The results of the present study indicate that decreased numbers of CD8+ lymphocytes are associated with prostate tumors, and that there is a significant dysregulation of cytokines in histological regions that contain cancer. T-cell lymphocytes are an important element in the immune response and are regulated through a cascade of cellular and cytokine-based interactions. CD4+ lymphocytes, or T-helper cells, recognize antigens in conjunction with antigen presenting cells (APC) and produce cytokines that induce a wide response against foreign proteins on viruses or those on tumor cells [12
]. In contrast, CD8+ cytotoxic T lymphocytes directly attack and kill infected cells or cancers via binding of the CD8 glycoprotein and T-cell receptor to Class I MHC molecules on the target cells. CD8+ cells release cytotoxic perforin and granzymes that initiate a series of cell killing events that include formation of aqueous channels, activation of cellular cysteine proteases and caspases, and ultimately apoptosis of the infected or cancerous cell [13
]. A second way to induce apoptosis is via cell-surface interactions between the CD8+ cells and the surface protein FAS ligand
(FasL)(Apo1L)(CD95L) that binds to Fas (Apo1)(CD95) molecules expressed on the target cell [13
]. Engagement of Fas with FasL allows for recruitment of the death-induced silencing complex (DISC). The Fas-associated death domain (FADD) translocates with the DISC, allowing recruitment of procaspases 8 and 10 which then activate the effector caspases 3, 6, and 7, leading to cleavage of death substrates such as lamin A, lamin B1, lamin B2, PARP, and DNAPK. The final result is apoptosis of the target cell that expresses Fas [13
With respect to the current study, the observed decrease in CD8+ lymphocytes may facilitate tumor development by removing this important tumor-cytotoxic aspect of the immune response. This result is consistent with the previous work of Naoe and colleagues [14
] who assessed MHC class I expression and lymphocytes in both benign and malignant prostate tissue and found significantly fewer CD8+ T lymphocytes associated with prostate tumor cells than in benign prostate, including a significant correlation between the number of CD8+ T lymphocytes and MHC class I expression. The authors concluded this relationship might be important in cancer growth. Our study extends this observation further by analyzing CD8+ lymphocytes in the context of a detailed histopathological examination, and further analyzing cytokine profiles at the transcript level.
Overall, the cytokine mRNA pattern showed mostly down-regulation of genes in tumor cells and up-regulation in the tumor stroma. The directionality of immune-related mRNA changes is the same as we have previously observed in microdissection-based global gene expression studies of both prostate cancer and esophageal squamous cell carcinoma; more down-regulated transcripts in tumor cells as compared to corresponding normal epithelium, and more up-regulated mRNAs in tumor associated stroma versus normal quiescent stroma [11
]. Changes in genes encoding inflammatory proteins included up-regulation of PAWR and IGSF4 and down regulation of SOCS3, CEBPB, IL6ST, CLU and IFI16 in the tumor cells. In the tumor-associated stroma, up-regulation of IGSF4D, CD24, and TNFSF10 was seen, and down-regulation of HLA-C, IGKC, and C3 was observed .
The data on CXCR4 were among the most interesting findings in the study as these results differed from some reports in the literature and there is extensive evidence that CXCR4 is involved in development or progression of multiple different tumor types [15
]. CXCR4 is a seven transmembrane G protein-coupled receptor that plays a role in many physiological processes involving cell migration and cell fate, such as vascular formation in the gastrointestinal tract and other sites of angiogenesis, stem cell homing, neural development, and immune cell trafficking [15
]. CXCR4's ligand, CXCL12/ SDF-1, is constitutively expressed in many cell types, including endothelium, keratinocytes, sweat glands, and sites of chronic inflammation [17
]. The signaling mechanism by which CXCR4 promotes cell migration through CXCL12 is mediated through the Ga13
-Rho pathway [16
]. Apart from leukocytes, in which CXCR4 is ubiquitously present, expression of CXCR4 is low or absent in many normal tissues, including breast, ovary, and colon. However, CXCR4 is implicated in various pathological conditions, including metastatic spread and human immunodeficiency virus infection, and CXCL12 is expressed in common sites of metastasis (lung, liver, bone). CXCR4 is also frequently expressed by human cancer cells and to date more than 20 different tumor types are known to up-regulate CXCR4 protein [18
]. In contrast, our data showed decreased levels of CXCR4 mRNA in microdissected tumor cells. A recent report by Singh et al studying CXCR4 in prostate cancer provides a potential mechanistic explanation for this finding [19
]. These investigators found that CXCR4 induces caspase-mediated apoptosis in prostate tumor cells when stimulated by gp-120-IIIB, thus diminished levels of CXCR4 could have a protective effect. Based on these findings, additional study of CXCR4 and its ligands are warranted in prostate cancer.
In summary, by using an open, non-directed histomathematics analysis technique, we were able to identify differential expression in the inflammatory infiltrate in human prostate cancer. This report highlights the importance of quantitative, high throughput approaches in pathological specimens, efforts that can lead to new insight into disease pathogenesis.