STAT3 In Group Research Articles

Th17 Cell Subset levels in Peripheral Blood of Patients with Aplastic Anemia and Their Clinical Significance

To study the level and clinical value of Th17 cell subset in the peripheral blood of the patients with aplastic anemia (AA). Eighty-five patients with aplastic anemia in our hospital from May 2017 to February 2018 were selected. Among them, 51 patients with poor curative effect were enrolled in group A. and the 34 patients with good curative effect were enrolled in group B, the 35 healthy persons were selected as controls. The levels of serum IL-17, IL-6, IL-4 and IFN-β were analyzed by ELISA; the levels of peripheral blood Th17 cell subset were analyzed by flow cytometry; the expression levels of RORγt and STAT3 mRNA in lymphocytes were analyzed by RT-PCR; the expression levels of RORγt and STAT3 protein in lymphocytes were analyzed by Western blot. There was no significant difference in sex, age, WBC count and complications between group A and group B (P＞0.05). Non-severe aplastic anemia (NSAA) in group B accounted for 23 cases, and the NSAA ratio (23/34) was significantly higher than that in group A (20/51) (P＜0.05). The Hb level (86.25±7.9 g/L) and Plt count (54.7 ± 6.3×109/L) in group B were significantly higher than those in group A (P＜0.05). ELISA showed that the levels of IL-17 and IL-6 in group A were significantly higher than those in control group (P＜0.05); the levels of IL-17 and IL-6 in group B were significantly lower than those in group A (P＜0.05); the levels of IL-4 and IFN-β in group A were significantly lower than those in control group (P＜0.05); the levels of IL-4 and IFN-βin group B were significantly higher than those in group A (P＜0.05). Flow cytometry showed that the proportion of Th17 cells in peripheral blood of group A was higher than that of group B (P＜0.05). RT-PCR showed that the levels of RORγt and STAT3 mRNA in group A were significantly higher than those in control group (P＜0.05), and the mRNA levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). Western blot showed that the protein levels of RORγt and STAT3 in group A were significantly higher than those in control group (P＜0.05), and the protein levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). The Th17 cells in peripheral blood of AA patients increase, and the inflammatory reaction in the patients are enhanced. It may be related with the rise of RORγt and STAT3 gene expression, which provides a research direction for clinical treatment of AA.

Th17 Cell Subset levels in Peripheral Blood of Patients with Aplastic Anemia and Their Clinical Significance

To study the level and clinical value of Th17 cell subset in the peripheral blood of the patients with aplastic anemia (AA). Eighty-five patients with aplastic anemia in our hospital from May 2017 to February 2018 were selected. Among them, 51 patients with poor curative effect were enrolled in group A. and the 34 patients with good curative effect were enrolled in group B, the 35 healthy persons were selected as controls. The levels of serum IL-17, IL-6, IL-4 and IFN-β were analyzed by ELISA; the levels of peripheral blood Th17 cell subset were analyzed by flow cytometry; the expression levels of RORγt and STAT3 mRNA in lymphocytes were analyzed by RT-PCR; the expression levels of RORγt and STAT3 protein in lymphocytes were analyzed by Western blot. There was no significant difference in sex, age, WBC count and complications between group A and group B (P＞0.05). Non-severe aplastic anemia (NSAA) in group B accounted for 23 cases, and the NSAA ratio (23/34) was significantly higher than that in group A (20/51) (P＜0.05). The Hb level (86.25±7.9 g/L) and Plt count (54.7 ± 6.3×109/L) in group B were significantly higher than those in group A (P＜0.05). ELISA showed that the levels of IL-17 and IL-6 in group A were significantly higher than those in control group (P＜0.05); the levels of IL-17 and IL-6 in group B were significantly lower than those in group A (P＜0.05); the levels of IL-4 and IFN-β in group A were significantly lower than those in control group (P＜0.05); the levels of IL-4 and IFN-βin group B were significantly higher than those in group A (P＜0.05). Flow cytometry showed that the proportion of Th17 cells in peripheral blood of group A was higher than that of group B (P＜0.05). RT-PCR showed that the levels of RORγt and STAT3 mRNA in group A were significantly higher than those in control group (P＜0.05), and the mRNA levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). Western blot showed that the protein levels of RORγt and STAT3 in group A were significantly higher than those in control group (P＜0.05), and the protein levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). The Th17 cells in peripheral blood of AA patients increase, and the inflammatory reaction in the patients are enhanced. It may be related with the rise of RORγt and STAT3 gene expression, which provides a research direction for clinical treatment of AA.

