A Macrophages-Enriched Head and Neck Tumor Spheroid Model to Study Foslip® Behavior in Tumor Microenvironment.

Purpose: The tumor microenvironment (TME) is composed of various stromal components, including immune cells such as tumor-associated macrophages (TAMs), which play a crucial role in cancer initiation and progression. TAMs can exhibit either a tumor-suppressive M1 or a tumor-promoting M2 phenotype. First, we aimed to develop a 3D human heterotypic model consisting of head and neck squamous cell carcinoma (HNSCC) cells and different subtypes of macrophages to replicate the interactions between immune cells and cancer cells. We further investigated the behavior of Foslip®, a liposomal formulation of temoporfin, using a macrophage-enriched 3D model. Methods: Monocytes were differentiated into M1 and M2 macrophages, which represent two distinct subtypes. Following histological and molecular characterization, these macrophages were used to establish a 3D spheroid model of HNSCC enriched with either polarized macrophages or conditioned media. Flow cytometry and fluorescence microscopy were used to assess the accumulation and distribution of Foslip®. The cytotoxic effect of Foslip®-mediated photodynamic therapy (PDT) was evaluated using flow cytometry. Results: We developed heterotypic spheroids characterized by a mixed phenotype of evenly distributed macrophages. In this 3D co-culture model, both M1 and M2 macrophages showed significantly higher accumulation of Foslip® compared to the cancer cells. Although this differential accumulation did not drastically affect the overall PDT efficiency, spheroids generated with conditioned media exhibited a significant enhancement in photo-induced cell death, suggesting that the microenvironment could modulate the response to Foslip®-PDT. Conclusion: 3D models of HNSCC cells and macrophages provide valuable insights into the complex response of HNSCC cells to PDT using Foslip® in vitro. This model can be used to screen immunomodulatory nanomedicines targeting TAMs in solid head and neck tumors, either alone or in combination with standard therapies.

Phototoxicity of temoporfin-loaded cyclodextrin nanosponges in stroma-rich three-dimensional models of head and neck cancer.

Photodynamic therapy is a multistage treatment, in which cancerous and precancerous cells are destroyed by light activation of a drug (photosensitizer). For a long time, high cellular uptake of the photosensitizer was an important indication of efficient PDT, while the role of photosensitizer penetration was unexplored. Recently, we have demonstrated that nanosponges based on hypercrosslinked ß-cyclodextrin polymer (ß-CDp) can increase drug penetration at the cost of their cellular uptake in multicellular spheroids, paving the way for studying the impact of penetration on PDT response. In the present work, we used ß-CDp nanosponges to deliver temoporfin to the depth of stroma-rich head and neck cancer multicellular spheroids and then assess PDT response. Encapsulation of temoporfin in ß-CDp nanosponges resulted in increased penetration and more uniform distribution of temoporfin in spheroids, however, was also associated with a two-fold reduction of cellular uptake compared to the free drug. Nevertheless, we demonstrated that ß-CDp nanosponges possess similar PDT efficiency as the free drug in stroma-rich head and neck cancer multicellular spheroids. Overall, this study suggests that ß-CDp nanosponges are a strong candidate for in vivo studies as they have fewer "off-target" effects while providing a similar therapeutic response.

A Coculture Based, 3D Bioprinted Ovarian Tumor Model Combining Cancer Cells and Cancer Associated Fibroblasts.

Ovarian cancer remains a major public health issue due to its poor prognosis. To develop more effective therapies, it is crucial to set-up reliable models that closely mimic the complexity of the ovarian tumor's microenvironment. 3D bioprinting is currently a promising approach to build heterogenous and reproducible cancer models with controlled shape and architecture. However, this technology is still poorly investigated to model ovarian tumors. In this study, a 3D bioprinted ovarian tumor model combining cancer cells (SKOV-3) and cancer associated fibroblasts (CAFs) are described. The resulting tumor models show their ability to maintain cell viability and proliferation. Cells are observed to self-assemble in heterotypic aggregates. Moreover, CAFs are observed to be recruited and to circle cancer cells reproducing an in vivo process taking place in the tumor microenvironment. Interestingly, this approach also shows its ability to rapidly generate a high number of reproducible tumor models that can be subjected to usual characterizations (cell viability and metabolic activity; histology and immunological studies; and real-time imaging). Therefore, these ovarian tumor models can be an interesting tool for high throughput drug screening applications.

