Multifactorial diagnostic NIR imaging of CCK2R expressing tumors

AbstractOptical imaging-based diagnostics identify malignancies based on molecular changes instead of morphological criteria in a non-invasive, irradiation free process. The aim of this study was to improve imaging efficiency by the development of a new Cholecystokinin-2-receptor targeted fluorescent peptide that matches the clinical needs regarding biodistribution and pharmacokinetics while displaying superior target specificity. Furthermore we performed multifactorial imaging of Cholecystokinin-2-receptor and tumor metabolism, since simultaneous targeting of various tumor biomarkers could intensely increase tumor identification and characterization. Affinity and specificity of the fluorescent Cholecystokinin-2-receptor targeted minigastrin (dQ-MG-754) were tested in vitro. We conducted in vivo imaging of the dQ-MG-754 probe alone and in a multifactorial approach with a GLUT-1 targeted probe (IR800 2-DG) on subcutaneous xenograft bearing athymic nude mice up to 24 h after intravenous injection (n = 5/group), followed by ex vivo biodistribution analysis and histological examination. We found specific, high affinity binding (Kd = 1.77 nm ± 0.6 nm) of dQ-MG-754 to Cholecystokinin-2-receptor expressing cells and xenografts as well as favorable pharmacokinetics for fluorescence-guided endoscopy. We successfully performed multifactorial imaging for the simultaneous detection of the Cholecystokinin-2-receptor and GLUT-1 targeted probe. Prominent differences in uptake patterns of the two contrast agents could be detected. The results were validated by histological examinations. The multifactorial imaging approach presented in this study could facilitate cancer detection in diagnostic imaging and intraoperative and endoscopic applications. Especially the dQ-MG-754 probe bears great potential for translation to clinical endoscopy imaging, because it combines specific high affinity binding with renal elimination and a favorable biodistribution

Molecular View on the iRGD Peptide Binding Mechanism: Implications for Integrin Activity and Selectivity Profiles

Receptor-selective peptides are widely used as smart carriers for specific tumor-targeted delivery. A remarkable example is the cyclic nonapeptide iRGD (CRGDKPGDC, 1) that couples intrinsic cytotoxic effects with striking tumor-homing properties. These peculiar features are based on a rather complex multistep mechanism of action, where the primary event is the recognition of RGD integrins. Despite the high number of preclinical studies and the recent success of a phase I trial for the treatment of pancreatic ductal adenocarcinoma (PDAC), there is little information available about the iRGD three-dimensional (3D) structure and integrin binding properties. Here, we re-evaluate the peptide's affinity for cancer-related integrins including not only the previously known targets alpha v beta 3 and alpha v beta 5 but also the alpha v beta 6 isoform, which is known to drive cell growth, migration, and invasion in many malignancies including PDAC. Furthermore, we use parallel tempering in the well-tempered ensemble (PT-WTE) metadynamics simulations to characterize the in-solution conformation of iRGD and extensive molecular dynamics calculations to fully investigate its binding mechanism to integrin partners. Finally, we provide clues for fine-tuning the peptide's potency and selectivity profile, which, in turn, may further improve its tumor-homing properties

Efficient treatment of breast cancer xenografts with multifunctionalized iron oxide nanoparticles combining magnetic hyperthermia and anti-cancer drug delivery

