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Characterization of cervical cancer stem cell-like cells: phenotyping, stemness, and human papilloma virus co-receptor expression

Characterization of cervical cancer stem cell-like cells: phenotyping, stemness, and human... www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 22 Characterization of cervical cancer stem cell-like cells: phenotyping, stemness, and human papilloma virus co-receptor expression 1 1 1 Elizabeth Ortiz-Sánchez , Luz Santiago-López , Verónica B. Cruz-Domínguez , 1 2 1 Mariel E. Toledo-Guzmán , Daniel Hernández-Cueto , Saé Muñiz-Hernández , 3 4 5 Efraín Garrido , David Cantú De León and Alejandro García-Carrancá Subdirección de Investigación Básica, Instituto Nacional de Cancerología, Secretaría de Salud (SS), México City, Mexico Laboratorio de Marcadores Moleculares, Hospital Infantil de México “Federico Gómez”, SA, Mexico City, Mexico Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, Mexico Subdirección de Investigación Clínica, Instituto Nacional de Cancerología, Secretaría de Salud (SS), México City, Mexico Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM) and Instituto Nacional de Cancerología, Secretaría de Salud (SS), Mexico City, Mexico Correspondence to: Elizabeth Ortiz-Sánchez, email: elinfkb@yahoo.com.mx Correspondence to: Alejandro García-Carrancá, email: carranca@biomedicas.unam.mx Keywords: cervical cancer, cervospheres, cervical cancer stem cell phenotype, stemness markers, ALDH activity Received: September 23, 2015 Accepted: March 06, 2016 Published: March 20, 2016 AbstrAct Cancer stem cells (CSC) exhibit high tumorigenic capacity in several tumor models. We have now determined an extended phenotype for cervical cancer stem + + + cells. Our results showed increased CK-17, p63 , AII , CD49f expression in these bright cells, together with higher Aldehyde dehydrogenase (ALDH ) activity in Cervical CSC (CCSC) enriched in cervospheres. An increase in stem cell markers, represented by OCT-4, Nanog, and β-catenin proteins, was also observed, indicating that under our culture conditions, CCSC are enriched in cervospheres, as compared to monolayer bright cultures. In addition, we were able to show that an increased ALDH activity correlated with higher tumorigenic activity. Flow cytometry and immunflorescence assays demonstrated that CCSC in cervosphere cultures contain a sub-population of cells that contain Annexin II, a Human papillomavirus (HPV) co-receptor. Taken together, under our conditions there is an increase in the number of CCSC in + + + cervosphere cultures which exhibit the following phenotype: CK-17, p63 , AII , CD49f and high ALDH activity, which in turn correlates with higher tumorigenicity. The presence of Annexin II and CD49f in CCSC opens the possibility that normal cervical stem cells could be the initial target of infection by high risk HPV. after treatment, a phenomenon that could be explained, in INtrODUctION part, by the hierarchy theory of carcinogenesis, in which only Cancer Stem Cells (CSC) possess the capability Cervical cancer (CC) continues to be an important to promote and support tumor growth [4]. Additionally, human public health problem in developing countries disease relapse could also be a consequence of resistant [1, 2]. There are some ambulatory surgical procedures, cancer cell clonal selection, including stem and/or non- for example, cryosurgery and electro-surgery to remove stem cells, in which the accumulation of mutations in and cure premalignant lesions. However, for high-risk these cells can be associated with the ability to develop premalignant lesions and carcinomas, aggressive protocols anti-cancer therapy resistance resulting in tumor are the therapeutic options for patients, including chemo- progression [5]. These CSC can bypass drug cytotoxicity and radiotherapies (reviewed in [3]). Furthermore, the due to the presence of an efflux pump belonging to the majority of patients with CC exhibit tumor recurrence www.impactjournals.com/oncotarget 31943 Oncotarget Adenosine triphosphate (ATP)-dependent protein family, with Growth Factor Receptor (GFR) and with CD49f such as ABCG2, which has been observed to be increased (α-integrin), we suggest that these CCSC could be infected in several CSC [6]. Additionally, CSC can promote anti- by HPV. Finally, latent HPV infections could be explained apoptotic mechanisms to prevent drug effects. as part of the resting immune system, and if HPV is able to In addition to quiescence or the resting G0 cell- infect reserve cells, these could be “epithelial stem cells” cycle state, CSC share phenotype surface markers with (in our hypothetical model), present in the epithelial basal their normal counterparts [7-9]. However, because normal layer. This infection may be on standby status, in parallel cervical epithelial stem cell markers remain unknown, with the resting state of stem cells, until an event occurs general strategies are suggested to isolate CSC-enriched to initiate the malignant transformation program in order subsets and early progenitors, such as Side population (SP) to generate CCSC. However, we still lack evidence to and Aldehyde dehydrogenase activity (ALDH) assays. confirm that the CCSC can be generated by HPV infection Villanueva and collaborators have reported the presence of a normal epithelial stem cell. of a SP in SiHa and CaLo cervical cancer cell lines, in which these SP cells have shown properties of CSC, such rEsUL ts as the capacity to form colonies in clonogenic assays [10]. In our group, HeLa SP and CD49f (α-integrin) cells were also evaluated in HeLa cervospheres [11]. ALDH activity Phenotype characterization of cervospheres: has also been used to identify CSC. It has been reported putative cervical cancer stem cells that ALDH activity is related to drug detoxification by aldehyde oxidation, which in turn is related to chemo and HeLa (HPV-18), SiHa (HPV-16), Ca Ski (HPV- radioresistance of CSC and to the maintenance of the CSC 16) and C-33 A (HPV-negative) cell lines were cultured population. However, the role of this enzyme in stemness in Mammocult to promote self-renewal of the CCSC remains unknown. There are some reports that demonstrate present in cell lines, as well as to maintain their that ALDH activity is related to an increase of Hypoxia dedifferentiated state. The cervospheres are depicted transcription factor HIF-2α expression. This transcription in Figure 1. Different cervosphere morphologies can factor is related with the expression of OCT-4, a stem cell be observed, which could be related to the differences transcription factor necessary for maintaining a stemness among the cell lines tested. To characterize the cells that state (reviewed in [12]). make up the cervospheres, we analyzed the presence of Indeed, it has been observed that cells with high p63, Cytokeratin-17 (CK-17), and Annexin II (AII). In ALDH activity are able to induce greater tumor growth Figure 2, a discrete increase of p63 protein was observed compared to ALDH-negative subpopulations, thus, in cells from HeLa cervospheres (Mean Florescence high ALDH activity evaluation has been employed to Intensity [MFI] = 20.1±2.5 relative fluorescence units identify and to isolate CSC from several tumors, such [RFU]) compared to their monolayer counterpart cells as ovarian cancer [13], prostate cancer [14], lung cancer (MFI = 8.9±2.0 RFU). It has been demonstrated that p63 [15], breast cancer [16], leukemic stem cell cancer [17], is involved in morphogenesis and has been proposed as a gastrointestinal neuroendocrine tumors [18], head and stem cell marker for the epithelial cervix [21]. However, neck tumors [19], sarcoma [20], and more. under our conditions, the p63 protein did not increase In this work, we established sphere cultures from after SiHa, Ca Ski and C-33 A cervosphere formation as cervical cell lines (denominated cervospheres) utilizing the MFIs in these cases are almost the same (ie: in C-33 the commercial epithelial stem cell sphere conditioned A monolayer MFI = 2.36±1.3 RFU compared to MFI = medium Mammocult to enrich the cervical CSC pool 4.41±1.5 RFU in sphere condition). Additionally, CK-17 through the self-renewal capability of CSC. Using Flow has been suggested as a cervical stem cell marker [21, cytometry (FC), we analyzed some phenotype stem-cell 22]. The majority of our monolayer cells are CK-17 markers such as cytokeratine 17 (CK-17), p63 (a homolog (except C-33 A); however, there was an increase of CK- of p53 related with embryogenesis), and Annexin II (AII), 17 detection in all cervosphere cells tested, which could be a protein characterized as a HPV co-receptor. We also related with CCSC-like enrichment. Interestingly, similar demonstrated that our cervospheres