Single institution study of the immune landscape for canine oral melanoma based on transcriptome analysis of the primary tumor.

Introduction: Understanding a tumor's immune context is paramount in the fight against cancer. Oral melanoma in dogs serves as an excellent translational model for human immunotherapy. However, additional study is necessary to comprehend the immune landscape of dog oral melanomas, including their similarity to human melanomas in this context. Methods: This retrospective study utilizes formalin-fixed paraffin-embedded (FFPE) tissue samples to analyze RNA sequences associated with oral melanoma in dogs. Nanostring Technologies was used for conducting RNA sequencing. The focus is on understanding the differences between melanoma tumors restricted to the oral cavity (OL) and the same primary oral tumors with a history of metastasis to the lymph nodes or other organs (OM). Normal buccal mucosa samples are also included as a normal tissue reference. Results: In the OM patient group, gene signatures exhibit significant changes relative to the OL patient group, including significantly decreased expression of S100, BRAF, CEACAM1, BCL2, ANXA1, and tumor suppressor genes (TP63). Relative to the OL tumors, the OM tumors had significantly increased expression of hypoxia-related genes (VEGFA expression), cell mobility genes (MCAM), and PTGS2 (COX2). The analysis of the immune landscape in the OM group indicates a shift from a possible "hot" tumor suppressed by immune checkpoints (PDL1) to significantly heightened expression not only of those checkpoints but also the inclusion of other immune blockades such as PD1 and IDO2. In addition, the OM group had significantly reduced expression of Toll-like receptors (TLR4) and IL-18 relative to the OL group, contributing to the tumor's immune escape. Additionally, signs of immune cell exhaustion are evident in both the OM and OL groups through significantly increased expression of TIGIT relative to normal tissue. Both the OM and OL groups had significantly increased expression of the immune cell marker CD4 expression relative to normal tissue. Further, CD4 expression significantly decreased in OM relative to OL; however, this study cannot determine the specific cell types expressing CD4 in OM and OL tumors. Discussion: This preliminary study reports significant changes in gene expression for oral melanoma between canine patients with localized disease relative to those with metastatic disease. In the future, a more in-depth investigation involving immunohistochemistry analysis and single-cell RNA expression is necessary to confirm our findings.

A single local delivery of paclitaxel and nucleic acids via an immunoactive polymer eliminates tumors and induces antitumor immunity.

Despite recent advances in cancer therapy, hard-to-reach, unidentified tumors remain a significant clinical challenge. A promising approach is to treat locatable and accessible tumors locally and stimulate antitumor immunity in situ to exert systemic effects against distant tumors. We hypothesize that a carrier of immunotherapeutics can play a critical role in activating antitumor immunity as an immunoadjuvant and a local retainer of drug combinations. Here, we develop a polyethyleneimine-lithocholic acid conjugate (2E'), which forms a hydrophobic core and cationic surface to codeliver hydrophobic small molecules and anionic nucleic acids and activates antigen-presenting cells via the intrinsic activities of 2E' components. 2E' delivers paclitaxel and small-interfering RNA (siRNA) targeting PD-L1 (or cyclic dinucleotide, [CDN]) to induce the immunogenic death of tumor cells and maintain the immunoactive tumor microenvironment, and further activates dendritic cells and macrophages, leveraging the activities of loaded drugs. A single local administration of 2E' or its combination with paclitaxel and PD-L1­targeting siRNA or CDN induces strong antitumor immunity, resulting in immediate regression of large established tumors, tumor-free survival, an abscopal effect on distant tumors, and resistance to rechallenge and metastasis in multiple models of murine tumors, including CT26 colon carcinoma, B16F10 melanoma, and 4T1 breast cancer. This study supports the finding that local administration of immunotherapeutics, when accompanied by the rationally designed carrier, can effectively protect the host from distant and recurrent diseases.

Development of hot-melt extruded drug/polymer matrices for sustained delivery of meloxicam.

