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.

Peptide-mediated targeting of Quantum Dots in a 3D model of head and neck cancer.

BACKGROUND: Oral squamous cell carcinoma (OSCC) treatment mainly relies on surgery. The status of surgical margin is a major prognostic factor for patients as positive margins are associated with lower survival. However, the anatomical particularities of this area complicate margin establishment. Fluorescence guided surgery (FGS) could be employed as an intraoperative technique to improve tumor resection and margin investigation. Quantum dots (QDs) serve as ideal contrast agents in this technique due to their brightness and stability. Since αVß6 integrin is overexpressed in OSCC, coupling QDs with A20FMDV2 peptide (QDs-A20) targeting the αVß6 integrin constitute a real opportunity. This study investigates the accumulation of QDs-A20 in 2D and 3D tongue cancer models, as well as QDs coupled to a scrambled version of this peptide (QDs-Scr) or without peptide (QDs-SPP), for imaging purposes. METHODS: CdSeCdS/ZnS quantum dots were coated with sulfobetaine polymers (QDs-SPP) and conjugated to A20FMDV2 peptide (QDs-A20) or its scrambled version (QDs-Scr). Two-dimensional (2D) and three-dimensional (3D) tongue cancer cells HSC-3 were employed to test the effectiveness of intracellular accumulation of all types of QDs. Targeting ability of each QDs was assessed by flow cytometry, while the depth of penetration into cancerous spheroids was assessed by fluorescence microscopy. RESULTS: QDs coating with sulfobetaines polymers (QDs-SPP) completely prevented their internalization by HSC-3 cells in 2D and 3D models, making QDs stealthy and preventing their non-specific accumulation. Conversely, peptides conjugated QDs (QDs-A20 & QDs-Scr) labeled HSC-3 monolayers and managed to label spheroid periphery up to 23 µm deep. However, no difference in accumulation was found between these two QDs whereas only A20 peptide could potentially target αVß6 integrin. It appears that peptide conjugation increased QDs zeta potential, promoting their adsorption and subsequent endocytosis by cells, independently from αVß6 integrin. CONCLUSIONS: The present study highlighted the impact of peptide conjugation on QDs internalization in 2D and 3D tongue cancer cell models. QDs-SPP were stealthy and did not accumulate in cells. Peptides conjugated QDs could be used as contrast agents, but in a passive targeting approach. Modifications to surface chemistry are required to target αVß6 integrin through active targeting. This study also highlights the need for controls such as scrambled peptides, the absence of which can lead to misinterpretation of results.

DDB2 expression lights the way for precision radiotherapy response in PDAC cells, with or without olaparib.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Therapeutic options for PDAC are primarily restricted to surgery in the early stages of the disease or chemotherapy in advanced disease. Only a subset of patients with germline defects in BRCA1/2 genes can potentially benefit from personalized therapy, with the PARP inhibitor olaparib serving as a maintenance treatment for metastatic disease. Although the role of radiotherapy in PDAC remains controversial, the use of radiosensitizers offers hope for improving cancer management. Previously, we have shown that damage-specific DNA binding protein 2 (DDB2) is a potential prognostic and predictive biomarker for chemotherapy response in PDAC. In this study, we investigated the function of DDB2 in radiotherapy response, with and without radiosensitization by olaparib in PDAC cells. Our findings demonstrated DDB2 resistance to radiation effects, thereby improving cell survival and enhancing the repair of ionizing radiation-induced DNA double-strand breaks. We observed that DDB2 expression enhances the cell cycle arrest in the G2 phase by phosphorylating Chk1 and Chk2 cell cycle checkpoints. Additionally, we identified a novel link between DDB2 and PARP1 in the context of radiotherapy, which enhances the expression and activity of PARP1. Our findings highlight the potential of low-DDB2 expression to potentiate the radiosensitization effect of olaparib in PDAC cells. Collectively, this study provides novel insights into the impacts of DDB2 in the radiotherapy response in PDAC, enabling its employment as a potential biomarker to predict resistance to radiation. Furthermore, DDB2 represents a significant step forward in precision radiotherapy by widening the scope of patients who can be benefiting from olaparib as a radiosensitizer. Hence, this research has the potential to enrich the limited use of radiotherapy in the care of patients with PDAC.

