Biology subjects

van de Stolpe, A.

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

3 recordsLinked to original sources

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↗