Choline kinase alpha (ChoKa) is typically over-expressed in hypoxic conditions and is regulated by HIF-1α3 4, and is also responsible for increased phosphocholine (PC) in tumours. 4 Inhibition of ChoKa has been shown to have therapeutic effects, however, the relationship of choline metabolism to hypoxia is not well understood.

Methods

GBM lines (F98, 9L, U87, U251) were cultured and were incubated for 3 days in 21% O₂ (normoxia) or 1% O₂ (hypoxia) in a hypoxic workstation. Cell viability and proliferation was measured by trypan blue exclusion and cell invasion under normoxic conditions was assessed using a 3D spheroid model in a Matrigel matrix and imaged on a spinning disk confocal microscope.
Choline metabolism was assessed using high resolution ¹H NMR. Cells grown under hypoxic or normoxic conditions were treated with 10μM JAS239/0.1% DMSO or 0.1% DMSO control and incubated for 4 hours. Metabolites were extracted via acetonitrile extraction and 1D ¹H NMR spectra were acquired on a Bruker Avance III HD 700MHz spectrometer using a Carr-Purcell-Meiboom-Gill (CPMG) sequence. 

Results/Discussion

¹H NMR spectra was acquired (figure 1a & 1b) and the % difference in PC:GPC ratio in hypoxic conditions, compared to normoxic cells was calculated (figure 1c). The F98 cells (rat GBM) had the largest reduction in PC:GPC compared to 9L (p<0.005) and U87 (p<0.0001) lines.
The efficacy of JAS239, a ChoK inhibitor, in hypoxic and normoxic conditions. was measured using PC/GPC ratio. A decrease in PC/GPC was observed in both normoxic and hypoxic conditions (figure 1d). However, largest and significant decreases were noted in hypoxic 9L and U251 cell lines (figure 1d).
Since increased choline metabolism is a marker of proliferation, 4 we tested the effects of hypoxia on proliferation and viability (figure 2a). Proliferation was reduced in all cell lines; however, this was more pronounced in the rat (F98 & 9L) lines although viability was not affected. In normoxic conditions JAS239 also led to a decrease in the cells ability to invade into the surrounding matrix (figure 2b).

Conclusions

We observed that hypoxia significantly reduces the PC:GPC ratio in addition to a marked reduction in cellular proliferation. Since hypoxia did not affect cell viability, this suggests prolonged hypoxia may induce a cell cycle arrest. JAS239 efficacy was higher in the hypoxic 9L and U251, which are less aggressive GBM lines indicating an interplay between hypoxia, invasion and choline metabolism.

AcknowledgmentAll NMR data was aquired at the NMR centre, University of Liverpool. Cell imaging was aquired at the Centre of Cell Imaging, University of Liverpool. 
References
[1] Masoud, G. N., & Li, W. (2015). HIF-1α pathway: Role, regulation and intervention for cancer therapy. Acta Pharmaceutica Sinica B. https://doi.org/10.1016/j.apsb.2015.05.007
[2] Bansal, A., Harris, R. A., & DeGrado, T. R. (2012). Choline phosphorylation and regulation of transcription of choline kinase α in hypoxia. Journal of Lipid Research, 53(1), 149–157. https://doi.org/10.1194/jlr.M021030
[3] Glunde, K., Shah, T., Winnard, P. T., Raman, V., Takagi, T., Vesuna, F., … Bhujwalla, Z. M. (2008). Hypoxia regulates choline kinase expression through hypoxia-inducible factor-1α signaling in a human prostate cancer model. Cancer Research, 68(1), 172–180. https://doi.org/10.1158/0008-5472.CAN-07-2678
[4] Arlauckas, S. P., Popov, A. V., & Delikatny, E. J. (2016). Choline kinase alpha - Putting the ChoK-hold on tumor metabolism. Progress in Lipid Research, 63, 28–40. https://doi.org/10.1016/j.plipres.2016.03.005
[5] Al-Mutawa, Y. K., Herrmann, A., Corbishley, C., Losty, P. D., Phelan, M., & Sée, V. (2018). Effects of hypoxic preconditioning on neuroblastoma tumour oxygenation and metabolic signature in a chick embryo model. Bioscience Reports, 38(4), 1–15. https://doi.org/10.1042/BSR20180185
Figure 1.
Example spectra of F98 cells treated with 0.1% DMSO (A) or 10µM JAS239 (B) under hypoxic conditions. Figure 1 C shows the % change in PC:GPC ratio of hypoxic versus normoxic cells. The % decrease was more pronounced in rat F98 and 9L (p=0.0024) lines compared to human U87 (p<0.001) and U251 (p=0.087) cells. The effect of JAS239 on PC:GPC ratio between normoxic and hypoxic conditions (1D) shows PC:GPC is significantly reduced in 9L (p=0.0002) and U251 (p=0.0037) hypoxic cells compared to normoxic cells. F98 and U87 cells show similar changes in PC:GPC in both normoxic and hypoxic conditions.
Figure 2.

Hypoxia reduces cell proliferation (2A) to a larger degree in rat F98 and 9L cell lines compared to the human U87 and U251 GBM lines without affecting cell viability. In normoxic conditions JAS239 appears to reduce U87 cell invasion (2B) over the course of 48 hours compared to control.