showed a stemness to CD49f expression in cervical stem cell-like cells, tested state characterized by the presence of OCT-4 and Nanog previously [11], AII is another HPV co-receptor found to transcription factors. be increased in HPV-infected cervosphere cells but less in Taken together, we demonstrated, to our knowledge + + + C-33 A cells, a negative HPV CC cell line and in HaCaT for the first time, that the profile of CD49 AII p63 , + bright cells, a non-tumorigenic immortalized cell line. CK-17 and ALDH activity can be considered as a phenotype of putative Cervical cancer stem cells (CCSC). Additionally, because we demonstrated that these cells express AII, a co-receptor necessary for Human papillomavirus (HPV) entry into cells together www.impactjournals.com/oncotarget 31944 Oncotarget t able 1: t umorigenic capability of cervical cancer stem cell-like cells in nu/nu mice. HeLa siHa # cells Monolayer Sphere ALDH+ ALDH- Monolayer Sphere ALDH+ ALDH- 1X10 0/5 0/5 3/5 0/5 0/5 0/5 4/5 0/5 1x10 0/5 5/5 5/5 0/5 0/5 4/5* 5/5 0/5 1X10 0/5** 5/5*** NT NT 0/5 5/5** NT NT bright ALDH+ (ALDH cells: high ADLH activity) low ALDH- (ALDH cells: poor ALDH activity) NT: not tested * Tumor growing (≈ 0.5 cm) was observed 30 days after inoculation. ** Tumor growing (≈ 0.5 cm) was observed in 1/5 mice 60 days after inoculation. *** Tumor growing (≈ 1 cm) was observed 21 days after inoculation. stemness markers are present in cervospheres, a that HeLa monolayer cells lack OCT-4 messenger RNA (mRNA) and protein expression [23]. However, this tiny cell culture enriched in cervical stem cells increase could be due to the sphere stem cell-like tissue culture condition, in that we also were able to detect Transcription factors OCT-4 and Nanog are OCT-4 mRNA in HeLa cervospheres by Q-RT-PCR conventional markers used to demonstrate cell stemness. (Supplementary Figure 1). Figure 3 shows that there is an increase of Nanog protein Since the Wnt/β-catenin cell signaling pathway detected in HeLa and SiHa cervospheres, compared with increases Nanog expression [24], we evaluated β-catenin their monolayer cells. In the case of OCT-4 protein, its protein in the cervospheres. Figure 3 shows that β-catenin increase was clearly detected in SiHa cervospheres, protein is clearly increased in SiHa cervospheres and there was a discreet increase of OCT-4 detected in compared to monolayer counterpart cells (MFI = 17.4±2.3 HeLa cervosphere cells. This is an expected result as data and 6.4±1.4 RFU, respectively). In addition, β-catenin was published by the Schöler group in 2008, demonstrated Figure 1: Morphological differences between cervospheres enriched with cancer stem cells derived from human cervical cancer cell lines. Optical microscopy images show the morphological differences of cervospheres derived from HeLa A. SiHa b. Ca Ski c. and C-33 A D. cell lines cultured in Mammocult serum-free media under tissue culture conditions for 7 days using a 40X objective (Olympus CK31 microscope). www.impactjournals.com/oncotarget 31945 Oncotarget also increased to a lesser extended in HeLa cervospheres ALDH activity was increased in cervospheres, compared to HeLa cells cultured under monolayer tissue another cervical stem cell-like property and culture conditions (MFI = 28±1.1 and 22.6±1.3 RFU, phenotype respectively). The teratocarcinoma NCCIT cell line was used as positive control for stemness markers. In addition to evaluating putative stem cell markers such as p63, CK-17, and AII proteins, we determined ALDH activity in these cells. Figure 4 shows an increase of Aldehyde dehydrogenase (ALDH) activity in HeLa and SiHa cervosphere cells, indicated by the percentage of cells that showed intensification of fluorescence intensity Figure 2: Putative cervical cancer stem cell phenotype present in cervospheres. Monolayer (pink line) and cervosphere cells (green line) derived from HeLa, SiHa, Ca Ski, C-33 A and HaCat cell lines were incubated with specific antibodies to detect p63, CK-17, AII and CD49f proteins, using flow cytometry. Dark lines show isotope control. HaCat cell line was used as non-tumorigenic cells control and HaCat cells do not form spheres in our cell culture conditions. Data is representative of at least three independent experiments. Ten thousand cells are recorded for their analysis using the FloJo software. RFU (Relative fluorescence units) www.impactjournals.com/oncotarget 31946 Oncotarget (21.9±1.8 % and 35.8±2.96 %, respectively) compared to and SiHa cervospheres are more tumorigenic compared their monolayer counterparts (5.73±1.1% and 4.24±0.93%, to cells originating from whole spheres (see Table 1), as respectively). tested by in vivo assays. The tumor development after bright Specifically, in SiHa cervospheres, a subpopulation challenge with 10,000 ALDH cells was faster and is clearly shifted to the right and two ALDH- positive greater compared with xenotransplants derived from bright low bright subpopulations: ALDH and ALDH are observed. monolayer cells. Actually, SiHa ALDH cell tumors bright bright Interestingly, the ALDH cells derived from HeLa grow faster than HeLa ALDH cell tumors. In contrast, Figure 3: stemness markers are increased in cervosphere cells. Specific OCT-4, Nanog and β-catenin antibodies were used to detect the stemness markers present in the cervospheres enriched in cervical cancer stem cells by flow cytometry. Data are representative of three independent experiments. Pink and green lines represent the assays in monolayer and cervosphere cells, respectively. Black TM ® lines show Isotype controls. Ten thousand cells are recorded in BD FAC Scan and then analyzed in FloJo software. RFU (Relative fluorescence units) www.impactjournals.com/oncotarget 31947 Oncotarget brigth bright HeLa ALDH cell tumors were larger than ALDH DIscUssION tumors derived from SiHa cervospheres (data not shown). Thus, all results demonstrate, once again, that the CC Since we studied cells with stemness and putative stem/tumor initiating cell phenotype includes an increase stem cell markers in cervical cancer cell lines growing in ALDH enzyme activity. in monolayer tissue culture conditions, it is necessary to claim for them the name stemloids, a term used to describe Annexin II, another HPV co-receptor present in proliferating cells with self-renewal capacity [9]. This term emphasizes the absence of quiescence cells (stem) cervosphere cells with high tumorigenic capability in proliferating cell lines. Villanueva and colaborators showed the presence of a G subpopulation of cells Because CD49f is a co-receptor that is increased in (quiescence) in a side population derived from the HeLa cervosphere cells, it was important to determine whether cell line, additionally demonstrating their self-renewal these cells derived from SiHa cervopheres showed an capacity as shown by an increase in side population cells increase of HPV AII co-receptor on their surface (Figure after serial sorting assays [10]. We must be careful to use 2). In Figure 5, AII was clearly and specifically detected the term cervical cancer stem cells (CCSC), however, on the surface of cervosphere cells with high tumorigenic since our spheres are cultured without fetal bovine serum capability (Table 1), using confocal microscopy. This (which contains differentiation stimuli) and we using a confocal analysis demonstrated, to our knowledge for the commercial medium for enriching stem cells with EGF first time, that the cervical cancer stem/tumor initiating and bFGF, thus, we suggest that our spheres are enriched cell-like cells contain the AII HPV co-receptor on their for cervical tumorigenic cells and they could named surface, making them target cells for HPV entry and cervical cancer stem cell-like cells, characterized by infection (Figure 5). Moreover, we also evaluated the stemness and cervical stem markers. Therefore, in this presence of p63, CK-17, and AII proteins on cells derived work we propose an extended putative CCSC phenotype. from biopsies of patients with CC. Interestingly, in a tissue In addition to CD44 and CD133, previously studied sample of a patient with a benign lesion, AII protein was by other authors under different models, we include the undetected (Supplementary Figure 2). detection of CD49f, CK-17, p63 and AII proteins as putative CCSC markers in enriched cultures. Mazuko and collaborators demonstrated, in vivo, a protective Figure 4: ALDH activity is increased in cervical cancer stem cell enriched cultures. The ALDEFLUOR kit was used to evaluate the percentage of cells derived from HeLa and SiHa cervospheres compared to their monolayer counterparts. Dot blots are TM ® representative of at least three independent assays. Ten thousand cells are recorded in BD FAC-Scan and then analyzed in FloJo