A 3D Bio-Printed-Based Model for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a disease with a very poor prognosis, characterized by incidence rates very close to death rates. Despite the efforts of the scientific community, preclinical models that faithfully recreate the PDAC tumor microenvironment remain limited. Currently, the use of 3D bio-printing is an emerging and promising method for the development of cancer tumor models with reproducible heterogeneity and a precisely controlled structure. This study presents the development of a model using the extrusion 3D bio-printing technique. Initially, a model combining pancreatic cancer cells (Panc-1) and cancer-associated fibroblasts (CAFs) encapsulated in a sodium alginate and gelatin-based hydrogel to mimic the metastatic stage of PDAC was developed and comprehensively characterized. Subsequently, efforts were made to vascularize this model. This study demonstrates that the resulting tumors can maintain viability and proliferate, with cells self-organizing into aggregates with a heterogeneous composition. The utilization of 3D bio-printing in creating this tumor model opens avenues for reproducing tumor complexity in the future, offering a versatile platform for improving anti-cancer therapy models.

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.

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.

DDB2 represses epithelial-to-mesenchymal transition and sensitizes pancreatic ductal adenocarcinoma cells to chemotherapy.

Introduction: Damage specific DNA binding protein 2 (DDB2) is an UV-indiced DNA damage recognition factor and regulator of cancer development and progression. DDB2 has dual roles in several cancers, either as an oncogene or as a tumor suppressor gene, depending on cancer localization. Here, we investigated the unresolved role of DDB2 in pancreatic ductal adenocarcinoma (PDAC). Methods: The expression level of DDB2 in pancreatic cancer tissues and its correlation with patient survival were evaluated using publicly available data. Two PDAC cell models with CRISPR-modified DDB2 expression were developed: DDB2 was repressed in DDB2-high T3M4 cells (T3M4 DDB2-low) while DDB2 was overexpressed in DDB2-low Capan-2 cells (Capan-2 DDB2-high). Immunofluorescence and qPCR assays were used to investigate epithelial-to-mesenchymal transition (EMT) in these models. Migration and invasion properties of the cells were also determined using wound healing and transwell assays. Sensitivity to 5-fluorouracil (5-FU), oxaliplatin, irinotecan and gemcitabine were finally investigated by crystal violet assays. Results: DDB2 expression level was reduced in PDAC tissues compared to normal ones and DDB2-low levels were correlated to shorter disease-free survival in PDAC patients. DDB2 overexpression increased expression of E-cadherin epithelial marker, and decreased levels of N-cadherin mesenchymal marker. Conversely, we observed opposite effects in DDB2 repression and enhanced transcription of SNAIL, ZEB1, and TWIST EMT transcription factors (EMT-TFs). Study of migration and invasion revealed that these properties were negatively correlated with DDB2 expression in both cell models. DDB2 overexpression sensitized cells to 5-fluorouracil, oxaliplatin and gemcitabine. Conclusion: Our study highlights the potential tumor suppressive effects of DDB2 on PDAC progression. DDB2 could thus represent a promising therapeutic target or biomarker for defining prognosis and predicting chemotherapy response in patients with PDAC.

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.

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.

The alteration of temoporfin distribution in multicellular tumor spheroids by ß-cyclodextrins.

To be effective anticancer drugs must penetrate tissue efficiently, reaching all target population of cancer cells in a concentration sufficient to exert a therapeutic effect. This study aimed to investigate the ability of methyl-ß-cyclodextrin (Me-ß-CD) and 2-hydroxypropyl-ß-cyclodextrin (Hp-ß-CD) to alter the penetration and diffusion of temoporfin (mTHPC) in HT29 multicellular tumor spheroids. mTHPC had а nonhomogenous distribution only on the periphery of spheroids. The presence of ß-CDs significantly altered the distribution of mTHPC consisting in the increase of both the depth of photosensitizer penetration and accumulation in HT29 spheroids. We suggest that this improvement is related to the nanoshuttle mechanism of ß-CD action, when ß-CDs facilitate mTHPC transportation to the cells in the inner layers of spheroids. As a result of mTHPC distribution improvement, ß-CDs enhance mTHPC photosensitizing activity towards HT29 multicellular tumor spheroids. The observed effects strongly depend on the type of ß-CD. Thus, varying the type of ß-CD we can finely tune the possibility of using mTHPC for diagnostic (delimitation of tumor margins) or therapeutic purposes.