Dynamin impacts homology-directed repair and breast cancer response to chemotherapy.

After the initial responsiveness of triple-negative breast cancers (TNBCs) to chemotherapy, they often recur as chemotherapy-resistant tumors, and this has been associated with upregulated homology-directed repair (HDR). Thus, inhibitors of HDR could be a useful adjunct to chemotherapy treatment of these cancers. We performed a high-throughput chemical screen for inhibitors of HDR from which we obtained a number of hits that disrupted microtubule dynamics. We postulated that high levels of the target molecules of our screen in tumors would correlate with poor chemotherapy response. We found that inhibition or knockdown of dynamin 2 (DNM2), known for its role in endocytic cell trafficking and microtubule dynamics, impaired HDR and improved response to chemotherapy of cells and of tumors in mice. In a retrospective analysis, levels of DNM2 at the time of treatment strongly predicted chemotherapy outcome for estrogen receptor-negative and especially for TNBC patients. We propose that DNM2-associated DNA repair enzyme trafficking is important for HDR efficiency and is a powerful predictor of sensitivity to breast cancer chemotherapy and an important target for therapy.

Rare-Earth-Doped Nanoparticles for Short-Wave Infrared Fluorescence Bioimaging and Molecular Targeting of αVß3-Expressing Tumors.

The use of short-wave infrared (SWIR) light for fluorescence bioimaging offers the advantage of reduced photon scattering and improved tissue penetration compared to traditional shorter wavelength imaging approaches. While several nanomaterials have been shown capable of generating SWIR emissions, rare-earth-doped nanoparticles (REs) have emerged as an exceptionally bright and biocompatible class of SWIR emitters. Here, we demonstrate SWIR imaging of REs for several applications, including lymphatic mapping, real-time monitoring of probe biodistribution, and molecular targeting of the αvß3 integrin in a tumor model. We further quantified the resolution and depth penetration limits of SWIR light emitted by REs in a customized imaging unit engineered for SWIR imaging of live small animals. Our results indicate that SWIR light has broad utility for preclinical biomedical imaging and demonstrates the potential for molecular imaging using targeted REs.

SDF-1 Blockade Enhances Anti-VEGF Therapy of Glioblastoma and Can Be Monitored by MRI.

Despite the approval of antiangiogenic therapy for glioblastoma multiforme (GBM) patients, survival benefits are still limited. One of the resistance mechanisms for antiangiogenic therapy is the induction of hypoxia and subsequent recruitment of macrophages by stromal-derived factor (SDF)-1α (CXCL-12). In this study, we tested whether olaptesed pegol (OLA-PEG, NOX-A12), a novel SDF-1α inhibitor, could reverse the recruitment of macrophages and potentiate the antitumor effect of anti-vascular endothelial growth factor (VEGF) therapy. We also tested whether magnetic resonance imaging (MRI) with ferumoxytol as a contrast agent could provide early information on macrophage blockade. Orthotopic human G12 glioblastomas in nude mice and rat C6 glioblastomas were employed as the animal models. These were treated with bevacizumab or B-20, both anti-VEGF antibodies. Rats were MR imaged with ferumoxytol for macrophage detection. Tumor hypoxia and SDF-1α expression were elevated by VEGF blockade. Adding OLA-PEG to bevacizumab or B-20 significantly prolonged the survival of rodents bearing intracranial GBM compared with anti-VEGF therapy alone. Intratumoral CD68+ tumor associated macrophages (TAMs) were increased by VEGF blockade, but the combination of OLA-PEG + VEGF blockade markedly lowered TAM levels compared with VEGF blockade alone. MRI with ferumoxytol as a contrast agent noninvasively demonstrated macrophage reduction in OLA-PEG + anti-VEGF-treated rats compared with VEGF blockade alone. In conclusion, inhibition of SDF-1 with OLA-PEG inhibited the recruitment of TAMs by VEGF blockage and potentiated its antitumor efficacy in GBM. Noninvasive MRI with ferumoxytol as a contrast agent provides early information on the effect of OLA-PEG in reducing TAMs.

Colony stimulating factor 1 receptor inhibition delays recurrence of glioblastoma after radiation by altering myeloid cell recruitment and polarization.

