Inhibition of the CCR6-CCL20 axis prevents regulatory T cell recruitment and sensitizes head and neck squamous cell carcinoma to radiation therapy.

BACKGROUND:  Radioresistance of HNSCCs remains a major challenge for effective tumor control. Combined radiotherapy (RT) and immunotherapy (IT) treatment improved survival for a subset of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To overcome radioresistance and improve treatment outcomes, an understanding of factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs. In this study, we investigated how radiation modulates Treg infiltration in tumors through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity. METHODS:  Human and mouse HNSCC cell lines with different immune phenotypes were irradiated at doses of 2 or 10 Gy. Conditioned media, RNA and protein were collected for assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cells were implanted into the buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for analysis of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Mass-spectrometry was performed to analyze proteomic changes in the tumor microenvironment after anti-CCL20 treatment. RESULTS:  Cal27 and MOC2 HNSCCs had a gene signature associated with Treg infiltration, whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed TME. In vitro, tumor irradiation at 10 Gy significantly induced CCL20 in Cal27 and MOC2 cells relative to control. The increase in CCL20 was associated with increased Treg migration. Neutralization of CCL20 reversed radiation-induced migration of Treg cells in vitro and decreased intratumoral Tregs in vivo. Furthermore, inhibition of CCL20 resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. This effect was further enhanced after combination with RT compared to either treatment alone. CONCLUSION:  Our results suggest that radiation promotes CCL20 secretion by tumor cells which is responsible for the attraction of Tregs. Inhibition of the CCR6-CCL20 axis prevents infiltration of Tregs in tumors and suppresses tumor growth resulting in improved response to radiation.

Downregulation of KRAB zinc finger proteins in 5-fluorouracil resistant colorectal cancer cells.

Radio-chemotherapy with 5-flu orouracil (5-FU) is the standard of care treatment for patients with colorectal cancer, but it is only effective for a third of them. Despite our understanding of the mechanism of action of 5-FU, drug resistance remains a significant limitation to the clinical use of 5-FU, as both intrinsic and acquired chemoresistance represents the major obstacles for the success of 5-FU-based chemotherapy. In order to identify the mechanism of acquired resistance, 5-FU chemoresistance was induced in CRC cell lines by passaging cells with increasing concentrations of 5-FU. To study global molecular changes, quantitative proteomics and transcriptomics analyses were performed on these cell lines, comparing the resistant cells as well as the effect of chemo and radiotherapy. Interestingly, a very high proportion of downregulated genes were annotated as transcription factors coding for Krüppel-associated box (KRAB) domain-containing zinc-finger proteins (KZFPs), the largest family of transcriptional repressors. Among nearly 350 KRAB-ZFPs, almost a quarter were downregulated after the induction of a 5-FU-resistance including a common one between the three CRC cell lines, ZNF649, whose role is still unknown. To confirm the observations of the proteomic and transcriptomic approaches, the abundance of 20 different KZFPs and control mRNAs was validated by RT-qPCR. In fact, several KZFPs were no longer detectable using qPCR in cell lines resistant to 5-FU, and the KZFPs that were downregulated only in one or two cell lines showed similar pattern of expression as measured by the omics approaches. This proteomic, transcriptomic and genomic analysis of intrinsic and acquired resistance highlights a possible new mechanism involved in the cellular adaptation to 5-FU and therefore identifies potential new therapeutic targets to overcome this resistance.

Cerebrovascular inflammation promotes the formation of brain metastases.