software. www.impactjournals.com/oncotarget 31948 Oncotarget effect of inhibiting the stem cell receptor CD44RI using suggesting that our cervospheres are a CCSC enriched an antibody against this isoform, in mice previously culture compared to monolayer cells. challenged with 1x10 cells from ME180, a human Initially, in the sphere formation assay, we can cervical cancer cell line [25]. Furthermore, Feng and observe that the four cervical cell lines tested possess the collaborators evaluated the presence of CD44 and CK-17 capacity to form cervospheres. However, these spheres in tumorigenic cervospheres injected into mice challenged show, to a certain degree, different morphology, size, with 100,000 total cervosphere cells [26]. In contrast, and compactness (Figure 1). It was clearly shown that the tumorigenicity of cervospheres grown under our Ca Ski cervospheres are more compact compared with conditions show tumor growth capability using 10,000 other cervospheres. Furthermore, HeLa, SiHa, and C-33 cells, a small amount compared to previous reports, A cervospheres demonstrated a relaxed morphology under Figure 5: Annexin II HPV co-receptor is expressed on the cell surface of cervosphere cells. SiHa cervospheres were fixed and paraffin embedded. Four µm sections were incubated with specific AII antibodies (green). Confocal images were taken under 100 X objectives. A. Nomarski image, b. Annexin II detection (green color) and c. Merge. Axiovision Image was used for image acquisition. Scale bars, 10 µm. www.impactjournals.com/oncotarget 31949 Oncotarget our conditions. These disparate morphologies could be CD44 expression and ALDH activity. In this work, these related with their different cell line origin. Thus, HeLa are proteins were also assessed with CD49f and Annexin II derived from HPV-18 infected adenocarcinoma, Ca Ski protein detection. and SiHa are HPV-16 infected squamous carcinoma, and The first contact between HPV and the host cell is C-33 A is a HPV-negative carcinoma. By testing HeLa initiated by heparan sulfates and glycosaminoglycans. and SiHa cervospheres using a cell line passage number On the surface, after HPV interaction with CD49f (an less than 15, we observed discrete morphological changes. alpha-integrin), a subsequent HPV Annexin II co-receptor Furthermore, the cervosphere compactness could be cell is required for full virus-cell interaction. Furthermore, line origin associated as well (data not shown). HPV is coated with Clathrin, caveolin, and cholesterol to In our cervospheres, we demonstrated that there engender dynamic endocytosis. In the cytosol, the viral are increases in p63, CK-17, and Annexin II (AII) genome is released to continue the viral cell cycle [35]. In proteins in cells belonging to HeLa, SiHa, Ca Ski, and this work, we demonstrated the presence of both HPV co- C-33 A cervospheres compared with their monolayer receptors in the subpopulation enriched with CCSC-like counterparts (Figure 2). The p63 and CK-17 proteins cells. First, we observed a vast presence of CD49f in cells have been considered putative normal cervical stem cell cultured under non-stem cell conditions in the presence markers [21] due to their role in morphogenesis. P63 is a of fetal bovine serum (FBS) (Figure 2). Furthermore, transcription factor belonging to the p53 family of normal an increase of CD49f positive cells with more surface epithelial stem cells and its role has been demonstrated in expression of CD49f molecules was observed in maintaining the immature epithelial state in endometrium cervospheres, in which the subpopulation of cancer cells, and progenitor cells of breast and cervical epithelia stem cell-like cells is enriched, compared to monolayer [27]. Specifically, ∆Np63 is an isoform that lacks a cultures. This was also confirmed by tumorigenic in p53-like transactivation domain, usually expressed in vivo assays (Table 1). It has been discussed that the poor squamous and glandular epithelial tissues, where it is ability of tumor-sustaining cells can be a consequence of involved in stem cell renewal. Employing a mammary the limited capability of human cancer cells to proliferate stem cell model using serial sphere-formation assays, it in foreign microenvironments (reviewed in [9]. However, bright was demonstrated that Np63 is a key protein related to we observed that a small amount of ALDH cells are self-renewal [28]. Furthermore, it was demonstrated that able to induce tumor growth compared to monolayer p63 is also implicated in the Sonic-Hg signaling pathway, cells, indicating that these cells adapted in the nude mice in a manner that is likely stemness-responsible, through microenvironment. the induction of Bmi-1, a protein necessary for stem-cell Interestingly, in addition to the increase of CD49f, proliferation [28-30]. we observed a clear increase of Annexin II (AII), the Additionally, we observed an increase in cytokeratin other HPV co-receptor, in the CCSC-like enriched 17, which has also been implicated as an epithelial stem sphere cultures from SiHa, Ca Ski (HPV16), and HeLa cell marker [21]. Using immunohistochemical analysis, (HPV18) cells, in comparison to monolayer cultures CK-17 was detected in a small number of cells located (Figure 2). In contrast, the presence of AII in C-33 A cells in the basal layer, additionally, CK-17 expression was was less compared to the other cancer cells. These data increased in parallel with high, premalignant and cancer were confirmed by confocal images which illustrate the lesions. Remarkably, CK-17 negative premalignant presence of AII HPV co-receptor on the surface of the specimens were obtained from patients who did not CCSC-like that are enriched in our cervospheres. The progress to CC [31], while patients with CK-17 positive phenotype marker assay was also tested in CC biopsies. cells had a greater probability of progression, suggesting Interestingly, CK-17, p63, and CD49f proteins were its relevance in cervical carcinogenesis. Recently, 1,750 detected in cells derived from tumor biopsies. However, patients with different grades of premalignant and cancer AII protein was present only in malignant cervical lesions lesions were included in a study in which the authors and not in benign lesions, suggesting that AII could be a validated the use of CK-17 positive cells as a poor key co-receptor for HPV cell infection, including cervical prognostic marker [32]. stem cells (Supplementary Figure 2). Martens and collaborators in 2009 suggested the In addition to FC assays, the presence of HPV AII presence of two subpopulations of reserve cells related to receptor on the surface of CCSC-like cells was also shown the expression of p63 and CK-17. CK-17 and p63positive by immunofluorescence, suggesting that normal cervical cells are progenitor cells that give rise to endo- and ecto- cells, as part of the reserve cells of the cervical epithelium, cervical epithelial cells. In contrast, CK-17 negative cells are able to be infected by HPV (Figure 5). However, we are reserve cells from which only endocervical epithelial are unable to suggest that HPV infection converts normal cells arise [33]. CK-17 positive cells were also detected cervical stem cells into CC stem cells. in immature squamous metaplasia [34]. Therefore, our In order to characterize the cells that make up data make a contribution of putative CCSC markers for the cervospheres tested, stemness markers were also future studies, enhancing previous publications studying detected by FC assay. For the specific cases of HeLa and www.impactjournals.com/oncotarget 31950 Oncotarget SiHa cervospheres, an increase of transcription factors the origin of the cervical cancer and maintaining tumor OCT-4 and Nanog was detected. We suggest that the growth. disparate-detection level of these stemness markers in HeLa and SiHa cells can be related to their different cell MAtErIALs AND MEtHODs origin (deriving from simple and squamous epithelia, respectively) (Figure 3). The β-catenin protein is also related to the self-renewal capacity of stem cells due t issue culture to its role in the Wnt/β-catenin cell signaling pathway. The target genes of β-catenin are involved in OCT-4 Human CC cell lines, HeLa (adenocarcinoma, HPV- transcription, a key protein for the transcription of Nanog 18) and SiHa (squamous cell carcinoma, HPV-16) were and SOX-2 required for self-renewal and for maintaining purchased from ATCC (American Type Culture Collection, the undifferentiated state. It is interesting to observe that Manassas, VA, USA). Ca Ski (epidermoid carcinoma the greatest total β-catenin detected correlated with the HPV-16), and C-33 A (carcinoma, HPV-negative) cervical higher OCT-4 protein in SiHa cervosphere cells compared cell lines and the NCCIT teratocarcinoma cell line