Bmi1-positive cells in the lingual epithelium could serve as cancer stem cells in tongue cancer

Abstract

We recently reported that the polycomb complex protein Bmi1 is a marker for lingual epithelial stem cells (LESCs), which are involved in the long-term maintenance of lingual epithelial tissue in the physiological state. However, the precise role of LESCs in generating tongue tumors and Bmi1-positive cell lineage dynamics in tongue cancers are unclear. Here, using a mouse model of chemically (4-nitroquinoline-1-oxide: 4-NQO) induced tongue cancer and the multicolor lineage tracing method, we found that each unit of the tumor was generated by a single cell and that the assembly of such cells formed a polyclonal tumor. Although many Bmi1-positive cells within the tongue cancer specimens failed to proliferate, some proliferated continuously and supplied tumor cells to the surrounding area. This process eventually led to the formation of areas derived from single cells after 1-3 months, as determined using the multicolor lineage tracing method, indicating that such cells could serve as cancer stem cells. These results indicate that LESCs could serve as the origin for tongue cancer and that cancer stem cells are present in tongue tumors.

Figure 1. Morphological observations of tongue cancer and histological analysis of normal tongue and tongue cancer model.

(a) Schematic representation of the timing of 4-NQO administration. (b) Morphological observations of normal tongues and tongues of cancer model mice. The red arrow indicates papilloma. The blue arrow indicates invasive carcinoma. (c) H&E staining of normal tongues and the filiform papillae (d) H&E staining of tongue cancer (e) Immunohistochemical staining of tongue cancer for Krt14 and Ki67. Scale bars: (b) 1 mm; (c) 1 mm (left), 100 μm (right); (d) 1 mm (left), 100 μm (right) (e) 100 μm.

Figure 2. Rosa26^CreERT2/rbw mice were labeled with tamoxifen and induced by 4-NQO after the induction of tongue cancer.

Schematic representation of the outcome of multi-color lineage tracing in the Rosa26^CreERT2/+ tongue cancer model and timing of 4–NQO and tamoxifen treatment. (b,c) Rosa26^CreERT2/rbw mice were injected with tamoxifen after inducing tongue cancer and analyzed at the indicated time points (day 1 and day 7). Scale bars: (b) 50 μm (right), 100 μm (left); (c) 50 μm (right), 100 μm (left and middle).

Figure 3. Fate of cells derived from Bmi1-positive cells in tongue cancer.

(a) Schematic representation of the outcome of multi-color lineage tracing in the Bmi1^creER/+/Rosa26^rbw/+ tongue cancer model. (b,c,d) Bmi1^creER/+/Rosa26^rbw/+ mice were injected with tamoxifen and analyzed at the indicated time points (day 7, day 14, and day 28). (e) The ratio of single Bmi1-positive cells in the tumor (Bmi1^creER/+/Rosa26^rbw/+ mice, N = 6; day 7: n = 39 clones, day 14: n = 29 clones, day 28: n = 123 clones). *p < 0.01. Scale bars: (b) 50 μm (left), 100 μm (right); (c) 50 μm (left), 100 μm (right); (d) 100 μm.