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

Quantum Dots (QDs) are fluorescent nanoparticles known for their exceptional optical properties, i.e., high fluorescence emission, photostability, narrow emission spectrum, and broad excitation wavelength. These properties make QDs an exciting choice for bioimaging applications, notably in cancer imaging. Challenges lie in their ability to specifically label targeted cells. Numerous studies have been carried out with QDs coupled to various ligands like peptides, antibodies, aptamers, etc., to achieve efficient targeting. Most studies were conducted in vitro with two-dimensional cell monolayers (n = 8902) before evolving towards more sophisticated models. Three-dimensional multicellular tumor models better recapitulate in vivo conditions by mimicking cell-to-cell and cell-matrix interactions. To date, only few studies (n = 34) were conducted in 3D in vitro models such as spheroids, whereas these models could better represent QDs behavior in tumors compared to monolayers. Thus, the purpose of this review is to present a state of the art on the studies conducted with Quantum Dots on spheroid models for imaging and phototherapy purposes.

Antitumor Effect and Induced Immune Response Following Exposure of Hexaminolevulinate and Blue Light in Combination with Checkpoint Inhibitor in an Orthotopic Model of Rat Bladder Cancer.

Previous studies have found that use of hexaminolevulinate (HAL) and blue light cystoscopy (BLC) during treatment of bladder cancer had a positive impact on overall survival after later cystectomy, indicating a potential treatment effect beyond improved diagnostic accuracy. The aim of our study was to determine whether HAL and BL mimicking clinically relevant doses in an orthotopic rat model could have therapeutic effect by inducing modulation of a tumor-specific immune response. We also assessed whether administration with a checkpoint inhibitor could potentiate any effects observed. Rats were subjected to HAL BL alone and in combination with anti-PD-L1 and assessed for anti-tumor effects and effects on immune markers. Positive anti-tumor effect was observed in 63% and 31% of rats after, respectively, 12 and 30 days after the procedure, together with a localization effect of CD3+ and CD8+ cells after 30 days. Anti-tumor effect at 30 days increases from 31% up to 38% when combined with intravesical anti-PD-L1. In conclusion, our study demonstrated treatment effects with indications of systemic immune activation at diagnostic doses of HAL and blue light. The observed treatment effect seemed to be enhanced when used in combination with intravesically administrated immune checkpoint inhibitor.

Fluorescence imaging analysis of distribution of indocyanine green in molecular and nanoform in tumor model.

BACKGROUND: The efficient intraoperative identification of tumors requires the development of highly specific near-infrared (NIR) probes as contrast agents. One of the most effective dyes existing in clinic oncology is Indocyanine Green (ICG). However, ICG has a rapid excretion, thus ruling out its extended accumulation in pathological tissues therefore limiting its clinical applications. ICG colloid solution (ICG NPs) consists predominantly of J-aggregates and to a lesser extent of H-aggregates and monomers. In the present study we assessed the spectral properties of ICG nanoforms in preclinical models. METHODS: We used optical spectroscopy and video fluorescence navigation to monitor accumulation and distribution of ICG monomers and ICG NPs in various tissues in mice with xenografted laryngopharyngeal carcinoma after intravenous drugs injection. RESULTS: After i.v. injection, the molecular form of ICG was not retained in the tumor and its circulation cycle averaged 5 min. Alternatively, the nanoform of the drug had a different pharmacokinetics, reaching maximum accumulation 24 h after intravenous injection. Moreover, once in the circulation, we observed a progressive accumulation in the tumor of both ICG H-aggregates and ICG monomers, but not J-aggregates. CONCLUSION: Spectral characteristics of ICG NPs indicated the presence of several fractions, namely, J- and H-aggregates along with molecular forms. These fractions had different fluorescence spectra, allowing us to track the transformation of the drug in vivo conditions. After ICG NPs administration, J-aggregates induce accumulation of monomeric forms in the tumor, enabling extended intraoperative diagnostic, and as such further studies of J-aggregates for theranostic applications in oncological surgery are of great interest.

