Original Article
Elevated Intrinsic Cancer Stem Cell Population in Human Papillomavirus-Associated Head and Neck Squamous Cell Carcinoma Manchao Zhang, PhD1,2; Bhavna Kumar, MS1,2; Longzhu Piao, PhD1,2; Xiujie Xie, PhD1,2; Alessandra Schmitt, MD3; Nicole Arradaza, MS4; Michael Cippola, MD1,2; Matthew Old, MD1,2; Amit Agrawal, MD1,2; Enver Ozer, MD1,2; David E. Schuller, MD1,2; Theodoros N. Teknos, MD1,2; and Quintin Pan, PhD1,2
BACKGROUND: Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC), particularly the development of oropharyngeal squamous cell carcinoma (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies, and it is postulated that they are responsible for disease recurrence and=or progression. Because the prognoses of patients with HPV16-positive and HPV-negative HNSCC are distinct, the authors sought to determine whether differences in the number of CSCs could account for this clinical observation. METHODS: CSC populations in HPV16-positive and HPVnegative HNSCC were assessed using a proprietary assay based on expression of the enzyme aldehyde dehydrogenase (ALDH), an in vitro tumorsphere formation assay, and an in vivo limiting cell dilution in nonobese diabetic=severe combined immunodeficiency mice. A high-density tissue microarray was stained with ALDH1, a CSC marker, to determine the association between CSCs and HPV16-positive=HPV-negative OPSCC. RESULTS: HPV16-positive HNSCC had a greater intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 has been identified as a major mechanism for the elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using tumors from patients with HPV16-positive and HPV-negative OPSCC indicated that the CSC frequency was 62.5-fold greater in an HPV16-positive OPSCC tumor than in an HPV-negative OPSCC tumor. Primary tumors from patients with HPV16-positive OPSCC were associated with elevated tumor ALDH1 staining, further extending the association between HPV16 and CSCs. CONCLUSIONS: The current data and the clinical observation that patients with HPV16-positive HNSCC respond more favorably to current treatment paradigms than patients with HPV-negative HNSCC support the suggestion that CSC phenotype is not homogeneous. Therefore, the reliance on the CSC number may be insufficient to accurately assess the potential of a particular C 2014 American Cancer Society. tumor for disease recurrence and=or progression. Cancer 2014;000:000–000. V KEYWORDS: cancer-initiating cells, cancer stem cells, head and neck cancer, human papillomavirus, ALDH1, prognostic biomarker.
INTRODUCTION Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, with an annual incidence of approximately 600,000 cases worldwide.1 Alcohol and tobacco use are the hallmark etiologic factors for HNSCC. However, the pathogenesis of HNSCC is changing because of the recognition that human papillomavirus (HPV) is a major risk factor for the development of HNSCC, in particular oropharyngeal squamous cell carcinoma (OPSCC). High-risk HPV16 is by far the most frequent HPV type detected in HNSCC.2 Epidemiologic data indicate that the prevalence of HPV-associated HNSCC has rapidly increased by about 3-fold in the past 3 decades in the United States and Europe.3-5 In light of these observations, it has been suggested that an epidemic of HPV-positive HNSCC will emerge in the near future.5,6 Cancer stem cells (CSCs) are a small subset of cancer cells within the tumor that have the exclusive capacity to divide and expand the CSC pool or to differentiate into heterogeneous, nontumorigenic cells that constitute the bulk of the tumor. There is emerging evidence that CSCs are refractory to chemotherapy and radiation, suggesting that CSCs may be responsible for disease relapse and progression.7-11 To date, the effect of HPV16 infection on the CSC population is unknown. Our results demonstrate that HPV16-positive OPSCC tumors have a higher intrinsic CSC population than HPV-negative HNSCC tumors. Elevated CSC numbers in HPV16-positive OPSCC are partly because of E6-mediated
