EMIM 2018 ControlCenter

Online Program Overview Session: PW-11

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Discover: Understand Tumour Biology

Session chair: Susanne Kossatz - New York, USA; Michal Neeman - Rehovot, Israel
 
Shortcut: PW-11
Date: Thursday, 22 March, 2018, 11:30 AM
Room: Banquet Hall | level -1
Session type: Poster Session

Abstract

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# 120

Carbon sources for citrate secreted by human prostate cancer cell lines (#554)

A. Heerschap1, V. Breukels1, C. Jansen1, J. Schalken1, F. V. Heijster1

1 Radboud University Nijmegen; Department of Radiology & Nuclear Medicine, Nijmegen, Netherlands

Introduction

A reduced 1H signal of citrate (Cit) in MRSI of human prostates is a hallmark in the detection and characterization of cancer in this organ [1]. Previous cell studies suggest that Cit carbons originate from aspartate as an anaplerotic route to supplement Krebs cycle carbons lost by by Cit secretion [2]. We investigated carbon sources for Cit, secreted by androgen sensitive, PSA producing prostate cancer cell lines, using 13C glucose, pyruvate, aspartate, and glutamine and 1H/13C NMR of cell growth medium to establish metabolic pathways for Cit production and potential imaging biomarkers.   

Methods

The prostate cell lines LNCaP and VCaP were grown in medium supplemented with different 13C-labeled substrates for 48h. 4-mL aliquots of the medium were lyophilized, dissolved in D2O and adjusted to pH 7.4. Then 1H-NMR spectra at 500 MHz and 13C-NMR spectra with WALTZ64 1H-decoupling were obtained.

Results/Discussion

1H NMR spectra of the medium of both cell lines showed clear signals for Cit. [2-13C] pyruvate and [1,6-13C]) glucose showed label in Cit C1/3 and C2/3/4 (Fig 1). U-13C aspartate showed no label for any Cit peak. 5-13C Glu resulted in label at the C1/5 position of secreted Cit, but not at the C6 position.

Quantitative analysis of the labelling profile of Cit revealed that 10 – 20 % of the carbons in Cit originate from pyruvate or glucose carbons entering the TCA cycle via pyruvate carboxylase (PC). Furthermore it is estimated that 20 – 25% of Cit carbons can come from carbons in glutamine. However, the absence of labeling at the C6 position indicates that this occurs via an extramitochondrial pathway.

Conclusions

This study shows that cancerous epithelial prostate cells may still produce and secrete Cit and that in contrast to previous studies aspartate does not appear to be an important anaplerotic substrate to keep the TCA cycle going. Instead anaplerosis seems to occur via the pyruvate carboxylate route and Cit may also be produced extra-mitochondrially by reductive carboxylation. Although these results have to be confirmed in the whole prostate they indicate potential carbon routes for citrate production that have to be taken into account in the choice of metabolic biomarkers for prostate cancer

References

1. Thiele et al. NMR Biomed , 2014; Tayari et al. Invest Rad 2017 2. Costello, Lao and Franklin. Prostate 16. 1990

Fig 1. Part of 13C MR spectrum of cell medium
The 13C2 signal of citrate in the cell medium observed after growth of cells in 13C1,6 glucose. A coupling pattern is visible due to processing of carbons via the Krebs cycle. A quantitative analysis of the coupling indicates that about 1 out of 2 synthetized citrate molecules proceeds via the Krebs cycle and 1 is secreted.     
Keywords: prostate, cancer, citrate, carbon source
# 121

Response evaluation in soft-tissue osteosarcoma metastases using fluoride-18 (18F)-PET/CT radiomics analysis for 223Ra-therapy (#515)

K. Kairemo1, A. Rao2, H. Macapinlac1, V. Subbiah3

1 MD Anderson Cancer Center, Nuclear Medicine, Houston, Texas, United States of America
2 MD Anderson Cancer Center, Biostatistics, Houston, Texas, United States of America
3 MD Anderson Cancer Center, Investigational Cancer Therapeutics, Houston, Texas, United States of America

Introduction

Bone forming osteosarcoma metastases are common in extra-osseous sites especially when metastastic in the lungs. Since bone forming tumors do not shrink in response to therapy,  intra-tumoral and inter-tumoral changes are challenging to evaluate using conventional  imaging modalities.  Sodium fluoride-18 (Na18F)-PET/CT better characterizes bone forming metastases than FDG-PET CT scan.

Methods

A dose-escalation trial of 223RaCl2 (50, 75, and 100 kBq 223RaCl2 /kg ) enrolled pts age 15y+ w advanced osteosarcma. Molecular imaging with technetium (Tc)-99m bone scan, FDG PET or sodium fluoride-18 (NaF) PET was done at baseline and at restaging.  We developed a PET radiomics method for analyzing NaF, i.e. radioactive 18F-  -atom concentration in soft-tissues, approximately 1000 concentration data points  for 18F-  per 1 cm3 metastatic tumor.  We analysed data from the SUV intensity values, we obtained (i) the histogram of intensities, (ii) entropy value. The fractions of voxels were plotted against intensity bins.

Results/Discussion

We applied this method to two lung metastases and two soft-tissue metastases, before and after two cycles of Ra-223-treatment. The metastatic tumors were 80 -120 cm3 in size. This alpha-therapy is effective only in the close vicinity of the target cell, i.e. 4-5 cell diameter range. All these metastases demonstrated mixed type of response, where RECIST nor PERCIST type of criteria do not apply.

In these metastatic sites there were abundant conformational changes which could be considered as therapeutic changes. These can be explained e.g. by changes in entropy, kurtosis. The configuration, amount of distortion etc. can give us guidance of responding tumor parts.

Conclusions

Radiomics can inform intra-tumoral and inter tumoral heterogeneity in response of bone forming osteosarcoma to alpha particle therapy. With the help of this new PET radiomics method for 18F-  -atom concentration, this can be studied in detail,  bone formation regions can be identified. Characteristic changes in entropy may be used to quantify the treatment response.  Further larger validated studies are warranted.

Keywords: osteosarcoma, soft-tissue metastases, fluoride-18-PET, Ra-223 treatment, radiomics, alpha-radiation, response evaluation, phase I trial
# 122

FLT PET-CT imaging to determine treatment effectiveness on an Acute Myeloid Leukemia (AML) patient-derived xenograft (PDX) (#451)

M. Mayoral Safont1, M. Popa1, 6, H. Espedal3, T. C. H. Adamsen4, ?. Bruserud2, 5, B. T. Gjertsen1, 2, E. McCormack1, 2

1 University of Bergen, Center for Cancer Biomarkers CCBIO, Department of Clinical Science, Faculty of Medicine and Dentistry, Bergen, Norway
2 Haukeland University Hospital, Department of Internal Medicine, Hematology Section, Bergen, Norway
3 University of Bergen, Department of Biomedicine,Translational Cancer Research, Bergen, Norway
4 Haukeland University Hospital, Center for Nuclear Medicine/PET, Bergen, Norway
5 University of Bergen, Department of Clinical Science, Leukemia Research Group, Faculty of Medicine and Dentistry, Bergen, Norway
6 Kinn Therapeutics, Bergen, Norway

Introduction

Animal models for AML serve for making the disease accessible to experimentation impossible in human patients. The establishment of PDX models have served for understanding AML and to establish basis for preclinical development of novel therapeutics. It has previously been published an application of fluorescently labelled Monoclonal antibodies to demonstrate preclinical efficacy1. A caveat of this approach is that it is not quantitative, complicated by background antibody uptake and limited in clinical translatability.

