Membrane-derived particles shed by PSMA-positive cells function as pro-angiogenic stimuli in tumors.

Cell membrane-derived particles (Mp) are rounded membrane-enclosed particles that are shed from tumor cells. Mp are formed from tumor membranes and are capable of tumor targeting and immunotherapeutic agents because they share membrane homology with parental cells; thus, they are under consideration as a drug delivery vehicle. Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein with enzymatic functionality, is highly expressed in Mp and extracellular vesicles (EV) from prostate cancer (PCa) with poor clinical prognosis. Although PSMA expression was previously shown in EV and Mp isolated from cell lines and from the blood of patients with high-grade PCa, no pathophysiological effects have been linked to PCa-derived Mp. Here, we compared Mp from PSMA-expressing (PSMA-Mp) and PSMA-non-expressing (WT-Mp) cells side by side in vitro and in vivo. PSMA-Mp can transfer PSMA and new phenotypic characteristics to the tumor microenvironment. The consequence of PSMA transfer to cells and increased secretion of vascular endothelial growth factor-A (VEGF-A), pro-angiogenic and pro-lymphangiogenic mediators, with increased 4E binding protein 1 (4EBP-1) phosphorylation.

Ambient Light Resistant Shortwave Infrared Fluorescence Imaging for Preclinical Tumor Delineation via the pH Low-Insertion Peptide Conjugated to Indocyanine Green.

Shortwave infrared (900-1,700 nm) fluorescence imaging (SWIRFI) has shown significant advantages over visible (400-650 nm) and near-infrared (700-900 nm) fluorescence imaging (reduced autofluorescence, improved contrast, tissue resolution, and depth sensitivity). However, there is a major lag in the clinical translation of preclinical SWIRFI systems and targeted SWIRFI probes. Methods: We preclinically show that the pH low-insertion peptide conjugated to indocyanine green (pHLIP ICG), currently in clinical trials, is an excellent candidate for cancer-targeted SWIRFI. Results: pHLIP ICG SWIRFI achieved picomolar sensitivity (0.4 nM) with binary and unambiguous tumor screening and resection up to 96 h after injection in an orthotopic breast cancer mouse model. SWIRFI tumor screening and resection had ambient light resistance (possible without gating or filtering) with outstanding signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) values at exposures from 10 to 0.1 ms. These SNR and CNR values were also found for the extended emission of pHLIP ICG in vivo (>1,100 nm, 300 ms). Conclusion: SWIRFI sensitivity and ambient light resistance enabled continued tracer clearance tracking with unparalleled SNR and CNR values at video rates for tumor delineation (achieving a tumor-to-muscle ratio above 20). In total, we provide a direct precedent for the democratic translation of an ambient light resistant SWIRFI and pHLIP ICG ecosystem, which can instantly improve tumor resection.

Translatable Drug-Loaded Iron Oxide Nanophore Sensitizes Murine Melanoma Tumors to Monoclonal Antibody Immunotherapy.

Macrophages comprise a significant portion of the immune cell compartment within tumors and are known contributors to tumor pathology; however, cancer immunotherapies targeting these cells are not clinically available. The iron oxide nanoparticle, ferumoxytol (FH), may be utilized as a nanophore for drug delivery to tumor-associated macrophages. We have demonstrated that a vaccine adjuvant, monophosphoryl lipid A (MPLA), can be stably captured within the carbohydrate shell of ferumoxytol without chemical modification of either the drug or the nanophore. This drug-nanoparticle combination (FH-MPLA) activated macrophages to an antitumorigenic phenotype at clinically relevant concentrations. In the immunotherapy-resistant B16-F10 model of murine melanoma, FH-MPLA treatment induced tumor necrosis and regression in combination with agonistic α-CD40 monoclonal antibody therapy. FH-MPLA, composed of clinically approved nanoparticle and drug payload, represents a potential cancer immunotherapy with translational relevance. FH-MPLA may be useful as an adjunctive therapy to existing antibody-based cancer immunotherapies which target only lymphocytic cells, reshaping the tumor immune environment.

Detection of Shortwave-Infrared Cerenkov Luminescence from Medical Isotopes.

