Combined Replenishment of miR‐34a and let‐7b by Targeted Nanoparticles Inhibits Tumor Growth in Neuroblastoma Preclinical Models

Abstract

Neuroblastoma (NB) tumor substantially contributes to childhood cancer mortality. The design of novel drugs targeted to specific molecular alterations becomes mandatory, especially for high-risk patients burdened by chemoresistant relapse. The dysregulated expression of MYCN, ALK, and LIN28B and the diminished levels of miR-34a and let-7b are oncogenic in NB. Due to the ability of miRNA-mimics to recover the tumor suppression functions of miRNAs underexpressed into cancer cells, safe and efficient nanocarriers selectively targeted to NB cells and tested in clinically relevant mouse models are developed. The technology exploits the nucleic acids negative charges to build coated-cationic liposomes, then functionalized with antibodies against GD 2 receptor. The replenishment of miR-34a and let-7b by NB-targeted nanoparticles, individually and more powerfully in combination, significantly reduces cell division, proliferation, neoangiogenesis, tumor growth and burden, and induces apoptosis in orthotopic xenografts and improves mice survival in pseudometastatic models. These functional effects highlight a cooperative down-modulation of MYCN and its down-stream targets, ALK and LIN28B, exerted by miR-34a and let-7b that reactivate regulatory networks leading to a favorable therapeutic response. These findings demonstrate a promising therapeutic efficacy of miR-34a and let-7b combined replacement and support its clinical application as adjuvant therapy for highrisk NB patients.

Cell surface Nucleolin represents a novel cellular target for neuroblastoma therapy

“…PEGASEMP™ can inhibit NB cell proliferation and reduce NB cell viability in vitro, and can delay tumor growth in two different animal models of NB. Altogether, these results demonstrate that cell surface NCL might be used for the selective targeting of NB cells, through intravenous administration of nanoparticles encapsulating not only chemotherapeutic agents, but also retinoids or macromolecules like siRNA or miRNA mimics molecules, specific for NB oncogene silencing or for tumor suppressor miRNA replacement, respectively, as previously demonstrated [ 15 , 23 , 26 ].…”

“…Human (IMR-32, HTLA-230, SH-SY5Y and SK-N-AS) and murine (NXS2) neuroblastoma (NB) cell lines were grown in complete Dulbecco’s Modified Eagle Medium (DMEM) medium, as previously described [ 12 – 15 ]. In some experiments, IMR-32 cells were infected with retrovirus expressing the firefly luciferase (luc) gene, as previously reported [ 13 ].…”

“…In some experiments, IMR-32 cells were infected with retrovirus expressing the firefly luciferase (luc) gene, as previously reported [ 13 ]. Luciferase activity of IMR-32-luc cells was confirmed by bio-luminescent imaging (BLI, Lumina-II, Caliper Life Sciences, Hopkinton, MA) after a 10 min incubation with 150 μg/mL d-luciferin (Caliper Life Sciences) diluted in cell culture medium, as previously described [ 13 – 15 ]. Normal human dermal fibroblasts (FIBRO) were purchased from American Type Culture Collection (ATCC, Manassas, VA); human skin keratinocytes cells (HaCaT) were kindly provided by Dr. Flavio Curnis (San Raffaele Scientific Institute, Italy); both cell lines were grown in complete RPMI-1640 medium, as previously described [ 12 ].…”

“…For the pseudo-metastatic model, HTLA-230 cell line (4 × 10 6 cells in 200 μL culture medium) was inoculated in the tail vein of four-week-old nu/nu mice, as described [ 12 , 15 , 16 ]. For the orthotopic model, IMR-32 (wild-type and luc-transfected) or SH-SY5Y cell lines (1 × 10 6 cells in 10 μL culture medium) were inoculated in the left adrenal gland of five-week-old nu/nu mice, as described [ 12 , 15 , 17 ]. No mice died as a result of the surgery.…”

“…Mice were weighed 24 h after each treatment. Moreover, in the systemic chronic toxicity experiment, mice were anesthetized with xylazine 24 h after the last day of treatment and blood was collected, as previously reported [ 15 , 22 – 25 ]. Hematological levels of red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), reticulocytes (RET), platelets (PLT) and white blood cells (WBC) as well as clinical chemistry levels of serum albumin (ALB), phosphatase alkaline (ALP), glutamic-pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), lactate dehydrogenase (LDH), creatine phosphokinase (CK) and creatinine (CREA) and urea were quantified.…”