Preclinical in vivo imaging modalities have complementary relative strengths in sensitivity, resolution, quantification and molecular versus anatomical detections. The use of these modalities can be combined in studies of oncology, infec-tion, and probe/tracer development to leverage their relative strengths. As an example, optical molecular imaging and microCT imaging have complementary, synergistic strengths. Optical molecular imaging offers a relatively high throughput platform, where multiple experimental animals can be imaged simultaneously. Further, using disease models with biolumi-nescent or fluorescent transfected organisms (e.g. tumor cells, stem cells, and/or infectious agents), optical molecular imag-ing can provide highly sensitive, relatively early in vivo detec-tions. Bioluminescence Imaging (BLI) is particularly well suited for sensitive cell progression imaging owing to the excellent Signal to Noise Ratio (SNR) achieved with luciferase report-ers (Conley-LaComb et al., 2016). In a complementary fashion, microCT imaging provides excellent resolution of anatomical features, given the inherent density differences of bone, adi-pose, and lung tissues (Postnov et al., 2009).

In this study, the relative synergies of in vivo BLI, Fluorescence Imaging (FLI), and micro-CT imaging was assessed in a classic orthotopic lung cancer model from onset to late stage progres-sion. The tumor progression study was performed using a dual bioluminescent/fluorescent reporter human lung carcinoma cell line (A549-FLuc-Neo/iRFP-Puro; Imanis Life Sciences, Rochester, MN, USA) in athymic nude female mice (Envigo

RMS, Inc. Indianapolis, IN, USA; see Table 1). Pulmonary tumor foci were initiated by i.v. tail vein injection. Imaging was performed weekly to assess limits of detection and resolution for each modality. Ex vivo imaging was also performed. Optical imaging was performed using the In-Vivo Xtreme II optical/X-ray imaging system (Bruker BioSpin, Billerica, MA, USA), and microCT imaging was completed using the SkyScan 1176 & 1272 scanners (Bruker microCT, Kontich, Belgium). Image system settings and study methods were selected to optimize lung imaging results and are outlined in Table 1.

Lung BLI signals were detected in all test mice (n = 4) from the earliest stages (2 hours post challenge) to study comple-tion, while microCT imaging provided excellent anatomical details of tumor location, volume, and tissue environment starting at mid-late stage tumor development (4 weeks post challenge). Historically, there has been some emphasis on the concept that a key synergy of optical and microCT lies in their image registration/fusion; approaches for combined optical + microCT imaging and registration, however, typically require long scan times and compromises in microCT imaging quality, limiting the traditional strengths for both optical (throughput) and microCT (resolution). Furthermore, the points of useful optical and microCT tumor detection during progressions differ significantly. Here, it could be demonstrated that optical/X-ray imaging performed using the In-Vivo Xtreme II supplied excellent functional/anatomical longitudinal tumor monitoring while maintaining high throughput imaging/processing – the

Monitoring Lung Tumor Progression from Onset to Late Stage, and ex vivo using Complementary Bioluminescence-, Fluorescence-, and microCT Imaging Authors: Van Praagh A.; Bahadur A.; Saaussan, M. and Sasser TA.

Bruker Preclinical Imaging, 15 Fortune Dr., Billerica, MA 01821 US

details not feasible using more limited non-gated microCT low resolution methods. Ex vivo microCT imaging was also done using the Skyscan 1272 at 5 microns to show high resolution details of the tumor distribution.

Figure 2key strengths of in vivo small animal optical imaging. Mid-late stage tumor burden was successfully imaged using the 4D ret-rospective synchronization option using the dedicated microCT scanner model SkyScan 1176 and SkyScan 3D rendering soft-ware which provided complementary anatomical/physiological

Table 1: Tumor Progression Study. From 2 hours to 9 weeks after injection of orthotopic lung tumors (A549-FLuc-Neo/iRFP-Puro cell line).

Tumor Model Mice: Hsd: Athymic Nude-Foxn1nu, females, 9-10 weeks (Envigo RMS, Inc. Indianapolis, IN, USA)

Tumor: human lung carcinoma cell line: A549-FLuc-Neo/iRFP-Puro (Imanis Life Sciences, Rochester, MN, USA)

Route of injection: Tail vein

Bioluminescence/X-ray (in vivo) Vasculature or blood perfusion markers

Instrument: Bruker In-Vivo Xtreme II (Bruker BioSpin, Billerica, MA, USA)

Luminescence setting: 2 min, 4 x 4 binning, f/1.1, 19 cm FOV, at 10 min post i.v. Luciferin (Targeting Systems, Inc., El Cajon, CA, USA)