Abstract B69: Activation of the IL6/STAT3 pathway in childhood ependymoma is associated with a pro-inflammatory tumor microenvironment and a poor prognosis

Abstract To aid development of immunotherapy for poor outcome Group A ependymoma (EPN) in children we sought to identify the molecular pathway underlying the adverse pro-inflammatory phenotype associated with this molecular subgroup. We have previously shown the importance of immunological factors in EPN outcome and that an anti-tumor immunophenotype was critical in survival whereas a pro-inflammatory phenotype was associated with a poor outcome. Moreover we showed a strong correlation between these immunophenotypes and the newly recognized molecular subgroups of posterior fossa EPN. We showed that Group A EPN have an adverse pro-inflammatory immunophenotype at presentation which adversely affects outcome even at relapse. We contrasted the gene expression profiles of patient tumor samples from Group A EPN to the more clinically favorable Group B EPN, as well as a broader panel of pediatric and adult brain tumors and normal brain, to identify potential molecular pathways that might be associated with the Group A immunophenotype. The results of these studies were validated using functional assays in short-term cultures of patient tumor samples and novel EPN cell lines. Strikingly we found a robust expression signature implicating the interleukin-6/signal transduced and activator of transcription 3 (IL6/STAT3) pathway in the Group A tumors. Comparison of transcriptomic profiles of EPN Group A (n=17) and Group B (n=20) demonstrated significant overexpression both IL6 (19.7-fold) and STAT3 (1.6 fold) in Group A. High overexpression of STAT3 driven genes IL8 (30-fold), SOCS3 (14-fold) and BIRC3 (8.5-fold) provided additional proof of IL6/STAT3 pathway activation in Group A. Furthermore, IL6 and IL8 gene expression was shown to be significantly higher (FDR<0.05) than any other type of pediatric or adult brain tumor or normal brain, suggesting that this phenotype is unique to Group A EPN. Western blot analysis of whole tumor samples from Groups A and B showed a significantly elevated ratio of pSTAT3 to total STAT3 in the Group A tumors compared to the Group B, denoting activation of STAT3. Measurement of IL6 secretion from primary ex-vivo tumor cells, isolated by flow sorting of disaggregated surgical tumor samples, showed a significantly higher secretion by tumors cells from Group A patients compared to Group B. To further investigate the role of the IL6/STAT3 pathway, we utilized a novel EPN cell line (811) established from a multiple recurrent EPN, with the transcriptome characteristics of a Group A EPN. This cell line secretes large amounts of IL6, considerably more than glioblastoma (GBM) cell line U87 in which the IL6/STAT3 pathway has been extensively studied. This cell line induced a contact-independent polarization of myeloid cells to a pro-inflammatory phenotype. HLA-matched donor CD14+ that we treated with 811 conditioned media showed a 9.2-fold increase in IL8 secretion (p=0.027). Demonstrating the mitogenic role of STAT3 activation in EPN, we showed 811 is more sensitive to STAT3 inhibition by S31-201 (IC50 = 81uM) than breast cancer and GBM cell lines. PhosphoSTAT3 and the downstream targets of the pathway, SOCS3 and Mcl-1 are significantly reduced in a dose dependent manner in response to S31-201. S3I-201 treatment of 811 and an EPN short-term cell line were shown to cause a significant dose-dependent increase in Caspase-3/7 cleavage demonstrating apoptosis as a result of STAT3 inhibition. To conclude: The IL6/STAT3 pathway is active in Group A EPN and is important in maintaining tumor growth in a pro-inflammatory microenvironment. Effective design of Group A-targeted immunotherapy for children with EPN may require alleviation of this potentially immunosuppressive pathway. Citation Format: Andrea Griesinger, Andrew M. Donson, Diane Birks, Vladimir Amani, Rebecca Josephson, Kathleen Dorris, Philip Reigan, Michael Handler, Nicholas Foreman. Activation of the IL6/STAT3 pathway in childhood ependymoma is associated with a pro-inflammatory tumor microenvironment and a poor prognosis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B69.

EP-04 * ACTIVATION OF THE IL6/STAT3 PATHWAY IN CHILDHOOD EPENDYMOMA IS ASSOCIATED WITH A PRO-INFLAMMATORY TUMOR MICROENVIRONMENT AND A POOR PROGNOSIS

To aid development of immunotherapy for poor outcome Group A ependymoma (EPN) in children we sought to identify the molecular pathway underlying the adverse pro-inflammatory phenotype associated with this molecular subgroup. We previously showed the importance of immunological factors and that an anti-tumor immunophenotype was critical in survival whereas a pro-inflammatory phenotype was associated with a poor outcome. We contrasted gene expression profiles of patient tumor samples from Group A EPN to the more clinically favorable Group B, identifying potential molecular pathways associated with the Group A immunophenotype. Strikingly, we found a robust expression signature implicating the IL6/STAT3 pathway in Group A tumors. Comparison of transcriptomic profiles of Group A and Group B demonstrated significant overexpression both IL6 and STAT3 in Group A. High overexpression of STAT3 driven IL8 and SOCS3 was also seen in Group A. Western blot analysis of tumor samples from Groups A and B showed a significantly elevated ratio of pSTAT3:STAT3 in Group A, denoting activation of STAT3. Measurement of IL6 secretion from primary ex-vivo tumor cells showed a significantly higher secretion by Group A than Group B. To further investigate the role of the IL6/STAT3 pathway, we utilized a novel EPN cell line (811) with the transcriptome characteristics of Group A. Similar to Group A tumors, 811 secretes high levels of IL-6 and has elevated STAT3 activation. Loss of STAT3 resulted in reduction of downstream targets and apoptosis. The elevated IL6 from 811 induced a contact-independent polarization of myeloid cells to a pro-inflammatory phenotype. CD14+ cells treated with 811 conditioned media showed a significant increase in IL8 secretion. The IL6/STAT3 pathway is active in Group A EPN and is important in maintaining tumor growth in a pro-inflammatory microenvironment. Effective Group A-targeted immunotherapy for children with EPN may require alleviation of this potentially immunosuppressive pathway.