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. 2013 Aug 6;109(3):658-66.
doi: 10.1038/bjc.2013.356. Epub 2013 Jul 9.

Ewing sarcoma dissemination and response to T-cell therapy in mice assessed by whole-body magnetic resonance imaging

L Liebsch  1 , S Kailayangiri, L Beck, B Altvater, R Koch, C Dierkes, M Hotfilder, N Nagelmann, C Faber, H Kooijman, J Ring, V Vieth, C Rossig
Affiliations

Ewing sarcoma dissemination and response to T-cell therapy in mice assessed by whole-body magnetic resonance imaging

L Liebsch et al. Br J Cancer. .

Abstract

Background: Novel treatment strategies in Ewing sarcoma include targeted cellular therapies. Preclinical in vivo models are needed that reflect their activity against systemic (micro)metastatic disease.

Methods: Whole-body magnetic resonance imaging (WB-MRI) was used to monitor the engraftment and dissemination of human Ewing sarcoma xenografts in mice. In this model, we evaluated the therapeutic efficacy of T cells redirected against the Ewing sarcoma-associated antigen GD2 by chimeric receptor engineering.

Results: Of 18 mice receiving intravenous injections of VH-64 Ewing sarcoma cells, all developed disseminated tumour growth detectable by WB-MRI. All mice had lung tumours, and the majority had additional manifestations in the bone, soft tissues, and/or kidney. Sequential scans revealed in vivo growth of tumours. Diffusion-weighted whole-body imaging with background signal suppression effectively visualised Ewing sarcoma growth in extrapulmonary sites. Animals receiving GD2-targeted T-cell therapy had lower numbers of pulmonary tumours than controls, and the median volume of soft tissue tumours at first detection was lower, with a tumour growth delay over time.

Conclusion: Magnetic resonance imaging reliably visualises disseminated Ewing sarcoma growth in mice. GD2-retargeted T cells can noticeably delay tumour growth and reduce pulmonary Ewing sarcoma manifestations in this aggressive disease model.