Introduction: Tumor cells can effectively be killed by heat, e.g. by using magnetic hyperthermia. The main challenge in the field, however, is the generation of therapeutic temperatures selectively in the whole tumor region. We aimed to improve magnetic hyperthermia of breast cancer by using innovative nanoparticles which display a high heating potential and are functionalized with a cell internalization and a chemotherapeutic agent to increase cell death. Methods: The superparamagnetic iron oxide nanoparticles (MF66) were electrostatically functionalized with either Nucant multivalent pseudopeptide (N6L; MF66-N6L), doxorubicin (DOX; MF66-DOX) or both (MF66-N6LDOX). Their cytotoxic potential was assessed in a breast adenocarcinoma cell line MDA-MB-231. Therapeutic efficacy was analyzed on subcutaneous MDA-MB-231 tumor bearing female athymic nude mice. Results: All nanoparticle variants showed an excellent heating potential around 500 W/g Fe in the alternating magnetic field (AMF, conditions: H = 15.4 kA/m, f = 435 kHz). We could show a gradual inter- and intracellular release of the ligands, and nanoparticle uptake in cells was increased by the N6L functionalization. MF66-DOX and MF66-N6LDOX in combination with hyperthermia were more cytotoxic to breast cancer cells than the respective free ligands. We observed a substantial tumor growth inhibition (to 40% of the initial tumor volume, complete tumor regression in many cases) after intratumoral injection of the nanoparticles in vivo. The proliferative activity of the remaining tumor tissue was distinctly reduced. Conclusion: The therapeutic effects of breast cancer magnetic hyperthermia could be strongly enhanced by the combination of MF66 functionalized with N6L and DOX and magnetic hyperthermia. Our approach combines two ways of tumor cell killing (magnetic hyperthermia and chemotherapy) and represents a straightforward strategy for translation into the clinical practice when injecting nanoparticles intratumorallyThe described work was carried out within the project, Multifunctional Nanoparticles for the Selective Detection and Treatment of Cancer (Multifun), which is funded by the European Seventh Framework Program (FP7/2007-2013) under grant agreement number 262943. We thank Dr Vijay Patel and Liquids Research Ltd (Mentec, Deiniol Road, Bangor, Gwynedd, North Wales, UK,) for the supply of MF66 MNP. We gratefully acknowledge Julia Göring and Susann Burgold for technical assistance in carrying out in vivo experiments and Yvonne Ozegowski for animal handling. We thank Francisco J. Teran (Unidad Asociada de Nanobiotecnología CNB-CSIC & IMDEA Nanociencia, Madrid) for helpful discussions. AS and ALC acknowledge financial support from Ministerio de Economia y Competitividad (grants: SAF-15440 and BIO2012-34835) and IMDEA Nanociencia. This work was partially founded by the Comunidad de Madrid NANOFRONTMAG-CM project (S2013/MIT-2850) (IMDEA-Nanociencia)

An 89Zr-HDL PET Tracer Monitors Response to a CSF1R Inhibitor

The immune function within the tumor microenvironment has become a prominent therapeutic target, with tumor-associated macrophages (TAMs) playing a critical role in immune suppression. We propose an 89Zr-labeled high-density lipoprotein (89Zr-HDL) nanotracer as a means of monitoring response to immunotherapy. Methods: Female MMTV-PyMT mice were treated with pexidartinib, a colony-stimulating factor 1 receptor (CSF1R) inhibitor, to reduce TAM density. The accumulation of 89Zr-HDL within the tumor was assessed using PET/CT imaging and autoradiography, whereas TAM burden was determined using immunofluorescence. Results: A significant reduction in 89Zr-HDL accumulation was observed in PET/CT images, with 2.9% ± 0.3% and 3.7% ± 0.2% injected dose/g for the pexidartinib- and vehicle-treated mice, respectively. This reduction was corroborated ex vivo and correlated with decreased TAM density. Conclusion: These results support the potential use of 89Zr-HDL nanoparticles as a PET tracer to quickly monitor the response to CSF1R inhibitors and other therapeutic strategies targeting TAMs.We thank the Small Animal Imaging Core, the Radiochemistry and Molecular Imaging Probes Core, and the Molecular Cytology Core at Memorial Sloan Kettering Cancer Center. This work was supported by National Institutes of Health grants R01 CA204441, P30 CA008748 and R01 CA220234. The authors thank the Tow Foundation and Memorial Sloan Kettering Cancer Center's Center for Molecular Imaging & Nanotechnology (CMINT), the Imaging and Radiation Sciences Program and the MSK Molecularly Targeted Intraoperative Imaging Fund.S

Acid specific dark quencher QC1 pHLIP for multi-spectral optoacoustic diagnoses of breast cancer

Breast cancer is the most common type of malignant growth in women. Early detection of breast cancer, as well as the identification of possible metastatic spread poses a significant challenge because of the structural and genetic heterogeneity that occurs during the progression of the disease. Currently, mammographies, biopsies and MRI scans are the standard of care techniques used for breast cancer diagnosis, all of which have their individual shortfalls, especially when it comes to discriminating tumors and benign growths. With this in mind, we have developed a non-invasive optoacoustic imaging strategy that targets the acidic environment of breast cancer. A pH low insertion peptide (pHLIP) was conjugated to the dark quencher QC1, yielding a non-fluorescent sonophore with high extinction coefficient in the near infrared that increases signal as a function of increasing amounts of membrane insertion. In an orthotopic murine breast cancer model, pHLIP-targeted optoacoustic imaging allowed us to differentiate between healthy and breast cancer tissues with high signal/noise ratios. In vivo, the sonophore QC1-pHLIP could detect malignancies at higher contrast than its fluorescent analog ICG-pHLIP, which was developed for fluorescence-guided surgical applications. PHLIP-type optoacoustic imaging agents in clinical settings are attractive due to their ability to target breast cancer and a wide variety of other malignant growths for diagnostic purposes. Intuitively, these agents could also be used for visualization during surgery

