Background
Renal cell carcinoma (RCC) is one of the commonest malignancies of the genitourinary tract accounting for 61,560 new cases and 38,270 deaths in the United States per annum [
1]. Patients with RCC still face a dismal clinical outcome owing to high rate of metastasis both at initial presentation and after radical nephrectomy despite considerable improvements have been made in diagnosis, surgical techniques and adjuvant therapies in last decades [
2]. Therefore, it is important to understand the molecular mechanism involved in the essence and origin of RCC. Identification of novel biomarkers associated with disease progression and metastasis of RCC and combination of their application with traditional diagnostic and prognostic parameters would contribute to development of effective strategies for the prevention, early diagnosis and treatment of RCC.
It has been assumed that tumor initiating cells (TICs) constitute a reservoir of self-sustaining cells with ability to self-renew and maintain the tumor [
3‐
5]. A series of studies indicate that TICs may be the origin of local recurrence and distant metastases, if they were not completely eradicated by conventional treatments [
6‐
11]. Evidence is accumulating that TICs have been isolated from several types of human tumors, such as breast cancer and brain tumors [
6,
12]. However, the literature of investigating stem cells of kidney cancers is limited and lack of TIC-specific cell surface antigen markers. Currently, there are data to demonstrate that “sphere forming cells” or “spheroids” are commonly found and are useful to enrich the potential TIC subpopulations when the specific TIC makers have not been defined, as is the case for most TICs [
13,
14]. Therefore, in the present study, we isolated spheroid cells from RCC cell line (SN12C) and determined whether these cells acquired TICs characteristics, including self-renewing capacity and tumorigenic capacity.
Methods
Cell culture
Human RCC cell line, SN12C was used for this study which was obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). SN12C were cultured in DMEM (Gibco, California, USA) medium. Both media contained 10 % fetal bovine serum (FBS, Gibco).
Spheroid cells were derived by placing in serum-free medium (SFM) consisting of DMEM/F-12 medium with 20 ng/ml EGF (Invitrogen), 20 ng/ml bFGF (Invitrogen) and B27 (Invitrogen) on poly (2-hydroxyethylmethacrylate) (poly-HEMA; Sigma-Aldrich)-precoated plates. After primary spheroid body reached the size of approximately 100–200 cells per spheroid body, the spheroid bodies were dissociated at the density of 1000 cells per milliliter and 100 single cell suspension (100 μl) was seeded in each well of a 96-well ultra-low attachment plate (Corning) in serum-free medium described above. Two weeks later, wells were analyzed for subspheroid body formation.
Evaluation of tumorigenicity and histologic staining
All animal studies adhered to protocols approved by the local Institutional Animal Care and Use Committee. Five-week-old female BALB/C nude mice were purchased from the Animal Institute of Peking Union Medical College (PUMC). To assess tumorigenicity of spheroid cells and adherent cells, firstly, 104,105,106,107 cells of SN12C adherent cells were injected respectively into the right armpit, right inguen, left inguen and left armpit of the nude mouse(n = 3); 103,104,105,106 cells of SN12C spheroid cells were injected respectively into the right armpit, left armpit, left inguen and right inguen of the nude mouse(n = 3); secondly, 105 cells of spheroid cells and adherent cells were injected s.c. respectively into the left and right inguen of the same nude mouse (n = 3), to comparatively evaluate their tumorigenic potential. Mice were sacrificed by cervical dislocation at a tumor diameter of 1 cm or at 6 months post-transplantation. H&E staining was done on 4 μm paraffin-embedded sections following standard protocols.
Immunohistochemistry
Tumor specimens were fixed with buffered formalin and embedded in paraffin. Sections (5 mm) were placed on glass slides, heated at 60 °C for 20 min, and then deparaffinized with xylene and ethanol. For antigen retrieval, tumor specimens mounted on glass slides were immersed in preheated antigen retrieval solution for 20 min and allowed to cool for 20 min at room temperature. After the inactivation of endogenous peroxidase, rat anti mouse (1:50 dilution) or mouse anti human CD34 (1:200 dilution) (santa cruz) were then added, and incubated overnight at 4 °C. The second antibody was detected with a biotinylated anti-rat or anti-mouse IgG (DAKO). Diaminobenzidine tetrahydrochloride was then added for development for 10 min, followed by counterstaining with hematoxylin solution. CD34/PAS double-stained was used to validate vasculogenic mimicry (VM). It was identified by the detection of PAS-positive loops surrounding with tumor cells (not endothelial cells), with or without red blood cells in it. Microvessel density (MVD) was determined by light microscopy examination of CD34-stained sections at the “hot spot”. The fields of greatest neovascularization were identified by scanning tumor sections at low power (×100). The average vessel count of three fields (×400) was regarded as the MVD.
