Bioluminescent Imaging of Animal Models for Human Colorectal Cancer Tumor Growth and Metastatic Dissemination to Clinically Significant Sites

Special Article - Cancer Imaging

J Mol Biol & Mol Imaging. 2015;2(2): 1019.

Bioluminescent Imaging of Animal Models for Human Colorectal Cancer Tumor Growth and Metastatic Dissemination to Clinically Significant Sites

Fernández Y1,2,6*, Foradada L1,2,6, García-Aranda N1,2,6, Mancilla S1,2,6, Suárez-López L2,6, Céspedes MV3,6, Herance JR4,2, Arango D5,6, Mangues R3,6, Schwartz S Jr2,6 and Abasolo I1,2,6*

¹Functional Validation & Preclinical Research (FVPR), CIBBIM-Nanomedicine, Hospital Universitari Vall d’Hebron (HUVH) - Vall d’Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain

²Drug Delivery & Targeting, CIBBIM-Nanomedicine, HUVH-VHIR, UAB, Barcelona, Spain

³Grup d’Oncogènesi i Antitumorals of the Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

4Institut d’Alta Tecnologia-PRBB, Barcelona, Spain

5Molecular Oncology, CIBBIM-Nanomedicine, HUVHVHIR, UAB, Barcelona, Spain

6Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain

*Corresponding author: Fernández Y, Functional Validation & Preclinical Research (FVPR), CIBBIMNanomedicine, Hospital Universitari Vall d’Hebron (HUVH) - Vall d’Hebron Institut de Recerca (VHIR), Edifici Collserola – Lab 202; Passeig Vall d’Hebron, Spain,

Abasolo I, Functional Validation & Preclinical Research (FVPR), CIBBIM-Nanomedicine, Hospital Universitari Vall d’Hebron (HUVH) - Vall d’Hebron Institut de Recerca (VHIR), Edifici Collserola – Lab 202; Passeig Vall d’Hebron, 119-129; Spain

Received: May 07, 2015; Accepted: June 19, 2015; Published: June 22, 2015


Purpose: Development of clinically-relevant mouse models which mimic natural tumor progression and metastatic dissemination of human colorectal cancer (CRC) is an essential requirement to better understand the mechanisms of cancer metastasis and to improve clinical therapeutics. A new era of modeling cancer metastasis involves the use of imaging technologies to monitor tumor growth and colonization after inoculation of cancer cells into the animals. This study reports on new experimental mouse models which mimics human CRC disease using noninvasive bioluminescent imaging (BLI).

Procedures: Luciferase-expressing HT-29 and HCT 116 cells were injected subcutaneously, orthotopically into the cecal wall, intrasplenically and intracardiacly into the left ventricle of nude mice. Tumor growth and metastatic dissemination patterns were monitored and quantified via in vivo and Ex vivo BLI, and compared to tumor volume or histopathology. BLI results were validated using positron emission tomography (PET).

Results: Subcutaneous model validated BLI as a powerful tool for noninvasive monitoring and quantification of tumor growth and treatment efficacy, and for identifying new metastatic foci. Orthotopic colon model resembled the clinical pattern of CRC metastases that includes lymphatic, hematologic and coelomic dissemination. Furthermore, the intrasplenic and intracardiac models resulted in hepatic and bone-marrow metastases, respectively, sites with high clinical relevance in CRC. Importantly, in all models, BLI allowed the longitudinal follow-up of the CRC metastatic disease as it happens.

Conclusions: We provide improved and biologically relevant CRC experimental mouse models monitorable by BLI. These models are a valuable aid for the investigation on molecular mechanisms driving metastatic human CRC as well as on novel therapeutic strategies.

Keywords: Bioluminescent imaging; Positron emission tomography; Subcutaneous; Intracecal; Intrasplenic and intracardiac mouse models; Metastasis; Colorectal cancer


BLI: Bioluminescent Imaging; CRC: Colorectal Cancer; FDG: 2-deoxy-2-[18F]fluoro-D-glucose; Fluc: Firefly Luciferase; Fluc2: Firefly Luciferase 2; IC: Intracardiac; PET: Positron Emission Tomography; ph/s: Photons/second; SC: Subcutaneously; SEM: Standard Error of the Mean; SOI: Surgical Orthotopic Implantations


Colorectal cancer (CRC) is one of the leading causes of cancer deaths in the western world. Metastatic dissemination of primary tumors is directly related to patient’s survival and accounts for about 90% of all colon cancer deaths [1, 2]. Hence, the main problem in the treatment of CRC is not so much eradication of the primary tumor, but rather the formation of incurable metastases. The most common sites of metastasis in CRC patients are lymph nodes (55%), liver (45%) and lungs (22.5%) [3, 4]. Moreover, bone metastasis have previously been uncommonly reported [5], but its incidence has increased significantly in patients that have received multiple systemic treatments [6]. When such an event occurs, it is usually a late manifestation of the disease.

