Tumor-associated primo vascular system is derived from xenograft, not host

Abstract

The primo vascular system (PVS), which is composed of very small primo-vessels (PV) and primo-nodes (PN), has recently emerged as a third component of circulatory system. Here, we report the presence of a tumor derived PVS in murine xenografts of human histiocytic lymphoma (U937) in close proximity to the tumor. Within this system, PNs are small (~ 500–600 μM diameter) membranous sac-like structures which contain numerous small cells which can be demonstrated by DAPI staining. Hematoxylin and Eosin (H&E) staining of the peri-tumoral PVS shows the presence of loose structures lined by fibroblasts but filled with dense fibers, cells, lacunae and nerve-like structures. The origin and type of cells within the PVS was characterized by immunostaining with antibodies for CD68, CD45 and lysozyme. The results of these studies reveal that the PVS of the xenograft originates from the human U937 tumor cells. qRT-PCR analysis of mRNA isolated from PVS cells reveals a striking predominance of human, rather than mouse, sequences. Of particular interest, human stem cell specific transcription factors were overexpressed, most notably KLF4, an upstream regulator of NANOG which maintains the pluripotent and undifferentiated state of stem cells. These results suggest that the cells present within the PVS are derived from the human xenograft and suggests that the primo-vessels associated with the xenografted tumor may provide a safe haven for a select population of cancer stem cells. Further understanding of the biological properties of these cells may allow the development of new anti-cancer interventions.

Introduction

Almost fifty years ago, Bong-Han Kim, a physicist from North Korea, first reported the existence of a third vascular compartment in addition to the blood and lymphatic systems. He identified these novel anatomical vessels as ‘meridian primo-vessels’ (now known as superficial Bonghan ducts) and proposed that their distribution mirrored acupuncture meridians (Kim, 1963). This system is now known as the primovascular system. Since Kim's early work, there have been numerous descriptions of the primovascular system which is comprised of primo-nodes (PN) and primo-vessels (PV). The presence of PVs and PNs, which are strongly stained with trypan blue, has been reported in various tissues in mice, rats and rabbits (Johng et al., 2007, Lee et al., 2004, Lee et al., 2007, Lee et al., 2008, Shin et al., 2005, Yoo et al., 2008). PNs and PVs have also been found in adipose tissue [7,8] which, interestingly, is a source of pluripotent stem cells (Gimble and Guilak, 2003). The fluid dynamics and role of PV nodes in this network are not well established but some evidence suggests the existence of a fluid-containing conducting system throughout this vascular compartment (Sung et al., 2008).

Important to the research reported here, the PVS has also been found on the surface of subcutaneous tumors. It has been suggested that the PVS exists not only in and around tumors but also within the tumors (Yoo et al., 2009, Yoo et al., 2010). Using trypan blue staining or near infra-red quantum dot (NIR-QD) tracing, the PVS has been found in ovarian cancer, lung cancer and melanoma mouse models alongside lymphatic vessels and blood vessels. The PVS has been distinguished from other vascular systems by immunostaining which shows an absence of LYVE-1, an antigen specific for lymphatic endothelial cells (Yoo et al., 2010). It has been suggested that the PVS might contribute to tumor growth and metastasis (Yoo et al., 2009), but direct evidence for this is lacking.

We have used a human histiocytic lymphoma cell line, U937 (Sundstrom and Nilsson, 1976), which expresses the histiocyte-associated markers CD68 (macrophage specific) and CD45, and produces lysozyme, to characterize the PVS of xenografted tumors (Reid et al., 1995). A very small fraction of U937 cells are CD133 positive and, thus, considered to be ‘stem like’ cells while the vast majority of U937 cells are non-stem-like (Wang et al., 2010). We have also confirmed the presence of CD133 positive cells in U937 cells (data not shown). The de novo formation of a PVS which seems to originate from tumor cells implies the involvement of a population of stem like tumor cells which are capable of differentiation to form these ducts.

Recent studies have documented the presence of self-renewing, stem-like cells within tumors (Jordan et al., 2006). These cells have been called cancer stem cells (CSCs) or “tumor initiating cells”. CSCs are defined as cells that can divide to give rise to additional cancer stem cells but can also produce the phenotypically differentiated tumor cell types that form the bulk of the tumor. Evidence for the existence of CSCs was first obtained in leukemia, in which case it was clear that only a small subset of malignant cells was able to perpetuate the cancer after serial transplantation from one mouse to another. It is now clear that CSCs constitute only a small minority of neoplastic cells, varying from less than 0.1% in acute leukemia to 1–10% in breast cancer.

It has become clear that CSCs arise as a result of Epithelial to Mesenchymal Transition (EMT). EMT is involved in many physiological biologic processes including embryonic development, wound healing and cancer progression. During EMT, epithelial cells lose cell-cell contacts and undergo cytoskeletal remodeling and polarity changes, resulting in acquisition of a mesenchymal morphology as well as enhanced migratory ability. Importantly, EMT is reversible and these cells can undergo the reverse process (MET) resulting in a polarized epithelium. While both EMT and MET play central roles in embryogenesis, it has been proposed that EMT plays an important role in tumor growth and metastasis.

The EMT state has been associated with the loss of epithelial characteristics including apical basal polarity and expression of E-cadhedrin and the acquisition of mesenchymal characteristics including loss of polarity and increased expression of the transcription factors slug, snail and twist, and mesenchymal proteins including vimentin and fibronectin. In adult tissues, TGF-beta can induce EMT which is characterized by down-regulation of epithelial markers such as E-cadhedrin and up-regulation of EMT inducing factors such as twist, snail and slug (Kurrey et al., 2009). Furthermore, a number of developmental pathways, including the Wnt and HGF-c-Met pathways which are frequently dysregulated in cancer cells, are also regulators of EMT. The changes in activity of these pathways reflect dynamic changes in the levels of transcription factors, including KLF4, NANOG, OCT4 and SOX2 (Boyer et al., 2005, Loh et al., 2006).

Many of the traits of high-grade malignancy, including motility, invasiveness and self-renewal, have now been attributed to the minority subpopulations of CSCs within carcinomas. This has led to speculation that CSCs are responsible both for tumor progression (growth) and metastasis. Although CSCs may represent a tiny fraction of the total cell number of individual tumors they may be the critical drivers of tumor behavior.

The overall goal of the present work was to confirm the existence of PVS in a murine xenograft model and to determine whether it is of tumor or host origin. We have provided strong evidence of the existence of a tumor-derived PVS in and around the fascia of tumor xenografts. We have also characterized the cellular and molecular aspects of the tumor-associated PVS, unexpectedly demonstrating that the tumor-associated PVS harbors a unique population of tumor-derived cells which express high levels of stem cell-specific transcription factors. This provides the first evidence that the PVS may play an important role as a stem cell “niche”.