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Despite advances in cancer therapies, nanomedicine approaches including the treatment of glioblastoma (GBM), the most common, aggressive brain tumor, remains inefficient. These failures are likely attributable to the complex and not yet completely known biology of this tumor, which is responsible for its strong invasiveness, high degree of metastasis, high proliferation potential, and resistance to radiation and chemotherapy. The intimate connection through which the cells communicate between them plays an important role in these biological processes. In this scenario, tunneling nanotubes (TnTs) are recently gaining importance as a key feature in tumor progression and in particular in the re-growth of GBM after surgery. In this context, we firstly identified structural differences of TnTs formed by U87-MG cells, as model of GBM cells, in comparison with those formed by normal human astrocytes (NHA), used as a model of healthy cells. Successively, we have studied the possibility to exploit U87-MG TnTs as drug-delivery channels in cancer therapy, using liposomes composed of cholesterol/sphingomyelin and surface functionalized with mApoE and chlorotoxin peptides (Mf-LIP) as nanovehicle model. The results showed that U87-MG cells formed almost exclusively thick and long protrusions, whereas NHA formed more thin and short TnTs. Considering that thick TnTs are more efficient in transport of vesicles and organelles, we showed that fluorescent-labeled Mf-LIP can be transported via TnTs between U87-MG cells and with less extent through the protrusions formed by NHA cells. Our results demonstrate that nanotubes are potentially useful as drug-delivery channels for cancer therapy, facilitating the intercellular redistribution of this drug in close and far away cells, thus reaching isolated tumor niches that are hardly targeted by simple drug diffusion in the brain parenchyma. Moreover, the differences identified in TnTs formed by GBM and NHA cells can be exploited to increase treatment precision and specificity.

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Hydrogels are useful platforms as three-dimensional (3D) scaffolds for cell culture, drug-release systems, and regenerative medicine applications. Here, we propose a novel chemical cross-linking approach by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione or diethyl squarate for the preparation of 5 and 10% w/v gelatin-based hydrogels. Hydrogels showed good swelling properties, and the 5% gelatin-based hydrogel proved suitable as a 3D cell culture scaffold for the chondrocyte cell line C28/I2. In addition, diffusion properties of different sized molecules inside the hydrogel were determined.

Ovarian carcinoma is a major cause of cancer-related death in women. Besides late diagnosis, treatment often fails to produce a persistent disease control. The efficacy of the platinum drug-based therapy is limited by drug resistance. Thus, an ideal therapy for women with ovarian carcinoma is still missing. Because the karyopherin XPO1/CRM1 contributes to the regulation of the cellular localization of the transcription factor FOXO1 which participates in apoptosis regulation, the aim of this study was to examine if interference with XPO1 to improve FOXO1 nuclear localization may be exploited to kill efficiently ovarian carcinoma cells and to improve cisplatin efficacy. Here, we employed preclinical pharmacology approaches including growth inhibition assays, western blot analyses, gene knockdown by siRNA, quantitative Real-time PCR, immunofluorescence analyses and tests in in vivo models. The drug interaction was analyzed using the Chou and Talalay method. When cell sensitivity to KPT-330 of a panel of ovarian carcinoma cell lines was examined a marked sensitivity to the XPO1 inhibitor was found. The effect of the combination of cisplatin and KPT-330 was investigated in the IGROV-1 cells, using simultaneous or sequential schedules. According to the combination index values, when KPT-330 exposure followed cisplatin exposure the most favourable drug interaction was observed. In IGROV-1 cells, a modulation of proteins involved in apoptosis (p53, Bax) and in cell cycle progression (p21) was found, besides G1 and G2/M accumulation after exposure to KPT-330 and to the cisplatin/KPT-330 combination, respectively. KPT-330-treated cells exhibited FOXO1 nuclear staining, in keeping with the capability of the compound to inhibit FOXO1 nuclear export. Knock-down experiments by RNA interference indicated that FOXO1-silenced cells displayed a reduced sensitivity to KPT-330, but no significant changes in cisplatin sensitivity. FOXO1 silencing tended to reduce the efficacy of the drug combination at selected cisplatin concentrations. An analysis of the antitumor efficacy of KPT-330, indicated the capability of KPT-330 to significantly inhibit tumor growth when IGROV-1 cells were subcutaneously injected in immunodeficient mice or growth intraperitoneally. Our findings support that the XPO1 inhibitor KPT-330 is a promising agent for the treatment of ovarian carcinoma. The effect of the KPT-330-cisplatin combination appears to be dependent on the treatment schedule, as a synergistic interaction occurs when cells are treated with cisplatin followed by KPT-330. Such an interaction can be modulated by silencing of FOXO1, which results in reduced sensitivity to KPT-330. Our results suggest that therapeutic regimens selected on the basis of the molecular tumor features should be used to achieve a personalized therapy tailored to the specific characteristics of each patient. Citation Format: Simone Stucchi, Michelandrea De Cesare, Cristina Corno, Nives Carenini, Emilio Ciusani, Nadia Zaffaroni, Laura Gatti, Paola Perego. Role of FOXO1 in response of ovarian carcinoma cells to the XPO1/CRM1 inhibitor KPT-330/selinexor in combination with cisplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1070. doi:10.1158/1538-7445.AM2017-1070

Cannabidiol (CBD) is a non-psychoactive phytocannabinoid that has been discussed for its safety and efficacy in cancer treatments. For this reason, we have inquired into its use on triple-negative human breast cancer. Analyzing the biological effects of CBD on MDA-MB-231, we have demonstrated that both CBD dosage and serum concentrations in the culture medium influence its outcomes; furthermore, light scattering studies demonstrated that serum impacts the CBD aggregation state by acting as a surfactant agent. Pharmacological studies on CBD in combination with chemotherapeutic agents reveal that CBD possesses a protective action against the cytotoxic effect exerted by cisplatin on MDA-MB-231 grown in standard conditions. Furthermore, in a low serum condition (0.5%), starting from a threshold concentration (5 µM), CBD forms aggregates, exerts cytostatic antiproliferative outcomes, and promotes cell cycle arrest activating autophagy. At doses above the threshold, CBD exerts a highly cytotoxic effect inducing bubbling cell death. Finally, IGF-1 and EGF antagonize the antiproliferative effect of CBD protecting cells from harmful consequences of CBD aggregates. In conclusion, CBD effect is strongly associated with the physical state and concentration that reaches the treated cells, parameters not taken into account in most of the research papers.