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The deployment of hyperthermia-based treatments for cancer therapy has captured the attention of different researchers worldwide. In particular, the application of light-responsive nanomaterials to mediate hyperthermia has revealed promising results in several pre-clinical assays. Unlike conventional therapies, these nanostructures can display a preferential tumor accumulation and thus mediate, upon irradiation with near-infrared light, a selective hyperthermic effect with temporal resolution. Different types of nanomaterials such as those based on gold, carbon, copper, molybdenum, tungsten, iron, palladium and conjugated polymers have been used for this photothermal modality. This progress report summarizes the different strategies that have been applied so far for increasing the efficacy of the photothermal therapeutic effect mediated by nanomaterials, namely those that improve the accumulation of nanomaterials in tumors (e.g. by changing the corona composition or through the functionalization with targeting ligands), increase nanomaterials' intrinsic capacity to generate photoinduced heat (e.g. by synthesizing new nanomaterials or assembling nanostructures) or by optimizing the parameters related to the laser light used in the irradiation process (e.g. by modulating the radiation wavelength). Overall, the development of new strategies or the optimization and combination of the existing ones will surely give a major contribution for the application of nanomaterials in cancer PTT.

Scalable and reproducible production of 3D cellular spheroids is highly demanded, by pharmaceutical companies, for drug screening purposes during the pre-clinical evaluation phase. These 3D cellular constructs, unlike the monolayer culture of cells, can mimic different features of human tissues, including cellular organization, cell-cell and cell-extracellular matrix (ECM) interactions. Up to now, different techniques (scaffold-based and -free) have been used for spheroids formation, being the Liquid Overlay Technique (LOT) one of the most explored methodologies, due to its low cost and easy handling. Additionally, during the last few decades, this technique has been widely investigated in order to enhance its potential for being applied in high-throughput analysis. Herein, an overview of the LOT advances, practical approaches, and troubleshooting is provided for those researchers that intend to produce spheroids using LOT, for drug screening purposes. Moreover, the advantages of the LOT over the other scaffold-free techniques used for the spheroids formation are also addressed. Highlights • 2D cell culture drawbacks are summarized;• spheroids mimic the features of human tissues;• scaffold-based and scaffold-free technologies for spheroids production are discussed; • advantages of LOT over other scaffold-free techniques are highlighted; • LOT advances, practical approaches and troubleshooting are underlined.

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