For effective resolution of regional subacute inflammation and prevention of biofouling formation, we have developed a polymeric implant that can release meloxicam, a selective cyclooxygenase (COX)-2 inhibitor, in a sustained manner. Meloxicam-loaded polymer matrices were produced by hot-melt extrusion, with commercially available biocompatible polymers, poly(ε-caprolactone) (PCL), poly(lactide-co-glycolide) (PLGA), and poly(ethylene vinyl acetate) (EVA). PLGA and EVA had a limited control over the drug release rate partly due to the acidic microenvironment and hydrophobicity, respectively. PCL allowed for sustained release of meloxicam over two weeks and was used as a carrier of meloxicam. Solid-state and image analyses indicated that the PCL matrices encapsulated meloxicam in crystalline clusters, which dissolved in aqueous medium and generated pores for subsequent drug release. The subcutaneously implanted meloxicam-loaded PCL matrices in rats showed pharmacokinetic profiles consistent with their in vitro release kinetics, where higher drug loading led to faster drug release. This study finds that the choice of polymer platform is crucial to continuous release of meloxicam and the drug release rate can be controlled by the amount of drug loaded in the polymer matrices.

Nanocapsules modify membrane interaction of polymyxin B to enable safe systemic therapy of Gram-negative sepsis.

Systemic therapy of Gram-negative sepsis remains challenging. Polymyxin B (PMB) is well suited for sepsis therapy due to the endotoxin affinity and antibacterial activity. However, the dose-limiting toxicity has limited its systemic use in sepsis patients. For safe systemic use of PMB, we have developed a nanoparticulate system, called D-TZP, which selectively reduces the toxicity to mammalian cells but retains the therapeutic activities of PMB. D-TZP consists of an iron-complexed tannic acid nanocapsule containing a vitamin D core, coated with PMB and a chitosan derivative that controls the interaction of PMB with endotoxin, bacteria, and host cells. D-TZP attenuated the membrane toxicity associated with PMB but retained the ability of PMB to inactivate endotoxin and kill Gram-negative bacteria. Upon intravenous injection, D-TZP protected animals from pre-established endotoxemia and polymicrobial sepsis, showing no systemic toxicities inherent to PMB. These results support D-TZP as a safe and effective systemic intervention of sepsis.

Investigating auranofin for the treatment of infected diabetic pressure ulcers in mice and dermal toxicity in pigs.

Bacterial infection of pressure ulcers (PUs) are a notable source of hospitalization for individuals with diabetes. This study evaluated the safety profile and efficacy of auranofin to treat diabetic PUs infected with methicillin-resistant Staphylococcus aureus (MRSA). PUs were infected with MRSA in diabetic TALLYHO/JngJ mice and then treated with topical auranofin (2%), topical mupirocin (2%), or oral clindamycin (30 mg/kg) for four days. PUs were harvested post-treatment to enumerate bacterial burden and determine expression of cytokines/growth factors. Landrace cross pigs were exposed topically to auranofin (1%, 2%, and 3%) for 4-14 days and evaluated for signs of localized or systemic toxicity. Auranofin eradicated MRSA in PUs within four days (7.92-log10 reduction) in contrast to mupirocin (2.15-log10 reduction) and clindamycin (0.73-log10 reduction). Additionally, auranofin treatment resulted in decreased expression of pro-inflammatory cytokines and increased expression of biomarkers associated with re-epithelization of wounded tissue, confirmed with histopathologic analysis. No significant histopathologic lesions were present on porcine skin sites exposed to topical auranofin. Additionally, minimal accumulation of plasma gold and no systemic toxicity was observed in pigs exposed to topical auranofin. Auranofin appears to be a potent and safe topical agent to further investigate for treatment of mild-to-moderate MRSA-infected diabetic PUs.

Nanosac, a Noncationic and Soft Polyphenol Nanocapsule, Enables Systemic Delivery of siRNA to Solid Tumors.