Medical radioisotopes produce Cerenkov luminescence (CL) from charged subatomic particles (ß+/-) traveling faster than light in dielectric media (e.g., tissue). CL is a blue-weighted and continuous emission, decreasing proportionally to increasing wavelength. CL imaging (CLI) provides an economic PET alternative with the advantage of also being able to image ß- and α emitters. Like any optical modality, CLI is limited by the optical properties of tissue (scattering, absorption, and ambient photon removal). Shortwave-infrared (SWIR, 900-1700 nm) CL has been detected from MeV linear accelerators but not yet from keV medical radioisotopes. Methods: Indium-gallium-arsenide sensors and SWIR lenses were mounted onto an ambient light-excluding preclinical enclosure. An exposure and processing pipeline was developed for SWIR CLI and then performed across 6 radioisotopes at in vitro and in vivo conditions. Results: SWIR CL was detected from the clinical radioisotopes 90Y, 68Ga, 18F, 89Zr, 131I, and 32P (biomedical research). SWIR CLI's advantage over visible-wavelength (VIS) CLI (400-900 nm) was shown via increased light penetration and decreased scattering at depth. The SWIR CLI radioisotope sensitivity limit (8.51 kBq/µL for 68Ga), emission spectrum, and ex vivo and in vivo examples are reported. Conclusion: This work shows that radioisotope SWIR CLI can be performed with unmodified commercially available components. SWIR CLI has significant advantages over VIS CLI, with preserved VIS CLI features such as radioisotope radiance levels and dose response linearity. Further improvements in SWIR optics and technology are required to enable widespread adoption.

Visualizing surface marker expression and intratumoral heterogeneity with SERRS-NPs imaging.

Cell surface marker expression in tumors dictates the selection of therapeutics, therapy response, and survival. However, biopsies are invasive, sample only a small area of the tumor landscape and may miss significant areas of heterogeneous expression. Here, we investigated the potential of antibody-conjugated surface-enhanced resonance Raman scattering nanoparticles (SERRS-NPs) to depict and quantify high and low tumoral surface marker expression, focusing on the surface markers epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in an intracerebral and peripheral setting with an inter- and intratumoral comparison of Raman signal intensities. Methods: ICR-Prkdc mice were injected with glioblastoma, epidermoid carcinoma, or breast tumor cell lines intracerebrally and peripherally. SERRS-NPs were functionalized with cetuximab or trastuzumab and administered via tail vein injection. Raman imaging was performed 18 hours post-injection in excised tumors and in vivo through the skull. Tumors were then fixed and processed for immunohistochemical evaluation. Results: Confirmed by MRI and immunohistochemistry for EGFR and HER2, our results demonstrate that antibody-conjugated SERRS-NPs go beyond the delineation of a tumor and offer clear and distinct Raman spectra that reflect the distribution of the targeted surface marker. The intensity of the SERRS-NP signal accurately discriminated high- versus low-expressing surface markers between tumors, and between different areas within tumors. Conclusion: Biopsies can be highly invasive procedures and provide a limited sample of molecular expression within a tumor. Our nanoparticle-based Raman imaging approach offers the potential to provide non-invasive and more comprehensive molecular imaging and an alternative to the current clinical gold standard of immunohistochemistry.

Ultrasmall Downconverting Nanoparticle for Enhanced Cerenkov Imaging.

Cerenkov imaging provides an opportunity to expand the application of approved radiotracers and therapeutic agents by utilizing them for optical approaches, which opens new avenues for nuclear imaging. The dominating Cerenkov radiation is in the UV/blue region, where it is readily absorbed by human tissue, reducing its utility in vivo. To solve this problem, we propose a strategy to shift Cerenkov light to the more penetrative red-light region through the use of a fluorescent down-conversion technique, based upon europium oxide nanoparticles. We synthesized square-shape ultrasmall Eu2O3 nanoparticles, functionalized with polyethylene glycol and chelate-free radiolabeled for intravenous injection into mice to visualize the lymph node and tumor. By adding trimethylamine N-oxide during the synthesis, we significantly increased the brightness of the particle and synthesized the (to-date) smallest radiolabeled europium-based nanoparticle. These features allow for the exploration of Eu2O3 nanoparticles as a preclinical cancer diagnosis platform with multimodal imaging capability.

Gold/alpha-lactalbumin nanoprobes for the imaging and treatment of breast cancer.

