Biology subjects

van de Stolpe, A.

Publications and source records attributed to van de Stolpe, A..

4 recordsLinked to original sources

bioRxiv2020

Characterization of immunoactive and immunotolerant CD4+ T cells in breast cancer by measuring activity of signaling pathways that determine immune cell function

Cancer immunotolerance can be reversed by checkpoint blockade immunotherapy in some patients, but response prediction remains a challenge. CD4+ T cells play an important role in activating adaptive immune responses against cancer. Conversion to an immune suppressive state impairs the anti-cancer immune response and is mainly effected by CD4+ Treg cells. A number of signal transduction pathways activate and control functions of CD4+ T cell subsets. As previously described, assays have been developed which enable quantitative measurement of the activity of signal transduction pathways (e.g. TGF{beta}, NF{kappa}B, PI3K-FOXO, JAK-STAT1/2, JAK-STAT3, Notch) in a cell or tissue sample. Using these assays, pathway activity profiles for various CD4+ T cell subsets were defined and cellular mechanisms underlying breast cancer-induced immunotolerance investigated in vitro. Results were used to measure the immune response state in a clinical breast cancer study. MethodsSignal transduction pathway activity scores were measured on Affymetrix expression microarray data of resting and immune-activated CD4+ T cells, immune-activated CD4+ T cells incubated with breast cancer tissue supernatants, CD4+ Th1, Th2, and Treg cells, and of clinical study samples in which CD4+ T cells were derived from blood, lymph node and cancer tissue from primary breast cancer patients (n=10). ResultsIn vitro CD4+ T cell activation induced PI3K, NF{kappa}B, JAK-STAT1/2, and JAK-STAT3 pathway activity. Simultaneous incubation with primary cancer supernatant reduced PI3K and NF{kappa}B, and partly reduced JAK-STAT3, pathway activity, while simultaneously increasing TGF{beta} pathway activity; characteristic of an immune tolerant state. CD4+ Th1, Th2, and Treg cells all had a specific pathway activity profile, with activated immune suppressive Treg cells characterized by high NF{kappa}B, JAK-STAT3, TGF{beta}, and Notch pathway activity scores. An immune tolerant pathway profile was identified in CD4+ T cells from tumor infiltrate of a subset of primary breast cancer patients which could be contributed to activated Treg cells. A Treg pathway profile was also identified in blood samples. ConclusionSignaling pathway assays can be used to quantitatively measure the functional immune response state of lymphocyte subsets in vitro and in vivo. Clinical results suggest that in primary breast cancer the adaptive immune response of CD4+ T cells has frequently been replaced by immunosuppressive Treg cells, potentially causing resistance to checkpoint inhibition. In vitro study results suggest that this effect is mediated by soluble factors from cancer tissue (e.g. TGF{beta}). Signaling pathway activity analysis on TIL and/or blood samples is expected to improve predicting and monitoring response to checkpoint inhibitor immunotherapy.

cancer biology↗

bioRxiv2020

Heterogeneity in signaling pathway activity within primary and between primary and metastatic breast cancer

BackgroundTargeted drug treatment aims to block tumor driving signaling pathways, and is generally based on analysis of one primary tumor (PT) biopsy. Phenotypic heterogeneity within primary and between primary and metastatic lesions was investigated. MethodsActivity of androgen and estrogen receptor, PI3K-FOXO, Hedgehog, TGF{beta}, and Wnt signaling pathways was measured in breast cancer samples using a novel mRNA-based assay platform. Macro-scale heterogeneity analysis was performed on multiple spatially distributed PT tissue blocks from 17 luminal A-like, 9 luminal B-like, and 9 ER-negative primary breast cancers; micro-scale heterogeneity analysis was performed on four "quadrant" samples of a single tissue block of respectively 9, 4, and 4 matched PT. Samples from 6 PT with matched lymph node (LN, n=23) and 9 PT with distant metastatic sites (DS, n=12) were analyzed. Statistical variance analysis was performed with linear mixed models. A "checkerboard" model was introduced to explain the observed heterogeneity in PT. ResultsWithin PT, macro-scale heterogeneity in signaling pathway activity was similar to micro-scale heterogeneity, with a possible exception of the PI3K pathway. Variation was significantly higher on microscale for Hedgehog and TGF{beta} pathways. While pathway activity scores correlated significantly between different locations in the PT, positive correlations decreased between PT and LN, and even more between PT and DS metastases, including the emergence of a negative correlation for the ER pathway. ConclusionWith a possible exception of the PI3K pathway, variation in signaling pathway activity within a single PT tissue block was generally representative for the whole PT, but not for DS or LN metastases. The higher variation in TGF{beta} and HH pathway activity on microscale suggested the presence of multiple small cancer cell clones. While analysis of multiple sub-samples of a single biopsy block may be sufficient to predict PT response to some targeted therapies, such as hormonal therapy, metastatic breast cancer treatment requires analysis of metastatic biopsies. The findings on phenotypic intra-tumor heterogeneity are compatible with currently emerging ideas on a Big Bang type of cancer evolution.

