Endothelial interleukin-6 defines the tumorigenic potential of primary human cancer stem cells.

Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin-6 (IL-6), we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival, and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling.

Orosphere assay: a method for propagation of head and neck cancer stem cells.

BACKGROUND: Recent evidence suggests that head and neck squamous cell carcinomas (HNSCCs) harbor a small subpopulation of highly tumorigenic cells, designated cancer stem cells. A limiting factor in cancer stem cell research is the intrinsic difficulty of expanding cells in an undifferentiated state in vitro. METHODS: Here, we describe the development of the orosphere assay, a method for the study of putative head and neck cancer stem cells. An orosphere is defined as a nonadherent colony of cells sorted from primary HNSCCs or from HNSCC cell lines and cultured in 3-dimensional soft agar or ultralow attachment plates. Aldehyde dehydrogenase activity and CD44 expression were used here as stem cell markers. RESULTS: This assay allowed for the propagation of head and neck cancer cells that retained stemness and self-renewal. CONCLUSION: The orosphere assay is well suited for studies designed to understand the pathobiology of head and neck cancer stem cells.

Xenograft tumors vascularized with murine blood vessels may overestimate the effect of anti-tumor drugs: a pilot study.

Recent evidence demonstrated that endothelial cells initiate signaling events that enhance tumor cell survival, proliferation, invasion, and tumor recurrence. Under this new paradigm for cellular crosstalk within the tumor microenvironment, the origin of endothelial cells and tumor cells may have a direct impact on the pathobiology of cancer. The purpose of this pilot study was to evaluate the effect of endothelial cell species (i.e. murine or human) on xenograft tumor growth and response to therapy. Tumor xenografts vascularized either with human or with murine microvascular endothelial cells were engineered, side-by-side, subcutaneously in the dorsum of immunodefficient mice. When tumors reached 200 mm(3), mice were treated for 30 days with either 4 mg/kg cisplatin (i.p.) every 5 days or with 40 mg/kg sunitinib (p.o.) daily. Xenograft human tumors vascularized with human endothelial cells grow faster than xenograft tumors vascularized with mouse endothelial cells (P<0.05). Notably, human tumors vascularized with human endothelial cells exhibited nuclear translocation of p65 (indicative of high NF-kB activity), and were more resistant to treatment with cisplatin or sunitinib than the contralateral tumors vascularized with murine endothelial cells (P<0.05). Collectively, these studies suggest that the species of endothelial cells has a direct impact on xenograft tumor growth and response to treatment with the chemotherapeutic drug cisplatin or with the anti-angiogenic drug sunitinib.

Endothelial derived factors inhibit anoikis of head and neck cancer stem cells.

Recent evidence demonstrated that cancer stem cells reside in close proximity to blood vessels in human head and neck squamous cell carcinomas (HNSCC). These findings suggest the existence of a supporting perivascular niche for cancer stem cells. The purpose of this study was to evaluate the effect of endothelial cell-secreted factors on the behavior of head and neck cancer stem-like cells (HNCSC). HNCSC were identified by sorting UM-SCC-22A (cell line derived from a primary squamous cell carcinoma of the oropharynx) and UM-SCC-22B (derived from the metastatic lymph node of the same patient) for CD44 expression and ALDH (aldehyde dehydrogenase) activity. HNCSC (ALDH+CD44+) and control (ALDH-CD44-) cells were cultured in ultra-low attachment plates in presence of conditioned medium from primary human endothelial cells. ALDH+CD44+ generated more orospheres than control cells when cultured in suspension. The growth factor milieu secreted by endothelial cells protected HNCSC against anoikis. Mechanistic studies revealed that endothelial cell-secreted vascular endothelial growth factor (VEGF) induces proliferation of HNCSC derived from primary UM-SCC-22A, but not from the metastatic UM-SCC-22B. Likewise, blockade of VEGF abrogated endothelial cell-induced Akt phosphorylation in HNCSC derived from UM-SCC-22A while it had a modest effect in Akt phosphorylation in HNCSC from UM-SCC-22B. This study revealed that endothelial cells initiate a crosstalk that protect head and neck cancer stem cells against anoikis, and suggest that therapeutic interference with this crosstalk might be beneficial for patients with head and neck cancer.

Metronomic dosing of BH3 mimetic small molecule yields robust antiangiogenic and antitumor effects.

Bcl-2 is an antiapoptotic protein that has also been found to function as a proangiogenic signaling molecule. Improvements in antiangiogenic therapy can be engendered by metronomic dosing. Thus, we hypothesized that BH3-mimetic drugs that antagonize Bcl-2 family proteins may exert a greater efficacy when dosed metronomically. To examine this hypothesis, we employed AT101, an orally available and well-tolerated BH3-mimetic drug that has been established as effective. In a mouse xenograft model of human squamous cell carcinomas (SCC) that includes a humanized vasculature, we explored the effects of docetaxel in combination with either daily (metronomic) or weekly (bolus) doses of AT101. In addition, we explored the effect of single or combination therapy on angiogenesis and survival of endothelial or SCC cells in vitro. Metronomic AT101 therapy increased mouse survival, decreased tumor mitotic index, and decreased tumor microvessel density, compared with bolus therapy. Therapeutic potentiation was achieved by similar overall drug exposure and without altering systemic toxicities. Combinations of AT101 and docetaxel produced additive toxicity in both endothelial and SCC tumor cells. Notably, subapoptotic concentrations of AT101 potently inhibited the angiogenic potential of endothelial cells. Taken together, our findings unveil the efficacious benefits that can be achieved by metronomic delivery of BH3-mimetic drugs, in particular suggesting that SCC patients with might benefit from low-dose continuous administration of these drugs.

Endothelial cell-initiated signaling promotes the survival and self-renewal of cancer stem cells.

Recent studies have demonstrated that cancer stem cells play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). However, little is known about functional interactions between head and neck cancer stem-like cells (CSC) and surrounding stromal cells. Here, we used aldehyde dehydrogenase activity and CD44 expression to sort putative stem cells from primary human HNSCC. Implantation of 1,000 CSC (ALDH+CD44+Lin-) led to tumors in 13 (out of 15) mice, whereas 10,000 noncancer stem cells (ALDH-CD44-Lin-) resulted in 2 tumors in 15 mice. These data demonstrated that ALDH and CD44 select a subpopulation of cells that are highly tumorigenic. The ability to self-renew was confirmed by the observation that ALDH+CD44+Lin- cells sorted from human HNSCC formed more spheroids (orospheres) in 3-D agarose matrices or ultra-low attachment plates than controls and were serially passaged in vivo. We observed that approximately 80% of the CSC were located in close proximity (within 100-µm radius) of blood vessels in human tumors, suggesting the existence of perivascular niches in HNSCC. In vitro studies demonstrated that endothelial cell-secreted factors promoted self-renewal of CSC, as demonstrated by the upregulation of Bmi-1 expression and the increase in the number of orospheres as compared with controls. Notably, selective ablation of tumor-associated endothelial cells stably transduced with a caspase-based artificial death switch (iCaspase-9) caused a marked reduction in the fraction of CSC in xenograft tumors. Collectively, these findings indicate that endothelial cell-initiated signaling can enhance the survival and self-renewal of head and neck CSC.