to HeLa cervosphere cells (Figure 3). were kindly donated by Dr. Alejandro García-Carrancá Continuing with CCSC-like characterization, the (Instituto Nacional de Cancerología, Mexico City), while high activity of the ALDH enzyme employed for isolating the HaCaT (non-tumorigenic skin keratinocyte) cell line cancer stem cells in several tumors was also evaluated in was kindly donated by Dr. Nobert Fusenig. These cell SiHa and HeLa cervospheres, demonstrating an increase bright lines were authenticated by Laboratorio de Diagnóstico in an ALDH subpopulation in this enriched stem cell- Genómico at Instituto Nacional de Medicina Genómica conditioned culture compare to monolayer cell cultures and by University of Colorado Cancer Center. NCCIT (Figure 4). Due to this high ALDH activity, these cells teratocarcinoma cells were used as positive control for were sorted and injected subcutaneously (s.c.) into nude stemness markers. Cells were cultured in DMEM media mice. The in vivo assay demonstrated that cells with high (Life Technologies Corporation, Carlsbad, CA, USA) ALDH activity (Table 1) are more tumorigenic compared dim supplemented with 10% (v/v) FBS, 50 U/mL Penicillin, to ALDH cells, as was also observed by the Liu group and 50 µg/mL Streptomycin. Cells were cultured at 37°C, [36]. This data suggested that CCSC are present in the bright 5% CO . Finally, HeLa and SiHa cell lines under 25-cell ALDH subpopulation, and it is a marker that can be passage were employed for the assays. used for target therapy. Several leading authors in CSC, stress the need for cervospheres specific chemotherapies to eliminate CSC in patients. The conventional chemotherapy kills proliferating cancer cells and not cancer stem cells; there is possible Prior to initiating the sphere culture derived from clone selection of a resistant proliferating cancer cell CC cell lines (cervospheres), monolayer cells must be with enough proliferation capabilities to promote disease found in healthy condition at 70‒80% confluence. These recurrence and kill a patient [5, 9]. A combination therapy cells were harvested, counted, and washed with Phosphate must be designed to eliminate cancer proliferating cells, buffer solution (PBS) to remove the remainder of FBS. therapy-resistant cancer cells and also cancer stem cells. One thousand cells per mL of Mammocult medium (Stem It is known that normal and cancer stem cells are identical Cell Technologies, Vancouver, BC, Canada) were seeded except for their tumorigenic capacity; therefore greater in ultra-low adherence dishes and 6-well plates (Corning, efforts are needed for genomic, epigenetic and proteomic Inc., Corning, NY, USA). Cells were cultured under tissue studies to identify a specific gene, protein or pathway culture conditions for 7 days. Sphere formation was present only in cancer stem cells and not in normal stem monitored daily. cells. In summary, we have suggested an additional Flow cytometry + + + + phenotype for CCSC: CD49f , AII , CK-17 p63 bright ALDH . Furthermore, it is clear that the behavior and/ or phenotype could be different between cancer stem Phenotyping assays cells from adenocarcinomas and those from squamous Cervospheres were collected and placed in a 15- carcinomas. Because HPV-16 and HPV-18 exhibit a mL tube, where they were allowed to remain for 10 min. specific difference in gene expression regulation, we are After some time had elapsed, supernatant was removed able to suggest that the CCSC present in these cultures and the bottom sphere cells were washed with PBS and are also different. Taken together, we demonstrated that collected as previously mentioned. Pelleted cervospheres CCSC-like cells are positive for both HPV receptors, were suspended in PBS and disaggregated by mechanic CD49f and for AII, suggesting that cervical stem cells pipetting. For each primary antibody, 5 × 10 cells were could have been HPV-infected and may be responsible for www.impactjournals.com/oncotarget 31951 Oncotarget incubated with anti-p63, anti-CK-17, Annexin II (AII) the cells were rehydrated in decreasing concentrations (all of these by Santa Cruz Biotechnology, Inc., Dallas, of xylol-ethanol, and antigens were released with citrate TX, USA) in flow buffer (1X PBS, 0.05% BSA) on ice. buffer and washed in PBS. Then, sphere cuts were blocked After 30 min, cells were washed with flow buffer and spun in 1% albumin solution for 30 min at room temperature. down at 500 g (r = 11 cm) for 5 min at room temperature. After washing in PBS, samples were incubated with the Then, cells were incubated with FITC-coupled secondary primary antibody for 2 h, were washed, and were further antibody for 30 min on ice. After some time had elapsed, incubated with the secondary antibody for 1 h (Alexa cells were once again washed in flow buffer and fixed with Fluor 480 or 568 according to the case). Sections were 4% p-formaldehyde in PBS. For CD49f detection, 5 × 10 then washed and laid on Vectashield mounting medium cells were incubated with anti-CD49f-PE (BD Bioscience, (Vector Lab, USA) to be observed under confocal CA, USA) on ice for 30 min. Then, cells were washed microscopy (LSM 5 Carl Zeiss México) under 100X with flow buffer and fixed. Cells were also incubated with objective. Axiovision Image Software was used for isotype controls. Staining cells were read in BD FAC- software. Scan™ (BD Bioscience). At least, ten thousand events were recorded for each flow cytometer measurement. In vivo tumorigenic assays FlowJo software was utilized for analyzing data. BALB/c nu/nu female mice were used in this work stemness markers to test the tumorigenic capacity of CCSC-like enriched cultures. These animals were under 4‒6 weeks of age, Cells from monolayer and cervospheres were and were obtained from the Instituto Nacional de Ciencias collected, washed, and counted. For antibody- treatment Médicas y Nutrición “Salvador Zubirán” (INNSZ) incubation, 5 × 10 cells were permeabilized by incubation (“Salvador Zubirán” National Institute for Medical with methanol for 15 min on ice. Then, cells were washed Sciences and Nutrition). Mice were xenotransplanted with flow buffer and incubated with primary antibody anti- subcutaneously (s.c). with HeLa and SiHa monolayer cells OCT-4, Nanog, and β-catenin. After 30 min of incubation and their respective cervosphere cells at different amounts, on ice, cells were washed and then incubated with the using six mice per group. Additionally, other mice groups secondary antibody FITC-conjugated for an additional were challenged via subcutaneously (s.c) with previously 30 min on ice. Finally, cells were washed and suspended sorted ALDH high-activity cells. Mice were monitored for in 4% p-formaldehyde, end-reading them employing at least 3 months. For subcutaneous tumors in mice, the TM BD FAC-Scan flow cytometry (BD Bioscience). maximal allowable size is 2 cm in diameter (Tumor Policy Then, at least 10,000 events were recorded for each FC for Mice and Rats from Boston University Research measurement. Compliance). ALDH activity statistical analysis ALDH activity was evaluated by using the Data are represented as the mean ± Standard ALDEFLUOR kit (Stem Cell Technologies). Briefly, deviations (SD) of at least three independent experiments. cells were harvested, washed, counted, and suspended The Student t test was used to determine statistical in ALDEFLUOR buffer at a density of 1 × 10 cells/ significance ( p < 0.05) using Microsoft Excel 2011. mL. Cells were incubated with 1.5 µM ALDEFLUOR substrate. One half was co-incubated with the ALDH AcKNOWLEDGMENts inhibitor DEAB. Both conditions were incubated at 37°C in a water bath. After 45 min, cells were spin down and We thank Miriam Guido, Miguel Tapia Ramírez and suspended in ALDEFLUOR buffer. Cells were acquired Carlos Castellanos for their technical assistance in cell TM in a BD FAC-Scan cytometer . 10,000 events were culture, Confocal Microscopy and Flow Cytometry core recorded. For cell sorting, BD FAC-SAria II was used and facilities (IIB-UNAM), respectively. We would also like to cells were recovered under serum replacement to maintain thank Federico Centeno Cruz (INMEGEN) and Elizabeth their viability for in vivo assays. Langley McCarron (INCan) for their critical discussion of this work. Immunofluorescence cONFLIcts OF INtErEst Sphere cells were fixed with 4% formaldehyde, The authors declare no conflict of interest. dehydrated, and paraffin-embedded. Serial cuts of 4 µm were obtained for immunofluorescence assays. 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Characterization of cervical cancer stem cell-like cells: phenotyping, stemness, and human papilloma virus co-receptor expression

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Copyright: © 2016 Ortiz-Sánchez et al.
