Stroma-Rich Co-Culture Multicellular Tumor Spheroids as a Tool for Photoactive Drugs Screening

Abstract

Conventional 3D multicellular tumor spheroids of head and neck squamous cell carcinoma (HNSCC) consisting exclusively of cancer cells have some limitations. They are compact cell aggregates that do not interact with their extracellular milieu, thus suffering from both insufficient extracellular matrix (ECM) deposition and absence of different types of stromal cells. In order to better mimic in vivo HNSCC tumor microenvironment, we have constructed a 3D stroma-rich in vitro model of HNSCC, using cancer-associated MeWo skin fibroblasts and FaDu pharynx squamous cell carcinoma. The expression of stromal components in heterospheroids was confirmed by immunochemical staining. The generated co-culture FaDu/MeWo spheroids were applied to study penetration, distribution and antitumor efficacy of photoactive drugs such as Temoporfin and Chlorin e6 used in the photodynamic therapy flow cytometry and fluorescence microscopy techniques. We also investigated the distribution of photodiagnostic agent Indocyanine Green. We demonstrated that the presence of stroma influences the behavior of photoactive drugs in different ways: (i) No effect on Indocyanine Green distribution; (ii) lower accumulation of Chlorin e6; (iii) better penetration and PDT efficiency of Temoporfin. Overall, the developed stroma-rich spheroids enlarge the arsenal of in vitro pre-clinical models for high-throughput screening of anti-cancer drugs.

Effect of stroma on the behavior of temoporfin-loaded lipid nanovesicles inside the stroma-rich head and neck carcinoma spheroids

“…1 a). Using the liquid overlay technique [ 8 ], we formed monoculture F5 spheroids (5000 FaDu cells per well), and co-culture spheroids, denoted as F5M2 (5000 FaDu + 2000 MeWo cells) and F5M5 (5000 FaDu + 5000 MeWo cells). After 5 days, FaDu spheroids had a uniform spherical shape with a high cell density most probably due to the strong expression of E-Cadherin adhesion protein (Fig.…”

“…1 d). Based on our previous work, where we presented the images of spheroids stained with Picro Sirius Red (collagen staining) [ 8 ], we performed a semi-quantitative analysis of collagen expression (Fig. 1 e).…”

“…Peripheral fluorescence pattern of Foslip ® also persisted in FaDu/MeWo spheroids, which are rich in ECM components. mTHPC is tightly sequestered in cells, significantly decreasing the probability of intracellular transport of mTHPC within spheroids and, as such, resulting in heterogeneous accumulation of mTHPC only in the outermost peripherical cell layers [ 8 , 14 , 19 ]. In contrast, mTHPC-DCL easily penetrates into FaDu and FaDu/MeWo spheroids from 3 h incubation, demonstrating higher mTHPC fluorescence in the core of spheroids (Fig.…”

“…Most of 3D in vitro tumor models are focusing only on tumor cells, missing stroma components, e.g., extracellular matrix (ECM) [ 6 ], which are considered as a physical barrier for NPs penetration into the tumors [ 5 , 7 ]. Recently, we reported the optimization of sophisticated 3D co-culture tumor spheroids consisting of head and neck squamous carcinoma cells (HNSCC) and cancer-associated fibroblasts (CAFs), capable of recapitulating various levels of ECM expression in the HNSCC [ 8 ]. Using this model, we have demonstrated that the presence of stroma content in 3D HNSCC spheroids influences the behavior of photoactive drugs in different ways.…”

Fibroblasts modulate the tumor cell motility and their mTOR/S6K1 phosphorylation status in vitro

“…That is why, several approaches have been developed to study the modulating effect of stroma on tumor cells using the in vitro models: the first is the study of the effect of factors that can be produced by fibroblasts (a number of chemokines, growth factors, extracellular matrix components etc. ), the second is the application of medium conditioned by fibroblasts [13] and the third is the co-cultivation of fibroblasts and tumor cells [14]. In the first case, the experimental conditions are controlled, but they are only partially similar to the conditions of the organism.…”