Brain metastases are the most prevalent intracranial malignancy. Patient outcome is poor and treatment options are limited. Hence, new avenues must be explored to identify potential therapeutic targets. Inflammation is a known critical component of cancer progression. Intratumoral inflammation drives progression and leads to the release of circulating tumor cells (CTCs). Inflammation at distant sites promotes adhesion of CTCs to the activated endothelium and then initiates the formation of metastases. These interactions mostly involve cell adhesion molecules expressed by activated endothelial cells. For example, the vascular cell adhesion molecule-1 (VCAM-1) is known to promote transendothelial migration of cancer cells in different organs. However, it is unclear whether a similar mechanism occurs within the specialized environment of the brain. Our objective was therefore to use molecular imaging to assess the potential role of VCAM-1 in promoting the entry of CTCs into the brain. First, magnetic resonance imaging (MRI) and histological analyses revealed that cerebrovascular inflammation induced by intracranial injection of lipopolysaccharide significantly increased the expression of VCAM-1 in the Balb/c mouse brain. Next, intracardiac injection of 4T1 mammary carcinoma cancer cells in animals with cerebrovascular inflammation yielded a higher brain metastasis burden than in the control animals. Finally, blocking VCAM-1 prior to 4T1 cells injection prevented this increased metastatic burden. Here, we demonstrated that by contributing to CTCs adhesion to the activated cerebrovascular endothelium, VCAM-1 improves the capacity of CTCs to form metastatic foci in the brain.

Diffusion MRI monitoring of specific structures in the irradiated rat brain.

PURPOSE: The analysis of biological and mesoscopic structures properties by diffusion MRI (dMRI) in brain after radiation therapy remains challenging. In our study, we described the consequences associated with an unwanted dose to healthy tissue, assessing radiation-induced brain alterations of living rats with dMRI compared to histopathology and behavioral assays. METHODS: The right primary motor cortex M1 of the rat brain was targeted by stereotactic radiosurgery with a mean radiation dose of 41 Gy. Multidirectional single b-value dMRI data of the whole brain were acquired with a 7T small-animal scanner before irradiation until 110 days post-irradiation. Diffusion tensor imaging metrics, such as fractional anisotropy (FA), mean diffusivity (MD), axial (AD), and radial diffusivity (RD) were compared to brain alterations detected by immunohistochemistry and motor performances measured by a behavioral test. RESULTS: Between days 90 and 110, radiation necrosis was observed into the white matter spreading into M1 . Results showed a reduction of FA in the corpus callosum and in the striatum, which was driven by an increase in RD from 90 to 110 days post-irradiation, whereas only RD increased in M1 . Values of RD and AD increased in the irradiated hippocampus, while FA remained constant. Moreover, an increased MD, AD and RD was observed in the hippocampus that was probably related to inflammation as well as reactive astrogliosis after 110 days post-irradiation. Finally, rats did not exhibit locomotor deficits. CONCLUSIONS: dMRI metrics can assess brain damage; the sensitivity of dMRI metrics depends on the brain region.

Liposomal formulations of carboplatin injected by convection-enhanced delivery increases the median survival time of F98 glioma bearing rats.

BACKGROUND: Effectiveness of chemotherapy for treating glioblastoma (GBM) brain tumors is hampered by the blood-brain barrier which limits the entry into the brain of most drugs from the blood. To bypass this barrier, convection-enhanced delivery (CED) was proposed to directly inject drugs in tumor. However, the benefit of CED may be hampered when drugs diffuse outside the tumor to then induce neurotoxicity. Encapsulation of drugs into liposome aims at increasing tumor cells specificity and reduces neurotoxicity. However, the most appropriate liposomal formulation to inject drugs into brain tumor by CED still remains to be determined. In this study, four liposomal carboplatin formulations were prepared and tested in vitro on F98 glioma cells and in Fischer rats carrying F98 tumor implanted in the brain. Impact of pegylation on liposomal surface and relevance of positive or negative charge were assessed. RESULTS: The cationic non-pegylated (L1) and pegylated (L2) liposomes greatly improved the toxicity of carboplatin in vitro compared to free carboplatin, whereas only a modest improvement and even a reduction of efficiency were measured with the anionic non-pegylated (L3) and the pegylated (L4) liposomes. Conversely, only the L4 liposome significantly increased the median survival time of Fisher rats implanted with the F98 tumor, compared to free carboplatin. Neurotoxicity assays performed with the empty L4' liposome showed that the lipid components of L4 were not toxic. These results suggest that the positive charge on liposomes L1 and L2, which is known to promote binding to cell membrane, facilitates carboplatin accumulation in cancer cells explaining their higher efficacy in vitro. Conversely, negatively charged and pegylated liposome (L4) seems to diffuse over a larger distance in the tumor, and consequently significantly increased the median survival time of the animals. CONCLUSIONS: Selection of the best liposomal formulation based on in vitro studies or animal model can result in contradictory conclusions. The negatively charged and pegylated liposome (L4) which was the less efficient formulation in vitro showed the best therapeutic effect in animal model of GBM. These results support that relevant animal model of GBM must be considered to determine the optimal physicochemical properties of liposomal formulations.