Immunofluorescence
The primary antibodies were CD133-PE (1:50, Miltenyi Biotec), CD44 (1:400; Santa Cruz), CD105 (1:100; Santa Cruz), Oct4 (1:100; Santa Cruz) and Nanog (1:200; Santa Cruz). The second antibody was incubated with FITC-labeled goat anti-mouse IgG (1:200; SantaCruz). All were prepared according to the Manufacturer’s protocols. Cells were fixed with 4 % of paraformaldehyde and when needed, permeabilized with 0.05 % of Triton X-100 in PBS at room temperature for 20 min. Samples were blocked with 1 % of bovine serum albumin (Sigma) and incubated with appropriate primary antibody at 37 °C for 1 h. After washing extensively, they were incubated with second antibody at 37 °C for 1 h. After immunolabeling and washing procedure, nuclei were then counterstained with 4′6-diamidino-2-phenylindole (DAPI; Sigma). Coverslips were viewed under fluorescence microscopy (Olympus LX51, Tokyo, Japan) and photos were taken using 100-fold magnification.
Flow cytometry
Monolayer cells were detached from culture plates with 0.25 % trypsin (Gibco), and spheroids were collected and dissociated by 5–10 min digestion with trypsin. Viable cell number was determined using Trypan blue staining (Invitrogen). Antibodies analyses were carried out as previously described [
15]. The following anti-human monoclonal antibodies were used for flow cytometry: CD133-PE, CD44, CD105, Nanog, Oct4 and FITC conjugated goat anti-mouse IgG. Appropriate isotype controls were used for each antibody.
Reverse transcription polymerase chain reaction (RT-PCR)
We examined the expression of CD44, renal stem/progenitor genes (CD133, CD105), and pluripotency gene (Oct4, Nanog) by semiquantitative reverse transcription–polymerase chain reactions (RT-PCR). The primers are provided in Table
1. Total RNA was extracted from spheroid cells and adherent cells following manufacturer’s instructions. RNA was quantified by spectrophotometry at OD260. The target cDNA was amplified using Platinum Taq DNA Polymerase (Invitrogen) for 28–30 cycles in 25 μl reactions. Aliquots of 8 μl of the amplification products were separated by electrophoresis on 1 % agarose gels and using β-actin as a loading control. Each analysis reaction was performed in duplicates or triplicates.
Table 1
Premier sequences and product sizes
β-Actin | 5′- CGGGAAATCGTGCGTGAC -3′ | 5′- GAAGGAAGGCTGGAAGAGTG -3′ | 183 bp |
CD44 | 5′- AATCCCTGCTACCACTTT -3′ | 5′- TTTCTTCATTTGGCTCCC -3′ | 191 bp |
CD133 | 5′- TTACGGCACTCTTCACCT -3′ | 5′- TATTCCACAAGCAGCAAA -3′ | 172 bp |
CD105 | 5′- TTTGGTGCCTTCCTCATCG -3′ | 5′- GGTTGGTGCTGCTGCTCT -3′ | 136 bp |
Nanog | 5′- CCTATGCCTGTGATTTGT -3′ | 5′- GTTGTTTGCCTTTGGGAC -3′ | 160 bp |
OCT4 | 5′- GGTTCTATTTGGGAAGGTGTT -3′ | 5′- TGCTGGGCGATGTGGCTGA -3′ | 279 bp |
CD31 | 5′- GCCAACTTCACCATCCAG -3′ | 5′- CACCCTCAGAACCTCACTTA -3′ | 420 bp |
CD34 | 5′- TTGCTGCCTTCTGGGTTC -3′ | 5′- CATTTGATTTCTGCCTTGAT -3′ | 458 bp |
Western blot
The whole cell lysates were resolved by sodium dodecyl sulphatepolyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Blots were blocked and incubated with the monoclonal antibody CD133 (1:100), CD44 (1:1000), CD105 (1:500), Oct4 (1:500), Nanog (1:1000), E-cad(1:50; CST), and Vim(1:500; Santa Cruz) and followed by incubation with a secondary antibody (1:2000, Santa Cruz, CA, USA). Blots were developed using an enhanced chemiluminescence detection kit. For protein loading analyses, a monoclonal β-actin antibody (1:2000, Santa Cruz) was used as control.
Proliferation assay
In order to compare the proliferation rates between spheroid cells and adherent cells, 1000 spheroid cells and adherent cells were exposed to DMEM/F12 medium containing 10 % FBS, half of the medium was changed every 3 days and the number of the cells was measured and the cell growth curve was drawn accordingly. The proliferation assays were done in triplicate.