Corresponding author: Quintin Pan, PhD, Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Medical Center, 442 Tzagournis Medical Research, 420 West 12th Avenue, Columbus, OH, 43210; Fax: (614) 247-1917; [email protected] 1 Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, Columbus, Ohio; 2Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, Ohio; 3Department of Pathology, The Ohio State University Wexner Medical Center, Columbus, Ohio; 4Center for Biostatistics, The Ohio State University, Columbus, Ohio
DOI: 10.1002/cncr.28538, Received: August 16, 2013; Revised: October 21, 2013; Accepted: November 14, 2013, Published online Month 00, 2013 in Wiley Online Library (wileyonlinelibrary.com)
Cancer
Month 00, 2013
1
Original Article
p53 inactivation. In a large cohort of OPSCC patients, HPV16-positive OPSCC is associated with increased aldehyde dehydrogenase 1 (ALDH1) staining in the tumor and stromal cells. The clinical observation that patients with HPV-positive OPSCC have a better prognosis than those with HPV-negative OPSCC provides initial evidence that the CSC phenotype may be more critical than the CSC number as a determinant of patient outcome. Our work suggests that CSC phenotype is not static and challenges the current dogma of homogeneity in the CSC population. MATERIALS AND METHODS Cell Lines
SCC15 and CAL27 cells were purchased from the American Type Culture Collection (Manassas, Va). UMSCC47 and UMSCC-74A cells were obtained from Dr. Thomas Carey at the University of Michigan. UDSCC2 cells were provided by Dr. Henning Bier at Heinrich-Heine University (Dusseldorf, Germany). UPCI:SCC090 cells were provided by Dr. Susanne Gollin at the University of Pittsburgh. SCC15 cells were grown in a 1:1 mixture of Ham F-12 and Dulbecco Modified Eagle Medium (DMEM) supplemented with 0.4 mg=mL hydrocortisone, 10% fetal bovine serum (FBS), 2 mM Lglutamine, 100 mg=mL streptomycin, and 100 U=mL penicillin. UD-SCC2, CAL27, UMSCC-47, UMSCC74A, and UPCI:SCC090 cells were grown in DMEM containing 10% FBS, 2 mM glutamine, 100 mg=mL streptomycin, and 100 U=mL penicillin. Cell lines were authenticated using short tandem repeat profile analysis. HPV-Negative and HPV16-Positive HNSCC Patient Tumors
Fresh previously untreated, HPV-negative (n 5 3) and HPV16-positive (n 5 3) OPSCC tumors were collected with a protocol approved by the Institutional Review Board at The Ohio State University. Informed consent was obtained from all patients who were included in the study. Tumors were digested in collagenase=hyaluronidase (STEMCELL Technologies Inc., Vancouver, BC, Canada) for 6 to 8 hours with gentle agitation at 30 C, followed by brief trypsinization, then they were filtered through a 40-lM cell strainer. Single cell suspensions were depleted for fibroblasts (Anti-Fibroblast microbeads; Miltenyi Biotec Inc., Auburn, Calif), CD45 cells (Anti-CD45 microbeads; Miltenyi Biotec Inc.), and Lineage cells (Lineage Cell Depletion Kit; Miltenyi Biotec Inc.) to enrich for tumor cells. Enriched HNSCC patient tumor cells were stained with propidium 2
iodide and sorted by fluorescence-activated cell sorting (FACS) to eliminate dead cells. Subsequently, purified HNSCC patient tumor cells were assessed for CSCs using the ALDEFLUOR (STEMCELL Technologies Inc.) and tumorsphere formation efficiency assays. ALDEFLUOR Assay
Enriched HNSCC patient tumor cells and HNSCC cell lines were incubated in buffer containing ALDH substrate (BAAA) in the presence or absence of diethylaminobenzaldehyde, a specific ALDH inhibitor, to serve as the negative control. The sorting gate for CSCs that had high ALDH activity (ALDHhigh) was established using the negative control as the baseline. FACS analyses were performed using BD FACS Calibur (BD Biosciences Corporation, Franklin Lakes, NJ) at The Ohio State University Comprehensive Cancer Center Analytical Cytometry Core. Tumorsphere Formation Assay
Enriched HNSCC patient tumor cells and HNSCC cell lines were seeded on a low-attachment plate in a defined, serum-free culture medium12 at a density of 1000 cells per well. Tumorspheres were allowed to grow for 7 days. Tumorsphere formation efficiency was calculated as the number of tumorspheres formed divided by the original number of cells seeded. Heterotransplantation of OPSCC Nonobese Diabetic=Severe Combined Immunodeficiency Cells
