Search

PDF file - 51KB, Body weights of MDA-MB-231 bearing mice treated with E-3810, 5FU, DDP and their combinations.

PDF file - 63KB, Circulating factors in plasma of mice bearing MDA-MB-231 tumors untreated or treated with E-3810 (E), brivanib (B), sunitinib (S), paclitaxel (PTX) and their combinations as described in Material amd Methods.

PDf file - 30KB, Survival curves of MDA-MB-231 bearing mice treated with E-3810, PTX and their combination.

PDF file - 41KB, Dose response curve of PTX in MDA-MB-231 cells alone or in combination with E-3810, sunitinb and brivanib.

PDF file - 101KB

Trabectedin (ET743) and lurbinectedin (PM01183) limit the production of inflammatory cytokines that are elevated during cancer cachexia. Mice carrying C26 colon adenocarcinoma display cachexia (i.e., premature death and body wasting with muscle, fat and cardiac tissue depletion), high levels of inflammatory cytokines and subsequent splenomegaly. We tested whether such drugs protected these mice from cachexia. Ten-week-old mice were inoculated with C26 cells and three days later randomized to receive intravenously vehicle or 0.05 mg/kg ET743 or 0.07 mg/kg PM01183, three times a week for three weeks. ET743 or PM01183 extended the lifespan of C26-mice by 30% or 85%, respectively, without affecting tumor growth or food intake. Within 13 days from C26 implant, both drugs did not protect fat, muscle and heart from cachexia. Since PM01183 extended the animal survival more than ET743, we analyzed PM01183 further. In tibialis anterior of C26-mice, but not in atrophying myotubes, PM01183 restrained the NF-&kappa, B/PAX7/myogenin axis, possibly reducing the pro-inflammatory milieu, and failed to limit the C/EBP&beta, /atrogin-1 axis. Inflammation-mediated splenomegaly of C26-mice was inhibited by PM01183 for as long as the treatment lasted, without reducing IL-6, M-CSF or IL-1&beta, in plasma. ET743 and PM01183 extend the survival of C26-bearing mice unchanging tumor growth or cachexia but possibly restrain muscle-related inflammation and C26-induced splenomegaly.

Background: In order to investigate the mechanisms of acquired resistance to trabectedin, trabectedin-resistant human myxoid liposarcoma (402-91/T) and ovarian carcinoma (A2780/T) cell lines were derived and characterised in vitro and in vivo. Methods: Resistant cell lines were obtained by repeated exposures to trabectedin. Characterisation was performed by evaluating drug sensitivity, cell cycle perturbations, DNA damage and DNA repair protein expression. In vivo experiments were performed on A2780 and A2780/T xenografts. Results: 402-91/T and A2780/T cells were six-fold resistant to trabectedin compared with parental cells. Resistant cells were found to be hypersensitive to UV light and did not express specific proteins involved in the nucleotide excision repair (NER) pathway: XPF and ERCC1 in 402-91/T and XPG in A2780/T. NER deficiency in trabectedin-resistant cells was associated with the absence of a G2/M arrest induced by trabectedin and with enhanced sensitivity (two-fold) to platinum drugs. In A2780/T, this collateral sensitivity, confirmed in vivo, was associated with an increased formation of DNA interstrand crosslinks. Conclusions: Our finding that resistance to trabectedin is associated with the loss of NER function, with a consequent increased sensitivity to platinum drugs, provides the rational for sequential use of these drugs in patients who have acquired resistance to trabectedin.

