Noninvasive assessment and quantification of tumour vascularisation using MRI and CT in a tumour model with modifiable angiogenesis – An animal experimental prospective cohort study

Background To investigate vascular-related pathophysiological characteristics of two human lung cancers with modifiable vascularisation using MRI and CT. Methods Tumour xenografts with modifiable vascularisation were established in 71 rats (approval by the Animal Care Committee was obtained) by subcutaneous transplantation of two human non-small-cell lung cancer (NSCLC) cells (A549, H1299) either alone or co-transplanted with vascular growth promoters. The vascularity of the tumours was assessed noninvasively by MRI diffusion-weighted-imaging (DWI), T2-weighted, and time-of-flight (TOF) sequences) as well as contrast-enhanced CT (CE-CT), using clinical scanners. As a reference standard, histological examinations (CD-31, fluorescent beads) were done after explantation. Results Microvessel density (MVD) was higher in co-transplanted tumours (171 ± 19 number/mm2) than in non-co-transplanted tumours (111 ± 11 number/mm2; p = 0.002). Co-transplanted tumours showed higher growth rates and larger tumour vessels at TOF-MRI as well as larger necrotic areas at CE-CT. In co-transplanted tumours, DWI revealed higher cellularity (lower minimal ADCdiff 166 ± 15 versus 346 ± 27 mm2/s × 10−6; p < 0.001), highly necrotic areas (higher maximal ADCdiff 1695 ± 65 versus 1320 ± 59 mm2/s × 10−6; p < 0.001), and better-perfused tumour stroma (higher ADCperf 723 ± 36 versus 636 ± 51 mm2/s × 10−6; p = 0.005). Significant correlations were found using qualitative and quantitative parameters: maximal ADCperf and MVD (r = 0.326); maximal ADCdiff and relative necrotic volume on CE-CT (r = 0.551); minimal ADCdiff and MVD (r = −0.395). Conclusions Pathophysiological differences related to vascular supply in two human lung cancer cell lines with modifiable vascularity are quantifiable with clinical imaging techniques. Imaging parameters of vascularisation correlated with the results of histology. DWI was able to characterise both the extent of necrosis and the level of perfusion.


Key points
Rat xenograft tumours with modifiable angiogenesis can be investigated using clinical scanners DWI characterises necrosis, perfusion, and vascularisation in tumours with variable vascularisation Differentiated views on ADC diff give hints for histological traits of tumour vascularisation CE-CT and TOF-MRI visualise parameters reflecting vascularisation DWI, CE-CT, TOF-MRI noninvasively deliver data characterising specific parameters of tumour pathophysiology related to vascularity Background Angiogenesis is of fundamental importance for growing tumours [1] and only by switching to an angiogenic phenotype, followed by the development of new vascularisation, solid tumours can overcome a critical size of 1-2 mm 3 [2,3]. Because of their own blood supply, cancer cells in solid tumours become independent from nutrition by diffusion of oxygen and nutrients from their surroundings. This lays the foundation for both the growth of the primary tumour and the spreading of tumour cells through the body. Characterisation of the vascularity of tissues in patients through imaging predicts response to therapy and correlates with prognosis [4].
Magnetic resonance imaging (MRI) of tumour vascularisation in vivo can be facilitated by time-of-flight (TOF) techniques for imaging the supplying tumour vessels and by diffusion-weighted imaging (DWI) [5] measuring the degree of the motion of free water in the imaging target [6][7][8][9] by calculating the apparent diffusion coefficient (ADC). Higher ADCs indicate fewer or less functional cell membranes frequently found in areas of necrosis or lower cellularity [10,11]. Larger b-values in DWI acquisition generate a greater sensitivity for diffusion while with lower b-values gross motions within the target tissue, such as perfusion as well as extravascular and extracellular microcirculation, contribute increasingly to the received signal [11][12][13].
Contrast-enhanced computed tomography (CE-CT) delivers other parameters connected to vascularisation. Contrast enhancement is influenced by both vascularisation and the extravascular space and can be easily quantified [14]. However, results of dynamic CE-CT (DCE-CT) show considerable variations due to theoretical and technical limitations [15].
The reference standard for quantification of tumour vascularisation is immunohistology with different antibodies [16]. The use of intravenously administered fluorescent beads is another possibility to quantify vascularisation and perfusion [17]. Correlation with the reference standard is desirable to compare the results of different imaging modalities that investigate characteristics of vascularity.
The aim of this study was to investigate human lung cancer xenografts with different degrees of vascularity (tumour-induced and amplified), using different clinically available imaging techniques and to correlate these results with histology and immunohistology.