Radiation-induced lung metastasis development is MT1-MMP-dependent in a triple-negative breast cancer mouse model.

BACKGROUND: The prognosis of triple-negative breast cancer (TNBC) is still difficult to establish. Some TNBC benefit from radiotherapy (RT) and are cured, while in other patients metastases appear during the first 3 years after treatment. In this study, an animal model of TNBC was used to determine whether the expression of the cell membrane protease MT1-MMP in cancer cells was associated with radiation-stimulated development of lung metastases. METHODS: Using invasion chambers, irradiated fibroblasts were used as chemoattractants to assess the invasiveness of TNBC D2A1 cell lines showing downregulated expression of MT1-MMP, which were compared with D2A1-wt (wild-type) and D2A1 shMT1-mock (empty vector) cell lines. In a mouse model, a mammary gland was irradiated followed by the implantation of the downregulated MT1-MMP D2A1, D2A1-wt or D2A1 shMT1-mock cell lines. Migration of D2A1 cells in the mammary gland, number of circulating tumour cells and development of lung metastases were assessed. RESULTS: The reduction of MT1-MMP expression decreased the invasiveness of D2A1 cells and blocked the radiation enhancement of cancer cell invasion. In BALB/c mice, irradiation of the mammary gland has stimulated the invasion of cancer cells, which was associated with a higher number of circulating tumour cells and of lung metastases. These adverse effects of radiation were prevented by downregulating the MT1-MMP. CONCLUSIONS: This study shows that the MT1-MMP is necessary for the radiation enhancement of lung metastasis development, and that its expression level and/or localisation could be evaluated as a biomarker for predicting the early recurrence observed in some TNBC patients.

Understanding the continuum of radionecrosis and vascular disorders in the brain following gamma knife irradiation: An MRI study.

PURPOSE: The radiation dose delivered to brain tumors is limited by the possibility to induce vascular damage and necrosis in surrounding healthy tissue. In the present study, we assessed the ability of MRI to monitor the cascade of events occurring in the healthy rat brain after stereotactic radiosurgery, which could be used to optimize the radiation treatment planning. METHODS: The primary somatosensory forelimb area (S1FL) and the primary motor cortex in the right hemisphere of Fischer rats (n = 6) were irradiated with a single dose of Gamma Knife radiation (Leksell Perfexion, Elekta AG, Stockholm, Sweden). Rats were scanned with a small-animal 7 Tesla MRI scanner before treatment and 16, 21, 54, 82, and 110 days following irradiation. At every imaging session, T2 -weighted (T2 w), Gd-DTPA dynamic contrast-enhanced MRI (DCE-MRI), and T2*-weighted ( T2* w) images were acquired to measure changes in fluid content, blood vessel permeability, and structure, respectively. At days 10, 110, and 140, histopathology was performed on brain sections. Locomotion and spatial memory ability were assessed longitudinally by behavioral tests. RESULTS: No vascular changes were initially observed. After 54 days, a small necrotic volume in the white matter below the S1FL, surrounded by an area presenting significant vascular permeability, was revealed. Between 54 and 110 days, the necrotic volume increased and was accompanied by the formation of a ring-like region, where a mixture of necrosis and permeable blood vessels were observed, as confirmed by histology. Behavioral changes were only observed after day 82. CONCLUSION: Together, DCE-MRI and T2* w images supported by histology provided a coherent picture of the phenomena involved in the formation of new, leaky blood vessels, which was followed by the detection of radionecrosis in a preclinical model of brain irradiation. Magn Reson Med 78:1420-1431, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Convection-enhancement delivery of liposomal formulation of oxaliplatin shows less toxicity than oxaliplatin yet maintains a similar median survival time in F98 glioma-bearing rat model.