Keywords: data analysis, sample collection, figure making
129

Chemo-sensitivity of ovarian cancer enhanced by nanotechnology-based cisplatin intracellular delivery

Barbara Bortot1, Erica Valencic1, Danilo Licastro2, Chiara Agostinis1, Roberta Bulla3, Matteo Crosera4, Gianpiero Adami4, Enrico Rampazzo5, Giuseppe Ricci1, 6, Gabriella Zito1, Federico Romano1, Rita Ceccherini7, Giovanni Maria Severini1, Stefania Biffi1

1 Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
2 CBM Scrl, Trieste, Italy
3 Department of Life Sciences, University of Trieste, Trieste, Italy
4 Department of Chemical and Pharmaceutical Sciences, University of Trieste, Trieste, Italy
5 Department of Chemistry, Bologna, Italy
6 Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy
7 SC Centro Sociale Oncologico (ASS1), Trieste, Italy

Introduction

Epithelial Ovarian Cancer (EOC) is the deadliest gynecologic malignancy [1]. The reason for the high death rate is the diagnosis at an advanced stage with the widely metastatic peritoneal disease [1]. The primary treatment is based on various combinations of optimum surgical debulking with chemotherapy [2,3]. The unmet clinical need in EOC management stems from the fact that chemotherapy fails to eradicate all the cancer cells in the peritoneal cavity. Herein, the putative role of intracellular cisplatin concentration in regulating chemosensitivity of ovarian cancer was investigated.

Methods

PLGA nanoparticles were produced with an average hydrodynamic diameter of 70 nm under class 100 cleanroom conditions. SKOV3-luc cells were used as an in-vitro model and to establish a xenografted tumor model. Cell cytotoxicity was assessed after exposure of cells (SKOV3-luc and primary cancer cells derived from ascites of patients) to cisplatin or nanoparticle-cisplatin, analyzing cell viability, apoptosis, and cell proliferation. The effect of treatments on EMT and CSC-like phenotype was studied by analyzing a panel of markers by flow cytometry. Intracellular platinum concentration was determined by ICS-MS. The gene expression profiling was performed using RNA-seq. Biodistribution, tumor accumulation of CY5.5-labelled-nanoparticles, and in-vivo efficiency were analyzed by optical imaging.

Results/Discussion

We demonstrate that platinum concentration functions as a critical factor in driving features of EMT. High intracellular platinum concentration significantly enhanced apoptosis and prevented EMT phenotype in ovarian tumor cells. Several pathways were associated with chemosensitivity, and we identified genes consistently up-regulated or down-regulated shared by the nanoparticle-treated samples compared to the free drug. Furthermore, the in-vivo biodistribution study showed that intravenously injected nanoparticles accumulated in tumor tissues due to the enhanced permeability and retention effect.

Conclusions

PLGA nanoparticles loaded with cisplatin have great potential as a new therapeutic approach for patients with ovarian cancer. Furthermore, considering the capacity of these polymeric nanoparticles to load other drugs and different imaging probes, they provide a strategy to improve the in-vivo imaging of ovarian cancer mass and to visualize the therapeutic delivery site.

Acknowledgment

The research was funded by a Ministry of Health “Ricerca Corrente” Grant to IRCSS Burlo Garofolo, n°19/2016, and by "Bando POR FESR 2014-2020. Attività 1.3.b", project title: "TICheP: Tecnologie Innovative per la Chemioterapia Personalizzata".

References
[1] Jayson, GC, Kohn, EC, Kitchener, HC, Ledermann, JA 2014, ‘Ovarian cancer’, Lancet Lond. Engl., 384, 1376–1388.
[2] Di Lorenzo, G, Ricci, G, Severini, GM, Romano, F, Biffi, S 2018 ‘Imaging and therapy of ovarian cancer: clinical application of nanoparticles and future perspectives’, Theranostics, 8, 4279–4294.
[3] Castro-Mesta, JF, González-Guerrero, JF, Barrios-Sánchez, P, Villarreal-Cavazos, G 2016 ‘Bases and foundations of the treatment of peritoneal carcinomatosis: Review article’. Med. Univ.,18, 98–104.
Keywords: Ovarian cancer, chemosensitivity, nanoparticle, cisplatin, RNAseq
130

Mapping the dynamics of HURPin space and time during mitosis

Stylianos Didaskalou1, Christos Efstathiou1, Ilona Kesisova1, Avgi Tsolou1, Andreas Girod2, Margy Koffa1

1 Democritus University of Thrace, Department of Molecular Biology and Genetics, Alexandroupolis, Greece
2 University of Luxembourg, LSRU-FSTC, Luxembourg, Luxembourg

Introduction

The mitotic spindle ensures faithful chromosome segregation. Defects in spindle assembly may lead to aneuploidy, a hallmark of cancer. HURP is a Ran-regulated MAP that stabilizes K-fibers and is essential for proper chromosome congression and segregation1. HURP shows a gradient accumulation in the proximity of the chromosomes, regulated by the Aurora A kinase2. We found recently that HURP interacts with other MAPs exhibiting a distinct accumulation on the spindle. To understand how Ran generates a dynamic mitotic spindle, we examined the spatiotemporal dynamics of HURP during cell division.

Methods

HURP dynamics were studied by photoactivation and FRAP experiments. PA-GFP-HURP were was photoactivated at three different zones on the spindle. To analyze data we plotted the intensity distribution3 followed by the position of maximum intensity as a function of time. For FRAP experiments we used cells expressing EGFP-HURP in the presence or absence of an Aurora A inhibitor. Fluorescence recovery was analyzed in the three zones used for photoactivation experiments.

Results/Discussion

HURP photoactivated at the chromosome zone moves poleward, faster than MT flux, indicating a dynein-mediated movement. HURP photoactivated at the pole zone moves towards the MT (+) end, indicating a kinesin-mediated movement. Molecules activated at the interzone showed a biphasic movement, with HURP moving initially towards the pole and then towards the chromosomes. Analysis of FRAP showed that HURP molecules as they diffuse from the unbleached half-spindle, they bind to MTs in the vicinity of chromosomes and move towards the pole. Upon HURP phosphorylation by Aurora A at the interzone, HURP changes direction and finally accumulates in the vicinity of the chromosomes.