The imaging of drugs inside tissues is pivotal in oncology to assess whether a drug reaches all cells in an adequate enough concentration to eradicate the tumor. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) is one of the most promising imaging techniques that enables the simultaneous visualization of multiple compounds inside tissues. The choice of a suitable matrix constitutes a critical aspect during the development of a MALDI-MSI protocol since the matrix ionization efficiency changes depending on the analyte structure and its physico-chemical properties. The objective of this study is the improvement of the MALDI-MSI technique in the field of pharmacology; developing specifically designed nanostructured surfaces that allow the imaging of different drugs with high sensitivity and reproducibility. Among several nanomaterials, we tested the behavior of gold and titanium nanoparticles, and halloysites and carbon nanotubes as possible matrices. All nanomaterials were firstly screened by co-spotting them with drugs on a MALDI plate, evaluating the drug signal intensity and the signal-to-noise ratio. The best performing matrices were tested on control tumor slices, and were spotted with drugs to check the ion suppression effect of the biological matrix. Finally; the best nanomaterials were employed in a preliminary drug distribution study inside tumors from treated mice.

Background: Juvenile myelomonocytic leukaemia (JMML) and chronic myelomonocytic leukaemia (CMML) are myelodysplastic myeloproliferative (MDS/MPN) neoplasms with unfavourable prognosis and without effective chemotherapy treatment. Trabectedin is a DNA minor groove binder acting as a modulator of transcription and interfering with DNA repair mechanisms; it causes selective depletion of cells of the myelomonocytic lineage. We hypothesised that trabectedin might have an antitumour effect on MDS/MPN. Methods: Malignant CD14+ monocytes and CD34+ haematopoietic progenitor cells were isolated from peripheral blood/bone marrow mononuclear cells. The inhibition of CFU-GM colonies and the apoptotic effect on CD14+ and CD34+ induced by trabectedin were evaluated. Trabectedin's effects were also investigated in vitro on THP-1, and in vitro and in vivo on MV-4-11 cell lines. Results: On CMML/JMML cells, obtained from 20 patients with CMML and 13 patients with JMML, trabectedin - at concentration pharmacologically reasonable, 1-5 nM - strongly induced apoptosis and inhibition of growth of haematopoietic progenitors (CFU-GM). In these leukaemic cells, trabectedin downregulated the expression of genes belonging to the Rho GTPases pathway (RAS superfamily) having a critical role in cell growth and cytoskeletal dynamics. Its selective activity on myelomonocytic malignant cells was confirmed also on in vitro THP-1 cell line and on in vitro and in vivo MV-4-11 cell line models. Conclusions: Trabectedin could be good candidate for clinical studies in JMML/CMML patients.

Mass Spectrometry Imaging (MSI) is a widespread technique used to qualitatively describe in two dimensions the distribution of endogenous or exogenous compounds within tissue sections. Absolute quantification of drugs using MSI is a recent challenge that just in the last years has started to be addressed. Starting from a two dimensional MSI protocol, we developed a three-dimensional pipeline to study drug penetration in tumors and to develop a new drug quantification method by MALDI MSI. Paclitaxel distribution and concentration in different tumors were measured in a 3D model of Malignant Pleural Mesothelioma (MPM), which is known to be a very heterogeneous neoplasm, highly resistant to different drugs. The 3D computational reconstruction allows an accurate description of tumor PTX penetration, adding information about the heterogeneity of tumor drug distribution due to the complex microenvironment. The use of an internal standard, homogenously sprayed on tissue slices, ensures quantitative results that are similar to those obtained using HPLC. The 3D model gives important information about the drug concentration in different tumor sub-volumes and shows that the great part of each tumor is not reached by the drug, suggesting the concept of pseudo-resistance as a further explanation for ineffective therapies and tumors relapse.

The penetration of anticancer drugs in solid tumors is important to ensure the therapeutic effect, so methods are needed to understand drug distribution in different parts of the tumor. Mass spectrometry imaging (MSI) has great potential in this field to visualize drug distribution in organs and tumor tissues with good spatial resolution and superior specificity. We present an accurate and reproducible imaging method to investigate the variation of drug distribution in different parts of solid tumors. The method was applied to study the distribution of paclitaxel in three ovarian cancer models with different histopathological characteristics and in colon cancer (HCT116), breast cancer (MDA-MB-231) and malignant pleural mesothelioma (MPM487). The heterogeneous drug penetration in the tumors is evident from the MALDI imaging results and from the images analysis. The differences between the various models do not always relate to significant changes in drug content in tumor homogenate examined by classical HPLC analysis. The specificity of the method clarifies the heterogeneity of the drug distribution that is analyzed from a quantitative point of view too, highlighting how marked are the variations of paclitaxel amounts in different part of solid tumors.

