Abstract 2371: Breast cancer induces tolerogenic state of healthy activated CD4+ lymphocytes, characterized by reduced PI3K, NFκB, JAK-STAT, Notch, and increased TGFβpathway activity

Abstract

Tumor cells can induce immunotolerance, which is reversed by checkpoint blockade immunotherapy in some patients, although response prediction remains a challenge. CD4+ T cells play an important role in activating adaptive immune responses with their conversion to a suppressed state impairing anti-tumor immune responses. CD4+ T cells function by activating and controlling various signal transduction pathways. Over the past decade we have developed tests that quantitatively measure functional activities of signal transduction pathways (e.g. Hedgehog, Wnt, TGFβ Notch, NFκB, PI3K, JAK-STAT 1/2 and 3, and MAPK). They are based on Bayesian computational model inference of pathway activities from measurements (expression microarray, qPCR) of mRNA levels of target genes of the transcription factor associated with the respective signalling pathways1,2. These tests were extensively biologically validated, including on immune cells, and can be used to characterize their functional activity status. In the present study, this approach was used to investigate cellular mechanisms underlying breast cancer-induced immunosuppression of CD4+ T cells. Method: Generation of Affymetrix gene expression data has been previously described (J Clin Invest 2013;123(7):2873-92) and data is publically available (GEO dataset GSE36766). Briefly, breast cancer tissue sections from fresh surgical specimens were mechanically dissociated in X-VIVO 20. Following activation with anti-CD3/CD28, CD4+ T cells from healthy donor blood were incubated with primary tumor supernatants (SN) and compared to controls. Signaling pathway activities were measured using Affymetrix expression data from the individual CD4+ T cell treatment groups. Results: CD4+ T cell activation resulted in induction of PI3K, NFkB, JAK-STAT1/2, JAK-STAT3, Notch, and parallel decrease in TGFβ pathway activities. Incubation with primary tumor SN did not affect pathway activity in non-activated CD4+ T cells, but reduced activity of PI3K, NFκB, JAK-STAT1/2, JAK-STAT3, Notch, while increasing TGFβ pathway activity in activated CD4+ T cells. Conclusion: A soluble factor(s) from breast tumor tissues increases TGFβ and reduces effector immune pathway activity in activated CD4+ T cells and thereby can induce an immunotolerant state. Investigation into the nature of this soluble factor(s) is in progress. These data demonstrate that signaling pathway assays can be used to quantitatively measure the functional state of immune responses in CD4+ lymphocytes. The ultimate goal is to apply this approach for predicting and monitoring immunotherapy responses and identifying novel drug targets that can reverse tumor-induced immunosuppression. Ref: 1. Verhaegh W, et al. Cancer Res 2014;74(11):2936-45; 2. Ooijen H. van, et al. Am J Pathol 2018;188(9):1956-1972. Citation Format: Anja Van De Stolpe, Wim Verhaegh, Arie van Doorn, Grégory Noël, Chunyan Gu-Trantien, Karen Willard-Gallo. Breast cancer induces tolerogenic state of healthy activated CD4+ lymphocytes, characterized by reduced PI3K, NFκB, JAK-STAT, Notch, and increased TGFβpathway activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2371.

RNA Based Approaches to Profile Oncogenic Pathways From Low Quantity Samples to Drive Precision Oncology Strategies

“…With respect to cancer, measurement of STP activity in a tumor infiltrate (TIL), or possibly in blood samples, may be similarly informative on the immune-active versus immune-tolerant state of the immune response. Initial results suggest that in primary breast cancer the adaptive T cell response has already been switched off, while in vitro study results suggest that this may be mediated by soluble factors from cancer tissue (e.g., TGFβ) that reduce effector immune pathway activity (PI3K, JAK-STAT, NFκB pathways) and increase activity of the immune suppressive TGFβ pathway (Gu-Trantien et al, 2013;van de Stolpe et al, 2019b). Investigation of the value of STP analysis to predict response to checkpoint inhibitor immunotherapy is underway.…”