Author:

Sylvie Kossodo, PhD | Director, Scientific Development

Date:

January 2020

Deep Tissue 3D Imaging for In Vivo Biodistribution and Monitoring of Disease Processes

Fluorescence Molecular Tomography (FMT) is a non-invasive imaging modality designed to three-dimensionally visualize and quantify near-infrared fluorescence (NIRF) signal in tissues.[1] FMT offers a robust and sensitive alternative to traditional bio-distribution studies using invasive procedures or radiolabeling. It can be used to evaluate the spatio-temporal bio-distribution of test agents, including determination of specificity of tumor/tissue targeting. In this Tech Spotlight we will present the principles of FMT and highlight advantages this service offers in biodistribution studies and molecular target assessment of disease in tumors.

For FMT imaging, animals are injected with NIRF-tagged probes or test articles. These can be passive circulating agents, targeted to specific cellular ligands or activated by biologic processes. The choice for fluorescence imaging in the NIRF spectrum (600-900 nm) allows one to maximize tissue penetration and minimize absorption by physiologically abundant absorbers such as hemoglobin (<600 nm) or water (>900 nm). In the pre-clinical setting, anesthetized test mice are placed in an imaging cassette, sequentially illuminated in the FMT2500TMLX system by focused excitation lasers and the emanating light is captured by a CCD camera. Collected fluorescence data are reconstructed, quantified by the TrueQuant® software and the signal (in pmoles) calculated within 3D regions of interest drawn encompassing the whole body, tumor, or target tissue. Fluorescence calculated for each target tissue can be reported in pmoles or as % injected dose/gr.

2020_0107_TS_FMT_Fig 1: Naïve SKH1-Elite mice were injected with a Cy7-labeled test article and whole body imaging performed at different times post-injection.
Fig. 1: Naïve SKH1-Elite mice were injected with a Cy7-labeled test article and whole body imaging performed at different times post-injection.

Previous studies have shown the ability of quantitative FMT imaging to non-invasively visualize and measure the temporal bio-distribution of NIRF-labeled molecules.[2] The ability to accurately quantify dynamic changes in agent distribution over time is critical when screening for new therapeutics. Temporal quantification of NIRF signal in tissues is exemplified in Figure 1. Mice were injected intravenously (i.v.) with a Cy7-tagged molecule and imaged at different times post-injection. High levels of probe accumulation were observed in the liver, spleen and intestines while other organs showed extremely low or no detectable fluorescence signal. The fluorescently labeled test article showed a peak liver accumulation ~48 hours post-injection, followed by a continuous signal decrease over time and a 100% clearance by 336 hours post-administration. FMT bio-distribution assessment, by virtue of its safety and ease of use, may be ideal in lead identification and optimization of small molecule and larger imaging or therapeutic bio-molecules.

FMT and NIRF imaging agents also allow the characterization of key biochemical processes within a tumor. These processes are essential for monitoring tumor progression, designing new treatments and altering treatment options. An example of this application is shown in Figure 2. This study was designed to assess the feasibility of cross-platform, multi-modality imaging using FMT and computed tomography (CT) in female NIH III mice bearing disseminated 5TGM-1-luc murine multiple myelomas. Thirty days after tumor injection, mice were injected i.v. with IntegriSense 750 (targeting avb3 integrin, up-regulated in tumor cells and activated endothelial cells[3]) and imaged 24h later on the FMT. Micro-CT images were acquired immediately after FMT imaging. CT alone was insufficient to detect abdominal tumor foci while FMT identified the presence of tumors in the spine, skull cap and abdomen, underscoring the ability of FMT to not only detect tumor burden but also quantify the expression of specific markers such as integrins in vivo in a non-invasive manner.

Fig. 2: 5TGM-1-luc bearing NIH III mice were injected with IntegriSense 750 and whole body imaging performed 24h later using FMT and CT.
Fig. 2: 5TGM-1-luc bearing NIH III mice were injected with IntegriSense 750 and whole body imaging performed 24h later using FMT and CT.

A clear limitation of cell line-derived xenografts is that in the sub-cutaneous setting they do not maintain the morphology of the human tumor histotype that they represent. Patient-derived xenografts (PDX) have been shown to overcome this limitation and are known to closely resemble the original tumors of the patients. Their use has become prevalent in the assessment of efficacy of anti-tumor drugs or to elucidate the characteristics of cancer cells and their micro-environment. Unlike cell lines which can be modified to express luciferase (and tumor burden assessed by bio-chemiluminescence imaging), PDX tumors are not generally luciferase-enabled. While this might be considered a limitation of PDX models, the utilization of NIRF probes has been shown to overcome this. NIRF imaging probes targeting tumor cells, endothelial cells, inflammatory cells and/or other components of the tumor, can be used to image PDX tumors in vivo, particularly when implanted orthotopically in deep-tissue locations. This is illustrated in Figure 3 in which Transferrin-Vivo 750 was used to detect transferrin receptor levels associated with an altered metabolic need for iron in tumor and inflammatory cells. Female SCID mice implanted orthotopically with a patient-derived xenograft model in the pancreas were injected with Transferrin-Vivo 750 fifty days post-tumor implantation and imaged 24h later. As shown in Figure 3, a strong fluorescent signal (quantified at 1.6 nmoles) clearly showed the presence of the tumor in the pancreas, highlighting the uniqueness of FMT for studying alterations in cell metabolism in deep tissues.

