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. 2012 Oct;61(10):1617-26.
doi: 10.1007/s00262-012-1219-3. Epub 2012 Feb 19.

In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model

Alessa Pardo  1 , Michael Stöcker, Florian Kampmeier, Georg Melmer, Rainer Fischer, Theo Thepen, Stefan Barth
Affiliations

In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model

Alessa Pardo et al. Cancer Immunol Immunother. 2012 Oct.

Abstract

Purpose: Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.

Methods: We transfected A-431 tumour cells with the far red-emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A' (ETA'). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.

Results: The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA' resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.

Conclusions: We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA'.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Treatment with 425(scFv)-ETA’ and Doxorubicin. a Treatment was started at day 0 and performed in two cycles consisting each of 4 single injections. With each injection, mice received i.v. 10 μg 425(scFv)-ETA’, 100 μg Doxorubicin or PBS. Pictures were taken, and the tumour growth was calculated with the CRi Maestro System at all indicated time points. b All mice were imaged at different time points during, in between and after the treatment cycles. Based on the fluorescence signal, the tumour area could be determined, which is then calculated by the Maestro Software. The images show the tumour area of one representative mouse of each group at 3 different time points (days 0, 13 and 26). White light images of the mice and fluorescence images were overlaid. c The diagram shows the mean tumour size of 6 mice per group calculated by the Maestro Software throughout both treatment cycles. For comparison of the tumour growth between all groups, the mean tumour sizes on day 0 were set to 100%, while all other values were adjusted. The decelerated tumour growth of the group treated with 425(scFv)-ETA’ compared to the PBS control group was confirmed statistically significant (p < 0.05). d The diagram shows the mean tumour size of 6 mice per group measured with the vernier calliper and calculated according to the formula r1*r2*π throughout both treatment cycles. For comparison of the tumour growth between all groups, the mean tumour sizes on day 0 were set to 100%, while all other values were adjusted. The decelerated tumour growth of the group treated with 425(scFv)-ETA’ compared to the PBS control group was confirmed statistically significant (p < 0.05)
Fig. 2
Fig. 2
Spectral libraries of Kat2 and 425(scFv)SNAP-747. Spectral libraries are used for a the measurement of the Kat2 signal and b for unmixing of the Kat2 and 425(scFv)SNAP-747 signals. The library is calculated from a cube acquired with multiple filter settings: yellow (630–850 nm) and deep red (730–950 nm). The red lines represent the Kat2 signal and the blue lines the BG-747 signal in both filter settings
Fig. 3
Fig. 3
Visualisation of A431scM3-Kat2 cells in vivo. In vitro-transfected and sorted A431scM3-Kat2 cells were subcutaneously injected into the right hind leg of BALB/c nu/nu mice and visualised with the CRi Maestro system after 11 days of tumour growth. Measurement was taken with the yellow filter set (630–850 nm). Red fluorescent cells can be visualised without any background signal. Here, the picture of the fluorescence signal is merged with the white light picture of the mouse
Fig. 4
Fig. 4
Targeting of A431scM3-Kat2 with 425(scFv)SNAP-747. With BG-747 labelled 425(scFv)SNAP was i.v. applied into A431scM3-Kat2-bearing mice. The pictures shown here were taken 10 h after application. Measurement was taken with the yellow (630–850 nm) and deep red filter set (730–950 nm). a Composite image with all fluorescence signals (red A431scM3-Kat2, blue 425(scFv)SNAP-747, white background), b background signal, c pure A431scM3-Kat2 signal, d pure 425(scFv)SNAP-747 signal
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