Methods

Positron emission tomography-Computed tomography (PET-CT) is a nuclear medicine technique used in both preclinical and human subjects. In our study we used the radiotracer 3-deoxy-3-18F-fluorothymidine (18F-FLT) as a labeled analogue of thymidine for in vivo visualization and quantification of cell proliferation in PDX models of AML. 18F-FLT enters the cell and undergoes phosphorylation by thymidine kinase 1 (TK1) staying intracellular. TK1 activity is increased in proliferating cells, compared to those in resting state. Using this technique we analysed the applicability of using 18F-FLT to monitor AML PDX disease development and determination of relative efficacies of 2 nucleoside drug analogues Cytarabine and Elacytarabine.

 

Results/Discussion

PET/CT imaging of 18F-FLT activity in AML PDX models permitted both spatio-temporal disease quantification and visualisation (Figure 1). Correlated 18F-FLT PET/CT imaging showed higher uptake of 18F-FLT in mice treated with cytarabine i.e. greater cell proliferation, particularly in the spleen and bone marrow. Employing this technique in addition to survival studies in 10 AML PDX models we demonstrate elacytarabine exhibited longer survival than treatment with cytarabine in AML PDX models.

Conclusions

PET/CT imaging of 18F-FLT activity is a quantitative modality useful in drug screening approaches of AML, and may be useful in other haematological malignancies that demonstrate high proliferation rates.

References

1McCormack, E. et al. Multiplexed mAbs: a new strategy in preclinical time-domain imaging of acute myeloid leukemia. Blood. 2013; 121: e34-e42.

 

 

Acknowledgement

The study has received economic support from Novo Nordisk Fonden, Helse-Vest RHF, Helse Bergen HF, Norwegian Cancer Society, NFR/CCBIO.

Figure 1.

PET images of mice engrafted with AML patient cells at Day 20 (after injection of cells) (A,C) and Day 36 (B,D) for cytarabine treatment, and Day 20 (E,G) and Day 36 (F,H) for elacytarabine treatment. Presented as Axial and Coronal view. A higher uptake of FLT can be seen in spleen (B,D) and spine (B) of mouse treated with cytarabine.

SUV of FLT in spleen expressed as a Mean and Maximum uptake (I).

# 123

Investigation of PSMA receptor regulation upon drug administration (#206)

A. Stolzenburg1, K. Lückerath1, 2, A. Buck1

1 University Hospital Würzburg, Department of Nuclear Medicine, Würzburg, Bavaria, Germany
2 University of California Los Angeles, Department of Molecular & Medical Pharmacology, Los Angeles, California, United States of America

Introduction

Prostate cancer (PCa) is the second most frequent malignant tumors in men. [1]. Therefore, precise diagnostics is essential for patient counselling and therapy management especially with respect a potential development of drug resistances [2]. For staging and diagnosis of PCa patients, 68Ga-PSMA PET/CT is used more frequently [3, 4]. However, properties of the target protein PSMA with regard to pharmacological and -dynamic interactions on receptor expression are not known. Thus, the aim of our study was to analyze drug-induced changes and the impact of resistance formation on PSMA expression.

Methods

In our study, we applied PCa drugs (e.g. Docetaxel, Abiraterone, Enzalutamide) or supportive medication like Dexamethasone on different PCa cell lines (LNCaP, LNCaP C4-2, PC-3) for 72 h to simulate patient’s treatment options either as stand-alone or combination therapy. Subsequently, we used flow cytometry to analyze PSMA expression in these treated cell lines. Besides that, we establish a Docetaxcel-(Doc.)-resistant prostate cell line to study receptor expression in different stages of a possible resistance formation. Therefor we treated C4-2 cells repeatedly with increasing Docetaxcel concentration (from 5 nM to 20 nM).

Results/Discussion

Our analysis revealed a large variability in PSMA expression depending on particular cell line and PCa drug application, illustrating the heterogeneity of this disease. We proved a significant increase in receptor expression when applying e.g. Abiraterone (median 88.4 vs. 41.8 in untreated C4-2 cells, p = 0.005), whereas the application of other drugs like Dexamethasone resulted in a decline (median 33.4 vs. 41.8; C4-2 cells). In addition, repeated treatment with Doc. led to a resistance formation in PCa cell line C4-2 meaning growth was no more affected by Doc. application. Interestingly, we noted a long term-depending increase in PSMA expression in these cells (median expression 42.7 vs. 142.6; p = 0.016), resulting in a plateau status after several cycles with Doc. After stopping treatment, PSMA expression remained stable during the follow up of 3 months.

Conclusions

Together, our investigation depicts new insights in drug induces changes on PSMA expression, which might have great potential for clinical practice like the PCa therapy management before a radio-ligand therapy.

References

  1. Aus, G., et al., EAU guidelines on prostate cancer. European urology, 2005. 48(4): p. 546-551.
  2. Seruga, B., A. Ocana, and I.F. Tannock, Drug resistance in metastatic castration-resistant prostate cancer. Nature reviews Clinical oncology, 2011. 8(1): p. 12-23.
  3. Bluemel, C., et al., 68Ga-PSMA-PET/CT in patients with biochemical prostate cancer recurrence and negative 18F-choline-PET/CT. Clinical nuclear medicine, 2016. 41(7): p. 515-521.
  4. Maurer, T., et al., Current use of PSMA-PET in prostate cancer management. Nature Reviews Urology, 2016. 13(4): p. 226-235.

Acknowledgement

We thank Gabriele Riehl for technical assistance.

Keywords: prostate cancer, PSMA, docetaxel, resistance formation
# 124

In vivo assessment of VCAM-1 expression by SPECT/CT imaging in mice models of human triple negative breast cancer. (#335)

C. Montemagno1, L. Dumas1, P. Cavaillès2, M. Ahmadi1, S. Bacot1, M. Debiossat1, S. Hernot3, N. Devoogdt3, P. Perret1, L. Riou1, D. Fagret1, C. Ghezzi1, A. Broisat1

1 UMR INSERM 1039, Laboratoire Radiopharmaceutiques Biocliniques, Grenoble, France
2 UMR5525, CNRS-Université Grenoble Alpes, TIMC-IMAG, BNI team, Grenoble, France
3 ICMI-BEFY, Vrije Universiteit Brussel, Laboratory of In vivo Cellular and Molecular Imaging, Brussels, Belgium

Introduction

Recent progress in breast cancer and metastasis research has led to the identification of genes and mechanisms that mediates metastatic colonization (1). Among them Vascular Cell Adhesion Molecule 1 (VCAM-1) could play a key role. VCAM-1-tumor expression promotes lungs-metastasis and is associated with clinical early recurrence and poor outcome in breast cancer (2,3). Our objective was to perform the in vivo imaging of VCAM-1 in triple negative breast cancer (TNBC) using either subcutaneous tumor xenograft model or lung metastasis model.