Conclusions

We propose a model where HURP is able to interact with other Ran-regulated MAPs that accumulate near the spindle poles, through a transient complex, regulated in space and time. Mapping the dynamic interaction profile of the Ran-regulated MAPs will help us to better understand the complex process of spindle assembly.

Acknowledgment

We would like to thank the CIBIT microscopy facilities in MBG-DUTH, EMBL-Heidelberg, and LSRU-Luxembourg

References
[1] Koffa, M. D. et al. Curr. Biol. 16, 743–754 (2006).
[2] Kesisova IA et al. PLoS One, 8(3):e58485. (2013).
[3] Uteng, M., et al. J. Cell Biol. 182, 715–726 (2008).
Keywords: Mitosis, MAPs, Aneuploidy
131

In Vitro - In Silico integration of PML-mediated Glioblastoma evolution

Maria Tampakaki1, 2, Mariam-Eleni Oraiopoulou1, Eleftheria Tzamali1, Giorgos Tzedakis1, Stylianos Psycharakis3, Giannis Zacharakis3, Vangelis Sakkalis1, Joseph Papamatheakis4, 5

1 Foundation for Research and Technology-Hellas, Institute of Computer Science, Heraklion, Greece
2 University of Crete, School of Medicine, Heraklion, Greece
3 Foundation for Research and Technology-Hellas, Institute of Electronic Structure and Laser, Heraklion, Greece
4 Foundation for Research and Technology-Hellas, Institute of Molecular Biology and Biotechnology, Heraklion, Greece
5 University of Crete, Department of Biology, Heraklion, Greece

Introduction

The promyelocytic leukemia protein (PML) is a cell regulator, expressed in all tissues1 and modifications in its expression have been associated with various types of cancer2,3. In brain, PML regulates both neuronal migration and invasion of Glioblastoma (GB) cells via the EZH2 protein4. In order to examine the effect of PML in GB physiology, we studied the growth and invasive properties of GB PML-overexpressing cells using optical and fluorescence microscopy. We additionally applied a cancer predictive computational model to investigate further the PML mechanisms of action in GB invasion.

Methods

The U87MG GB cell line was lentivirusly transfected to conditionally overexpress the PML protein (U87MG-PML OE) conjugated with the fluorophore DsRed. 3D spheroids were generated using the hanging drop technique and cultured in invasion allowing or inhibiting conditions. Brightfield photo-micrographs were captured every 24h. The sensitivity to DZNeP, an EZH2 inhibitor, was also tested by estimating the growth elimination over time. Light Sheet Fluorescence and Confocal Microscopy were used to visualize the PML expression (DsRed), the cell somata (H2DC-FDA) and the cell death pattern (Draq7). A computational individual cell-based model was further parametrized based on the biological observations. A computational parameter study was performed to estimate the effect of PML on GB evolution.

Results/Discussion

U87MG-PML OE cells exhibited significant differences compared to the U87MG-PML control regarding their growth and invasive properties. They generated smaller spheroids, indicating lower proliferative rate. Their invasive pattern was common in both cell lines adopting the typical starburst morphology5, while presenting different migration dynamics Fig 1a-d. The presence of PML did not alter the effect of DZNeP on tumour growth, however, it affected significantly the invasive properties Fig 1 e&f. The U87MG-PML control spheroids maintained their invasive pattern under DZNeP presence, whereas the invasive capacity of the U87MG-PML OE cells was almost completely inhibited. The computational model indicated that PML regulates the promptness of one cell to detach from the tumour core and migrate without affecting the speed of cellular distribution Fig 2. Our overall results propose that PML inhibits tumour growth, while it promotes migration through the PML-EZH2 pathway in human gliomas4.

Conclusions

GB expansion is attributed to both excessive proliferation and local spreading. Our results are in line with previous findings1,4 indicating that these two functions are modulated by distinct cellular mechanisms. Unravelling further the role of PML in GB progression could set PML as a therapeutic target aiming at eliminating multiple sub-clones depending on their proliferative and/or invasive phenotype within the heterogeneous GB tumour.

Acknowledgment

Authors would like to thank Angela Pasparaki and Takis Makatounakis for all the help they provided. This work was supported by the project “BIOIMAGING-GR” (MIS5002755) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund). Also supported by the project “KRIPIS ΙΙ-VITAD (MIS 5002469)”, as well as the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under the HFRI PhD Fellowship grant (GA. no. 130178/I2/31-7-2017).

References
[1] Guan, D. & Kao, H. Y. The function, regulation and therapeutic implications of the tumor suppressor protein, PML. Cell and Bioscience 5, (2015).
[2] Lee, H. E. et al. Loss of promyelocytic leukemia protein in human gastric cancers. Cancer Lett. 247, 103–109 (2007).
[3] Martín-Martín, N. et al. Stratification and therapeutic potential of PML in metastatic breast cancer. Nat. Commun. 7, (2016).
[4] Amodeo, V. et al. A PML/Slit Axis Controls Physiological Cell Migration and Cancer Invasion in the CNS. Cell Rep. 20, 411–426 (2017).
[5] Oraiopoulou, M.-E. et al. Integrating in vitro experiments with in silico approaches for Glioblastoma invasion: the role of cell-to-cell adhesion heterogeneity. Sci. Rep. 8, 16200 (2018).
3D growth and invasion of the U87MG-PML control and OE cells at 72h.

