Establishment and characterization of an osteopontin-null cutaneous squamous cell carcinoma cell line

Abstract

Osteopontin (OPN) is a secreted glycoprotein implicated to function in cancer development and metastasis. Although elevated expression of OPN are observed in cancer cells of various types, in some cases, only the cells in the stromal region surrounding the tumor express OPN, suggesting distinct functional roles for this protein derived from host cells and from cancer cells. To provide a model for addressing the functions and mechanisms of host-derived OPN in cancer progression and metastasis, a cutaneous squamous cell carcinoma cell line (ONSC) that lacks the OPN gene, Spp1, was established. This line of cells was derived from a squamous cell carcinoma that developed in a female, OPN-null mouse subjected to two-stage skin carcinogenesis. Morphologically, ONSC cells resemble epithelial cells, and they express the epithelial markers, K1, K14, and p63, as confirmed by immunohistochemical analyses. Genomic analyses indicate the presence of mutated H-Ras and p53 genes. ONSC cells form colonies in soft agar and, subcutaneously injected into athymic nude mice, develop into squamous cell carcinomas that metastasize to the lungs. Lacking OPN expression, these squamous cell carcinoma cells provide a model to address the function of host OPN in the context of cancer progression and metastasis.

Figure 1

Characterization of ONSC cells as OPN-null SCCs. (A) ONSC cells lack the Spp1 gene. PCR analyses were performed with DNA purified from ONSC cells and from OPN heterozygous (+/-) mice. Lane M, DNA molecular marker; 600 bp indicates OPN+ alleles; 500 bp indicates OPN- alleles; Spp1, OPN gene. (B) Northern blot analysis of OPN mRNA. Total RNA extracted from JB6 Cl41.5a, CC4B, and ONSC cells was probed with radiolabeled OPN or β-actin cDNA, used as a loading control (lower panel). The observed OPN in the lower panel is due to non-stripped radiolabeled OPN probe. (C) Comparison of cell morphology with epidermal cell lines. ONSC, an OPN-null cutaneous SCC line; CC4A, an undifferentiated spindle cell carcinoma cell line; CC4B, a well-differentiated cutaneous SCC cell line; JB6 Cl41.5a, a preneoplastic mouse epidermal-like cell line. Magnification, ×200. (D) Immunohistochemical analyses for the expression of p63, K1 and K14 in ONSC cells. ONSC cells were immunostained with anti-p63, K1, and K14 antibodies or their isotype controls (IgG). Magnification, ×200; ×400 (inserted panel).

Figure 2

Time course of tumor development and tumor size and histology of SCCs from athymic nude mice injected s.c. with ONSC cells. (A) Time course of ONSC tumor development. ONSC cells (2×10⁶) were injected into the lower dorsal regions of female nude mice. Tumors were measured each week (except wk 3) for 7 wk. The curved line represents mean tumor volumes at each week (n=8/group/wk). (B) Histology of SCC in athymic nude mice injected with ONSC cells. Athymic nude mice were injected s.c. with ONSC cells. (a and b) Well differentiated SCC at 1 wk after injection of ONSC cells. (c and d) SCC metastasized to the lung of athymic nude mice. (a and c), magnification 10x; (b d), magnification 40x. □, magnified regions in b and d; ○, region with SCC in the lung.