Methods

MDA-MB-231 (VCAM-1+) and control HCC70 (VCAM-1-) TNBC cells were employed. First, these two cell lines were xenograft on BALB/c Nude mice (one cell line per hindlimb). Mice were then injected with the single-domain antibody (sdAb) fragment 99mTc-cAbVCAM1-5 (n=6) or with an irrelevant control sdAb (99mTc-Ctle, n=6) and tumor uptake was assessed in vivo by SPECT. Then, for experimental metastasis model, MDA-MB-231 were injected intravenously via the tail vein of SCID mice, and 8 weeks post injection, 99mTc-cAbVCAM1-5 (n=5) or 99mTc-Ctle (n=4) SPECT imaging was performed. Furthermore, RT-qPCR were performed in order to discriminate between human TNBC cells VCAM-1 (hVCAM-1) and potential murine VCAM-1 (mVCAM-1) expression induced by inflammatory processes.

Results/Discussion

In vivo in the subcutaneous tumor xenograft model MDA-MB-231 99mTc-cAbVCAM1-5 uptake was 2-fold higher than that of HCC70 (1.7 ± 0.3 vs 0.9 ± 0.3 %ID/cm3, P<0.01), and 4-fold higher than 99mTc-Ctl uptake (0.4 ± 0.1 % ID/cm3, P<0.01). Similarly, 99mTc-cAbVCAM1-5 uptake in MDA-MB-231 lung metastasis was ~3-fold higher than that of 99mTc-Ctl (1.7 ± 0.4 vs 0.6 ± 0.1 %ID/cm3, P=0.015). Ex vivo analysis confirmed that in MDA-MB-231 tumor VCAM-1 mRNA mainly originated from human tumor cells rather than from mouse endothelial or inflammatory cells. Indeed, hVCAM-1 mRNA was ~3-4-fold higher than mVCAM-1 (P<0.01).

Conclusions

99mTc-cAbVCAM1-5 allowed imaging of VCAM-1 breast positive lesions in subcutaneous xenograft model and in an experimental model of lung metastasis. RT-qPCR analysis reflected human TNBC cells VCAM-1 expression rather than murine inflammatory process. 99mTc-cAbVCAM1-5 is therefore a suitable tool to evaluate the role of VCAM-1 as a marker of tumor aggressiveness in murine model of TNBC and could be useful to characterize breast cancer aggressiveness in clinical practice.

References

1.         Minn AJ, Gupta GP, Siegel PM, et al. Genes that mediate breast cancer metastasis to lung. Nature. 2005;436:518.

2.         Chen Q, Massague J. Molecular Pathways: VCAM-1 as a Potential Therapeutic Target in Metastasis. Clin Cancer Res. 2012;18:5520-5525.

3.         Maimaiti Y, Wang C, Mushajiang M, et al. Overexpression of VCAM-1 is correlated with poor survival of patients with breast cancer. Int J Clin Exp Pathol. 2016;9:7451–7457.

Figure 1. In vivo biodistribution of 99mTc-cAbVCAM1-5 in mice bearing HCC70 and MDA-MB-231 tumors
(A) SPECT/CT images of HCC70 (Yellow arrows) and MDA-MB-231 (White arrows) bearing mice after i.v. injection of 99mTc-cAbVCAM1-5 or 99mTc-Ctl. Kidney (K), Liver (Li). (B) Quantification of 99mTc-cAbVCAM1-5 and 99mTc-Ctl tumor uptake. ** P<0.01 vs MDA-MB-231 + 99mTc-Ctl; # P<0.05, ## P<0.01 vs HCC70 + 99mTc-cAbVCAM1-5. (C) Ex vivo assessment of VCAM-1 expression by RT-qPCR. *** P<0.001 vs mVCAM-1
Figure 2. In vivo biodistribution of 99mTc-cAbVCAM1-5 in a pulmonary experimental metastasis model.
(A) SPECT/CT images of MDA-MB-231 lung metastases 1h after i.v. injection of 99mTc-cAbVCAM1-5 or 99mTc-Ctl. Lungs (Lu), Liver (Li), Kidney (K). (B) Quantification of 99mTc-cAbVCAM1-5 and 99mTc-Ctl lungs uptake. * P<0.05 vs 99mTc-Ctl. (C) Ex vivo assessment of mouse VCAM-1 (mVCAM-1) and human VCAM-1 (hVCAM-1) expression by RT-qPCR. *** P<0.001 vs mVCAM-1
Keywords: Triple negative breast cancer, VCAM-1, SPECT imaging, sdAbs
# 125

Light Sheet Fluorescence Microscopy imaging of primary Glioblastoma 3D cultures treated with Temozolomide and Doxorubicin (#412)

M. - E. Oraiopoulou1, 2, S. E. Psycharakis3, E. Parasiraki4, 5, E. Tzamali1, G. Tzedakis1, A. F. Vakis2, 6, V. Sakkalis1, J. Papamatheakis4, 5, G. Zacharakis3

1 Foundation for Research and Technology-Hellas, Institute of Computer Science, Heraklion, Greece
2 University of Crete, Department 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
6 University General Hospital of Heraklion, Neurosurgery Clinic, Heraklion, Greece

Introduction

Adjuvant Temozolomide (TMZ) chemotherapy is considered the front line Glioblastoma (GB) treatment along with maximal safe surgical resection and radiotherapy. The latest trends in GB clinical trials usually refer to Doxorubicin (DOX); yet it is unable to adequately overpass the blood brain barrier. GB prognosis remains poor mainly because of the high inter- and intra-tumoral heterogeneity and post-surgery relapse. Here, we present preclinical in vitro results on the cell death that TMZ and DOX induce in patient-derived GB 3D cultures measured with Light Sheet Fluorescence Microscopy (LSFM).

Methods

An own-established primary GB cell culture was used, together with the well-studied U87MG GB cell line which served as control. 3D spheroids were generated using the hanging drop technique and treated with a range of TMZ and DOX concentrations, based on the IC50 values previously estimated in 2D. Optical Microscopy was used to monitor the growth pattern and LSFM was used to visualize the drug penetration and cell death in the GB spheroids up to approximately 12 days after treatment.

Results/Discussion

Though both the U87MG and the primary GB spheroids were found sensitive at the drugs tested, DOX in general was found to be effective in less concentrated doses. According to the LSFM images, DOX had a low diffusion rate overtime and was able to accumulatively cause necrosis. On the other hand, in TMZ-treated spheroids growth-inhibiting effects were observed in a dose-response relationship.

Inline with the relevant literature, both drug effects were related to growth inhibition. For the purposes of this study, in order to further discriminate disabling cell division from eventually leading to cell death we used 3D cell culturing combined to LSFM imaging. This way, not only the 3D spheroids used more closely resemble real tumours by forming drug gradients compared to monolayer cultures, but also the LSFM technique enabled optical sectioning with various excitation lasers for multispectral high resolution 3D imaging.

