Tumor-Specific Delivery of 5-Fluorouracil-Incorporated Epidermal Growth Factor Receptor-Targeted Aptamers as an Efficient Treatment in Pancreatic Ductal Adenocarcinoma Models.

BACKGROUNDS & AIMS: Fluoropyrimidine c (5-fluorouracil [5FU]) increasingly represents the chemotherapeutic backbone for neoadjuvant, adjuvant, and palliative treatment of pancreatic ductal adenocarcinoma (PDAC). Even in combination with other agents, 5FU efficacy remains transient and limited. One explanation for the inadequate response is insufficient and nonspecific delivery of 5FU to the tumor. METHODS: We designed, generated, and characterized 5FU-incorporated systematic evolution of ligands by exponential enrichment (SELEX)-selected epidermal growth factor receptor (EGFR)-targeted aptamers for tumor-specific delivery of 5FU to PDAC cells and tested their therapeutic efficacy in vitro and in vivo. RESULTS: 5FU-EGFR aptamers reduced proliferation in a concentration-dependent manner in mouse and human pancreatic cancer cell lines. Time-lapsed live imaging showed EGFR-specific uptake of aptamers via clathrin-dependent endocytosis. The 5FU-aptamer treatment was equally effective in 5FU-sensitive and 5FU-refractory PDAC cell lines. Biweekly treatment with 5FU-EGFR aptamers reduced tumor burden in a syngeneic orthotopic transplantation model of PDAC, in an autochthonously growing genetically engineered PDAC model (LSL-KrasG12D/+;LSL-Trp53flox/+;Ptf1a-Cre [KPC]), in an orthotopic cell line-derived xenograft model using human PDAC cells in athymic mice (CDX; Crl:NU-Foxn1nu), and in patient-derived organoids. Tumor growth was significantly attenuated during 5FU-EGFR aptamer treatment in the course of follow-up. CONCLUSIONS: Tumor-specific targeted delivery of 5FU using EGFR aptamers as the carrier achieved high target specificity; overcame 5FU resistance; and proved to be effective in a syngeneic orthotopic transplantation model, in KPC mice, in a CDX model, and in patient-derived organoids and, therefore, represents a promising backbone for pancreatic cancer chemotherapy in patients. Furthermore, our approach has the potential to target virtually any cancer entity sensitive to 5FU treatment by incorporating 5FU into cancer cell-targeting aptamers as the delivery platform.

Fabrication and characterization of a multimodal 3D printed mouse phantom for ionoacoustic quality assurance in image-guided pre-clinical proton radiation research.

Objective.Image guidance and precise irradiation are fundamental to ensure the reliability of small animal oncology studies. Accurate positioning of the animal and the in-beam monitoring of the delivered radio-therapeutic treatment necessitate several imaging modalities. In the particular context of proton therapy with a pulsed beam, information on the delivered dose can be retrieved by monitoring the thermoacoustic waves resulting from the brief and local energy deposition induced by a proton beam (ionoacoustics). The objective of this work was to fabricate a multimodal phantom (x-ray, proton, ultrasound, and ionoacoustics) allowing for sufficient imaging contrast for all the modalities.Approach.The phantom anatomical parts were extracted from mouse computed tomography scans and printed using polylactic acid (organs) and a granite/polylactic acid composite (skeleton). The anatomical pieces were encapsulated in silicone rubber to ensure long term stability. The phantom was imaged using x-ray cone-beam computed tomography, proton radiography, ultrasound imaging, and monitoring of a 20 MeV pulsed proton beam using ionoacoustics.Main results.The anatomical parts could be visualized in all the imaging modalities validating the phantom capability to be used for multimodal imaging. Ultrasound images were simulated from the x-ray cone-beam computed tomography and co-registered with ultrasound images obtained before the phantom irradiation and low-resolution ultrasound images of the mouse phantom in the irradiation position, co-registered with ionoacoustic measurements. The latter confirmed the irradiation of a tumor surrogate for which the reconstructed range was found to be in reasonable agreement with the expectation.Significance.This study reports on a realistic small animal phantom which can be used to investigate ionoacoustic range (or dose) verification together with ultrasound, x-ray, and proton imaging. The co-registration between ionoacoustic reconstructions of the impinging proton beam and x-ray imaging is assessed for the first time in a pre-clinical scenario.

Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond.

Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damage response factors on the protein level, distinct transcriptomic alterations, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in glioblastoma cells in vitro. In vivo, HSP90 inhibition by NW457 improved the therapeutic outcome of fractionated CBCT-based irradiation in an orthotopic, syngeneic GBM mouse model, both in terms of tumor progression and survival. Nevertheless, in view of the promising in vitro results the in vivo efficacy was not as strong as expected, although apart from the radiosensitizing effects HSP90 inhibition also reduced irradiation-induced GBM cell migration and tumor invasiveness. Hence, our findings identify the combination of HSP90 inhibition and radiotherapy in principle as a promising strategy for GBM treatment whose performance needs to be further optimized by improved inhibitor substances, better formulations and/or administration routes, and fine-tuned treatment sequences.