Fresh, previously untreated, HPV-negative (n 5 1) and HPV16-positive (n 5 1) OPSCC tumors were collected with a protocol approved by the Institutional Review Board at The Ohio State University. Informed consent was obtained from all patients who were included in the study. All mouse procedures were approved by the Animal Care and Use Committee at The Ohio State University. HPV-negative and HPV16-positive HNSCC heterotransplantation lines were established by subcutaneous implantation of solid tissue fragments into 6-week-old to 8-week-old nonobese diabetic=severe combined immunodeficiency (NOD=SCID) mice (National Cancer Institute, Bethesda, Md). HNSCC tumors were cut into small fragments and implanted subcutaneously on the dorsal flank of NOD=SCID mice. Recipient NOD=SCID mice were anesthetized with a ketamine-xylazine mixture before tumor implantation. Once tumors reached a volume of approximately 500 mm3, tumors were resected and serially passaged in naive NOD=SCID mice using the same protocol described above. Cancer
Month 00, 2013
HPV16 and Cancer Stem Cells/Zhang et al
Figure 1. The cancer stem cell (CSC) population is elevated in human papillomavirus 16 (HPV16)-associated head and neck squamous cell carcinoma (HNSCC). (a,b) CSCs are measured in tumors from patients with HPV-negative (HPV2) and HPV16-positive (HPV161) oropharyngeal squamous cell carcinoma (OPSCC). (a) Cells with high aldehyde dehydrogenase (ALDH) activity (ALDHhigh) were assessed using the ALDEFLUOR assay (STEMCELL Technologies Inc., Vancouver, BC, Canada). (b) Tumorsphere formation efficiency (TFE) was calculated as the number of tumorspheres formed divided by the initial number of cells seeded. Data are presented as the mean 6 standard error of the mean. An asterisk indicates P
Elevated Intrinsic Cancer Stem Cell Population in Human Papillomavirus-Associated Head and Neck Squamous Cell Carcinoma Manchao Zhang, PhD1,2; Bhavna Kumar, MS1,2; Longzhu Piao, PhD1,2; Xiujie Xie, PhD1,2; Alessandra Schmitt, MD3; Nicole Arradaza, MS4; Michael Cippola, MD1,2; Matthew Old, MD1,2; Amit Agrawal, MD1,2; Enver Ozer, MD1,2; David E. Schuller, MD1,2; Theodoros N. Teknos, MD1,2; and Quintin Pan, PhD1,2
BACKGROUND: Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC), particularly the development of oropharyngeal squamous cell carcinoma (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies, and it is postulated that they are responsible for disease recurrence and=or progression. Because the prognoses of patients with HPV16-positive and HPV-negative HNSCC are distinct, the authors sought to determine whether differences in the number of CSCs could account for this clinical observation. METHODS: CSC populations in HPV16-positive and HPVnegative HNSCC were assessed using a proprietary assay based on expression of the enzyme aldehyde dehydrogenase (ALDH), an in vitro tumorsphere formation assay, and an in vivo limiting cell dilution in nonobese diabetic=severe combined immunodeficiency mice. A high-density tissue microarray was stained with ALDH1, a CSC marker, to determine the association between CSCs and HPV16-positive=HPV-negative OPSCC. RESULTS: HPV16-positive HNSCC had a greater intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 has been identified as a major mechanism for the elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using tumors from patients with HPV16-positive and HPV-negative OPSCC indicated that the CSC frequency was 62.5-fold greater in an HPV16-positive OPSCC tumor than in an HPV-negative OPSCC tumor. Primary tumors from patients with HPV16-positive OPSCC were associated with elevated tumor ALDH1 staining, further extending the association between HPV16 and CSCs. CONCLUSIONS: The current data and the clinical observation that patients with HPV16-positive HNSCC respond more favorably to current treatment paradigms than patients with HPV-negative HNSCC support the suggestion that CSC phenotype is not homogeneous. Therefore, the reliance on the CSC number may be insufficient to accurately assess the potential of a particular C 2014 American Cancer Society. tumor for disease recurrence and=or progression. Cancer 2014;000:000–000. V KEYWORDS: cancer-initiating cells, cancer stem cells, head and neck cancer, human papillomavirus, ALDH1, prognostic biomarker.
