Until recently, the intrinsically high level of cross-talk between immune cells, the complexity of immune cell development, and the pleiotropic nature of cytokine signaling have hampered progress in understanding the mechanisms of immunosuppression by which tumor cells circumvent native and adaptive immune responses. One technology that has helped to shed light on this complex signaling network is the cytokine antibody array, which facilitates simultaneous screening of dozens to hundreds of secreted signal proteins in complex biological samples. The combined applications of traditional methods of molecular and cell biology with the high-content, high-throughput screening capabilities of cytokine antibody arrays and other multiplexed immunoassays have revealed a complex mechanism that involves multiple cytokine signals contributed not just by tumor cells but by stromal cells and a wide spectrum of immune cell types. This review will summarize the interactions among cancerous and immune cell types, as well as the key cytokine signals that are required for tumors to survive immunoediting in a dormant state or to grow and spread by escaping it. Additionally, it will present examples of how probing secreted cell-cell signal networks in the tumor microenvironment (TME) with cytokine screens have contributed to our current understanding of these processes and discuss the implications of this understanding to antitumor therapies.

ClC-1 plays an important part in the maintenance of membrane potential in the mammalian skeletal muscle. To investigate the phosphorylation sites responsible for the effect of PKC (protein kinase C) activator, we constructed 21 different ClC-1 mutants with mutations at predicted phosphorylation sites for PKC. The functional experiments were performed on both wild-type and mutant proteins (17 point mutants and 4 double mutants) expressed in Xenopus oocytes with two-electrode voltage-clamp recording. PMA (12-myristate 13-acetate), a PKC activator, caused a right shift of half-maximum activation potential (V_1/2) significantly in the wild-type (from -42.9±4.4 to -13.7±1.7 mV; n = 8, P < 0.05) and most of the single mutants except the S892P (from -39.5±4.5 to -35.7±5.7 mV; n = 6) and S892D (from -10.2±4.9 to -9.6±3.5 mV; n = 4). S892D, a mutant mimicking PKC-mediated phosphorylation at position 892, can also mimic the effect of wild-type treated with PMA in V_1/2 value (-10.2±4.9 mV vs -13.7±1.7 mV, n = 4 - 8). However, S892A still had a significant response to PMA indicating that other sites responsible for PMA might exist. Thus double mutants are generated for the following analysis. The V_1/2 of double mutants, T891A/S892A, S892A/T893A and T891A/T893A, show no significant difference between before and after PMA treatment. We hypothesize that this structural modification results in the observed alteration of the gating properties of ClC-1 by PMA. In summary, our observations show that a C-terminal region Thr891-Ser892-Thr893, at least in part, responsible for the effect of PMA on ClC-1.

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PurposeOur objective was to develop a system to simultaneously and quantitatively measure the expression levels of the insulin-like growth factor (IGF) family proteins in numerous samples and to apply this approach to profile the IGF family proteins levels in cancer and adjacent tissues from patients with hepatocellular carcinoma (HCC).Experimental DesignAntibodies against ten IGF family proteins (IGF-1, IGF-1R, IGF-2, IGF-2R, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, and Insulin) were immobilized on the surface of a glass slide in an array format to create an IGF signaling antibody array. Tissue lysates prepared from patient's liver cancer tissues and adjacent tissues were then applied to the arrays. The proteins captured by antibodies on the arrays were then incubated with a cocktail of biotinylated detection antibodies and visualized with a fluorescence detection system. By comparison with standard protein amount, the exact protein concentrations in the samples can be determined. The expression levels of the ten IGF family proteins in 25 pairs of HCC and adjacent tissues were quantitatively measured using this novel antibody array technology. The differential expression levels between cancer tissues and adjacent tissues were statistically analyzed.ResultsA novel IGF signaling antibody array was developed which allows the researcher to simultaneously detect ten proteins involved in IGF signal pathway with high sensitivity and specificity. Using this approach, we found that the levels of IGF-2R and IGFBP-2 in HCC tissues were higher than those in adjacent tissues.ConclusionOur IGF signaling antibody array which can detect the expression of ten IGF family members with high sensitivity and specificity will undoubtedly prove a powerful tool for drug and biomarker discovery.

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