John H. Owen

Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehydrogenase

In accord with the cancer stem cell (CSC) theory, only a small subset of cancer cells are capable of forming tumors. We previously reported that CD44 isolates tumorigenic cells from head and neck squamous cell cancer (HNSCC). Recent studies indicate that aldehyde dehydrogenase (ALDH) activity may represent a more specific marker of CSCs.

Metastatic Potential of Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma

OBJECTIVE to design in vitro and in vivo models of metastasis to study the behavior of cancer stem cells (CSCs) in head and neck squamous cell carcinoma (HNSCC). DESIGN cells were sorted for CD44 expression using flow cytometry. Sorted cells were used in an in vitro invasion assay. For in vivo studies, CSCs and non-CSCs were injected into the tail veins of mice, and lungs were either harvested or imaged to evaluate for lesions. RESULTS in vitro, CD44(high) cells were more motile but not more invasive than CD44(low) cells. In vivo, 8 of 17 mice injected with CD44(high) cells and 0 of 17 mice injected with CD44(low) cells developed lung lesions. Two of the lesions arose from CSCs from a primary tumor and 6 from CSCs from HNSCC cell lines. CONCLUSIONS in vitro, CSCs do not have an increased ability to invade through basement membrane, but they migrate more efficiently through a porous barrier. In contrast, CSCs efficiently formed lung lesions in vivo, whereas non-CSCs did not give rise to any distant disease. This phenomenon could be due to the enhanced migratory capacity of CSCs, which may be more important than basement membrane degradation in vivo.

UM-SCC-104: A New human papillomavirus-16–positive cancer stem cell–containing head and neck squamous cell carcinoma cell line†

Few human papillomavirus (HPV)(+) head and neck squamous cell carcinoma (HNSCC) cell lines exist. We established University of Michigan‐squamous cell carcinoma‐104 (UM‐SCC‐104), a new HPV(+) HNSCC cell line from a recurrent oral cavity tumor, and characterized it for the presence of cancer stem cells (CSCs).

Head and neck cancer stem cells: The side population†

The cancer stem cell (CSC) theory concludes that a subpopulation of cancer cells, the cancer stem cells, can self‐renew and are responsible for tumor growth. Previous studies have identified cells able to efflux Hoechst 33342 dye as the side population (SP). SP cells and CSCs share many characteristics, suggesting the SP isolated from malignant tumors contains CSCs.

Cancer stem cells: Mediators of tumorigenesis and metastasis in head and neck squamous cell carcinoma

Cancer stem cells (CSCs) represent a subpopulation of cells responsible for tumor growth. Their role in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and metastasis remains uncertain.

Cancer stem cell vaccine inhibits metastases of primary tumors and induces Humoral immune responses against cancer stem cells

The inability to target cancer stem cells (CSC) may be a significant factor contributing to treatment failure. We have developed a strategy to target the CSC populations in melanoma and squamous cell carcinoma using CSC lysate-pulsed dendritic cells (DCs). The CSC-DC vaccine was administered in the adjuvant setting after localized radiation therapy of established tumors. Using mouse models we demonstrated that DCs pulsed with CSCs enriched by virtue of their expression of the CSC marker ALDH (termed CSC-DC) significantly inhibited tumor growth, reduced development of pulmonary metastases and prolonged survival. The effect was associated with downregulation of chemokine (C-C motif) receptors CCR7 and CCR10 in tumor cells and decreased expression of the chemokine (C-C motif) ligands CCL21, CCL27 and CCL28 in lung tissue. The CSC-DC vaccine significantly reduced ALDHhigh CSC frequency in primary tumors. Direct targeting of CSCs was demonstrated by the specific binding of IgG produced by ALDHhigh CSC-DC vaccine-primed B cells to ALDHhigh CSCs, resulting in lysis of these target CSCs in the presence of complement. These data suggest that the CSC-DC vaccine approach may be useful in the adjuvant setting where local and systemic relapse are high after conventional treatment of cancers.

