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Development of Metastatic and Nonmetastatic Tumor Lines from A TheProstate70:1636^1644(2010) Development of Metastatic and Non-Metastatic Tumor Lines From a Patient’s Prostate Cancer SpecimenöIdentification of a Small Subpopulation With Metastatic Potentialin the PrimaryTumor Dong Lin,1 Jane Bayani,2 Yuwei Wang,1 Marianne D. Sadar,3 Maisa Yoshimoto,4 Peter W. Gout,1 Jeremy A. Squire,4 and Yuzhuo Wang1,5,6* 1Department of Cancer Endocrinology,BC Cancer Agency,Vancouver,BC,Canada 2Department of Laboratory Medicine and Pathobiology,University of Toronto,Toronto,ON,Canada 3Department of Genome Sciences Centre, BCCancer Agency,Vancouver,BC,Canada 4Department of Pathology and Molecular Medicine, Queen’s University and Kingston General Hospital, Kingston,ON,Canada 5Department of Urologic Sciences, University of British Columbia,Vancouver, BC,Canada 6The LivingTumor Laboratory at theVancouver Prostate Centre,Vancouver,BC,Canada BACKGROUND. Metastasis is the major cause of prostate cancer deaths. Tumor hetero- geneity in both primary and metastatic prostate cancers is a major hurdle in elucidating the mechanisms of metastasis in this disease. To circumvent this obstacle and improve our understanding of prostate cancer metastasis, we developed multiple tumor tissue lines from one patient’s primary prostate cancer specimen to examine differences in metastatic ability. METHODS. Pieces of tissue from different foci of a patient’s primary prostate tumor were grafted into subrenal capsules of NOD-SCID mice and transplantable tumor sublines were established by serial passage. The metastatic ability of each subline was tested via orthotopic grafting into mice. Chromosomal alterations exclusively presented in a metastatic subline were examined by SKY and investigated for their presence in parental tissues by fluorescence in situ hybridization. RESULTS. Three transplantable sublines were developed, resembling the primary tumor histologically, exhibiting poor differentiation, and different growth rates. Importantly, the LTL-220N and LTL-221N sublines were non-metastatic, whereas the LTL-220M subline was spontaneously metastatic in vivo. SKY analysis showed limited but unique chromosomal alterations in each subline. Some chromosomal alterations, exclusively present in the metastatic LTL-220M subline, were also observed in a small portion of the parental cancer tissues. CONCLUSION. The results indicate that in primary prostate tumors metastatic potential can be confined to a minority of cancer cells. Subrenal capsule xenograft methodology can be used to dissect heterogeneous cancer cells in a patient’s primary tumor and sublines derived from such cells provide valuable tools for investigating mechanisms underlying prostate cancer metastasis. Prostate 70: 1636–1644, 2010. # 2010 Wiley-Liss, Inc. KEY WORDS: prostate cancer; metastasis; xenograft; FISH Additional supporting information may be found in the online *Correspondence to: Yuzhuo Wang, PhD, Department of Cancer version of this article. Endocrinology, BC Cancer Agency—Research Centre, 675 West 10th Dong Lin and Jane Bayani contributed equally to this work. Avenue, Vancouver, BC, Canada V5Z 1L3. E-mail: [email protected] Grant sponsor: Canadian Institutes of Health Research, Canada; Received 5 February 2010; Accepted 27 April 2010 Grant sponsor: NCIC, Canadian Prostate Cancer Research Initiative. DOI 10.1002/pros.21199 Published online 16 June 2010 in Wiley Online Library (wileyonlinelibrary.com). ß2010Wiley-Liss,Inc. Metastatic Clone in Primary Prostate Cancer 1637 INTRODUCTION metastatic potential of the disease is associated with The majority of prostate cancer deaths is caused by a minority of the cells in a primary tumor or with metastatic dissemination of the primary tumor [1]. the bulk of the tumor. To achieve this, a number of Metastasis is a complex multi-step process thought to transplantable prostate cancer sublines were develop- be driven by changes in the expression of multiple ed via subrenal capsule grafting in NOD-SCID mice genes caused by genetic or/and epigenetic alterations from different foci of a prostate cancer. Tissue invasive [2,3]. Prostate cancer has been recognized as a multi- or metastatic abilities of the sublines were then focal disease that generally consists of a dominant determined in vivo via orthotopic grafting and moni- cancer and one or more independent cancers of smaller toring organs/tissues of the hosts. Each subline was volume with different histological features and a wide then investigated for chromosomal alterations. Such spectrum of biological behavior [4–7]. The histological, alterations, exclusively present in a metastatic subline, biological, and genetic heterogeneity of multifocal were further investigated in parental cancer tissues by prostate cancers suggests that they