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1949-2553
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1949-2553
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10.18632/oncotarget.8218
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Abstract

www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 22 Characterization of cervical cancer stem cell-like cells: phenotyping, stemness, and human papilloma virus co-receptor expression 1 1 1 Elizabeth Ortiz-Sánchez , Luz Santiago-López , Verónica B. Cruz-Domínguez , 1 2 1 Mariel E. Toledo-Guzmán , Daniel Hernández-Cueto , Saé Muñiz-Hernández , 3 4 5 Efraín Garrido , David Cantú De León and Alejandro García-Carrancá Subdirección de Investigación Básica, Instituto Nacional de Cancerología, Secretaría de Salud (SS), México City, Mexico Laboratorio de Marcadores Moleculares, Hospital Infantil de México “Federico Gómez”, SA, Mexico City, Mexico Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, Mexico Subdirección de Investigación Clínica, Instituto Nacional de Cancerología, Secretaría de Salud (SS), México City, Mexico Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM) and Instituto Nacional de Cancerología, Secretaría de Salud (SS), Mexico City, Mexico Correspondence to: Elizabeth Ortiz-Sánchez, email: elinfkb@yahoo.com.mx Correspondence to: Alejandro García-Carrancá, email: carranca@biomedicas.unam.mx Keywords: cervical cancer, cervospheres, cervical cancer stem cell phenotype, stemness markers, ALDH activity Received: September 23, 2015 Accepted: March 06, 2016 Published: March 20, 2016 AbstrAct Cancer stem cells (CSC) exhibit high tumorigenic capacity in several tumor models. We have now determined an extended phenotype for cervical cancer stem + + + cells. Our results showed increased CK-17, p63 , AII , CD49f expression in these bright cells, together with higher Aldehyde dehydrogenase (ALDH ) activity in Cervical CSC (CCSC) enriched in cervospheres. An increase in stem cell markers, represented by OCT-4, Nanog, and β-catenin proteins, was also observed, indicating that under our culture conditions, CCSC are enriched in cervospheres, as compared to monolayer bright cultures. In addition, we were able to show that an increased ALDH activity correlated with higher tumorigenic activity. Flow cytometry and immunflorescence assays demonstrated that CCSC in cervosphere cultures contain a sub-population of cells that contain Annexin II, a Human papillomavirus (HPV) co-receptor. Taken together, under our conditions there is an increase in the number of CCSC in + + + cervosphere cultures which exhibit the following phenotype: CK-17, p63 , AII , CD49f and high ALDH activity, which in turn correlates with higher tumorigenicity. The presence of Annexin II and CD49f in CCSC opens the possibility that normal cervical stem cells could be the initial target of infection by high risk HPV. after treatment, a phenomenon that could be explained, in INtrODUctION part, by the hierarchy theory of carcinogenesis, in which only Cancer Stem Cells (CSC) possess the capability Cervical cancer (CC) continues to be an important to promote and support tumor growth [4]. Additionally, human public health problem in developing countries disease relapse could also be a consequence of resistant [1, 2]. There are some ambulatory surgical procedures, cancer cell clonal selection, including stem and/or non- for example, cryosurgery and electro-surgery to remove stem cells, in which the accumulation of mutations in and cure premalignant lesions. However, for high-risk these cells can be associated with the ability to develop premalignant lesions and carcinomas, aggressive protocols anti-cancer therapy resistance resulting in tumor are the therapeutic options for patients, including chemo- progression [5]. These CSC can bypass drug cytotoxicity and radiotherapies (reviewed in [3]). Furthermore, the due to the presence of an efflux pump belonging to the majority of patients with CC exhibit tumor recurrence www.impactjournals.com/oncotarget 31943 Oncotarget Adenosine triphosphate (ATP)-dependent protein family, with Growth Factor Receptor (GFR) and with CD49f such as ABCG2, which has been observed to be increased (α-integrin), we suggest that these CCSC could be infected in several CSC [6]. Additionally, CSC can promote anti- by HPV. Finally, latent HPV infections could be explained apoptotic mechanisms to prevent drug effects. as part of the resting immune system, and if HPV is able to In addition to quiescence or the resting G0 cell- infect reserve cells, these could be “epithelial stem cells” cycle state, CSC share phenotype surface markers with (in our hypothetical model), present in the epithelial basal their normal counterparts [7-9]. However, because normal layer. This infection may be on standby status, in parallel cervical epithelial stem cell markers remain unknown, with the resting state of stem cells, until an event occurs general strategies are suggested to isolate CSC-enriched to initiate the malignant transformation program in order subsets and early progenitors, such as Side population (SP) to generate CCSC. However, we still lack evidence to and Aldehyde dehydrogenase activity (ALDH) assays. confirm that the CCSC can be generated by HPV infection Villanueva and collaborators have reported the presence of a normal epithelial stem cell. of a SP in SiHa and CaLo cervical cancer cell lines, in which these SP cells have shown properties of CSC, such rEsUL ts as the capacity to form colonies in clonogenic assays [10]. In our group, HeLa SP and CD49f (α-integrin) cells were also evaluated in HeLa cervospheres [11]. ALDH activity Phenotype characterization of cervospheres: has also been used to identify CSC. It has been reported putative cervical cancer stem cells that ALDH activity is related to drug detoxification by aldehyde oxidation, which in turn is related to chemo and HeLa (HPV-18), SiHa (HPV-16), Ca Ski (HPV- radioresistance of CSC and to the maintenance of the CSC 16) and C-33 A (HPV-negative) cell lines were cultured population. However, the role of this enzyme in stemness in Mammocult to promote self-renewal of the CCSC remains unknown. There are some reports that demonstrate present in cell lines, as well as to maintain their that ALDH activity is related to an increase of Hypoxia dedifferentiated state. The cervospheres are depicted transcription factor HIF-2α expression. This transcription in Figure 1. Different cervosphere morphologies can factor is related with the expression of OCT-4, a stem cell be observed, which could be related to the differences transcription factor necessary for maintaining a stemness among the cell lines tested. To characterize the cells that state (reviewed in [12]). make up the cervospheres, we analyzed the presence of Indeed, it has been observed that cells with high p63, Cytokeratin-17 (CK-17), and Annexin II (AII). In ALDH activity are able to induce greater tumor growth Figure 2, a discrete increase of p63 protein was observed compared to ALDH-negative subpopulations, thus, in cells from HeLa cervospheres (Mean Florescence high ALDH activity evaluation has been employed to Intensity [MFI] = 20.1±2.5 relative fluorescence units identify and to isolate CSC from several tumors, such [RFU]) compared to their monolayer counterpart cells as ovarian cancer [13], prostate cancer [14], lung cancer (MFI = 8.9±2.0 RFU). It has been demonstrated that p63 [15], breast cancer [16], leukemic stem cell cancer [17], is involved in morphogenesis and has been proposed as a gastrointestinal neuroendocrine tumors [18], head and stem cell marker for the epithelial cervix [21]. However, neck tumors [19], sarcoma [20], and more. under our conditions, the p63 protein did not increase In this work, we established sphere cultures from after SiHa, Ca Ski and C-33 A cervosphere formation as cervical cell lines (denominated cervospheres) utilizing the MFIs in these cases are almost the same (ie: in C-33 the commercial epithelial stem cell sphere conditioned A monolayer MFI = 2.36±1.3 RFU compared to MFI = medium Mammocult to enrich the cervical CSC pool 4.41±1.5 RFU in sphere condition). Additionally, CK-17 through the self-renewal capability of CSC. Using Flow has been suggested as a cervical stem cell marker [21, cytometry (FC), we analyzed some phenotype stem-cell 22]. The majority of our monolayer cells are CK-17 markers such as cytokeratine 17 (CK-17), p63 (a homolog (except C-33 A); however, there was an increase of CK- of p53 related with embryogenesis), and Annexin II (AII), 17 detection in all cervosphere cells tested, which could be a protein characterized as a HPV co-receptor. We also related with CCSC-like enrichment. Interestingly, similar