PREPARATION AND EVALUATION OF A 3D LIVER TUMOR SPHEROID MODEL VIA THE USE OF A COMPOSITE COLLAGEN AND POLYMER HYDROGEL

Master'sMASTER OF SCIENC

Hypoxia- and MicroRNA-Induced Metabolic Reprogramming of Tumor-Initiating Cells

Colorectal cancer (CRC), the second most common cause of cancer mortality in theWestern world, is a highly heterogeneous disease that is driven by a rare subpopulation of tumorigenic cells, known as cancer stem cells (CSCs) or tumor-initiating cells (TICs). Over the past few years, a plethora of di erent approaches, aimed at identifying and eradicating these self-renewing TICs, have been described. A focus on the metabolic and bioenergetic di erences between TICs and less aggressive di erentiated cancer cells has thereby emerged as a promising strategy to specifically target the tumorigenic cell compartment. Extrinsic factors, such as nutrient availability or tumor hypoxia, are known to influence the metabolic state of TICs. In this review, we aim to summarize the current knowledge on environmental stress factors and how they a ect the metabolism of TICs, with a special focus on microRNA (miRNA)- and hypoxia-induced e ects on colon TICs

Hypoxia-induced Autophagy Drives Colorectal Cancer Initiation and Progression by Activating the PRKC/PKC-EZR (Ezrin) Pathway

In solid tumors, cancer stem cells (CSCs) or tumor-initiating cells (TICs) are often found in hypoxic niches. Nevertheless, the influence of hypoxia on TICs is poorly understood. Using previously established, TIC-enriched patient-derived colorectal cancer (CRC) cultures, we show that hypoxia increases the self-renewal capacity of TICs while inducing proliferation arrest in their more differentiated counterpart cultures. Gene expression data revealed macroautophagy/autophagy as one of the major pathways induced by hypoxia in TICs. Interestingly, hypoxia-induced autophagy was found to induce phosphorylation of EZR (ezrin) at Thr567 residue, which could be reversed by knocking down ATG5, BNIP3, BNIP3L, or BECN1. Furthermore, we identified PRKCA/PKCα as a potential kinase involved in hypoxia-induced autophagy-mediated TIC self-renewal. Genetic targeting of autophagy or pharmacological inhibition of PRKC/PKC and EZR resulted in decreased tumor-initiating potential of TICs. In addition, we observed significantly reduced in vivo tumor initiation and growth after a stable knockdown of ATG5. Analysis of human CRC samples showed that p-EZR is often present in TICs located in the hypoxic and autophagic regions of the tumor. Altogether, our results establish the hypoxia-autophagy-PKC-EZR signaling axis as a novel regulatory mechanism of TIC self-renewal and CRC progression. Autophagy inhibition might thus represent a promising therapeutic strategy for cancer patients

Effects of environmental stress factors on colon cancer and its microenvironment

Over the last decade, cancer-associated fibroblasts (CAFs) have risen to increased prominence as one of the key drivers in the pro-tumorigenic tumor microenvironment (TME). Their incredible heterogeneity means, however, that detailed analysis of these cells remains elusive and requires the usage of complex technologies such as single-cell sequencing. Using these approaches, numerous subtypes of cancer-associated fibroblasts have been identified, mainly based on their gene expression profiles. In this thesis, we highlight the IL1-family, especially IL1B and IL1R1, as an important driver in the pro-tumorigenic nature of fibroblast. We show how IL1B and IL1R1 are upregulated in CRC CAFs when contrasted to normal, non-tumor-associated, fibroblasts and how IL1R1 expression is strongly elevated in one of the CAF subtypes we identified using single-cell sequencing – cyCAF2. We also identified that IL1R1 correlates strongly with two of the markers characteristic of this subtype, FAP and CXCL12, and that it shows elevated signs of IL1β-derived signaling, based on gene signature analysis. From a functional perspective, we show that IL1β-activated fibroblasts induce the secretion of pro-inflammatory cytokines such as IL6, CXCL8, CXCL5, and CXCL1 and act in a pro-tumorigenic manner on tumor cells in three-dimensional co-culture systems. Furthermore, we show that IL1B can be upregulated in CAFs by, at the moment, unknown secreted factors from tumor spheroids. We also found that inhibition of basal IL1 cross-talk between CAFs and tumor spheres via IL1-inhibition results in lowered pro-tumorigenic activity of CAFs and that this can be recapitulated in vivo using ColVIcre+ IL1R1fl/fl conditional knockout mice. Finally, we highlight how IL1β stimulation in primary CAFs can modulate immune cell proliferation and the upregulation of immunomodulatory proteins such as PD-L1 and PD-L2 and may, therefore, alter the effectiveness of immune checkpoint therapy. Altogether, the results presented in this thesis suggest that inhibition of IL1β-signaling in CAFs may be a promising therapeutic option in CRC patients