The figures depict confocal microscopy scans of the growth (a & c) and the invasive condition (b & d) for the control and the PML OE spheroids. The respective LSFM scans for the growth condition of the control and the PML OE spheroids are shown within the frames. e. & f. depict confocal scans of the PML OE in the growth and the invasive condition under DZNeP treatment. Scale bar is set at 100um. Dead nuclei are shown in red, PML expression is shown in green and the cell somata are shown in blue.

In silico (left) compared to the in vitro (right) experiments after 144h of free invasion.

In a & b, the PML OE invasive pattern is illustrated wo/w DZNeP treatment, respectively. In order to simulate the invasive pattern under DZNeP treatment, the initial phenotypic ratio of the spheroids had to be altered from 2 Motile : 1 Adhesive to 1 Motile : 4 Adhesive cells.

Keywords: Glioblastoma, in silico modeling, LSFM, confocal, brain cancer
132

Radiopharmacokinetic analysis of [18F]FAZA dynamic PET imaging acquisitions, for highlighting tumor profiles

Claire Provost1, 2, Hamid Mammar3, Camille Schmitt1, Olivier Madar1, Laurence Champion2

1 Institut Curie, Radiopharmacology, Saint-Cloud, France
2 Institut Curie, Nuclear Medicine, Saint-Cloud, France
3 Institut Curie, Radio-therapy, Orsay, France

Introduction

Functional PET can be useful for a better understanding of specific processes activated by the tumor. Tumor hypoxia is known to induce resistant to therapy, aggressive phenotype and propensity for being invasive. [18F]FAZA is a radiolabeled nitroimidazole analogue for hypoxia PET imaging. Most images are analysed in static acquisitions, after the complete distribution of tracer. However, some studies used dynamic acquisition to evaluate the biodistribution evolution. The aim of this study was to investigate dynamic PET to analyze radiopharmacokinetics of [18F]FAZA in different profile tumors.

Methods

In the context of a clinical trial (NCT02802969), patients were enrolled for chordomas suspicion on MRI and underwent [18F]FAZA PET/CT. PET agent were injected intravenously with 5MBq/kg, early imaging at injection time with dynamic acquisition centered on the tumor was performed over a period of 20 minutes. The protocol for dynamic acquisition was constituted by 22 frames (6x10sec; 6x30sec; 5x60sec; 5x120sec). The reconstructed spatial resolution was 4x4mm2 with a 4mm slice thickness also. Blood input function was defined in arteries at the early time points (first 40 second frame). A tumor volume of interest (VOI) was drawn in the most intense frame of dynamic acquisition. This two volumes were reported in all frame for generate time activity curves (TACs) of blood and tumor.

Results/Discussion

Diagnostic with pathology results have shown that two of enrolled patients were finally not chordomas but paraganglioma and pituitary adenoma, with this results we decided to investagate radipharmacocinetic profiles. These 3 tumors are all slow-growing tumors. In our analyses, profile of paraganglioma and pituitary adenoma have high vascularization, shown by an important uptake and a quick distribution in the tumor from the first 40 sec in dynamic acquisition. Paraganglioma seems more vascularized than pituitary adenoma because of a sharp slope. In the second part of TACs we observed that tumors seem non hypoxic, shown by a constant elimination of [18F]FAZA. In contrast, chordoma with a poor vascularization, shown by a low uptake in the tumor from the first 40 sec and an active hypoxic process, shown by a continuous low increase of the [18F]FAZA over time. Profiles seem discriminant between the 3 pathologies, thus useful for tumors which can appear similar on conventional imagery.

Conclusions

Through visual imaging and quantification of blood and tumor TACs, distinct tumor profiles have been highlighted. The first part of TACs can estimate tumor vascularization, the second part of TACs, after around 135 sec of biodistribution, can estimate the beginning of accumulation in hypoxic cells tumor of [18F]FAZA. In this study, early PET dynamic acquisitions were allowed to give some additional information than tardive static acquisitions.

References
[1] Halmos GB, Bruine de Bruin L 2014, Head and neck tumor hypoxia imaging by 18F-fluoroazomycin-arabinoside (18F-FAZA)-PET: a review, Clin Nucl Med. 39(1):44-8.
[2] Vaupel P, Harrison L 2004, Tumor hypoxia: causative factors, compensatory mechanisms, and cellular response. Oncologist.; 9 (Suppl 5):4-9.
[3] Srilatha PS, Rao L 2017 Locally Aggressive Primary Intraosseous Paraganglioma of Sacrum. J Clin Diagn Res. 11(9):ED09-ED11
[4] Verwer EE, Zegers CM, 2016 Pharmacokinetic modeling of a novel hypoxia PET tracer [(18)F]HX4 in patients with non-small cell lung cancer. EJNMMI Phys. 3(1):30.
[5] Li F, Joergensen JT, 2014 Kinetic modeling in PET imaging of hypoxia. Am J Nucl Med Mol Imaging. 4(6):490-506.
Time activity curves
Biodistribution of [18F]FAZA in 3 different tumor profils: in red chordoma sacrum, in green paraganglioma and in purple pituitary adenoma. 
Keywords: [18F]FAZA, PET, Dynamic acquisition, tumor profil
133

Ultrasound imaging to track in vivo mechanical deformations in mice colon

Laura Zamfirov1, Kévin Sollier2, Maria-Elena Fernandez-Sanchez2, Charlie Demené1, Emmanuel Farge2, Mickael Tanter1

1 ESPCI Paris, CNRS, PSL research university, Physics for Medicine Paris, Paris, France
2 PSL Research University, CNRS UMR 168, Institut Curie, Mechanics and Genetics of Embryonic and Tumour Development, Paris, France

Introduction

Recently, tumorigenesis in mice colon has been shown to be modulated by static and local mechanical forces in vivo [1]. Ultrasound (US) can be a versatile non-invasive tool to quantify natural mechanical forces in situ. In this context, peristaltic activity in mice colon was monitored in vivo at a mesoscopic scale using ultrafast ultrasound imaging. We showed that local deformations propagates as waves at a 1mm/s speed and that this propagation is strongly modulated by the cannabinoid receptor agonist Win 55,212-2.

