Anti-MHC class I alloantibodies have been implicated in the process of acute and chronic rejection because these Abs can bind to endothelial cells and transduce signals leading to the activation of cell survival and proliferation pathways. To characterize the role of the MHC class I-signaling pathway in the pathogenesis of Ab-mediated rejection, we developed a mouse vascularized heterotopic cardiac allograft model in which B6.RAG1 KO hosts (H-2Kb/Db) received a fully MHC-incompatible BALB/c (H-2Kd/Dd) heart transplant and were passively transfused with anti-donor MHC class I Ab. We demonstrate that cardiac allografts of mice treated with anti-MHC class I Abs show characteristic features of Ab-mediated rejection including microvascular changes accompanied by C4d deposition. Phosphoproteomic analysis of signaling molecules involved in the MHC class I cell proliferation and survival pathways were elevated in anti-class I-treated mice compared with the isotype control-treated group. Pairwise correlations, hierarchical clustering, and multidimensional scaling algorithms were used to dissect the class I-signaling pathway in vivo. Treatment with anti-H-2Kd Ab was highly correlated with the activation of Akt and p70S6Kinase (S6K). When measuring distance as a marker of interrelatedness, multidimensional scaling analysis revealed a close association between members of the mammalian target of rapamycin pathway including mammalian target of rapamycin, S6K, and S6 ribosomal protein. These results provide the first analysis of the interrelationships between these signaling molecules in vivo that reflects our knowledge of the signaling pathway derived from in vitro experiments.

Context.—In 1999, the World Health Organization redefined bronchioloalveolar carcinomas (BACs) as those neoplasms with only a pure lepidic growth pattern and no invasion. Objectives.—The present study examined 45 lung cancers with a BAC component (1) to determine whether these tumors would be classified as BACs by current World Health Organization standards, (2) to quantitate the BAC component within these tumors, and (3) to see if phenotypic differences exist between the so-called invasive and noninvasive regions of these tumors. Design.—Retrospective review of hematoxylin-eosin–stained slides and classification of histologic grade, tumor subtype, and percentage of pure BAC pattern, with further characterization by immunohistochemical staining for thyroid transcription factor 1, cytokeratin 7, cytokeratin 20, and Ki-67 antibodies. Results.—Only 7 (15.6%) of the 45 tumors examined could be classified as BAC by current strict World Health Organization criteria. Those tumors, classified as nonmucinous and mixed, showed similar immunohistochemical staining for cytokeratin 7, cytokeratin 20, and thyroid transcription factor 1; mucinous tumors showed disparate staining. Significant differences in immunohistochemical staining and tumor cell proliferation were seen for the regions of tumors designated as lepidic, infiltrative, and leading edge and for the regions of tumors with different histologic grades (ie, well, moderately, and poorly differentiated). Conclusions.—Nonmucinous and mixed BACs are phenotypically similar and show identical immunohistochemical staining patterns; mucinous tumors, on the other hand, show disparate immunohistochemical staining. Pulmonary neoplasms designated as adenocarcinomas with a BAC component represent a heterogenous group with a range of cell types, differentiation, growth, and immunophenotypes. Within an individual neoplasm, there are regional differences in these parameters as well.

A1 The zinc-finger E-box-binding transcriptional repressor Snail has been implicated in tumor progression of several malignancies. Best known as a transcriptional repressor of the adherens junction component E-cadherin, Snail has predominantly been associated with the epithelial-mesenchymal transition (EMT), invasion, and metastasis. However, the role of Snail in non-small cell lung cancer (NSCLC) is not yet defined. Immunohistochemistry of human lung adenocarcinoma and squamous cell carcinoma sections revealed specific nuclear staining of tumor cells in all patient samples. A human lung adenocarcinoma cell line H441 was stably transduced with a Snail expressing retroviral vector. Western analysis verified up-regulation of Snail and down-regulation of E-cadherin in the Snail over-expressing cells (H441-Snail) as compared to vector control cells (H441-V). In three-dimensional spheroid culture, H441V cells grew into large, tight spheroids while H441-Snail cells were markedly discohesive, reflecting a Snail-mediated phenotypic change consistent with EMT. To examine the effects of Snail over-expression in vivo, severe combined immunodeficiency (SCID) mice were injected subcutaneously with either H441-Snail cells or with H441-V cells. Mice were sacrificed 6 weeks later. The primary tumor burden in mice bearing H441-Snail tumors was five fold greater than the primary tumor burden of mice bearing H441-V tumors (p<0.005). To evaluate the incidence of metastases, these cell lines were tested in an orthotopic model. H441-V and H441-Snail cells were transthoracically injected into the left lung of SCID mice. Organs were then harvested and analyzed by flow cytometry gated on the human marker, CD49b. The incidence of metastases to the right lung, liver, bone marrow, and adrenal glands were significantly increased in the mice bearing H441-Snail tumors (p<0.05). Because H441-Snail tumors appeared grossly more hemorrhagic as compared to H441-V tumors at the time of harvest, primary tumors were homogenized and analyzed by ELISA for levels of two angiogenic factors known to play a role in NSCLC tumor angiogenesis: CXCL8 and CXCL5. H441-Snail tumors were associated with increased levels of CXCL8 (p<0.05) and CXCL5 (p<0.05) as compared to H441-V tumors. Based on these results, SCID mice injected with H441-Snail cells were treated with a CXCR2 (CXCL8 and CXCL5 receptor) blocking antibody. These mice exhibited reduced tumor burden and metastases as compared to mice bearing H441-Snail tumors that were treated with a control antibody. In summary, Snail contributes to tumor progression in NSCLC by inducing tumor angiogenesis, as evidenced by elevated levels of angiogenic factors (CXCL8 and CXCL5) and the reversal of increased tumor burden and metastases with CXCR2 blockade. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A1.

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