Therapeutic response differences between 2D and 3D tumor models of magnetic hyperthermia

Abstract

Magnetic hyperthermia-based cancer therapy (MHCT) has surfaced as one of the promising techniques for inaccessible solid tumors. It involves generation of localized heat in the tumor tissues on application of an alternating magnetic field in the presence of magnetic nanoparticles (MNPs). Unfortunately, lack of precise temperature and adequate MNP distribution at the tumor site under in vivo conditions has limited its application in the biomedical field. Evaluation of in vitro tumor models is an alternative for in vivo models. However, generally used in vitro two-dimensional (2D) models cannot mimic all the characteristics of a patient's tumor and hence, fail to establish or address the experimental variables and concerns. Considering that three-dimensional (3D) models have emerged as the best possible state to replicate the in vivo conditions successfully in the laboratory for most cell types, it is possible to conduct MHCT studies with higher clinical relevance for the analysis of the selection of magnetic parameters, MNP distribution, heat dissipation, action and acquired thermotolerance in cancer cells. In this review, various forms of 3D cultures have been considered and the successful implication of MHCT on them has been summarized, which includes tumor spheroids, and cultures grown in scaffolds, cell culture inserts and microfluidic devices. This review aims to summarize the contrast between 2D and 3D in vitro tumor models for pre-clinical MHCT studies. Furthermore, we have collated and discussed the usefulness, suitability, pros and cons of these tumor models. Even though numerous cell culture models have been established, further investigations on the new pre-clinical models and selection of best fit model for successful MHCT applications are still necessary to confer a better understanding for researchers.

Fig. 1. Schematic representation of the mechanism of magnetic hyperthermia at the sub-cellular level (A) using optimized nanosystems by altering their size, composition, shape and surface chemistry; (B) understanding the effect of loacalized heat at the sub-cellular level by disrupting the membrane integrity/structure, induction of DNA damage and apoptotic cell death, ROS generation, decreased mitochondrial membrane potential, upregulation of heat shock proteins (HSPs) and activation of immune cells in the tumor microenvironment.

Fig. 2. Representation of cellular morphology of rat glioma C6 cells under 2D and 3D cultural conditions as observed by scanning electron microscopy in our lab. Cells were cultured on a glass coverslip in case of 2D (left panel) or on agarose gel to form 3D spheroid cultures (right panel). Scale bar = 5 μm.

Fig. 3. Tumor microenvironment under different culture conditions i.e. (A) 2D, (B) 3D and (C) in vivo conditions.

Fig. 4. Characteristics of 3D cultures as represented by tumor spheroids. (A) Cells are organized in a heterogenous arrangement, mainly in three layers (dividing, senescent and necrotic cells) owing to differential supply of nutrients and oxygen within the cells packed in the inner core which resembles that observed in patient tumors. (B) Tightly packed cellular organization in spheroids hinders penetration capability of drugs or nanoparticles leading to non-uniform distribution in tumors. Cellular organisation also favors tumor re-growth from a small cellular cluster resulting from lesser damage to cells on AMF application. (C) Enhanced contact between various cells (cancer–cancer and cancer–stromal cells interaction) reproduces the integrin distribution and signal transduction pathways found under in vivo conditions. (D) Representation of differential gene expression levels obtained from cultures grown in 2D, 3D and in vivo conditions.

Chart 1. Live/dead assay to study the effect of MHCT on C6 spheroids. Live/dead assay on C6 tumor 3D cultures representing (A) untreated cells and (B) cells exposed to AMF (405 kHz and 168 G) for 20 min. The AMF exposure resulted in enhancement of central necrotic zone area in the spheroids as seen by increased uptake of propidium iodide (PI) dye. Live cells were stained green using Fluorescein diacetate (FDA; Invitrogen) and dead/necrotic cells were stained red using propidium iodide (PI; Invitrogen) Scale bar = 200 μm.

Fig. 5. Differential heat shock response under 2D and 3D culture conditions. (A) On application of AMF to cells cultured under 2D or 3D conditions, heat stress response is activated which induces differential expression of HSPs depending upon the culture conditions; (B) HSF1 denotes transcription factor for HSPs (heat shock factor 1); HSE denotes heat shock elements (DNA binding domain for HSF1); (C) heat stress under 2D conditions causes higher cell death as cells are exposed to AMF uniformly while under 3D conditions, cells acquire higher thermo-tolerance owing to enhanced expression of HSPs.

Chart 2. Expression analysis of HSPs under different culture conditions. Time-dependent comparative gene expression analysis of (A) HSP70 and (B) HSP90 as expressed in monolayers and 3D tumor spheroids of C6 glioma cells after exposure to AMF of strength 168 G and 405 kHz for 20 min.

Fig. 6. Successful implications of 3D tumor cell culture models for MHCT-based therapies in tumor spheroids (A–D); and scaffolds (E–G). (A) Reprinted with permission from ref. © (2017) Elsevier. (B) Reprinted with permission from ref. © (2019) Elsevier. (C) Reprinted with permission from ref. © (2019) American Chemical Society. (D) Reprinted with permission from ref. © (2020) Elsevier. (E) Reprinted with permission from ref. © (2016) American Chemical Society. (F) Reprinted with permission from ref. © (2018) American Chemical Society. (G) With permission from ref. © 2016 Royal Society of Chemistry.

Ruby Gupta

Deepika Sharma