Theranostic agents should ideally be renally cleared and biodegradable. Here, we report the synthesis, characterization and theranostic applications of fluorescent ultrasmall gold quantum clusters that are stabilized by the milk metalloprotein alpha-lactalbumin. We synthesized three types of these nanoprobes that together display fluorescence across the visible and near-infrared spectra when excited at a single wavelength through optical colour coding. In live tumour-bearing mice, the near-infrared nanoprobe generates contrast for fluorescence, X-ray computed tomography and magnetic resonance imaging, and exhibits long circulation times, low accumulation in the reticuloendothelial system, sustained tumour retention, insignificant toxicity and renal clearance. An intravenously administrated near-infrared nanoprobe with a large Stokes shift facilitated the detection and image-guided resection of breast tumours in vivo using a smartphone with modified optics. Moreover, the partially unfolded structure of alpha-lactalbumin in the nanoprobe helps with the formation of an anti-cancer lipoprotein complex with oleic acid that triggers the inhibition of the MAPK and PI3K-AKT pathways, immunogenic cell death and the recruitment of infiltrating macrophages. The biodegradability and safety profile of the nanoprobes make them suitable for the systemic detection and localized treatment of cancer.

High-resolution optoacoustic imaging of tissue responses to vascular-targeted therapies.

The monitoring of vascular-targeted therapies using magnetic resonance imaging, computed tomography or ultrasound is limited by their insufficient spatial resolution. Here, by taking advantage of the intrinsic optical properties of haemoglobin, we show that raster-scanning optoacoustic mesoscopy (RSOM) provides high-resolution images of the tumour vasculature and of the surrounding tissue, and that the detection of a wide range of ultrasound bandwidths enables the distinction of vessels of differing size, providing detailed insights into the vascular responses to vascular-targeted therapy. Using RSOM to examine the responses to vascular-targeted photodynamic therapy in mice with subcutaneous xenografts, we observed a substantial and immediate occlusion of the tumour vessels followed by haemorrhage within the tissue and the eventual collapse of the entire vasculature. Using dual-wavelength RSOM, which distinguishes oxyhaemoglobin from deoxyhaemoglobin, we observed an increase in oxygenation of the entire tumour volume immediately after the application of the therapy, and a second wave of oxygen reperfusion approximately 24 h thereafter. We also show that RSOM enables the quantification of differences in neoangiogenesis that predict treatment efficacy.

Cranberry A-type proanthocyanidins selectively target acute myeloid leukemia cells.

Most elderly patients affected with acute myeloid leukemia (AML) will relapse and die of their disease even after achieving complete remission, thus emphasizing the urgent need for new therapeutic approaches with minimum toxicity to normal hematopoietic cells. Cranberry (Vaccinium spp.) extracts have exhibited anticancer and chemopreventive properties that have been mostly attributed to A-type proanthocyanidin (A-PAC) compounds. A-PACs, isolated from a commercially available cranberry extract, were evaluated for their effects on leukemia cell lines, primary AML samples, and normal CD34+ cord blood specimens. Our results indicated potent and specific antileukemia activity in vitro. In addition, the antileukemia activity of A-PACs extended to malignant progenitor and stem cell populations, sparing their normal counterparts. The antileukemia effects of A-PACs were also observed in vivo using patient derived xenografts. Surprisingly, we found that the mechanism of cell death was driven by activation of NF-κB. Overall, our data suggest that A-PACs could be used to improve treatments for AML by targeting leukemia stem cells through a potentially novel pathway.

Non-invasive In Vivo Imaging of Cancer Using Surface-Enhanced Spatially Offset Raman Spectroscopy (SESORS).