Methods

Experiments were conducted in anesthetized wild-type mice, before and after intraperitoneal injection of an inhibitor of colonic peristaltic waves (WIN55,212-2; Sigma) [2]. A BMode imaging sequence was used to localize and select a sagittal plane of mice colon. Ultrafast US imaging was performed using a linear ultrasonic probe (128 elements, 15MHz, 110µm pitch) driven by a programmable ultrasound system (Verasonics). US plane-wave transmission with n = 6 tilted angles were fired at a 3-kHz pulse repetition frequency during 60s. For each acquisition, a space-time displacement map was derived along a segment corresponding to the colonic wall. The dispersion diagram k-ω was computed to prove the presence of propagative phenomena and quantify wave speeds and amplitudes.

Results/Discussion

In the pre-drug period (control), peristaltic activity in mice colon (n=4) was shown along the colon wall on space-time representation. This reflex was characterized by the propagation of regular successive waves, with a 40µm displacement amplitude, towards anterograde (oro-aboral) direction. The dispersion diagram confirmed the presence of this propagative phenomenon for a signal centered at 0.2 s-1. The average propagation speed is 1.02 mm/s. The intraperitoneal injection of WIN55,212-2 in these mice resulted in an attenuation of peristaltic activity. The energy spectral density of the dispersion diagram at the frequency of interest significantly decreased by twofold compared to control (2.5E+09 versus 4.5E+08, p< 0.05). The velocity of the contraction waves also decreased (0.7 mm/s). US ultrafast imaging enables to quantitatively characterize and monitor very small propagative deformation non-invasively.

Conclusions

These data show that US is a powerful tool to remotely monitor peristaltic waves and differentiate mechanical characteristics of tissue environment. It demonstrates the efficiency and benefit of ultrasound as a pre-clinical device to track mechanical deformation in vivo and better understand tumor growth. It is the first step towards an extensive analysis of the impact of mechanical forces on colonic cancer using ultrasound.

References
[1] Fernandez-Sanchez, ME, et al, Nature, Vol 523, 2015
[2] Mancinelli, R, et al, Life Sciences, 69, 2001
Keywords: ultrafast ultrasound, mechanical force, peristaltism, colon cancer
134

Simultaneous dual isotope PET-SPECT/CT imaging of a metastatic αvβ6 expressing syngeneic murine model of pancreatic cancer

Katie Dexter1, Nicholas F. Brown2, Julie Foster1, Roxana Kashani1, Juliette Chupin1, 3, Julie Cleaver1, Lauren Cutmore2, John F. Marshall2, Jane Sosabowski1

1 Queen Mary University of London, Centre for Biomarkers and Biotherapeutics, Barts Cancer Institute, London, United Kingdom
2 Queen Mary University of London, Centre for Tumour Biology, London, United Kingdom
3 Invicro, London, United Kingdom

Introduction

Integrin αvβ6 is expressed on approximately 90% of human pancreatic cancers and correlates with worse patient prognosis. Therefore αvβ6 is of great interest as a target for imaging and therapy in cancer [1].  Here we investigate the uptake of the αvβ6-specific 111In-DOTA-A20FMDV2 peptide and 18F-FDG using simultaneous SPECT-PET/CT imaging in αvβ6 high- and low-expressing primary and metastatic tumours of KPC derived cells lines. The KPC (KrasLSL.G12D/+;p53R172H/+;PdxCre) transgenic mouse is an established and clinically relevant immunocompetent model of pancreatic ductal adenocarcinoma [2].

Methods

KPC derived cell lines with either minimal or high αvβ6 expression were injected orthotopically into the pancreas of C57BL/6 mice. Tumour bearing mice with high (n=3) and low (n=3) αvβ6 expression were imaged at days 14-15 and 22 post-tumour inoculation. Healthy controls (n=3) were also included.
Mice were injected intravenously with 10-15 MBq of 111In-DOTA-A20FMDV2 3 hours prior to imaging and with 10-15 MBq of 18F-FDG 1 hour prior to imaging. 18F-FDG injections were performed under anaesthesia which was maintained during the uptake period, as was body temperature. The CT contrast agent gastrografin was used to opacify the intestinal tract to distinguish endogenous αvβ6 uptake in the gut from that of tumour. Mice underwent simultaneous SPECT-PET and CT in a MILabs VECTor6CTXUHR scanner.

Results/Discussion

All primary tumours and the majority of metastases in the high αvβ6 group were visualised via simultaneous 111In SPECT and 18F PET imaging.  This was validated through MRI and dissection. Co-localisation was evaluated in metastases using a qualitative scoring system.
111In and 18F co-localisation was seen in all primary tumours with high αvβ6 expression, whereas only 18F signal was detected in primary tumours with low αvβ6 expression.
In the high αvβ6 group, five metastases were investigated with three giving clear co-localisation and one showing moderate co-localisation. None of the metastases in the low αvβ6 group displayed 111In signal above background.
In the tumour-free controls no co-localisation was observed in the pancreatic area, although some clustering around the stomach and intestines was observed as would be expected due to endogenous αvβ6 expression.

Conclusions

Imaging with 111In-DOTA-A20FMDV2 and 18F-FDG in αvβ6 tumour bearing immune competent mice shows co-localisation of signal not only in the primary tumour but also in the majority of metastases. This indicates that metastases express αvβ6 and can be monitored using SPECT. We hypothesise that metastases without co-localisation have proportionally fewer αvβ6 receptors which we are investigating immunohistochemically.

