Enhanced noninvasive imaging of oncology models using the NIS reporter gene and bioluminescence imaging

Abstract

Noninvasive bioluminescence imaging (BLI) of luciferase-expressing tumor cells has advanced pre-clinical evaluation of cancer therapies. Yet despite its successes, BLI is limited by poor spatial resolution and signal penetration, making it unusable for deep tissue or large animal imaging and preventing precise anatomical localization or signal quantification. To refine pre-clinical BLI methods and circumvent these limitations, we compared and ultimately combined BLI with tomographic, quantitative imaging of the sodium iodide symporter (NIS). To this end, we generated tumor cell lines expressing luciferase, NIS, or both reporters, and established tumor models in mice. BLI provided sensitive early detection of tumors and relatively easy monitoring of disease progression. However, spatial resolution was poor, and as the tumors grew, deep thoracic tumor signals were massked by overwhelming surface signals from superficial tumors. In contrast, NIS-expressing tumors were readily distinguished and precisely localized at all tissue depths by positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging. Furthermore, radiotracer uptake for each tumor could be quantitated noninvasively. Ultimately, combining BLI and NIS imaging represented a significant enhancement over traditional BLI, providing more information about tumor size and location. This combined imaging approach should facilitate comprehensive evaluation of tumor responses to given therapies.

Fig. 1

In vitro reporter activity of A549-hNIS-Neo/Fluc-Puro cells. a The indicated number of A549-hNIS-Neo/Fluc-Puro cells were seeded in six-well plates and an ¹²⁵I uptake assay was performed in the presence or absence of the NIS inhibitor KClO₄. b The indicated number of A549-hNIS-Neo/Fluc-Puro or parental A549 cells were seeded in 96-well plates, and luminescence (Flux) was measured immediately after the addition of 3 mg/ml d-luciferin. c, d The indicated number of A549-hNIS-Neo/Fluc-Puro cells were mixed with parental A549 cells to a total of 1 × 10⁶ cells/tube and incubated for 30 min with [¹⁸F]-TFB. Following a wash to remove residual [¹⁸F]-TFB, the cell pellets were imaged by PET/CT d and the [¹⁸F]-TFB uptake from each tube was determined c. e The indicated number of A549-hNIS-Neo/Fluc-Puro cells were seeded in 96-well plates, and BLI was performed immediately after the addition of 3 mg/ml d-luciferin

Fig. 2

High-resolution tumor imaging with precise anatomical localization using NIS. a, b C57Bl/6 mice were implanted intravenously with LL/2-mNIS cells; a representative mouse is shown. LL/2-mNIS tumors were imaged by PET/CT using [¹⁸F]-TFB on day 27. Shown is a 3D rendering of tumor localization within the mouse (arrow indicates location of bone metastasis) a and 2D z-stack slices of the thoracic region (3-mm interval) b. c, d SCID beige mice were implanted intravenously with Nalm6-Fluc-hNIS cells; a representative mouse is shown. Nalm6-Fluc-hNIS tumors were imaged by BLI on day 26 c and by SPECT/CT using ¹²⁵I on day 25 d

Fig. 3

Discrimination of surface and deep-tissue tumors using NIS. a–c Balb/c mice were implanted intravenously with 4T1-Fluc-Neo cells; a representative mouse is shown. a Tumors were imaged by BLI. b Post-mortem autopsy and BLI was performed upon killing and removal of the skin. c Excised lung lobes were imaged by BLI. d, e Balb/c mice were implanted intravenously with 4T1-mNIS cells and PET/CT imaging was performed after 20 days using [¹⁸F]-TFB; a representative mouse is shown. d Global [¹⁸F]-TFB uptake in mice. e Tomographic representation of tumors from d, colored based on tumor locations

Fig. 4

In vivo correlation of Fluc and NIS signal to tumor volume. a Median uptake (kBq/g) of [¹⁸F]-TFB from individual 4T1-mNIS tumors from mouse in Fig. 3d and e. b, c Five SCID beige mice were implanted subcutaneously with 4T1-Fluc-Neo/mNIS-Puro cells. On days 9 and 16 after implantation tumors were measured with calipers and BLI imaging was performed. SPECT/CT imaging using [^99mTc]-pertechnetate was performed on days 10 and 17 after implantation. Tumor volumes determined by caliper measurements or CT were compared to [^99mTc]-pertechnetate uptake b and total tumor flux c

Fig. 5

Multi-modality BLI and NIS imaging of tumors. SCID beige mice were implanted intravenously with 4T1-Fluc-Neo/mNIS-Puro cells; a representative mouse is shown. a Tumors were tracked over time using BLI. b On day 20 after implantation, PET/CT imaging was performed using [¹⁸F]-TFB