J Cancer Res Clin Oncol DOI 10.1007/s00432-014-1883-0
ORIGINAL ARTICLE – CANCER RESEARCH
BMP‑2 inhibits tumor‑initiating ability in human renal cancer stem cells and induces bone formation Lin Wang · Paul Park · Frank La Marca · Khoi D. Than · Chia‑Ying Lin
Received: 3 November 2014 / Accepted: 22 November 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose We have previously shown that BMP-2 induces bone formation and inhibits tumorigenicity of cancer stem cells (CSCs) in a human osteosarcoma OS99-1 cell line. In this study, we sought to determine whether BMP-2 can similarly induce bone formation and inhibit the tumorigenicity of renal CSCs identified based on aldehyde dehydrogenase (ALDH) activity in renal cell carcinoma (RCC) cell lines and primary tumors. Methods Using a xenograft model in which cells from human RCC cell lines ACHN, Caki-2, and primary tumors were grown in NOD/SCID mice, renal CSCs were identified as a subset of ALDHbr cells. The ALDHbr cells possessed a greater colony-forming efficiency, higher proliferative output, increased expression of stem cell marker genes Oct3/4A, Nanog, renal embryonic marker Pax-2, and greater tumorigenicity compared to cells with low ALDH activity (ALDHlo cells), generating new tumors with as few as 25 cells in mice. Results In vitro, BMP-2 was found to inhibit the ALDHbr cell growth, down-regulate the expression of embryonic stem cell markers, and up-regulate the transcription of osteogenic markers. In vivo, all animals receiving a low Electronic supplementary material The online version of this article (doi:10.1007/s00432-014-1883-0) contains supplementary material, which is available to authorized users. L. Wang · P. Park (*) · F. La Marca · K. D. Than · C.‑Y. Lin Spine Research Laboratory, Department of Neurosurgery, University of Michigan Medical School, 1500 E. Medical Center Drive, Room 3552 TC, Ann Arbor, MI 48109‑5338, USA e-mail: [email protected]; [email protected] C.‑Y. Lin Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
number of ALDHbr cells (5 × 103) from ACHN, Caki-2, and primary tumor xenografts treated with 30 µg BMP-2 per animal showed limited tumor growth with significant bone formation, while untreated cells developed large tumor masses without bone formation. Conclusions These results suggest that BMP-2 inhibits the tumor-initiating ability of renal CSCs and induces osseous bone formation. BMP-2 may therefore provide a beneficial strategy for human RCC treatment by targeting the CSC-enriched population. Keywords BMP-2 · Aldehyde dehydrogenase · Cancer stem cells · Renal cell carcinoma
Introduction A growing body of evidence indicates that the capacity of a tumor to grow and propagate exclusively resides in a small subset of tumor cells termed cancer stem cells (CSCs) or tumor-initiating cells (Diehn et al. 2009; Vermeulen et al. 2008). CSCs are in many ways similar to normal stem cells and are thought to arise either when normal stem cells gain oncogenic mutations, which confer enhanced proliferation and lack of homeostatic control mechanisms, or alternatively when a progenitor or differentiated cell acquires mutations conferring dedifferentiation to a malignant stemlike cell (Stratford et al. 2011). Like normal stem cells, these cells are characterized by their ability for self-renewal and their capacity to form serially transplantable tumors in immunodeficient mice. The stem cell-like phenotype of CSCs and their rare number within the tumor may account for their ability to escape from standard chemo- or radiation therapies, thus leading to tumor recurrence and eventually metastasis, even when the primary lesion has been
13