demonstrated that our cervospheres showed a stemness to CD49f expression in cervical stem cell-like cells, tested state characterized by the presence of OCT-4 and Nanog previously [11], AII is another HPV co-receptor found to transcription factors. be increased in HPV-infected cervosphere cells but less in Taken together, we demonstrated, to our knowledge + + + C-33 A cells, a negative HPV CC cell line and in HaCaT for the first time, that the profile of CD49 AII p63 , + bright cells, a non-tumorigenic immortalized cell line. CK-17 and ALDH activity can be considered as a phenotype of putative Cervical cancer stem cells (CCSC). Additionally, because we demonstrated that these cells express AII, a co-receptor necessary for Human papillomavirus (HPV) entry into cells together www.impactjournals.com/oncotarget 31944 Oncotarget t able 1: t umorigenic capability of cervical cancer stem cell-like cells in nu/nu mice. HeLa siHa # cells Monolayer Sphere ALDH+ ALDH- Monolayer Sphere ALDH+ ALDH- 1X10 0/5 0/5 3/5 0/5 0/5 0/5 4/5 0/5 1x10 0/5 5/5 5/5 0/5 0/5 4/5* 5/5 0/5 1X10 0/5** 5/5*** NT NT 0/5 5/5** NT NT bright ALDH+ (ALDH cells: high ADLH activity) low ALDH- (ALDH cells: poor ALDH activity) NT: not tested * Tumor growing (≈ 0.5 cm) was observed 30 days after inoculation. ** Tumor growing (≈ 0.5 cm) was observed in 1/5 mice 60 days after inoculation. *** Tumor growing (≈ 1 cm) was observed 21 days after inoculation. stemness markers are present in cervospheres, a that HeLa monolayer cells lack OCT-4 messenger RNA (mRNA) and protein expression [23]. However, this tiny cell culture enriched in cervical stem cells increase could be due to the sphere stem cell-like tissue culture condition, in that we also were able to detect Transcription factors OCT-4 and Nanog are OCT-4 mRNA in HeLa cervospheres by Q-RT-PCR conventional markers used to demonstrate cell stemness. (Supplementary Figure 1). Figure 3 shows that there is an increase of Nanog protein Since the Wnt/β-catenin cell signaling pathway detected in HeLa and SiHa cervospheres, compared with increases Nanog expression [24], we evaluated β-catenin their monolayer cells. In the case of OCT-4 protein, its protein in the cervospheres. Figure 3 shows that β-catenin increase was clearly detected in SiHa cervospheres, protein is clearly increased in SiHa cervospheres and there was a discreet increase of OCT-4 detected in compared to monolayer counterpart cells (MFI = 17.4±2.3 HeLa cervosphere cells. This is an expected result as data and 6.4±1.4 RFU, respectively). In addition, β-catenin was published by the Schöler group in 2008, demonstrated Figure 1: Morphological differences between cervospheres enriched with cancer stem cells derived from human cervical cancer cell lines. Optical microscopy images show the morphological differences of cervospheres derived from HeLa A. SiHa b. Ca Ski c. and C-33 A D. cell lines cultured in Mammocult serum-free media under tissue culture conditions for 7 days using a 40X objective (Olympus CK31 microscope). www.impactjournals.com/oncotarget 31945 Oncotarget also increased to a lesser extended in HeLa cervospheres ALDH activity was increased in cervospheres, compared to HeLa cells cultured under monolayer tissue another cervical stem cell-like property and culture conditions (MFI = 28±1.1 and 22.6±1.3 RFU, phenotype respectively). The teratocarcinoma NCCIT cell line was used as positive control for stemness markers. In addition to evaluating putative stem cell markers such as p63, CK-17, and AII proteins, we determined ALDH activity in these cells. Figure 4 shows an increase of Aldehyde dehydrogenase (ALDH) activity in HeLa and SiHa cervosphere cells, indicated by the percentage of cells that showed intensification of fluorescence intensity Figure 2: Putative cervical cancer stem cell phenotype present in cervospheres. Monolayer (pink line) and cervosphere cells (green line) derived from HeLa, SiHa, Ca Ski, C-33 A and HaCat cell lines were incubated with specific antibodies to detect p63, CK-17, AII and CD49f proteins, using flow cytometry. Dark lines show isotope control. HaCat cell line was used as non-tumorigenic cells control and HaCat cells do not form spheres in our cell culture conditions. Data is representative of at least three independent experiments. Ten thousand cells are recorded for their analysis using the FloJo software. RFU (Relative fluorescence units) www.impactjournals.com/oncotarget 31946 Oncotarget (21.9±1.8 % and 35.8±2.96 %, respectively) compared to and SiHa cervospheres are more tumorigenic compared their monolayer counterparts (5.73±1.1% and 4.24±0.93%, to cells originating from whole spheres (see Table 1), as respectively). tested by in vivo assays. The tumor development after bright Specifically, in SiHa cervospheres, a subpopulation challenge with 10,000 ALDH cells was faster and is clearly shifted to the right and two ALDH- positive greater compared with xenotransplants derived from bright low bright subpopulations: ALDH and ALDH are observed. monolayer cells. Actually, SiHa ALDH cell tumors bright bright Interestingly, the ALDH cells derived from HeLa grow faster than HeLa ALDH cell tumors. In contrast, Figure 3: stemness markers are increased in cervosphere cells. Specific OCT-4, Nanog and β-catenin antibodies were used to detect the stemness markers present in the cervospheres enriched in cervical cancer stem cells by flow cytometry. Data are representative of three independent experiments. Pink and green lines represent the assays in monolayer and cervosphere cells, respectively. Black TM ® lines show Isotype controls. Ten thousand cells are recorded in BD FAC Scan and then analyzed in FloJo software. RFU (Relative fluorescence units) www.impactjournals.com/oncotarget 31947 Oncotarget brigth bright HeLa ALDH cell tumors were larger than ALDH DIscUssION tumors derived from SiHa cervospheres (data not shown). Thus, all results demonstrate, once again, that the CC Since we studied cells with stemness and putative stem/tumor initiating cell phenotype includes an increase stem cell markers in cervical cancer cell lines growing in ALDH enzyme activity. in monolayer tissue culture conditions, it is necessary to claim for them the name stemloids, a term used to describe Annexin II, another HPV co-receptor present in proliferating cells with self-renewal capacity [9]. This term emphasizes the absence of quiescence cells (stem) cervosphere cells with high tumorigenic capability in proliferating cell lines. Villanueva and colaborators showed the presence of a G subpopulation of cells Because CD49f is a co-receptor that is increased in (quiescence) in a side population derived from the HeLa cervosphere cells, it was important to determine whether cell line, additionally demonstrating their self-renewal these cells derived from SiHa cervopheres showed an capacity as shown by an increase in side population cells increase of HPV AII co-receptor on their surface (Figure after serial sorting assays [10]. We must be careful to use 2). In Figure 5, AII was clearly and specifically detected the term cervical cancer stem cells (CCSC), however, on the surface of cervosphere cells with high tumorigenic since our spheres are cultured without fetal bovine serum capability (Table 1), using confocal microscopy. This (which contains differentiation stimuli) and we using a confocal analysis demonstrated, to our knowledge for the commercial medium for enriching stem cells with EGF first time, that the cervical cancer stem/tumor initiating and bFGF, thus, we suggest that our spheres are enriched cell-like cells contain the AII HPV co-receptor on their for cervical tumorigenic cells and they could named surface, making them target cells for HPV entry and cervical cancer stem cell-like cells, characterized by infection (Figure 5). Moreover, we also evaluated the stemness and cervical stem markers. Therefore, in this presence of p63, CK-17, and AII proteins on cells derived work we propose an extended putative CCSC phenotype. from biopsies of patients with CC. Interestingly, in a tissue In addition to CD44 and CD133, previously studied sample of a patient with a benign lesion, AII protein was by other authors under different models, we include the undetected (Supplementary Figure 2). detection of CD49f, CK-17, p63 and AII proteins as putative CCSC markers in enriched cultures. Mazuko and collaborators demonstrated, in vivo, a protective Figure 4: ALDH activity is increased in cervical cancer stem cell enriched cultures. The ALDEFLUOR kit was used to evaluate the percentage of cells derived from HeLa and SiHa cervospheres compared to their monolayer counterparts. Dot blots are TM ® representative of at least three independent assays. Ten