Human and murine squamous cell carcinomas (SCC) have been reported to produce proinflammatory cytokines IL-1alpha, IL-6, GM-CSF, and IL-8 or KC. Production of individual members of the proinflammatory cytokine family has been associated with increased tumor growth or metastasis in a variety of neoplasms. In this study, we determined whether the expression of these cytokines occurs as a result of the events of cellular transformation or culture, or is promoted by interaction of neoplastic cells with factors or cells in the host environment. We compared the expression of proinflammatory cytokines following the spontaneous transformation of murine keratinocytes in vitro, and following the formation of tumors and metastases from these transformed keratinocytes in syngeneic recipients in vivo. Using sensitive ELISA assays, we found that cultures of the in vitro transformed Balb/c SCC line Pam 212 do not produce elevated levels of proinflammatory cytokines IL-1alpha, IL-6, GM-CSF and KC, indicating that transformation or culture alone is insufficient to account for the level of cytokine expression detected in patient and experimental tumors. In contrast, Pam reisolates from primary and metastatic tumors were obtained which constitutively produce markedly elevated levels of cytokines IL-1alpha, IL-6, KC and GM-CSF. The increase in the expression of these cytokines by SCC in vivo occurred independent of T and B lymphocyte-mediated immunity, since increases in expression of the cytokines was observed in lines reisolated from immunodeficient athymic nude and SCID Balb/c congenic mice. The increased expression of cytokines appeared to result from additional events in vivo, rather than due to selection of a pre-existing cytokine-producing subpopulation, since clones of the parental cell line expressed lower cytokine levels than cloned reisolates, and clones of the non-secreting parental cell line that formed tumors in vivo secreted elevated levels of cytokines following reisolation. We conclude that the development of SCC that express proinflammatory cytokines is promoted by tumor-host interaction(s) that are independent of specific T and B cell immunity.

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Molecular changes occurring with tumor formation and metastasis need to be identified in order to define novel markers and targets for chemoprevention and therapy. Cell lines from a multistage model of murine squamous cell carcinoma were analyzed for differences in gene expression using mRNA differential display. mRNA was isolated from primary keratinocytes, an in vitro transformed keratinocyte line (Pam 212), and three metastatic cell lines derived from Pam 212 following tumor progression in vivo. cDNA was synthesized by reverse transcription and amplified by PCR using 72 primer combinations to screen and compare approximately 3,600 sequences. Five cDNAs with a differential expression pattern confirmed by Northern blot analysis were cloned and sequenced, revealing homology with known genes. The gene encoding tropomyosin ␣ was preferentially expressed in primary keratinocytes; genes for tyrosine kinase Yes-associated protein (YAP65) and ribosomal protein L18a were preferentially expressed in transformed and metastatic tumor cell lines; and genes for the Gro-␣ family cytokine KC and antigen Sp17 exhibited increased expression in the three metastatic cell lines. The structure and function of the genes identified suggest that they may possibly be linked to cell shape and motility, signal transduction, protein synthesis, growth, granulocyte chemotaxis, and angiogenesis. This study demonstrates the ability of mRNA differential display to detect altered gene expression in this tumor progression model of murine squamous cell carcinoma, and the potential usefulness of this approach for identification of candidate genes as chemoprevention markers and targets. J. Cell. Biochem. Suppls. 28/29:90-100. 1998 Wiley-Liss, Inc. †

The purpose of this study was to develop an experimental model of squamous cell carcinoma that can be used to identify molecular and immunologic changes associated with primary events in malignant transformation, and those associated with metastatic tumor progression in the presence of host homeostatic and immunologic factors. Metastatic variants were derived following in vivo tumor progression of the in vitro transformed squamous cell carcinoma line Pam 212. The parental and metastatic cell lines exhibited similar morphologic features and molecular markers of an epithelial lineage, including an epithelial morphology in culture, cell surface expression of integrin alpha6beta4, and expression of mRNA of cytokeratins K6 and K14. When the growth and metastatic phenotype of the parental and reisolate cell lines was compared, the reisolate cell lines were found to exhibit a greater rate of growth and incidence of metastasis than the parental cell line when reimplanted in vivo. The difference in the growth rate of the parental cell line and the variants observed in vivo was not detected when growth of these lines was compared in vitro, suggesting that the growth advantage and selection of these variants requires tumor-host interaction. The metastatic variants exhibited a similar growth advantage in normal immunocompetent and SCID Balb/c mice, indicating that the growth advantage in vivo is not due to T or B lymphocyte-dependent immune factor(s). We conclude that metastatic variants derived following in vivo tumor progression of an in vitro transformed squamous cell carcinoma line exhibit a differential growth advantage in vivo that requires the host environment. Comparison of these in vitro transformed and in vivo derived metastatic variant cell lines with phenotypic differences in growth and metastasis should prove useful for dissecting the role of tumor and host factor(s) in malignant transformation and metastatic tumor progression of squamous cell carcinoma.