References
[1] Reader, Claire S., et al., 2019, 'The integrin αvβ6 drives pancreatic cancer through diverse mechanisms and represents an effective target for therapy.' The Journal of pathology
[2] Hingorani, Sunil R., et al., 2005, 'Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.'Cancer cell 7.5: 469-483.
Transaxial PET-SPECT/CT slice of a primary tumour with high αvβ6 expression
Transaxial PET-SPECT/CT slice of a metastasis with αvβ6 expression
Keywords: SPECT/CT, PET/CT, Pancreatic Cancer, Metastases, integrin αvβ6
135

The impact of multidrug resistance on tumor microenvironment remodeling in murine 4T1 breast cancer models

Elena Rama1, Okan Tezcan1, Fabian Kiessling1, Twan Lammers1

1 University Clinic RWTH Aachen, Experimental Molecular Imaging, Aachen, Germany

Introduction

Multidrug resistance (MDR) is a major limiting event in chemotherapy. MDR results from changes in several cellular mechanisms such as altered gene expression profiles and signaling networks. These cellular MDR events were evaluated in the direction of tumor microenvironment remodeling. To examine this, we developed sensitive and resistant breast tumor-bearing mouse models. We evaluated the deposition of the extracellular matrix (ECM) components and the density of microvascularization in tumors. Lastly, we studied the accumulation and distribution of nanoparticles in both tumors.

Methods

Multidrug resistant 4T1 cells were established by stepwise selection using doxorubicin dose increments. Sensitive and resistant 4T1 cells were orthotopically injected into mammary fat pad of mice. When tumors reached 10-12 mm, mice were injected with rhodamine lectin and sacrificed. Tumors were excised for the fluorescence-based immunohistochemistry (IHC) analyses. To characterize the extracellular matrix deposition in both tumors, antibodies against collagen-1 (col-1), hyaluronan (HA) and collagen-4 (col-4) were used. The IHC analysis was performed also for the characterization of vascularization by using antibodies against endothelial cells (CD31) and pericytes (αSMA). CT-FMT and fluorescence microscopy were employed to visualize the nanoparticle accumulation and distribution.

Results/Discussion

Histological analyses revealed that the deposition of ECM components, col-1 (Fig.1A), HA (Fig.1B) and col-4 (Fig.1C) was significantly higher in resistant 4T1 tumors than the sensitive ones. The IHC analysis also showed that the density of the microvessels was higher in resistant 4T1 tumors as compared to the sensitive 4T1 tumors (Fig. 1D). The results from CT-FMT imaging showed a higher polymer and liposome accumulation in resistant 4T1 tumors than in sensitive 4T1 tumors (Fig.2A). The fluorescence microscopy analyses demonstrated that, despite having a higher accumulation, the distribution of nanoparticles was poor in resistant 4T1 tumors (Fig.2B). These findings demonstrate that cellular multidrug resistance signaling affects tumor microenvironment remodeling, specifically the composition of the ECM and the microvascularization. These remodeling features of multidrug resistant cells might further limit the success of nanoparticle delivery to MDR tumors.

Conclusions

Our findings show a relative increase in ECM deposition in multidrug resistant 4T1 tumors. We also supply evidence that the amount of angiogenic vessels in MDR tumors is higher than in sensitive tumors. This indicates that when cancer cells are continuously exposed to doxorubicin via stepwise dose increases, they present with a different microenvironment, which may affect the drug delivery to and into tumors.

Acknowledgment

This work was supported by DAAD (57048249) and Deutsche Forschungsgemeinschaft (DFG)  (403039938).

References
[1] Tezcan O, Ojha T, Storm G, Kiessling F, Lammers T. Targeting cellular and microenvironmental multidrug resistance.
[2] Chen Y, Tezcan O, Li D, Beztsinna N, Lou B, Etrych T, Ulbrich K, Metselaar JM, Lammers T, Hennink WE. Overcoming multidrug resistance using folate receptor-targeted and pH-responsive polymeric nanogels containing covalently entrapped doxorubicin. Nanoscale. 2017;9(29):10404-19.

Figure 1. Microenvironment characterization of 4T1 sensitive and resistant tumor models through IHC:

Representative images show the ECM components, collagen-1 (A), hyaluronan (B) and collagen-4 (C) in sensitive and resistant 4T1 tumors. These findings demonstrate that the deposition of ECM components is significantly higher in 4T1 resistant tumors. The analyses for microvessel density reveals a stronger angiogenic profile in in resistant 4T1 tumors as compared to the sensitive 4T1 tumors (D). All images were captured from periphery (P) and core (C) regions of each tumor section (*p<0.05, **p<0.001 and ***p<0.0001).

Figure 2. Accumulation and distribution of polymers and liposomes through CT-FMT and fluorescence mi

CT-FMT images show the accumulation of polymers and liposomes in sensitive and resistant 4T1 tumors in 48h and 72h (A). The accumulation of polymers and liposomes was higher in 4T1 resistant tumors than in sensitive 4T1 tumors. Fluorescence microscopy images demonstrate the distribution of polymers and liposomes (B). These results show that the distribution of polymer and liposomes were poor in resistant 4T1 tumor as compared to sensitive ones (*p<0.05, **p<0.001 and ***p<0.0001).

