A Method of Bone-Metastatic Tumor Progression Assessment in Mice Using Longitudinal Radiography

Abstract

Many types of solid tumors metastasize to the bone, where it causes significant morbidity and mortality in patients with advanced disease. Bone metastases are not only incurable but also affect bone health which impairs patients’ quality of life. In order to understand the mechanisms and develop effective treatments for bone-metastatic disease, it is first necessary to develop animal models that permit the assessment of tumor growth in the bone and progressive structural changes of the bone simultaneously. Longitudinal analysis of bone tumor progression is generally performed by bioluminescent imaging; however, this method is not able to assess progressive structural changes of the bone. Here, we describe a simple method for assessment of bone lesions using a scoring system that takes into account disease burden and bone destruction using longitudinal radiographs.

1. Introduction

Bone metastases are a common cause of morbidity and mortality in many types of solid tumors, particularly prostate cancer, breast cancer, and lung cancer. Clinically, prostate cancer commonly presents as osteoblastic lesions [1], while breast cancer and lung cancer develop osteolytic lesions [2]. It is also common for bone metastases to be a mix of both osteoblastic and osteolytic phenotypes [2]. These lesions result in skeletal-related events (SREs), such as bone pain, fracture, hypercalcemia, and spinal cord compression, which significantly impair patient quality of life [3, 4]. Therefore, understanding how bone-metastatic cancer cells disrupt normal bone physiology may be as crucial to patient care as revealing the mechanisms of disease progression in bone.

In in vivo animal studies, bioluminescent imaging is commonly used to measure tumor growth, especially in nonpalpable tissues, such as bone. However, bioluminescent imaging does not allow the assessment of tumor growth and structural changes in bone, simultaneously. Additionally, luminescent signals are unable to penetrate deep tissue without a critical mass of cells and high expression of luciferase [5]. In order to achieve high expression of luciferase, lentiviral vectors are commonly used to transduce the luciferase gene into cancer cell lines through a nonspecific mechanism of genome insertion that can cause off-target effects. Additionally, single-cell sorting or cloning methods, which have been used to maximize luciferase expression and prevent in vivo gene silencing, may also exacerbate the off-target effects of lentiviral gene transduction [6].

Alternatively, disease progression and bone density can be assessed simultaneously through the use of magnetic resonance imaging (MRI) or radiography. Although MRI and micro-computed tomography (μCT) analyses can provide very detailed assessments of bone-metastatic lesions, longitudinal follow-up with these technologies is cost-prohibitive. A more accessible tool for monitoring bone-metastatic cancer cells is an ordinary X-ray image. Osteoblastic and osteolytic lesions are clearly visible by X-ray if a baseline radiograph is acquired, and sufficient follow-up imaging is performed. Routine radiograph can provide an easy and cost-effective way to observe deep into tissue and track bone tumor progression without genetically modifying the cancer cell line of interest.

Here, we describe a method to score and monitor bone-metastatic growth/progression and bone destruction longitudinally with radiographic analysis based on scoring metrics, previously developed [7-9], in mouse models in which cancer cells are inoculated directly into the bone by intrafemoral injection.

2. Materials

1. MultiFocus 10 × 15 Digital Radiography System (Fig. 1a, Faxitron Bioptics; Tuscon, AZ).

2. Isoflurane vaporizer.

3. Inoculum: cancer cell line suspended in small volume (5 μL) of sterile Hank’s Balanced Salt Solution without Calcium and Magnesium (Gibco™ 14170112).

4. 28g internal injector (Plastics One C3131, 11 mm).

Fig. 1.

Longitudinal radiograph analysis of tumor-induced bone remodeling in the mouse femur, (a) The image of MultiFocus 10 × 15 Digital Radiography System, (b) Radiographs were collected prior to inoculation (DO) and weekly (D7, 14, 21, and 28) following intrafemoral injection of 5 μL Hank’s buffered salt solution (Sham, first column) or prostate cancer cells in mice. A blunt 28 gauge injector needle attached to a 25 μL Hamilton syringe with joint connector tubing (Eicom) is inserted between the distal condyles of femur for injecting the cancer cells. Five-week-old athymic nude male mice were injected with 2 × 10⁴ osteolytic human PC3 cell line (column 2). Five-week-old male C57BL6 mice were injected with 5 × 10³ mixed syngeneic RM-1 mouse prostate cancer cells (column 3). Representative radiographs showing the various progression scores are displayed in the bottom left of each panel and the time point after inoculation in the upper right of the panel. Note the progressive increases in pitted osteolytic lesions (arrows), mid-diaphysis of PC3-inoculated mice that ultimately results in erosion of cortical bone (arrowhead). In RM-1-inoculated mice, the osteolysis is most prominent in the distal metaphysis and results in progressive medullary and cortical bone erosion. The RM-1 cell line also produces extraperiosteal sclerotic lesions evident as slightly radio-opaque extracortical regions (outlined by dashed lines) in some mice