eradicated (Dean et al. 2005). Therefore, there is a pressing need to develop CSC-targeted strategies to eliminate these stem cell-like tumor cells, which might prevent cancer relapse. Renal cell carcinoma (RCC) is the most common malignant tumor arising from kidney, accounting for about 65,150 new cases and 13,680 new deaths in the USA in 2013 (Siegel et al. 2013). The incidence of RCC has been steadily increasing for decades. The major cause of death from RCC is metastases that have been notoriously resistant to conventional chemotherapies (Costa and Drabkin 2007). In recent years, the existence of stem cell-like tumor cells in RCC has also been identified in a subpopulation of cells that have superior tumor-initiating, self-renewal, and differentiation capabilities. These cells have been detected in spherical clones under anchorage-independent, serumstarved culture conditions, as side population (SP) cells based on efflux of Hoechst 33342 dye, as CD105+ cells sorted using mesenchymal stem cell marker CD105, or as CXC chemokine receptor 4 (CXCR4)+ cells (Bussolati et al. 2008; Gassenmaier et al. 2013; Huang et al. 2013; Nishizawa et al. 2012; Zhong et al. 2010). More recently, Ueda et al. (2013) revealed that aldehyde dehydrogenase (ALDH)-1+ cells from SP cells identify CSCs in a human RCC cell line. Identifying CSC markers in RCC will potentially lead to novel CSC-targeted therapies for patients with RCC. To date, however, there has been only one investigation indicating that interleukin-15 directs epithelial differentiation of all human renal CD105+ CSCs in vitro, but the effect of interleukin-15 on CSCs in vivo has not been characterized (Azzi et al. 2011). No study has reported the use of bone morphogenetic protein-2 (BMP-2) to induce differentiation of cancer stem cell-like tumor cells in RCC. BMP-2 was originally identified as a potent osteoinductive growth factor that promotes bone and cartilage formation in vivo, but is now considered as a multifunctional cytokine. Besides inducing bone and cartilage formation, BMP-2 has also been found to play important roles in the regulation of various cellular processes including cell differentiation, proliferation, morphogenesis, cellular survival, and apoptosis (Kang et al. 2010). It was recently reported that BMP-2 plays different roles on cancer cells, depending on tissue type and environment. BMP-2 has been shown to stimulate growth of pancreatic carcinoma, lung carcinoma, and prostate cancer cells in the absence of androgen (Ide et al. 1997; Kleeff et al. 1999; Langenfeld et al. 2003). Conversely, BMP-2 inhibits the growth of other cancer types including breast, myeloma, gastric, colon, and prostate (Beck et al. 2006; Brubaker et al. 2004; Ghosh-Choudhury et al. 2000; Ide et al. 1997; Kawamura et al. 2000; Wen et al. 2004). In a previous study from our group, BMP-2 has been shown to inhibit tumorigenicity of
13
J Cancer Res Clin Oncol
CSCs in human osteosarcoma OS99-1 cells, inhibit tumor growth of human RCC, and induce bone formation (Wang et al. 2011a, 2012, 2013). However, there are no published studies on the impact of recombinant human BMP-2 (rhBMP-2) on tumor-initiating ability in human renal CSCs. rhBMP-2 has achieved United States Food and Drug Administration approval for use in treatment of certain spinal fusions, tibial fractures, and is commercially available for bone repair (Laflamme et al. 2010). rhBMP-2 might be used to treat RCC bone metastasis to facilitate bone growth after surgical resection if rhBMP-2 could exert an inhibitory effect on the growth of human renal CSCs and induce bone formation. In the present study, we evaluated whether BMP-2 can be used to inhibit the tumor-initiating ability of human renal CSCs in vitro and induce bone formation in vivo. We chose to identify renal CSCs using the ALDH marker based on our previous results.