Analysis of Fluorescence Decay Kinetics of Indocyanine Green Monomers and Aggregates in Brain Tumor Model In Vivo.

Spectroscopic approach with fluorescence time resolution allows one to determine the state of a brain tumor and its microenvironment via changes in the fluorescent dye's fluorescence lifetime. Indocyanine green (ICG) is an acknowledged infra-red fluorescent dye that self-assembles into stable aggregate forms (ICG NPs). ICG NPs aggregates have a tendency to accumulate in the tumor with a maximum accumulation at 24 h after systemic administration, enabling extended intraoperative diagnostic. Fluorescence lifetime analysis of ICG and ICG NPs demonstrates different values for ICG monomers and H-aggregates, indicating promising suitability for fluorescent diagnostics of brain tumors due to their affinity to tumor cells and stability in biological tissue.

Ionizing radiations induce shared epigenomic signatures unraveling adaptive mechanisms of cancerous cell lines with or without methionine dependency.

BACKGROUND: Although radiation therapy represents a core cancer treatment modality, its efficacy is hampered by radioresistance. The effect of ionizing radiations (IRs) is well known regarding their ability to induce genetic alterations; however, their impact on the epigenome landscape in cancer, notably at the CpG dinucleotide resolution, remains to be further deciphered. In addition, no evidence is available regarding the effect of IRs on the DNA methylome profile according to the methionine dependency phenotype, which represents a hallmark of metabolic adaptation in cancer. METHODS: We used a case-control study design with a fractionated irradiation regimen on four cancerous cell lines representative of HCC (HepG2), melanoma (MeWo and MeWo-LC1, which exhibit opposed methionine dependency phenotypes), and glioblastoma (U251). We performed high-resolution genome-wide DNA methylome profiling using the MethylationEPIC BeadChip on baseline conditions, irradiated cell lines (cumulative dose of 10 Gy), and non-irradiated counterparts. We performed epigenome-wide association studies to assess the effect of IRs and methionine-dependency-oriented analysis by carrying out epigenome-wide conditional logistic regression. We looked for epigenome signatures at the locus and single-probe (CpG dinucleotide) levels and through enrichment analyses of gene ontologies (GO). The EpiMet project was registered under the ID#AAP-BMS_003_211. RESULTS: EWASs revealed shared GO annotation pathways associated with increased methylation signatures for several biological processes in response to IRs, including blood circulation, plasma membrane-bounded cell projection organization, cell projection organization, multicellular organismal process, developmental process, and animal organ morphogenesis. Epigenome-wide conditional logistic regression analysis on the methionine dependency phenotype highlighted several epigenome signatures related to cell cycle and division and responses to IR and ultraviolet light. CONCLUSIONS: IRs generated a variation in the methylation level of a high number of CpG probes with shared biological pathways, including those associated with cell cycle and division, responses to IRs, sustained angiogenesis, tissue invasion, and metastasis. These results provide insight on shared adaptive mechanisms of the epigenome in cancerous cell lines in response to IR. Future experiments should focus on the tryptic association between IRs, the initiation of a radioresistance phenotype, and their interaction with methionine dependency as a hallmark of metabolic adaptation in cancer.

Modulation of Temoporfin Distribution in Blood by ß-Cyclodextrin Nanoshuttles.

Photodynamic therapy represents a more targeted and less invasive alternative cancer treatment to traditional modalities. Temoporfin, as with many photosensitizers, is given by injection into a vein, and its subsequent fate is largely determined by the binding to plasma proteins and interaction with endothelial and blood cells. Thus, it is essential to be able to control and to alter the biodistribution of temoporfin in blood. In the present study, we evaluated the effect of co-administration of temoporfin with randomly methylated ß-CD (Me-ß-CD) on the distribution of temoporfin in the main subpopulations of blood cells of healthy donors using absorbance spectrophotometry and flow cytometry. We showed that cell-bound temoporfin fraction in blood strongly depends on the concentration of Me-ß-CD. In fact, the accumulation of temoporfin in white blood cells was more sensitive than that in red blood cells, due to the higher volume of membranous organelles in white blood cells. Finally, we demonstrated that Me-ß-CD significantly increases cellular uptake of temoporfin cancer human Burkitt's lymphoma Raji cells. The presence of Me-ß-CD resulted in a spotted pattern of temoporfin distribution in the plasma membrane compartment. Our results clearly demonstrated that ß-CDs derivatives provide new options to modulate temoporfin biodistribution in blood.

