Microarray profiling of mononuclear peripheral blood cells identifies novle candidate genes related to chemoradiation response in rectal cancer

Preoperative chemoradiation significantly improves oncological outcome in locally advanced rectal cancer. However there is no effective method of predicting tumor response to chemoradiation in these patients. Peripheral blood mononuclear cells have emerged recently as pathology markers of cancer and other diseases, making possible their use as therapy predictors. Furthermore, the importance of the immune response in radiosensivity of solid organs led us to hypothesized that microarray gene expression profiling of peripheral blood mononuclear cells could identify patients with response to chemoradiation in rectal cancer. Thirty five 35 patients with locally advanced rectal cancer were recruited initially to perform the study. Peripheral blood samples were obtained before neaodjuvant treatment. RNA was extracted and purified to obtain cDNA and cRNA for hybridization of microarrays included in Human WG CodeLink bioarrays. Quantitative real time PCR was used to validate microarray experiment data. Results were correlated with pathological response, according to Mandard´s criteria and final UICC Stage (patients with tumor regression grade 1–2 and downstaging being defined as responders and patients with grade 3–5 and no downstaging as non-responders). Twenty seven out of 35 patients were finally included in the study. We performed a multiple t-test using Significance Analysis of Microarrays, to find those genes differing significantly in expression, between responders (n = 11) and non-responders (n = 16) to CRT. The differently expressed genes were: BC 035656.1, CIR, PRDM2, CAPG, FALZ, HLA-DPB2, NUPL2, and ZFP36. The measurement of FALZ (p = 0.029) gene expression level determined by qRT-PCR, showed statistically significant differences between the two groups. Gene expression profiling reveals novel genes in peripheral blood samples of mononuclear cells that could predict responders and non-responders to chemoradiation in patients with locally advanced rectal cancer. Moreover, our investigation added further evidence to the importance of mononuclear cells’ mediated response in the neoadjuvant treatment of rectal cancer.This investigation was supported by the Fundación Investigación Biomédica Mutua Madrileña. MC, CC and AB were supported by projects P08-TIC-4299 and CTS2200 of Junta de Andalucía, TIN2009-13489 of DGICT, Madrid, and GREIB PYR_2010-02 and 2010_05 of University of Granada

Midkine is a NF-κB-inducible gene that supports prostate cancer cell survival

BackgroundMidkine is a heparin-binding growth factor that is over-expressed in various human cancers and plays important roles in cell transformation, growth, survival, migration, and angiogenesis. However, little is known about the upstream factors and signaling mechanisms that regulate midkine gene expression.MethodsTwo prostate cancer cell lines LNCaP and PC3 were studied for their expression of midkine. Induction of midkine expression in LNCaP cells by serum, growth factors and cytokines was determined by Western blot analysis and/or real-time quantitative reverse-transcription - polymerase chain reaction (RT-PCR). The cell viability was determined by the trypan blue exclusion assay when the LNCaP cells were treated with tumor necrosis factor alpha (TNFalpha) and/or recombinant midkine. When the LNCaP cells were treated with recombinant midkine, activation of intracellular signalling pathways was determined by Western blot analysis. Prostate tissue microarray slides containing 129 cases (18 normal prostate tissues, 40 early stage cancers, and 71 late stage cancers) were assessed for midkine expression by immunohistochemical staining.ResultsWe identified that fetal bovine serum, some growth factors (epidermal growth factor, androgen, insulin-like growth factor-I, and hepatocyte growth factor) and cytokines (TNFalpha and interleukin-1beta) induced midkine expression in a human prostate cancer cell line LNCaP cells. TNFalpha also induced midkine expression in PC3 cells. TNFalpha was the strongest inducer of midkine expression via nuclear factor-kappa B pathway. Midkine partially inhibited TNFalpha-induced apoptosis in LNCaP cells. Knockdown of endogenous midkine expression by small interfering RNA enhanced TNFalpha-induced apoptosis in LNCaP cells. Midkine activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in LNCaP cells. Furthermore, midkine expression was significantly increased in late stage prostate cancer, which coincides with previously reported high serum levels of TNFalpha in advanced prostate cancer.ConclusionThese findings provide the first demonstration that midkine expression is induced by certain growth factors and cytokines, particularly TNFalpha, which offers new insight into understanding how midkine expression is increased in the late stage prostate cancer

Polyfunctional T-Cell Responses Are Disrupted by the Ovarian Cancer Ascites Environment and Only Partially Restored by Clinically Relevant Cytokines

Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21

Tumor-Infiltrating T Cells Correlate with NY-ESO-1-Specific Autoantibodies in Ovarian Cancer

BACKGROUND: Tumor-infiltrating CD8+ T cells are correlated with prolonged progression-free and overall survival in epithelial ovarian cancer (EOC). A significant fraction of EOC patients mount autoantibody responses to various tumor antigens, however the relationship between autoantibodies and tumor-infiltrating T cells has not been investigated in EOC or any other human cancer. We hypothesized that autoantibody and T cell responses may be correlated in EOC and directed toward the same antigens. METHODOLOGY AND PRINCIPAL FINDINGS: We obtained matched serum and tumor tissue from 35 patients with high-grade serous ovarian cancer. Serum samples were assessed by ELISA for autoantibodies to the common tumor antigen NY-ESO-1. Tumor tissue was examined by immunohistochemistry for expression of NY-ESO-1, various T cell markers (CD3, CD4, CD8, CD25, FoxP3, TIA-1 and Granzyme B) and other immunological markers (CD20, MHC class I and MHC class II). Lymphocytic infiltrates varied widely among tumors and included cells positive for CD3, CD8, TIA-1, CD25, FoxP3 and CD4. Twenty-six percent (9/35) of patients demonstrated serum IgG autoantibodies to NY-ESO-1, which were positively correlated with expression of NY-ESO-1 antigen by tumor cells (r = 0.57, p = 0.0004). Autoantibodies to NY-ESO-1 were associated with increased tumor-infiltrating CD8+, CD4+ and FoxP3+ cells. In an individual HLA-A2+ patient with autoantibodies to NY-ESO-1, CD8+ T cells isolated from solid tumor and ascites were reactive to NY-ESO-1 by IFN-gamma ELISPOT and MHC class I pentamer staining. CONCLUSION AND SIGNIFICANCE: We demonstrate that tumor-specific autoantibodies and tumor-infiltrating T cells are correlated in human cancer and can be directed against the same target antigen. This implies that autoantibodies may collaborate with tumor-infiltrating T cells to influence clinical outcomes in EOC. Furthermore, serological screening methods may prove useful for identifying clinically relevant T cell antigens for immunotherapy

The Tumor-Immune Microenvironment and Response to Radiation Therapy

Chemotherapy and radiation therapy (RT) are standard therapeutic modalities for patients with cancer, including breast cancer. Historic studies examining tissue and cellular responses to RT have predominantly focused on damage caused to proliferating malignant cells leading to their death. However, there is increasing evidence that RT also leads to significant alterations in the tumor microenvironment, particularly with respect to effects on immune cells infiltrating tumors. This review focuses on tumor-associated immune cell responses following RT and discusses how immune responses may be modified to enhance durability and efficacy of RT

Improved estimates of the physical properties of the O-star binary V1007 Sco = HD 152248 and notes on several other binaries in the NGC 6231 cluster

Context.In spite of the importance of massive O-type stars for astrophysics, their accurate masses and other fundamental properties are still a matter of debate. Determining them reliably is hampered by various factors (stellar winds and other forms of circumstellar matter), and the agreement of derived properties with the model predictions is far from satisfactory. Careful studies of O-type binaries, especially of those in stellar clusters, are therefore desirable. Aims.Having obtained new series of electronic spectra and $U\!B{}V$ photometry of V1007 Sco, we analysed these data in an effort to check whether the observed properties of V1007 Sco indeed disagree with the prediction of stellar evolutionary models. We briefly analysed data for a few other binaries in NGC 6231, too. Methods.Spectral reductions were carried out with the MIDAS program, photometry reduced using the HEC22 program, the orbital elements were derived with the FOTEL program and the final solutions obtained with the program PHOEBE. Results.Our analysis led to an accurate determination of the apsidal advance, $\dot\omega = (0.00884\pm0.00012)$ deg d-1, based on a simultaneous solution of all usable radial-velocity and photometric data. This implies an apsidal period of 111.5 years. It is also demonstrated that the orbital inclination must be close to 67°. We arrived at the following preliminary values for masses and radii: $M_1 = (29.5\pm0.4)$ $M_{\odot}$, $M_2 = (30.1\pm0.4)$ $M_{\odot}$, $R_1 = (15.8\pm0.7)$ $R_{\odot}$, and $R_2 = (15.3\pm0.5)$ $R_{\odot}$. These values clearly indicate a $\log g$ of about 3.5 [CGS], implying that the stars are giants and not supergiants, as the standard spectral classification criteria indicate