Nanotechnology enhanced oxidative drug treatment of 3D culture of KRAS mutant colorectal cancer cell lines

Grant funding 2023-2025

The prevalence of colorectal cancer (CRC) has been dramatically growing at an alarming rate globally in recent years. In 2020 1.93 million new CRC cases were diagnosed, and 0.94 mil deaths, which represents approximately 10% of the global cancer incidence (total 19.29 million new cases) and 9.4% of all cancer caused deaths. CRC is the third leading cause of cancer related deaths worldwide. Recent analysis of the total incidence of CRC throughout Kazakhstan shows an increase in the rate of incidence CRC cases in stages I and II since 2011[1]. Around 40–52% of CRC cases have Kirsten rat sarcoma (KRAS) gene mutation that leads to hyperactivation of the mitogen-activated protein kinase pathways that promotes cancer metastasis and resistance. This results in a distinctive cellular metabolism with an unbalanced redox state due to high glucose intake leading to elevated generation of reactive oxygen species (ROS)[2]. Our research team has found that the oxidizing drug Arsenic Trioxide (ATO) in combination with Vitamin C or D-Vitamin C (DVC) induced a potent cytotoxic effect in KRAS mutant CRC[3]. The combined treatment induces suicidal ROS production by mitochondria (SRPM) leading to cell apoptosis, however exact mechanism remains poorly understood. Most recent studied have been done on individual cells (2D cell culture) or directly on animal (mouse) models, which limits the understanding the impact of the solid tumor environment on the efficacy of the treatment in human body. This project will examine the effect of ATO/DVC on CRC in 2D and 3D cell culture environment which is expected to better mimic the collective effect of the cancer cell tissue and to understand the processes that promote resistance. Our main goal is to investigate the targeting mechanism of the ATO/DVC oxidative drug combination on CRC. Characterizing the suicidal generation of ROS induced in KRAS mutant CRC by the ATO/DVC will be valuable in understanding of the mechanism of cell death. We will explore the mitochondrial metabolism in both 2D cell culture and in cell clusteroids under identical conditions. Our project team has developed robust technique for high throughput preparation of colorectal tumoroids which allows a wide range of experiments to be done under the same ATO/DVC treatment conditions.

We aim to explore the difference in the mitochondrial activity in 3D CRC culture environments as well as cell viability and apoptotic response compared with individual CRC. One would expect to have a radial distribution of the oxidative drug throughout the cell clusteroids, hypoxic conditions reduced glucose intake in the core of the cell cluster which would decrease the therapy effectiveness in solid tumors. We also aim to develop a novel protease-functionalized nanocarrier for delivery of ATO/DVC which is expected to increase the effectiveness of the therapy in solid tumors. Although very efficient in delivery of high local concentrations of anticancer agents, nanocarriers have limited dept of penetration in solid tumor because they get stuck in the collagen network of the extracellular matrix (ECM). Our idea here is to functionalize the surface of ATO/DVC nanocarriers with a collagenase which would help it to digest its way through ECM in between the CRC and allow them to penetrate deeply in the core of the solid tumor. In our studies the solid tumors will be modelled with large cell clusteroids. We will explore the effects of this active nano-formulation of ATO/DVC on both individual CRC and cell clusteroids under identical conditions. This new anticancer nanotechnology is expected to increase the treatment efficacy and reduce the overall concentration of oxidative drug, which is acting indiscriminately and has high toxicity in free form. Similar nanoform can also be applied to other types of solid tumors and help to decrease the side effects in patients and improve outcomes.