Validation of a Three-Dimensional Head and Neck Spheroid Model to Evaluate Cameras for NIR Fluorescence-Guided Cancer Surgery.

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.

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

BACKGROUND: Despite the highly expected clinical application of nanoparticles (NPs), the translation of NPs from lab to the clinic has been relatively slow. Co-culture 3D spheroids account for the 3D arrangement of tumor cells and stromal components, e.g., cancer-associated fibroblasts (CAFs) and extracellular matrix, recapitulating microenvironment of head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated how the stroma-rich tumor microenvironment affects the uptake, penetration, and photodynamic efficiency of three lipid-based nanoformulations of approved in EU photosensitizer temoporfin (mTHPC): Foslip® (mTHPC in conventional liposomes), drug-in-cyclodextrin-in-liposomes (mTHPC-DCL) and extracellular vesicles (mTHPC-EVs). RESULTS: Collagen expression in co-culture stroma-rich 3D HNSCC spheroids correlates with the amount of CAFs (MeWo cells) in individual spheroid. The assessment of mTHPC loading demonstrated that Foslip®, mTHPC-DCL and mTHPC-EVs encapsulated 0.05 × 10- 15 g, 0.07 × 10- 15 g, and 1.3 × 10- 15 g of mTHPC per nanovesicle, respectively. The mid-penetration depth of mTHPC NPs in spheroids was 47.8 µm (Foslip®), 87.8 µm (mTHPC-DCL), and 49.7 µm (mTHPC-EVs), irrespective of the percentage of stromal components. The cellular uptake of Foslip® and mTHPC-DCL was significantly higher in stroma-rich co-culture spheroids and was increasing upon the addition of serum in the culture medium. Importantly, we observed no significant difference between PDT effect in monoculture and co-culture spheroids treated with lipid-based NPs. Overall, in all types of spheroids mTHPC-EVs demonstrated outstanding total cellular uptake and PDT efficiency comparable to other NPs. CONCLUSIONS: The stromal microenvironment strongly affects the uptake of NPs, while the penetration and PDT efficacy are less sensitive to the presence of stromal components. mTHPC-EVs outperform other lipid nanovesicles due to the extremely high loading capacity. The results of the present study enlarge our understanding of how stroma components affect the delivery of NPs into the tumors.

Advanced co-culture 3D breast cancer model for investigation of fibrosis induced by external stimuli: optimization study.

Radiation-induced fibrosis (RIF) is the main late radiation toxicity in breast cancer patients. Most of the current 3D in vitro breast cancer models are composed by cancer cells only and are unable to reproduce the complex cellular homeostasis within the tumor microenvironment to study RIF mechanisms. In order to account complex cellular interactions within the tumor microenvironment, an advanced 3D spheroid model, consisting of the luminal breast cancer MCF-7 cells and MRC-5 fibroblasts, was developed. The spheroids were generated using the liquid overlay technique in culture media into 96-well plates previously coated with 1% agarose (m/v, in water). In total, 21 experimental setups were tested during the optimization of the model. The generated spheroids were characterized using fluorescence imaging, immunohistology and immunohistochemistry. The expression of ECM components was confirmed in co-culture spheroids. Using α-SMA staining, we confirmed the differentiation of healthy fibroblasts into myofibroblasts upon the co-culturing with cancer cells. The induction of fibrosis was studied in spheroids treated 24 h with 10 ng/mL TGF-ß and/or 2 Gy irradiation. Overall, the developed advanced 3D stroma-rich in vitro model of breast cancer provides a possibility to study fibrosis mechanisms taking into account 3D arrangement of the complex tumor microenvironment.