Results of clinical trials with oxaliplatin in treating glioblastoma are dismal. Previous works showed that intravenous (i.v.) delivery of oxaliplatin did not increase the survival of F98 glioma-bearing Fisher rats. Low accumulation of the drug in tumor cells is presumed to be responsible for the lack of antitumor effect. In the present study, convection-enhanced delivery (CED) was used to directly inject oxaliplatin in brain tumor implanted in rats. Since CED can led to severe toxicity, the liposomal formulation of oxaliplatin (Lipoxal™) was also assessed. The maximum tolerated dose (MTD) of oxaliplatin was 10 µg, while that of Lipoxal™ was increased by 3-times reaching 30 µg. Median survival time (MeST) of F98 glioma-bearing rats injected with 10 µg oxaliplatin by CED was 31 days, 7.5 days longer than untreated control (p = 0.0002); while CED of 30 µg Lipoxal™ reached the same result. Compared to previous study on i.v. delivery of these drugs, their injection by CED significantly increased their tumoral accumulations as well as MeSTs in the F98 glioma bearing rat model. The addition of radiotherapy (15 Gy) to CED of oxaliplatin or Lipoxal™ increased the MeST by 4.0 and 3.0 days, respectively. The timing of radiotherapy (4 h or 24 h after CED) produced similar results. However, the treatment was better tolerated when radiotherapy was performed 24 h after CED. In conclusion, a better tumoral accumulation was achieved when oxaliplatin and Lipoxal™ were injected by CED. The liposomal encapsulation of oxaliplatin reduced its toxic, while maintaining its antitumor potential.

Stimulation of triple negative breast cancer cell migration and metastases formation is prevented by chloroquine in a pre-irradiated mouse model.

BACKGROUND: Some triple negative breast cancer (TNBC) patients are at higher risk of recurrence in the first three years after treatment. This rapid relapse has been suggested to be associated with inflammatory mediators induced by radiation in healthy tissues that stimulate cancer cell migration and metastasis formation. In this study, the ability of chloroquine (CQ) to inhibit radiation-stimulated development of metastasis was assessed. METHODS: The capacity of CQ to prevent radiation-enhancement of cancer cell invasion was assessed in vitro with the TNBC cell lines D2A1, 4T1 and MDA-MB-231 and the non-TNBC cell lines MC7-L1, and MCF-7. In Balb/c mice, a single mammary gland was irradiated with four daily doses of 6 Gy. After the last irradiation, irradiated and control mammary glands were implanted with D2A1 cells. Mice were treated with CQ (vehicle, 40 or 60 mg/kg) 3 h before each irradiation and then every 72 h for 3 weeks. Migration of D2A1 cells in the mammary gland, the number of circulating tumor cells and lung metastasis were quantified, and also the expression of some inflammatory mediators. RESULTS: Irradiated fibroblasts have increased the invasiveness of the TNBC cell lines only, a stimulation that was prevented by CQ. On the other hand, invasiveness of the non-TNBC cell lines, which was not enhanced by irradiated fibroblasts, was also not significantly modified by CQ. In Balb/c mice, treatment with CQ prevented the stimulation of D2A1 TNBC cell migration in the pre-irradiated mammary gland, and reduced the number of circulating tumor cells and lung metastases. This protective effect of CQ was associated with a reduced expression of the inflammatory mediators interleukin-1ß, interleukin-6, and cyclooxygenase-2, while the levels of matrix metalloproteinases-2 and -9 were not modified. CQ also promoted a blocking of autophagy. CONCLUSION: CQ prevented radiation-enhancement of TNBC cell invasion and reduced the number of lung metastases in a mouse model.