arise independently fluorescence in situ hybridization (FISH) to identify the from different clones [8–11]. Experimental assays cells in the primary tumor which carried the same based on a mouse cancer cell line demonstrated that chromosomal alterations. only a small portion of cancer cells were endowed with metastasis-promoting functions, indicating that meta- MATERIALS AND METHODS static lesions are derived from descendants of a rare cell Materials and Animals in the primary tumor [11]. On the other hand, advances in molecular profiling of cancers suggest that the Chemicals, stains, solvents, and solutions were metastatic potential of human tumors is encoded in obtained from Sigma–Aldrich Canada Ltd (Oakville, the bulk of a primary tumor, thus challenging the above ON, Canada), unless otherwise indicated. Male 6- to hypothesis [12]. It is still controversial as to whether 8-week-old NOD-SCID mice were bred by the BC a minority or a majority of cancer cells in primary Cancer Research Centre Animal Resource Centre (BC tumors has metastatic potential. Cancer Agency, Vancouver, Canada). Mice were Studies of prostate cancer metastasis at the cellular housed in groups of three in microisolators with free and tissue levels have been impeded by a lack of access to food and water and their health was optimal experimental models. While established cul- monitored daily. Animal care and experiments were tured cancer cell lines representing different stages of carried out in accordance with the guidelines of the cancer progression can be very useful for identifying Canadian Council on Animal Care. mechanisms underlying metastasis, they do not adequately mimic clinical disease [13,14]. Efforts have Prostate CancerTissue Acquisition therefore focused on use of prostate cancer specimens Prostate cancer tissue specimens were obtained via from patients. However, the significant differences in prostatectomy from a 70-year-old male, with informed microenvironment between primary and secondary consent, at the Urology Research Unit (Carlton Centre, prostate cancers and the typical heterogeneity of such San Fernando, Trinidad). The patient was diagnosed specimens (e.g., consisting of both non-metastatic with advanced prostate cancer (Gleason grade 5 þ 5) and potentially metastatic subpopulations) make it and had not received neoadjuvant therapy. No detect- difficult to identify genes underlying the development able metastasis was observed prior to prostatectomy. of metastasis [15]. To overcome the above hurdles, we The specimens obtained from different foci were recently developed experimental prostate cancer mod- examined independently and confirmed to have els that resemble the clinical situation and allow the same histopathological grade by pathologists. The establishment of transplantable prostate cancer sub- samples were shipped overnight, immersed in cold lines that differ in metastatic ability and as such can be Hanks’ balanced salt solution supplemented with useful for investigating development of metastasis at antibiotics, to Vancouver (Canada). the cellular and molecular levels. The model is based on subrenal capsule grafting of pieces of a patient’s Subrenal Capsule Grafting and Development of primary prostate cancer tissue into immuno-deficient TransplantableTumorTissue Lines mice leading to establishment of multiple transplant- able tumor lines retaining major growth and histopa- Xenografting was performed as previously des- thological features of the original cancer [16–19]. cribed [16]. In brief, within 24 hr of its arrival, tumor The present study was aimed at establishing tumor tissue was cut into small pieces about 1 Â 3 Â 3mm3 in lines with different metastatic ability from a patient’s size. The tumor pieces were grafted under the kidney primary prostate cancer and examining whether capsules of male NOD-SCID mice supplemented with The Prostate 1638 Lin et al. testosterone (10 mg/mouse) via subcutaneously incubated for 30 min at room temperature with avidin– implanted testosterone pellets. After 90 days of growth biotin complex (Vector Laboratories, Burlingame, CA). (or earlier if required by the health status of the hosts), Following a further 30 min of washing in PBS, the animals were sacrificed by CO2 for necropsy. immunoreactivity was visualized using 3,3’-diamino- Tumors were harvested, measured, photographed, benzidine tetrahydrochloride (DAB) in PBS and 0.03% and fixed for histopathological analysis. Some of hydrogen peroxide. Sections were counterstained with the rapidly growing tumors were cut into pieces 5% (w/v) Harris hematoxylin and dehydrated in and maintained for up to five transplant generations graded alcohols. Control sections were processed by serial subrenal capsule transplantation into in parallel with rabbit non-immune IgG (Dako,
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