Hypoxia- and MicroRNA-Induced Metabolic Reprogramming of Tumor-Initiating Cells

Colorectal cancer (CRC), the second most common cause of cancer mortality in the Western world, is a highly heterogeneous disease that is driven by a rare subpopulation of tumorigenic cells, known as cancer stem cells (CSCs) or tumor-initiating cells (TICs). Over the past few years, a plethora of different approaches, aimed at identifying and eradicating these self-renewing TICs, have been described. A focus on the metabolic and bioenergetic differences between TICs and less aggressive differentiated cancer cells has thereby emerged as a promising strategy to specifically target the tumorigenic cell compartment. Extrinsic factors, such as nutrient availability or tumor hypoxia, are known to influence the metabolic state of TICs. In this review, we aim to summarize the current knowledge on environmental stress factors and how they affect the metabolism of TICs, with a special focus on microRNA (miRNA)- and hypoxia-induced effects on colon TICs

Tumor suppressor miR-215 counteracts hypoxia-induced colon cancer stem cell activity

Cancer stem cells, also known as tumor-initiating cells (TICs), are a population of aggressive and self-renewing cells that are responsible for the initiation and progression of many cancers, including colorectal carcinoma. Intratumoral hypoxia, i.e. reduced oxygen supply following uncontrolled proliferation of cancer cells, is thought to support TIC activity by inducing specific hypoxia-responsive mechanisms that are not yet entirely understood. Using previously established and fully characterized patient-derived TIC cultures, we could observe increased sphere and colony formation under hypoxic conditions. Mechanistically, microRNA (miRNA)-profiling experiments allowed us to identify miR-215 as one of the main hypoxia-induced miRNAs in primary colon TICs. Through stable overexpression of miR-215, followed by a set of functional in vitro and in vivo investigations, miR-215 was pinpointed as a negative feedback regulator, working against the TIC-promoting effects of hypoxia. Furthermore, we could single out LGR5, a bona fide marker of non-neoplastic intestinal stem cells, as a downstream target of hypoxia/miR-215 signaling. The strong tumor- and TIC-suppressor potential of miR-215 and the regulatory role of the hypoxia/miR-215/LGR5 axis may thus represent interesting points of attack for the development of innovative anti-CSC therapy approaches

Fibroblasts in the Tumor Microenvironment

The implications of a tumor microenvironment in cancer initiation and progression have drawn interest in recent years. Within the tumor stroma, fibroblasts represent a predominant cell type and are responsible for the majority of extracellular components within the tumor microenvironment, such as matrix and soluble factors. A switch from quiescent fibroblasts to cancer-associated fibroblasts triggers a large variety of pro-tumorigenic signals that support tumor progression and shape the surrounding pathological stroma, with the remodeling of tissue architecture and repression of the local immune response. The heterogeneous nature of cancer-associated fibroblasts and their multiple functions are subject of active research as they could represent promising targets for cutting-edge therapeutic approaches to cancer and the tumor microenvironment