**2. IC10 and IC50 in HEp-2 and Ca9-22 cells**

We used the same IC10 and IC50 concentrations (Table 1) (Kogashiwa et al. 2010) as our previous study. At IC10 concentrations, anti-proliferative effect was not observed.


Table 1 IC10 or IC50 values in two cells

IC10 or IC50 values after 1 hour drug exposure followed by a 96 hours incubation in two head and neck cell lines.

Microtubule and Cdc42 are the Main Targets of

repeated 4 times.

were repeated 4 times.

Docetaxel's Suppression of Invasiveness of Head and Neck Cancer Cells 153

Fig. 2. 3D gel culture of HEp-2 cell spheroids at IC50 concentration.

**5. Tubulin bundle was formed by docetaxel treatment** 

HEp-2 cell spheroids were treated with cisplatin or docetaxel at IC50 followed by 96-hour incubation. bars= 250 µm. 4 replicate were used in each experiments and experiments were

Taxanes, including docetaxel, function as a mitotic spindle toxin by inhibiting microtubule turnover. They bind to microtubules and enhance tubulin polymerization. We hypothesized that docetaxel may exert similar effect on cytosolic, non-centrisome associated microtubules resulting in decreased cell motility. We therefore examined the structure of microtubules as well as actin filaments. Consistent with previous observation, filopodia formation was less in docetaxel treatment compared to cisplatin treatment. But no gross abnormality was found in actin filament structure between the treatments. On the other hand, tubulin bundle formation was noted in docetaxel treatment but not in cisplatin treatment (Fig.3.). Then, we attempted to find the mechanism that connect deformed microtubule and decreased filopodia formation.

Fig. 3. Staining of α-tubulin (by antibody) in HEp-2 cells treated by cisplatin, docetaxel or no treatment at IC50. Bars=50 µm. 3 replicate were used in each experiments and experiments

#### **3. Docetaxel inhibits the migration of head and neck cancer cells**

To assess cell migration a wound healing assay was employed. These results have been reported (Kogashiwa et al. 2010). Briefly, both in HEp-2 cell and CA9-22 cell, wound closure relative to no treatment condition is significantly reduced in docetaxel treatment while cisplatin treatment does not affect the cell migration (Fig.1.) (Kogashiwa et al. 2010).

Fig. 1. Migration assay at IC10 in two head and neck cancer cell lines. Migration rate is compared to control cell migration rate. Each data point represents mean ± SE. \*\*\*p < 0.001. 15 replicate were used in each experiments and experiments were repeated 4 times.

#### **4. Docetaxel inhibits the invasiveness of multicellular tumor spheroids.**

The similar results are obtained in three-dimensional multicellular tumor spheroid culture (Kogashiwa et al. 2010). At IC10 determined in monolayer culture, either cisplatin or docetaxel does not affect filopodia formation. However, at IC50 determined in monolayer culture, docetaxel, but not cisplatin, significantly decreases filopodia formation in HEp-2 cells in spheroid culture (Fig.2.) (Kogashiwa et al. 2010). Taken together, In the previous study, we have shown that docetaxel, but not cisplatin inhibits cell migration both in 2D and 3D culture.

To assess cell migration a wound healing assay was employed. These results have been reported (Kogashiwa et al. 2010). Briefly, both in HEp-2 cell and CA9-22 cell, wound closure relative to no treatment condition is significantly reduced in docetaxel treatment while

**3. Docetaxel inhibits the migration of head and neck cancer cells** 

Fig. 1. Migration assay at IC10 in two head and neck cancer cell lines.

repeated 4 times.

3D culture.

Migration rate is compared to control cell migration rate. Each data point represents mean ± SE. \*\*\*p < 0.001. 15 replicate were used in each experiments and experiments were

The similar results are obtained in three-dimensional multicellular tumor spheroid culture (Kogashiwa et al. 2010). At IC10 determined in monolayer culture, either cisplatin or docetaxel does not affect filopodia formation. However, at IC50 determined in monolayer culture, docetaxel, but not cisplatin, significantly decreases filopodia formation in HEp-2 cells in spheroid culture (Fig.2.) (Kogashiwa et al. 2010). Taken together, In the previous study, we have shown that docetaxel, but not cisplatin inhibits cell migration both in 2D and

**4. Docetaxel inhibits the invasiveness of multicellular tumor spheroids.** 

cisplatin treatment does not affect the cell migration (Fig.1.) (Kogashiwa et al. 2010).

Fig. 2. 3D gel culture of HEp-2 cell spheroids at IC50 concentration. HEp-2 cell spheroids were treated with cisplatin or docetaxel at IC50 followed by 96-hour incubation. bars= 250 µm. 4 replicate were used in each experiments and experiments were repeated 4 times.