Convection-enhancement delivery of platinum-based drugs and Lipoplatin(TM) to optimize the concomitant effect with radiotherapy in F98 glioma rat model.

The prognosis for patients with glioblastoma remains poor with current treatments. Although platinum-based drugs are sometimes offered at relapse, their efficacy in this setting is still disputed. In this study, we use convection-enhanced delivery (CED) to deliver the platinum-based drugs (cisplatin, carboplatin, and Lipoplatin(TM) - liposomal formulation of cisplatin) directly into the tumor of F98 glioma-bearing rats that were subsequently treated with γ radiation (15 Gy). CED increased by factors varying between 17 and 111, the concentration of these platinum-based drugs in the brain tumor compared to intra-venous (i.v.) administration, and by 9- to 34-fold, when compared to intra-arterial (i.a.) administration. Furthermore, CED resulted in a better systemic tolerance to platinum drugs compared to their i.a. injection. Among the drugs tested, carboplatin showed the highest maximum tolerated dose (MTD). Treatment with carboplatin resulted in the best median survival time (MeST) (38.5 days), which was further increased by the addition of radiotherapy (54.0 days). Although the DNA-bound platinum adduct were higher at 4 h after CED than 24 h for carboplatin group, combination with radiotherapy led to similar improvement of median survival time. However, less toxicity was observed in animals irradiated 24 h after CED-based chemotherapy. In conclusion, CED increased the accumulation of platinum drugs in tumor, reduced the toxicity, and resulted in a higher median survival time. The best treatment was obtained in animals treated with carboplatin and irradiated 24 h later.

Cancer radiotherapy based on femtosecond IR laser-beam filamentation yielding ultra-high dose rates and zero entrance dose.

Since the invention of cancer radiotherapy, its primary goal has been to maximize lethal radiation doses to the tumor volume while keeping the dose to surrounding healthy tissues at zero. Sadly, conventional radiation sources (γ or X rays, electrons) used for decades, including multiple or modulated beams, inevitably deposit the majority of their dose in front or behind the tumor, thus damaging healthy tissue and causing secondary cancers years after treatment. Even the most recent pioneering advances in costly proton or carbon ion therapies can not completely avoid dose buildup in front of the tumor volume. Here we show that this ultimate goal of radiotherapy is yet within our reach: Using intense ultra-short infrared laser pulses we can now deposit a very large energy dose at unprecedented microscopic dose rates (up to 10(11) Gy/s) deep inside an adjustable, well-controlled macroscopic volume, without any dose deposit in front or behind the target volume. Our infrared laser pulses produce high density avalanches of low energy electrons via laser filamentation, a phenomenon that results in a spatial energy density and temporal dose rate that both exceed by orders of magnitude any values previously reported even for the most intense clinical radiotherapy systems. Moreover, we show that (i) the type of final damage and its mechanisms in aqueous media, at the molecular and biomolecular level, is comparable to that of conventional ionizing radiation, and (ii) at the tumor tissue level in an animal cancer model, the laser irradiation method shows clear therapeutic benefits.

The role of pore size and mechanical properties on the accumulation, retention and distribution of F98 glioblastoma cells in macroporous hydrogels.