One of the major drawbacks that limits the clinical application of nanoparticles is the lack of preliminary investigations related to their biocompatibility, biodegradability and biodistribution. In this work, biodegradable PEGylated polymer nanoparticles (NPs) have been synthesized by using macromonomers based on poly(ε-caprolaconte) oligomers. More in detail, NPs have been produced by adopting a surfactant-free semibatch emulsion polymerization process using PEG chains as a stabilizing agent. The NPs were also labeled with rhodamine B covalently bound to the NPs to quantitatively study their biodistribution in vivo. NPs were investigated in both in vitro and in vivo preclinical systems to study their biodistribution in mice bearing B16/F10 melanoma, as well as their biocompatibility and biodegradability. The NP concentration was evaluated in different tissues at several times after intravenous injection. The disappearance of the NPs from the plasma was biphasic, with distribution and elimination half-lives of 30 min and 15 h, respectively. NPs were retained in tumors and in filter organs for a long time, were still detectable after 7 d and maintained a steady concentration in the tumor for 120 h. 48 h after injection, 70± 15% of the inoculated NPs were excreted in the feces. The favorable tumor uptake, fast excretion and absence of cytotoxicity foster the further development of produced NPs as drug delivery carriers. © 2014 IOP Publishing Ltd.

To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) to trabectedin and the suggested selectivity for specific subtypes, we have developed and characterized three MRCL xenografts, namely ML017, ML015 and ML004 differing for the break point of the fusion gene FUS-CHOP, respectively of type I, II and III. FUS-CHOP binding to the promoters of some target genes such as Pentraxin 3 or Fibronectin 1, assessed by chromatin immunoprecipitation, was strongly reduced in the tumor 24 h after the first or the third weekly dose of trabectedin, indicating that the drug at therapeutic doses causes a detachment of the FUS-CHOP chimera from its target promoters as previously shown in vitro. Moreover, the higher sensitivity of MRCL types I and II appears to be related to a more prolonged block of the transactivating activity of the fusion protein. Doxorubicin did not affect the binding of FUS-CHOP to target promoters. Histologically, the response to trabectedin in ML017 and ML015 was associated with a marked depletion of non-lipogenic tumoral cells and vascular component, as well as lipidic maturation as confirmed by PPARγ2 expression in western Blot. By contrast, in ML004 no major changes either in the cellularity or in the amount of mature were found, and consistently PPARγ2 was null. In conclusion, the data support the view that the selective mechanism of action of trabectedin in MRCL is specific and related to its ability to cause a functional inactivation of the oncogenic chimera with consequent derepression of the adypocytic differentiation.

E-3810 is a novel small molecule that inhibits VEGF receptor-1, -2, and -3 and fibroblast growth factor receptor-1 tyrosine kinases at nmol/L concentrations currently in phase clinical II. In preclinical studies, it had a broad spectrum of antitumor activity when used as monotherapy in a variety of human xenografts. We here investigated the activity of E-3810 combined with different cytotoxic agents in a MDA-MB-231 triple-negative breast cancer xenograft model. The molecule could be safely administered with 5-fluorouracil, cisplatin, and paclitaxel. The E-3810–paclitaxel combination showed a striking activity with complete, lasting tumor regressions; the antitumor activity of the combination was also confirmed in another triple-negative breast xenograft, MX-1. The activity was superior to that of the combinations paclitaxel+brivanib and paclitaxel+sunitinib. Pharmacokinetics studies suggest that the extra antitumor activity of the combination is not due to higher paclitaxel tumor levels, which in fact were lower in mice pretreated with all three kinase inhibitors, and the paclitaxel plasma levels excluded reduced drug availability. Pharmacodynamic studies showed that E-3810, brivanib, and sunitinib given as single agents or in combination with paclitaxel reduced the number of vessels, but did not modify vessel maturation. Reduced tumor collagen IV and increased plasma collagen IV, associated with increased matrix metalloproteinases (MMP), particularly host MMP-9, indicate a proteolytic remodeling of the extracellular matrix caused by E-3810 that in conjunction with the cytotoxic effect of paclitaxel on the tumor cells (caspase-3/7 activity) may contribute to the striking activity of their combination. These data support the therapeutic potential of combining E-3810 with conventional chemotherapy. Mol Cancer Ther; 12(2); 131–40. ©2012 AACR.