The biological processes that drive metastatic progression involve the success of cells in tissue invasion, intravasation, survival in the blood-stream and lymph, extravasation, and growth within a secondary organ [7]. It is therefore important to study tumor development and possible metastasis in biologically relevant environments, like the tissue from which they were derived or the tissue to which they metastasize. Thus, proper modeling of the early phases of spontaneous colorectal metastasis formation requires growing a metastatic tumor orthotopically in the intestine (e.g. cecum or rectum) [8]. Alternatively, in experimental metastasis models, early stages, including local invasion at the site of the primary tumor and gaining access to lymphatic or blood vessels, are bypassed by injection of tumor cells directly into systemic circulation. The site of injection in some cases defines which metastases will be developed. Thus, lateral tail-vein injection tends to mainly cause pulmonary metastases, whereas injection into the portal vein or spleen will usually elicit liver metastases [9], and intracardiac (i.c.) injection into the left ventricle of the heart introduces tumor cells to the arterial circulation leading to the colonization of cells to specific sites of the skeleton [10].

Traditionally, the follow-up over time of tumor burden and metastases development in orthotopic and experimental metastasis mouse models is limited to a specific endpoint which precludes longitudinal studies of metastases development in vivo. Observation of tumor growth kinetics as well as the determination of endpoints is therefore complex. The course of tumor development in time is then assessed by comparing groups of animals that were euthanized at different time points. Because of substantial inter-individual variation, large numbers of animals and laborious efforts are required, rendering the use of orthotopic and experimental metastasis models highly impracticable [11]. Nevertheless, access to technologies able to noninvasively detect molecular and biological processes in small animals such as bioluminescent optical imaging offer a new approach to overcome these drawbacks [12, 13]. Bioluminescent imaging (BLI) refers to light produced by an enzymatic reaction, usually between Photinus pyralis firefly luciferase (Fluc) enzyme and its substrate D-luciferin, in a reaction that requires oxygen and ATP [14]. The BLI backgrounds are extremely low, due to the fact that rodents do not naturally produce light, resulting in an excellent signal-to-noise ratio, and representing an extremely sensitive mean of detecting labeled cells [15]. Moreover, bioluminescence technology is fast and easy to perform, done in high-throughput, relatively inexpensive and suited for small animals compared to other preclinical functional imaging modalities available such as positron emission tomography (PET), which is radioactive and require a high degree of operational expertise, can be labor-intensive and is often cost prohibitive [16].

The elucidation of the molecular mechanisms of tumorigenesis and the development of therapeutic strategies to treat metastasized colorectal carcinoma require biologically relevant and adequate animal models in which tumor and metastasis progression of cancer cells are generated under well-controlled conditions. Thus, the aim of this study was to develop and provide to the scientific community more relevant animal models that are suitable to study CRC disease. In this study, the application of BLI to track and quantify tumor growth and therapeutic efficacy was first validated using subcutaneous mouse models, and then extended to orthotopic and experimental models. Orthotopic models were employed to reproduce the physiological colon environment and its spontaneous metastasis patterns, whereas experimental intrasplenic and intracardiac models were used to study colorectal liver and bone metastases, respectively.

Materials and Methods


Human CRC cell line HT-29 and HCT 116 were obtained from the ATCC (Rockville, MD, USA), and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Lonza, Verviers, Belgium) and antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA) at 37ºC in a humidified atmosphere containing 5% CO2.

Firefly luciferase and firefly luciferase 2 genes were cloned into the pcDNA3.1 vector (Invitrogen) and pGL4 (Promega Biotech Ibérica, Madrid, Spain), respectively. Cells were transfected using Lipofectamine 2000 (Invitrogen) and selected with 500 μg/mL of geneticin (Invitrogen). The HT-29.Fluc-C4 and HCT 116.Fluc2-C9 cell variants with the highest bioluminescent light emission were selected (Supplementary Information 1). These cell variants did not show changes in either growth rate or morphology in comparison with the parental cells (data not shown). Regarding to the luciferase expression of Fluc or Fluc2, in our hands, both luciferase genes worked similarly.