Glioblastoma (GBM) accounts for half of all central nervous system tumors. Once the tumor is removed, many GBM cells remain present near the surgical cavity and infiltrate the brain up to a distance of 20-30 mm, resulting in recurrence a few months later. GBM remains incurable due to the limited efficiency of current treatments, a result of the blood-brain barrier and sensitivity of healthy brain tissues to chemotherapy and radiation. A new therapeutic paradigm under development to treat GBM is to attract and accumulate GBM cells in a cancer cell trap inserted in the surgical cavity after tumor resection. In this work, porous gels were prepared using porous polylactide molds obtained from melt-processed co-continuous polymer blends of polystyrene and polylactide, with an average pore size ranging from 5 µm to over 500 µm. In order to efficiently accumulate and retain GBM brain cancer cells within a macroporous sodium alginate-based hydrogel trap, the pores must have an average diameter superior to 100 µm, with the best results obtained at 225 µm. In that case, the accumulation and retention of F98 GBM cells were more homogeneous, especially when functionalized with RGD adhesion peptides. At an alginate concentration of 1% w/v, the compression modulus reaches 15 kPa, close to the average value of 1-2 kPa reported for brain tissues, while adhesion and retention were also superior compared to 2% w/v gels. Overall, 1% w/v gels with 225 µm pores functionalized with the RGD peptide display the best performances.

Optimization of the route of platinum drugs administration to optimize the concomitant treatment with radiotherapy for glioblastoma implanted in the Fischer rat brain.

Treatment of glioblastoma with platinum compounds modestly improves progression-free survival and may cause toxic effects which prevent use at higher dose that would otherwise improve the antineoplastic effect. To reduce toxicity, we propose to encapsulate the platinum drug in a liposome. We have also tested three methods of drug administration (intra-venous, intra-arterial and intra-arterial combined with blood brain barrier disruption) to determine which one optimizes the tumor cell uptake, limits the toxicity and delivers the best concomitance effect with radiotherapy. Cisplatin, oxaliplatin, their respective liposomal formulations, Lipoplatin™ and Lipoxal™, and carboplatin were assessed in F98 glioma, orthotopically implanted in Fischer rats. We found that the modest accumulation of drugs in tumor cells after intra-venous injection was significantly improved when the intra-arterial route was used and further increased after the transient opening of the blood brain barrier with mannitol. The liposomal formulations have largely reduced the toxicity and have allowed a better exploitation of the anti-cancer activity of platinum agent. Although the liposomes Lipoplatin™ and Lipoxal™ have shown a similar ability to that of carboplatin, to accumulate in brain tumors, the highest additive effect with radiotherapy was obtained with carboplatin. We conclude that the intra-arterial infusion of carboplatin or Lipoxal™ in concomitance with radiation therapy leads to the best tumor control as measured by an increase of mean survival time in Fischer rats implanted with the F98 glioma with a benefit in survival time of 13.4 and 6.5 days respectively compared to intra-venous.

Reconciling two opposing effects of radiation therapy: stimulation of cancer cell invasion and activation of anti-cancer immunity.

PURPOSE: The damage caused by radiation therapy to cancerous and normal cells inevitably leads to changes in the secretome profile of pro and anti-inflammatory mediators. The inflammatory response depends on the dose of radiation and its fractionation, while the inherent radiosensitivity of each patient dictates the intensity and types of adverse reactions. This review will present an overview of two apparently opposite reactions that may occur after radiation treatment: induction of an antitumor immune response and a protumoral response. Emphasis is placed on the molecular and cellular mechanisms involved. CONCLUSIONS: By understanding how radiation changes the balance between anti- and protumoral effects, these forces can be manipulated to optimize radiation oncology treatments.

In vivo behavior of [64Cu]NOTA-terpyridine platinum, a novel chemo-radio-theranostic agent for imaging, and therapy of colorectal cancer.

