178 in mice (Eulderink 1972). Nowadays xenograft models are widely used. In this model human cells are injected and grow in immunodeﬁcient mice. Advantages are the speed and certainty of tumor development. The premier limita- tion is the lack of functional T lymphocytes in nude mice resulting in a non physiological tumor response. The widely used hamster cheek pouch model in which 7,12-dimethlbenz(a)anthrance is used to induce tumors has a disadvantage because the tumors are unalike the human counterparts (Kanojia and Vaidya 2006). After the intro- duction of 4-nitroquinoline-1-oxide (4-NQO) for inducing tumors in rats (Wallenius and Lekholm 1973), it has been extensively used in both rats and mouse and can useful for analyzing the effects of anti-tumor agents (Suzuki et al. 2006). The 4NQO oral squamous cell carcinoma model is a lengthy multi-step process in which eventual invasive squamous cell carcinoma is attained after several months. Once established the tumors are suitable for studying the therapeutic effect of various therapies including gene therapy. Intratumoral injection of recombinant adeno-viral vectors is mainly used in preclinical models (St George 2003). Therefore at the time of injection a minimum tumor volume must be attained and the realization of squamous cell carcinoma must be assured. Up till now, in the 4NQO mouse model, this time point is unknown. The present study was set up to investigate the time needed for sufﬁcient tumor volume to be developed enabling intra-tumoral injection of anticancer agents such as gene therapy. As important, we have identiﬁed that the generated mouse oral tumors resembles human oral tumor formation. Materials and methods Animals and treatment with carcinogen Thirty-two 7- and 8-weeks-old male CBA mice weighing between 23 and 27 g were purchased from Harlan in Zeist, The Netherlands. The experiments were approved in advance by the Leiden University Animal Welfare Com- mittee. All animals were kept at standard laboratory con- ditions of alternating 12-h periods of light and darkness and were fed sterilized laboratory chow and water ad libitum. The carcinogen 4-nitroquinoline-1-oxide (Fig. 1) was dissolved in propylene glycol to a ﬁnal concentration of 5 mg/ml. For each session a fresh aliquot was used. Anesthesia was achieved by inhalation of halothane vapour and the tongue was stroked once with a no 3 camel hair brush (Hawkins et al. 1994), which had been dipped in the 4NQO solution. The mice were restrained from drinking the ﬁrst hour after 4NQO application. The mice were treated with 4NQO during 16 weeks with a frequency of 3 123 Fig. 1 Chemical structure of 4-nitroquinoline 1-oxide (4NQO) J Mol Hist (2009) 40:177–181 weekly treatments. All mice were carefully inspected daily and weighed weekly. Pathological examination and immunohistochemistry After the ﬁnal 4NQO application the oral mucosa of each individual mouse was examined under inhalation anesthe- sia every week. The volume of exophytic tumors was calculated each week by measuring three dimensions (length, height and width). After 24 weeks following the start of the experiment 6 mice were sacriﬁced and tongue tissue was removed and ﬁxed in freshly made 4% buffered formalin. Subsequently there were groups of 6 mice sac- riﬁced after 28, 32, 36 and 40 weeks. After parafﬁn-embedding, serial sections of 4 lm width were made and one in ﬁve subsequent sections were stained with hematoxylin-eosin (HE). The epithelia was examined by a pathologist in our institution and classiﬁed as either normal epithelia, mild dysplasia, moderate dys- plasia, sever dysplasia or invasive squamous cell carci- noma. Dysplastic epithelia and invasive squamous cell carcinoma were subsequently investigated by immunohis- tochemistry with cyclin D1 and E-cadherin. The following antibodies were used from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): the rabbit polyclonal antibody against cyclin D1 (H-295, dilution 1:100) and a rabbit polyclonal antibody against E-cadherin (H-108, dilution 1:50). After the slides were deparafﬁnized and rehydrated, antibodies were applied according to the manufacture’s protocol. Results Histological ﬁndings The ﬁrst macroscopic changes were seen 19 weeks after starting the experiment. After 24 weeks some tongues did not show any pathological changes (Fig. 2a) although at the same phase hyperplasia without atypia and mild dys- plastic changes were detected (Fig. 2b). Four weeks further on, advance in atypia was found and moderate (Fig. 2c)