Conclusions

Our results are in agreement with variable drug responsiveness of individual GBs and interestingly, in favor of the option of a TMZ-DOX therapeutic scheme to disable proliferation and increase cytotoxicity against GB. We are currently extending these studies to parametrize and validate GB predictive computational algorithms to further support this hypothesis.

Acknowledgement

Authors would like to thank Evangelos Liapis for all the help he provided, Elias Drakos for his collaboration, and Katerina Manolitsi for her advisory comments, as well as Despina Tsoukatou, Georgios Vrentzos and Venediktos Makatounakis for the expert technical assistance. This work was supported by the Grants “Skin-DOCTor” implemented under the "ARISTEIA" Action of the "OPERATIONAL PROGRAMME EDUCATION AND LIFELONG LEARNING", co-funded by the European Social Fund (ESF) and National Resources and from the EU Marie Curie Initial Training Network “OILTEBIA”, as well as the General Secretariat for Research and Technology (GSRT) and the Hellenic Foundation for Research and Innovation (HFRI).

Keywords: Light Sheet Fluorescence Microscopy (LSFM), Glioblastoma, primary cell cultures, Temozolomide, Doxorubicin, 3D cell cultures, preclinical drug screening
# 126

Preclinical PET-MRI and BLI for the Characterization of Tumor Development and Microenvironment (#24)

S. Belderbos1, K. Govaerts1, A. Croitor Sava1, J. Wouters1, B. B. Manshian1, C. M. Deroose2, S. J. Soenen1, W. Gsell1, U. Himmelreich1

1 KU Leuven, Biomedical MRI/MoSAIC, Imaging and Pathology, Leuven, Belgium
2 UZ Leuven, Nuclear Medicine and Molecular Imaging/MoSAIC, Imaging and Pathology, Leuven, Belgium

Introduction

Due to few nanoparticles (NPs) reaching the tumor and large tumor heterogeneity, nanomaterial-based medicine has found little translation to clinic in cancer applications. Understanding dynamic changes in tumor microenvironment could aid in selecting the right time window for NP-therapy. In vivo preclinical multimodal imaging, i.e. combining magnetic resonance imaging (MRI), positron emission tomography (PET) and bioluminescence imaging (BLI), allows longitudinal follow-up of tumor vascularization, metabolism, cellularity and necrosis, hereby facilitating translation of NP-therapy to clinic.

Methods

Human ovarian SKOV-3 xenografts were generated after subcutaneous injection (inj.) of 107 firefly luciferase-positive cells in Swiss nude mice. Animals were scanned weekly using a 9.4T MRI (n=6) or 7T PET-MRI (n=3) and the IVIS Spectrum to determine total photon flux (BLI). Following MR scans were acquired: 2D T2-weighted RARE, diffusion-weighted RARE, dynamic contrast enhanced sequences after inj. of Magnevist®, and T1 and T2 parametric maps. On the PET-MRI, a 1h static 18F-FDG scan was acquired simultaneously with a 3D T2 T2-weighted RARE one hour after intravenous inj. of 2.66 - 7.28 MBq of tracer. T2 and apparent diffusion coefficient (ADC) parametric maps were clustered into three groups using a k-means clustering algorithm to determine the presence of different tumor tissue types.

Results/Discussion

Mean standardized uptake values (SUVmean) and total photon flux increased until week (w) 5 and 6, respectively. Magnevist® was taken up by tumors at all time points. Preliminary results indicate the presence of leaky blood vessels. T2 and ADC values decreased until w4, indicating a dense growing tumor mass, while they increased afterwards, which is representative of edema and necrosis (Fig. 1A). Significant correlation was found between ADC and T2 values (p < 0.0001). Clustering of their respective parametric maps (Fig. 1B) show a largely homogeneous tumor at initial time points (w2 – w4), as there are little differences in average values between clusters. From w5 on, large differences in average values within clusters are seen and the three clusters have similar importance, showing tumor heterogeneity. At later time points, regions with high T2 and ADC values correspond to those with low SUVmean, while at initial time points all clusters correspond to low SUVmean (Fig. 1A).

Conclusions

In conclusion, we developed a multimodal imaging approach, which allows longitudinal follow-up of tumor development and its microenvironment. All three imaging modalities are necessary as the metabolic activity of the tumors does not correspond to the actively growing tumor areas at initial time points. Our results further indicate that, due to the presence of an actively growing tumor, which is metabolically active, vascularized and contains leaky blood vessels, w4 after SKOV-3 tumor cell inj. is a suitable therapeutic time window for this ovarian cancer model to test new NP-based therapies.

Acknowledgement

The research leading to these results received funding from the KU Leuven Funding Program ‘In Vivo Molecular Imaging Research’ (IMIR) and the IWT Strategic Basic Research Grant n°140061 ('NanoCoMIT') from the Flemish Government. SB also received support from the Academische Stichting Leuven.

Evolution of tumor metabolism and microenviroment

A) Corresponding 18F-FDG PET-MRI, T2 and ADC maps. High T2 and ADC values at later time points correspond to low SUVmean, while at early time points tumors display low SUVmean, T2 and ADC values. B) Workflow for clustering a tumor: after manually delineating the tumor, a mask was created and a k-means clustering algorithm was applied to define 3 clusters. End-stage tumors were heterogeneous.

Keywords: Tumor xenografts, Tumor microenvironment, PET-MRI, BLI, Multimodal Imaging
# 127

Therapy monitoring and cell tracking of immune cells in cancer with Fluorine-19 MR Imaging (#251)

W. Reichardt1, 2, 3, D. von Elverfeldt1, E. Fischer1, C. Weidensteiner1

1 University of Freiburg, Medical Physics, Department of Radiology, Faculty of Medicine, Freiburg, Baden-Württemberg, Germany
2 German Consortium for Translational Cancer Research (DKTK), Heidelberg, Baden-Württemberg, Germany
3 German Cancer Research Center (DKFZ), Heidelberg, Baden-Württemberg, Germany

Introduction

The tracking of immune cells in vivo is getting increasingly important with the rise of  immune therapy1. Antibodies can activate a variety of immune cells. This leads to a massive infiltration of activated immune cells into the tumor and lymph nodes. Per-fluorinated carbons (PFC) which are taken up primarily by monocytes after i.v. injection can serve as a tracer for immune cell tracking. Therefore the signal from the exogenous tracer can be detected and localized with high sensitivity using 19F MRI if the number of cells loaded with PFC-tracer in the region of interest is sufficient³.

Methods

MR Imaging: A receive surface coil was built by the Medical Physics Group of the University Medical Center Freiburg and operated with a double-tuned 19F-1H birdcage resonator for transmission. Imaging (Fig.1) was performed on a 9.4 T Bruker animal scanner (Bruker, Ettlingen, Germany). 50 nl of the PFC was placed next to the tumor in a vial as a reference. The 19F-MRI signal intensities were normalized to the reference. Animal Model: A subcutaneous tumor model was generated in mice. After randomization five animals were treated with vehicle, 10 animals were treated with a combination of  anti-PD-L1  and anti-CD40 immune-stimulating antibodies.Three days after treatment all animals received the contrast agent PFC  (120µl  i.v.) . Five days after treatment animals were analyzed by MRI.

