Enhanced L1CAM expression on pancreatic tumor endothelium mediates selective tumor cell transmigration.

L1 cell adhesion molecule (L1CAM) is a transmembrane cell adhesion molecule initially defined as a promigratory molecule in the developing nervous system that appears to be also expressed in some endothelial cells. However, little is known about the functional role of L1CAM on endothelial cells. We observed that L1CAM expression was selectively enhanced on endothelium associated with pancreatic adenocarcinoma in situ and on cultured pancreatic tumor-derived endothelial cells in vitro. L1CAM expression of endothelial cells could be augmented by incubation with immunomodulatory cytokines such as tumor necrosis factor alpha, interferon gamma, or transforming growth factor beta 1. Antibodies to L1CAM and the respective ligand neuropilin-1 blocked tube formation and stromal cell-derived factor 1beta induced transmigration of tumor endothelial cells in vitro. L1CAM expression on tumor-derived-endothelial cells enhanced Panc1 carcinoma cell adhesion to endothelial cell monolayers and transendothelial migration. Our data demonstrate a functional role of L1CAM expression on tumor endothelium that could favor metastasis and angiogenesis during tumor progression.

Detection and functional analysis of tumor infiltrating T-lymphocytes (TIL) in liver metastases from colorectal cancer.

BACKGROUND: Tumor-infiltrating T lymphocytes (TIL) play an important role in primary colorectal cancer, but their activity in liver metastases has not yet been investigated. The aim of this study was to examine whether tumor-selective infiltration, activation, and cytotoxic activity of TIL can be demonstrated in situ in colorectal liver metastases. METHODS: TIL were obtained from liver metastases and corresponding normal liver tissue of 16 patients with colorectal liver metastases. Characterization of TIL in situ was performed by multicolor flowcytometric analysis. Presence of tumor antigen-reactive T cells was evaluated by interferon gamma Elispot analysis. RESULTS: TIL in colorectal liver metastases responding against tumor antigens were present in most patients. Although the proportions of CD3(+) T cells were comparable in liver metastasis and normal liver tissue, metastases contained significantly enhanced proportions of CD4(+) cells (49% vs. 22%, P < .001). Among all CD4(+) T helper cells, the proportion of activated (CD4(+)CD25(+)) effector cells was significantly increased in liver metastases (15.0% vs. 7.8%, P = .003). Metastases showed significantly higher proportions of activated (CD69(+) [70.1% vs. 49.8%, P = .02] and CD25(+) [4.1% vs. .6%, P = .06]) and cytotoxically active (CD107a(+)) CD8(+) TIL (3.2% vs. 1.3%, P = .03). Importantly, the presence of activated T helper cells correlated with the frequencies of cytotoxic T lymphocytes that exerted cytotoxic activity in situ (P = .02). CONCLUSION: CD4(+) and CD8(+) TIL are selectively activated in liver metastases, and cytotoxic T lymphocytes exert tumor-selective cytotoxic activity in situ in the presence of activated T helper cells, suggesting the requirement of in-situ-activated T helper cells for efficient cytotoxic T lymphocytes effector function.

High frequencies of functional tumor-reactive T cells in bone marrow and blood of pancreatic cancer patients.

Pancreatic cancer is characterized by aggressive growth and treatment resistance. New approaches include immunotherapeutic strategies but the type and extent of spontaneous immune responses against tumor antigens remains unclear. A dominance of TH2 cytokines in patients' sera reported previously suggests systemic tumor-induced immunosuppression, potentially inhibiting the induction of tumor-reactive T cells. We characterized the localization, frequencies, and functional potential of spontaneously induced memory T cells specific for individual tumor antigens or the tumor-associated antigen mucin-1 in the peripheral blood and bone marrow of 41 pancreatic cancer patients. We found high numbers of tumor-reactive T cells in all bone marrow samples and in 50% of the blood samples. These cells secreted the TH1 cytokine IFN-gamma rather than TH2 cytokines upon stimulation with tumor antigens. Although consistently induced during pancreatic cancer, T cells specific for pancreatic antigens were not detected during chronic pancreatitis, suggesting that their evaluation may be of diagnostic use in both diseases. Freshly isolated T cells from cancer patients recognized autologous tumor cells and rejected them in vitro and in a xenotransplant model in vivo, suggesting their therapeutic potential. Thus, tumor antigen-specific T cell responses occur regularly during pancreatic cancer disease and lead to enrichment of tumor cell-reactive memory T cells in the bone marrow. The bone marrow can therefore be considered an important organ for antitumor immune responses in pancreatic cancer.

