AptBCis1, An Aptamer-Cisplatin Conjugate, Is Effective in Lung Cancer Leptomeningeal Carcinomatosis

Abstract

Treatment of lung cancer leptomeningeal carcinomatosis (LM) remains challenging partly due to the biological nature of the blood-brain barrier (BBB). Cisplatin has limited effects on LM, and it is notorious for neurotoxicity. Aptamers are small oligonucleotides considered as antibody surrogates. Here we report a DNA therapeutics, AptBCis1. AptBCis1 is a cisplatin-conjugated, BBB-penetrating, and cancer-targeting DNA aptamer. Its backbone, AptB1, was identified via in vivo SELEX using lung cancer LM orthotopic mouse models. The AptB1 binds to EAAT2, Nucleolin, and YB-1 proteins. Treatment with AptBCis1 1 mg/kg (equivalent to cisplatin 0.35 mg/kg) showed superior tumor suppressive effects compared to cisplatin 2 mg/kg in mice with lung cancer LM diseases. The cerebrospinal fluid platinum concentration in the AptBCis1 group was 10% of that in the cisplatin group. The data suggested the translational potential of AptBCis1 in lung cancer with LM and in cancers in which platinum-based chemotherapy remains as the standard of care.

Keywords: aptamer; blood−brain barrier; cisplatin; in vivo SELEX; leptomeningeal carcinomatosis; lung cancer.

Figure 1

Establishment of a leptomeningeal carcinomatosis (LM) mouse model. (a) Scheme illustrating the direct inoculation of luciferase-expressing cells through cisterna magna to establish a LM orthotopic mouse model. (b) Monitoring of the mouse by IVIS. Prominent BLI signals over the brain and the spinal cord were observed at Day 6 post tumor cell inoculation, and the mouse was sacrificed for IHC confirmation. (c) Tumor cells (red arrows) observed in the ventricular space of the brain (left panel) and the spinal cord (right panel) in IHC studies.

Figure 2

Identification of AptB1, a BBB-penetrating and cancer-targeting aptamer by in vivo SELEX. (a) Scheme illustrating the process of in vivo SELEX. (b) The aptamers were divided into group I or II based on the QGRS prediction. The group I aptamer contained G-quadruplex structure and the group II did not. The LM mouse I administered with Cy5-labeled group I aptamers showed fluorescent signals over the brain/spine in a crescendo pattern at 2 and 4 h after injection. The lesions are outlined with black circles. (c) Confocal microscopy images revealing group I aptamer signals (red) within the tumor cells (green) on the leptomeninges. (d) Strong Cy5 fluorescent signals emitted from the brain and the spine were detected in the LM mouse administered with AptB1, as shown in the IVIS imaging and the quantified bar chart. The lesions are outlined with black circles. (e) The confocal microscopy images revealed AptB1 signals (pink) within the tumor cell (green) on the leptomeninges. (f) The Mfold prediction of AptB1 secondary structures.

Figure 3

Detection of AptB1 in the CSF. (a) The CSF was sampled directly from the cisterna magna 30 min after AptB1 injection through the tail vein. (b) Gross picture of mouse cisterna magna (blue triangle). (c) The CSF was sampled from the cisterna magna with a capillary. (d) The AptB1 was amplified from the CSF and the plasma. The PCR cycle number was 20. (e) Accuracy of the amplified AptB1 sequences was confirmed by Sanger sequencing. (f) Concentrations of the CSF and the plasma AptB1 were determined by qPCR. SD: standard deviation.

Figure 4

AptBCis1, an aptamer–cisplatin conjugate, showed antitumor effects in lung cancer LM diseases. (a) The native polyacrylamide gel electrophoresis result showed successful conjugation of the AptB1 and the cisplatin. (b) The scheme illustrated the timeline of tumor cell inoculation and IV drug treatment (AptBCis1 or cisplatin). (c) The mice were monitored by IVIS; BLI signal intensity implicated corresponding tumor burden. Mice in the cisplatin group showed stronger BLI signals than mice in the AptBCis1 group on Day 14 post tumor inoculation (n = 8 in each group). The lesions are outlined with yellow circles. (d) Significant body weight reduction in the cisplatin group (**P < 0.01). Formulation of body weight change: (Day X/Day 2) %. (e) The IVIS images were taken at Day 2 and Day 8 post tumor inoculation; cisplatin or AptBCis1 was given on Days 2, 3, 5, and 7 post tumor inoculation. The mice were sacrificed on Day 8, and the brains were subjected to immunofluorescent studies. The lesions are outlined with yellow circles. (f) The confocal microscopy images showed better preserved tumor cell contours (green; upper panel: luciferase; lower panel: EGFR) and lower percentage of γH2AX-positive cells (red) in the cisplatin group. (g) A lower percentage of γH2AX-positive cells was observed in the cisplatin group. A total of 1200 or 450 cells, respectively, was analyzed in the cisplatin or the AptBCis1 group. Asterisks denote statistically significant differences. **P < 0.01 (unpaired t test).

Figure 5

AptBCis1 suppressed tumor growth at lower platinum concentrations. (a) The plasma and the CSF platinum concentrations were measured by ICP-MS. (b) The scheme illustrated the timeline of subcutaneous tumor cell inoculation and drug treatment in the lung cancer subcutaneous xenograft mouse model. The SELEX buffer, AptB1, AptBCis1, oligo-cisplatin or cisplatin was given via tail vein at Day 6, 7, 9, 11, 13, and 15 post tumor inoculation (n = 4 for each group). (c) Tumor size was measured daily, and the mice were sacrificed on Day 23. (d) Tumor gross pictures. (e) Body weight reduction was more obvious in the cisplatin 2 mg/kg group. Asterisks denote statistically significant differences. *P < 0.05, **P < 0.01 (unpaired t test).

Figure 6

AptB1 interacting with EAAT2, Nucleolin, and YB-1. (a) Results of AptB1-AP/MS study revealed three candidate AptB1-interacting proteins: EAAT2, YB-1, and Nucleolin. (b) AP-immunoblots verified the interaction between AptB1 and EAAT2, Nucleolin, as well as YB-1 in the PC9 cells and in the mouse brain. (c) The confocal microscopy images showed colocalization (yellow) of AptB1 (red) and Nucleolin (green, upper panel) or YB-1 (green, lower panel) in the PC9 cells. (d) AptB1-treated cells were fractionated into cytosol and nucleus fractions. GAPDH is a cytosolic marker, and Histone H3 is a nucleus marker. The AptB1 sequences were successfully amplified in both cellular fractions. (e) The Coomassie Blue stain and the immunoblots showed the purified GST and GST-YB-1 proteins. (f) The YB-1 exonuclease assay results supported the role of YB-1 as an exonuclease for AptB1. (g) Scheme illustrating the proposed mechanism of AptBCis1 as therapeutics for lung cancer with and without LM.