Head and Neck Cancer Stem Cells The Effect of HPV—An In Vitro and Mouse Study

Objectives To determine if the behavior of cancer stem cells (CSCs) is affected by human papillomavirus (HPV) status. Study Design An in vitro and in vivo analysis of HPV and CSCs. Setting University laboratory. Subjects and Methods We isolated CSCs from HPV-positive and HPV-negative cell lines. Two HPV-negative cell lines underwent lentiviral transduction of E6/E7. Chemoresistence was determined using colony formation assays. Native HPV-positive and HPV E6/E7-transduced cells were compared for lung colonization after tail vein injection in NOD/SCID mice. Results The proportion of CSC is not significantly different in HPV-positive or HPV-negative head and neck squamous cell carcinoma (HNSCC) cell lines. The HNSCC CSCs are more resistant to cisplatin than the non-CSCs, but there were no significant differences between HPV-positive and HPV-negative cells. The HPV-negative cancer cells yielded low colony formation after cell sorting. After transduction with HPV E6/E7, increased colony formation was observed in both CSCs and non-CSCs. Results from tail vein injections yielded no differences in development of lung colonies between HPV E6/E7-transduced cells and nontransduced cells. Conclusions Human papillomavirus status does not correlate with the proportion of CSCs present in HNSCC. The HPV-positive cells and those transduced with HPV E6/E7 have a greater clonogenicity than HPV-negative cells. The HNSCC CSCs are more resistant to cisplatin than non-CSCs. This suggests that common chemotherapeutic agents may shrink tumor bulk by eliminating non-CSCs, whereas CSCs have mechanisms that facilitate evasion of cell death. Human papillomavirus status does not affect CSC response to cisplatin therapy, suggesting that other factors explain the better outcomes for patients with HPV-positive cancer.

In vitro evaluation of Sialyl Lewis X relationship with head and neck cancer stem cells

Objective To evaluate in vitro the potential links between sialyl Lewis X (sLeX) and cancer stem cells (CSC) in head and neck squamous cell carcinoma (HNSCC). HNSCC is an aggressive malignancy with high mortality mainly due to metastasis. CSC have emerged as important players in HNSCC metastasis. sLeX is a tetrasaccharide carbohydrate known to play a key role in metastatic dissemination by promoting binding of the tumor cells to the endothelium. Study Design Experimental, in vitro. Setting Laboratory of Head and Neck Cancer Metastasis, University of Michigan. Subjects and Methods A panel of stage- and anatomic-site specific primary and metastatic HNSCC cell lines was assessed by flow cytometry to quantify sLeX relative expression levels. Serum-free conditioned media from the same HNSCC lines was collected over a time course of 72 hours and assessed by Western blot for secreted sLeX expression. Representative HNSCC cell lines were cultured as floating orospheres (condition that enhance CSC growth) or under normal adherent conditions and characterized by flow cytometry for CSC markers (CD44, aldehyde dehydrogenase [ALDH]) comparatively with sLeX expression. Results sLeX is predominantly expressed in carcinomas originating from the oral cavity. Secreted sLeX is also found to be high in oral carcinomas and increased over the analyzed time course. Floating orospheres were strongly positive for CD44 and ALDH, confirming CSC enrichment of the orospheres. Tumor cells grown as orospheres are 95% to 100% positive for sLeX compared to 10% to 40% of adherent counterpart. Conclusion These studies provide the first evidence of sLeX relationship with CSC in HNSCC.

Integration of high-risk human papillomavirus into cellular cancer-related genes in head and neck cancer cell lines: HPV integration into cancer genes in HNSCC cell lines

Human papillomavirus (HPV)‐positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV‐positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16‐ and HPV18‐positive squamous cell carcinoma lines.

Therapeutic efficacy of cancer stem cell vaccines in the adjuvant setting.

Dendritic cell (DC)-based vaccine strategies aimed at targeting cancer stem-like cells (CSC) may be most efficacious if deployed in the adjuvant setting. In this study, we offer preclinical evidence that this is the case for a CSC-DC vaccine as tested in murine models of SCC7 squamous cell cancer and D5 melanoma. Vaccination of mice with an ALDH(high) SCC7 CSC-DC vaccine after surgical excision of established SCC7 tumors reduced local tumor relapse and prolonged host survival. This effect was augmented significantly by simultaneous administration of anti-PD-L1, an immune checkpoint inhibitor. In the minimal disease setting of D5 melanoma, treatment of mice with ALDH(high) CSC-DC vaccination inhibited primary tumor growth, reduced spontaneous lung metastases, and increased host survival. In this setting, CCR10 and its ligands were downregulated on ALDH(high) D5 CSCs and in lung tissues, respectively, after vaccination with ALDH(high) D5 CSC-DC. RNAi-mediated attenuation of CCR10 blocked tumor cell migration in vitro and metastasis in vivo T cells harvested from mice vaccinated with ALDH(high) D5 CSC-DC selectively killed ALDH(high) D5 CSCs, with additional evidence of humoral immunologic engagement and a reduction in ALDH(high) cells in residual tumors. Overall, our results offered a preclinical proof of concept for the use of ALDH(high) CSC-DC vaccines in the adjuvant setting to more effectively limit local tumor recurrence and spontaneous pulmonary metastasis, as compared with traditional DC vaccines, with increased host survival further accentuated by simultaneous PD-L1 blockade. Cancer Res; 76(16); 4661-72. ©2016 AACR.