Keywords: Tumor microenvironment heterogeneity, Multidrug resistance, CT-FMT imaging, Fluorescence microscopy, Nanoparticle delivery
136

Characterization of the sigma-1 receptors status with (S)-(−)-[18F]fluspidine PET of an orthotopic mouse model of glioblastoma to assess its potential in glioblastoma management

Magali Toussaint1, Mathias Kranz1, Winnie Deuther-Conrad1, Marianne Patt2, Osama Sabri2, Peter Brust1

1 Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Department of Neuroradiopharmaceuticals, Research site Leipzig, Leipzig, Germany
2 University Hospital Leipzig, Department of Nuclear Medicine, Leipzig, Germany

Introduction

The sigma-1 receptor (S1R) is a chaperone protein of the mitochondrion-associated endoplasmic reticulum membrane and regarded as potential therapeutic target for a variety of malignant tumors including glioblastoma. Noninvasive assessment of changes in the availability of S1R could help to better understand the pathophysiology of glioblastoma and to improve diagnosis or treatment follow-up. We aim to evaluate the potential of (S)-(−)-[18F]fluspidine, a highly specific S1R radioligand, to characterize the expression of S1R in an orthotopic glioblastoma model in mouse with small-animal PET/MRI.

Methods

U87 human glioblastoma cells were stereotactically implanted into the striatum of three female nude mice (8 weeks old). At a median tumor size of 27 mm3 (determined with MR) 60 min dynamic PET scans were performed after i.v. injection of (S)-(-)-[18F]fluspidine (9.1 ± 2.1 MBq; Am: 140 ± 50 GBq/µmol, EOS). Time-activity curves (TACs) from the tumor and the contralateral regions were analyzed (PMOD v3.9). Peak-to-end ratios (P/E; Peak: SUV mean from 2-9 min, end: SUV mean from 45-60 min) were used to compare regions. Statistics: unpaired two-tailed Student’s T-test (P < 0.05).
To determine the S1R affinity (KD) and density (Bmax) in the tumor and the contralateral region, in vitro autoradiography was performed with (S)-(-)-[18F]fluspidine using cryosections of tumor bearing mice.

Results/Discussion

Autoradiography showed an equal affinity of [18F]fluspidine for S1R in the contralateral and tumor region (KD: 17.5 ± 1.3 vs. 18.0 ± 4.9 nM), but a higher Bmax in the tumor (490 ± 43 vs. 1756 ± 40 fmol/mg prot). PET TACs reflected significantly different kinetic profiles in the tumor and the contralateral side (P/E: 3.10 ± 0.48 vs. 1.81 ± 0.16, p < 0.05) (Figure 1). Interestingly, the tumor regions were characterized not only by a lower initial uptake compared to the contralateral side (SUV2-9 min p.i.: 0.89 vs. 1.17, p < 0.05) but also by a slower washout resulting in equivalent SUVs in both regions at 45 min p.i. (SUV45-60 min p.i.: 0.38 vs. 0.34 respectively). This slower radiotracer washout from the tumor compared to the contralateral side is assumed to be caused by the higher S1R site density found in the tumor, along with the specific pathophysiology of the tumor itself (neovascularization, oncotic pressure).

Conclusions

The PET investigation revealed a significant difference in the pharmacokinetics of (S)-(-)-[18F]fluspidine between the brain tumor and the contralateral region, probably related to different S1R availabilities. These results show the suitability of (S)-(-)-[18F]fluspidine for the non-invasive determination of the S1R status in an orthotopic glioblastoma model.

Figure 1

A) peak-to-end ratio of tumor and contralateral region, B) representative axial PET/MR fusion image of the peak (time frame. 2 – 9 min) and of the end (time frame: 45 – 60 min). Statistics: unpaired two-tailed Student’s T-test (*P < 0.05).

Keywords: Sigma-1 receptor, Positron emission tomography, orthotopic glioblastoma model
137

Modulating macrophage polarization and the tumor microenvironment using iron oxide nanoparticles

Karolin Roemhild1, 2, Kim Lindelauf1, Seyed M. Dadfar1, Sven Thoröe-Boveleth3, Saskia von Stillfried2, Thomas Kraus3, Fabian Kiessling1, 4, Ruth Knüchel-Clarke2, Twan Lammers1

1 University Hospital RWTH Aachen, Institute of Experimental Molecular Imaging, Aachen, Germany
2 University Hospital RWTH Aachen, Institute of Pathology, Aachen, Germany
3 University Hospital RWTH Aachen, Institute of Occupational, Social and Environmental Medicine, Aachen, Germany
4 Fraunhofer MEVIS, Institute for Medical Image Computing, Aachen, Germany

Introduction

Macrophages (MΦ), as part of the tumor microenvironment (TME), are a key player in tumor growth and progression1 and demonstrate a suitable target for anti-cancer therapy, especially in the field of nanomedicine2. In this regard, iron oxide nanoparticles (IONP) have been investigated for their ability to repolarize MΦ and thereby modulate the TME to further improve cancer treatment in tumors with limited treatment options. Amongst these, triple negative breast cancer (TNBC) is characterized by the absence of suitable targets. Our aim is to activate MΦ with IONP to further stimulate the TME.

Methods

Various iron oxide nanoparticle formulations have been evaluated in means of in vitro toxicity via XTT and macrophage uptake utilizing IPC-MS measurements and Prussian blue staining. Furthermore, their ability of polarizing macrophages towards a pro-inflammatory phenotype was investigated by qPCR and flow cytometry. To observe macrophage induced TME changes, hetero-spheroids consisting of triple negative breast cancer cells and macrophages have been co-cultured. For analyzes, heterospheroid sections have been immunofluorescently stained and image acquisition was conducted with fluorescent microscopy.