thousand cells are recorded in BD FAC-Scan and then analyzed in FloJo software. www.impactjournals.com/oncotarget 31948 Oncotarget effect of inhibiting the stem cell receptor CD44RI using suggesting that our cervospheres are a CCSC enriched an antibody against this isoform, in mice previously culture compared to monolayer cells. challenged with 1x10 cells from ME180, a human Initially, in the sphere formation assay, we can cervical cancer cell line [25]. Furthermore, Feng and observe that the four cervical cell lines tested possess the collaborators evaluated the presence of CD44 and CK-17 capacity to form cervospheres. However, these spheres in tumorigenic cervospheres injected into mice challenged show, to a certain degree, different morphology, size, with 100,000 total cervosphere cells [26]. In contrast, and compactness (Figure 1). It was clearly shown that the tumorigenicity of cervospheres grown under our Ca Ski cervospheres are more compact compared with conditions show tumor growth capability using 10,000 other cervospheres. Furthermore, HeLa, SiHa, and C-33 cells, a small amount compared to previous reports, A cervospheres demonstrated a relaxed morphology under Figure 5: Annexin II HPV co-receptor is expressed on the cell surface of cervosphere cells. SiHa cervospheres were fixed and paraffin embedded. Four µm sections were incubated with specific AII antibodies (green). Confocal images were taken under 100 X objectives. A. Nomarski image, b. Annexin II detection (green color) and c. Merge. Axiovision Image was used for image acquisition. Scale bars, 10 µm. www.impactjournals.com/oncotarget 31949 Oncotarget our conditions. These disparate morphologies could be CD44 expression and ALDH activity. In this work, these related with their different cell line origin. Thus, HeLa are proteins were also assessed with CD49f and Annexin II derived from HPV-18 infected adenocarcinoma, Ca Ski protein detection. and SiHa are HPV-16 infected squamous carcinoma, and The first contact between HPV and the host cell is C-33 A is a HPV-negative carcinoma. By testing HeLa initiated by heparan sulfates and glycosaminoglycans. and SiHa cervospheres using a cell line passage number On the surface, after HPV interaction with CD49f (an less than 15, we observed discrete morphological changes. alpha-integrin), a subsequent HPV Annexin II co-receptor Furthermore, the cervosphere compactness could be cell is required for full virus-cell interaction. Furthermore, line origin associated as well (data not shown). HPV is coated with Clathrin, caveolin, and cholesterol to In our cervospheres, we demonstrated that there engender dynamic endocytosis. In the cytosol, the viral are increases in p63, CK-17, and Annexin II (AII) genome is released to continue the viral cell cycle [35]. In proteins in cells belonging to HeLa, SiHa, Ca Ski, and this work, we demonstrated the presence of both HPV co- C-33 A cervospheres compared with their monolayer receptors in the subpopulation enriched with CCSC-like counterparts (Figure 2). The p63 and CK-17 proteins cells. First, we observed a vast presence of CD49f in cells have been considered putative normal cervical stem cell cultured under non-stem cell conditions in the presence markers [21] due to their role in morphogenesis. P63 is a of fetal bovine serum (FBS) (Figure 2). Furthermore, transcription factor belonging to the p53 family of normal an increase of CD49f positive cells with more surface epithelial stem cells and its role has been demonstrated in expression of CD49f molecules was observed in maintaining the immature epithelial state in endometrium cervospheres, in which the subpopulation of cancer cells, and progenitor cells of breast and cervical epithelia stem cell-like cells is enriched, compared to monolayer [27]. Specifically, ∆Np63 is an isoform that lacks a cultures. This was also confirmed by tumorigenic in p53-like transactivation domain, usually expressed in vivo assays (Table 1). It has been discussed that the poor squamous and glandular epithelial tissues, where it is ability of tumor-sustaining cells can be a consequence of involved in stem cell renewal. Employing a mammary the limited capability of human cancer cells to proliferate stem cell model using serial sphere-formation assays, it in foreign microenvironments (reviewed in [9]. However, bright was demonstrated that Np63 is a key protein related to we observed that a small amount of ALDH cells are self-renewal [28]. Furthermore, it was demonstrated that able to induce tumor growth compared to monolayer p63 is also implicated in the Sonic-Hg signaling pathway, cells, indicating that these cells adapted in the nude mice in a manner that is likely stemness-responsible, through microenvironment. the induction of Bmi-1, a protein necessary for stem-cell Interestingly, in addition to the increase of CD49f, proliferation [28-30]. we observed a clear increase of Annexin II (AII), the Additionally, we observed an increase in cytokeratin other HPV co-receptor, in the CCSC-like enriched 17, which has also been implicated as an epithelial stem sphere cultures from SiHa, Ca Ski (HPV16), and HeLa cell marker [21]. Using immunohistochemical analysis, (HPV18) cells, in comparison to monolayer cultures CK-17 was detected in a small number of cells located (Figure 2). In contrast, the presence of AII in C-33 A cells in the basal layer, additionally, CK-17 expression was was less compared to the other cancer cells. These data increased in parallel with high, premalignant and cancer were confirmed by confocal images which illustrate the lesions. Remarkably, CK-17 negative premalignant presence of AII HPV co-receptor on the surface of the specimens were obtained from patients who did not CCSC-like that are enriched in our cervospheres. The progress to CC [31], while patients with CK-17 positive phenotype marker assay was also tested in CC biopsies. cells had a greater probability of progression, suggesting Interestingly, CK-17, p63, and CD49f proteins were its relevance in cervical carcinogenesis. Recently, 1,750 detected in cells derived from tumor biopsies. However, patients with different grades of premalignant and cancer AII protein was present only in malignant cervical lesions lesions were included in a study in which the authors and not in benign lesions, suggesting that AII could be a validated the use of CK-17 positive cells as a poor key co-receptor for HPV cell infection, including cervical prognostic marker [32]. stem cells (Supplementary Figure 2). Martens and collaborators in 2009 suggested the In addition to FC assays, the presence of HPV AII presence of two subpopulations of reserve cells related to receptor on the surface of CCSC-like cells was also shown the expression of p63 and CK-17. CK-17 and p63positive by immunofluorescence, suggesting that normal cervical cells are progenitor cells that give rise to endo- and ecto- cells, as part of the reserve cells of the cervical epithelium, cervical epithelial cells. In contrast, CK-17 negative cells are able to be infected by HPV (Figure 5). However, we are reserve cells from which only endocervical epithelial are unable to suggest that HPV infection converts normal cells arise [33]. CK-17 positive cells were also detected cervical stem cells into CC stem cells. in immature squamous metaplasia [34]. Therefore, our In order to characterize the cells that make up data make a contribution of putative CCSC markers for the cervospheres tested, stemness markers were also future studies, enhancing previous publications studying detected by FC assay. For the specific cases of HeLa and www.impactjournals.com/oncotarget 31950 Oncotarget SiHa cervospheres, an increase of transcription factors the origin of the cervical cancer and maintaining tumor OCT-4 and Nanog was detected. We suggest that the growth. disparate-detection level of these stemness markers in HeLa and SiHa cells can be related to their different cell MAtErIALs AND MEtHODs origin (deriving from simple and squamous epithelia, respectively) (Figure 3). The β-catenin protein is also related to the self-renewal capacity of stem cells due t issue culture to its role in the Wnt/β-catenin cell signaling pathway. The target genes of β-catenin are involved in OCT-4 Human CC cell lines, HeLa (adenocarcinoma, HPV- transcription, a key protein for the transcription of Nanog 18) and SiHa (squamous cell carcinoma, HPV-16) were and SOX-2 required for self-renewal and for maintaining purchased from ATCC (American Type Culture Collection, the undifferentiated state. It is interesting to observe that Manassas, VA, USA). Ca Ski (epidermoid carcinoma the greatest total β-catenin detected correlated with the HPV-16), and C-33 A (carcinoma, HPV-negative) cervical