The OTUD6B-LIN28B-MYC axis determines the proliferative state in multiple myeloma

Deubiquitylases (DUBs) are therapeutically amenable components of the ubiquitin machinery that stabilize substrate proteins. Their inhibition can destabilize oncoproteins that may otherwise be undruggable. Here, we screened for DUB vulnerabilities in multiple myeloma, an incurable malignancy with dependency on the ubiquitin proteasome system and identified OTUD6B as an oncogene that drives the G1/S-transition. LIN28B, a suppressor of microRNA biogenesis, is specified as a bona fide cell cycle-specific substrate of OTUD6B. Stabilization of LIN28B drives MYC expression at G1/S, which in turn allows for rapid S-phase entry. Silencing OTUD6B or LIN28B inhibits multiple myeloma outgrowth in vivo and high OTUD6B expression evolves in patients that progress to symptomatic multiple myeloma and results in an adverse outcome of the disease. Thus, we link proteolytic ubiquitylation with post-transcriptional regulation and nominate OTUD6B as a potential mediator of the MGUS-multiple myeloma transition, a central regulator of MYC, and an actionable vulnerability in multiple myeloma and other tumors with an activated OTUD6B-LIN28B axis.</p

Discriminating radiation injury from recurrent tumor with [18F]PARPi and amino acid PET in mouse models

Background Radiation injury can be indistinguishable from recurrent tumor on standard imaging. Current protocols for this differential diagnosis require one or more follow-up imaging studies, long dynamic acquisitions, or complex image post-processing; despite much research, the inability to confidently distinguish between these two entities continues to pose a significant dilemma for the treating clinician. Using mouse models of both glioblastoma and radiation necrosis, we tested the potential of poly(ADP-ribose) polymerase (PARP)-targeted PET imaging with [18F]PARPi to better discriminate radiation injury from tumor. Results In mice with experimental radiation necrosis, lesion uptake on [18F]PARPi-PET was similar to contralateral uptake (1.02 ± 0.26 lesion/contralateral %IA/ccmax ratio), while [18F]FET-PET clearly delineated the contrast-enhancing region on MR (2.12 ± 0.16 lesion/contralateral %IA/ccmax ratio). In mice with focal intracranial U251 xenografts, tumor visualization on PARPi-PET was superior to FET-PET, and lesion-to-contralateral activity ratios (max/max, p = 0.034) were higher on PARPi-PET than on FET-PET. Conclusions A murine model of radiation necrosis does not demonstrate [18F]PARPi avidity, and [18F]PARPi-PET is better than [18F]FET-PET in distinguishing radiation injury from brain tumor. [18F]PARPi-PET can be used for discrimination between recurrent tumor and radiation injury within a single, static imaging session, which may be of value to resolve a common dilemma in neuro-oncology

Target engagement imaging of PARP inhibitors in small-cell lung cancer

Insufficient chemotherapy response and rapid disease progression remain concerns for smallcell lung cancer (SCLC). Oncologists rely on serial CT scanning to guide treatment decisions, but this cannot assess in vivo target engagement of therapeutic agents. Biomarker assessments in biopsy material do not assess contemporaneous target expression, intratumoral drug exposure, or drug-target engagement. Here, we report the use of PARP1/2-targeted imaging to measure target engagement of PARP inhibitors in vivo. Using a panel of clinical PARP inhibitors, we show that PARP imaging can quantify target engagement of chemically diverse small molecule inhibitors in vitro and in vivo. We measure PARP1/2 inhibition over time to calculate effective doses for individual drugs. Using patient-derived xenografts, we demonstrate that different therapeutics achieve similar integrated inhibition efficiencies under different dosing regimens. This imaging approach to non-invasive, quantitative assessment of dynamic intratumoral target inhibition may improve patient care through realtime monitoring of drug delivery

Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity in vitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.This work was funded by the American Association for Cancer Research, Lustgarten Foundation, and Stand Up to Cancer as a Pancreatic Cancer Collective New Therapies Challenge grant (grant no. SU2C-AACR-PCC-01-18)