Wesseling-Rozendaal, Y. | bioRxiv Science

bioRxiv2021

Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells

Stem cell research is emerging both as a scientifically and clinically relevant area. One of the current challenges in stem cell research and regenerative medicine is assessment of the pluripotency state of induced pluripotent stem (iPS) cells. Once a stem cell differentiation process is initiated the challenge is how to assess the state of differentiation, and the purity of the differentiated cell population. Stem cell potency and differentiation states are determined by tightly coordinated activity of developmental signaling pathways, such as the Notch, Hedgehog, TGF{beta}, Wnt, PI3K, MAPK-AP1, and NF{kappa}B pathways. Source of the stem cells and culture protocols may influence stem cell phenotype, with potential consequences for pluripotency and in general for experimental reproducibility. Human pluripotent embryonic (hES) and iPS stem cell lines under different culture conditions, organ derived multipotent stem cells, and differentiated cell types, were phenotyped with respect to functional activity of developmental signaling pathways. MethodsWe previously reported on the development and validation of a novel assay platform for quantitative measurement of activity of multiple signal transduction pathways (STP) simultaneously in a single sample, based on interpreting a preselected set of target mRNA expression levels. Assays were used to calculate Notch, Hedgehog, TGF{beta}, Wnt, PI3K, MAPK-AP1, and NF{kappa}B signal transduction pathway activity scores for individual cell samples, using publicly available Affymetrix expression microarray data. ResultsCulture conditions (e.g. mouse versus human feeder) influenced pluripotent stem cell pathway activity profiles. hES and iPS stem cell lines cultured in the same lab under similar conditions showed minimal variation in pathway activity profile despite different genetic backgrounds, while across different labs larger variations were measured, even for the same stem cell line. Pathway activity scores for PI3K, MAPK, Hedgehog, Notch, TGF{beta}, and NF{kappa}B pathways rapidly decreased upon pluripotent stem cell differentiation, while increasing for the Wnt pathway. Further differentiation to intestinal progenitor cells resulted in higher PI3K, Wnt and Notch pathway activity. In multipotent intestinal crypt stem cells obtained from intestinal mucosa samples, similar Notch and even higher Wnt pathway activity were measured, which disappeared upon differentiation to mucosal cells. ConclusionResults support the validity of using these STP assays for quantitative phenotyping of stem cells and differentiated derivatives, and enabled definition of a pluripotency profile with high PI3K, MAPK, Hedgehog, TGF{beta}, Notch, and NF{kappa}B, and low Wnt pathway activity scores. Measurement of combined signaling pathway activity scores is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture and differentiation. It enables controlled manipulation of signaling pathway activity using pathway targeting compounds. An envisioned additional utility may lie in quality control for regenerative medicine purposes.

developmental biology↗

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↗