Stromal fibroblasts shape the myeloid phenotype in normal colon and colorectal cancer and induce CD163 and CCL2 expression in macrophages

Colorectal cancer (CRC) accounts for about 10% of cancer deaths worldwide. Colon carcinogenesis is critically influenced by the tumor microenvironment. Cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) represent the major components of the tumor microenvironment. TAMs promote tumor progression, angiogenesis and tissue remodeling. However, the impact of the molecular crosstalk of tumor cells (TCs) with CAFs and macrophages on monocyte recruitment and their phenotypic conversion is not known in detail so far. In a 3D human organotypic CRC model, we show that CAFs and normal colonic fibroblasts are critically involved in monocyte recruitment and for the establishment of a macrophage phenotype, characterized by high CD163 expression. This is in line with the steady recruitment and differentiation of monocytes to immunosuppressive macrophages in the normal colon. Cytokine profiling revealed that CAFs produce M-CSF, and IL6, IL8, HGF and CCL2 secretion was specifically induced by CAFs in co-cultures with macrophages. Moreover, macrophage/CAF/TCs co-cultures increased TC invasion. We demonstrate that CAFs and macrophages are the major producers of CCL2 and, upon co-culture, increase their CCL2 production twofold and 40-fold, respectively. CAFs and macrophages expressing high CCL2 were also found in vivo in CRC, strongly supporting our findings. CCL2, CCR2, CSF1R and CD163 expression in macrophages was dependent on active MCSFR signaling as shown by M-CSFR inhibition. These results indicate that colon fibroblasts and not TCs are the major cellular component, recruiting and dictating the fate of infiltrated monocytes towards a specific macrophage population, characterized by high CD163 expression and CCL2 production

Investigation of the role of microtubule dynamics in paracrine signaling and cell invasion in human cancer cells

Metastasis is a critical characteristic of aggressive human cancer, which is determined by the acquirement of increased cancer cell migration and invasion capabilities. Recent published work indicated that supernumerary centrosomes, which are frequently seen in cancer cells, induce a paracrine signaling contributing to enhanced cell invasion in breast epithelial cells. Interestingly, our lab has identified a correlation between increased microtubule plus end assembly rates in interphase cells and enhanced cell migration and invasion in colorectal cancer and melanoma cell lines suggesting that microtubule dynamics might be involved in the regulation of cancer cell migration and invasion. This Ph.D. thesis focused on the investigation of the role of supernumerary centrosomes on microtubule plus end assembly in melanoma and colorectal cancer cells and its involvement in paracrine signaling leading to increased microtubule growth rates and cell migration and invasion. I demonstrated that colorectal cancer and melanoma cells that exhibit increased microtubule plus end growth rates in interphase show increased cell migration and invasion. Moreover, rescue experiments demonstrated a requirement of increased microtubule dynamics for migration and invasion in these cancer cells. Interestingly, these cancer-associated phenotypes were accompanied by the presence of a high proportion of cells with supernumerary centrosomes. In fact, induction of supernumerary centrosomes was sufficient to trigger increased microtubule plus end assembly rates in interphase. Intriguingly, the ability to increase microtubule plus end assembly rates could be transferred onto non-invasive cells by paracrine signaling and this was found to be mediated by shedding microvesicles in a microtubule dynamics dependent manner. Finally, evidence was obtained that microvesicle-mediated increase of microtubule dynamics might involve HER2/ERK signaling. The data presented in this Ph.D. thesis indicate a requirement of abnormally increased microtubule dynamics for cancer cell migration and invasion and thus, for metastasis. Furthermore, the shedding of cancer-relevant microvesicles and the induction of paracrine signaling dependents on microtubule plus end assembly rates in interphase and seems to be associated with an increased activity of HER2/ERK signaling. Thus, inhibition of HER2/ERK signaling or direct suppression of microtubule dynamics, e.g., by the treatment with microtubule targeting drugs including Taxol, may offer new possibilities for targeting metastasis in human cancer.2022-09-1

The p53/p73 - p21CIP1 tumor suppressor axis guards against chromosomal instability by restraining CDK1 in human cancer cells

Whole chromosome instability (W-CIN) is a hallmark of human cancer and contributes to the evolvement of aneuploidy. W-CIN can be induced by abnormally increased microtubule plus end assembly rates during mitosis leading to the generation of lagging chromosomes during anaphase as a major form of mitotic errors in human cancer cells. Here, we show that loss of the tumor suppressor genes TP53 and TP73 can trigger increased mitotic microtubule assembly rates, lagging chromosomes, and W-CIN. CDKN1A, encoding for the CDK inhibitor p2

Exclusion from spheroid formation identifies loss of essential cell-cell adhesion molecules in colon cancer cells

Many cell lines derived from solid cancers can form spheroids, which recapitulate tumor cell clusters and are more representative of the in vivo situation than 2D cultures. During spheroid formation, a small proportion of a variety of different colon cancer cell lines did not integrate into the sphere and lost cell-cell adhesion properties. An enrichment protocol was developed to augment the proportion of these cells to 100% purity. The basis for the separation of spheroids from non-spheroid forming (NSF) cells is simple gravity-sedimentation. This protocol gives rise to sub-populations of colon cancer cells with stable loss of cell-cell adhesion. SW620 cells lacked E-cadherin, DLD-1 cells lost -catenin and HCT116 cells lacked P-cadherin in the NSF state. Knockdown of these molecules in the corresponding spheroid-forming cells demonstrated that loss of the respective proteins were indeed responsible for the NSF phenotypes. Loss of the spheroid forming phenotype was associated with increased migration and invasion properties in all cell lines tested. Hence, we identified critical molecules involved in spheroid formation in different cancer cell lines. We present here a simple, powerful and broadly applicable method to generate new sublines of tumor cell lines to study loss of cell-cell adhesion in cancer progression.(VLID)463741