Methods
To examine the influence of vascularisation on imaging parameters, a vascularisation-modifiable xenograft tumour model consisting of human cancer cell lines was used in this study. By the co-administration of vascular endothelial cells as vascular growth promoters, the vascularisation in tumours of the same cell line was manipulated [18][19][20]. This allows both to investigate the influence of vascular structures and vascular perfusion and to exclude influences of different cancer cell lines on imaging parameters. In this way, the differences related to the size of cells or nuclei, to the tumour stroma and microenvironment, to gene expression, and other cell-line characteristics should be minimised.

Cells, animals, and tumour transplantation
The human non-small cell lung cancer (NSCLC) cell lines A549 and H1299 were investigated and rat glomerular endothelial (RGE) cells were used to modify vascularity in these tumours.
The approval by the local Animal Care Committee was obtained in accordance with the institutional guidelines and the national animal welfare regulations. Four to 6-week-old athymic nude rats receiving food and water ad libitum were used. To enable tumour growth, all rats received one uniform whole-body irradiation of 4 Gy [21,22]. Short anaesthesia during irradiation and CE-CT was performed by intraperitoneal injection of ketamine and xylazine [23], long anaesthesia during MRI was done with gas narcosis using desflurane. Tumour cells were transplanted 48 h after irradiation by subcutaneous injection of 200 μl of tumour cell suspension into the right lower limb at the femur. The rats in the animal experimental prospective cohort study were assigned randomly to one of four groups (Fig. 1) and correspondingly received 5 × 10 6 tumour cells, 2 × 10 6 RGE cells, 160 ng recombinant human vascular endothelial growth factor 165 (rHu-VEGF-165), and 320 ng recombinant human fibroblast growth factor b (rHu-FGF-b). After injection, animals were inspected every 2 days and the tumour's length and width were measured by a caliper. As soon as any tumour had reached a size of 20 mm in one dimension, this tumour-bearing rat was subjected to the multimodal imaging protocol.

Imaging techniques
The imaging of tumours included MRI, DWI, CE-CT, and histological examinations. For CE-CT a positron emission tomography (PET)/CT Biograph 16 (Siemens AG, Erlangen, Germany) was used. A catheter implanted into the jugular vein was used for intravenous application of contrast material (Ultravist 370, Bayer Vital GmbH, Leverkusen, Germany) and beads. The CT protocol was: unenhanced CT scan; CE-CT after intravenous injection of 1 ml of contrast material using a small-animal injection pump (Medtron, Saarbrücken, Germany). The CT scan started 20 s after injection start. The scan specifications were 80 kV and 100 mAs; 512 2 pixels with a voxel resolution of 420 μm × 420 μm × 750 μm.
For MRI, a 1.5-T system and an eight-channel clinical knee coil (Magnetom Avanto, Siemens AG, Erlangen) were used. Rats were placed on a custom-made bed for gas narcosis. The following sequences were performed: T2-weighted turbo spin-echo (voxel size 0.

Histology and immunohistology
Two hundred microlitres of suspension containing 2.4 × 10 7 fluorescent beads (diameter 2.5 μm, excitation wave length 633 nm; G. Kisker GbR, Steinfurt, Germany) were injected via the jugular vein. After imaging and after two min of beads' circulation the rats were sacrificed and the organs as well as the tumours were removed [17].
Representative slices (slice thickness 10 μm) of the liver and kidneys (to verify appropriate application of beads) as well as the tumours were stained with hematoxylin-eosin. Tumour slices were additionally stained with CD-31 antibody to quantify microvessel density (MVD). The quantification of beads embolised into the vascularity of tumours was examined by fluorescent microscopy.

