Abstract 500: Liposomal two-tailed topotecan (L-2TT) induces more apoptosis than topotecan hydrochloride on neuroblastoma xenografts

Abstract

Abstract Background: Neuroblastoma (NB) is the second most common malignancy diagnosed in infants, accounting for 15% of pediatric tumor deaths. High risk NB have a <50% 5-year survival and often resist treatment, with acute and long-term toxicities. Topotecan, part of the standard of care in NB is a Topoisomerase1 inhibitor with a short half-life in circulation (30 minutes). We esterified phospholipid to topotecan at the phenolic hydroxyl group, producing the prodrug 2T-T. 2T-T was loaded onto liposome membranes (termed L-2TT); thus preventing drug leakage. We hypothesized L-2TT would be more efficient at inducing apoptosis than topotecan in NB models (in vitro and in vivo) in part due to its extended circulation time and endocytic uptake. Methods: IC50 was determined on 8 NB cell lines using a WST assay, comparing equal dosages of L-2TT to topotecan (empty liposomes were assessed as controls). NB cells were imaged with DiD-tagged liposomes together with lysotracker green, under confocal microscopy 24 hrs. Non-tumor bearing mice received increasing L-2TT doses, assessing weight, body score over five days. Drug circulation time was estimated by following DiR-liposomes in vivo (IVIS) over 72 hrs. Nude mice received 1 × 10^6 NGP cells (NB cells) intrarenally. When tumors reached 1 gram, xenografts received 10mg/kg L-2TT (containing 3.33mg/kg topotecan) via retroorbital injection, 3.33 mg/kg topotecan-hcl or no drug. Tumors were harvested 24 hrs. later, immunohistochemical analysis (apoptosis marker TUNEL), counting the % of positive cells of total tumor area. Some mice received DiR-liposomes together with L-2TT, fixed in PFA and 50mm sections captured total DiR under confocal microscopy. Finally, we interrogated L-2TT vs. Topotecan-HCl tumors via RNAseq for chemoresistance pathways. Results: IC50 values of L-2TT were lower than topotecan-HCl by almost 3-fold in 7/8 cell lines (empty liposomes at equal concentrations had no effect). DiD-tagged liposomes formed a punctate pattern and colocalized with lysotracker green, confirming liposomes are endocytosed by NB cells in vitro. L-2TT caused no changes adverse effects on mice. Drug circulation time remained stable over 48 hours and was still detectable at 72hrs after liposomal delivery. L-2TT resulted in 3-fold higher tumor apoptosis than Topotecan-hcl (p<0.0001), and itself 3-fold higher than controls (p<0.001, n=5). Mice receiving L-2TT+L-DiR resulted in 2-fold higher DiR signal than L-DiR only (p<0.05, n=3. Finally, RNAseq suggests L-2TT elicits less chemoresistance pathways than topotecan. Conclusions: L-2TT is more potent than topotecan in vitro and in vivo. Mechanisms for these effects could be longer circulation times, different tumor cell uptake, as well as changes in tumor vascular permeability. These drug modifications could provide potent alternative for NB treatment as well as other solid tumor treatment. Citation Format: Sonia L. Hernandez, Isabella Iwanicki, Paula Viza-Gomes, Lydia Wu, Mendi Marquez, Fernando Flores-Guzman, Rachael Sundland, Michaelann Tartis. Liposomal two-tailed topotecan (L-2TT) induces more apoptosis than topotecan hydrochloride on neuroblastoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 500.