Chemotherapy resistance-associated epithelial to endothelial transition in gastric cancer

Gastric cancer (GC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related death. Despite decreased incidence in the last decades and progress in early detection of the disease, most of GC cases are still diagnosed at advanced stages. The only curative treatment remains surgery, but chemo- and radio-therapy are widely used to downstage, prevent metastasis and recurrence. Chemoresistance is a major problem in GC due to limited therapeutic options and it is responsible for treatment failure. We used a primary adenocarcinoma cell line (AGS) and made it resistant to three different chemotherapeutic drugs (5-fluorouracil, cisplatin and paclitaxel), separately. After evaluating the resistance of each line obtained to the different chemotherapy drugs and verifying that there was no cross-resistance, we tried to identify differences between resistant lines and the parental one that could be exploited to restore chemosensitivity. We evaluated factors associated with stemness and found an up-regulation in the resistant cell lines. This led us to investigate whether there was a greater propensity to vasculogenic mimicry in AGS resistant lines. The 5FU-resistant AGS cell line showed a marked capacity for vasculogenic mimicry, a phenomenon described and associated to bad prognosis in cancers like melanomas, but only recently discovered in gastric carcinoma. In addition, 5FU-resistant line acquired CD31 positivity, a marker that is normally associated with endothelial cells, demonstrating that 5FU-resistant AGS cells have undergone an epithelial-to-endothelial transition. We also tested an anti-angiogenic/anti-vasculogenic mimicry agent on 5FU-resistant AGS cells, that restored its chemosensitivity to 5-Fluorouracil. Preliminary studies also investigated metabolic alterations occurring in AGS-chemoresistant cell lines with respect to the chemosensitive AGS-wt cell line. We have detected alterations in AGS-chemoresistant cell lines that could possibly be exploited to revert chemoresistance. I also spent 8 months in the Netherlands, at the Hubrecht Institute, with the purpose of learning how to cultivate and manipulate stomach organoids derived from human biopsies. There I followed a project aimed to differentiate, in gastric organoids, endocrine cells (secreting somatostatin, ghrelin, serotonin, histamine and gastrin) that are found in the organ but only seldomly detected in organoid culture. For this purpose, it was necessary to transduce the pro-endocrine transcription factor Neurogenin 3 by lentivirus, which allowed a strong enrichment of the culture in endocrine cells but not for all lineages. New constructs were therefore cloned for the overexpression of other transcriptional factors that could be involved in endocrine differentiation. These were equally transduced into stomach organoid cultures. However, this approach did not lead to the desired result. Simultaneously, a gastric organoid line with an endogenous tagging for the gastrin gene was created by CRISPR/Cas9, so that when gastrin is expressed, the cell produces also a fluorescent protein that allows its easy identification. The realization of this cell line allowed a fast-screening of different culture conditions that could induce differentiation towards gastrin-secreting endocrine lineage. This led in a short time to the discovery of molecules that induced differentiation in gastrin-secreting cells, testifying the feasibility of the proposed method.