BACKGROUND: Glioblastoma (GBM) may initially respond to treatment with ionizing radiation (IR), but the prognosis remains extremely poor because the tumors invariably recur. Using animal models, we previously showed that inhibiting stromal cell-derived factor 1 signaling can prevent or delay GBM recurrence by blocking IR-induced recruitment of myeloid cells, specifically monocytes that give rise to tumor-associated macrophages. The present study was aimed at determining if inhibiting colony stimulating factor 1 (CSF-1) signaling could be used as an alternative strategy to target pro-tumorigenic myeloid cells recruited to irradiated GBM. METHODS: To inhibit CSF-1 signaling in myeloid cells, we used PLX3397, a small molecule that potently inhibits the tyrosine kinase activity of the CSF-1 receptor (CSF-1R). Combined IR and PLX3397 therapy was compared with IR alone using 2 different human GBM intracranial xenograft models. RESULTS: GBM xenografts treated with IR upregulated CSF-1R ligand expression and increased the number of CD11b+ myeloid-derived cells in the tumors. Treatment with PLX3397 both depleted CD11b+ cells and potentiated the response of the intracranial tumors to IR. Median survival was significantly longer for mice receiving combined therapy versus IR alone. Analysis of myeloid cell differentiation markers indicated that CSF-1R inhibition prevented IR-recruited monocyte cells from differentiating into immunosuppressive, pro-angiogenic tumor-associated macrophages. CONCLUSION: CSF-1R inhibition may be a promising strategy to improve GBM response to radiotherapy.

Tumor-specific targeting by Bavituximab, a phosphatidylserine-targeting monoclonal antibody with vascular targeting and immune modulating properties, in lung cancer xenografts.

Bavituximab is a chimeric monoclonal antibody with immune modulating and tumor-associated vascular disrupting properties demonstrated in models of non-small cell lung cancer (NSCLC). The molecular target of Bavituximab, phosphatidylserine (PS), is exposed on the outer leaflet of the membrane bi-layer of malignant vascular endothelial cells and tumor cells to a greater extent than on normal tissues. We evaluated the tumor-targeting properties of Bavituximab for imaging of NSCLC xenografts when radiolabeled with (111)In through conjugation with a bifunctional chelating agent, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In vitro binding of (111)In-DOTA-Bavituximab to PS was determined by enzyme-linked immunosorbent assay (ELISA). Biodistribution of (111)In-DOTA-Bavituximab was conducted in normal rats, which provided data for dosimetry calculation. Single-photon emission computed tomography/computed tomography (SPECT/CT) imaging was performed in athymic nude rats bearing A549 NSCLC xenografts. At the molar conjugation ratio of 0.54 DOTA per Bavituximab, the PS binding affinity of (111)In-DOTA-Bavituximab was comparable to that of unmodified Bavituximab. Based on the quantitative SPECT/CT imaging data analysis, (111)In-DOTA-Bavituximab demonstrated tumor-specific uptake as measured by the tumor-tomuscle ratio, which peaked at 5.2 at 72 hr post-injection. In contrast, the control antibody only presented a contrast of 1.2 at the same time point.These findings may underlie the diagnostic efficacy and relative low rates of systemic vascular and immune-related toxicities of this immunoconjugate. Future applications of (111)In-DOTA-bavituximab may include prediction of efficacy, indication of tumor immunologic status, or characterization of radiographic findings.

Efficient Radioisotope Energy Transfer by Gold Nanoclusters for Molecular Imaging.

Beta-emitting isotopes Fluorine-18 and Yttrium-90 are tested for their potential to stimulate gold nanoclusters conjugated with blood serum proteins (AuNCs). AuNCs excited by either medical radioisotope are found to be highly effective ionizing radiation energy transfer mediators, suitable for in vivo optical imaging. AuNCs synthesized with protein templates convert beta-decaying radioisotope energy into tissue-penetrating optical signals between 620 and 800 nm. Optical signals are not detected from AuNCs incubated with Technetium-99m, a pure gamma emitter that is used as a control. Optical emission from AuNCs is not proportional to Cerenkov radiation, indicating that the energy transfer between the radionuclide and AuNC is only partially mediated by Cerenkov photons. A direct Coulombic interaction is proposed as a novel and significant mechanism of energy transfer between decaying radionuclides and AuNCs.

Phosphatidylserine-targeted molecular imaging of tumor vasculature by magnetic resonance imaging.

Phosphatidylserine (PS), normally restricted to the inner leaflet of the plasma membrane, becomes exposed on the outer surface of viable endothelial cells in tumor vasculature, but not in normal blood vessels. In the present study, we report the use of PGN635, a novel human monoclonal antibody that specifically targets PS, for in vivo molecular MRI of tumor vasculature. The F(ab')2 fragments of PGN635 were conjugated to polyethylene glycol (PEG) coated iron oxide nanoparticles (IO). Targeting specificity of the PS-targeted Nanoprobe, IO-PGN635F(ab')2 was first confirmed by in vitro MRI and histological staining. In vivo longitudinal MRI was then performed before and after i.v. injection of IO-PGN635F(ab')2 into mice bearing 4T1 breast tumors. T2-weighted MR images at 9.4 T revealed inhomogeneous signal loss in tumor as early as 2 h post injection. Furthermore, ionizing radiation induced a significant increase in PS exposure on tumor vascular endothelial cells, resulting in significantly enhanced and sustained tumor contrast (p < 0.05). Spatially heterogeneous MRI contrast correlated well with histological staining of tumor vascular endothelium. Our studies suggest that PS exposed within the lumen of tumor vasculature is a highly specific and useful biomarker for targeted MRI contrast agents.