Results/Discussion

Statistics (Fig.2) showed a difference in the mean normalized signal intensity between the two groups indicating differences in immune cell infiltration after antibody treatment compared to control in s.c. tumor models in immune competent mice using PFCs as tracer. Additional analysis of tumor, liver and spleen showed a large difference in the spleen volume between the two groups while there was no significant difference in the normalized spleen intensity or the normalized liver intensity values. Tumor volume was smaller in the treated group. The results for the tumor volume indicate a tumor shrinking effect of the therapy. However, the mean normalized tumor signal intensity was lower in the treated group. This finding was not expected after treatment with immune-stimulating antibodies. However, as the spleen recorded a significant increase in volume while showing constant signal intensity it seems likely that a large part of activated immune cells is migrating to the spleen.

Conclusions

A 19F-MRI coil was built and successfully tested at a small bore animal scanner Bruker Biospec 9.4T. In vitro and in vivo test scans using phantoms and mice were performed to test and optimize the MR-protocol. The results showed that it is possible to detect dynamic changes in the migratory pattern and activation of immune cells in tumor models undergoing immune therapy. This might be useful to differentiate pseudoprogression due to the invasion off immune cells from real progression of a tumor.

References

  1. Ahrens ET, Bulte JW. Tracking immune cells in vivo using magnetic resonance imaging. Nat Rev Immunol. 2013 Oct;13(10):755-63. doi: 10.1038/nri3531. Epub 2013 Sep 10. Review. PubMed PMID: 24013185; PubMed Central PMCID: PMC3886235.
  2. Schwab, L, Goroncy, L, Palaniyandi, S., Gautam, S., Triantafyllopoulou, A., Mocsai, A, Reichardt, W., Karlsson, FJ, Radhakrishnan, SV, Hanke, K, Schmitt-Graeff, A, Freudenberg, M, von Loewenich, FD, Wolf, P, Leonhardt, F, Baxan, N, Pfeifer, D, Schmah, O, Schönle, A, Martin, SF, Mertelsmann, R, Duyster, J, Finke, J., Prinz, M., Henneke, P., Häcker, H., Hildebrandt, G.C, Häcker, G, Zeiser, R. Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance GvHD via tissue damage. Nature Medicine 20: 648-54, 2014
  3. Janjic JM, Srinivas M, Kadayakkara DK, Ahrens ET. Self-delivering nanoemulsions for dual fluorine-19 MRI and fluorescence detection. J Am Chem Soc. 2008; 130:2832–2841.
Figure 1:
Overlay of T2 RARE Image and the 19F image. Note the distribution of PFC positive cells in the tumor primarily in the rim. Next to the tumor the reference can be seen. 19F images were acquired with a RARE sequence: TR/TE/FA = 3000ms/76ms/90° matrix 64x64, FOV= 40 mmx40 mm, 12 axial slices with 3 mm thickness (30min.) 50 nl of the PFC was placed next to the tumor in a vial as a reference.
Figure 2:
a) Box plot of normalized tumor signal ROI values in both groups. b) Box plot of tumor volume values in both groups

 
# 128

Spatiotemporal assessment of spontaneous metastasis formation using multimodal in vivo imaging in HER2+ and triple negative metastatic breast cancer xenograft models in mice (#16)

I. B. Fricke1, 2, R. De Souza1, 3, L. Costa Ayub3, G. Francia4, R. Kerbel4, D. A. Jaffray1, 2, 5, J. Zheng1, 2

1 University Health Network, TECHNA Institute for the Advancement of Technology for Health, Toronto, Ontario, Canada
2 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Toronto, Ontario, Canada
3 University of Toronto, Leslie Dan Faculty of Pharmacy, Toronto, Ontario, Canada
4 University of Toronto, Biological Sciences Platform, Sunnybrook Research Institute, Department of Medical Biophysics, Toronto, Ontario, Canada
5 Princess Margaret Cancer Centre, Radiation Medicine Program, Toronto, Ontario, Canada

Introduction

Preclinical breast cancer models frequently fail to recapitulate the clinical course of metastatic disease, resulting in drug development studies that are poorly predictive of clinical outcome1. Thus, this study aims to characterize the spatiotemporal pattern of metastasis formation in the two highly metastatic MDA-MB-231-derived triple negative (TN) LM2-4 and HER2+ LM2-4H2N breast cancer cell lines2 using BLI, CT, fluorescence imaging, and [18F]FDG-PET, thereby providing a toolset for monitoring spontaneous metastasis formation and quantification of disease burden and treatment response.

Methods

LM2-4 (n=17), LM2-4H2N (n=17) and MDA-MB-231 (MDA; n=5) tumors were established in the right inguinal mammary fat pad (MFP) of female SCID mice and were resected 14-16 days (d) later. Metastasis formation was monitored longitudinally using BLI. Metabolic activity of the primary and metastatic lesions in mice bearing the LM2-4 or LM2-4H2N was assessed by [18F]FDG-PET. Metastatic burden at study endpoint was assessed by CT and fluorescence imaging (Ex/Em 785/820nm) following intravenous administration of a dual-modality liposome agent3.

Results/Discussion

Comparable temporal metastasis patterns were observed using BLI for the highly metastatic LM2-4 & LM2-4H2N cell lines, while metastasis formed about 10d later for MDA. 21d post primary tumor resection, metastases were detected in 86% of LM2-4, 69% of LM2-4H2N, and 60% of MDA inoculated mice, predominantly in the axillary region, contralateral MFP, and liver/lung. LM2-4 & LM2-4H2N tumors displayed high metabolism based on [18F]FDG-PET uptake (SUVmax=3.1±0.7). Lung metastases were detected as the [18F]FDG-PET uptake increased significantly between pre- and post-metastasis scan (∆SUVmax: LM2-4=1.5±0.7, LM2-4H2N=1.1±0.3). Using a liposomal dual-modality agent, CT and fluorescence confirmed BLI detected lesions and identified additional nodules in the intraperitoneal cavity and lung.

Conclusions

Non-invasive imaging can be used to assess the spatiotemporal pattern of metastasis formation, overcoming major disadvantages of pure endpoint studies and enables accurate randomization of animal groups for investigating treatment outcome. Our results suggest that [18F]FDG-PET and liposome contrast enhanced CT can complement BLI by providing greater sensitivity and 3D spatial information on metastatic disease burden. The described metastatic tumor models and the imaging toolset can be used to assess the efficacy of new chemotherapy regimens for TN and HER2+ metastatic breast cancer.

References

1Kerbel R, Cancer J, 2015.

2Munoz R, Cancer Res, 2006.

3Zheng J, Biomaterials, 2015.

Acknowledgement

This work was supported in-part by the Fidani Chair in Radiation Physics, Dr. Jinzi Zheng’s TECHNA Faculty Start-up Fund, and a MITACS Accelerate Post-doctoral Fellowship awarded to Dr. Inga Fricke. The authors would like to thank Dr. Stefano Serra for his pathology expertise, and Linyu Fan and Michael Dunne for their valuable technical support.

