Malignant phyllodes tumors display mesenchymal stem cell …

Abstract Introduction

Although breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors.

Paraffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation.

Immunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH+ cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH- cells. GD2+ cells showed a 3.9-fold greater capacity than GD2- cells. ALDH+/GD2+cells displayed 12.8-fold greater mammosphere forming ability than ALDH-/GD2- cells. In vivo, the tumor-initiating frequency of ALDH+/GD2+ cells were up to 33-fold higher than that of ALDH+ cells, with as few as 50 ALDH+/GD2+ cells being sufficient for engraftment. Moreover, we provided the first evidence for the induction of ALDH+/GD2+ cells to differentiate into neural cells of various lineages, along with the observation of neural differentiation in clinical specimens and xenografts of malignant phyllodes tumors. ALDH+ or ALDH+/GD2+ cells could also be induced to differentiate into adipocytes, osteocytes or chondrocytes.

Our findings revealed that malignant phyllodes tumors possessed many characteristics of MSC, and their CSCs were enriched in ALDH+ and ALDH+/GD2+ subpopulations.

Breast phyllodes tumors (PTs) are rare neoplasms [1], representing less than 1% of all primary breast tumors in western countries [2]. However, an incidence rate of 6.92% was reported in a Singaporean study, suggesting its higher frequency among Asian women [3]. The World Health organization classified breast PTs into benign, borderline and malignant histopathologically [4]. However, there are occasional discrepancies between the clinical behavior and histopathological parameters of PTs, and the progression rate and outcomes of PTs remain unpredictable [1]. So far, there is no effective therapy other than surgery [5]. While all grades of breast PTs have the potential for local recurrence, only borderline and malignant PTs were shown to metastasize to other organs, such as lungs, bone and liver [6]. The metastatic PTs may show a resemblance to osteogenic sarcoma, chondrosarcoma, liposarcoma, leiomyosarcoma or rhabdomyosarcoma [7], which is attributed to the inherent heterogeneity within the primary PTs [1]. However, there has been no report of neural differentiation of malignant PTs. The versatile property of PTs to convert into various sarcoma types is reminiscent of the features of mesenchymal stem cells (MSCs). It has been well-documented that MSCs may differentiate into adipocytes, osteocytes and chondrocytes [8]. Subsequent studies demonstrated that MSCs can even be induced to neuron-like cells differentiation [9]. This led us to hypothesize that malignant PTs may possess MSC-like properties. Recently, GD2, a disialoganglioside has been identified as a marker for stem cells of MSCs [10] and breast cancer [11]. It will be of interest to determine whether GD2 is expressed in PTs and their stem cells.

Cancer stem cells (CSCs) have the capacity to create bulk tumors through self-renewal and differentiation [12]. A successful cancer therapy must thus eliminate these cells. The identification and isolation of CSCs thus become important in the treatment of malignant PTs. Although several markers have been successfully used to enrich cancer stem cells from various cancers, CSC markers for PTs have yet to be deciphered. In this study, we investigated the expression of a variety of markers in malignant PTs and searched for CSC markers for PTs.

All human breast cancer specimens were obtained from patients with malignant PT who had undergone initial surgery at the Tri-Service General Hospital (Taipei, Taiwan), National Taiwan University Hospital (Taipei, Taiwan), Chunghua Christian Hospital (Chunghua, Taiwan). Samples were fully encoded to protect patient confidentiality and were utilized under a protocol approved by the Institutional Review Board of Human Subjects Research Ethics Committees of Academia Sinica (Taipei, Taiwan) and collaborating medical centers. We have confirmed that informed written consent was obtained from those patients who provided fresh tumor specimens and that the IRB exempted the informed consent from patients who provided paraffin-embedded tissue sections.

Female NOD-SCID (non-obese diabetic-severe combined immunodeficiency; Tzu Chi University, Hualien, Taiwan mice were purchased from Jackson Lab, Bar Harbor, ME, USA) and housed under specific pathogen-free conditions in the Animal Center of the Institute of Cellular and Organismic Biology of Sinica. We developed an orthotropic xenograft model as described by Kuperwasser et al.[13]. Briefly, fat pads were cleared and injected with a mixture of human primary cancer cells, human mammary stromal cells and Matrigel (BD 356237, 2.5mg/ml, USA). The human mammary stromal cells were obtained from patient BC515 who had undergone initial surgery. The tumor specimens were sliced to square (1 mm2) then subjected to enzymatic digestion by being incubated in RPMI1640 medium containing collagenase (Sigma C5138, 1,000 U/ml, USA), hyaluronidase (Sigma H3884, 300 U/ml), and DNase I (Sigma DN25, 100 g/ml) at 37C for one hour. After filtration through a 100-m cell strainer (BD Biosciences, USA), primary breast tumor cells were collected and resuspended in RPMI1640 medium supplemented with 5% FBS, and then injected into mammary fat pads of NOD-SCID mice. The animals were monitored weekly for tumor growth. Tumor cells from the xenografted mice were harvested in a similar manner and injected into other mice for serial passages. Mice were treated in accordance with the Institutional Animal Care and Use Committee of the Academia Sinica guidelines for experiments and approved by a committee of the same office.

Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded tissue. Sections (3 m) on coated slides were deparaffinized and rehydrated then subjected to antigen retrieval by autoclave or microwave in alkaline buffer pH9 (antigen Retrieval AR10, BioGenex, Fremont, CA, USA)
for 10 minutes. After antigen retrieval, sections were treated with H2O2 to block the endogenous peroxidase activity. After washing out the H2O2, the sections were incubated with diluted primary antibodies as indicated by the manufacturer at room temperature for one hour, followed by staining with Super Sensitive Polymer-HRP Detection System (BioGenex), counter-staining with Mayers hematoxylin and mounted in glycerin. The primary antibodies used included the following: CD44 (DF1485, DAKO, USA), CD29 (O.N.98, US Biological), CD106 (3H1814, US Biological), CD166 (MOG/07, Novocastra, USA) CD105 (SN6h, DAKO), CD90 (3F102, US Biological), GD2 (14G2a, Bio Technetics, San Diego, CA 92121), ALDH1 (44/ALDH, it recognizes all ALDH1 isoforms, BD, USA), Oct-4 (240408, Santa Cruz, USA), CD117 (polyclonal c-Kit, DAKO), CD10 (56C6, BioCarta, USA), P53 (DO-7, DAKO), P63 (4A4, DAKO), Ki-67 (MIB-1, DAKO), bcl-2 Oncoprotein (124, DAKO), Globo-H (MBr1, ALEXIS, USA), CD34 (QBEnd 10, DAKO), vimentin (Vim3B4, DAKO), collagen type II (polyclonal: 1 fibrillar collagen NC1 and 1VWFC, Abcam, UK), nestin (196908, R&D and polyclonal, Santa Cruz Biotechnology, USA), III-tubulin (Tuj-1, R&D and polyclonal, Millipore, USA) and glial fibrillary acidic protein (GFAP) (273807, R&D and polyclonal, Millipore). Sections were examined by pathologists.

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Malignant phyllodes tumors display mesenchymal stem cell ...