Rationale: The goal of imaging tumors at depth with high sensitivity and specificity represents a significant challenge in the field of biomedical optical imaging. 'Surface enhanced Raman scattering' (SERS) nanoparticles (NPs) have been employed as image contrast agents and can be used to specifically target cells in vivo. By tracking their unique "fingerprint" spectra, it becomes possible to determine their precise location. However, while the detection of SERS NPs is very sensitive and specific, conventional Raman spectroscopy imaging devices are limited in their inability to probe through tissue depths of more than a few millimetres, due to scattering and absorption of photons by biological tissues. Here, we combine the use of "Spatially Offset Raman spectroscopy" (SORS) with that of "surface-enhanced resonance Raman spectroscopy" (SERRS) in a technique known as "surface enhanced spatially offset resonance Raman spectroscopy" (SESO(R)RS) to image deep-seated glioblastoma multiforme (GBM) tumors in vivo in mice through the intact skull. Methods: A SORS imaging system was built in-house. Proof of concept SORS imaging was achieved using a PTFE-skull-tissue phantom. Imaging of GBMs in the RCAS-PDGF/N-tva transgenic mouse model was achieved through the use of gold nanostars functionalized with a resonant Raman reporter to create SERRS nanostars. These were then encapsulated in a thin silica shell and functionalized with a cyclic-RGDyK peptide to yield integrin-targeting SERRS nanostars. Non-invasive in vivo SORS image acquisition of the integrin-targeted nanostars was then performed in living mice under general anesthesia. Conventional non-SORS imaging was used as a direct comparison. Results: Using a low power density laser, GBMs were imaged via SESORRS in mice (n = 5) and confirmed using MRI and histopathology. The results demonstrate that via utilization of the SORS approach, it is possible to acquire clear and distinct Raman spectra from deep-seated GBMs in mice in vivo through the skull. SESORRS images generated using classical least squares outlined the tumors with high precision as confirmed via MRI and histology. Unlike SESORRS, conventional Raman imaging of the same areas did not provide a clear delineation of the tumor. Conclusion: To the best of our knowledge this is the first report of in vivo SESO(R)RS imaging. In a relevant brain tumor mouse model we demonstrate that this technique can overcome the limitations of conventional Raman imaging with regards to penetration depth. This work therefore represents a significant step forward in the potential clinical translation of SERRS nanoparticles for high precision cancer imaging.

DNA-enabled rational design of fluorescence-Raman bimodal nanoprobes for cancer imaging and therapy.

Recently, surface-enhanced Raman scattering nanoprobes have shown tremendous potential in oncological imaging owing to the high sensitivity and specificity of their fingerprint-like spectra. As current Raman scanners rely on a slow, point-by-point spectrum acquisition, there is an unmet need for faster imaging to cover a clinically relevant area in real-time. Herein, we report the rational design and optimization of fluorescence-Raman bimodal nanoparticles (FRNPs) that synergistically combine the specificity of Raman spectroscopy with the versatility and speed of fluorescence imaging. DNA-enabled molecular engineering allows the rational design of FRNPs with a detection limit as low as 5 × 10-15 M. FRNPs selectively accumulate in tumor tissue mouse cancer models and enable real-time fluorescence imaging for tumor detection, resection, and subsequent Raman-based verification of clean margins. Furthermore, FRNPs enable highly efficient image-guided photothermal ablation of tumors, widening the scope of the NPs into the therapeutic realm.

Phase I trial of plerixafor combined with decitabine in newly diagnosed older patients with acute myeloid leukemia.

Acute myeloid leukemia carries a dismal prognosis in older patients. The objective of this study was to investigate the safety and efficacy of decitabine combined with the CXCR4 antagonist plerixafor in newly diagnosed older patients with acute myeloid leukemia and to evaluate the effects of plerixafor on leukemia stem cells. Patients were treated with monthly cycles of decitabine 20 mg/m2 days 1-10 and escalating doses of plerixafor (320-810 mcg/kg) days 1-5. Sixty-nine patients were treated, with an overall response rate of 43%. Adverse karyotype did not predict response (P=0.31). Prior hypomethylating agent treatment was the strongest independent predictor of adverse overall survival (hazard ratio 3.1; 95%CI: 1.3-7.3; P=0.008) and response (14% in previously treated patients, 46% in treatment naïve; P=0.002). As expected, the most common toxicities were myelosuppression and infection. Plerixafor induced mobilization of leukemia stem and progenitor cells, but did not cause clinically significant hyperleukocytosis. Reduction in leukemia stem cells appeared to correlate with duration of response. Plerixafor can be safely added to decitabine in poor-prognosis, elderly acute myeloid leukemia patients. The maximum tolerated dose of the combination was 810 mcg/kg. While mobilization of leukemia stem cells was observed in some patients, the clinical benefit of adding plerixafor was uncertain. This trial was registered at clinicaltrials.gov identifier: 01352650.

Dual-Modality Surface-Enhanced Resonance Raman Scattering and Multispectral Optoacoustic Tomography Nanoparticle Approach for Brain Tumor Delineation.