Image analysis
Histology and bead counts (beads: number of beads/field of view) served as standard of reference and were analysed within the tumours, the liver, and the kidneys. For CD-31 staining, tumour slices were digitally recorded at 20-fold magnification and the number of CD-31 positively stained structures were evaluated as published before, with MVD measured as the number of stained structures/mm 2 [16].
For analysis of CE-CT data, a contour was drawn around the contrast-enhanced tumour in all slices, including the outer rim of the tumour, the skin, and unenhanced lacunas but excluding bone and vessels without contact to the tumour. Limits of density values for analysis of the entire tumour were -50 ≤ HU ≤ +350. These contours were copied to the plain CT series and mean HU values in the tumours before and after contrast injection were measured.
The MRI protocol contained morphological and functional techniques. T2-weighted imaging served for colocalisation and tumour volumetry, the latter was obtained by manual segmentation. For visualisation of vessels, TOF-angiography was done and an ordinal scale was applied to the resulting three-dimensional image. The comparison to the three-dimensional datasets of the opposite femoral region was quantified according to the following ordinal scale: tumours received zero points if no differences in the visualisation of the vessels were detectable, one point if an increased vascularisation was visible, e.g. by separate short vessels, and two points if large circumscribable vessels travelled through the tumour. We did not use contrast-enhanced angiography studies (neither CT angiography nor MR angiography) for qualitative vessel visualisation since contrast bolus timing for appropriate imaging is dependent on circulation parameters that vary to a very high degree in sedated rats. To evaluate the quantity of small vessels and related motion in the extracellular space, the acquired b-values of DWI were utilised to separate apparent diffusion coefficients (ADCs) [11] for diffusion (ADC diff ) and perfusion (ADC perf ) [24]. Basically, for each slice, the images with bvalues of 500, 750, and 1000 s/mm 2 were aggregated to calculate eight-bit coloured ADC diff maps and images with bvalues of 50, 100, and 150 s/mm 2 were aggregated and subtracted from ADC diff maps to calculate eight-bit coloured ADC perf maps. These were registered to the T2-weighted image stacks and the contours of the tumours were copied to the ADC maps to extract the minimum, maximum, and mean ADC diff and ADC perf .

Statistical analysis
All statistical analyses were carried out with PASW 18 (Predictive Analytics SoftWare, IBM, Armonk, NY, USA) by comparisons between co-transplanted and non-cotransplanted tumours separated for each cell line as well as averaged for both cell lines. The Kolmogorov-Smirnov test was used to test for normality. The Mann-Whitney U test was used to analyse non-normally distributed data, whereas the t test was used for normally distributed data. Spearman's rank correlation coefficients were calculated to evaluate correlations. The χ 2 test was used to compare the results of TOF sequences between the groups. A p value lower than 0.050 was considered to indicate a statistically significant difference.

Cells, animals, and tumour transplantation
Both tumour cell lines H1299 and A549 revealed significantly faster growth of the tumours in the co-transplanted group as compared to the non-co-transplanted group (Fig. 2). Tumours in animals with co-transplantation reached imaging size earlier (Table 1). While bead embolisation in reference organs was not different between the groups, the vascularisation in tumours quantified by MVD and count of beads embolised into the tumours showed significant differences (Table 1). MVD and bead count were higher in co-transplanted tumours. For both cell lines, the central regions of the co-transplanted tumours often showed clusters of necrotic regions as well as vessels with larger diameters often closely connected to clouds of CD-31-positive small vessels (Fig. 3). Both necrotic areas and CD-31-positive vessels with a wide diameter were only rarely found in the centre of non-co-transplanted tumours (Fig. 3).

T2-weighted and TOF sequences
Higher volumes in the co-transplanted tumours than in non-co-transplanted tumours were found ( Table 2). TOF sequences allowed the visualisation of vessels in all xenograft tumours and revealed larger vessels in cotransplanted tumours (Table 2).

DWI, ADC diff , and ADC perf values
All ADC parameters were acquired in the kidneys in all groups for reference and no significant difference for any ADC parameter was found (data not shown). DWI revealed significantly lower minimal ADC diff values and significantly higher maximal ADC diff values in co-transplanted tumours as compared to non-co-transplanted tumours ( Table 2, Fig. 5a). At DWI perfusion analysis, significantly higher mean and maximal ADC perf values were found ( Table 2, Fig. 5b) in co-transplanted as compared to non-cotransplanted tumours. The mean ADC perf values in H1299 tumours were significantly larger in co-transplanted than in non-co-transplanted tumours (p = 0.001) while in A549 tumours this difference was not significant ( Table 2).

