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STAT3 decoy oligonucleotide-carrying microbubbles with pulsed ultrasound for enhanced therapeutic effect in head and neck tumors.

ABSTRACT: Signal transducer and activator of transcription-3 (STAT3) is an oncogenic transcription factor implicated in carcinogenesis, tumor progression, and drug resistance in head and neck squamous cell carcinoma (HNSCC). A decoy oligonucleotide targeting STAT3 offers a promising anti-tumor strategy, but achieving targeted tumor delivery of the decoy with systemic administration poses a significant challenge. We previously showed the potential for STAT3 decoy-loaded microbubbles, in conjunction with ultrasound targeted microbubble cavitation (UTMC), to decrease tumor growth in murine squamous cell carcinoma. As a next step towards clinical translation, we sought to determine the anti-tumor efficacy of our STAT3 decoy delivery platform against human HNSCC and the effect of higher STAT3 decoy microbubble loading on tumor cell inhibition. STAT3 decoy was loaded on cationic lipid microbubbles (STAT3-MB) or loaded on liposome-conjugated lipid microbubbles to form STAT3-loaded liposome-microbubble complexes (STAT3-LPX). UTMC treatment efficacy with these two formulations was evaluated in vitro using viability and apoptosis assays in CAL33 (human HNSCC) cells. Anti-cancer efficacy in vivo was performed in a CAL33 tumor murine xenograft model. UTMC with STAT3-MB caused significantly lower CAL33 cell viability compared to UTMC with STAT3-LPX (56.8±8.4% vs 84.5±8.8%, respectively, p<0.05). In vivo, UTMC with STAT3-MB had strong anti-tumor effects, with significantly less tumor burden and greater survival compared to that of UTMC with microbubbles loaded with a mutant control decoy and untreated control groups (p<0.05). UTMC with STAT3 decoy-loaded microbubbles significantly decreases human HNSSC tumor progression. These data set the stage for clinical translation of our microbubble platform as an imaged-guided, targeted delivery strategy for STAT3 decoy, or other nucleotide-based therapeutics, in human cancer treatment.

SUBMITTER: Ramasamy T

PROVIDER: S-EPMC7673576 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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STAT3 decoy oligonucleotide-carrying microbubbles with pulsed ultrasound for enhanced therapeutic effect in head and neck tumors.

Ramasamy Thiruganesh T Chen Xucai X Qin Bin B Johnson Daniel E DE Grandis Jennifer R JR Villanueva Flordeliza S FS

PloS one 20201118 11

Signal transducer and activator of transcription-3 (STAT3) is an oncogenic transcription factor implicated in carcinogenesis, tumor progression, and drug resistance in head and neck squamous cell carcinoma (HNSCC). A decoy oligonucleotide targeting STAT3 offers a promising anti-tumor strategy, but achieving targeted tumor delivery of the decoy with systemic administration poses a significant challenge. We previously showed the potential for STAT3 decoy-loaded microbubbles, in conjunction with ul ...[more]

PMID: 33206698

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Project description:Magnetic resonance imaging (MRI)-guided pulsed focused ultrasound (pFUS) combined with microbubbles (MB) contrast agent infusion has been shown to transiently disrupt the blood-brain barrier (BBBD), increasing the delivery of neurotherapeutics to treat central nervous system (CNS) diseases. pFUS interaction with the intravascular MB results in acoustic cavitation forces passing through the neurovascular unit (NVU), inducing BBBD detected on contrast-enhanced MRI. Multiple pFUS+MB exposures in Alzheimer's disease (AD) models are being investigated as a method to clear amyloid plaques by activated microglia or infiltrating immune cells. Since it has been reported that pFUS+MB can induce a sterile inflammatory response (SIR) [1-5] in the rat, the goal of this study was to investigate the potential long-term effects of SIR in the brain following single and six weekly sonications by serial high-resolution MRI and pathology. Methods: Female Sprague Dawley rats weighing 217±16.6 g prior to sonication received bromo-deoxyuridine (BrdU) to tag proliferating cells in the brain. pFUS was performed at 548 kHz, ultrasound burst 10 ms and initial peak negative pressure of 0.3 MPa (in water) for 120 s coupled with a slow infusion of ~460 µL/kg (5-8×107 MB) that started 30 s before and 30 s during sonication. Nine 2 mm focal regions in the left cortex and four regions over the right hippocampus were treated with pFUS+MB. Serial high-resolution brain MRIs at 3 T and 9.4 T were obtained following a single or during the course of six weekly pFUS+MB resulting in BBBD in the left cortex and the right hippocampus. Animals were monitored over 7 to 13 weeks and imaging results were compared to histology. Results: Fewer than half of the rats receiving a single pFUS+MB exposure displayed hypointense voxels on T2*-weighted (w) MRI at week 7 or 13 in the cortex or hippocampus without differences compared to the contralateral side on histograms of T2* maps. Single sonicated rats had evidence of limited microglia activation on pathology compared to the contralateral hemisphere. Six weekly pFUS+MB treatments resulted in pathological changes on T2*w images with multiple hypointense regions, cortical atrophy, along with 50% of rats having persistent BBBD and astrogliosis by MRI. Pathologic analysis of the multiple sonicated animals demonstrated the presence of metallophagocytic Prussian blue-positive cells in the parenchyma with significantly (p<0.05) increased areas of activated astrocytes and microglia, and high numbers of systemic infiltrating CD68+ macrophages along with BrdU+ cells compared to contralateral brain. In addition, multiple treatments caused an increase in the number of hyperphosphorylated Tau (pTau)-positive neurons containing neurofibrillary tangles (NFT) in the sonicated cortex but not in the hippocampus when compared to contralateral brain, which was confirmed by Western blot (WB) (p<0.04). Conclusions: The repeated SIR following multiple pFUS+MB treatments could contribute to changes on MR imaging including persistent BBBD, cortical atrophy, and hypointense voxels on T2w and T2*w images consistent with pathological injury. Moreover, areas of astrogliosis, activated microglia, along with higher numbers of CD68+ infiltrating macrophages and BrdU+ cells were detected in multiple sonicated areas of the cortex and hippocampus. Elevations in pTau and NFT were detected in neurons of the multiple sonicated cortex. Minimal changes on MRI and histology were observed in single pFUS+MB-treated rats at 7 and 13 weeks post sonication. In comparison, animals that received 6 weekly sonications demonstrated evidence on MRI and histology of vascular damage, inflammation and neurodegeneration associated with the NVU commonly observed in trauma. Further investigation is recommended of the long-term effects of multiple pFUS+MB in clinical trials.

Project description:BACKGROUND: The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-?B, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear. RESULTS: The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-?B, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-?B-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death. CONCLUSIONS: The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.

Dataset Information

STAT3 decoy oligonucleotide-carrying microbubbles with pulsed ultrasound for enhanced therapeutic effect in head and neck tumors.

Publications

STAT3 decoy oligonucleotide-carrying microbubbles with pulsed ultrasound for enhanced therapeutic effect in head and neck tumors.

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