Longitudinal MRI contrast enhanced monitoring of early tumour development with manganese chloride (MnCl2) and superparamagnetic iron oxide nanoparticles (SPIOs) in a CT1258 based in vivo model of prostate cancer

Abstract

Background: Cell lines represent a key tool in cancer research allowing the generation of neoplasias which resemble initial tumours in in-vivo animal models. The characterisation of early tumour development is of major interest in order to evaluate the efficacy of therapeutic agents. Magnetic resonance imaging (MRI) based in-vivo characterisation allows visualisation and characterisation of tumour development in early stages prior to manual palpation. Contrast agents for MRI such as superparamagnetic iron oxide nanoparticles (SPIOs) and manganese chloride (MnCl2) represent powerful tools for the in-vivo characterisation of early stage tumours. In this experimental study, we labelled prostate cancer cells with MnCl2 or SPIOs in vitro and used 1 T MRI for tracing labelled cells in-vitro and 7 T MRI for tracking in an in-vivo animal model.

Methods: Labelling of prostate cancer cells CT1258 was established in-vitro with MnCl2 and SPIOs. In-vitro detection of labelled cells in an agar phantom was carried out through 1 T MRI while in-vivo detection was performed using 7 T MRI after subcutaneous (s.c.) injection of labelled cells into NOD-Scid mice (n = 20). The animals were scanned in regular intervals until euthanization. The respective tumour volumes were analysed and corresponding tumour masses were subjected to histologic examination.

Results: MnCl2in-vitro labelling resulted in no significant metabolic effects on proliferation and cell vitality. In-vitro detection-limit accounted 105 cells for MnCl2 as well as for SPIOs labelling. In-vivo 7 T MRI scans allowed detection of 103 and 104 cells. In-vivo MnCl2 labelled cells were detectable from days 4-16 while SPIO labelling allowed detection until 4 days after s.c. injection. MnCl2 labelled cells were highly tumourigenic in NOD-Scid mice and the tumour volume development was characterised in a time dependent manner. The amount of injected cells correlated with tumour size development and disease progression. Histological analysis of the induced tumour masses demonstrated characteristic morphologies of prostate adenocarcinoma.

Conclusions: To the best of our knowledge, this is the first study reporting direct in-vitro MnCl2 labelling and 7 T based in-vivo MRI tracing of cancer cells in a model of prostate cancer. MnCl2 labelling was found to be suitable for in-vivo tracing allowing long detection periods. The labelled cells kept their highly tumourigenic potential in-vivo. Tumour volume development was visualised prior to manual palpation allowing tumour characterisation in early stages of the disease.

Figure 1

1T MRI scan of MnCl₂and SPIOs labelled cells in a 1% agar phantom. The numbers of labelled cells accounted: 7 × 10⁵, 2.5 × 10⁵, 10⁵, 5 × 10⁴, 2.5 × 10⁴, 10⁴, 5 × 10³, 10³. As controls 1 × 10⁵ unlabelled cells, the culture medium, 1 M MnCl₂, and Endorem solution were used. A: T1 weighted MRI. SPIOs labelled cells (left two lanes), the controls with un-labelled cells and the culture medium showed comparable signals. 1 M MnCl₂ solution showed a strong signal enhancement. MnCl₂ labelled cells (right two lanes) were detected to a limit of 10⁵ cells. B: T2* weighted MRI scan. MnCl₂ labelled cells (right two lanes), the controls, and the culture medium showed comparable signals. Endorem solution showed a strong signal extinction. SPIOs labelled cells (left two lanes) were detected to a limit of 10⁵cells.

Figure 2

7T MRI of a male NOD-scid mouse after subcutaneous injection of 10⁴MnCl₂labelled CT1258 cells. The MRI scans were performed on the same day as the injection (day 1). A: FSE T1 weighted MRI Scan. B: FSE T2 weighted MRI scan. Arrows: localisation of the injected MnCl₂ labelled cells in T1 (A) and T2 (B) weighted MRI. In T1 weighted MRI (A) the MnCl₂ labelled cells were detected due to a strong signal enhancement.

Figure 3

7T MRI scans of a male NOD-scid mouse after subcutaneous injection of 10⁴SPIOs labelled CT1258 cells. The MRI scans were performed on the same day as the injection (day 1). A: FSE T1 weighted MRI Scan. B: FSE T2 weighted MRI scan. C: FISP T2* weighted MRI scan. Arrows: localisation of the injected SPIOs labelled cells in T1 (A), T2 (B) and T2* (C) weighted MRI. In T2 (B) and T2* (C) weighted MRI the SPIOs labelled cells were detected due to a strong signal extinguishment.

Figure 4

Longitudinalin vivo7 T MRI scans of two male NOD-scid mice after subcutaneous injection of 10⁴CT1258 cells labelled with either MnCl₂or SPIOs at the day 1, 4, 9 and 28 after injection.A-D: in vivo MRI scans of an animal which received MnCl₂ labelled cells. A: FSE T1 weighted MRI scan on the day of injection (day 1). B: FSE T1 weighted MRI scan on day 4 after injection. C: FSE T1 weighted MRI scan on day 9 after injection. D: FSE T1 weighted MRI scan on day 28 after injection. The MnCl₂ labelled cells were not detectable and a tumour mass developed at the site of injection. E-H: in vivo MRI scans of an animal which received SPIOs labelled cells. E: FSE T2 weighted MRI scan on the day of injection (day 1). F: Turbo RARE T2 weighted MRI scan on day 4 after injection. G: Turbo RARE T2 weighted MRI scan on day 9 after injection. The cells were not detected and there were no signs of tumour development. H: FSE T1 weighted MRI scan on day 28 after injection. White arrows: localisation of labelled cells. Red arrow: localisation of the developed tumour at the site of injection.

Figure 5

T1 weighted MRI scan, three dimensional reconstruction and histological analyses of a tumour induced by injection of MnCl₂labelled CT1258 cells into a male NOD-scid mouse.A: RARE T1 weighted in vivo MRI scan 24 days after sc injection of the labelled cells. On the right abdominal flank, a tumour mass developed. No signs of metastasis were detected. B: Three dimensional graphical analysis of the tumour structure 24 days after injection of the labelled cells. C and D: Histological analysis of the obtained tumour mass. The obtained tumour showed the characteristic appearance and morphology of a prostate adenocarcinoma. The analysis displayed pleomorphic cells with a high mitotic index. The centre of the obtained tumour was highly necrotic and the tumour infiltrated into the subcutaneous fat tissue. The tumour developed an incomplete fibrous capsule. White arrow: localisation of the developed tumour at the site of injection.