NIR Imaging of the Integrin-Rich Head and Neck Squamous Cell Carcinoma Using Ternary Copper Indium Selenide/Zinc Sulfide-Based Quantum Dots.

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 αvß6 integrin-rich head and neck squamous cell carcinoma (HNSCC). Integrin αvß6 is usually not detectable in nonpathological tissues, but is highly upregulated in HNSCC. QD-A20 showed αvß6 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.

Cyclodextrin nanosponge as a temoporfin nanocarrier: Balancing between accumulation and penetration in 3D tumor spheroids.

As the intertissue delivery of hydrophobic temoporfin (mTHPC) remains inefficient, we propose the use of cyclodextrin-based nanosponges as a smart, advanced system for improved mTHPC delivery. Recently, we demonstrated that cyclodextrins (CDs) allow mTHPC to penetrate into tumor spheroids via a nanoshuttle mechanism. However, the CD complexes were very sensitive to the dilution, thus limiting their translation invivo. Hypercrosslinked CD monomers in a three-dimensional network (namely, CD nanosponges), however, may form both inclusion and non-inclusion complexes with drug molecules, providing controlled release and prolonged exposure to the drug. In the present work, we demonstrate that epichlorohydrin-crosslinked CD nanosponges based on ß-CD (ßCDp) and carboxymethyl-ß-CD (CMßCDp) monomers efficiently encapsulated mTHPC. We calculated the apparent binding constants between mTHPC and CD polymers (K=(6.3-8.8) × 106M-1 and K=(1.2-1.7) × 106M-1 for ßCDp and CMßCDp, respectively) using fluorescence titration curve fitting. The encapsulation of mTHPC in a CD polymer matrix had slower photosensitizer (PS) release compared to monomer CD units, providing deep penetration of mTHPC in 3D tumor spheroids in a concentration-dependent manner. However, the improvement of mTHPC penetration in 3D human pharynx squamous cell carcinoma (FaDu) spheroids using CD polymers was strongly accompanied by the inhibition of PS cellular uptake, demonstrating the delicate balance between the accumulation and the penetration of PS in FaDu spheroids. In summary, mTHPC-loaded CD nanosponges are a strong candidate for further invivo study in preclinical models, which could be considered as an advanced smart system for mTHPC delivery.

mTHPC-Loaded Extracellular Vesicles Significantly Improve mTHPC Diffusion and Photodynamic Activity in Preclinical Models.

Extracellular vesicles (EVs), derived from the cell, display a phospholipid bilayer membrane that protects the cargo molecules from degradation and contributes to increasing their stability in the bloodstream and tumor targeting. EVs are interesting in regard to the delivery of photosensitizers (PSs) used in the photodynamic therapy (PDT), as they allow us to overcome the limitations observed with liposomes. In fact, liposomal formulation of meta-tetra(hydroxyphenyl)chlorin (mTHPC) (Foslip®), one of the most potent clinically approved PSs, is rapidly destroyed in circulation, thus decreasing in vivo PDT efficacy. mTHPC-EV uptake was evaluated in vitro in a 3D human colon HT-29 microtumor and in vivo study was performed in HT-29 xenografted mice. The obtained data were compared with Foslip®. After intravenous injection of the mTHPC formulations, biodistribution, pharmacokinetics and PDT-induced tumor regrowth were evaluated. In a 3D model of cells, mTHPC-EV uptake featured a deeper penetration after 24h incubation compared to liposomal mTHPC. In vivo results showed a considerable improvement of 33% tumor cure with PDT treatment applied 24h after injection, while 0% was observed after Foslip®/PDT. Moreover, 47 days were required to obtain ten times the initial tumor volume after mTHPC-EVs/PDT compared to 30 days for liposomal mTHPC. In conclusion, compared to Foslip®, mTHPC-EVs improved mTHPC biodistribution and PDT efficacy in vivo. We deduced that a major determinant factor for the improved in vivo PDT efficacy is the deep mTHPC intratumor penetration.

