In a previous study, we employed a histomathematical analysis strategy to measure protein levels in the prostate cancer microenvironment and observed a decrease in the number of T lymphocytes in the vicinity of tumor epithelium relative to normal epithelium and prostatic intraepithelial neoplasia (PIN), while benign prostate hyperplasia (BPH) showed an intermediate value between that of the cancer and non-cancer cells (Figure 1) [9]. These results were generated using CD3 antigen as a pan-T cell marker, thus we could not determine the identity of the tumor-dysregulated lymphocytes at that time. The aim of the present study was to further investigate this phenomenon by measuring the lymphocyte sub-types associated with the epithelial phenotypes in prostate cancer specimens, and to investigate the cytokines that mediate the process. Both quantitative immuno-histochemistry and expression microarray analysis of microdissected cell populations were utilized.

Five whole-mount prostate tissues were collected anonymously from patients undergoing radical prostatectomy at the Catholic University Hospital in Santiago Chile. A complete transverse section of each excised prostate was embedded in O.C.T. medium, and then flash frozen at -80°C. The frozen tissue blocks were anonymized and shipped to the National Cancer Institute as part of an IRB-approved protocol. Five localized primary prostate cancer cases with low to moderate Gleason grades were selected on the basis of both adequate tumor size and the ability to laser capture microdissect tumor-associated stroma without contamination by infiltrating tumor or inflammatory cells.

Five mm thick tissue sections were immunostained with three antibodies according to a standard immunohistochemistry (IHC) protocol (DAKO Cytomation EnVision system-HRP. Carpinteria, CA). Sections were incubated for 1 hour at RT with one of the following primary antibodies: CD4, mouse anti-human (Zymed labs, San-Francisco CA, cat # 08-0101), pre-diluted; CD8, mouse anti-human (DAKO Cytomation, Carpinteria, CA, cat #M0716), 1:20; CD20, mouse anti-human (DAKO Cytomation, Carpinteria, CA, cat #M0755), 1:200. Slides were counterstained with hematoxylin and positively stained cells were identified by the presence of a brown precipitate. Negative cells were identified by the absence of a brown precipitate and the presence of only blue counterstain.

A detailed experimental protocol appears elsewhere [11]. In short: Recut histological sections from five frozen whole-mounted cases were laser capture microdissected (PixCell lle, Arcturus Engineering, Inc., Mountain View, CA) in four separate histopathologically-defined regions: tumor epithelium; tumor-associated stroma; normal epithelium; and normal stroma. Linearly amplified RNA samples were hybridized to Affymetrix U133 plus 2.0 GeneChips (Affymetrix Inc., Santa Clara, CA). Raw data signal intensities were quantified using the Gene Chip Operating Software (GCOS) provided by Affymetrix. Intensities were normalized to remove background signal and filtered such that only perfectly matched probe sets were retained. The data were then analyzed by comparing tumor epithelium to normal epithelium and tumor stroma to normal stroma. Data sets were uploaded into the National Cancer Institute's microarray analysis program (mAdb) to perform molecular ontology and pathway analysis. Differentially expressed transcripts specifically related to immune function were then identified.

In general, the same protocol that was used in our previous histomathematical report was utilized [9]. Briefly, a pathologist (JWG) analyzed the IHC stained tissue sections from all ten cases in the study and photographed four representative epithelial regions: a) Normal epithelium; b) Benign prostate hyperplasia (BPH); c) Prostatic intraepithelial neoplasia (PIN); c) Carcinoma

Qualitatively, IHC for CD4 revealed occasional to abundant immuno-positive lymphocytes in the epithelial compartment and the surrounding stroma of all four components analyzed. In contrast, IHC for CD8 cells showed only a few scattered immuno-positive lymphocytes in the epithelium and stroma of normal, BPH, PIN, and tumor (Figure 2). CD20-positive cells (B lymphocytes) were rarely observed in the stroma overall and were not seen associated with any of the epithelial components except for cancer where rare immuno-positive cells were identified.

Quantitative analyses of these results are summarized in Figure 3 and Table 1. CD4 positive cells were observed at a similar abundance level in all sub-regions of the prostate. In contrast, CD8 positive cells were significantly less common in regions containing cancer and BPH as opposed to normal epithelium and PIN. Reductions in CD8+ lymphocytes were noted for both the overall histological field (tumor and stroma) as well as the epithelial components of BPH and cancer (Figure 4). CD20 positive cells were generally scarce throughout the prostate and were not significantly associated with any of the epithelial regions (Figure 3).

We next assessed expression levels of immune-related transcripts in normal and tumor areas to examine the associated cytokine profile. Data from previous global mRNA measurements from five prostatectomy specimens with cancer were re-analyzed specifically for immune-related features [11]. Both the epithelial and stromal components were individually assessed. Twenty-four genes related to immune function were differentially expressed in the tumor epithelium relative to the normal epithelium, four up-regulated and twenty down-regulated (Table 2). Nine immune-related transcripts were differentially expressed in the tumor-associated stroma (six up-regulated, three down-regulated). The two most highly up-regulated immune-related transcripts in the tumor epithelium were chemokine ligand 14 (CXCL14, 2.55 fold difference), and interleukin 17 receptor B (IL17RB, 2.08 fold difference). CXCL14 was also the most highly dysregulated transcript in the tumor-associated stroma (3.26 fold difference). In contrast, CXCR4 was the third-most negatively regulated transcript (-1.98 fold difference) in the tumor epithelium behind suppressor of cytokine signaling 3 (SOCS3.-2.21 fold difference) and CCAAT/enhancer binding protein (CEBPB.-2.21 fold difference).

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 [15-19]. 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, 16]. 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.