Podocalyxin is crucial for the growth of oral squamous cell carcinoma cell line HSC-2

Oral cancers constitute approximately 2% of all cancers, with the most common histological type being oral squamous cell carcinoma (OSCC), representing 90% of oral cancers. Although diagnostic technologies and therapeutic techniques have progressed, the survival rate of patients with OSCC is still 60%, whereas the incidence rate has increased. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is detected in normal tissues such as heart, breast, and pancreas as well as in many cancers, including lung, renal, breast, colorectal, and oral cancers. This glycoprotein is associated with the progression, metastasis, and poor outcomes of oral cancers. PODXL overexpression was strongly detected using our previously established anti-PODXL monoclonal antibody (mAb), PcMab-47, and its mouse IgG2a-type, 47-mG2a. In previous studies, we also generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines, in order to investigate the function of PODXL in the proliferation of oral cancer cells. The growth of SAS/PODXL-KO cell lines was observed to be lower than that of parental SAS cells. For this study, PODXL-KO OSCC cell lines were generated using HSC-2 cells, and the role of PODXL in the growth of OSCC cell lines in vitro was assessed. Decreased growth was observed for HSC-2/PODXL-KO cells compared with HSC-2 parental cells. The influence of PODXL on tumor growth of OSCC was also investigated in vivo, and both the tumor volume and the tumor weight were observed to be significantly lower for HSC-2/PODXL-KO than that for HSC-2 parental cells. These results, taken together, indicate that PODXL plays an important role in tumor growth, both in vitro and in vivo.


Introduction
Globally, oral cancers constitute approximately 2% of all cancers [1]. They can be histologically classified into the following types: squamous cell carcinoma, adenoid carcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, and osteosarcoma. Approximately 90% of oral cancer burden is borne by oral squamous cell carcinoma (OSCC) [2]. Although diagnostic technologies and therapeutic techniques have progressed by leaps and bounds in recent decades, the survival rate of patients with OSCC has not improved. Consequently, the 5year survival rate of patients with OSCC is still 60% [3]; however, the incidence rate of OSCC is increasing [4,5].
In our previous study, we established specific and sensitive anti-PODXL mAbs: PcMab-47 (mouse IgG 1 , kappa) [20], 47-mG 2a (mouse IgG 2a -type of PcMab-47), and 47-mG 2a -f (core fucose-deficient type of 47-mG 2a ) [21]. We demonstrated that 47-mG 2a -f significantly reduced tumor growth in OSCC xenograft models. [21]. To investigate the function of PODXL in the growth of oral cancer cells, we previously generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines. The growth of SAS/PODXL-KO cell lines (BINDS-01) was observed to be lower than that of parental SAS cells. In this study, we further established PODXL-KO OSCC cell lines using HSC-2 (oral squamous cell carcinoma from oral cavity) cells and investigated the role of PODXL in the growth of OSCC cell lines both in vitro and in vivo.

In vitro proliferation assay
In vitro cell proliferation was measured using Cell Cloning Kit-8 (CCK-8; Dojindo, Kumamoto, Japan), which can assess cell viability. Cells were plated (1500, 3000, and 6000 cells/100 μL/well) in quintuple wells in 96-well plates and were incubated for 48 h. After adding 10 μL of CCK-8 to each well, the plates were incubated for 4 h at 37°C. Subsequently, the absorbance was recorded at 450 nm using iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA). The mean absorbance of the 5-well set was obtained at 48 h after cell seeding. All data were expressed as the mean ± SEM. Statistical significance was analyzed using Tukey-Kramer's test. P-values < 0.05 were considered statistically significant.

In vivo proliferation assay
Five-week-old, female BALB/c nude mice were purchased from CLEA Japan (Tokyo, Japan). Seven-week-old mice were used for the in vivo proliferation assay. Cells  tumor volume was calculated using the following formula: volume = W 2 × L/2, where W is the short diameter, and L is the long diameter. All mice were euthanized 21 days after cell implantation, and the tumor weight was measured. All data were expressed as mean ± SEM. Statistical analysis was performed using Tukey-Kramer's test. P-values < 0.05 were considered statistically significant.

Results and discussion
For this study, CRISPR/Cas9 plasmids targeting human PODXL were used to generate three PODXL-KO OSCC cell lines: HSC-2/PODXL-KO #1, #2, and #3. Before initiating the study, PODXL expression was confirmed in these cell lines. As shown in Fig. 1, anti-human PODXL mAb, PcMab-47 reacted with the HSC-2 parental cell line, but did not react with any of the three HSC-2/PODXL-KO cell lines.
Next, we investigated the potential involvement of PODXL in the stimulation of in vitro OSCC cell growth. At every plated cell density (1500, 3000, and 6000 cells/well), the growth of HSC-2/PODXL-KO #1 and #2 cells was significantly lower than that of HSC-2 cells (Fig. 2). In contrast, HSC-2/PODXL-KO #3 showed significantly less growth than HSC-2 cells only when at a plated cell density of 1500 cells/well. These results indicate that while PODXL is an important factor for in vitro HSC-2 cell growth, many other factors may be involved in the induction or inhibition of HSC-2 cell growth.
Taken together, our results indicate that PODXL plays an important role in the growth of tumor in oral cancers. The HSC-2/PODXL-KO cell lines, which were generated in this study, are promising tools that can be used in future studies to elucidate the function of PODXL in the proliferation of oral cancer.