Preclinical neuroblastoma models for a pharmacological study of a new MYCN oncogene inhibitor

Background. Neuroblastoma is the most deadly solid tumor of childhood. In the 25% of cases it is associated with MYCN amplification (MA), resulting in the disregulation of several genes involved in cancer progression, chemotherapy resistance and poor prognosis causing the disregulation of several genes involved in cancer progression and chemotherapy resistance and resulting in a poor prognosis. Moreover, in this contest, therapy-related p53 mutations are frequently found in relapsed cases conferring an even stronger aggressiveness. For this reason, the actual therapy requires new antitumor molecules. Therefore, rapid, accurate, and reproducible preclinical models are needed to evaluate the evolution of the different subtypes and the efficacy of new pharmacological strategies. Procedures. We report the real-time tumorigenesis of MA Neuroblastoma mouse models: transgenic TH-MYCN mice and orthotopic xenograft models with either p53wt or p53mut, by non-invasive micro PET and bioluminescent imaging, respectively. Characterization of MYCN amplification and expression was performed on every collected sample. We tested the efficacy of a new MYCN inhibitor in vitro and in vivo. Results. MicroPET in TH-MYCN mice permitted the identification of Neuroblastoma at an early stage and offered a sensitive method to follow metabolic progression of tumors. The MA orthotopic model harboring multitherapy-related p53 mutations showed a shorter latency and progression and a stronger aggressiveness respect to the p53wt model. The presence of MA and overexpression was confirmed in each model and we saw a better survival in the TH-MYCN homozigous mice treated with the inhibitor. Conclusions. The mouse models obtained show characteristics of non-invasiveness, rapidity and sensitivity that make them suitable for the in vivo preclinical study of MA-NB. In particular, our firstly reported p53mut BLI xenograft orthotopic mouse model offers the possibility to evaluate the role of multitherapy-related p53 mutations and to validate new p53 independent therapies for this highly aggressive Neuroblastoma subtype. Moreover, we have shown potential clinical suitability of an antigene strategy through its cellular and molecular activity, ability to specifically inhibit transcription and in vivo efficacy with no evidence of toxicity.

Use of VEGFR-2 targeted microbubbles (BR55, Bracco imaging) for the early ultrasound evaluation of response to antiangiogenic treatment in a xenograft model of hepatocarcinoma

Aim: To evaluate the early response to treatment to an antiangiogenetic drug (sorafenib) in a heterotopic murine model of hepatocellular carcinoma (HCC) using ultrasonographic molecular imaging. Material and Methods: the xenographt model was established injecting a suspension of HuH7 cells subcutaneously in 19 nude mice. When tumors reached a mean diameter of 5-10 mm, they were divided in two groups (treatment and vehicle). The treatment group received sorafenib (62 mg/kg) by daily oral gavage for 14 days. Molecular imaging was performed using contrast enhanced ultrasound (CEUS), by injecting into the mouse venous circulation a suspension of VEGFR-2 targeted microbubbles (BR55, kind gift of Bracco Swiss, Geneve, Switzerland). Video clips were acquired for 6 minutes, then microbubbles (MBs) were destroyed by a high mechanical index (MI) impulse, and another minute was recorded to evaluate residual circulating MBs. The US protocol was repeated at day 0,+2,+4,+7, and +14 from the beginning of treatment administration. Video clips were analyzed using a dedicated software (Sonotumor, Bracco Swiss) to quantify the signal of the contrast agent. Time/intensity curves were obtained and the difference of the mean MBs signal before and after high MI impulse (Differential Targeted Enhancement-dTE) was calculated. dTE represents a numeric value in arbitrary units proportional to the amount of bound MBs. At day +14 mice were euthanized and the tumors analyzed for VEGFR-2, pERK, and CD31 tissue levels using western blot analysis. Results: dTE values decreased from day 0 to day +14 both in treatment and vehicle groups, and they were statistically higher in vehicle group than in treatment group at day +2, at day +7, and at day +14. With respect to the degree of tumor volume increase, measured as growth percentage delta (GPD), treatment group was divided in two sub-groups, non-responders (GPD>350%), and responders (GPD<200%). In the same way vehicle group was divided in slow growth group (GPD<400%), and fast growth group (GPD>900%). dTE values at day 0 (immediately before treatment start) were higher in non-responders than in responders group, with statistical difference at day 2. While dTE values were higher in the fast growth group than in the slow growth group only at day 0. A significant positive correlation was found between VEGFR-2 tissue levels and dTE values, confirming that level of BR55 tissue enhancement reflects the amount of tissue VEGF receptor. Conclusions: the present findings show that, at least in murine experimental models, CEUS with BR55 is feasable and appears to be a useful tool in the prediction of tumor growth and response to sorafenib treatment in xenograft HCC.

Generation and characterization of mouse models of Small cell lung cancer and Basal cell carcinoma for the preclinical evaluation of new therapies

Background. Human small cell lung cancer (SCLC) accounting for approximately 15-20% of all lung cancers, is an aggressive tumor with high propensity for early regional and distant metastases. Although the initial tumor rate response to chemotherapy is very high, SCLC relapses after approximately 4 months in ED and 12 months in LD. Basal cell carcinoma (BCC) is the most prevalent cancer in the western world, and its incidence is increasing worldwide. This type of cancer rarely metastasizes and the death rate is extraordinary low. Surgery is curative for most of the patients, but for those that develop locally advanced or metastatic BCC there is currently no effective treatment. Both types of cancer have been deeply investigated and genetic alterations, MYCN amplification (MA) among the most interesting, have been found. These could become targets of new pharmacological therapies. Procedures. We created and characterized novel BLI xenograft orthotopic mouse models of SCLC to evaluate the tumor onset and progression and the efficacy of new pharmacological strategies. We compared an in vitro model with a transgenic mouse model of BCC, to investigate and delineate the canonical HH signalling pathway and its connections with other molecular pathways. Results and conclusions. The orthotopic models showed latency and progression patterns similar to human disease. Chemotherapy treatments improved survival rates and validated the in vivo model. The presence of MA and overexpression were confirmed in each model and we tested the efficacy of a new MYCN inhibitor in vitro. Preliminar data of BCC models highlighted Hedgehog pathway role and underlined the importance of both in vitro and in vivo strategies to achieve a better understanding of the pathology and to evaluate the applicability of new therapeutic compounds