For systemic delivery of small interfering RNA (siRNA) to solid tumors, the carrier must circulate avoiding premature degradation, extravasate and penetrate tumors, enter target cells, traffic to the intracellular destination, and release siRNA for gene silencing. However, existing siRNA carriers, which typically exhibit positive charges, fall short of these requirements by a large margin; thus, systemic delivery of siRNA to tumors remains a significant challenge. To overcome the limitations of existing approaches, we have developed a carrier of siRNA, called "Nanosac", a noncationic soft polyphenol nanocapsule. A siRNA-loaded Nanosac is produced by sequential coating of mesoporous silica nanoparticles (MSNs) with siRNA and polydopamine, followed by removal of the sacrificial MSN core. The Nanosac recruits serum albumin, co-opts caveolae-mediated endocytosis to enter tumor cells, and efficiently silences target genes. The softness of Nanosac improves extravasation and penetration into tumors compared to its hard counterpart. As a carrier of siRNA targeting PD-L1, Nanosac induces a significant attenuation of CT26 tumor growth by immune checkpoint blockade. These results support the utility of Nanosac in the systemic delivery of siRNA for solid tumor therapy.

Evaluation of ebselen in resolving a methicillin-resistant Staphylococcus aureus infection of pressure ulcers in obese and diabetic mice.

Pressure ulcers (PUs) are a source of morbidity in individuals with restricted mobility including individuals that are obese or diabetic. Infection of PUs with pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), impairs ulcers from healing. The present study evaluated ebselen as a topical antibacterial to treat MRSA-infected PUs. Against two different S. aureus strains, including MRSA USA300, resistance to ebselen did not emerge after 14 consecutive passages. Resistance to mupirocin emerged after only five passages. Additionally, ebselen was found to exert a modest postantibiotic effect of five hours against two MRSA strains. Ebselen was subsequently evaluated in MRSA-infected PUs in two models using obese and diabetic mice. In obese mice, topical ebselen (89.2% reduction) and oral linezolid (84.5% reduction) similarly reduced the burden of MRSA in infected PUs. However, in diabetic mice, topical ebselen (45.8% reduction in MRSA burden) was less effective. Histopathological evaluation of ulcers in diabetic mice determined that ebselen treatment resulted in fewer bacterial colonies deep within the dermis and that the treatment exhibited evidence of epithelial regeneration. Topical mupirocin was superior to ebselen in reducing MRSA burden in infected PUs both in obese (98.7% reduction) and diabetic (99.3% reduction) mice. Ebselen's antibacterial activity was negatively impacted as the bacterial inoculum was increased from 105 CFU/mL to 107 CFU/mL. These results suggest that a higher dose of ebselen, or a longer course of treatment, may be needed to achieve a similar effect as mupirocin in topically treating MRSA-infected pressure ulcers.

Structural disruption of the blood-brain barrier in repetitive primary blast injury.

BACKGROUND: Blast-induced traumatic brain injury (bTBI) is a growing health concern due to the increased use of low-cost improvised explosive devices in modern warfare. Mild blast exposures are common amongst military personnel; however, these women and men typically do not have adequate recovery time from their injuries due to the transient nature of behavioral symptoms. bTBI has been linked to heterogeneous neuropathology, including brain edema, neuronal degeneration and cognitive abnormalities depending on the intensity of blast overpressure and frequency. Recent studies have reported heterogeneity in blood-brain barrier (BBB) permeability following blast injury. There still remains a limited understanding of the pathologic changes in the BBB following primary blast injuries. In this study, our goal was to elucidate the pathologic pattern of BBB damage through structural analysis following single and repetitive blast injury using a clinically relevant rat model of bTBI. METHODS: A validated, open-ended shock tube model was used to deliver single or repetitive primary blast waves. The pathology of the BBB was assessed using immunofluorescence and immunohistochemistry assays. All data were analyzed using the one-way ANOVA test. RESULTS: We have demonstrated that exposure to repetitive blast injury affects the desmin-positive and CD13-positive subpopulations of pericytes in the BBB. Changes in astrocytes and microglia were also detected. CONCLUSION: This study provides analysis of the BBB components after repetitive blast injury. These results will be critical as preventative and therapeutic strategies are established for veterans recovering from blast-induced traumatic brain injury.