INTRODUCTION Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, with an annual incidence of approximately 600,000 cases worldwide.1 Alcohol and tobacco use are the hallmark etiologic factors for HNSCC. However, the pathogenesis of HNSCC is changing because of the recognition that human papillomavirus (HPV) is a major risk factor for the development of HNSCC, in particular oropharyngeal squamous cell carcinoma (OPSCC). High-risk HPV16 is by far the most frequent HPV type detected in HNSCC.2 Epidemiologic data indicate that the prevalence of HPV-associated HNSCC has rapidly increased by about 3-fold in the past 3 decades in the United States and Europe.3-5 In light of these observations, it has been suggested that an epidemic of HPV-positive HNSCC will emerge in the near future.5,6 Cancer stem cells (CSCs) are a small subset of cancer cells within the tumor that have the exclusive capacity to divide and expand the CSC pool or to differentiate into heterogeneous, nontumorigenic cells that constitute the bulk of the tumor. There is emerging evidence that CSCs are refractory to chemotherapy and radiation, suggesting that CSCs may be responsible for disease relapse and progression.7-11 To date, the effect of HPV16 infection on the CSC population is unknown. Our results demonstrate that HPV16-positive OPSCC tumors have a higher intrinsic CSC population than HPV-negative HNSCC tumors. Elevated CSC numbers in HPV16-positive OPSCC are partly because of E6-mediated
Corresponding author: Quintin Pan, PhD, Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Medical Center, 442 Tzagournis Medical Research, 420 West 12th Avenue, Columbus, OH, 43210; Fax: (614) 247-1917; [email protected] 1 Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, Columbus, Ohio; 2Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, Ohio; 3Department of Pathology, The Ohio State University Wexner Medical Center, Columbus, Ohio; 4Center for Biostatistics, The Ohio State University, Columbus, Ohio
DOI: 10.1002/cncr.28538, Received: August 16, 2013; Revised: October 21, 2013; Accepted: November 14, 2013, Published online Month 00, 2013 in Wiley Online Library (wileyonlinelibrary.com)
Cancer
Month 00, 2013
1
Original Article
p53 inactivation. In a large cohort of OPSCC patients, HPV16-positive OPSCC is associated with increased aldehyde dehydrogenase 1 (ALDH1) staining in the tumor and stromal cells. The clinical observation that patients with HPV-positive OPSCC have a better prognosis than those with HPV-negative OPSCC provides initial evidence that the CSC phenotype may be more critical than the CSC number as a determinant of patient outcome. Our work suggests that CSC phenotype is not static and challenges the current dogma of homogeneity in the CSC population. MATERIALS AND METHODS Cell Lines
SCC15 and CAL27 cells were purchased from the American Type Culture Collection (Manassas, Va). UMSCC47 and UMSCC-74A cells were obtained from Dr. Thomas Carey at the University of Michigan. UDSCC2 cells were provided by Dr. Henning Bier at Heinrich-Heine University (Dusseldorf, Germany). UPCI:SCC090 cells were provided by Dr. Susanne Gollin at the University of Pittsburgh. SCC15 cells were grown in a 1:1 mixture of Ham F-12 and Dulbecco Modified Eagle Medium (DMEM) supplemented with 0.4 mg=mL hydrocortisone, 10% fetal bovine serum (FBS), 2 mM Lglutamine, 100 mg=mL streptomycin, and 100 U=mL penicillin. UD-SCC2, CAL27, UMSCC-47, UMSCC74A, and UPCI:SCC090 cells were grown in DMEM containing 10% FBS, 2 mM glutamine, 100 mg=mL streptomycin, and 100 U=mL penicillin. Cell lines were authenticated using short tandem repeat profile analysis. HPV-Negative and HPV16-Positive HNSCC Patient Tumors