Role of tumor endothelium in CD4+ CD25+ regulatory T cell infiltration of human pancreatic carcinoma.

BACKGROUND: Regulatory T (Treg) cells have been detected in human carcinomas and may play a role in preventing the rejection of malignant cells. METHODS: We quantified Treg cells and the expression of the addressins and the respective ligands that attract them in blood and in human pancreatic tumors and adjacent nonmalignant tissues from 47 patients. The capacity of Treg cells to adhere to and transmigrate through autologous endothelial cells was tested in vitro using spheroid adhesion assays and in vivo using a xenotransplant NOD/SCID model and in the presence and absence of antibodies to addressins. All statistical tests were two-sided. RESULTS: More Treg cells infiltrated pancreatic carcinomas than adjacent nonmalignant pancreatic tissues (120 cells per mm2 versus 80 cells per mm2, difference = 40 cells per mm2, 95% confidence interval [CI] = 21.2 cells per mm2 to 52.1 cells per mm2; P<.001). In contrast to conventional CD4+ T cells, more blood-derived Treg cells adhered to (1.0% versus 5.2%, difference = 4.2%, 95% CI = 2.7% to 5.6%; P<.001) and transmigrated through (3332 cells versus 4976 cells, difference = 1644 cells, 95% CI = 708 cells to 2580 cells; P = .008) autologous tumor-derived endothelial cells in vitro and in vivo (458 cells versus 605 cells, difference = 147 cells, 95% CI = 50.8 to 237.2 cells; P = .04). Tumor-derived endothelial cells expressed higher levels of addressins--including mucosal adressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD62-E, and CD166--than endothelial cells from normal tissue. Experiments using antibodies to addressins showed that transmigration was mediated by interactions of addressins, including MAdCAM-1, VCAM-1, CD62-E, and CD166 with their respective ligands, beta7 integrin, CD62L, and CD166, which were expressed specifically on Treg cells. CONCLUSIONS: Tumor-induced expression of addressins on the surface of endothelial cells allows a selective transmigration of Treg cells from peripheral blood to tumor tissues.

Tumor infiltrating T lymphocytes in colorectal cancer: Tumor-selective activation and cytotoxic activity in situ.

OBJECTIVE: To examine whether tumor-selective infiltration, activation, and cytotoxic activity of tumor infiltrating T lymphocytes (TIL) can be demonstrated in situ in colorectal cancer samples. SUMMARY BACKGROUND DATA: Recent studies indicated a correlation between the presence of TIL and an improved prognosis in colorectal cancer. However, tumor-selective activation and cytotoxic activity of CD8 TIL in situ in colorectal cancer patients have not yet been examined. METHODS: Tumor samples from 49 patients and corresponding normal mucosa samples from 23 patients with colorectal cancer (UICC stages II-IV) were examined for TIL. Two-color fluorescence immunohistochemistry and multicolor flowcytometric (FACS) analysis were used for quantification of CD8 T cells and measurement of their activation status (CD69-expression) and cytotoxic activity (CD107a-expression) in situ. Presence of tumor antigen-reactive T cells in tumor, blood, and bone marrow was evaluated by IFN-gamma Elispot analysis. RESULTS: While absolute numbers of CD8 T cells were similar, CD4 T helper cells were significantly increased in tumor tissue compared with normal mucosa. There was a significantly higher proportion of activated and cytotoxically active CD8 TIL in colorectal cancer compared with normal mucosa. Increased activation, cytotoxic activity, and functional reactivity of TIL were correlated with the presence of functional tumor antigen-reactive T cells in the blood and bone marrow. The proportion of activated TIL decreased significantly with higher tumor stage. CONCLUSIONS: Tumor-selective activation and cytotoxic activity of CD8 TIL and tumor-selective migration of CD4 T helper cells were demonstrated in colorectal cancer for the first time. Our data support the immunogenicity of colorectal cancer and suggest clinical significance of tumor-specific immune responses.