cancer biology↗

bioRxiv2020

Quantitative measurement of activity of JAK-STAT signaling pathways in blood samples and immune cells to predict innate and adaptive cellular immune response to viral infection and accelerate vaccine development.

The host immune response determines the clinical course of a viral infection, for example in case of COVID-19 infection. The effectiveness of vaccination also depends on the induced immune response. Currently there is no method to measure the cellular immune response in blood samples. The functional activity of cells of innate and adaptive immune system is determined by coordinated activity of signaling pathways, especially the JAK-STAT pathways. Using a previously described approach we developed mRNA-based tests to measure activity of these signaling pathways, and show that they can be used to measure in a quantitative manner the cellular innate and adaptive immune response to a viral infection or vaccine in whole blood, PBMC, and specific immune cell type samples. Pathway activity level and range in healthy individuals was established, enabling interpretation of a pathway activity score on a patient sample without the need for a reference sample. Evidence is presented that the pathway activity analysis may also be useful for in vitro vaccine development and assessment of vaccine immunogenicity. Other envisioned applications lie in development of immunomodulatory drugs and drug response prediction and monitoring. Tests are expected to be of value in the COVID-19 crisis. In addition to the described Affymetrix microarray-based pathway tests for measuring host immune response, qPCR-based versions are in development; the latter can in principle be performed within three hours in routine hospital labs.

immunology↗

bioRxiv2020

A novel dual antibody staining assay to measure estrogen receptor transcriptional activity

Activity of the canonical estrogen receptor (ER) pathway is equivalent to functional activity of the nuclear ER transcription factor. To assess transcriptional activity of ER, for biomedical research and diagnostic purposes ER monoclonal antibodies are routinely used to identify nuclear ER staining in cells and tissue samples, however it remained unclear whether this is sufficiently predictive for transcriptional activity of ER, and thus for ER pathway activity. Using ER positive breast cancer cell lines (MCF7 and T47D) in which the transcriptional activity status of ER was quantified using an mRNA based ER pathway activity assay, the relation between ER activity and nuclear ER staining with ER monoclonal antibodies (MoAb) was investigated. While the presence of ER in the cell nucleus is a prerequisite for ER activity, it was not predictive for ER transcriptional activity, confirming earlier findings. There were remarkable differences in behaviour of the used MoAbs: EP1 and 1D5 MoAbs showed reduced nuclear staining when ER was transcriptionally active, while staining with H4624 MoAb was independent of ER activity. To improve discrimination between active and inactive nuclear ER based on ER staining, a method was developed which consists of dual ER MoAb immunofluorescent staining, followed by generation of a digital image with a standard digital pathology scanner, and application of a cell nucleus detection algorithm and per cell calculation of the nuclear H4624/EP1 fluorescence intensity ratio, where a high H4624/EP1 ratio predicts an active ER. In this method the EP1 MoAb can in principle be replaced by the 1D5 MoAb. We hypothesize that the EP1 and 1D5 monoclonal antibodies (MoAb) recognize an ER epitope which becomes hidden upon transcriptional activation of ER, while the H4624 MoAb binds an ER epitope which remains accessible when ER is activated. The method is expected to be of value to better assess ER activity by means of staining.

cell biology↗