Correlation analyses
The following correlations were found to be significant considering all tumours: the correlation between maximal ADC perf and MVD (p = 0.010; r = 0.326; Fig. 6), the correlation between mean ADC perf and contrast enhancement in whole tumour in CE-CT (p = 0.009; r = 0.294; Fig. 7), the correlation between maximal ADC diff   Fig. 9). For technical reasons, the computer-based calculation of the necrotic volume at CE-CT resulted in a value of 100% for one rat which is the outlying value in Fig. 8. Excluding this data point from the correlation analysis increases the correlation between the maximal ADC diff and relative necrotic volume estimated in CE-CT to p < 0.001; r = 0.594 (Spearman's rank).

Discussion
The aim of this study was to evaluate imaging parameters quantifying vascularity in human cancer xenografts with two levels of vascularisation. The amount of vascularisation was determined ex vivo by a reference standard, i.e. specific histological and immunohistological methods. To provide translational results, these tumours were investigated in vivo with scanners used in clinical patient care. Clinically applied imaging techniques were utilised and optimised for small-animal imaging.  The selected human lung cancer cell lines are frequently used in preclinical xenograft research, typically on mice. In this study the authors decided to use a rat model. These animals can carry larger tumour sizes that mandate induction of relevant vascularisation to overcome restrictions of oxygen diffusion distance and additionally will develop relevant amounts of necrosis.
Moreover, these tumour sizes are appropriate for evaluation with clinical scanners and imaging protocols. Two different lung cancer cell lines were utilised and both of them were investigated either with or without coinjection of vascular growth promoters (i.e. vascular growth factors and rat endothelial cells) creating two different levels of vascular development in the growing Co-transplantation resulted in faster tumour growth. Since the amount of transplanted tumour cells was constant, the faster growth rate can be explained by a better supply of oxygen and nutrients as well as an improved evacuation of lactate and end products of metabolism Fig. 7 Spearman's rank correlation between contrast enhancement in whole tumour in contrast-enhanced computed tomography and mean ADC perf ; p = 0.009; r = 0.294 Fig. 8 Spearman's rank correlation between relative necrotic volume on contrast-enhanced computed tomography and maximal ADC diff ; p < 0.001; r = 0.551 due to better vascularisation. An important condition for fast tumour growth is the angiogenic switch that is dependent on endothelial precursor cells [25]. Beside all other influences, co-transplantation with vascular growth factors and rat endothelial cells increased the probability of a successful switch to an angiogenic phenotype which led to better vascularisation in these tumours. This was proven in our study by the increases of MVD and the larger amount of embolised beads within the tumours. Interestingly, the co-transplanted tumours grew so fast that the development of central necrosis was higher in this group even though the number of larger vessels was also higher. In fact, CE-CT showed wide hypodense areas within a contrastenhancing tumour parenchyma at the tumour rim and around larger central vessels in co-transplanted tumours. Using TOF sequences, co-transplanted tumours revealed the development of larger vessels in contrast to non-cotransplanted tumours. In summary, better vascularisation boosts fast growth which at the same time can increase necrosis by uncoupling regions from blood supply due to fast growth. Conversely, this means that regions of necrosis may be an indirect indicator of better vascularisation.
Adding a functional MRI technique, like quantitative DWI, with the calculation of ADC values, increases the diagnostic information. ADC diff describes various properties of tumours. Higher ADC diff values indicate higher diffusion; that is equating with fewer or less functional cell membranes frequently found in areas of necrosis or lower cellularity. As known from biology and neurology, necrosis is a continuum extending from viability to cell to tissue death. Therefore, a differentiated view on the ADC diff is required since the latter allows for detecting different levels of necrosis.
Furthermore, ADC diff conveys indirect information concerning the viable part of the tumours and the condition of the tumour microenvironment [26]. The peak value of ADC diff (high maximal ADC diff ) reflects the degree of necrosis, whereas the nadir of ADC diff (low minimal ADC diff ) informs about the condition and cellular density of the viable tumour cells. Both permit direct and indirect interpretations on the level of vascularisation, blood supply, and the rate of growth. The range of the ADC diff values provides an additional source of information for a differentiated interpretation of ADC diff . The maximal ADC diff in a tumour may reflect the extent of necrosis in certain regions. As mentioned before, better vascularisation induces faster tumour growth that may impede the balance between nutrient supply and nutrient demand which may trigger regional critical shortness in blood supply leading to increased necrosis [27,28]. In addition, fast cellular growth rate is paralleled by the unorganised development of pathological vessels leading to critical shortage of oxygen in highly vascularised tumours. Such shortage leads to necrosis in relevant