Mechanism of PRL2 phosphatase-mediated PTEN degradation and tumorigenesis.

Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) levels are frequently found reduced in human cancers, but how PTEN is down-regulated is not fully understood. In addition, although a compelling connection exists between PRL (phosphatase of regenerating liver) 2 and cancer, how this phosphatase induces oncogenesis has been an enigma. Here, we discovered that PRL2 ablation inhibits PTEN heterozygosity-induced tumorigenesis. PRL2 deficiency elevates PTEN and attenuates AKT signaling, leading to decreased proliferation and increased apoptosis in tumors. We also found that high PRL2 expression is correlated with low PTEN level with reduced overall patient survival. Mechanistically, we identified PTEN as a putative PRL2 substrate and demonstrated that PRL2 down-regulates PTEN by dephosphorylating PTEN at Y336, thereby augmenting NEDD4-mediated PTEN ubiquitination and proteasomal degradation. Given the strong cancer susceptibility to subtle reductions in PTEN, the ability of PRL2 to down-regulate PTEN provides a biochemical basis for its oncogenic propensity. The results also suggest that pharmacological targeting of PRL2 could provide a novel therapeutic strategy to restore PTEN, thereby obliterating PTEN deficiency-induced malignancies.

Dynamic transition of the blood-brain barrier in the development of non-small cell lung cancer brain metastases.

Invasion of the brain by non-small cell lung cancer (NSCLC) results in a shift of the blood-brain barrier (BBB) to the insufficiently characterized blood-tumor barrier (BTB). Effective drug delivery through the BTB is one of the greatest therapeutic obstacles in treating brain metastases. Using an experimental model, we defined key changes within the BTB and the BBB in the brain around the tumor (BAT) region over time. Brain-seeking NSCLC cells were delivered into the circulation of athymic-nude mice via intracardiac injection and developing brain metastases were evaluated over six-weeks. Components of the BBB and BTB were analyzed using immunofluorescence microscopy and compared using a mixed model of regression. Our results demonstrate a dynamic time-dependent BTB phenotype. Capillaries of the BAT and BTB were dilated with increased CD31 expression compared to controls. Expression of collagen IV, a pan-basement membrane component, was significantly decreased in the BTB compared to the BBB. There was also a significant increase in the desmin-positive pericyte subpopulation in the BTB compared to the BBB. The most striking changes were identified in astrocyte water channels with a 12.18-fold (p < 0.001) decrease in aquaporin-4 in the BTB; the BAT was unchanged. Analysis of NSCLC brain metastases from patient samples similarly demonstrated dilated capillaries and loss of both collagen IV and aquaporin-4. These data provide a comprehensive analysis of the BTB in NSCLC brain metastasis. Astrocytic endfeet, pericytes, and the basement membrane are potential therapeutic targets to improve efficacy of chemotherapeutic delivery into NSCLC brain metastases.

Agreement in Histological Assessment of Mitotic Activity Between Microscopy and Digital Whole Slide Images Informs Conversion for Clinical Diagnosis.

Validating digital pathology as substitute for conventional microscopy in diagnosis remains a priority to assure effectiveness. Intermodality concordance studies typically focus on achieving the same diagnosis by digital display of whole slide images and conventional microscopy. Assessment of discrete histological features in whole slide images, such as mitotic figures, has not been thoroughly evaluated in diagnostic practice. To further gauge the interchangeability of conventional microscopy with digital display for primary diagnosis, 12 pathologists examined 113 canine naturally occurring mucosal melanomas exhibiting a wide range of mitotic activity. Design reflected diverse diagnostic settings and investigated independent location, interpretation, and enumeration of mitotic figures. Intermodality agreement was assessed employing conventional microscopy (CM40×), and whole slide image specimens scanned at 20× (WSI20×) and at 40× (WSI40×) objective magnifications. An aggregate 1647 mitotic figure count observations were available from conventional microscopy and whole slide images for comparison. The intraobserver concordance rate of paired observations was 0.785 to 0.801; interobserver rate was 0.784 to 0.794. Correlation coefficients between the 2 digital modes, and as compared to conventional microscopy, were similar and suggest noninferiority among modalities, including whole slide image acquired at lower 20× resolution. As mitotic figure counts serve for prognostic grading of several tumor types, including melanoma, 6 of 8 pathologists retrospectively predicted survival prognosis using whole slide images, compared to 9 of 10 by conventional microscopy, a first evaluation of whole slide image for mitotic figure prognostic grading. This study demonstrated agreement of replicate reads obtained across conventional microscopy and whole slide images. Hence, quantifying mitotic figures served as surrogate histological feature with which to further credential the interchangeability of whole slide images for primary diagnosis.

