BMC Cancer 2009, 9:251 http://www.biomedcentral.com/1471-2407/9/251 been described. Elevated expression of TAA90K has been detected in breast, gastric, lung and pancreatic cancers [32-35] and, in the cases of lung cancer and malignant mesothelioma [33,34], protein levels correlated with metastasis and predicted survival or disease manifesta- tion. In a recent study [36], we have also detected elevated TAA90K expression in colorectal adenocarcinoma. Although the function of aberrantly expressed TAA90K in cancer is unclear, it has been shown to mediate β1- integrin-dependent cell adhesion [37], interact with vari- ous extracellular proteins including extracellular matrix proteins and galectins [36-38], and stimulate expression of promatrilysin [39], previously implicated in colon can- cer progression [40]. However, the primary and physiological role of TAA90K appears to be, according to the current knowledge, in immune regulation. This glycoprotein has been shown to be a potent immune stimulator with positive effects on the generation of cytotoxic effector cells (NK/LAK) from human peripheral blood mononuclear cells (PBMC) [41,42]. In fact, 90 K in human milk may be protective against acute respiratory infections [43]. The protective effect seems to be related to the amount of 90 K ingested during the first few days, as milk 90 K concentrations sub- sequently decline rapidly in all mothers [44]. These find- ings suggested that 90 K initiates a defense mechanism in the infant early in life. In recent years, cyclophilin C-associated protein (CyCAP) has been identified as the murine orthologue of TAA90K with up to 80% homology in their amino acid sequence [45,46]. CyCAP is a widely expressed secreted glycopro- tein that modulates the host response to bacterial endo- toxin [47]. In response to endotoxin, CyCAP-deficient mice overproduced interleukin 12 and interferon-γ sys- temically and tumor necrosis factor α locally, proinflam- matory molecules that also promote T helper 1 responses. Furthermore, macrophages stimulated in vitro with endo- toxin in serum deficient CyCAP-null cells secreted more TNF-α, supporting the proposal that CyCAP specifically down-modulates endotoxin signaling [47]. In this report, we have examined the contribution of CyCAP to colon hyperplasia and tumorigenesis in vivo before and after injection of the colon carcinogen azoxymethane as a possible animal model suitable for elucidating the role of TAA90K in colon tumorigenesis and progression, thereby providing a level of biological understanding not readily available in clinical disease. Methods Animals and azoxymethane treatment Female Balb/C mice (6–8 weeks) were obtained from the Animal Resources Center (University of Saskatchewan, Saskatoon, SK). Breeding pairs of Balb/C CyCAP knock- out mice were obtained from Dr. I. Weissman (Stanford University, San Francisco, CA) and used to set up an in- house breeding colony. Forty seven animals were tested routinely for the presence of pathogens. In particular, mice were free of Citrobacter freundii, a bacterium associ- ated with transmissible intestinal hyperplasia [48]. All experimental procedures were carried in accordance with guidelines from the Canadian Council on Animal Care. Forty seven female Balb/c wild-type and CyCAP knock- out mice (6–8 weeks old) were administered 6 weekly subcutaneous injections of azoxymethane (10 mg/kg) (Midwest Research Institute, MO) [49] for measurements of ACF or colon tumor incidence, respectively, using pro- cedures described previously [50]. Six weeks (ACF meas- urement) or five months (colon tumor measurement) post-injection, animals were sacrificed, the intestine removed and washed in ice-cold PBS. Colons were cut longitudinally, laid flat between two pieces of 3 mm filter paper and fixed for at least two hours in formalin (10% formaldehyde in phosphate buffered saline pH 7.0). For ACF and tumor measurements, colons were stained for 5 min in a solution of 1% methylene blue in PBS and destained for 5 min in PBS. Stained colons were examined microscopically for the presence of ACF, as described by Bird et al. [51] and also for the presence of tumors. The size of tumors (length in mm × width in mm) was meas- ured at a magnification of 40× using a gradicule (Precision Instruments, Vancouver, BC) calibrated with a slide micrometer. After the data were collected, the fixed colons of 5 untreated and 5 azoxymethane-treated wild-type and CyCAP knock-out animals were embedded in paraffin. Hematoxylin and eosin-stained five micron-thick serial sections were examined histologically. Measurements and cell counts All measurements and cell counts were performed on his- tologically non-tumorous mucosa, with no definite path- ological findings. The exact absolute length of each colonic crypt was measured in μm and the exact absolute number of cells per each crypt was recorded. In addition, only areas away from the tumors and lymphoid aggre- gates were measured to avoid so-called traditional mucosa or reactive changes. The thickness of the crypts was meas- ured in microns in 60 to 246 well-oriented crypts (all available well-oriented crypts were measured) using a cal- ibrated camera (Nikon Digital Sight DS-SM, Japan) mounted on the microscope (Nikon Eclipse 80i, Japan). The presence of the full thickness of the crypt with open lumen, to avoid tangentionally-sectioned crypts, was the only positive selection criterion. The same method was used for establishing the crypt proliferation fraction as measured by the number of Ki-67+ cells divided by the total number of cells per crypt. Immunohistochemical tests for Ki-67, cyclin D1, c-myc, lysozyme, and β-catenin were done as previously described [52,53]. In the majority Page 3 of 11 (page number not for citation purposes)