3. Methods

1. Turn on the X-ray system (Faxitron) and perform the necessary steps for operation (see Note 1).

2. Anesthetize mouse with isoflurane.

3. Place the mouse prone on the stage with its nose in a secured nosecone.

4. Position the mouse so that the bone to be imaged is centered on the stage (see Note 2).

5. Place the stage at the level for the desired magnification (see Note 3).

6. Radiograph the mouse using the same settings throughout the entire experiment (see Note 4).

7. Inoculate the animal (see Note 5), and repeat the imaging process at regular intervals, of both the contralateral and ipsilateral bones (see Note 6).

8. Save the images in a de-identified manner.

9. Provide the images to a blinded observer to perform longitudinal scoring using the following scale for assessing the degree of osteolysis (see Note 7).

   1. 0 = Bones with no lesions
   2. 1 = Bones with one to three small pits of radiolucent lesions
   3. 2 = Bones with three to six small pits of radiolucent lesions
   4. 3 = Bones with obvious loss of medullary bone and erosion of the cortical bone
   5. 4 = Bones with full thickness unicortical bone loss
   6. 5 = Bones with full thickness bicortical bone loss and displaced skeletal fracture

10. For cancer cells that produce mixed osteolytic and osteosclerotic lesions, tumor-induced new bone formation is often apparent as intramedullary or extraperiosteal slightly radiopaque regions that have a spongy appearance (Fig. 1b, column 3). The degree of tumor-induced bone formation can be quantified according to previously published methods [10].

11. Representative radiographs of intrafemorally injected Sham, PC3, and RM-1 prostate cancer cells can be found in Fig. 1b. Over time, sham-injected bones remain without osteolytic or sclerotic lesions (score = 0). PC3-injected femurs start similar to sham at day 0 and 7 (score = 0), but develop visible osteolytic lesions by day 14 (score = 1). These lesions increase in number by day 21 (score = 2), and in size by day 28 (score = 3) when the cortical and medullary bones are clearly eroded. In RM-1-inoculated mice, osteolysis is evident in the distal meta-physis as soon as day 7 (score = 1) and progressively erodes medullary and cortical bone erosion over time [day 14 (score = 2), day 21 (score = 3)] until reaching full thickness unicortical bone loss by day 28 (score = 4). Additionally, an extraperiosteal sclerotic lesion is observed on day 21, which grows in size by day 28.

4. Notes

1. The automatic system used in our lab (Faxitron) is turned on with a key and images are captured on an attached desktop computer using Faxitron Vision Software. After both the machine and desktop are turned on, the Faxitron Vision Software is launched, and an automatic initialization and calibration sequence are performed. After these steps are completed, the system is ready to digitally capture images.

2. It may be necessary to secure the animal to the stage with tape if the images are developed blurry due to breathing artifacts.

3. A magnification of 4× or 5× is recommended. It is usually possible to capture the ipsilateral and contralateral limbs in the same image at 4× magnification, which can be a helpful reference for scoring; however, a 5× magnification of the ipsilateral limb is usually preferable. If the time is available, we recommend taking one image with both limbs at 4× magnification, followed by another image of just the ipsilateral limb at 5× magnification.

4. The settings we use for mice on our instrument are 28 kV for 8 s. It may be helpful to first set the machine to fully automated mode to see what the suggested settings are for your conditions. After an automated image is captured, it will be necessary to manually set the machine for subsequent images, as only images captured at the same settings are comparable.

5. For direct intrafemoral injection, we recommend verifying needle placement by radiograph before inoculation to ensure that the inoculum is deposited inside the bone (Fig. 1b, Sham D0). Detailed protocols for intrafemoral injection can be found elsewhere [11].

6. For slow-growing tumors, perform baseline radiographs before implantation and radiograph animals at least once a week following implantation. For fast-growing tumors, perform baseline radiographs before implantation and radiograph animals at least twice a week following implantation.

7. It may be acceptable to organize the de-identified images in the sequence in which they are taken, from baseline to the end of the experiment. The most quantitative way to perform the analysis would be to completely randomize the images, but this level of de-identification may not be necessary for most experiments.