Materials and methods Human cell lines and tumor specimens Human RCC cell lines used in these studies have been previously described (Wang et al. 2012). ACHN and Caki-2 cell lines were purchased from American Type Culture Collection (Manassas, VA). Cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Gibco, Carlsbad, CA) supplemented with 10 % FBS (Gibco) in a humidified atmosphere of 5 % CO2 in air at 37 °C and used when in the log phase of growth. Following approval by the University of Michigan Institutional Review Board, freshly harvested primary metastatic RCC tissue was obtained from consenting patients undergoing surgical resection at the University of Michigan Hospital. Xenografts, tumor dissociation, ALDEFLUOR cell analysis, and flow cytometry sorting Immunodeficient non-obese diabetic (NOD)/severe combined immunodeficient (SCID) (NOD/SCID) mice (5– 6 weeks old) were purchased from Harlan Laboratories (Indianapolis, IN). All animal studies were performed according to the protocol approved by the Institutional Animal Care and Use Committee of the University of Michigan. Primary patient tumor xenografts were established by injection of dissociating fresh tumor tissue in Matrigel (BD Biosciences, San Jose, CA) subcutaneously into the right lower abdominal area of NOD/SCID mice. Tumor cells from six patients were injected into mice, but only two established a xenograft that was subsequently used for ALDEFLUOR (StemCell Technologies, Vancouver, BC,
J Cancer Res Clin Oncol
Canada) cell analysis (Supplementary Table S1). Basic experimental procedures for tumor dissociation, ALDEFLUOR cell analysis, and flow cytometry to isolate ALDHbr cells and ALDHlo cells from ACHN, Caki-2, and primary tumor xenografts are detailed elsewhere (Wang et al. 2011a, b). Tumorigenicity of ALDHbr and ALDHlo cells in NOD/SCID mice and serial transplantation To assess differences in tumorigenicity of ALDHbr and ALDHlo cells sorted from xenografts, freshly sorted cells were washed, suspended in 200 μL of serum-free PBS/Matrigel mixture (1:1 volume), and then injected subcutaneously into the right and left lower abdominal area of NOD/SCID mice. Co-injection with Matrigel has been shown to increase tumor formation and yield optimal tumor growth. Tumor growth was monitored weekly for 32 weeks. Xenografted tumors were extracted, minced with scissors, mixed with 1 mg/mL collagenase type II (Sigma-Aldrich Co., St. Louis, MO), incubated for 3–4 h, passed through a 40-μm cell strainer, and then washed twice with DMEM/F12/10 % FBS medium. Cells were re-sorted into ALDHbr and ALDHlo fractions, and then, sorted cells were re-injected into the animals as the primary transplantation. The entire procedure was repeated with the formed tumors to conduct secondary and tertiary transplantations. Cell proliferation assay Freshly sorted ALDHbr and ALDHlo cells from xenografts were cultured at a density of 5 × 103 cells per well in a 96-well plate in triplicate with 100 μL of culture medium and allowed to grow for 9–14 days. To investigate the effects of BMP-2 on cell growth, freshly sorted ALDHbr cells from ACHN, Caki-2, and primary tumor xenografts were washed and cultured in 10 % DMEM/F12 medium for expansion and then inoculated at 1 × 104 cells per well for 24 h. Cells were then cultured in 1 % serum-containing medium for another 24 h after washing in PBS. Cells were treated with 10, 100, or 300 ng/mL BMP-2 (GenScript Corporation, Piscataway, NJ) diluted in 1 % serum-containing medium or vehicle control for 24, 48, and 72 h. Cell proliferation was assessed as described previously (Wang et al. 2011b). Briefly, at a specific time point during cultivation, the medium was discarded and replaced with 100 μL PBS, and then, 20 μL Celltiter96 AQueous One Solution reagent (Promega, Madison, WI) was added to each well and incubated at 37 °C for 2 h. Absorbance at 490 nm was recorded using Bio-Tek ELx800 Absorbance Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT).
