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.

The efficient intraoperative identification of cancers requires the development of the bright, minimally-toxic, tumor-specific near-infrared (NIR) probes as contrast agents. Luminescent semiconductor quantum dots (QDs) offer several unique advantages for in vivo cellular imaging by providing bright and photostable fluorescent probes. Here, we present the synthesis of ZnCuInSe/ZnS core/shell QDs emitting in NIR (~750 nm) conjugated to NAVPNLRGDLQVLAQKVART (A20FMDV2) peptide for targeting α_vβ₆ integrin-rich head and neck squamous cell carcinoma (HNSCC). Integrin α_vβ₆ is usually not detectable in nonpathological tissues, but is highly upregulated in HNSCC. QD-A20 showed α_vβ₆ integrin-specific binding in two-dimension (2D) monolayer and three-dimension (3D) spheroid in vitro HNSCC models. QD-A20 exhibit limited penetration (ca. 50 µm) in stroma-rich 3D spheroids. Finally, we demonstrated the potential of these QDs by time-gated fluorescence imaging of stroma-rich 3D spheroids placed onto mm-thick tissue slices to mimic imaging conditions in tissues. Overall, QD-A20 could be considered as highly promising nanoprobes for NIR bioimaging and imaging-guided surgery.

The balance between the amount of drug delivered to tumor tissue and the homogeneity of its distribution is a challenge in the efficient delivery of photosensitizers (PSs) in photodynamic therapy (PDT) of cancer. To date, many efforts have been made using various nanomaterials to efficiently deliver temoporfin (mTHPC), one of the most potent photosensitizers. The present study aimed to develop double-loaded matryoshka-type hybrid nanoparticles encapsulating mTHPC/cyclodextrin inclusion complexes in mTHPC-loaded liposomes. This system was expected to improve the transport of mTHPC to target tissues and to strengthen its accumulation in the tumor tissue. Double-loaded hybrid nanoparticles (DL-DCL) were prepared, characterized, and tested in 2D and 3D in vitro models and in xenografted mice in vivo. Our studies indicated that DL-DCL provided deep penetration of mTHPC into the multicellular tumor spheroids via cyclodextrin nanoshuttles once the liposomes had been destabilized by serum proteins. Unexpectedly, we observed similar PDT efficiency in xenografted HT29 tumors for liposomal mTHPC formulation (Foslip^®) and DL-DCL.

Photodynamic therapy (PDT), an approved anticancer treatment, is reported as a potent inducer of programmed cell death (PCD) by both apoptosis and autophagy. The present study investigated the kinetics of both autophagy and caspase activation in MCF-7 cells submitted to mTHPC-PDT upon condition of treatment promoting ER accumulation of mTHPC. Fluence-dependent immediate cytochrome c (cyt C) release followed by caspase-9 and -7 activation at 1 h post-PDT evidenced a mitochondrial oxidative stress triggered by high light doses leading to >90% of cell death. ER oxidative stress was monitored by the induction of the glucose-related protein chaperone GRP78. From 6 h post-PDT, GRP78 induction was accompanied by the conversion of LC3-I into LC3-II, the hallmark of autophagosome formation. The formation of acid vesicles evidenced by fluorescence microscopy was obvious from 22 h post-PDT. Twenty-four hours post-PDT, cyt C release decreased and caspase-9 cleavage disappeared, while the expression of cleaved caspase-7 remained significant. At the same time, the profiles of GRP78, cleaved caspase-7 and LC3-II expression were similar irrespective of light doses. In contrast to an inhibitor of caspase activation Z-VAD-FMK, the use of autophagy inhibitor, Wortmannin, impaired cytotoxicity along with an increase in caspase-7 activation. These results demonstrate a valuable contribution of autophagy to cell death in mTHPC-photosensitized MCF-7 cells.

Near-infrared (NIR) fluorescence-guided surgery is an innovative technique for the real-time visualization of resection margins. The aim of this study was to develop a head and neck multicellular tumor spheroid model and to explore the possibilities offered by it for the evaluation of cameras for NIR fluorescence-guided surgery protocols. FaDu spheroids were incubated with indocyanine green (ICG) and then included in a tissue-like phantom. To assess the capability of Fluobeam® NIR camera to detect ICG in tissues, FaDu spheroids exposed to ICG were embedded in 2, 5 or 8 mm of tissue-like phantom. The fluorescence signal was significantly higher between 2, 5 and 8 mm of depth for spheroids treated with more than 5 µg/mL ICG (p < 0.05). The fluorescence intensity positively correlated with the size of spheroids (p < 0.01), while the correlation with depth in the tissue-like phantom was strongly negative (p < 0.001). This multicellular spheroid model embedded in a tissue-like phantom seems to be a simple and reproducible in vitro tumor model, allowing a comparison of NIR cameras. The ideal configuration seems to be 450 μm FaDu spheroids incubated for 24 hours with 0.05 mg/ml of ICG, ensuring the best stability, toxicity, incorporation and signal intensity.

Background: Approximately 900 children/adolescents are treated with radiotherapy (RT) every year in France. However, among the 80% of survivors, the cumulative incidence of long-term morbidity - including second malignancies - reach 73.4% thirty years after the cancer diagnosis. Identifying a priori the subjects at risk for RT sequelae is a major challenge of paediatric oncology. Individual radiosensitivity (IRS) of children/adolescents is unknown at this time, probably with large variability depending on the age when considering the changes in metabolic functions throughout growth. We previously retrospectively showed that unrepaired DNA double strand breaks (DSB) as well a delay in the nucleoshuttling of the pATM protein were common features to patients with RT toxicity. We aim to validate a high performance functional assay for IRS prospectively. Methods/design: ARPEGE is a prospective open-label, non-randomized multicentre cohort study. We will prospectively recruit 222 children/adolescents who require RT as part of their routine care and follow them during 15 years. Prior RT we will collect blood and skin samples to raise a primary dermal fibroblast line to carry out in blind the IRS assay. As a primary objective, we will determine its discriminating ability to predict the occurrence of unusual early skin, mucous or hematological toxicity. The primary endpoint is the measurement of residual double-strand breaks 24 h after ex vivo radiation assessed with indirect immunofluorescence (gamma H2AX marker). Secondary endpoints include the determination of pATM foci at 10 min and 1 h (pATM marker) and micronuclei at 24 h. In parallel toxicity including second malignancies will be reported according to NCI-CTCAE v4.0 reference scale three months of the completion of RT then periodically during 15 years. Confusion factors such as irradiated volume, skin phototype, previous chemotherapy regimen, smoking, comorbities (diabetes, immunodeficiency, chronic inflammatory disease...) will be reported. Discussion: ARPEGE would be the first study to document the distribution of IRS in the pediatric subpopulation. Screening hypersensitive patients would be a major step forward in the management of cancers, opening a way to personalized pediatric oncology.