Signatures associated with rejection or recurrence in HER-2/neu-positive mammary tumors

Abstract

We have previously shown T-cell-mediated rejection of the neu-overexpressing mammary carcinoma cells (MMC) in wild-type FVB mice. However, following rejection of primary tumors, a fraction of animals experienced a recurrence of a neu antigen-negative variant (ANV) of MMC (tumor evasion model) after a long latency period. In the present study, we determined that T cells derived from wild-type FVB mice can specifically recognize MMC by secreting IFN-gamma and can induce apoptosis of MMC in vitro. Neu transgenic (FVBN202) mice develop spontaneous tumors and cannot reject it (tumor tolerance model). To dissect the mechanisms associated with rejection or tolerance of MMC tumors, we compared transcriptional patterns within the tumor microenvironment of MMC undergoing rejection with those that resisted it either because of tumor evasion/antigen loss recurrence (ANV tumors) or because of intrinsic tolerance mechanisms displayed by the transgenic mice. Gene profiling confirmed that immune rejection is primarily mediated through activation of IFN-stimulated genes and T-cell effector mechanisms. The tumor evasion model showed combined activation of Th1 and Th2 with a deviation toward Th2 and humoral immune responses that failed to achieve rejection likely because of lack of target antigen. Interestingly, the tumor tolerance model instead displayed immune suppression pathways through activation of regulatory mechanisms that included in particular the overexpression of interleukin-10 (IL-10), IL-10 receptor, and suppressor of cytokine signaling (SOCS)-1 and SOCS-3. These data provide a road map for the identification of novel biomarkers of immune responsiveness in clinical trials.

Figure 1. T cells derived from wild-type FVB mice will induce apoptosis in MMC in vitro but fail to reject MMC in FVBN202 mice following AIT

A) Flow cytometry analysis of MMC after 24 hrs culture with splenocytes of FVB mice following three color staining. Gated neu positive cells were analyzed for the detection of annexin V+ and PI+ apoptotic cells. Data are representative of quadruplicate experiments. B) Donor T cells were enriched from the spleen of FVB mice using nylon wool column following the rejection of MMC. FVBN202 mice (n=4) were injected with CYP followed by inoculation with MMC (4 × 10⁶ cells/mouse), and tail vein injection of donor T cells. Control groups were challenged with MMC in the presence or absence of CYP treatment. Tumor growth was monitored twice weekly.

Figure 2. Gene expression profiling and gene oncology pathway analyses in tumor regressing and tumor non-regressing groups

(A) Unsupervised cluster visualization of genes differentially expressed among regressing tumors (pink bar) and non regressing tumors (blue bar = evasion model; turquoise bar = tolerogenic model). MMC tumors were harvested 10 days after challenge and hybridized to 36k oligo mouse arrays. 11256 genes with at least 3fold ratio change and 80% presence call among all samples were projected using log2 intensity. (B) Supervised cluster analysis (Student t test, p< 0.001 and fold change >3) comparing regressing tumors (pink bar) and non regressing tumors (blue bar = evasion model; turquoise bar = tolerogenic model). 2449 differentially expressed genes have been selected for further analysis. (C) Gene Ontology databank was queried to assign genes to functional categories and up-regulated genes within the tumor regression group to functional categories.

Figure 3. Gene expression profiling and gene oncology pathway analyses in tolerance and evasion models

(A) Supervised cluster analysis (Student t test, p< 0.001 and fold change >3) comparing evasion (blue bar) and tolerogenic group (turquoise bar). 1326 differentially expressed genes have been visualized including also tumor regression samples (pink bar). Gene ontology pathway analysis projecting either up-regulated pathways in evasion group (B, 854 genes) or tolerogenic tumor models (C, 475 genes).