Soft agar assay Freshly sorted ALDHbr and ALDHlo cells from xenografts were seeded in 6-well plates coated with a 1 % agarose bottom layer in culture medium containing 10 % FBS, a middle layer of 0.6 % agarose including 5 × 103 cells, and a top layer of culture medium only. Plates were incubated at 37 °C for 4 weeks and fixed with formalin for 20 min. Plates were then stained with 0.005 % crystal violet for 1 h, and colonies were visualized by trans-UV illumination and counted using analysis software Quantity One (Bio-Rad, Hercules, CA). Quantitative reverse transcription PCR (qRT‑PCR) analysis of Oct3/4A, Nanog, and Pax‑2 mRNA To assess mRNA expression levels of embryonic stem cell markers Oct3/4A, Nanog, and renal embryonic marker Pax-2 in ALDHbr and ALDHlo cells from xenografts, total RNA was extracted using RNeasy Mini Kit (Qiagen Inc., Valencia, CA). Reverse transcription was made using SuperScript First-Strand Synthesis Kit (Invitrogen, Carlsbad, CA). To investigate the effects of BMP-2 on gene expression of Oct3/4A, Nanog, and Pax-2, freshly sorted ALDHbr cells were washed, cultured for expansion, and then inoculated at 1 × 105 cells in a 6-well culture plate. After 24-h incubation, the medium was replaced with 1 % serum-containing medium for 24 h and then replaced with 0 and 300 ng/mL BMP-2 diluted in 1 % serum-containing medium. After 48 h, total RNA was extracted as described above. qRT-PCR assay was performed as previously reported (Wang et al. 2011b). The Eppendorf Mastercycler Realplex Detection System (Eppendorf, Germany) was used for conducting qRT-PCR. In vivo co‑treatment experiments and assessment of bone formation To examine the tumor inhibitory effect of BMP-2 on tumorigenic ALDHbr cells from xenografts, 1 × 104 freshly sorted ALDHbr cells were mixed with Affi-Gel blue beads (Bio-Rad) loaded with 30 μg/animal of BMP-2 at 37 °C for 1 h. Sorted ALDHbr cells treated with the same volume of vehicle were used as a control, and then, ALDHbr cells with BMP-2 or vehicle were subcutaneously injected into NOD/SCID mice. After the mice were euthanized, radiographs were obtained using Faxitron X-ray unit (Field Emission Corp., McMinnville, OR). For microcomputed tomography (micro-CT) analysis, specimens were scanned at 8.93 um voxel resolution on a micro-CT scanner (EVS Corp., London, ON, Canada), with a total of 667 slices per scan. GEMS MicroView software (GE Healthcare Biosciences, Piscataway, NJ) was used to make
13
a three-dimensional reconstruction from the set of scans. For histomorphometry, specimens were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) and Masson’s trichrome staining to show collagen type I protein in the newly formed bone. Non-decalcified sections were stained with von Kossa staining to identify calcification during osteogenesis in the tumor. Statistical analysis Data were expressed as mean ± SD. Statistically significant differences were determined by one-way ANOVA and Student’s t test where appropriate, and defined as p
ORIGINAL ARTICLE – CANCER RESEARCH
BMP‑2 inhibits tumor‑initiating ability in human renal cancer stem cells and induces bone formation Lin Wang · Paul Park · Frank La Marca · Khoi D. Than · Chia‑Ying Lin
Received: 3 November 2014 / Accepted: 22 November 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose We have previously shown that BMP-2 induces bone formation and inhibits tumorigenicity of cancer stem cells (CSCs) in a human osteosarcoma OS99-1 cell line. In this study, we sought to determine whether BMP-2 can similarly induce bone formation and inhibit the tumorigenicity of renal CSCs identified based on aldehyde dehydrogenase (ALDH) activity in renal cell carcinoma (RCC) cell lines and primary tumors. Methods Using a xenograft model in which cells from human RCC cell lines ACHN, Caki-2, and primary tumors were grown in NOD/SCID mice, renal CSCs were identified as a subset of ALDHbr cells. The ALDHbr cells possessed a greater colony-forming efficiency, higher proliferative output, increased expression of stem cell marker genes Oct3/4A, Nanog, renal embryonic marker Pax-2, and greater tumorigenicity compared to cells with low ALDH activity (ALDHlo cells), generating new tumors with as few as 25 cells in mice. Results In vitro, BMP-2 was found to inhibit the ALDHbr cell growth, down-regulate the expression of embryonic stem cell markers, and up-regulate the transcription of osteogenic markers. In vivo, all animals receiving a low Electronic supplementary material The online version of this article (doi:10.1007/s00432-014-1883-0) contains supplementary material, which is available to authorized users. L. Wang · P. Park (*) · F. La Marca · K. D. Than · C.‑Y. Lin Spine Research Laboratory, Department of Neurosurgery, University of Michigan Medical School, 1500 E. Medical Center Drive, Room 3552 TC, Ann Arbor, MI 48109‑5338, USA e-mail: [email protected]; [email protected] C.‑Y. Lin Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
