Experimental orthotopic prostate tumor in nude mice: Techniques for local cell inoculation and three-dimensional ultrasound monitoring

Abstract

Objectives

Orthotopic prostate cancer models are of great importance for cancer research. Orthotopic models in mice have been described previously. However, these studies lack a detailed methodological description and fail to define standards for local cell inoculation. Herein, we studied the effect of different protocols on tumor growth and report for the first time the use of high resolution ultrasound for monitoring of tumor growth.

Materials and methods

Orthotopic inoculation of DU 145 MN1 prostate cancer cells was performed in 30 nude mice varying (1) the amount of cells (5 × 10⁵ vs. 5 × 10⁴), (2) the number of puncture sites, and (3) the addition of matrigel. Surgical complications such as recoil of cells through the injection canal and rupture of the prostatic capsule were monitored. Animals were tracked by ultrasound imaging after 4, 5, and 6 weeks. Autopsy and histology confirmed local tumor growth.

Results

A take rate of 27/30 (90%) was observed. Growth of orthotopic prostate tumors was increased after inoculation of a large amount of cells under the capsule of 1 dorsal prostate lobe, but inoculation of small amounts of cells still induced local tumors. Noninvasive ultrasound examination allowed to identify orthotopic tumor formation and to monitor tumor growth in vivo. Addition of matrigel did not accelerate tumor growth. Complications like recoil (6.8%) or rupture of the prostate capsule (1.4%) were rare.

Conclusions

Inoculation of DU 145 MN1 cells under the prostate capsule with a defined procedure results in very high take rates. Ultrasound screening is feasible to repetitively monitor tumor growth.

Introduction

Prostate cancer is the most common cancer in men and a major cause of cancer-related morbidity and mortality [1], [2]. Despite a large amount of prostate cancer research accomplished over the last decades, only very few findings have influenced the clinical management of the disease. A major limitation in prostate cancer research is the lack of relevant preclinical models, which allow studying the molecular mechanisms of tumorigenesis. In fact, advanced in vitro and in vivo models are an indispensable requirement for the development of effective prevention and therapeutic intervention strategies.

Most of the studies in prostate cancer research rely on the use of permanent cell lines, especially PC-3, DU 145, and LNCaP. It remains unclear why it is so difficult to establish new prostate cancer cell lines derived from primary cell cultures [3]. To overcome the well-known limitations of using permanent cell lines in tumor research, several animal models have been developed and xenografts in mice have been described even for prostate cancer [4]. Most frequently, heterotopic, subcutaneously implanted tumor models were used. These models have the advantage of an easy inoculation of tumor cells and in vivo tumor growth monitoring. More realistic animal models to mimic human prostate cancer, however, are achieved by orthotopic implantation of the tumor cells into the animal host because this more likely resembles the native environment for the tumor cells [5], [6].

Unfortunately, orthotopic inoculation of tumor cells into the small prostate gland of nude mice requires distinct technical skills, and recoil of cells outside the prostate or rupture of the prostatic capsule are common complications [7]. Independently from the advantages of these models in preclinical research, most studies are additionally hampered by a lack of standardization. Especially the methods of orthotopic tumor cell application, the quality and number of inoculated tumor cells, as well as the addition of extracellular co-factors have never been investigated in detail. The mouse prostate consists of ventral, dorsolateral, and anterior lobes containing encapsulated prostatic ducts [8] with significantly differing morphogenesis [9], raising the question whether there is a preferable lobe for tumor cell inoculation and engraftment.

A further drawback, which demands for new methods and techniques, is the lack of noninvasive and repetitive monitoring of orthotopic tumor growth. Thus, in order to understand cancer progression in animal models and to achieve therapeutic success, new imaging methods and reporter systems are required [4].

Whereas measurement of tumor size is easily achieved by the use of calipers after sacrifice of the animals at the end of the observation period [10], monitoring of tumor take rate and tumor growth during tumor development demands for less invasive technologies [11], [12]. Live cell imaging using fluorescent dyes [13] has been used, as well as magnetic resonance imaging (MRT), which allow repeated measurements in the same animal. Whilst these technologies require sophisticated equipment and a prolonged time of narcosis [14], the introduction of ultrasound imaging using high-frequency wavelength combines a short narcosis with a powerful resolution and 3D-imaging capabilities [15]. In the present study, we analyzed in an orthotopic prostate cancer model in nude mice different modes of DU 145 MN1 cell inoculation, and elucidated the potential of repeated high-resolution ultrasound examination as a method to quantitatively monitor local tumor engraftment and growth.