Fluorescence/X-ray (in vivo) Fluorescence setting: Ex/Em 690/750 nm, 20 secs, 2 x 2 binning, f/1.1, 19 cm FOV, for iRFP in vivo tumor imaging

Fluorescence (ex vivo) Instrument: Bruker In-Vivo Xtreme II

Fluorescence setting: Ex/Em 690/750 nm, 120 secs, no Bin, f1.1, 7.2 cm FOV, for iRFP ex vivo tumor imaging

microCT Imaging (in vivo) Instrument: Bruker SkyScan 1176 (Bruker microCT, Kontich, Belgium)

Settings: 35 µm resolution, with gating

microCT Imaging (ex vivo) Instrument: Bruker SkyScan 1272 (Bruker microCT, Kontich, Belgium)

Settings: 5 µm resolution

MRI Instrument: Bruker ICON 1 Tesla (Bruker BioSpin MRI GmbH, Ettlingen, Germany)

Settings: RARE factor = 8, TE = 45 msec, TR = 1600 msec, Nslices = 10, Avg = 8, Image = avg of all echoes, Image matrix = 128 x 128, FOV = 25 x 25 mm, Slice thickness = 1 mm, Trigger ON (respiratory gated), Scan time > 2 min, 33 sec

Optical bioluminescence detection supplied early detection starting at 2 hours post challenge and through late stage tumor progression (Fig. 1). In vivo fluorescent detections (not shown) for tumor iRFP signal were only possible during mid-late stage

tumor progression, probably owing to a relatively lower SNR with in vivo FLI relative to in vivo BLI. Starting at week 4, lung tumor location and volume were readily discerned by both MRI and microCT (Fig. 1).

Figure 1. BLI, MRI, and microCT in vivo imaging of orthotopic lung tumors (A549-FLuc-Neo/iRFP-Puro cell line) in a representative longitudinal study (2 hours to 9 weeks post challenge). Row 1) BLI signal (fire; LUM) was detected at study onset (2 hours after injection) through study completion. Row 2) BLI signal (fire; LUM) was registered with X-ray. MRI (row 3) and microCT (row 4) revealed tumor location and volume (red arrows) starting at mid-stage tumor progression.

Figure 1

survival from initial engraftment to time point 24 hours, or due to a reduced rate of the luciferase enzymatic reaction due to a reduced concentration (within the lung environment relative to cell culture) of co-factors (e.g. ATP) critical for the luciferase enzymatic reaction in tumor cells. Similarly, during late stage tumor development from week 8 to 9, lung tumor BL signal again declined. Others have reported that luciferase signal reductions during late stage tumor progression can occur as a result of extensive cellular necrosis within poorly vascular-ized regions of large tumor masses. In fact, microCT imaging results (further below) revealed a reduction in vasculature in tumor regions in some tumor animals at week 9.

Figure 2BLI supplied high throughput, quantitative (photon/sec) tumor progression monitoring throughout the study (Fig. 2). Tradi-tional orthotopic lung cancer models are often executed via tail vein injection. Without BLI, it can be difficult to be fully confi-dent that tumor cell injections have been properly executed. Thus the early detection/validation by BLI of cell engraftment at target sites at the study onset, within 2 hours post challenge as demonstrated here, allows researchers to confirm the suc-cess of tumor injections, and to generate new experiment ani-mals if needed. Madero-Visbal et al. (2012) reported previously that BL signal, in an A549-FLuc lung tumor progression model, positively correlated with increased tumor burden. Consistent with this report, we observed a mostly steady increase in BL signal with tumor progression, with two interesting excep-tions. There was an apparent decline in BL signal from 2 to 24 hours post-injection in all mice (Fig. 2, bottom). Specula-tively, this decrease in signal is due to a some reduced cell

Figure 2. Bioluminescence imaging of orthotopic lung tumor (A549-FLuc-Neo/iRFP-Puro cell line) mice over time. Four tumor mice (n = 4) and one non-tumor mouse (control) were studied over a time course up to 9 weeks post challenge. Top) Throughput time course, BL signals (fire color coded) from tumor challenged mice and control mouse, and registered with X-ray at week 9. Bottom) Quantification of optical signal (photons/sec) from challenged mice (n = 4) over the study time course (2 hours to 9 weeks post challenge).

Figure 2

and semi-automated tumor segmentation protocols. Here, microCT imaging supplied excellent details of tumor distribu-tion, volume, and even tumor environment during mid-late stage tumor development (Fig. 3). MicroCT imaging offered a unique combined view of tumor distribution and pulmonary vascular environment, even in the absence of contrast agent for enhanced imaging.

