Periodic Reporting for period 2 - IntratumoralNiche (Defining heterocellular signalling within the intratumoral stem cell niche of colorectal cancer) Reporting period: 2020-07-01 to 2021-12-31 Summary of the context and overall objectives of the project Purpose: Cells in a tumor are highly heterogeneous. The role and consequence of having multiple cell typeswithin a cancer is mostly centered towards the function of cancer stem cells (CSCs) since they are the drivingforces of tumor growth. However, even the most malignant cancers contain differentiated tumor cells. Is there a functional role for these cells,for instance by providing signaling cues that support CSC function. In other words, do colorectal tumors build their own stem cell supporting niche?Importance for society:My overarching aim is to identify and characterize the cell-cell signaling pathways between cancer stem cells and differentiated tumor cells (intratumoral stem cell niche) that are vital for colorectal tumor growth and metastasis formation.Objectives:-Identify cell-cell signaling pathways that act between cancer stem cells and differentiated tumor cells-Develop new technological strategies to characterize signaling activities in real-time and with single-cell resolution in 3-dimensional tumor organoids of patients-Functional investigation of candidate genes that are essential components of the intratumoral stem cell niche Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We are mapping and characterizing the signaling pathways during outgrowth experiments of patients-tumor organoids. These are a proxy for clonal liver metastases formation from primary colon cancer.We established real-time quantification of EGFR/MAPK signaling activity in single tumor cells of patient organoids. This enables us to study drug response in real-time, as well as revealed that the EGFR receptor is a strong amplifier of downstream signaling activity, even when this pathway contains frequent cancer mutations in KRAS or BRAF.To facilitate functional studies on many genes in patient organoids, we developed new CRISPR strategies for efficient knock-ins (e.g. fluorescent gene tags) and (conditional) knock-outs. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Developed strategies that provide insights into real-time signaling activity in tumor organoids and its quantitative assessment upon targeted therapies is a technological breakthrough. We aim to expand our toolbox to study more signaling pathways. patient organoids