Malignant pleural mesothelioma (MPM) incidence is increased over the past two decades and it is predicted to increase further in the next 20 years especially in developing countries where asbestos has not yet been banned. Surgery is not an option for the majority of patients due to the diffuse growth of MPM, making chemotherapy the treatment of choice, but prognosis remains very poor with a median survival time of 10-17 months. Trabectedin (ET-743, Yondelis) is a marine alkaloid used for the 2nd line therapy of ovarian cancer (combined with pegylated doxorubicin) and soft tissue sarcomas. It binds the DNA minor groove modulating transcription of cancer cells and has an anti-inflammatory effect modifying tumor microenvironment and reducing tumor associated macrophages. The aim was to test antitumor activity of trabectedin in newly established patient derived xenografts of MPM. MPM473, MPM484 and MPM487 xenografts were obtained in athymic nude mice by s.c. injection of 107 cells isolated from the pleural effusion of MPM patients and maintained by serial passages in mice. H&E staining and immunohistochemistry were performed. Mice were randomized when tumor reached about 200 mg to receive trabectedin, cisplatin, pemetrexed, gemcitabine, and imatinib. Antitumor activity was expressed as T/C% were T and C are the mean tumor weight of treated and control mice, respectively. The three xenografts reproduced the morphology and the histochemical profile of the human tumors. As reported in the table trabectedin was able to induce a 50% reduction in tumor growth in MPM473 and MPM 487 xenografts and only a marginal effect in MPM484 xenograft. TreatmentDose (mg/kg)ScheduleBest T/C% (Day)MPM473MPM484MPM487Cisplatin5i.v. q7dx361.9 (69)66.1 (69)67.3 (39)Gemcitabine100i.p. q4dx583.2 (41)67.7 (48)49.7 (56)Imatinib200p.o. qdx1568.9 (76)89.4 (55)87.1 (39)Pemetrexed250i.p. q4dx477.4 (52)76.8 (49)84.3 (39)Trabectedin0.15i.v. q7dx351.0 (73)63.6 (69)51.3 (56) In conclusion we report three new MPM xenografts able to mimic the pathological features of human MPM. Although trabectedin showed only a moderate antitumor activity in our experimental models, its efficacy is comparable or better than that of the other drugs currently used for this disease. Studies are in progress in syngenic models that are more appropriate to test also the ability of trabectedin to modulate the immune system. Citation Format: Simonetta Andrea Licandro, Roberta Frapolli, Ezia Bello, Roberta Libener, Sara Orecchia, Federica Grosso, Maurizio D’Incalci. Trabectedin activity in patient-derived mesothelioma xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3764.

