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

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

Project goal: The main goal is to develop novel therapies for KRAS mutant CRC with nanoforms of oxidative drugs ATO and DVC that work effectively in solid tumor environment simulated by 3D cell culture. We will use enzyme-functionalized nanocarriers that can partially degrade the extracellular matrix and enhance drug penetration.

Project description: 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.

Realisation period: 2023-2025

Expected results: 1) Our team expects to deliver better understanding of the differences in the ATO/DVC mechanism in 2D and 3D cell culture environment which would bring us closer to revealing the mechanism of resistance and toxic (side) effects in this anticancer therapy for CRC. The project will bring us closer in the understanding the mechanism of suicidal ROS generation upon application of oxidative drugs.
2) Development of novel therapies for CRC requires new drugs development (which are costly to get through clinical trials) or reformulation and novel methods of delivery of already approved drugs (as ATO/VC) as proposed in this project. We expect to produce novel class of smart nanocarriers of ATO/DVC which can penetrate CRC tissue (3D cell culture) and make the therapy more successful with lower side effects. This will be achieved by lowering the overall amount of ATO required for the nanoform therapy compared with free ATO administration. This may result in a revolutionary new anticancer therapy with potential application for CRC but also for other types of tumors.
3) Based on our results of study of the ATO/VC mechanisms of action in 2D and 3D cell culture as well as the development nanoform of delivery of these drugs the results will be published:
- at least 3 articles and/or reviews in peer-reviewed scientific journals, included in the Q1,Q2 and/or Q3 according to the IF in the Web of Science(WoS) and/or having a CiteScore percentile in the Scopus ≥50;
- either at least 2 articles and/or reviews in peer-reviewed journals included in the Q1,Q2 and/or Q3 by IF in the WoS and/or having a percentile in CiteScore in the Scopus ≥65;
- either at least 1 article or review in a peer-reviewed journals in the Q1 in the WoS or a CiteScore percentile in the Scopus ≥95. Open access policy endorsed by Nazarbayev University(NU) will be followed. The project has a planned budget for open access article processing fees. All manuscripts and published conference abstracts arising from this work will be deposited in the NU Digital Repository (NUR, nur.nu.edu.kz).
4) The project will also allow NU and NLA to further develop novel anticancer team capabilities, train a new generation of Kazakhstani researchers in this field and advance biomedical knowledge and the creation of modern nanotechnology-based therapies for colorectal cancer.
5) The project team will ensure that the results are disseminated by participation and presentations at one international and one national conference per year over the duration of the project. The team will also participate in organizing the 3rd and 4th Eurasian International Conferences on Antimicrobial and Biomedical Nanotechnologies, in 2023 and 2024 at NU, where our research assistants and the PI will be delivering research talks.

Methodology: We will employ 3D culture models of KRAS mutant CRC, . 3D culture of cell clusteroids will be fabricated by using a methodology recently developed by Prof Paunov group (see Task 2.2 below). 2D-cultured cells lose their polarity and have equal access to various compounds in the medium in disparity with in vivo circumstances as CRC in the colon do not grow as flat 2D cell structures in the human body. The key hypothesis here is that in 3D culture microenvironment can be mimicked in tumour cell clusteroids (tumoroids), where the nutrients and oxygen deficiency is observed in the cores of the clusters similarly to the 3D solid tumours found in colon. Cells in the tumours may have different responses to oxidative stress agents such as ATO and DVC, depending on their position within the tumour structure i.e. surface vs. tumour core. So far, most of studies in this area which focused on novel anticancer drugs development were typically performed using monolayer cultures (2D) of cancer cells and frequently leading to the failure of subsequent in vivo models because may not be representative of the real outcome. The proposed technique of generation of CRC clusteroids (tumoroids) is novel approach in the testing the efficiency of the oxidative drug. For this purpose, we will: (a) Produce 3D cell culture of HCT-116 and SW480 CRC cell lines. (b) 3D tumoroids and cells grown in 2D monolayers will be treated with ATO/DVC. After the treatment the 2D cell culture and the tumoroids will then be dispersed down to individual cells using trypsin and studied by flow cytometry to find the distribution of the live/dead cells in the treated clusters and assess mitochondrial membrane potential. This experimental set up would allow us to test bioactivity of ATO, DVC and combination of these oxidative compounds in core, hypoxic, regions of this 3D culture of cancer model. Results would provide answers to whether tumour cells in metabolically compromised microenvironments are able or not or to which extend to respond to this specific anticancer treatment combination.

The development of nanoparticle carriers filled with ATO would require collaboration with nanotechnology experts to design nano-carriers for mitochondria targeted controlled-release of ATO. The approach will be based on research from for anticancer therapy instead of antibiotic treatment. ATO will be conjugated or co-precipitated with biodegradable polymers like polylactic acid, PLGA or shellac, following coating with polyethylene glycol-derivatives for suppressing the immune response towards the nanocarriers and increasing blood circulation time. ATO/DVC-loaded P407-stabilized shellac NPs will be produced by co-precipitation with pH drop methods and further coated with Sainase or Alcalase (proteases). The formulated nanoparticles will be characterised by dynamic light scattering (DLS) and electron microscopy (TEM, SEM), EDS and the ATO encapsulation efficiency and release kinetics from the nanocarrier will be determined along with the enzymatic coating efficiency. irst exploratory experiments with novel nanoparticles will be tested in 2D and 3D models of CRC cell lines (HCT-116 and SW620). We will stain the cells with cell tracker dye and the nanoparticles will be also fluorescently tagged to allow us to colocalise them within the 3D cell cultures and examine their ability to penetrate in between the cells. We will optimise the nanocarrier ATO payload and the enzyme coating to ensure more uniform treatment of the CRC tissue with the oxidative drugs combination. The safety and efficacy in vitro will be investigated by assessing the ATO nano-formulation cytotoxicity, cell proliferation, migration, apoptosis, and other changes in the 3D culture cell behaviour. We will compare the effect of ATO/DVC in nanoparticle form at the same concentrations and treatment times without loading into nanoparticles and without protease coating. This will allow us to evaluate how much the protease coating improves the oxidative drug penetration within the cell clusteroids. This will be done by using CLSM as well as by dispersing the 3D cell culture by tripsin/EDTA treatment and using flow cytometry. Various markers will be evaluated to track cell viability (MTS assay, FDA assay), cell apoptosis (Annexin-V) as well as ROS production in mitochondria (MitoSox-Red) and overall (DCFDA assay).