PSMA PET Imaging in Glioblastoma: A Preclinical Evaluation and Theranostic Outlook.

BACKGROUND: Prostate specific membrane antigen (PSMA) PET imaging has recently gained attention in glioblastoma (GBM) patients as a potential theranostic target for PSMA radioligand therapy. However, PSMA PET has not yet been established in a murine GBM model. Our goal was to investigate the potential of PSMA PET imaging in the syngeneic GL261 GBM model and to give an outlook regarding the potential of PMSA radioligand therapy in this model. METHODS: We performed an 18F-PSMA-1007 PET study in the orthotopic GL261 model (n=14 GBM, n=7 sham-operated mice) with imaging at day 4, 8, 11, 15, 18 and 22 post implantation. Time-activity-curves (TAC) were extracted from dynamic PET scans (0-120 min p. i.) in a subset of mice (n=4 GBM, n=3 sham-operated mice) to identify the optimal time frame for image analysis, and standardized-uptake-values (SUV) as well as tumor-to-background ratios (TBR) using contralateral normal brain as background were calculated in all mice. Additionally, computed tomography (CT), ex vivo and in vitro 18F-PSMA-1007 autoradiographies (ARG) were performed. RESULTS: TAC analysis of GBM mice revealed a plateau of TBR values after 40 min p. i. Therefore, a 30 min time frame between 40-70 min p. i. was chosen for PET quantification. At day 15 and later, GBM mice showed a discernible PSMA PET signal on the inoculation site, with highest TBRmean in GBM mice at day 18 (7.3 ± 1.3 vs. 1.6 ± 0.3 in shams; p=0.024). Ex vivo ARG confirmed high tracer signal in GBM compared to healthy background (TBRmean 26.9 ± 10.5 vs. 1.6 ± 0.7 in shams at day 18/22 post implantation; p=0.002). However, absolute uptake values in the GL261 tumor remained low (e.g., SUVmean 0.21 ± 0.04 g/ml at day 18) resulting in low ratios compared to dose-relevant organs (e.g., mean tumor-to-kidney ratio 1.5E-2 ± 0.5E-2). CONCLUSIONS: Although 18F-PSMA-1007 PET imaging of GL261 tumor-bearing mice is feasible and resulted in high TBRs, absolute tumoral uptake values remained low and hint to limited applicability of the GL261 model for PSMA-directed therapy studies. Further investigations are warranted to identify suitable models for preclinical evaluation of PSMA-targeted theranostic approaches in GBM.

Contrast-enhanced, conebeam CT-based, fractionated radiotherapy and follow-up monitoring of orthotopic mouse glioblastoma: a proof-of-concept study.

BACKGROUND: Despite aggressive treatment regimens comprising surgery and radiochemotherapy, glioblastoma (GBM) remains a cancer entity with very poor prognosis. The development of novel, combined modality approaches necessitates adequate preclinical model systems and therapy regimens that closely reflect the clinical situation. So far, image-guided, fractionated radiotherapy of orthotopic GBM models represents a major limitation in this regard. METHODS: GL261 mouse GBM cells were inoculated into the right hemispheres of C57BL/6 mice. Tumor growth was monitored by contrast-enhanced conebeam CT (CBCT) scans. When reaching an average volume of approximately 7 mm3, GBM tumors were irradiated with daily fractions of 2 Gy up to a cumulative dose of 20 Gy in different beam collimation settings. For treatment planning and tumor volume follow-up, contrast-enhanced CBCT scans were performed twice per week. Daily repositioning of animals was achieved by alignment of bony structures in native CBCT scans. When showing neurological symptoms, mice were sacrificed by cardiac perfusion. Brains, livers, and kidneys were processed into histologic sections. Potential toxic effects of contrast agent administration were assessed by measurement of liver enzyme and creatinine serum levels and by histologic examination. RESULTS: Tumors were successfully visualized by contrast-enhanced CBCT scans with a detection limit of approximately 2 mm3, and treatment planning could be performed. For daily repositioning of the animals, alignment of bony structures in native CT scans was well feasible. Fractionated irradiation caused a significant delay in tumor growth translating into significantly prolonged survival in clear dependence of the beam collimation setting and margin size. Brain sections revealed tumors of similar appearance and volume on the day of euthanasia. Importantly, the repeated contrast agent injections were well tolerated, as liver enzyme and creatinine serum levels were only subclinically elevated, and liver and kidney sections displayed normal histomorphology. CONCLUSIONS: Contrast-enhanced, CT-based, fractionated radiation of orthotopic mouse GBM represents a versatile preclinical technique for the development and evaluation of multimodal radiotherapeutic approaches in combination with novel therapeutic agents in order to accelerate translation into clinical testing.