# 129

Nal-IRI Demonstrates Treatment Efficacy in Orthotopic Patient-Derived Pancreatic Tumor Models of High and Low Hypoxia (#25)

M. Ventura1, N. Bernards1, R. De Souza1, I. B. Fricke1, B. S. Hendriks2, J. B. Fitzgerald2, N. Paz2, H. Lee2, S. Klinz2, J. Zheng1, 3

1 TECHNA Institute for the Advancement of Technology for Health, University Health Network., Toronto, Ontario, Canada
2 Merrimack Pharmaceuticals, Inc., Cambridge, Massachusetts, United States of America
3 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

Introduction

High levels of hypoxia have been linked to aggressiveness, resistance to therapy, and poor prognosis of pancreatic tumors. Liposomal irinotecan (nal-IRI, ONIVYDE®), approved for the treatment of gemcitabine-refractory metastatic pancreatic cancer in combination with 5-fluorouracil and leucovorin, has previously shown potential in reducing hypoxia in preclinical models. Our study evaluates the anti-tumor activity of nal-IRI and its ability to modulate hypoxia in patient derived orthotopic models of pancreatic cancer, with high (OCIP51) and low (OCIP19) hypoxia.

Methods

Mice bearing orthotopic OCIP51 or OCIP19 tumors were randomized into: (i) control (n = 5 per tumor model) and (ii) treated, nal-IRI 20 mg/kg (n = 10 per tumor model) group. A total of four doses were administered and treatment efficacy was monitored by multiple MRI sessions. [18F]FAZA imaging for tumor hypoxia quantification was performed on day 0, 7, 14, and 21 post treatment initiation. [18F]FLT imaging for tumor cell proliferation evaluation was performed on day -1
and 16 for the OCIP51, and on days -1, 6, 13, and 20 for the OCIP19 model. 

Results/Discussion

At baseline, OCIP51 mice exhibited significantly higher tumor [18F]FAZA uptake (1.75 ± 0.12 %ID/g) compared to the OCIP19 model (0.75 ± 0.04 %ID/g). More evident was the difference in tumor hypoxic fraction, 0.02 ± 0.01 for the OCIP19 versus 0.71 ± 0.06 for the OCIP51 (p < 0.0001). Nal-IRI treated OCIP51 mice maintained significantly lower levels of hypoxia (p = 0.0003), smaller hypoxic fractions (p = 0.003), and smaller tumor volumes (p = 0.03) throughout the entire study, compared to the controls, and compared to the baseline characteristics. Differences in [18F]FAZA uptake were detectable 8 days before any significant change in tumor volume. OCIP19 mice also responded to therapy, but significance was reached only after 4 doses of nal-IRI. Moreover, for this model, tumor volume was controlled, but not accompanied by any reduction in hypoxia or hypoxic fraction. [18F]FLT uptake was comparable between both tumor models and between treated and untreated animals at any given time point.

Conclusions

In this study, nal-IRI reduced tumor volume in high hypoxic tumors (OCIP51) but not in low hypoxic tumors (OCIP19), suggesting that tumor hypoxia modulation might be a key mechanism through which nal-IRI acts, and potentially that tumors with a hypoxic microenvironment are more sensitive to nal-IRI. [18F]FAZA imaging of hypoxia also provided early prediction of treatment response, showing a significant reduction in the levels of hypoxia 8 days prior to significant changes in tumor volume.

# 130

Gemcitabine reduces tumor perfusion and drug delivery in PDAC (#174)

I. Heid1, F. Englert1, M. Trajkovic-Arsic2, 3, K. Steiger4, A. Steingeotter5, F. Lohöfer1, G. Kaissis1, L. Liotta6, B. Crone7, U. Karst7, M. Wildgruber8, H. Algül6, J. Siveke2, 3, R. Braren1

1 Klinikum rechts der Isar, Technical University of Munich, Institute of Radiology, Munich, Germany
2 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany
3 West German Cancer Center, University Hospital Essen, Division of Solid Tumor Translational Oncology, Essen, Germany
4 Technical University of Munich, Institute of Pathology, Munich, Germany
5 University Hospital Zurich, Division of Gastroenterology and Hepatology, Zurich, Switzerland
6 Klinikum rechts der Isar, Technical University of Munich, Molecular Gastroenterology, Medical Clinic II, Munich, Germany
7 University of Muenster, Institute of Inorganic and Analytical Chemistry, Muenster, Germany
8 Translational Research Imaging Center (TRIC), Institut für Klinische Radiologie, Muenster, Germany

Introduction

Ineffective drug delivery is one of the reasons for therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC). We classify PDAC into three subgroups based on tumor cellularity (amount of neoplastic cells, arrangement into clusters):  PDAClow, PDACmed , PDAChigh (1). Here we investigate perfusion in genetically engineered mouse models (GEM). We use GEM to assess changes in perfusion under the standard treatment with gemcitabine and correlate this finding with patient data.

Methods

A cohort of mice (n=48) with Ptf1awt/cre(C)Kraswt/G12D(K)Ela-Tgfa(T), CK;p53(P)wt/fl, CKPfl/fl, CKTPwt/R172H und CKTPwt/fl genotypes was subjected to DCE-MRI at 1.5 T Achieva, Philips with a 47-mm surface coil and measured by single-shot Look-Locker based radial T1 mapping technique using the golden cut principle with a bolus of 0.04 mmol/kg of Gd-DTPA (Magnevist®). In a subset of CKPfl/fl mice gemcitabine (gem, 120mg/kg, 2 weeks) treatment was monitored. Histopathological correlations (H&E, CD31, Ki67 and cisplatin concentrations in the murine (m)PDAClow measured by LA-ICP-MS) were performed. Change in CT-contrast agent uptake was monitored in human PDAC (n=61) before and 3 month after different types of chemotherapy regimen (gem, gem/abraxane, FOLFIRINOX).

Results/Discussion

Relative area under the curve tumor/muscle (AUC60) derived from Gd-DTPA concentration time curves correlated positively with stroma content (r=0.46, CI:0.2-0.66) and negatively with the amount of tumor cells (r=-0.42, CI:-0.63--0.17) in mPDAC. Differences were noted in AUC60 between mPDAClow=2.06±0.1 and mPDACmed=1.21±0.1 tumors (P=.0001, Fig. 1A). mPDACmed tumors reveal reduced perfusion independent of the treatment. However, mPDAClow tumors showed reduced perfusion under gem treatment (AUC60pre=2.7±0.6, AUC60post=2.0±0.2, P=.012) and no significant changes were noted in the vehicle group (Fig. 1B). Accordingly, penetration of cisplatin in mPDAClow tissue samples was reduced in gem pretreated tumors only (NaCl: 3233±652, vs gem: 1189±299, P=.018, Fig. 1C). 63 % of human (h)PDAC treated with gem and 74% treated with gem/abraxane showed reduced signal intensity upon treatment, compared to only 38% of patients treated with FOLIRINOX (Fig. 2)

Conclusions

mPDAClow lesions with low amount of tumor cells and high stroma content reveal higher perfusion compared to tumors with intermediate tumor cellularity. Gem treatment resulted in reduced perfusion and lower cisplatin penetration into mPDAClow. Similar findings were observed in hPDAC under gem or gem/abraxane treatment. Our findings suggest a negative impact of gem treatment on tumor perfusion and would warrant re-evaluation of combinatorial treatment regimen including gem.