Intramuscular administration of hexachloroplatinate reverses cyanide-induced metabolic derangements and counteracts severe cyanide poisoning.

Cyanide is a highly toxic industrial chemical that is widely used by manufactures. Smoke inhalation during household fires is the most common source of cyanide poisoning while additional risks to civilians include industrial accidents and terrorist attacks. Despite the risks to large numbers of individuals, an antidote capable of administration at scale adequate for a mass casualty, prehospital scenario does not yet exist. Previously, we demonstrated that intravenous cisplatin analogues accelerate recovery from cyanide poisoning in mice and rabbits. Of the dozens of platinum-based organometallic complexes tested, hexachloroplatinate (HCP) emerged as a promising lead compound, exhibiting strong affinity for cyanide and efficacy across model systems. Here, we show HCP is an antidote to lethal cyanide exposure and importantly is effective when delivered intramuscularly. The pharmacokinetic profile of HCP exhibited bioavailability in the systemic circulation 2.5 minutes post-treatment and subsequent renal clearance of HCP-cyanide. HCP restored parameters of cellular physiology including cytochrome oxidase redox state and TCA cycle metabolism. We next validated these findings in a large animal model (swine). Finally, preclinical safety studies in mice revealed minimal toxicity. Cumulatively, these findings demonstrate hexachloroplatinate is a promising lead compound for development of an intramuscular injectable cyanide antidote for mass casualty scenarios.

Epithelial-mesenchymal Transition Phenotypes in Vertebral Metastases of Lung Cancer.

Vertebral metastases of non-small cell lung cancer (NSCLC) are frequently diagnosed in the metastatic setting and are commonly identified in the thoracic vertebrae in patients. Treatment of NSCLC bone metastases, which are often multiple, is palliative, and the median survival times are 3 to 6 months. We have characterized spontaneous vertebral metastases in a brain metastases model of NSCLC and correlated these findings with epithelial-mesenchymal transition (EMT). Brain metastases were established in athymic nude mice following intracardiac injection of brain-seeking adenocarcinoma NSCLC cells. Thirty-nine percent of mice (14/36) developed spontaneous vertebral metastases, spinal cord compression, and hind-limb paralysis. Vertebral metastases consisted of an adenocarcinoma phenotype with neoplastic epithelial cells arranged in cords or acini and a mesenchymal phenotype with spindloid neoplastic cells arranged in bundles and streams. Quantitative and qualitative immunohistochemical and immunofluorescence assays demonstrated an increase in vimentin expression compared to cytokeratin expression in vertebral metastases. A correlation with EMT was supported by an increase in CD44 in vertebral metastases and parenchymal metastases. These data demonstrate a translational lung cancer metastasis model with spontaneous vertebral metastasis. The mesenchymal and epithelial phenotype of these spontaneous metastases coupled with EMT provide a conduit to improve drug delivery and overall patient survival.

Development of Liposomal Gemcitabine with High Drug Loading Capacity.