Bioluminescent imaging

BLI was performed using an IVIS® Spectrum imaging system (PerkinElmer, MA, USA) and the Living Image® 4.3 software (PerkinElmer) at the Molecular Imaging Platform of Vall d’Hebron Research Institute (VHIR) (Barcelona, Spain). For in vivo BLI, animals were given 150 mg/kg of D-luciferin (Promega) by intraperitoneal injection, and anesthetized using 1-3% isofluorane (Abbott Laboratories, IL, and USA). The light emitted from the bioluminescent cells was detected, digitalized and electronically displayed as a pseudocolor overlay onto a gray scale animal image. Regions of interest (ROI) from displayed images were drawn automatically around the bioluminescent signals and quantified as photons/second (ph/s). For ex vivo BLI, organs were removed 5-10 min after D-luciferin administration, incubated in 300 μg/mL D-luciferin solution, and imaged. All techniques were performed following procedures previously described in the literature [17-21].

Mouse models

Female athymic nude mice (Harlan Interfauna Iberica, Barcelona, Spain) were kept in pathogen-free conditions and used at 5-12 weeks of age. Animal care was handled in accordance with the Guide for the Care and Use of Laboratory Animals of the Vall Hebron University Hospital Animal Facility, and the experimental procedures were approved by the Animal Experimentation Ethical Committee at the institution. All in vivo experiments were performed at the CIBERBBN’s in vivo Experimental Platform of the Functional Validation & Preclinical Research (FVPR) area (Barcelona, Spain).

Subcutaneous: HT-29.Fluc-C4 or HCT 116.Fluc2-C9 cells (1×106) were injected subcutaneously (s.c.) on the rear flanks of the mice (5-6 weeks, n=12-15). Tumor growth was monitored twice a week for 4-7 weeks by conventional caliper measurements (D×d²/2, where D is the major diameter and d the minor diameter) and in vivo BLI of the dorsal or lateral mouse views. Primary tumors were excised and secondary metastases were followed by in vivo BLI. At the end of the experiment, selected tissues were analyzed by ex vivo BLI and processed for histopathology.

For the efficacy studies, HT-29 tumor-bearing mice were randomized based on tumor volume (median, 125 mm³) and tumor BLI (median, 4×108 ph/s) into 5-Fluorouracil (Sigma-Aldrich, Madrid, Spain) and PBS-control treatment groups (n=10/group). The 5-Fluorouracil was given at 50 mg/kg twice a week for 4 weeks by intravenous administration.

Orthotopic intracecum: The cecum of anesthetized mice (5-6 weeks, n=6-21) was exteriorized through a laparotomy, and 5×105 HT-29.Fluc-C4 or 2×106 HCT 116.Fluc2-C9 cells were injected under binocular lens into the cecal wall between the mucosa and the muscularis externa layers using a specially designed micropipette [22] or a 30-gauge needle attached to an insulin syringe. A proper implantation into the cecum was confirmed at day 0 by a localized and unique bioluminescent signal into the mice abdominal cavity. Mice successfully injected were imaged by BLI once a week from ventral and dorsal views. At termination, the gastrointestinal tract and the organs of interest were removed and examined by ex vivo BLI prior to the histological analyses. Moreover, two animals were imaged by PET (see section below).

Intrasplenic: To induce colorectal liver metastases the spleen of anesthetized mice (8-10 weeks, n=8-10) was exposed through a small left subcostal incision through the peritoneal wall, and 5×105 HT-29.Fluc-C4 or 1×106 HCT 116.Fluc2-C9 cells were injected into the spleen parenchyma. After 10 min, the splenic hilum was ligated and the spleen was removed to avoid intrasplenic tumor growth. The incision was closed and mice imaged to identify those with a successful intrasplenic injection, which were identified by a localized bioluminescent signal in the anatomic position of the liver. Metastatic growth was monitored once a week from ventral and dorsal views. Upon necropsy, liver, and other organs of interest were removed and examined by ex vivo BLI prior to histopathology.