References

1. Heid I et al. Clin Cancer Res, 2017 Mar 15;23(6):1461-1470.

Acknowledgement

We would like to thank M. Settles for programming the DCE data reconstructions and analysis soft wares as well as I. Skuratovska, J. Blumenberg and T. Schilling for experimental support.

Figure 1: Gemcitabine reduces tumor perfusion and drug delivery in stroma rich mPDAC.

A: AUC60 distribution in mPDAC population. B: Perfusion changes over two weeks under indicated treatment. C: Distribution of cisplatin in mPDAClow tumors. After 2 weeks of treatment cisplatin was injected i.p. 10 min prior death. Snap frozen tumor samples were analyzed via LA-ICP-MS. Scale bar 1mm. The mean concentrations in distinct ROIs are presented on the right (n=4 mice/treatment).

Figure 2: Gemcitabine reduces tumor perfusion in hPDAC.

Patients with first diagnosis of PDAC underwent CT in a portal venous contrast phase before and 3 months after chemotherapy as indicated. Regions of interest were drawn in the tumor. Hounsfield units (HU) values of tumor roi were normalized to HU values in the aorta. Paired student T-test was used for statistical analysis. N = patients with perfusion drop/all patients in this cohort.

Keywords: PDAC, DCE-MRI, tumor cellularity and perfusion, therapy, gemcitabine
# 131

Control multivalent targeting of integrin avß3 and Neuropilin 1 generates potent variable biological activities (#298)

T. Jia1, J. Ciccione2, J. Choi1, T. Jacquet1, M. Henry1, M. Amblard2, A. Mehdi2, J. Martinez2, G. Subra2, J. - L. Coll1

1 UGA, INSERM U1209, CNRS 5309, IAB, La Tronche, France
2 UMR5247 CNRS, Institut des Biomolécules Max Mousseron, Montpellier, France

Introduction

Tumor angiogenesis depends on the activation of integrins (especially integrin avß3) and receptor tyrosine kinases like VEGFR2. The objective of this work is to generate dual targeted nanoparticles (NPs) that block simultaneously integrin avß3 and Neuropilin-1 (NRP1), a co-receptor of VEGFR2. This is expected to augment the anti-angiogenic and anti-tumor activities, as compared to each “mono-therapy” separately and to improve the tumor recognition for diagnostic purposes.

Methods

A library of silica-based NP (40 nm) was generated that present different amount and ratio of the cRGD or ATWLPPR peptides ± a PEG coating. The cRGD peptide is specific for integrin avß3 while the ATWLPPR recognizes NRP1. We studied their affinity, selectivity, and biological activity on primary endothelial cells (ECs). In particular we investigated the molecular cascades triggered by the contact of these NP with the ECs

Results/Discussion

Sub-nanomolar concentrations of NPs grafted either with ATWLPPR alone or in combination with cRGD exhibit potent specific antagonist activity against VEGFR2/AKT signaling. However, a 1 nM concentration of cRGD/ATWLPPR-heteromultivalent particles (RGD/ATW-NPs) also blocks the phosphorylation of VEGFR2 while co-inducing an unexpected long-lasting activation of AKT via IGF-1R/IR-AKT/GSK3β/eNOS signaling that stimulates cell survival on serum-starved HUVECs. We also showed that their repeated IV administration was associated with the proliferation of human U87MG tumor cells engrafted in nude mice and a dilatation of the tumor blood vessels.

Conclusions

We present evidence for the complex cross-talk generated by the binding of the heteromultivalent NPs with avβ3-integrin and with NRP1. We show for the first time that these heteromultivalent NPs can trans-activate IGF1R and exert dose-dependent pro-survival activity. This study demonstrates the difficulties in designing targeted silica-based NPs for antiangiogenic therapies and the possible risks posed by undesirable side effects.

References

Jia, T., Choi, J., Ciccione, J., Henry, M., Mehdi, A., Martinez, J., Eymin, B., Subra, G., and Coll, J. L. (2017) Heteromultivalent targeting of integrin alphavbeta3 and neuropilin 1 promotes cell survival via the activation of the IGF-1/insulin receptors. Biomaterials 155, 64-79.

Ciccione, J., Jia, T., Coll, J.-L., Parra, K., Amblard, M., Jebors, S., Martinez, J., Mehdi, A., and Subra, G. (2016) Unambiguous and Controlled One-Pot Synthesis of Multifunctional Silica Nanoparticles. Chemistry of Materials 28, 885-889

Acknowledgement

This study was supported by INCA-PLBio16-085, by the “Fondation ARC”(Projet Labellisé 2016) and by the FLI program– ANR-11-INBS-0006. We acknowledge the OPTIMAL Grenoble small animal optical imaging facility.

Keywords: Angiogenesis, Tumor Targeting, Multiligand, heteromultivalent, nanoparticles
# 132

Imaging hypoxia-driven tumor cell invasion and lymphatic dissemination (#159)

R. Perera1, M. Barrett2, L. Füglister1, J. Xandry1, S. Proulx3, B. Weber2, M. Rudin1, 2, S. Lehmann1

1 ETH and University of Zürich, Institute for Biomedical Engineering, Zürich, Switzerland
2 University of Zurich, Institute of Pharmacology and Toxicology, Zürich, Switzerland
3 ETH, Institute of Pharmaceutical Sciences, Zürich, Switzerland

Introduction

Tumor hypoxia has been linked to metastasis (1), but how remains unclear. In hypoxia carcinoma cells switch from collective to single amoeboid cell migration (2), used by immune cells to enter lymphatic vessels (3), a major metastatic route. Localized furthest from the blood lymphatics may show a lower O2 level, possibly favoring survival of hypoxic cancer cells. Here we established microscopic imaging assays to i) confirm amoeboid migration in hypoxic melanoma cells, ii) study hypoxic cancer cell migration to and into lymphatic vessels in vitro and in vivo and iii) measure pO2 in lymphatics.

Methods

B16 melanoma cells, incubated in normoxia or hypoxia were embedded in 3D collagen matrices and their migration mode assessed using time-lapse microscopy and analysis of amoeboid cell markers by immunofluorescence. Transmigration/chemotaxis of B16 melanoma and 4T1 breast carcinoma cells to and across lymphatic endothelial cells (LEC) upon treatment in normoxia or hypoxia was studied using a transwell assay combined with confocal microscopy. B16 cells were stably transfected with a novel hypoxia-induced fluorescent reporter (FP635) construct to monitor their migration in mouse tumors by 2P microscopy. Phosphorescence lifetime imaging was performed to measure the pO2 within lymphatic vessels in the mouse ear following i.v. injection of a 2P-excitable oxygen-quenched phosphorescence probe (4).

