In vivo fluorescence imaging and urinary monoamines as surrogate biomarkers of disease progression in a mouse model of pheochromocytoma

Abstract

Pheochromocytoma (PHEO) is a rare but potentially lethal neuroendocrine tumor arising from catecholamine-producing chromaffin cells. Especially for metastatic PHEO, the availability of animal models is essential for developing novel therapies. For evaluating therapeutic outcome in rodent PHEO models, reliable quantification of multiple organ lesions depends on dedicated small-animal in vivo imaging, which is still challenging and only available at specialized research facilities. Here, we investigated whether whole-body fluorescence imaging and monitoring of urinary free monoamines provide suitable parameters for measuring tumor progression in a murine allograft model of PHEO. We generated an mCherry-expressing mouse PHEO cell line by lentiviral gene transfer. These cells were injected subcutaneously into nude mice to perform whole-body fluorescence imaging of tumor development. Urinary free monoamines were measured by liquid chromatography with tandem mass spectrometry. Tumor fluorescence intensity and urinary outputs of monoamines showed tumor growth-dependent increases (P < .001) over the 30 days of monitoring post-tumor engraftment. Concomitantly, systolic blood pressure was increased significantly during tumor growth. Tumor volume correlated significantly (P < .001) and strongly with tumor fluorescence intensity (rs = 0.946), and urinary outputs of dopamine (rs = 0.952), methoxytyramine (rs = 0.947), norepinephrine (rs = 0.756), and normetanephrine (rs = 0.949). Dopamine and methoxytyramine outputs allowed for detection of lesions at diameters below 2.3 mm. Our results demonstrate that mouse pheochromocytoma (MPC)-mCherry cell tumors are functionally similar to human PHEO. Both tumor fluorescence intensity and urinary outputs of free monoamines provide precise parameters of tumor progression in this sc mouse model of PHEO. This animal model will allow for testing new treatment strategies for chromaffin cell tumors.

Figure 1.

Biological properties of MPC-mCherry cells compared with nontransduced parent MPC cells in vitro; A and B, Morphological appearance and fluorescence under microscopy; nuclei stained with Hoechst 33258; scale bars, 30 μm. C, Growth of 2 × 10⁵ MPC and MPC-mCherry cells during 16 days post subculture quantified as protein of adherent cells per cm², presented as means ± SEM, n = 4. D, Positive proportional relation of MPC-mCherry cell number and corresponding fluorescence intensities at λ_Ex/Em = 550/600 nm.

Figure 2.

Progression of sc tumors in male and female nude mice after sc injection of 2 × 10⁶ MPC-mCherry cells. A, Overlays of x-ray and fluorescence images (λ_Ex/Em = 600/700 nm) of a representative animal at 1 h to 29 d post injection. B, Tumor fluorescence intensities in vivo over time. C, Tumor volumes over time; data are presented as mean ± SD, (male animals, n = 10; female animals, n = 10). D, Correlation analysis between tumor volume and tumor fluorescence intensity; n = 20; confidence interval, 95%; number of XY pairs, 136; p.i., post injection; px, pixel.

Figure 3.

Physiologic parameters during tumor progression in male and female nude mice after sc injection of 2 × 10⁶ MPC-mCherry cells. A–D, Urinary concentration of free monoamines −10 and −2 d before, and 6, 13, 20, and 27 d after cell injection; presented as mean ± SD (male animals, n = 10, female animals, n = 10). E, Changes in the urinary concentration pattern of free monoamines (net chart) presented as mean (male + female animals, n = 20). F, Systolic BP −10 and −4 d before and 3, 10, 17, 24, 31 d after cell injection presented as mean (male animals, n = 10; female animals, n = 10) and mean ± SD (male + female animals, n = 20); DA, dopamine; MTY, 3-methoxytyramine; NE, norepinephrine; NMN, normetanephrine; EPI, epinephrine; MN, metanephrine; p.i., post injection. **, P < 0.01; ***, P < 0.001.

Figure 4.

Analysis of covariance between urinary free monoamines and tumor volume. DA, dopamine; MTY, 3-methoxytyramine; NE, norepinephrine; EPI, epinephrine; NMN, normetanephrine. n = 20, confidence interval, 95%, number of XY pairs, 78.