number of ALDHbr cells (5 × 103) from ACHN, Caki-2, and primary tumor xenografts treated with 30 µg BMP-2 per animal showed limited tumor growth with significant bone formation, while untreated cells developed large tumor masses without bone formation. Conclusions These results suggest that BMP-2 inhibits the tumor-initiating ability of renal CSCs and induces osseous bone formation. BMP-2 may therefore provide a beneficial strategy for human RCC treatment by targeting the CSC-enriched population. Keywords BMP-2 · Aldehyde dehydrogenase · Cancer stem cells · Renal cell carcinoma
Introduction A growing body of evidence indicates that the capacity of a tumor to grow and propagate exclusively resides in a small subset of tumor cells termed cancer stem cells (CSCs) or tumor-initiating cells (Diehn et al. 2009; Vermeulen et al. 2008). CSCs are in many ways similar to normal stem cells and are thought to arise either when normal stem cells gain oncogenic mutations, which confer enhanced proliferation and lack of homeostatic control mechanisms, or alternatively when a progenitor or differentiated cell acquires mutations conferring dedifferentiation to a malignant stemlike cell (Stratford et al. 2011). Like normal stem cells, these cells are characterized by their ability for self-renewal and their capacity to form serially transplantable tumors in immunodeficient mice. The stem cell-like phenotype of CSCs and their rare number within the tumor may account for their ability to escape from standard chemo- or radiation therapies, thus leading to tumor recurrence and eventually metastasis, even when the primary lesion has been
13
eradicated (Dean et al. 2005). Therefore, there is a pressing need to develop CSC-targeted strategies to eliminate these stem cell-like tumor cells, which might prevent cancer relapse. Renal cell carcinoma (RCC) is the most common malignant tumor arising from kidney, accounting for about 65,150 new cases and 13,680 new deaths in the USA in 2013 (Siegel et al. 2013). The incidence of RCC has been steadily increasing for decades. The major cause of death from RCC is metastases that have been notoriously resistant to conventional chemotherapies (Costa and Drabkin 2007). In recent years, the existence of stem cell-like tumor cells in RCC has also been identified in a subpopulation of cells that have superior tumor-initiating, self-renewal, and differentiation capabilities. These cells have been detected in spherical clones under anchorage-independent, serumstarved culture conditions, as side population (SP) cells based on efflux of Hoechst 33342 dye, as CD105+ cells sorted using mesenchymal stem cell marker CD105, or as CXC chemokine receptor 4 (CXCR4)+ cells (Bussolati et al. 2008; Gassenmaier et al. 2013; Huang et al. 2013; Nishizawa et al. 2012; Zhong et al. 2010). More recently, Ueda et al. (2013) revealed that aldehyde dehydrogenase (ALDH)-1+ cells from SP cells identify CSCs in a human RCC cell line. Identifying CSC markers in RCC will potentially lead to novel CSC-targeted therapies for patients with RCC. To date, however, there has been only one investigation indicating that interleukin-15 directs epithelial differentiation of all human renal CD105+ CSCs in vitro, but the effect of interleukin-15 on CSCs in vivo has not been characterized (Azzi et al. 2011). No study has reported the use of bone morphogenetic protein-2 (BMP-2) to induce differentiation of cancer stem cell-like tumor cells in RCC. BMP-2 was originally identified as a potent osteoinductive growth factor that promotes bone and cartilage formation in vivo, but is now considered as a multifunctional cytokine. Besides inducing bone and cartilage formation, BMP-2 has also been found to play important roles in the regulation of various cellular processes including cell differentiation, proliferation, morphogenesis, cellular survival, and apoptosis (Kang et al. 2010). It was recently reported that BMP-2 plays different roles on cancer cells, depending on tissue type and environment. BMP-2 has been shown to stimulate growth of pancreatic carcinoma, lung carcinoma, and prostate cancer cells in the absence of androgen (Ide et al. 1997; Kleeff et al. 1999; Langenfeld et al. 2003). Conversely, BMP-2 inhibits the growth of other cancer types including breast, myeloma, gastric, colon, and prostate (Beck et al. 2006; Brubaker et al. 2004; Ghosh-Choudhury et al. 2000; Ide et al. 1997; Kawamura et al. 2000; Wen et al. 2004). In a previous study from our group, BMP-2 has been shown to inhibit tumorigenicity of
13
J Cancer Res Clin Oncol
CSCs in human osteosarcoma OS99-1 cells, inhibit tumor growth of human RCC, and induce bone formation (Wang et al. 2011a, 2012, 2013). However, there are no published studies on the impact of recombinant human BMP-2 (rhBMP-2) on tumor-initiating ability in human renal CSCs. rhBMP-2 has achieved United States Food and Drug Administration approval for use in treatment of certain spinal fusions, tibial fractures, and is commercially available for bone repair (Laflamme et al. 2010). rhBMP-2 might be used to treat RCC bone metastasis to facilitate bone growth after surgical resection if rhBMP-2 could exert an inhibitory effect on the growth of human renal CSCs and induce bone formation. In the present study, we evaluated whether BMP-2 can be used to inhibit the tumor-initiating ability of human renal CSCs in vitro and induce bone formation in vivo. We chose to identify renal CSCs using the ALDH marker based on our previous results.