To overcome resistance to chemotherapy for colorectal cancer, we propose to validate in vivo a novel terpyridine-platinum (TP) compound radiolabeled with the radio-theranostic isotope 64Cu. In vivo stability, biodistribution, PET imaging, tumor growth delay, toxicity and dosimetry of [64Cu]NOTA-C3-TP were determined. The current experimental studies show that [64Cu]NOTA-C3-TP is stable in vivo, rapidly eliminated by the kidneys and has a promising tumor uptake ranging from 1.8 ± 0.4 to 3.0 ± 0.2 %ID/g over 48 h. [64Cu]NOTA-C3-TP retarded tumor growth by up to 6 ± 2.0 days and improved survival relative to vehicle and non-radioactive [NatCu]NOTA-C3-TP over 17 days of tumor growth observation. This effect was obtained with only 0.4 nmol i.v. injection of [64Cu]NOTA-C3-TP, which delivers 3.4 ± 0.3 Gy tumoral absorbed dose. No evidence of toxicity, by weight loss or mortality was revealed. These findings confirm the high potential of [64Cu]NOTA-TP as a novel radio-theranostic agent.

Drug Delivery Systems in the Development of Novel Strategies for Glioblastoma Treatment.

Glioblastoma multiforme (GBM) is a grade IV glioma considered the most fatal cancer of the central nervous system (CNS), with less than a 5% survival rate after five years. The tumor heterogeneity, the high infiltrative behavior of its cells, and the blood-brain barrier (BBB) that limits the access of therapeutic drugs to the brain are the main reasons hampering the current standard treatment efficiency. Following the tumor resection, the infiltrative remaining GBM cells, which are resistant to chemotherapy and radiotherapy, can further invade the surrounding brain parenchyma. Consequently, the development of new strategies to treat parenchyma-infiltrating GBM cells, such as vaccines, nanotherapies, and tumor cells traps including drug delivery systems, is required. For example, the chemoattractant CXCL12, by binding to its CXCR4 receptor, activates signaling pathways that play a critical role in tumor progression and invasion, making it an interesting therapeutic target to properly control the direction of GBM cell migration for treatment proposes. Moreover, the interstitial fluid flow (IFF) is also implicated in increasing the GBM cell migration through the activation of the CXCL12-CXCR4 signaling pathway. However, due to its complex and variable nature, the influence of the IFF on the efficiency of drug delivery systems is not well understood yet. Therefore, this review discusses novel drug delivery strategies to overcome the GBM treatment limitations, focusing on chemokines such as CXCL12 as an innovative approach to reverse the migration of infiltrated GBM. Furthermore, recent developments regarding in vitro 3D culture systems aiming to mimic the dynamic peritumoral environment for the optimization of new drug delivery technologies are highlighted.

Effect of Chitosan on Alginate-Based Macroporous Hydrogels for the Capture of Glioblastoma Cancer Cells.

Glioblastoma multiforme is a type of brain cancer associated with a very low survival rate since a large number of cancer cells remain infiltrated in the brain despite the treatments currently available. This work presents a macroporous hydrogel trap, destined to be implanted in the surgical cavity following tumor resection and designed to attract and retain cancer cells, in order to eliminate them afterward with a lethal dose of stereotactic radiotherapy. The biocompatible hydrogel formulation comprises sodium alginate (SA) and chitosan (CHI) bearing complementary electrostatic charges and stabilizing the gels in saline and cell culture media, as compared to pristine SA gels. The highly controlled and interconnected porosity, characterized by X-ray microCT, yields mechanical properties comparable to those of brain tissues and allows F98 glioblastoma cells to penetrate the gels within the entire volume, as confirmed by fluorescence microscopy. The addition of a grafted -RGD peptide on SA, combined with CHI, significantly enhances the adhesion and retention of F98 cells within the gels. Overall, the best compromise between low proliferation and a high level of accumulation and retention of F98 cells was obtained with the hydrogel formulated with 1% SA and 0.2% CHI, without the -RGD adhesion peptide.

Cellular uptake and cytoplasm / DNA distribution of cisplatin and oxaliplatin and their liposomal formulation in human colorectal cancer cell HCT116.