parts of larger tumours. The formation of regions with severe necrosis in better-vascularised tumours can be the consequence. Such necrosis is not seen in smaller tumours [29] because these tumours show a slower growth rate with more adequate vascular development and do not develop critically supplied areas [28]. The histological findings in this study supported this fact and are in agreement with the current knowledge on the pathophysiology of tumour growth. In highly vascularised, large tumours, wide-diameter vessels, which lack further small branches, were found traversing through necrotic regions. Thus, nutrition in these regions was limited to diffusion from one large single vessel. In this respect, better-vascularised tumours exhibited areas with more necrosis. These areas were formerly viable tumour regions that lost their oxygen supply and became necrotic. This situation was found less often in slowergrowing tumours. To sum up, tumours with better vascularisation exhibit both severe necrosis and wellvascularised areas. Consequently, the existence of large necrotic areas in a tumour can be an indirect indicator for good vascularisation. The differentiated view on ADC diff could detect this indicator. More precisely, the maximal ADC diff (high maximal values in a tumour) suggests the existence of areas of high necrosis which is more often found in better-vascularised tumours. Hence, a higher maximal ADC diff suggests better-vascularised tumours.
The results of this study support this fact. DWI is capable of detecting these differences and was already found to correlate with MVD [30]. The ADC diff is much higher in better-vascularised tumours because of the existence of highly necrotic areas exhibiting high diffusion values [31]. The peak value of ADC diff (maximal ADC diff ) indicates focal areas of severe necrosis found in tumours with faster growth due to better vascularisation. The latter was insufficient at some point of tumour growth resulting in critical shortage of oxygen and nutrients and consecutive development of necrosis. This may indicate a poorer prognosis as Marconi et al. recently showed in women with advanced-stage cervical cancer [32].
The interpretation of the minimal ADC diff should also be meaningful. The two major factors for impeded diffusion as estimated by the lowest values of ADC diff are the severely reduced intracellular space (due to more organelles and larger nuclei) and extracellular space (due to high proliferation rate) in viable tumour tissue. In general, the lower ADC diff values correspond to higher cellularity and more compact tumour tissue. Impeded diffusion in tumours occurs if tumour cells can proliferate and form their typical tumour microenvironment. However, such proliferation requires oxygen and nutrient supply and, therefore, the existence of a vascular system within the tumour. As found herein, a low minimal ADC diff may indirectly indicate well-vascularised areas. The available academic literature gathered contradictory results related to this topic. In women suffering from cervical cancer, Nakamura et al. found that low ADC values in the tumours were related to poor prognosis [33] which supports the findings in this study, since patients with highly vascularised tumours frequently exhibit a poorer prognosis.
In summary, the range of the ADC diff is related to high vascularisation. More precisely, higher maximal ADC diff values are associated with high necrosis as a consequence of very fast tumour growth while very low minimal ADC diff values are associated with high cellularity, dense cell-cell contacts, and high protein expression as a consequence of good proliferation. Both fast growth and good proliferation may be surrogate parameters related to better vascularisation.
The ADC perf is capable of detecting perfusion in tissues directly and is related to vascularisation in tumours. In this tumour model, highly vascularised and wellperfused tumours exhibited higher ADC perf values, indicating increased tumour vascularisation. This was shown in both cell lines and was independent from the influence of the histological cell type. The results of this study show that the separation of the ADCs can provide physicians with more information. However, in addition to the suggestion by Koh et al. [12] that separating different b-values may contribute to this, it was shown herein that the differentiated analysis of minimal and maximal ADC values may increase the value of the imaging [34,35].
DWI can be used in extension to histology and other imaging modalities. The evaluation of vascularisation in tumours can be improved if different methods are used in combination. ADC diff , ADC perf , MVD, and other methods detect correlated characteristics of vascularisation in tumours. The low, but significant, correlation between MVD and maximal ADC perf supports the idea that both parameters are influenced by vascularisation properties. The reason for the comparatively weak correlation might be that both parameters are influenced by different aspects of vascularisation. MVD is predominantly influenced by the existence of vessels (which do not necessarily have to be well perfused), whereas the ADC perf is mainly influenced by the level of perfusion (which is not necessarily linked to the existence of intact vessel walls).
