Campbell and Moorehead BMC Cancer 2011, 11:480 http://www.biomedcentral.com/1471-2407/11/480 Invasion assays Recurrent cell lines were transfected with siRNA as described above. Cell invasion was assessed using 8 μm BioCoat™ Matrigel invasion chambers (BD Biosciences, Franklin Lakes, NJ, USA) 24 h after transfection. Cham- bers were placed in 12-well dishes and the top and bot- tom compartment were rehydreated with serum free and full serum media (DMEM containing only gluta- mine, sodium pyruvate, antibiotic/antimycotic and HEPES buffer) respectively for two hours at 37°C. Sub- sequently, 2.0 × 104 RJ348 or 1.0 × 105 RJ423 cells (pre- viously transfected) suspended in either 200 or 500 μL of serum free media were plated in the upper chamber, while 300 μL of fresh full serum media was added to the bottom chamber. Invasion was allowed to proceed for 24 h at 37°C. At this time, 500 μL of 5% gluteralde- hyde was added to the bottom chamber and invading cells were fixed for 10 min. Subsequently, wells were washed three times with distilled water and incubated with 0.5% toluidine blue for 20 min. Liquid was then aspirated from both chambers and the lower chamber was washed three times with water. A cotton swab was used to wipe the inner surface of the upper chamber to remove non-invasive cells. Invading cells were visualized and images were captured using an Olympus IX81 microscope (Olympus, Markham, ON) at 20 × magnifi- cation and Image Pro Plus V.5.0.2.9 software (MediaCy- bernetics, Bethesda, MD). Cells were counted from 3-4 fields of view and the average number of invading cells per field of view was expressed relative to respective controls. Results represent the average of three replicates. Statistics Results are presented as the mean ± standard error. Sta- tistical significance was assessed through either a stu- dent’s t-test (for analyzing two means) or analysis of variance (ANOVA) followed by a post hoc Tukey test (for more than two means). Results PDGFRs are overexpressed in IGF-IR-independent mammary tumors A previous study from our lab showed that downregula- tion of the IGF-IR transgene in established mammary tumors in MTB-IGFIR transgenic mice resulted in stable regression of a majority of the tumors, however, a small proportion of the tumors resumed growth and expressed only very low levels of IGF-IR [13]. A DNA microarray was then performed on wild type mammary tissue, mammary tumors induced by high levels of the IGF-IR transgene (also called primary mammary tumors or PMTs) and mammary tumors that resumed growth fol- lowing IGF-IR transgene downregulation that only Page 4 of 13 expressed low levels of IGF-IR (also called IGF-IR-inde- pendent recurrent spindle tumors or RSTs). The micro- array data indicated that both PDGFRa and PDGFRb were upregulated in the IGF-IR-independent, recurrent tumors compared to the IGF-IR-dependent, primary tumors [14]. Based on these findings it was hypothesized that PDGFR overexpression is important for tumor sur- vival following IGF-IR downregulation. As a first step, western blotting was performed for PDGFRa and PDGFRb in primary mammary tumors (IGF-IR-dependent) and IGF-IR-dependent recurrent tumors of MTB-IGFIR transgenic mice. Although there was some inter-tumor variability, the IGF-IR-independent recurrent spindle tumors displayed an overall increase in both PDGFRa and PDGFRb, with some tumors displaying especially high levels (Figure 1a). Expression of PDGFRs was also assessed using immunohistochemistry. In primary tumors, only sparse expression of both PDGFRa and b was observed (Figure 1b-e) while in IGF-IR-independent recurrent tumors, these receptors were often found to be highly expressed (Figure 1f-i). PDGFRs are inversely regulated with IGF-IR expression To determine if PDGFRs were rapidly upregulated in response to IGF-IR downregulation, MTB-IGFIR trans- genic mice with palpable mammary tumors were switched to normal food (not containing doxycycline). IGF-IR levels were previously shown to decrease dramati- cally at 24 h and 48 h following doxycycline withdrawal [13]. As shown in Figure 2a, both PDGFRa and PDGFRb were observed to increase at both time points evaluated (24 h and 48 h). Consistent with the western blot data, qRT-PCR analysis revealed that IGF-IR mRNA was sig- nificantly decreased in the mammary tumors following doxycycline withdrawal while the levels of PDGFRa and PDGFRb mRNA were elevated at least 2.9-fold (Table 1). To further validate the induction of PDGFRa and PDGFRb following IGF-IR transgene downregulation several cell lines were utilized. First, a cell line estab- lished from a primary, IGF-IR-dependent mammary tumor (RJ345) and two cell lines from unique IGF-IR- independent recurrent tumors (RJ348 and RJ423) were examined. The RJ345 cells maintained high levels of IGF-IR while the RJ348 and RJ423 cells expressed much lower levels of IGF-IR. Consistent with the in vivo find- ings, tumor cells expressing low levels of IGF-IR (RJ348 and RJ423) expressed high levels of PDGFRa and PDGFRb compared to the RJ345 cells (Figure 2b). Next, the RM11A cell line was examined in vitro and in vivo. The RM11A cell line was also established from a MTB-IGFIR mammary tumor and this cell line has been more extensively characterized [23]. The RM11A cells express high levels of IGF-IR when cultured in the presence of doxycycline and only express low levels of