Soft agar colony formation assay was performed by seeding cells in a layer of 0.35 % agar DMEM-F12 over a layer of 0.5 % agar/DMEM-F12. Cultures were maintained in a humidified incubator at 37 °C and 5 % CO
2. Additional complete media was added every 2 days to continuously supply growth supplements to the cells. On day 14 or day 21 after seeding, cells were fixed with pure ethanol containing 0.05 % crystal violet and CFE quantified by light microscopy [
16]. The clone formation efficiency was the ratio of the number of clones to the number of seeded cells.
Invasion assays
Transwell chambers (8 μm pore) were first coated with 10 μg/ml fibronectin overnight at 4 °C, washed in PBS and rehydrated with serum-free media for 30 min at 37 °C. The media were removed, and then the cells (1 × 105/chamber) were plated in the upper chambers of the serum-free media. The lower chambers contained media with 2.5 % FBS as a chemo attractant. Chambers were incubated at 37 °C and 5 % CO2. Migrated cells were stained with crystal violet solution and counted. All tests were done in duplicate.
Wound healing (Scratch) assay
Parental and spheroid cells were cultured for 24 h. Then cells were seeded into 6-well plates to 80–90 % confluence and the cell monolayer was scratched in a straight line with a 200 μl pipette tip to create a “scratch”. Debris was removed with PBS and then the culture was re-fed with fresh medium. Images were taken at 0 and 24 h after the scratch to calculate the cell migration rate.
Aldefluor assay by FACS
The ALDEFLUOR kit (Stem Cell Technologies, Durham, NC, USA) was used to analyze the population with a high ALDH enzymatic activity. Cells obtained from adherent or spheroid 109 cells were suspended in ALDEFLUOR assay buffer containing ALDH substrate and incubated during 40 min at 37 °C. As a negative control, for each sample of cells an aliquot was treated with 50 mmol/L diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor. FACS was performed using a FACS Aria flow cytometer (BD Biosciences).
Endothelial cell differentiation and characterization
We collected spheroid cells, washed with PBS, then added the induced medium which contained DMEM, 10 % FBS, VEGF (10 ng/mL), and bFGF (20 ng/mL). The culture were maintained for 14–21 days by induced medium and exchanged every 3 days. To examine the uptake of Dill-labeled ac-LDL (Bio-medical Technologies, Stoughton, MA), spheroid cells and differentiated endothelial cells were incubated with 10 μg/ml Dill-labeled ac-LDL for 4 h at 37 °C. After incubation, cells were washed three times with PBS, fixed with 3 % paraformaldehyde for 30 min. To investigate tube formation Assay, spheroid cells and differentiated endothelial cells (5000 cells) were seeded on the surface of the matrigel well, and were serum starved in DMEM medium for 2 h. Thereafter, they were exposed to DMEM supplemented with 5 % FBS for 24 h. Tube formation was visualized using a microscope.
Statistical analyses
All experiments were performed at least three times and representative results were presented (quantitative data expressed as the mean ± SD). Statistical significance was set at P < 0.05.
Discussion
Currently, there is data to demonstrate that spheroid body culture has been increasingly used as a method for enriching stem cells which relies on their property of anchorage-independent growth. Various types of potential TIC subpopulations from primary tumors have been reported to be isolated and enriched by the application of spheroid body culture [
17,
18]. The spheroid body-forming cells from primary tumors, such as ovarian cancer and breast cancer, showed stem-like properties and expressed their TIC markers [
14]. Since the markers of renal TICs were uncertain, it is unimaginable to isolate cells using all the markers. Also, to our knowledge, there have been few reports on the isolation and characterization of renal TICs by the method of spheroid culture, therefore, we isolated spheroid body cells by cultivating human RCC cell line SN12C in defined serum-free medium. In this report, we demonstrated that spheroid cells could generate greater numbers of new spheroid cells than the parental cells. These observations indicated that spheroid body-forming cells were capable of self-renewal and proliferation, which were important characteristics of TICs.
The gold standard assay of TICs is that the candidate population of cells can preferentially initiate tumor development in animals [
19]. The least stringent definition is that the prospectively purified TICs population is much more tumorigenic than the bulk or the tumor cell population in a suitable tumor development assay. Our findings suggested that 10
4 spheroid cells from primary tumor cells engrafted into mice and allowed full recapitulation of the original tumor, whereas 10
6 adherent cells remained non-tumorigenic, which indicated spheroid cells were more tumorigenic than the corresponding, and the tumor formation ability of the spheroid cells overall was at least 100-fold higher than that of adherent cells. Although the adherent cells could produce tumors when large numbers of cells were implanted, the tumors that formed regressed spontaneously, whereas the spheroid-derived tumors continued to grow, indicating that the adherent cells-derived tumors may lack the TICs required sustaining tumor growth. The initial formation of tumors from high numbers of adherent cells may be explained by the high intrinsic tumorigenicity of SN12C cells, a high fraction of which express TICs markers.