Figure 2MicroCT imaging is uniquely suited for lung tumor and lung infection imaging owing to the innate contrast of pulmonary tissues with X-ray. Furthermore, microCT image contrast is not dependent on the functional status of tissues being imaged. Several methods for lung tumor progression imaging using microCT have been reported (Choi et al., 2014, Kumar et al., 2012). Methods used vary, and include optimized gating protocols for correction of respiratory motion, and manual

Figure 3. In vivo microCT imaging revealed tumor location and numbers as well the vascular conditions of the surrounding environment. Data for a representative animal at weeks 4, 6, 7, and 9 post challenge is shown here. A) In vivo microCT imaging during mid-late stage tumor development revealed tumor (red) distribution shown in dorsal and ventral orientations (top and mid line images), as detected by software surface rendering (CTVol, SkyScan), with both opaque and transparent contrast settings (the latter showing lung vasculature; bottom line) B) Total lung tumor volume (mm3) measurements by microCT imaging, showing progression during mid-late stage of study (4 to 9 weeks post challenge).

Finally, ex vivo fluorescence imaging and ex vivo high resolution microCT lung imaging was per-formed (Fig. 4). While optical luciferase reporters offer very little potential for ex vivo imaging, fluo-rescent reporters offer ex vivo whole organ imag-ing potential and can even be used for fluorescent microscopy imaging at later stages. Tumor cell lines with dual BL/FL reporters can support tran-sitional studies from in vivo BLI/FLI to ex vivo FLI. Ex vivo organ image resolution and contrast is relatively better for both optical and microCT. For optical FLI, fluorescent signal is subject to less tissue scatter resulting in near pinpoint focal accuracy. Ex vivo microCT organ imaging is ame-nable to imaging with high resolution scanners that offer high geometric magnification and using imaging doses not feasible for whole animal in vivo imaging.

Figure 4. Ex vivo fluorescence and microCT imaging provided high resolution and high contrast imaging/analysis of tumors in isolated lung organ, from a representative, challenged mouse at 9 weeks post challenge. Left) Ex vivo FLI reveals tumor foci. Right) Ex vivo microCT imaging offers micron level resolution of tumor foci, using protocols and methods specific for ex vivo sample imaging/analysis.

Figure 3

Figure 4

Figure 2Imaging Throughput*

Resolution Quantitation Tumor Stage

Ex vivo Experimental Value

Bioluminescence 5-10 mice/minute Limited (mm) (~1 mm in vivo; ~0.1 mm ex vivo)

Photons/s, correlates well with viable tumor cell burden

O n s e t -Late

Limited Temporal analysis from onset to late stage High throughput imaging

Fluorescence 5-10 mice/minute Limited (mm) (~1 mm in vivo; ~0.1 mm ex vivo)

Photons/s, correlates well with total tumor cell burden (alive and dead cells)

Mid-Late Yes Combined tumor and molecu-lar detections Transitional imaging from in vivo to ex vivo

microCT 1 mouse/30 sec--10 minutes

4-100 μm (<0.5 μm ex vivo)

Tumor volume, loca-tion, and number

Mid-Late Yes Refined analysis for tumor burden location, volume, and host environment

MRI 1mouse/5-60 min 1-250 µm Tumor volume, loca-tion, and number

Mid-Late Yes Refined analysis for tumor burden location, volume, and host environment

ConclusionThese studies indicate that combined optical and microCT imaging could be useful to interrogate aspects of tumor biol-ogy and therapeutics throughout tumor development (Table 2). BLI provides high throughput tumor monitoring from early onset to late stage that could be leverage to ascertain temporal points for efficacy of candidate therapeutics, assess temporal aspects of tumor biology/development, and supply neces-sary statistical power using large sample numbers with high throughput. During mid-late stage tumor development, dedi-cated anatomical imaging, using high performance microCT imaging methods, supplies anatomical details in addition to the molecular signals. Here, precise focal tumor location, dis-tribution and volume are revealed. Furthermore, details of host tissue (e.g. vasculature) critical to tumor development can be obtained. While the focus for this study was to assess optical and microCT imaging methods for direct tumor monitoring, exogenous fluorescent or radioisotopic probes could be used in concert to assess other aspects of tumor biology and tracer/probe development. A range of fluorescent and radioisotopic reporters/probes for key tumor biomarkers (e.g. angiogenesis, apoptosis, and metabolism) in oncology are readily available.

Table 2. Complementary in vivo Tumor Progression Imaging Using BLI, FLI, and microCT

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