Trabectedin (ET-743, Yondelis) is a marine alkaloid isolated from the tunicate Ecteinascidia turbinata, approved in Europe and US for the 2nd line therapy of soft tissue sarcomas. It is particularly effective against myxoid liposarcoma, a specific histological subtype within the family of adults soft tissue sarcomas. More than 90% of usual myxoid/round cell liposarcomas (MRCLS) are characterized by the chromosomal translocation t(12;16) (q13; p11), which produces the FUS-CHOP oncogene. Different chimera subtypes seem to respond differently to trabectedin in clinical setting. Trabectedin was able to remove FUS-CHOP type I, II and III from its own target genes but it causes adipocytic maturation and tumor regression only in type I and II, not in type III MRCLS (Di Giandomenico et al, Oncogene, 2014 Oct 30; 33(44): 5201-10). Pioglitazone is a PPARgamma agonist with pro-differentiation effects in different cancer types included liposarcomas (Tontonoz P. et al, Proc Natl Acad Sci U S A. 1997 Jan 7;94(1): 237-41). The aim of the study is to evaluate the efficacy of the combination of trabectedin with pioglitazone in type III myxoid liposarcoma xenografts. ML006 and ML004 type III MRCLS xenografts mice were treated with trabectedin 0.15 mg/kg iv every seven days for three times or pioglitazone 150 mg/kg po daily for 28 days or with their combination. For antitumor activity evaluation, tumor growth was measured with a caliper and the tumor weights (1 mm3 = 1 mg) were calculated with the formula: length × (width)2/2. Fifteen days after the last dose of trabectedin and 4 h after the last dose of pioglitazone tumors were collected from a separate group of mice to perform histological and molecular analyses. Trabectedin and pioglitazone as single agents have a comparable antitumor activity on these models with T/C around 40-50% with no tumor regression observed. In the combination groups an impressive and long lasting tumor regression was observed in ML 006 xenograft, with an observed optimal T/C of 17%. In ML004 MRCLS model the combination of trabectedin and pioglitazone induced a minimal regression followed by a long lasting tumor stabilization, resulting in a T/C of 23%. The histological analyses showed adipocytic maturation in tumors treated with pioglitazone alone or in combination but not in samples from mice treated with trabectedin alone. In conclusion, the combination of trabectedin with pioglitazone was able to induce tumor regression and adipocytic maturation in type III myxoid liposarcoma xenografts that were only marginally sensitive to trabectedin alone. Chromatin Immunoprecipitation, gene expression profile and Western Blot analysis are on-going to unravel the molecular mechanism behind the potentiation of the antitumor efficacy observed with this combination. Citation Format: Ezia Bello, Roberta Frapolli, Simonetta Andrea Licandro, Silvia Brich, Laura Carrassa, Roberta Sanfilippo, Alessandro Gronchi, Paolo Casali, Silvana Pilotti, Maurizio D’Incalci. PPARgamma agonist promotes adipocytic differentiation and potentiates the activity of trabectedin in myxoid liposarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1183.