Difficulty in visualizing glioma margins intraoperatively remains a major issue in the achievement of gross total tumor resection and, thus, better clinical outcome of glioblastoma (GBM) patients. Here, the potential of a new combined optical + optoacoustic imaging method for intraoperative brain tumor delineation is investigated. A strategy using a newly developed gold nanostar synthesis method, Raman reporter chemistry, and silication method to produce dual-modality contrast agents for combined surface-enhanced resonance Raman scattering (SERRS) and multispectral optoacoustic tomography (MSOT) imaging is devised. Following intravenous injection of the SERRS-MSOT-nanostars in brain tumor bearing mice, sequential MSOT imaging is performed in vivo and followed by Raman imaging. MSOT is able to accurately depict GBMs three-dimensionally with high specificity. The MSOT signal is found to correlate well with the SERRS images. Because SERRS enables uniquely sensitive high-resolution surface detection, it could represent an ideal complementary imaging modality to MSOT, which enables real-time, deep tissue imaging in 3D. This dual-modality SERRS-MSOT-nanostar contrast agent reported here is shown to enable high precision depiction of the extent of infiltrating GBMs by Raman- and MSOT imaging in a clinically relevant murine GBM model and could pave new ways for improved image-guided resection of brain tumors.

MUC1 Aptamer Targeted SERS Nanoprobes.

Recently, surface-enhanced Raman scattering (SERS) nanoprobes (NPs) have shown promise in the field of cancer imaging due to their unparalleled signal specificity and high sensitivity. Here we report the development of a DNA aptamer targeted SERS NP. Recently, aptamers are being investigated as a viable alternative to more traditional antibody targeting due to their low immunogenicity and low cost of production. We developed a strategy to functionalize SERS NPs with DNA aptamers, which target Mucin1 (MUC1) in human breast cancer (BC). Thorough in vitro characterization studies demonstrated excellent serum stability and specific binding of the targeted NPs to MUC1. In order to test their in vivo targeting capability, we co-injected MUC1-targeted SERS NPs, and as controls non-targeted and blocked MUC1-targeted SERS NPs in BC xenograft mouse models. A two-tumor mouse model with differential expression of MUC1 (MDA-MB-468 and MDA-MB-453) was used to control for active versus passive targeting in the same animals. The results showed that the targeted SERS NPs home to the tumors via active targeting of MUC1, with low levels of passive targeting. We expect this strategy to be an advantageous alternative to antibody-based targeting and useful for targeted imaging of tumor extent, progression, and therapeutic response.

Galectin-3 modulates the EGFR signalling-mediated regulation of Sox2 expression via c-Myc in lung cancer.

Galectin-3 is a ubiquitous lectin exerting multiple cellular functions such as RNA splicing, protein trafficking and apoptosis. Its expression is positively correlated with the poor prognosis in lung cancer patients. Galectin-3 can promote cancer progression through its effects on cell proliferation, cell survival or cancer metastasis. However, the role of galectin-3 in the regulation of cancer stem-like cells (CSCs) is still unclear. Here, we investigated the hypothesis that galectin-3 might regulate lung CSCs via the EGF receptor (EGFR) signaling pathway. In our study, galectin-3 facilitated EGFR activation and enhanced the sphere formation activity of lung cancer cells. Furthermore, galectin-3 promoted Sox2 expression in an EGFR activation-dependent manner; importantly, forced expression of Sox2 blunted the effect of galectin-3 knockdown on lung cancer sphere formation ability. These results suggest that galectin-3 promotes EGFR activation leading to the upregulation of Sox2 expression and lung CSCs properties. Moreover, we showed that the carbohydrate-binding activity of galectin-3 was important for the regulation of EGFR activation, Sox2 expression and sphere formation. We have recently reported that c-Myc is a transcriptional activator of Sox2. We further found that galectin-3 enhanced c-Myc protein stability leading to increased c-Myc binding to the Sox2 gene promoter. We also examined the effect of the stemness factors, Oct4, Nanog and Sox2 on the expression of galectin-3. We found that Oct4 enhanced galectin-3 expression. Our results together suggest that galectin-3 enhances lung cancer stemness through the EGFR/c-Myc/Sox2 axis; Oct4, in turn, promotes galectin-3 expression, forming a positive regulatory loop in lung CSCs.

External vibration enhances macromolecular crowding for construction of aligned three-dimensional collagen fibril scaffolds.