Fresh previously untreated, HPV-negative (n 5 3) and HPV16-positive (n 5 3) OPSCC tumors were collected with a protocol approved by the Institutional Review Board at The Ohio State University. Informed consent was obtained from all patients who were included in the study. Tumors were digested in collagenase=hyaluronidase (STEMCELL Technologies Inc., Vancouver, BC, Canada) for 6 to 8 hours with gentle agitation at 30 C, followed by brief trypsinization, then they were filtered through a 40-lM cell strainer. Single cell suspensions were depleted for fibroblasts (Anti-Fibroblast microbeads; Miltenyi Biotec Inc., Auburn, Calif), CD45 cells (Anti-CD45 microbeads; Miltenyi Biotec Inc.), and Lineage cells (Lineage Cell Depletion Kit; Miltenyi Biotec Inc.) to enrich for tumor cells. Enriched HNSCC patient tumor cells were stained with propidium 2
iodide and sorted by fluorescence-activated cell sorting (FACS) to eliminate dead cells. Subsequently, purified HNSCC patient tumor cells were assessed for CSCs using the ALDEFLUOR (STEMCELL Technologies Inc.) and tumorsphere formation efficiency assays. ALDEFLUOR Assay
Enriched HNSCC patient tumor cells and HNSCC cell lines were incubated in buffer containing ALDH substrate (BAAA) in the presence or absence of diethylaminobenzaldehyde, a specific ALDH inhibitor, to serve as the negative control. The sorting gate for CSCs that had high ALDH activity (ALDHhigh) was established using the negative control as the baseline. FACS analyses were performed using BD FACS Calibur (BD Biosciences Corporation, Franklin Lakes, NJ) at The Ohio State University Comprehensive Cancer Center Analytical Cytometry Core. Tumorsphere Formation Assay
Enriched HNSCC patient tumor cells and HNSCC cell lines were seeded on a low-attachment plate in a defined, serum-free culture medium12 at a density of 1000 cells per well. Tumorspheres were allowed to grow for 7 days. Tumorsphere formation efficiency was calculated as the number of tumorspheres formed divided by the original number of cells seeded. Heterotransplantation of OPSCC Nonobese Diabetic=Severe Combined Immunodeficiency Cells
Fresh, previously untreated, HPV-negative (n 5 1) and HPV16-positive (n 5 1) OPSCC tumors were collected with a protocol approved by the Institutional Review Board at The Ohio State University. Informed consent was obtained from all patients who were included in the study. All mouse procedures were approved by the Animal Care and Use Committee at The Ohio State University. HPV-negative and HPV16-positive HNSCC heterotransplantation lines were established by subcutaneous implantation of solid tissue fragments into 6-week-old to 8-week-old nonobese diabetic=severe combined immunodeficiency (NOD=SCID) mice (National Cancer Institute, Bethesda, Md). HNSCC tumors were cut into small fragments and implanted subcutaneously on the dorsal flank of NOD=SCID mice. Recipient NOD=SCID mice were anesthetized with a ketamine-xylazine mixture before tumor implantation. Once tumors reached a volume of approximately 500 mm3, tumors were resected and serially passaged in naive NOD=SCID mice using the same protocol described above. Cancer
Month 00, 2013
HPV16 and Cancer Stem Cells/Zhang et al
Figure 1. The cancer stem cell (CSC) population is elevated in human papillomavirus 16 (HPV16)-associated head and neck squamous cell carcinoma (HNSCC). (a,b) CSCs are measured in tumors from patients with HPV-negative (HPV2) and HPV16-positive (HPV161) oropharyngeal squamous cell carcinoma (OPSCC). (a) Cells with high aldehyde dehydrogenase (ALDH) activity (ALDHhigh) were assessed using the ALDEFLUOR assay (STEMCELL Technologies Inc., Vancouver, BC, Canada). (b) Tumorsphere formation efficiency (TFE) was calculated as the number of tumorspheres formed divided by the initial number of cells seeded. Data are presented as the mean 6 standard error of the mean. An asterisk indicates P
