Immune prevention of mammary carcinogenesis in HER-2/neu transgenic mice: a microarray scenario

Abstract

Neoplastic transformation is a multistep process in which gene products of specific regulatory pathways are involved at each stage. Identification of these overexpressed or mutated gene products provides an unprecedented opportunity to address the immune system against defined antigens and eliminate transformed cells. Mice transgenic for these oncogenes (e.g. HER-2/neu, a prototype of deregulated oncogenic protein kinase membrane receptors) are ideal experimental models for assessing the potential of active immunization. The demonstration that vaccines can cure HER-2/neu transplantable tumors, prevent their onset and delay the progression of preneoplastic lesions in mice at risk suggests that efficient immunological inhibition of HER-2/neu carcinogenesis can be achieved by specific vaccination. To further explore this issue, halting of tumor progression in the mammary glands of BALB-neuT mice with two immunization protocols in two laboratories has been studied independently by DNA microarray analysis. Combination of the two sets of results revealed a clear correlation between them when the tumor mass was titrated by transcription profiling. It was also clear that both protocols induced a strong, polyclonal antibody response and halted tumor growth at a condition very similar to that at which the vaccination began. Differences in the expression profiles were mainly related to the expression levels of a few chemokines and T-cell-specific genes that may be in some way correlated with the efficacy of the vaccination. Last, combination of the expression data with the protection results indicated that chronic vaccination is needed to maintain an active IFN-gamma-mediated response in the mammary gland.

Fig. 1

Cross-study validation of data generated by Quaglino et al. [26] and De Giovanni et al. [; Astolfi, unpublished results] reveals significant agreement of the gene expression patterns in these two independent studies. Cross-experiment consistency of expression patterns was evaluated with an integrative correlation (IC) coefficient that quantifies cross-study reproducibility without relying on direct assimilation of expression measurements across experiments. a Histogram of the empirical distribution of the gene-specific correlation of correlations. b Scatterplot of the pairwise correlation comparison within the probe sets in the two datasets.

Fig. 2

Principal component analysis of the transcriptional profiling of gene expression linked to HER-2/neu neoplastic alteration and anti-HER-2/neu vaccination. a The panel shows the PCA experiment clustering generated using TMEV (http://www.tigr.org). Each light blue dot relates to the average of the experimental replicates for each gene in each experiment. Even though the data have been generated by two independent laboratories with slightly different approaches, the variance accounting for the differences between the groups is related to age, as the total variance is distributed around the blue axis. b Yellow dots showing transcriptional profiling at week 22 following vaccination at week 10 reveals that growth of the tumor mass is halted at week 10 (Cavallo’s laboratory). Since the profiles at week 15 and week 26 following vaccination at week 6 (red dots Lollini’s laboratory) resemble that of wk6nt mice, it is clear that vaccination time determines the stage at which tumor progression is halted.

Fig. 3

“The blind men and the elephant”: poem by John Godfrey Saxe.

Fig. 4

Whole mount of the fourth mammary gland of a 26-week-old BALB-neuT mouse vaccinated with allogeneic mammary carcinoma cells expressing HER-2/neu and engineered to produce IL-12. The black dot is the lymph node (LN). The gland is free from carcinoma and displays only some hyperplastic foci in the end buds.

Fig. 5

Hierarchical clustering of probe sets differentially expressed in vaccinated as opposed to nt mice. Probe sets were converted in virtual two-dye experiments by comparing all replicates of each group with the wk2pgr replicates. Virtual two-dye experiments of wk6nt, wk10nt, wk15vax, wk22pb and wk26vax, grouped by hierachical clustering, yielded five gene clusters characterizing the experimental groups. a Cluster strongly down-regulated in wk6nt and wk10nt mice, and encompassing three casein isoforms and WAP. b Cluster mainly up-modulated in Triplex-vaccinated mice and encompassing two chemokine genes, two MHC II antigens and one T-cell-specific GTPase. c Cluster with many “stromal” genes (protocollagens, actins and elastins) mainly expressed in wk6nt mice. d Cluster containing IFN-induced genes, up-modulated almost solely in triplex-vaccinated mice. e Cluster containing only Ig chain genes.

Fig. 6

Level of expression measured with microarrays of WAP and alpha, beta and gamma casein in mammary glands from non-transgenic BALB/c mice, pregnant mice, cell lines derived from tumors in HER-2/neu transgenic mice (1A-1E), mammary glands from HER-2/neu transgenic nt mice at different time points, and from vaccinated mice (Triplex, Prime-boost). Both vaccinations increase the level of expression of these genes, which are normally related to pregnancy and lactation.

Fig. 7

Virtual two-dye experiments showing Ig chain genes in wk6nt, wk10nt, wk15vax, wk26vax and wk22pb with respect to wk2pgn grouped by hierarchical clustering (Euclidean distance, average linkage). All the vaccinated mice show up-regulation of immunoglobulin genes with respect to nt mice. Triplex-vaccinated mice (wk15vax, wk26vax) whose mammary glands were collected with the lymph node display a higher Ig gene induction than wk22pb mice whose lymph node was not analyzed. This demonstrates that the B-cell response resides mainly in the lymph node rather than in the surrounding mammary tissue.