higher OCT-4 protein in SiHa cervosphere cells compared cell lines and the NCCIT teratocarcinoma cell line to HeLa cervosphere cells (Figure 3). were kindly donated by Dr. Alejandro García-Carrancá Continuing with CCSC-like characterization, the (Instituto Nacional de Cancerología, Mexico City), while high activity of the ALDH enzyme employed for isolating the HaCaT (non-tumorigenic skin keratinocyte) cell line cancer stem cells in several tumors was also evaluated in was kindly donated by Dr. Nobert Fusenig. These cell SiHa and HeLa cervospheres, demonstrating an increase bright lines were authenticated by Laboratorio de Diagnóstico in an ALDH subpopulation in this enriched stem cell- Genómico at Instituto Nacional de Medicina Genómica conditioned culture compare to monolayer cell cultures and by University of Colorado Cancer Center. NCCIT (Figure 4). Due to this high ALDH activity, these cells teratocarcinoma cells were used as positive control for were sorted and injected subcutaneously (s.c.) into nude stemness markers. Cells were cultured in DMEM media mice. The in vivo assay demonstrated that cells with high (Life Technologies Corporation, Carlsbad, CA, USA) ALDH activity (Table 1) are more tumorigenic compared dim supplemented with 10% (v/v) FBS, 50 U/mL Penicillin, to ALDH cells, as was also observed by the Liu group and 50 µg/mL Streptomycin. Cells were cultured at 37°C, [36]. This data suggested that CCSC are present in the bright 5% CO . Finally, HeLa and SiHa cell lines under 25-cell ALDH subpopulation, and it is a marker that can be passage were employed for the assays. used for target therapy. Several leading authors in CSC, stress the need for cervospheres specific chemotherapies to eliminate CSC in patients. The conventional chemotherapy kills proliferating cancer cells and not cancer stem cells; there is possible Prior to initiating the sphere culture derived from clone selection of a resistant proliferating cancer cell CC cell lines (cervospheres), monolayer cells must be with enough proliferation capabilities to promote disease found in healthy condition at 70‒80% confluence. These recurrence and kill a patient [5, 9]. A combination therapy cells were harvested, counted, and washed with Phosphate must be designed to eliminate cancer proliferating cells, buffer solution (PBS) to remove the remainder of FBS. therapy-resistant cancer cells and also cancer stem cells. One thousand cells per mL of Mammocult medium (Stem It is known that normal and cancer stem cells are identical Cell Technologies, Vancouver, BC, Canada) were seeded except for their tumorigenic capacity; therefore greater in ultra-low adherence dishes and 6-well plates (Corning, efforts are needed for genomic, epigenetic and proteomic Inc., Corning, NY, USA). Cells were cultured under tissue studies to identify a specific gene, protein or pathway culture conditions for 7 days. Sphere formation was present only in cancer stem cells and not in normal stem monitored daily. cells. In summary, we have suggested an additional Flow cytometry + + + + phenotype for CCSC: CD49f , AII , CK-17 p63 bright ALDH . Furthermore, it is clear that the behavior and/ or phenotype could be different between cancer stem Phenotyping assays cells from adenocarcinomas and those from squamous Cervospheres were collected and placed in a 15- carcinomas. Because HPV-16 and HPV-18 exhibit a mL tube, where they were allowed to remain for 10 min. specific difference in gene expression regulation, we are After some time had elapsed, supernatant was removed able to suggest that the CCSC present in these cultures and the bottom sphere cells were washed with PBS and are also different. Taken together, we demonstrated that collected as previously mentioned. Pelleted cervospheres CCSC-like cells are positive for both HPV receptors, were suspended in PBS and disaggregated by mechanic CD49f and for AII, suggesting that cervical stem cells pipetting. For each primary antibody, 5 × 10 cells were could have been HPV-infected and may be responsible for www.impactjournals.com/oncotarget 31951 Oncotarget incubated with anti-p63, anti-CK-17, Annexin II (AII) the cells were rehydrated in decreasing concentrations (all of these by Santa Cruz Biotechnology, Inc., Dallas, of xylol-ethanol, and antigens were released with citrate TX, USA) in flow buffer (1X PBS, 0.05% BSA) on ice. buffer and washed in PBS. Then, sphere cuts were blocked After 30 min, cells were washed with flow buffer and spun in 1% albumin solution for 30 min at room temperature. down at 500 g (r = 11 cm) for 5 min at room temperature. After washing in PBS, samples were incubated with the Then, cells were incubated with FITC-coupled secondary primary antibody for 2 h, were washed, and were further antibody for 30 min on ice. After some time had elapsed, incubated with the secondary antibody for 1 h (Alexa cells were once again washed in flow buffer and fixed with Fluor 480 or 568 according to the case). Sections were 4% p-formaldehyde in PBS. For CD49f detection, 5 × 10 then washed and laid on Vectashield mounting medium cells were incubated with anti-CD49f-PE (BD Bioscience, (Vector Lab, USA) to be observed under confocal CA, USA) on ice for 30 min. Then, cells were washed microscopy (LSM 5 Carl Zeiss México) under 100X with flow buffer and fixed. Cells were also incubated with objective. Axiovision Image Software was used for isotype controls. Staining cells were read in BD FAC- software. Scan™ (BD Bioscience). At least, ten thousand events were recorded for each flow cytometer measurement. In vivo tumorigenic assays FlowJo software was utilized for analyzing data. BALB/c nu/nu female mice were used in this work stemness markers to test the tumorigenic capacity of CCSC-like enriched cultures. These animals were under 4‒6 weeks of age, Cells from monolayer and cervospheres were and were obtained from the Instituto Nacional de Ciencias collected, washed, and counted. For antibody- treatment Médicas y Nutrición “Salvador Zubirán” (INNSZ) incubation, 5 × 10 cells were permeabilized by incubation (“Salvador Zubirán” National Institute for Medical with methanol for 15 min on ice. Then, cells were washed Sciences and Nutrition). Mice were xenotransplanted with flow buffer and incubated with primary antibody anti- subcutaneously (s.c). with HeLa and SiHa monolayer cells OCT-4, Nanog, and β-catenin. After 30 min of incubation and their respective cervosphere cells at different amounts, on ice, cells were washed and then incubated with the using six mice per group. Additionally, other mice groups secondary antibody FITC-conjugated for an additional were challenged via subcutaneously (s.c) with previously 30 min on ice. Finally, cells were washed and suspended sorted ALDH high-activity cells. Mice were monitored for in 4% p-formaldehyde, end-reading them employing at least 3 months. For subcutaneous tumors in mice, the TM BD FAC-Scan flow cytometry (BD Bioscience). maximal allowable size is 2 cm in diameter (Tumor Policy Then, at least 10,000 events were recorded for each FC for Mice and Rats from Boston University Research measurement. Compliance). ALDH activity statistical analysis ALDH activity was evaluated by using the Data are represented as the mean ± Standard ALDEFLUOR kit (Stem Cell Technologies). Briefly, deviations (SD) of at least three independent experiments. cells were harvested, washed, counted, and suspended The Student t test was used to determine statistical in ALDEFLUOR buffer at a density of 1 × 10 cells/ significance ( p < 0.05) using Microsoft Excel 2011. mL. Cells were incubated with 1.5 µM ALDEFLUOR substrate. One half was co-incubated with the ALDH AcKNOWLEDGMENts inhibitor DEAB. Both conditions were incubated at 37°C in a water bath. After 45 min, cells were spin down and We thank Miriam Guido, Miguel Tapia Ramírez and suspended in ALDEFLUOR buffer. Cells were acquired Carlos Castellanos for their technical assistance in cell TM in a BD FAC-Scan cytometer . 10,000 events were culture, Confocal Microscopy and Flow Cytometry core recorded. For cell sorting, BD FAC-SAria II was used and facilities (IIB-UNAM), respectively. We would also like to cells were recovered under serum replacement to maintain thank Federico Centeno Cruz (INMEGEN) and Elizabeth their viability for in vivo assays. Langley McCarron (INCan) for their critical discussion of this work. Immunofluorescence cONFLIcts OF INtErEst Sphere cells were fixed with 4% formaldehyde, The authors declare no conflict of interest. dehydrated, and paraffin-embedded. Serial cuts of 4 µm were obtained for immunofluorescence assays. 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OncotargetPubmed Central

Published: Mar 20, 2016

References