ICG-induced NIR fluorescence mapping in patients with head & neck tumors after the previous radiotherapy.

BACKGROUND: The distinction between tumor and healthy tissues is complicated in the areas previously subjected to radiation therapy (RT). This is related to the fact that tissues can undergo delayed and irreversible deterioration such as inflammation, vascular alteration and fibrosis. The trials related to the fluorescence -guided surgery (FSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) patients, previously subjected to RT, have not yet been reported. The present study addresses for the first time the possibilities of tumor near-infrared (NIR) imaging using Indocynaine Green (ICG) in irradiated areas. METHODS: Four patients with histologically confirmed HNSCC were included in this study. All included patients were previously treated with RT with at least 50 Gy. RT-radiation fields from original treatment fully encompassed the second tumor or recurrence. ICG was injected via cephalic vein 45 min before the images were captured using a NIR camera system Artemis. The images were also captured before ICG injection serving as background signal. The fluorescence intensity measurements were carried out using specially designed software. RESULTS: ICG fluorescence clearly demonstrated a significant difference in fluorescence intensity between healthy and tumor tissues in 2 of 4 patients. Histology post-resection analysis confirmed a complete tumor resection with safe surgical margins. No difference between tumor and surrounding healthy tissue was detected in patients with an epidermoid carcinoma developed from sclerohypertrophic lichen. CONCLUSIONS: In our pilot study, we clearly established the feasibility of using NIR FGS with ICG to delineate tumor and healthy tissues in irradiated areas in infiltrating lichen-free tumors.

Trials of a Fluorescent Endoscopic Video System for Diagnosis and Treatment of the Head and Neck Cancer.

This article presents the results of intraoperative fluorescent diagnostics via the endoscopic system for assessing the quality of photodynamic therapy (PDT) of head and neck cancer. The diagnosis and PDT procedures were performed on the five patients with malignant neoplasms of the vocal cords, lateral surface of the tongue, and trachea and cancer of the left parotid salivary gland. Molecular form of chlorin E6 (Ce6) was intravenously administered with a 1.0-1.1 mg/kg concentration for PDT. Fluorescent diagnostics (FD) was conducted before PDT and after PDT procedures. Control of PDT efficiency was carried out by evaluating the photobleaching of the drug (photosensitizer). The method of intraoperative fluorescent imaging allows determining the exact location of the tumor and its boundaries. The assessment of photosensitizer photobleaching in real time regime allows making quick decisions during PDT procedure, which helps improving the quality of patients' treatment. The results showed the convenience of endoscopic fluorescent video system in various nosologies of head and neck cancer. Therefore, this diagnostic approach will improve the effectiveness of cancer treatment.

NIR fluorescence-guided tumor surgery: new strategies for the use of indocyanine green.

Surgery is the frontline treatment for a large number of cancers. The objective of these excisional surgeries is the complete removal of the primary tumor with sufficient safety margins. Removal of the entire tumor is essential to improve the chances of a full recovery. To help surgeons achieve this objective, near-infrared fluorescence-guided surgical techniques are of great interest. The concomitant use of fluorescence and indocyanine green (ICG) has proved effective in the identification and characterization of tumors. Moreover, ICG is authorized by the Food and Drug Administration and the European Medicines Agency and is therefore the subject of a large number of studies. ICG is one of the most commonly used fluorophores in near-infrared fluorescence-guided techniques. However, it also has some disadvantages, such as limited photostability, a moderate fluorescence quantum yield, a high plasma protein binding rate, and undesired aggregation in aqueous solution. In addition, ICG does not specifically target tumor cells. One way to exploit the capabilities of ICG while offsetting these drawbacks is to develop high-performance near-infrared nanocomplexes formulated with ICG (with high selectivity for tumors, high tumor-to-background ratios, and minimal toxicity). In this review article, we focus on recent developments in ICG complexation strategies to improve near-infrared fluorescence-guided tumor surgery. We describe targeted and nontargeted ICG nanoparticle models and ICG complexation with targeting agents.

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

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.

Matryoshka-Type Liposomes Offer the Improved Delivery of Temoporfin to Tumor Spheroids.

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.