To further explore the TIC properties of spheroid body-forming cells, we evaluated the spheroid cells for their stemness characteristics. Our recent work has shown overexpression of stem cell-specific transcription factor such as CD44, CD133, Oct4, CD105 and Nanog is vital characteristic of TICs [
20‐
22]. Bussolati et al. reported the isolation of CD105+ cancer stem cells from renal carcinoma [
23]. In this study, we demonstrated that CD44, CD133, Oct4, CD105 and Nanog were overexpressed in spheroid cells compared with parental cells by immunofluorescent, FCM, RT-PCR and Western blot analysis with statistical significance. So it is conceivable that the expression of CD44, CD133, Oct4, CD105 and Nanog in spheroid cells will endow some cells with certain stem/progenitor cell properties. Strikingly, spheroid cells showed approximately threefold increase in CFE when compared with that of adherent cells on soft agar, suggesting that spheroid cells have a higher self-renew potential than adherent cells. Spheroid cells showed higher migration ability than their adherent cells both in migration and wound healing assays, which were in line with Yu et al.[
24], indicated that spheroid cells might promote more aggressive biological behavior. ALDHs are a superfamily of 17 intracellular enzymes that protect cells from the cytotoxic effects of peroxic aldehydes. Increased ALDH activity has also been found in stem cell populations in various types of cancer. ALDH activity may therefore provide a marker for normal and malignant stem as well as progenitor cells. Our study indicated that a high ALDH enzymatic activity is a function of RCC tumor-sphere.
Several intriguing studies have described that RCC cells developmental epithelial-to-mesenchymal transition (EMT) program to enrich their stemness features, thereby facilitating metastasis and drug resistance [
25]. This is an important function, because a loss of E-cadherin on the cell surface has been shown to play a role in tumor progression and metastasis. In our study, spheroid cells demonstrated down-regulation of E-cadherin, and up-regulation of Vimentin. So, the spheroid cells display the phenotype of EMT according to our results. And the biological behaviors of spheroid cells consistent with the mesenchymal cells, such as morphological character, lower adhesion but higher in vitro migratory/invasive capacity.
Recently many accumulative evidences have showed that TICs may participate in the angiogenesis process to form endothelial cells directly [
26]. It has been reported that two kinds of distinct tumor vessels play function in tumor blood-supply in malignant tumor tissues, one of which is VM, where vessels are exclusively composed of tumor cells; and the other is the endothelium vessel (EDV), where vessels are covered by the endothelium [
27]. We previously also showed definite relationship between TICs, microvessel density (MVD), and VM [
20]. In this study, we found spheroid cells not only formed xenograft tumors in vivo, histopathological studies have also found that some cells composing of tumor vessel lumen showed anti human CD34 positive, indicating these cells in the vessel wall of xenograft tumors were originated from the cells of human origin, not derived from host (murine) cells, which suggested that TICs might be the progenitor for tumor vasculature. This finding is consistent with recent reports that tumors grown from brain TICs, isolated based on the marker CD133, are more angiogenic than non-TIC-derived tumors [
28], and that C6 glioma-derived TICs, isolated by a sphere-forming assay, exhibit increased microvessel density and blood perfusion compared with non-TIC-derived tumors [
29]. Endothelial differentiation of spheroid cells were morphologically similar to HUEVC and, and displayed functional characteristics of fully mature endothelial cells. Vasculogenic potential of these cells was shown by assessing formation of networks when plated on a semi-solid substrate, and internalization of ac-LDL distinctly identified endothelial cells based on metabolic activity. These results suggest that spheroid cells are able to differentiate into functional endothelial cells in vitro and are suited for evaluation for vasculogenic potential within a tissue-engineered construct in vivo.
Together, these findings support the hypothesis that TICs promote tumor angiogenesis by secreting elevated levels of pro-angiogenic factors compared to non-TIC populations [
30]. Work is in progress in our laboratory to target the specific mechanisms by which spheroid cells increased angiogenesis. Further investigation of the molecular mechanisms responsible for the increased angiogenesis seen in TIC-derived kidney tumors may improve the efficacy of cancer therapies that target angiogenesis, either alone or in combination with chemotherapy [
31].