Intracardiac: Anesthetized mice (10-12 weeks, n=8) were injected with 3×106 HT-29.Fluc-C4 cells into the left ventricle of the heart by nonsurgical means. Mice were then imaged to identify those with a successful i.c. injection, which was detected by an immediate but transient systemic bioluminescent signal over the entire animal. Only mice with evidence of a proper injection were included. The development of metastases was monitored once a week from ventral and dorsal views until endpoint criteria were reached. Upon necropsy, all organs were excised and prepared for ex vivo BLI and histopathology.

Positron emission tomography

For PET imaging, 2-deoxy-2-[18F]fluoro-D-glucose (FDG) was prepared using an IBA 18/9 cyclotron and a routine FDG synthesis module. Animals were fasted for 2 h and isofluorane anesthetized immediately before the intravenous injection of 10 MBq FDG. Animals were maintained awaken throughout the FDG uptake time (40 min) and anesthetized again to acquired static images for 10 min within a microPET R4 scanner (Concorde Microsystems, Knoxville, TN, USA), using an energy window of 350-650 keV and a coincidence window of 6 ns. The resulting list-mode data was sorted into 3D sinograms and images reconstructed by an Ordered Subset Expectation Maximization – Maximum-a-posteriori (OSEM3DMAP) algorithm (18 iterations, 12 subsets) into a 128x128x63 (0.85x0.85x1.21 mm) matrix. Imaging data was corrected for nonuniformity response of the microPET, dead time count losses, and physical decay to the time of injection; no attenuation, scatter, or partial-volume averaging correction was applied. The same imaging procedure was applied for imaging ex vivo tissues. Parametric images based on standardized uptake value (activity concentration (MBq/ml) × body mass (g) / injected dose (MBq)) were generated for coronal, sagittal and transverse sections. For projected images, coronal sections covering the whole animal were fused using Amide software (Medical Image Data Examiner).


To confirm the presence of neoplastic cells, soft tissues samples were preserved in 4% formaldehyde solution and processed for histological analysis. Bones were fixed and decalcified using Decalcifier I (Surgipath Europe Ltd., Peterborough, UK). All tissues were paraffin embedded, sectioned, and stained with hematoxylin and eosin.

Results and Discussion

For many years, metastatic spread in experimental in vivo models could not be directly observed during the course of the disease. Tumor cells were injected at one site and metastatic dissemination determined post mortem. Advances in noninvasive imaging technologies have shed new light on the metastatic process, enabling now to watch metastasis as it develops. In this study, we have demonstrated that in vivo bioluminescent optical imaging allows the direct observation of cancer cells spreading from their site of origin and arriving at secondary sites longitudinally, offering early reads of disease progression and a rapid, sensitive and less invasive monitoring of neoplastic growth and metastases versus traditional cancer models. Specifically, we have provided novel mouse models to study metastatic CRC disease using the bioluminescent HT-29 and HCT 116 human colorectal cell lines.

Bioluminescent imaging as a reliable tool to monitor and quantify tumor progression and treatment efficacy, and to identify new metastatic sites noninvasively

In order to validate the use of BLI for measuring tumor progression, HT-29.Fluc-C4 cells were implanted s.c. and tumor growth was monitored from the same day of inoculation by in vivo BLI and compared to external caliper measurements (Figure 1A, B). Bioluminescent signal at the injection site was visible on the inoculation day (day 0) while traditional caliper measurements could not begin until day 6. Tumor bioluminescence increased over time up to day 20, and a strong correlation between mean tumor bioluminescence and mean tumor volume was observed from day 6 to 20 (r2=0.98, p=0.0015). These results indicate that the light emission from constitutively expressed Fluc is proportional to tumor cell burden, and changes of the bioluminescent signal over time accurately reflect tumor growth or regression. Moreover, the high sensitivity of BLI was effectively demonstrated in these subcutaneous tumor-bearing animals when tumors were immeasurable by means of a caliper, but quantification was feasible by photon emission calculation. Nonetheless, from day 20, tumor bioluminescence appears to plateau unlike the callipered tumor volume which continued to increase. Histopathological analyses of the HT-29 subcutaneous tumors further revealed that the bioluminescent plateau signal corresponds to tumors that have undergone central necrosis (Figure1C). Because bioluminescence is a function of the number of metabolically active tumor cells rather than a volumetric measure of the tumor mass, its ability to solely measure viable cells offers a great advantage and a more accurate value of tumor physiology over traditional volumetricbased measurements, which also include the contribution of dead or necrotic regions within a tumor.