Results/Discussion

The iron oxide nanoparticles under investigation show little to no toxic effects on murine mouse macrophages after 72 hours of incubation. The ICP-MS results together with the Prussian blue staining revealed increasing amounts of iron depending on prolonged incubation time, increasing concentration and particle type. Furthermore, the iron treated macrophages showed overexpression of pro-inflammatory, anti-tumoral markers as well as up-regulation of anti-inflammatory, pro-tumoral markers when incubated for 18 hours under serum free conditions. This effect was enhanced by a combined treatment of iron oxide nanoparticles with N-acetyl cysteine. The co-culture experiments with TNBC spheroids will further explore the impact of iron oxide nanoparticles on macrophage polarization and its potential influence on TME modulation.

Conclusions

The condition dependent uptake leading to an increase in iron amount in macrophages was shown. We further explored the phenotypical changes upon elevated intracellular iron in macrophages and observed a general up-regulation of pro- and anti-tumoral markers indicating a general activation of macrophage function. This will be further elucidated in co-culture experiments with TNBC cells and finally be combined with available immunotherapy.

Acknowledgment

We gratefully acknowledge financial support by the German Research Foundation (DFG) (GRK2375, grant 331065168).  

References
[1] Aras, S, Raza Zaidi, M, 2017, ‘TAMeless traitors: macrophages in cancer progression and metastasis’, Br J Cancer, 117(11), 1583-1591
[2] Andón, FT, et al., 2017, ‘Targeting tumor associated macrophages: The new challenge for nanomedicine.’, Semin Immunol, 34, 103-113
In vitro evaluation of different iron oxide nanoparticle formulations

1: A. Citrate coated and commercial available IONP (Ferumoxytol) have been tested in means of toxicity. J774A.1 have been incubated for 72 h with different concentrations of IONP and XTT assay has been performed to evaluate the viability of the cells. B. Primary human macrophages have been exposed to different IONP with increasing concentration. After 18 h of incubation Prussian blue staining was performed. C. Primary human macrophages have been treated with selected iron formulation under serum free conditions, with and without N-acetyl cysteine (NAC) for 18 h and further analyzed with qPCR.

Keywords: Iron oxide nanoparticles, Iron metabolism, Tumor microenvironment, Macrophage polarization
139

Interrogating Skin Cancer Molecular Pathology using Imaging Mass Spectrometry

Kelly Dimovska Nilsson1, Noora Neittaanmaki2, Marwa Munem1, Oscar Zaar3, John Paoli3, John S. Fletcher1

1 University of Gothenburg, Department of Chemistry and Molecular Biology, Gothenburg, Sweden
2 Sahlgrenska University Hospital, Department of Clinical Pathology, Gothenburg, Sweden
3 Sahlgrenska Academy, University of Gothenburg, Department of Dermatology and Venereology, Institute of Clinical Sciences, Gothenburg, Sweden

Introduction

Skin cancers are the most common cancers in the fair-skinned population worldwide. Annually, two to three million non-melanoma skin cancers, including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), and 132,000 malignant melanoma (MM) cases are reported worldwide. Considering the suffering that these cancers cause along with the economic costs, it is of great importance to attain a deeper biochemical understanding of skin cancer in order to improve diagnostics and treatments and thereby also patient outcome.

Methods

Time-of-fight secondary ion mass spectrometry (ToF-SIMS) was used to image human tissue sections of BCC, malignant melanoma and melanocytic nevi from patients at Sahlgrenska University Hospital in order to better understand the biochemistry of these cancers and to search for potential biomarkers. ToF-SIMS is starting to be of increasing value to clinicians and has been used on different tissue samples to successfully identify and localise chemical components to various areas of the tissue and answer disease related questions. The main advantage of ToF-SIMS is the label free detection of a large number of different molecules within one experiment on the same tissue section. ToF-SIMS is successfully used for analysing lipids behaviour in biological samples like breast cancer tissue [1].

Results/Discussion

The ToF-SIMS analysis provided detailed chemical information about individual lipid species and the spatial distribution of these within the tissue sections for all cancer types analysed. It was possible to observe differences between the layers of the skin, between healthy and cancerous tissue and in the case of BCC, it was possible to chemically differentiate between high and low aggressive cancerous tissue (Figure 1). There were also indications that hair follicles in cancerous areas of the tissue have a chemical profile more in line with cancerous cells. To understand what was seen in the ToF-SIMS data and to confirm where and from which structures or regions of the tissue the individual signals originated, the ToF-SIMS data was correlated with H&E stained images.

Conclusions

A close collaboration with a pathologist allowed for more clinically relevant conclusions to be drawn from the ToF-SIMS analysis. Using ToF-SIMS to image skin cancer tissue sections, it was possible to create a chemical map to better understand the local biochemistry and to differentiate between not only cancerous and healthy tissue but also within cancerous tissue where it is possible to distinguish between regions of different aggressiveness.

References
[1] Angerer, T. B., Magnusson Y., Landberg,G., and Fletcher, J. S., 2016, Lipid Heterogeneity Resulting from Fatty Acid Processing in the Human Breast Cancer Microenvironment Identified by GCIB-ToF-SIMS Imaging, Analytical Chemistry, 88 (23), 11946-11954
[2] Munem, M., Zaar,O., Dimovska Nilsson, K., Neittaanmaki, N., Paoli, P., and Fletcher, J. S., 2018, Chemical imaging of aggressive basal cell carcinoma using time-of-flight secondary ion mass spectrometry, Biointerphases, 13
Figure 1.
H&E-stained image and single ion images showing signal from ion species originating from tumour areas of varying aggressiveness. The boarder of the aggressive and highly aggressive tumour growth in the H&E stained images is indicated by red lines while demarcation of the aggressive tumour region and the normal tissue is provided by a blue line. Scale bar = 1600 µm. Adapted from Munem et al. 2018 [2].
Keywords: Skin cancer, Basal cell carcinoma, Malignant melanoma, Melanocytic nevi, Lipids