Highly specific PET imaging of prostate tumors in mice with an iodine-124-labeled antibody fragment that targets phosphatidylserine.

Phosphatidylserine (PS) is an attractive target for imaging agents that identify tumors and assess their response to therapy. PS is absent from the surface of most cell types, but becomes exposed on tumor cells and tumor vasculature in response to oxidative stresses in the tumor microenvironment and increases in response to therapy. To image exposed PS, we used a fully human PS-targeting antibody fragment, PGN635 F(ab')2, that binds to complexes of PS and ß2-glycoprotein I. PGN635 F(ab')2 was labeled with the positron-emitting isotope iodine-124 ((124)I) and the resulting probe was injected into nude mice bearing subcutaneous or orthotopic human PC3 prostate tumors. Biodistribution studies showed that (124)I-PGN635 F(ab')2 localized with remarkable specificity to the tumors with little uptake in other organs, including the liver and kidneys. Clear delineation of the tumors was achieved by PET 48 hours after injection. Radiation of the tumors with 15 Gy or systemic treatment of the mice with 10 mg/kg docetaxel increased localization in the tumors. Tumor-to-normal (T/N) ratios were inversely correlated with tumor growth measured over 28 days. These data indicate that (124)I-PGN635 F(ab')2 is a promising new imaging agent for predicting tumor response to therapy.

Near-infrared Optical Imaging of Exposed Phosphatidylserine in a Mouse Glioma Model.

Phosphatidylserine (PS) is normally intracellular but becomes exposed on the luminal surface of vascular endothelial cells in tumors. It also becomes exposed on tumors cells responding to therapy. In the present study, we optically imaged exposed PS in vivo using PGN635, a novel monoclonal antibody that binds PS. The F(ab')(2) fragment of PGN635 was labeled with the near-infrared (NIR) dye, IRDye800CW. In vivo dynamic NIR imaging was performed after injection of 800CW-PGN635 into mice bearing radiation-treated or untreated U87 glioma xenografts growing subcutaneously or orthotopically. NIR optical imaging revealed a clear tumor contrast in nonirradiated subcutaneous U87 gliomas after injection of 800CW-PGN635. The tumor contrast was visible as early as 4 hours later and was maximal 24 hours later (tumor-to-normal tissue ratio [TNR] = 2.8 ± 0.7). Irradiation enhanced the tumor contrast at 24 hours (TNR = 4.0 ± 0.3). Similar results were observed for orthotopic gliomas. Localization of 800CW-PGN635 to tumors was antigen specific because 800CW-Aurexis, a control probe of irrelevant specificity, did not localize to the tumors, and preadministration of unlabeled PGN635 blocked the uptake of 800CW-PGN635. Fluorescence microscopy confirmed that 800CW-PGN635 was binding to PS-positive vascular endothelial cells in nonirradiated gliomas. Irradiation of the gliomas increased PS exposure on both tumor vascular endothelial cells and tumor cells and gave rise to an increase in tumor contrast with 800CW-PGN635 that was predictive of the reduction in tumor growth. 800CW-PGN635 may be a useful new imaging probe for detection of exposed PS in tumors responding to therapy.

Increased exposure of phosphatidylethanolamine on the surface of tumor vascular endothelium.

We have previously shown that oxidative stress within the tumor microenvironment causes phosphatidylserine (PS) to redistribute from the inner to the outer membrane leaflet of the endothelial cells (EC) creating a highly specific marker for the tumor vasculature. Because the distribution of phosphatidylethanolamine (PE) and PS within the membrane is coregulated, we reasoned that PE would also be localized in the outer membrane leaflet of tumor EC. To demonstrate this, the PE-binding peptide duramycin was biotinylated and used to determine the distribution of PE on EC in vitro and in vivo. Exposure of cultured EC to hypoxia, acidity, reactive oxygen species, or irradiation resulted in the formation of membrane blebs that were intensely PE-positive. When biotinylated duramycin was intravenously injected into tumor-bearing mice, it preferentially localized to the luminal surface of the vascular endothelium. Depending on tumor type, 13% to 56% of the tumor vessels stained positive for PE. PE-positive vessels were observed in and around hypoxic regions of the tumor. With the exception of intertubular vessels of the kidney, normal vessels remained unstained. To test the potential of PE as a biomarker for imaging, duramycin was conjugated to the near-infrared fluorophore 800CW and used for optical imaging of RM-9 prostate carcinomas. The near-infrared probe was easily detected within tumors in live animals. These results show that PE, like PS, becomes exposed on tumor vascular endothelium of multiple types of tumors and holds promise as a biomarker for noninvasive imaging and drug targeting.