Materials and methods Human cell lines and tumor specimens Human RCC cell lines used in these studies have been previously described (Wang et al. 2012). ACHN and Caki-2 cell lines were purchased from American Type Culture Collection (Manassas, VA). Cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Gibco, Carlsbad, CA) supplemented with 10 % FBS (Gibco) in a humidified atmosphere of 5 % CO2 in air at 37 °C and used when in the log phase of growth. Following approval by the University of Michigan Institutional Review Board, freshly harvested primary metastatic RCC tissue was obtained from consenting patients undergoing surgical resection at the University of Michigan Hospital. Xenografts, tumor dissociation, ALDEFLUOR cell analysis, and flow cytometry sorting Immunodeficient non-obese diabetic (NOD)/severe combined immunodeficient (SCID) (NOD/SCID) mice (5– 6 weeks old) were purchased from Harlan Laboratories (Indianapolis, IN). All animal studies were performed according to the protocol approved by the Institutional Animal Care and Use Committee of the University of Michigan. Primary patient tumor xenografts were established by injection of dissociating fresh tumor tissue in Matrigel (BD Biosciences, San Jose, CA) subcutaneously into the right lower abdominal area of NOD/SCID mice. Tumor cells from six patients were injected into mice, but only two established a xenograft that was subsequently used for ALDEFLUOR (StemCell Technologies, Vancouver, BC,
J Cancer Res Clin Oncol
Canada) cell analysis (Supplementary Table S1). Basic experimental procedures for tumor dissociation, ALDEFLUOR cell analysis, and flow cytometry to isolate ALDHbr cells and ALDHlo cells from ACHN, Caki-2, and primary tumor xenografts are detailed elsewhere (Wang et al. 2011a, b). Tumorigenicity of ALDHbr and ALDHlo cells in NOD/SCID mice and serial transplantation To assess differences in tumorigenicity of ALDHbr and ALDHlo cells sorted from xenografts, freshly sorted cells were washed, suspended in 200 μL of serum-free PBS/Matrigel mixture (1:1 volume), and then injected subcutaneously into the right and left lower abdominal area of NOD/SCID mice. Co-injection with Matrigel has been shown to increase tumor formation and yield optimal tumor growth. Tumor growth was monitored weekly for 32 weeks. Xenografted tumors were extracted, minced with scissors, mixed with 1 mg/mL collagenase type II (Sigma-Aldrich Co., St. Louis, MO), incubated for 3–4 h, passed through a 40-μm cell strainer, and then washed twice with DMEM/F12/10 % FBS medium. Cells were re-sorted into ALDHbr and ALDHlo fractions, and then, sorted cells were re-injected into the animals as the primary transplantation. The entire procedure was repeated with the formed tumors to conduct secondary and tertiary transplantations. Cell proliferation assay Freshly sorted ALDHbr and ALDHlo cells from xenografts were cultured at a density of 5 × 103 cells per well in a 96-well plate in triplicate with 100 μL of culture medium and allowed to grow for 9–14 days. To investigate the effects of BMP-2 on cell growth, freshly sorted ALDHbr cells from ACHN, Caki-2, and primary tumor xenografts were washed and cultured in 10 % DMEM/F12 medium for expansion and then inoculated at 1 × 104 cells per well for 24 h. Cells were then cultured in 1 % serum-containing medium for another 24 h after washing in PBS. Cells were treated with 10, 100, or 300 ng/mL BMP-2 (GenScript Corporation, Piscataway, NJ) diluted in 1 % serum-containing medium or vehicle control for 24, 48, and 72 h. Cell proliferation was assessed as described previously (Wang et al. 2011b). Briefly, at a specific time point during cultivation, the medium was discarded and replaced with 100 μL PBS, and then, 20 μL Celltiter96 AQueous One Solution reagent (Promega, Madison, WI) was added to each well and incubated at 37 °C for 2 h. Absorbance at 490 nm was recorded using Bio-Tek ELx800 Absorbance Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT).
