180 Fig. 4 Volume of established tumors. Tumors were measured in three dimensions and the volume was calibrated in mm3. The volumes and ranges of each time point after the start of the treatment are shown Discussion Oral squamous cell carcinoma is one of the most common cancers in the world. Novel treatment is best studied in animal models that mimic the same clinical features as human squamous cell carcinomas. In this study we induced oral squamous cell carcinomas by applying 4NQO oraly during 16 weeks in mice. Pre- malignant laesions appeared 3 weeks after the last appli- cation of 4NQO. The typical tumor progression for squamous cell carcinoma was seen before invasive squa- mous cell carcinomas appeared. At 36 weeks two-thirds of the tumors were squamous cell carcinomas, 4 weeks fur- ther all 6 oral tumors were squamous cell carcinomas. These results are in accordance with earlier observed results in which squamous cell carcinomas were detected between 12 and 33 weeks after applying 4NQO during 16 weeks (Hawkins et al. 1994; Tang et al. 2004). Immunohishtochemistry was performed on the tissues to assess the similarities with human squamous cell carci- noma. Biomarkers have been studied to predict behaviour of tumor cells and are used to characterise tumors (Takes 2004). Cyclin D1 plays a pivotal role in transition from G1 to S phase and cyclin D1 expression pattern is associated tumor characteristics and survival (Bova et al. 1999). As is seen in human and other animal models (Niwa et al. 2001; Yoshida et al. 2005) cyclin D1 expression showed an increased expression in the oral squamous cell carcinoma in our mice. E-cadherin, a calcium dependent membrane protein that is essential for the formation of adherens junction between cells. There is a connection with chang- ing expression of E-cadherin and the transition from hyperplasia to invasive carcinoma (Perl et al. 1998). We observed less expression of E-cadherin in invasive section of the carcinoma and evenly distributed expression in earlier stages, a pattern that has been reported earlier in 123 J Mol Hist (2009) 40:177–181 human oral squamous cell carcinoma (Williams et al. 1998). Average tumor volume advanced from 6 mm3 after 20 weeks to 30 mm3 at the 40 weeks time point. For intra- tumor injection with an adenovirus it is desirable that the size of each axe of the tumor is at least 2 mm, though preferable higher. Eventual tumor size reduction after treatment is then feasible. Assuming that the 4NQO oral tumor model will not have the prospect to develop in to a large tumor volume such as the model in which tumor cells are injected subcutaneously in the ﬂank, tumor volume will 3 not easily exceed 100 mm . Previous experiments with intra-tumor injection of gene therapy showed that the injected volume must not surpass 30% of the volume of the tumor (van der Eb et al. 2002). Taking this in to account and knowing that the volume of an oral squamous cell carcinoma in mice is small we estimate that a tumor vol- ume of at least 15 mm3 is desirable. After 32 weeks the tumor volume surpassed this point and after 40 weeks the 3 volume averaged 30 mm . We estimate that a desirable tumor volume will be reached after these 40 weeks and the histopathology will reveal a squamous cell carcinoma, without hampering the animal’s welfare. The two most frequent used experimental procedures for investigating therapies are in vitro cell lines and the nude mouse. The 4NQO model has several advantages and dis- advantages over these laboratory experiments. Compared with the cell lines and the nude mouse model, the close similarity of the 4NQO model to the physiological process is a big beneﬁt. The biggest drawback of the nude mouse model is the lack of an immunocompetent component. The weakness of the hamster cheek pouch model is the earlier mentioned dissimilarity of the tumors with the human equivalent. Application of DMBA causes an inﬂammatory response and necrosis making it hard to study early squa- mous lesions (Nauta et al. 1996). No inﬂammatory reaction or necrosis was seen in our specimens. Other drawbacks of this model are that the cheek pouch has no anatomic counterpart in human, the epithelium of the cheek pouch is signiﬁcantly thinner than other parts of the oral mucosa of humans and mice and the tumors seem to progress from papillomas (MacDonald 1981; Nauta et al. 1995), which is uncommon in humans and was not seen in our samples. The clear disadvantage of the 4NQO model is that both the cell line and the nude mouse are less time consuming and that cell line experiments are less costly and most of the time readily available. In our conclusion taking both advantages and disad- vantages in to account the 4NQO oral tumor model has clear beneﬁts and seems suitable for therapeutic research applications.