Research paper
Lymphatic mapping of mice with systemic lymphoproliferative disorder: Usefulness as an inter-lymph node metastasis model of cancer

https://doi.org/10.1016/j.jim.2013.01.004Get rights and content

Abstract

Preclinical models of lymph node (LN) metastasis are fundamental to the study and design of new techniques for the diagnosis and treatment of LN metastasis. However, the identification of LNs and lymphatic vessels (LVs) in mice is challenging with conventional imaging modalities, since the LN diameter in normal mice is 1–2 mm. Here, we describe MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) inbred mice, which develop systemic swelling of LNs up to 10 mm in diameter, allowing investigation of the topography of LNs and LVs. Using a gross anatomy dissection approach, we identified 22 different LNs situated in the head and neck, limbs, thoracic and abdominal regions. Furthermore, four peripheral inter-LN vessels were found: from the subiliac LN (SiLN) to the proper axillary LN (PALN); from the parotid LN to the caudal deep cervical LN; and from the popliteal LN to both the sciatic LN and the SiLN. Metastasis to the PALN via LVs was induced by inoculating FM3A/Luc mouse mammary carcinoma cells into the SiLN. Our results demonstrate that the MXH10/Mo/lpr mouse strain is an excellent model in which to investigate lymphatic drainage and inter-LN metastasis of cancer. This paper unveils the anatomy of murine lymphatics to give new insights into the investigation of inter-LN metastasis of cancer, especially the mechanisms involved in the trafficking of cancer cells through inter-LN vessels. The results provide data that may prove very useful in the quest to develop better lymph drainage-based drug delivery systems.

Introduction

Lymph node (LN) metastasis is an extremely important factor for defining the prognosis of cancer. Various imaging modalities have been used to diagnose early LN metastasis, including computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and ultrasound (Benaron, 2002). However, a diagnostic imaging procedure has yet to be developed that can accurately diagnose metastasis to LNs that are less than 10 mm in diameter. In previous studies, we demonstrated that the combined use of high-frequency ultrasound and ultrasound contrast agents enabled us to visualize small blood vessels in cancer, a potentially useful method for the early diagnosis of cancer (Kodama et al., 2011, Li et al., in press-a). In addition, we developed an LN metastasis model using an MRL/MpJ-lpr/lpr (MRL/lpr) strain of mouse (Li et al., in press-b), which does not express the Fas gene involved in apoptosis (Watson et al., 1992, Wu et al., 1994). At three to four months of age, the LNs in these mice are enlarged to approximately 10 mm in diameter, a size similar to that found in humans. Furthermore, proliferation of nascent tumor blood vessels could be analyzed from the start of cancer metastasis, after the inflow of tumor cells from the afferent lymphatic vessel (LV) to the LN. Thus, this metastasis model is very well suited to the development of the next generation of ultrasound imaging modalities for the detection of early LN metastasis.

The MRL/lpr strain of mouse shows LN swelling throughout its whole body at three to four months of age (Murphy and Roths, 1978, Watanabe-Fukunaga et al., 1992). However, MRL/lpr mice spontaneously develop glomerulonephritis, vasculitis, arthritis and sialadenitis, which are similar to human collagen diseases, at the same age that they exhibit LN swelling (Andrews et al., 1978). Since the glomerulonephritis of MRL/lpr mice becomes fatal at five months, their period of use in LN metastasis experiments is limited.

In previous work, our group established recombinant inbred strains of MXH/lpr mice by intercrossing MRL/lpr and C3H/HeJ-lpr/lpr (C3H/lpr) strains; these were used for the analysis of susceptibility genes associated with a number of important diseases (Tanaka et al., 2010). From these recombinant inbred strains of mice, a sub-line of MXH-10/Mo-lpr/lpr (MXH10/Mo/lpr) mice was found to be unique; the sizes of most of the peripheral LNs were as large as 10 mm at only 2.5 to 3 months of age; furthermore, both the size and the time of onset of LN swelling were consistent and predictable. Moreover, these mice did not develop severe glomerulonephritis and vasculitis, compared with MRL/lpr mice, and thus their life span was longer than that of MRL/lpr mice. Consequently, breeding and maintenance of the MXH10/Mo/lpr mice was found to be easier than that of MRL/lpr mice, and based on these attributes, we determined that these mice would be suitable for various studies of murine lymph networks, especially in the field of experimental exploration of LN metastasis (Shao et al., 2012).