There are many techniques for preparing two-dimensional aligned fibril matrices. However, the critical problem associated with these techniques is the destruction of the native structure (e.g., the α-helix) of the proteins. Moreover, most of these techniques cannot create a three-dimensional (3D), aligned reconstituted collagen fibril matrix in one step. In this study, we used a simple device composed of a pneumatic membrane that generates a tunable vibration frequency to apply physical stimulation to fabricate a 3D, aligned collagen fibril matrix with the characteristic D-period structure of collagen in one step. Using second harmonic images, we demonstrated that the aligned, reconstituted collagen fibrils preserve the native collagen D-period structure. The average angular deviation of fibril alignment was reduced to 25.01 ± 4.2° compared with the 39.7 ± 2.19° of alignment observed for the randomly distributed fibril matrix. In addition, the ultimate tensile strength of the aligned matrix when force was applied in the direction parallel to the fiber orientation was higher than that of the randomly oriented matrix. The aligned reconstituted collagen fibril matrix also enhanced the expression of smoothelin (a specific marker of contractile phenotype) of thoracic aortic smooth muscle cell (A7r5) relative to the randomly distributed collagen fibril matrix.

Paper-based microfluidic sensing device for label-free immunoassay demonstrated by biotin-avidin binding interaction.

Efficient diagnosis is very important for the prevention and treatment of diseases. Rapid disease screening in ambulatory environment is one of the most pressing needs for disease control. Despite there are many methods to detect the results of immunoassays, quantitative measurement for rapid disease screening is still a great challenge for point-of-care applications. In this study, a fabrication method for depositing carbon nanotube bundles has been successfully developed for realization of functional paper-based microfluidic sensing device. Quantitative detection of label-free immunoassay, i.e., biotin-avidin binding interaction, was demonstrated by direct measurement of the current change of the biosensor after single application of the target analyte. Sensitivity of 0.33 µA/ng mL(-1) and minimal detectable analyte concentration of 25 ng/mL were achieved. The time necessary for the detection was 500 s which is a large reduction compared with the conventional immunoassay. Such paper-based biosensor has the benefits of portability, fast response, simple operation, and low cost and has the potential for the development of rapid disease screening devices.

Development of a paper-based carbon nanotube sensing microfluidic device for biological detection.

Carbon nanotube (CNT) has been utilized for the biological detection due to its extremely sensitive to biological molecules. A paper-based CNT sensing microfluidic device has been developed for the detection of protein, i.e., biotin-avidin, binding. We have developed a fabrication method that allows controlled deposition of bundled CNTs with well-defined dimensions to form sensors on paper. Then, polydimethyl siloxane (PDMS) was used to pattern the hydrophobic boundary on paper to form the reaction sites. The proposed fabrication method is based on vacuum filtration process with a metal mask covering on a filter paper for the definition of the dimension of sensor. The length, width, and thickness of the CNT-based sensors are readily controlled by the metal mask and the weight of the CNT powder used during the filtration process, respectively. Homogeneous deposition of CNTs with well-defined dimensions can be achieved. The CNT-based sensor on paper has been demonstrated on the detection of the protein binding. Biotin was first immobilized on the CNT's sidewall and avidin suspended solution was applied to the site. The result of the biotin-avidin binding was measured by the resistance change of the sensor, which is a label-free detection method. It showed the CNT is sensitive to the biological molecules and the proposed paper-based CNT sensing device is a possible candidate for point-of-care biosensors. Thus, electrical bio-assays on paper-based microfluidics can be realized to develop low cost, sensitive, and specific diagnostic devices.

Arp2/3 promotes junction formation and maintenance in the Caenorhabditis elegans intestine by regulating membrane association of apical proteins.

It has been proposed that Arp2/3, which promotes nucleation of branched actin, is needed for epithelial junction initiation but is less important as junctions mature. We focus here on how Arp2/3 contributes to the Caenorhabditis elegans intestinal epithelium and find important roles for Arp2/3 in the maturation and maintenance of junctions in embryos and adults. Electron microscope studies show that embryos depleted of Arp2/3 form apical actin-rich microvilli and electron-dense apical junctions. However, whereas apical/basal polarity initiates, apical maturation is defective, including decreased apical F-actin enrichment, aberrant lumen morphology, and reduced accumulation of some apical junctional proteins, including DLG-1. Depletion of Arp2/3 in adult animals leads to similar intestinal defects. The DLG-1/AJM-1 apical junction proteins, and the ezrin-radixin-moesin homologue ERM-1, a protein that connects F-actin to membranes, are required along with Arp2/3 for apical F-actin enrichment in embryos, whereas cadherin junction proteins are not. Arp2/3 affects the subcellular distribution of DLG-1 and ERM-1. Loss of Arp2/3 shifts both ERM-1 and DLG-1 from pellet fractions to supernatant fractions, suggesting a role for Arp2/3 in the distribution of membrane-associated proteins. Thus, Arp2/3 is required as junctions mature to maintain apical proteins associated with the correct membranes.