Mesothelioma, sometimes referred to as asbestos cancer, is known to be among the most aggressive and difficult to treat tumours. The disease attacks the mesothelium, a protective two-layered membrane that covers the internal organs of the body including the lungs, heart and abdominal organs. Mesothelioma can affect any of these layers, but is usually seen in the pleural (lung) or peritoneal (abdomen) mesothelium. The most commonly diagnosed form of this cancer is pleural mesothelioma, caused primarily by the inhalation of asbestos fibers. The prevalence of this cancer is estimated at less than 1% of all cancers, however its incidence is increasing, with an expected peak in the next 10-20 years. Mesotheliomas are tumours that have a poor prognosis due to limited treatment options. The most aggressive form is the sarcomatoid histotype, followed by the biphasic and the epithelioid histotypes.In order to setup in vivo models of human mesothelioma, translational related to human pathology in terms of histology, antigen expression and pharmacological response to therapy, we have established three mesothelioma in vivo models using primary cells derived from patients having different mesothelioma histotypes.We stably transfected MM-432 (sarcomatoid), MM-473 (epithelioid) and MM-487 (biphasic) mesothelioma cell lines with a luciferase expression vector. The selected high luc-espressing clones were inoculated intrapleurally in immunodeficient nude mice, and they successfully invaded and proliferated within the murine host. In particular, the epithelioid histotype presented tumour growth 100% of the time, after a short latency period. The biphasic histotype failed to present tumour growth in 30% of the cases, however was far more aggressive than the epithelial histotype upon establishment of tumour growth. Moreover, the ex-vivo Immunocytochemistry (i.e for CEA, EMA, Mesothelin, Podoplanin, calretinin) and biochemical characterization (i.e.EGFR, VEGF, Top1, TKs pathways) of the three histotypes, revealed histotype-related differences. Finally, the cytotoxic responses to a panel of antitumor drugs (i.e . Doxorubicin , Topotecan , Ciclofosfamide, Dacarbazina, Gemcitabine, Temozolomide, Bortezomib, 5-azacitidina, Paclitaxel, Etoposide, 5-FU, Cisplatin, and AZD-2281) revealed that the three original cell lines, compared to the corresponding luc-expressing clones, had similar sensitivities to drugs depending upon the tumor histotypes. Further, in vivo characterizations focused on chemotherapeutic responses of these models, are currently in progress. Citation Format: Claudio Pisano, Alison G. Cole, Marcella Barbarino, Erminia Bianchino, Mario Guglielmi, Carmela Melito, Grazia Mercadante, Alfonsina Porciello, Assunta Riccio, Ilaria La Porta, Sara Orecchia, Roberta Libener, Laura Mazzucco, Simonetta Andrea Licandro, Pasquale De Luca. Mice models of three human mesothelioma histotypes derived from primary patient tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5556. doi:10.1158/1538-7445.AM2014-5556

Introduction: Trabectedin (T) is a marine-derived anticancer drug with a peculiar mechanism of action, ascribed to a covalent modification of DNA by guanine-specific alkylation at the N2 position in the minor groove that induces DNA damage, affects transcription regulation and triggers cell differentiation and death. Trabectedin has been approved in the EU for the second line treatment of soft tissue sarcomas and ovarian carcinomas. In order to investigate the mechanisms of resistance to trabectedin, that generally occurs after prolonged treatment with the drug, two different trabectedin-resistant cell lines such as myxoid liposarcoma 402-91 and ovarian carcinoma A2780 were developed and characterized. Material and Methods: Resistant cell lines (402-91/T and A2780/T) were obtained by stepwise increase in drug concentration using a short exposure (1 hr) applied at 10-20 day intervals. Drug sensitivity was measured by colony assay, DNA content and cell cycle were evaluated by flow cytometric analysis and XPG expression by Western Blotting. Results: 402-91/T and A2780/T cell lines were 16 and 6 fold resistant to trabectedin as compared to parental cells respectively. Resistant cell lines maintained the same DNA content compared with the parental cells, while showed different cell cycle perturbations induced by trabectedin treatment; in fact the characteristic G2/M block was found only in sensitive cell lines. Both 402-91/T and A2780/T cell lines did not express XPG, a protein involved in NER pathway, and were found hypersensitive to UV light. This finding is in keeping with previous observations that NER deficiency is associated to a decreased sensitivity to trabectedin. Being NER activity crucial for the repair of platinum DNA adducts, we tested cisplatin cytotoxicity in 402-91/T and A2780/T cell lines and they were 2.5 and 11 fold more sensitive than the parental cell lines. The collateral sensitivity to cisplatin of trabectedin resistant cell lines is likely related to the important function of NER in the repair of the bulky adducts produced by the drug. Conclusion: The finding that resistance to trabectedin is associated to the loss of NER function, with consequent increased sensitivity to cisplatin, provides the rational to test the sequential combination of trabectedin and platinum complexes in the clinic. This strategy is currently under evaluation in ovarian cancer patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C93. Citation Format: Maurizio D'Incalci, Benedetta Colmegna, Sarah Uboldi, Roberta Frapolli, Simonetta Andrea Licandro, Eugenio Erba, Carlos M. Galmarini, Nadia Badri. Collateral sensitivity to cisplatin of trabectedin-resistant cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C93.