Liposomal formulations of cisplatin and oxaliplatin (Lipoplatin™ and Lipoxal™, respectively) were recently proposed to reduce systemic toxicity, while optimizing the anti-cancer effectiveness of these compounds. As the anti-neoplastic or radio-sensitizing activity of these drugs is attributed to their binding to DNA, we assessed the impact of the liposomal formulations on the time course of accumulation of these platinum compounds in the human colorectal cancer HCT116 cell lines and their distribution between cytoplasm and DNA. Their cytotoxicity was determined by colony formation assay. Intracellular platinum and platinum bound to DNA was measured by inductively coupled plasma mass spectrometry. Although, as a chemotherapeutic agent, cisplatin was as efficient as oxaliplatin after exposure for a short time, oxaliplatin and Lipoxal™ became more active than cisplatin against HCT116 cells after 24 h incubation. Lipoxal™ displayed a higher accumulation in the cytoplasm of HCT116 cells compared to free oxaliplatin, consistent with its proposed mechanism of fusion with the cell membrane. The distribution cytoplasm/DNA of free cisplatin and Lipoplatin™ were similar. Conversely, Lipoxal™ had a significantly different cytoplasm/DNA distribution from oxaliplatin: more than 95% of oxaliplatin transported by the liposome was trapped in the cytoplasm, even after 48 h incubation. Our study indicates that Lipoxal™ can largely improve the cellular uptake of oxaliplatin, but this was not followed by a similar increase in the DNA bound fraction.

High Cytotoxic Effect by Combining Copper-64 with a NOTA-Terpyridine Platinum Conjugate.

Terpyridine platinum (TP)-based chemotherapeutic agents target three-dimensional structures on DNA known as G-quadruplexes. We report the rational design and synthesis of a TP conjugate combined with copper-64 (64Cu), the decay characteristics of which include emission of ß- and Auger electrons for radiotherapy and ß+ particles for positron emission tomography (PET) imaging. The present experimental studies show that the novel [64Cu]Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-TP is stable, permitting selective killing of cancer cells. The antitumor activity of [64Cu]Cu-NOTA-TP at high apparent molar activity is in the low nanomolar range and 27,800-fold greater than that of natCu-NOTA-TP at 24 h post treatment. These results suggest that this combination of a cytotoxic TP agent with 64Cu has considerable potential for cancer treatment and PET imaging.

Design, Synthesis, and Cytotoxicity Assessment of [64Cu]Cu-NOTA-Terpyridine Platinum Conjugate: A Novel Chemoradiotherapeutic Agent with Flexible Linker.

Maximum benefits of chemoradiation therapy with platinum-based compounds are expected if the radiation and the drug are localized simultaneously in cancer cells. To optimize this concomitant effect, we developed the novel chemoradiotherapeutic agent [64Cu]Cu-NOTA-C3-TP by conjugating, via a short flexible alkyl chain spacer (C3), a terpyridine platinum (TP) moiety to a NOTA chelator complexed with copper-64 (64Cu). The decay of 64Cu produces numerous low-energy electrons, enabling the 64Cu-conjugate to deliver radiation energy close to TP, which intercalates into G-quadruplex DNA. Accordingly, the in vitro internalization kinetic and the cytotoxic activity of [64Cu]Cu-NOTA-C3-TP and its derivatives were investigated with colorectal cancer (HCT116) and normal human fibroblast (GM05757) cells. Radiolabeling by 64Cu results in a >55,000-fold increase of cytotoxic potential relative to [NatCu]Cu-NOTA-C3-TP at 72 h post administration, indicating a large additive effect between 64Cu and the TP drug. The internalization and nucleus accumulation of [64Cu]Cu-NOTA-C3-TP in the HCT116 cells were, respectively, 3.1 and 6.0 times higher than that for GM05757 normal human fibroblasts, which is supportive of the higher efficiency of the [64Cu]Cu-NOTA-C3-TP for HCT116 cancer cells. This work presents the first proof-of-concept study showing the potential use of the [64Cu]Cu-NOTA-C3-TP conjugate as a targeted chemoradiotherapeutic agent to treat colorectal cancer.