Cancer Biology

Chemoresistance, the ability of cancer cells to overcome therapeutic interventions, is an area of active research. Studies on intrinsic and acquired chemoresistance have partly succeeded in elucidating some of the molecular mechanisms in this elusive phenomenon. Hence, drug-resistant cellular models are routinely developed and used to mimic the clinical scenario in-vitro. In an attempt to identify the underlying molecular mechanisms that allow ovarian cancer cells to gradually acquire chemoresistance, we have developed isogenic cellular models of cisplatin and paclitaxel resistance (singularly and in combination) over six months, using a clinically relevant modified pulse method. These models serve as important tools to investigate the underlying molecular players, modulation in genetics, epigenetics, and relevant signaling pathways, as well as to understand the role of drug detoxification and drug influx-efflux pathways in development of resistance. These models can also be used as screening tools for new therapeutic molecules. Additionally, repurposing therapeutic agents approved for diseases other than cancer have gained significant attention in improving cancer therapy. To investigate the effect of metformin on acquirement of chemoresistance, we have also developed a combinatorial model of metformin and platinum-taxol, using two different strategies. All these models were subsequently used to study modulation in receptor tyrosine kinase pathways, cancer stem cell functionalities, autophagy, metastasis, metabolic signatures, and various biological processes during development of chemoresistance. Herein, we outline the protocols used for developing these intricate resistant cellular models.

Graphic abstract:

Schematic of the step-wise development of cellular chemoresistant model. Schema illustrating the modified pulse method for the development of individual and combinatorial resistant models. Two different cell lines (A2780 and OAW42) were exposed to drug treatment (either alone or in various combinations) of one concentration for three consecutive cycles (3×). Furthermore, the surviving cells were sub-cultured subsequently with increasing drug concentrations. After every treatment cycle, 50% of the cells were cryo-preserved for further experiments. Additionally, to check for the development of chemoresistance and to assess the changes in cell cycle during resistance development, cell viability assays and flow cytometry were performed.

This protocol illustrates a pipeline for modeling the nonlinear behavior of intracellular signaling pathways. At fixed spatial points, nonlinear signaling dynamics are described by ordinary differential equations (ODEs). At constant parameters, these ODEs may have multiple attractors, such as multiple steady states or limit cycles. Standard optimization procedures fine-tune the parameters for the system trajectories localized within the basin of attraction of only one attractor, usually a stable steady state. The suggested protocol samples the parameter space and captures the overall dynamic behavior by analyzing the number and stability of steady states and the shapes of the assembly of nullclines, which are determined as projections of quasi-steady-state trajectories into different 2D spaces of system variables. Our pipeline allows identifying main qualitative features of the model behavior, perform bifurcation analysis, and determine the borders separating the different dynamical regimes within the assembly of 2D parametric planes. Partial differential equation (PDE) systems describing the nonlinear spatiotemporal behavior are derived by coupling fixed point dynamics with species diffusion.

We have demonstrated that a specific population of ginger-derived nanoparticles (GDNP-2) could effectively target the colon, reduce colitis, and alleviate colitis-associated colon cancer. Naturally occurring GDNP-2 contains complex bioactive components, including lipids, proteins, miRNAs, and ginger secondary metabolites (gingerols and shogaols). To construct a nanocarrier that is more clearly defined than GDNP-2, we isolated lipids from GDNP-2 and demonstrated that they could self-assemble into ginger lipid-derived nanoparticles (GLDNP) in an aqueous solution. GLDNP can be used as a nanocarrier to deliver drug candidates such as 6-shogaol or its metabolites (M2 and M13) to the colon. To characterize the nanostructure of GLDNP, our lab extensively used atomic force microscopy (AFM) technique as a tool for visualizing the morphology of the drug-loaded GLDNP. Herein, we provide a detailed protocol for demonstrating such a process.