2020_0107_TS_FMT_Fig. 3: PDX model implanted orthotopically in the pancreas of a SCID mouse and imaged with Transferrin-Vivo750.
Fig. 3: PDX model implanted orthotopically in the pancreas of a SCID mouse and imaged with Transferrin-Vivo750.

FMT and disease-activated probes can also be used to quantify disease-associated enzyme activity in vivo. Cathepsin K (Cat K), a lysosomal cysteine protease with strong collagenolytic activity, is expressed predominantly in osteoclasts, chondrocytes and synovial fibroblasts and up-regulated in cancer cells that proteolyze extracellular matrix, contributing to invasiveness. Cat K 680 FAST is an imaging probe, optically silent until it is cleaved by Cathepsin K. We measured Cat K activity in peri-tibial bone metastasis in nude mice implanted with prostate PC-3M-luc-C6 tumors (Fig. 4). Tumor-associated Cathepsin K activity correlated with a decreased CT score, underscoring its role in tissue remodeling. The takeaway message is that FMT, in combination with disease-activated imaging agents, can be a powerful tool for investigating both bone resorption and pathologic conditions involving soft tissue calcification.

2020_0107_TS_FMT_Fig 4: Cathepsin K activity in a peri-tibial model of PC-3M-luc-C6 bone metastasis as compared to treated mice.
Fig. 4: Cathepsin K activity in a peri-tibial model of PC-3M-luc-C6 bone metastasis as compared to treated mice.

In addition to bio-distribution and cancer-related studies, FMT is uniquely positioned to image other deep-tissue diseases such as acute lung injury (ALI).[4] ALI is characterized by an alteration of the alveolar capillary barrier leading to fluid accumulation, loss of surfactant, and neutrophil infiltration. Activated neutrophils in turn release deleterious molecules such as neutrophil elastase, contributing to tissue damage, organ dysfunction, and further intensifying the inflammatory process. We used FMT to determine disease burden by quantifying the amount of neutrophil elastase activity (using Neutrophil Elastase 680 FAST) in the lungs of female BALB/c mice after LPS/fMLP administration (Figure 5). Animals with lung injury exhibited a 3.5-fold increase in lung neutrophil elastase activity as compared to healthy mice. In this application we were able to quantify inflammatory processes in vivo, critical when monitoring disease, designing new therapeutic approaches and measuring treatment response.

2020_0107_TS_FMT_Fig. 5: Measurement of neutrophil elastase activity in mice with acute lung disease as compared to healthy controls using Neutrophil Elastase 680 FAST.
Fig. 5: Measurement of neutrophil elastase activity in mice with acute lung disease as compared to healthy controls using Neutrophil Elastase 680 FAST.

Since its introduction in 2002,[5] FMT has provided an alternative to the use of established invasive or radiation-dependent non-invasive imaging modalities. FMT offers significant advantages, such as: 1) 3D imaging of deep tissues and tumors (localized and disseminated) and volumetric quantitation of fluorophores with picomolar sensitivity; 2) does not require the use of radiolabeled material (with its related costs, complexity, shorter half-lives and radioactive material handling/disposal) and, 3) does not depend on the expression of luciferase transgenes.

Contact the scientists at Covance to request the full data set or to learn more about our FMT imaging service and how it can be applied to your preclinical research.

1 Stucker F, Ripoll J and Rudin M. “Fluorescence Molecular Tomography: Principles and potential for pharmaceutical research.” Pharmaceutics 3.2 (2011): 229-274.

2 Vasquez KO, Casanvant C and Peterson JD. “Quantitative whole body biodistribution of fluorescent-labeled agents by non-invasive tomographic imaging.” PLoS One 6.6 (2011): e 20594.

3 Kossodo SPickarski M, et al. “Dual in vivo quantification of integrin-targeted and protease-activated agents in cancer using fluorescence molecular tomography (FMT).” Mol Imaging Biol 12.5 (2010): 488-99.

4 Kossodo SZhang J, et al. “Noninvasive in vivo quantification of neutrophil elastase activity in acute experimental mouse lung injury.” Int J Mol Imaging 2011 (2011):581406.

5 Ntziachristos V, Bremer C, et al. “In vivo tomographic imaging of near-infrared fluorescent probes.” Mol Imaging 1.2 (2002): 82-88.