INSIDIA 2.0 High-Throughput Analysis of 3D Cancer Models: Multiparametric Quantification of Graphene Quantum Dots Photothermal Therapy for Glioblastoma and Pancreatic Cancer

Abstract

Cancer spheroids are in vitro 3D models that became crucial in nanomaterials science thanks to the possibility of performing high throughput screening of nanoparticles and combined nanoparticle-drug therapies on in vitro models. However, most of the current spheroid analysis methods involve manual steps. This is a time-consuming process and is extremely liable to the variability of individual operators. For this reason, rapid, user-friendly, ready-to-use, high-throughput image analysis software is necessary. In this work, we report the INSIDIA 2.0 macro, which offers researchers high-throughput and high content quantitative analysis of in vitro 3D cancer cell spheroids and allows advanced parametrization of the expanding and invading cancer cellular mass. INSIDIA has been implemented to provide in-depth morphologic analysis and has been used for the analysis of the effect of graphene quantum dots photothermal therapy on glioblastoma (U87) and pancreatic cancer (PANC-1) spheroids. Thanks to INSIDIA 2.0 analysis, two types of effects have been observed: In U87 spheroids, death is accompanied by a decrease in area of the entire spheroid, with a decrease in entropy due to the generation of a high uniform density spheroid core. On the other hand, PANC-1 spheroids’ death caused by nanoparticle photothermal disruption is accompanied with an overall increase in area and entropy due to the progressive loss of integrity and increase in variability of spheroid texture. We have summarized these effects in a quantitative parameter of spheroid disruption demonstrating that INSIDIA 2.0 multiparametric analysis can be used to quantify cell death in a non-invasive, fast, and high-throughput fashion.

Quantum Dots Mediated Imaging and Phototherapy in Cancer Spheroid Models: State of the Art and Perspectives

“…U87 spheroids become smaller, which is correlated with a decrease of global area, core area, and entropy, while PANC-1 spheroids start disintegrating and become loose, increasing global area and entropy but reducing core area. All these underline the interest of a multiparametric analysis for NPs/drug screening on spheroids, and the ability of GQDs to destroy various types of spheroids by PTT [ 89 ].…”

“…In the work of Perini et al, U87 spheroids decreased in volume, while PANC-1 spheroids became larger, but less dense, as a result of progressive cell disaggregation after PTT. In addition, their study highlighted the potential of the INSIDIA 2.0 macro as a tool allowing a quantified analysis of different parameters specific to spheroids, such as their total area, core area, entropy, etc., helping to guide their evolution in response to the treatment [ 89 ]. Regarding PDT, study on spheroid models is essential.…”

“…This effect is further increased with combination of PDT/PTT with chemotherapy, demonstrating the highest cytotoxic effect. Please refer to experimental details [ 89 , 90 , 91 , 92 ] in the Table 2 of the main text.…”

Carboxylated Graphene Quantum Dots-Mediated Photothermal Therapy Enhances Drug-Membrane Permeability, ROS Production, and the Immune System Recruitment on 3D Glioblastoma Models.

“…As a fact, several studies reported that most of the tests carried out on 2D models show a signi cant change in their outcomes when compared to to 3D cultures. 10,[28][29][30][31][32][33] Therefore, a multidisciplinary approach for the treatment of glioblastoma, which guarantees effective tumor cell targeting and killing and is coupled with an e cient experimental model is mandatory. For this purpose, we exploited another feature of GQDs, which is their ability to absorb near-infrared (NIR) light and convert it into heat, exerting a photothermal effect.…”

“…9 We observed that this synergystic tumor killing e cacy of GQDs and chemotherapy was reduced on 3D glioblastoma spheroids indeed. 10 We therefore expolited another photophysical property of GQDs: the capability of absorbing near-infrared light and convert it into heat, namely phototherapy. Phototherapy, which comprises photothermal therapy (PTT) and photodynamic therapy (PDT), is a non-invasive treatment that can either sensitize cells to chemotherapy by inducing a local increase of temperature mediated by light and that can induce a local increase in the production of reactive oxygen species, respectively.…”