Lymphatic mapping is generally considered to be critical for investigations of LN metastasis. In order to make better use of the MXH10/Mo/lpr mouse as a model of LN metastasis, it is necessary to establish the anatomical locations and nomenclatures of the various LNs, and identify the lymphatic drainage routes between upstream and downstream LNs. In previous reports, the anatomical locations and nomenclatures of murine LNs have often been ignored and even assigned incorrectly (Cain and Rank, 1995, Deaglio et al., 1996, Anjuere et al., 1999). Such confusion about the anatomical locations and names of LNs leaves scope for misinterpretation of mouse lymphatic network data obtained by different research groups. Indeed, Van den Broeck and colleagues (Van den Broeck et al., 2006) have noted misuse of the anatomical names of the LNs of mice in previous publications. These authors examined the anatomical locations of the LNs of the BALB/cAnNCrl strain of mouse, and reported their anatomical nomenclature. In order to visualize mouse LNs that were difficult to identify macroscopically, the investigators injected dye and adjuvant into tissue near the LNs, using 12 different protocols, and identified 22 different LNs (Van den Broeck et al., 2006).

In the present study, we have examined the anatomical locations, sizes and nomenclatures of the LNs of MXH10/Mo/lpr mice, and the peripheral lymphatic drainage routes between upstream and downstream LNs, without the need for prior manipulation. In addition, based on the results we obtained and carried out experiments to explore the development of cancer metastasis between LNs.

Section snippets

Mice

All in vivo studies were approved by the Institutional Animal Care and Use Committee of Tohoku University.

Characteristics of MXH10/Mo/lpr mice

The MXH10/Mo/lpr mouse strain is a substrain of the recombinant inbred strain of the MXH/lpr mouse (Tanaka et al., 2010). MXH/lpr mice were generated using two different parental inbred strains as progenitors, MRL/lpr (H-2k haplotype) and C3H/lpr (H-2k), followed by an F1 intercross and more than 20 generations of strict brother–sister mating. The MXH/lpr mice were bred under specific

Weight of LNs

As the first step toward using MXH10/Mo/lpr mice as a model to study LN metastasis, the average weight of their LNs was measured. The mean weight of the proper and accessory axillary LNs from the bilateral axillary regions of the MXH10/Mo/lpr mice was 0.862 ± 0.256 g (n = 8), compared with a value for MRL/lpr mice of 1.095 ± 0.372 g (n = 22).

Pathological scores for glomerulonephritis and renal vasculitis

When compared with MRL/lpr mice, microscopic examination of the kidneys of MXH10/Mo/lpr mice revealed greatly reduced signs of glomerulonephritis and renal

Discussion

Accurate identification of LNs in mice is critical for studies of the diagnosis and treatment of LN metastasis. However, these small lymphatic organs are often difficult to identify in mice using standard dissection techniques. Consequently, rats (which are larger) have been used to characterize rodent lymphatic drainage, although there are distinct differences between species (Tilney, 1971, Harrell et al., 2008). The LNs of the rat have been classified into somatic nodes, which drain the skin

Acknowledgements

M. Shiro acknowledges a Grant-in-Aid for Scientific Research (B) (22390378) and a Grant-in-Aid for Challenging Exploratory Research (24659884). T. Kodama acknowledges a Grant-in-Aid for Scientific Research (B) (23300183) and a Grant-in-Aid for Challenging Exploratory Research (24650286). Special thanks are expressed to Masao Nose for pathological discussions and for assistance in preparing the manuscript.

Conflict of interest statement

No author has any financial or personal relationships with

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