Results/Discussion

The low elongation indexes measured in hypoxic B16 melanoma cells suggest these cells, in analogy to the 4T1 breast carcinoma model (2), to predominantly migrate in an amoeboid mode (Fig1A). Hypoxic 4T1 and B16 cells displayed a significantly higher capacity to migrate towards and across LEC monolayers plated on the bottom side of transwell inserts compared to normoxic cells (Fig1B, C). In vitro validation of the newly established reporter construct showed the fluorescent reporter to be strongly induced by hypoxia. First in vivo experiments allowed reliable tracking of hypoxic cancer cells. We are currently using 2P microscopy to confirm amoeboid migration of hypoxic tumor cells into lymphatic vessels in tumors implanted in the mouse dorsal skin fold chamber. Furthermore, phosphorescence life-time based pO2 measurements indicate lower pO2 values in lymphatic vessels compared to blood vessels (Fig2).

Conclusions

Our data identify hypoxia as a driver of amoeboid cell migration not only in epithelial cancers, but also in melanoma. Using a combination of in vitro cell assays and microscopic imaging we further found the hypoxic cell profile to enhance cancer cell migration to and across lymphatic endothelial cell barriers. Our in vivo pO2 measurements suggest lymphatic vessels to constitute a low oxygen environment which may favour hypoxic tumor cell survival in lymphatic vessels and their metastasis via the lymphatic route.

References

  1. Höckel and Vaupal, Journal of the National Cancer Institute, 2001
  2. Lehmann S et al., Current Biology, 2017
  3. Pflicke, H., Sixt, M. J. Exp. Med, 2009.
  4. Roussakis E, Spencer JA, Lin CP, Vinogradov SA. Analytical Chemistry. 2014

Acknowledgement

We would like to thank Prof. M.Detmar (ETH Zurich) for kindly providing the transgenic Prox1-reporter mice used in this work and for helpful discussion of the project. 

Fig1: Hypoxia enhances cancer cell migration across and towards lymphatic endothelial cell barriers

(A) Low Elongation Index (length/width) of B16 hypoxic cells at 6h and 10h indicate amoeboid morphology. Statistics: non-paired Mann-Whitney test. (B) Hypoxia enhanced 4T1- (left) and B16- (right) cell crossing through primary lymphatic endothelial cell barriers (pLEC) (C) Chemotaxis assay indicates that B16 cells migrate towards pLECs as a result of chemotaxis. Statistics:Student T-test

Fig2: pO2 measurements in vivo suggest lymphatic vessels to constitute a low oxygen environment

pO2 map (left) of respective fluorescent image. (Right) pO2 of lymphatic vessels (LVs) is significantly lower than blood vessels (BVs). Statistics: Nested Analysis

# 133

CXCR4 – watching chemokine receptor regulation in vitro (#269)

A. Bögelein1, A. Stolzenburg1, K. Lückerath1, 2, A. Buck1

1 University Hospital Würzburg, Department of Nuclear Medicine, Würzburg, Bavaria, Germany
2 University of California Los Angeles, Department of Molecular & Medical Pharmacology, Los Angeles, California, United States of America

Introduction

Chemokine receptors (CXCR) influence immune responses and cell migration in diverse conditions including inflammation or several diseases [1]. Radiolabeled peptides have been introduced for in vivo imaging of CXCR4 expression with PET technique in multiple myeloma (MM) patients. However, despite rising possibilities in imaging and theranostic approaches [2-4], only little is known about CXCR4 regulation on cellular levels. The aim of this study was to investigate receptor expression upon drug-administration in MM cells to address tumor heterogeneity [5] and improve CXCR4 receptor availability.

Methods

By adding various routinely applied myeloma drugs (e.g. Bortezomib, Doxorubicin, Dexamethasone) to different multiple myeloma cell lines (OPM-2, INA6, MM1.S) as well as patient-derived CD138+ MM cells, we simulated receptor regulation under more realistic circumstances of heavily pre-treated MM patients. After an incubation time of 48 hours, we evaluated CXCR4 expression by using flow cytometry analysis and cellular uptake experiments using the radiolabeled antagonist 68Ga-CPCR4 [2]. Moreover, we analyzed CXCR4 expression over time starting from one hour to 48 hours after drug application

Results/Discussion

The receptor expression analysis demonstrated a large variability for CXCR4. Besides a dose-dependence, we noted a time-dependent effect for CXCR4 receptor expression depending on the particular drug. Interestingly, despite differences in the baseline CXCR4 surface expression, general effects –meaning up or down regulation– upon drug admiration were similar through all analyzed myeloma cell lines. When applying proteasome inhibitors like Bortezomib (5nM), we detected CXCR4 down regulation (OPM-2 median 79.6 vs. 147.8 in untreated cells, p=0.002), which could also be confirmed in primary human MM cells. In contrast, treatment with Dexamethasone (OPM-2, 100µM: 697.1, p=0.01) and Doxorubicin (OPM-2, 5µM: 322.2, p=0.002) led to an increased CXCR4 expression on myeloma cell surface; treatment with both substances, enhanced the amount of CXCR4 receptor even more. Similar results were obtained by performing uptake experiment with 68Ga-CPCR4.

Conclusions

In summary, our study discloses a high inducible variability of CXCR4 in humanized cell culture and primary MM cells. With regard to recent studies, it would be favorable to address CXCR4 receptor enhancement directly before a potential Lu177-Pentixather PRRT [3, 6]. Based on our in vitro results, a combination of Doxorubicin and Dexamethasone increases CXCR4 surface expression multiple times. Hence, further preclinical in vivo experiment will prove the transferability and applicability of these results within the complex macro- and microenvironment of the body.

References

  1. Qin, L., et al., Crystal structure of the chemokine receptor CXCR4 in complex with a viral chemokine. Science, 2015. 347(6226): p. 1117-1122.
  2. Philipp-Abbrederis, K., et al., In vivo molecular imaging of chemokine receptor CXCR4 expression in patients with advanced multiple myeloma. Embo Molecular Medicine, 2015. 7(4): p. 477-487.
  3. Lapa, C., et al., [Ga-68] Pentixafor-PET/CT for imaging of chemokine receptor CXCR4 expression in multiple myeloma - Comparison to [F-18] FDG and laboratory values. Theranostics, 2017. 7(1): p. 205-212.
  4. Herrmann, K., et al., First-in-human experience of CXCR4-directed endoradiotherapy with 177Lu-and 90Y-labeled pentixather in advanced-stage multiple myeloma with extensive intra-and extramedullary disease. Journal of nuclear medicine, 2016. 57(2): p. 248-251.
  5. Bolli, N., et al., Heterogeneity of genomic evolution and mutational profiles in multiple myeloma. Nature communications, 2014. 5.
  6. Lapa, C., et al., CXCR4-directed endoradiotherapy induces high response rates in extramedullary relapsed Multiple Myeloma. Theranostics, 2017. 7(6): p. 1589.

Acknowledgement

We are grateful to G. Riehl for excellent technical assistance. We thank the Radiopharmacy staff members in the department of Nuclear Medicine (Universitätsklinikum Würzburg) especially A. Schirbel and S. Samnick for their support and we are grateful to use the CPCR4 kindly granted by HJ. Wester (Technische Universität München). This work received support from the Wilhelm-Sander-Stiftung (grant no. 2013.906.1).

Keywords: multiple myeloma, cxcr4, 68Ga-CPCR4