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Figures

Figure 1
Figure 1
Visualisation of disseminated growth of Ewing sarcoma xenografts in mice by WB-MRI. VH-64 cells (2 × 106) were injected into the tail veins of 18 NOD/scid mice, followed by WB-MRI after 3 weeks, and then at weekly intervals. T2 axial sequences were used for detection of lung tumours, and T2 axial, sagittal, and STIR sequences were used for detection of manifestations at all other sites. (A) Numbers of mice developing detectable tumour manifestations at the indicated locations. (B) Total numbers of tumours per individual tumour site. (C) Boxplots of median tumour volumes at first detection at the various locations. The lines indicate the median sizes, and boxes represent the 25th and 75th percentiles. Circles symbolise outliers, asterisks show extreme values. (D) Light microscopy of haematoxylin and eosin (HE)-stained sections (left panel) and immunohistochemistry analysis by huCD99 staining (right panel) of tumours from the indicated organ and tissue sites. Altogether, the Ewing sarcoma origin was confirmed by histology and/or immunohistochemistry in four lungs with multiple lesions, five bone lesions (three in legs, two in pelvis and vertebral column, respectively), and four renal tumours.
Figure 2
Figure 2
The kinetics of disseminated tumour growth in xenografted mice. (A) The volumes of individual tumours were determined in T2 axial (lung tumours) and T2 sagittal (all others) WB sequences in NOD/scid mice at time points 1, 2, 3, and 4. Extrapulmonary tumour manifestations were categorised by localisation to leg bones, skeletal trunk bones (pelvis, vertebral column), kidneys and/or suprarenal gland, and soft tissue. For lungs, each line corresponds to one mouse, as volumes of all tumour lesions were combined for each affected lung. For all other sites, each line reflects one individual tumour. Lung tumour volumes increased between TPs 1 and 2 (P<0.001) and TPs 2 and 3 (P<0.001). Femur/tibia tumours and pelvic tumours increased between TPs 1 and 2 (P<0.001 and P=0.031, respectively). Kidney tumours increased between TPs 1 and 2 (P<0.001), 2 and 3 (P<0.001), and 3 and 4 (P<0.001). (B) Lung tumours were counted in T2 axial WB sequences in NOD/scid mice at TPs 1, 2, 3, and 4. Each line represents one mouse. In addition, boxplots represent the tumour number of the evaluable mice at each TP. Brackets represent statistically noticeable differences (P⩽0.05) of the Wilcoxon-signed rank tests. Circles symbolise outliers; asterisks show extreme values. (C) Sequential MRI scans of one individual mouse demonstrate the progressive pulmonary involvement over time. The number and size of metastases that are detected as hyperintense manifestations in the dark lung in axial T2 images increases over time. Photodocumentation (to the right) of mice upon autopsy confirms the MRI findings, showing white-blueish metastases within the reddish lung tissue.
Figure 3
Figure 3
Whole-body MRI with DWIBS of Ewing sarcoma-engrafted mice. Sequential WB-MRI and DWIBS scans were performed in the cohort of 18 mice starting 3 weeks after tumour inoculation. (A) T2 sagittal MRI sequences (left and right panel) and DWIBS (central panel) images of a single mouse at sequential examinations performed 20–41 (TPs 1–4) days after tumour inoculation, as indicated. The left and right panels illustrate the growth of a kidney and a femur tumour, respectively. Images of DWIBS performed at the same time points demonstrate the two tumour manifestations in the femoral bone (larger arrow) and kidney (smaller arrow). T2 sagittal sequences were used to define the presence of tumour manifestations. The following regions were separately analysed for the presence or absence of tumours: right and left lower limbs, pelvic bones/vertebral column, left and right kidneys and suprarenal glands, and soft tissues. (B) Sensitivity, specificity, and positive (ppV) and negative predictive values (npV) of DWIBS were calculated for each localisation in comparison with standard T2 sagittal sequences. Numbers in brackets represent 95% confidence intervals. (C) Boxplots of median tumour volumes at all sites detectable and non-detectable by DWIBS, assessed by volumetry on T2 sagittal MR images. The bracket represents the result of the exact Mann–Whitney U-test. Circles symbolise outliers; asterisks show extreme values.
Figure 4
Figure 4
MRI documentation of in vivo activity of 14.G2a-28ζ T cells against disseminated tumour xenografts. A cohort of nine mice were intravenously injected with 2 × 106 VH-64 cells per mouse, followed by intravenous transfer of 1 × 107 human 14.G2a-28ζ gene-modified T cells (group A). Control mice (n=9) received analogous injections of non-transduced T cells (group B). Mice were followed up by sequential T2 sagittal and axial, STIR and DWIBS WB-MRI scans. Tumour numbers were assessed at the time of termination. (A) Total numbers of mice of groups A and B with at least one tumour at indicated tumour sites. (B) Total numbers of tumours at the indicated tumour sites in groups A and B. The bracket represents a statistically highly noticeable result (P⩽0.01) of the exact χ2-goodness-of-fit test. (C) Boxplots of median tumour volumes at first detection at the various locations in groups A and B. Brackets represent statistically noticeable differences (P⩽0.05) between the groups determined by exact Mann–Whitney U-tests. (D) Growth of tumours at the individual sites in group A (upper panel) and group B (lower panel) over time. Volumes of lung tumours per mouse were combined and presented as the total lung tumour burden per mouse in mm3. For all other localisations, each line corresponds to one individual tumour. (E) Boxplots of median numbers (left panel) and volumes (right panel) of lung tumours at the indicated TPs. Brackets indicate differences between the groups determined by exact Mann–Whitney U-tests. At TPs 1 and 2, group A consisted of nine mice, and group B of seven mice. Owing to drop-out by termination or unexpected death during anaesthesia, the sizes of groups were reduced to n=4 (group A and n=3 (group B) at TP 3. Circles symbolise outliers; asterisks show extreme values. (F) Kaplan–Meier curves of overall survival after tumour cell injection in groups A and B, and P-value of the log-rank test. Overall survival is defined by killing of the animals, as explained in the Materials and Methods section or by unexpected death of few animals during the anaesthesia.
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