Gastric cancer spheroids and organoids: protein kinases and extracellular matrix architecture tracking and therapeutic impact

Abstract

Stomach cancer is fifth in incidence and third in mortality worldwide. The leading cause of gastric carcinogenesis is infection with Helicobater pylori, a bacterium which infects almost half the human population. Differences in virulence strains, other environmental factors and patient (genetic) characteristics contribute to cancer progression among those individuals infected with H pylori. The molecular mechanisms associated with H. pylori-induced carcinogenesis remain not fully understood, but include progression to atrophic gastritis, and subsequently intestinal metaplasia (IM), which is considered a precursor lesion. Patients are often diagnosed late, with fewer therapeutic options and poorer survival. For this reason, at the Erasmus MC, an extensive surveillance protocol is in place for patients with IM. In this project, cancer hallmarks will be the focus of the investigation: a) protein kinases associated with proliferation and resistance to death stimuli; b) extracellular matrix components (mainly, integrins and collagen) relevant to signaling pathways associated with tumor progression and fibrotic condition. The latter, among other aspects, plays a role in preventing the spread of drugs in the tumor mass, compromising therapeutic efficacy. For this purpose, 3D culture models (spheroid and organoid) of gastric cancer and its precursor lesions will be used. We have established 2 study fronts: in Brazil, analyzes will be carried out with spheroids whose data will serve to guide the validation in organoids, which will be performed by the group led by Prof. Fuhler (Netherlands). Spheroid mimics better interactions such as: cell-matrix, cell-cell, in addition to presenting zones (regions) with different gradients of nutrients, pH and oxygenation, as observed in vivo tumor. Spheroids derived from stomach cancer cells lines and IM patients will be cultured in separately or mixes with fibroblasts. Specifically, in relation to protein kinases: platelet-derived growth factor receptor, epidermal growth factor receptor, Scr kinase protein and mitogen-activated protein kinases, we will analyze their quantity and/or phosphorylation, as well as whether the location of these proteins, in both spheroid models, and how this may be influenced by the spheroid size, hypoxia gradient and inhibitors (used in the clinic or in the clinical screening phase) of these enzymes. Together, the ability of mitoxantrone to interfere in the spatial arrangement of the components (integrins and collagen) of the extracellular matrix will be investigated, and consequently, to improve the bioavailability of kinase inhibitors. In other words, the intended purpose of this study is to prevent or reorganize fibrotic regions of affecting the spheroid, a critical factor in the diffusion of drugs in the tumor mass. Thus, the present investigation may bring greater robustness to the screening of new drugs, assist in the prediction of effectiveness of drug response, in addition to being a useful tool for the rational development of formulations that allow accessibility of the drug to the molecular target (for example , kinase), if it has a heterogeneous location along the spheroid. In addition, another aspect that deserves to be highlightedis that the project may demonstrate new aspects, from the molecular point of view, to support innovative strategies in the development of antitumor drugs. (AU)