Soft agar assay Freshly sorted ALDHbr and ALDHlo cells from xenografts were seeded in 6-well plates coated with a 1 % agarose bottom layer in culture medium containing 10 % FBS, a middle layer of 0.6 % agarose including 5 × 103 cells, and a top layer of culture medium only. Plates were incubated at 37 °C for 4 weeks and fixed with formalin for 20 min. Plates were then stained with 0.005 % crystal violet for 1 h, and colonies were visualized by trans-UV illumination and counted using analysis software Quantity One (Bio-Rad, Hercules, CA). Quantitative reverse transcription PCR (qRT‑PCR) analysis of Oct3/4A, Nanog, and Pax‑2 mRNA To assess mRNA expression levels of embryonic stem cell markers Oct3/4A, Nanog, and renal embryonic marker Pax-2 in ALDHbr and ALDHlo cells from xenografts, total RNA was extracted using RNeasy Mini Kit (Qiagen Inc., Valencia, CA). Reverse transcription was made using SuperScript First-Strand Synthesis Kit (Invitrogen, Carlsbad, CA). To investigate the effects of BMP-2 on gene expression of Oct3/4A, Nanog, and Pax-2, freshly sorted ALDHbr cells were washed, cultured for expansion, and then inoculated at 1 × 105 cells in a 6-well culture plate. After 24-h incubation, the medium was replaced with 1 % serum-containing medium for 24 h and then replaced with 0 and 300 ng/mL BMP-2 diluted in 1 % serum-containing medium. After 48 h, total RNA was extracted as described above. qRT-PCR assay was performed as previously reported (Wang et al. 2011b). The Eppendorf Mastercycler Realplex Detection System (Eppendorf, Germany) was used for conducting qRT-PCR. In vivo co‑treatment experiments and assessment of bone formation To examine the tumor inhibitory effect of BMP-2 on tumorigenic ALDHbr cells from xenografts, 1 × 104 freshly sorted ALDHbr cells were mixed with Affi-Gel blue beads (Bio-Rad) loaded with 30 μg/animal of BMP-2 at 37 °C for 1 h. Sorted ALDHbr cells treated with the same volume of vehicle were used as a control, and then, ALDHbr cells with BMP-2 or vehicle were subcutaneously injected into NOD/SCID mice. After the mice were euthanized, radiographs were obtained using Faxitron X-ray unit (Field Emission Corp., McMinnville, OR). For microcomputed tomography (micro-CT) analysis, specimens were scanned at 8.93 um voxel resolution on a micro-CT scanner (EVS Corp., London, ON, Canada), with a total of 667 slices per scan. GEMS MicroView software (GE Healthcare Biosciences, Piscataway, NJ) was used to make
13
a three-dimensional reconstruction from the set of scans. For histomorphometry, specimens were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) and Masson’s trichrome staining to show collagen type I protein in the newly formed bone. Non-decalcified sections were stained with von Kossa staining to identify calcification during osteogenesis in the tumor. Statistical analysis Data were expressed as mean ± SD. Statistically significant differences were determined by one-way ANOVA and Student’s t test where appropriate, and defined as p