The correlation with the minimal ADC diff values is also in line with this concept. Well-vascularised tumours developed areas with high proliferation and high cellularity which may account for the correlation of MVD and the minimal ADC diff . This indicates that perfusion and vascularisation in tumours affect the DWI values obtained which was confirmed by the histological results of this study. Much more than the ADC perf , the minimal ADC diff and the MVD are influenced by further aspects of vascularisation. The authors speculate that the minimal ADC diff is linked indirectly to vascularisation, whereas MVD is connected much more closely to vascularisation. While this might explain the small magnitude of this correlation, a small, but significant, correlation can be also meaningful. As described before, highly vascularised fast-growing tumours reveal both well-perfused and necrotic areas. The correlation between CE-CT and DWI implies that both methods capture this situation. Necrotic areas were detected by high maximal ADC diff values. CE-CT visualised larger, comparatively necrotic, volumes and stronger contrast enhancement in vascularised areas. In this case, both parametersthe maximal ADC diff and the relative necrotic volume in CE-CT are directly influenced by the same factor: the level of necrosis. Both diffusion and contrast enhancement increase with increasing levels of necrosis. Accordingly, a moderately significant correlation was found. Thus, DWI and CE-CT complement each other and extend the insights in the tumour biology concerning vascularisation.
One limitation of this study is the utilisation of clinical scanners for small-animal imaging with reduced resolution as compared to dedicated preclinical scanners. To overcome this disadvantage, a rat animal model was used that allows the use of higher tumour volumes. The frequently used mice tumour models only permit smaller tumour sizes for animal welfare reasons. Tumours of this size are difficult to scan with clinical scanners [5]. In contrast, the rat tumours in this study were at least two centimetres in maximal extension. The pathophysiology related to vascularisation of tumours of this size may be much more comparable to tumours in patients since diffusion distances for oxygen and other nutrients cannot cover comparatively large tumour volumes, especially at the tumour margins. Tumour sizes investigated in this study are dependent on sufficient neovascularisationas the tumours in patients areand should provide information more comparable to the clinical situation. In diagnostic imaging of tumours of patients, the presence of large necrotic areas (hypodense in CE-CT in the venous phase) and highly vascularised peripheral areas (hyperdense in CE-CT in the arterial phase) is a frequent clinical finding. The applicability of clinical imaging in this model is demonstrated by the comparison to the histological and immunohistological results. Furthermore, even though we pushed the imaging protocols to their limits, we believe that the results found herein are a good basis for translating this to clinical patient care.
With respect to DWI applications in oncology, the reliable transferability of ADC values between different imaging systems has been under discussion for many years [36]. The definition of standardised technical protocols for DWI and standardised interpretation of results are still under evaluation and increasing consensus is developing [37]. In our study, we used standardised parameters that are applicable for clinical imaging and allow comparability to other imaging systems. Using the comparison with histology, this research added new information to the current research on how reliable ADC values for both diffusion and perfusion are. The use of two different cell lines each with two different stages of vascular development reduced the influences on imaging due to different histological traits of different cell lines like protein expression, size, and membrane permeability of the tumour cells. Consequently, differences in imaging parameters within the same tumour cell line are more likely caused by vascularisation rather than by characteristics of the cell line. This is an important advantage compared to other research investigating histologically diverse tumour lines. The investigated lung cancer cell lines are standard in preclinical research and are previously well described. Further clinical investigations will be developed taking advantage of the results found herein.
The present study established a human tumour xenograft cancer model with modifiable vascularisation and successfully evaluated parameters reflecting vascularisation in tumours by noninvasive clinical imaging. Results were correlated with histology and immunohistology. The amount of necrosis and the pathologic tumour vasculature in tumours were visualised using clinical scanners and were correlated with histological results. The results of the study showed that DWI is capable of assessing information not only on cell density but also indirectly on the vascularisation in tumours. The differentiated observation of DWI parameters increased the information obtainable from imaging and provide additional parameters that can be introduced to large-data analysis. ADC diff should be interpreted in its minimal, mean and maximal extension. The harness of established clinical methods (DWI, CE-CT, TOF) may by expanded by a better understanding of these imaging parameters. The results of this study could contribute to this. This may give more information concerning the aggressiveness of tumours, which may influence therapy [4,32,33]. Additionally, this study showed that clinical imaging units may well be used for preclinical imaging, which allows faster translation of results.