Liposomes are widely used for systemic delivery of chemotherapeutic agents to reduce their nonspecific side effects. Gemcitabine (Gem) makes a great candidate for liposomal encapsulation due to the short half-life and nonspecific side effects; however, it has been difficult to achieve liposomal Gem with high drug loading capacity. Remote loading, which uses a transmembrane pH gradient to induce an influx of drug and locks the drug in the core as a sulfate complex, does not serve Gem as efficiently as doxorubicin (Dox) due to the low p Ka value of Gem. Existing studies have attempted to improve Gem loading capacity in liposomes by employing lipophilic Gem derivatives or creating a high-concentration gradient for active loading into the hydrophilic cores (small volume loading). In this study, we combine the remote loading approach and small volume loading or hypertonic loading, a new approach to induce the influx of Gem into the preformed liposomes by high osmotic pressure, to achieve a Gem loading capacity of 9.4-10.3 wt % in contrast to 0.14-3.8 wt % of the conventional methods. Liposomal Gem showed a good stability during storage, sustained-release over 120 h in vitro, enhanced cellular uptake, and improved cytotoxicity as compared to free Gem. Liposomal Gem showed a synergistic effect with liposomal Dox on Huh7 hepatocellular carcinoma cells. A mixture of liposomal Gem and liposomal Dox delivered both drugs to the tumor more efficiently than a free drug mixture and showed a relatively good anti-tumor effect in a xenograft model of hepatocellular carcinoma. This study shows that bioactive liposomal Gem with high drug loading capacity can be produced by remote loading combined with additional approaches to increase drug influx into the liposomes.

Automated Computational Detection, Quantitation, and Mapping of Mitosis in Whole-Slide Images for Clinically Actionable Surgical Pathology Decision Support.

BACKGROUND: Determining mitotic index by counting mitotic figures (MFs) microscopically from tumor areas with most abundant MF (hotspots [HS]) produces a prognostically useful tumor grading biomarker. However, interobserver concordance identifying MF and HS can be poorly reproducible. Immunolabeling MF, coupled with computer-automated counting by image analysis, can improve reproducibility. A computational system for obtaining MF values across digitized whole-slide images (WSIs) was sought that would minimize impact of artifacts, generate values clinically relatable to counting ten high-power microscopic fields of view typical in conventional microscopy, and that would reproducibly map HS topography. MATERIALS AND METHODS: Relatively low-resolution WSI scans (0.50 µm/pixel) were imported in grid-tile format for feature-based MF segmentation, from naturally occurring canine melanomas providing a wide range of proliferative activity. MF feature extraction conformed to anti-phospho-histone H3-immunolabeled mitotic (M) phase cells. Computer vision image processing was established to subtract key artifacts, obtain MF counts, and employ rotationally invariant feature extraction to map MF topography. RESULTS: The automated topometric HS (TMHS) algorithm identified mitotic HS and mapped select tissue tiles with greatest MF counts back onto WSI thumbnail images to plot HS topographically. Influence of dye, pigment, and extraneous structure artifacts was minimized. TMHS diagnostic decision support included image overlay graphics of HS topography, as well as a spreadsheet and plot of tile-based MF count values. TMHS performance was validated examining both mitotic HS counting and mapping functions. Significantly correlated TMHS MF mapping and metrics were demonstrated using repeat analysis with WSI in different orientation (R 2 = 0.9916) and by agreement with a pathologist (R 2 = 0.8605) as well as through assessment of counting function using an independently tuned object counting algorithm (OCA) (R 2 = 0.9482). Limits of agreement analysis support method interchangeability. MF counts obtained led to accurate patient survival prediction in all (n = 30) except one case. By contrast, more variable performance was documented when several pathologists examined similar cases using microscopy (pair-wise correlations, rho range = 0.7597-0.9286). CONCLUSIONS: Automated TMHS MF segmentation and feature engineering performance were interchangeable with both observer and OCA in digital mode. Moreover, enhanced HS location accuracy and superior method reproducibility were achieved using the automated TMHS algorithm compared to the current practice employing clinical microscopy.

Surface Modification of Polymeric Nanoparticles with M2pep Peptide for Drug Delivery to Tumor-Associated Macrophages.

PURPOSE: Tumor-associated macrophages (TAMs) with immune-suppressive M2-like phenotype constitute a significant part of tumor and support its growth, thus making an attractive therapeutic target for cancer therapy. To improve the delivery of drugs that control the survival and/or functions of TAMs, we developed nanoparticulate drug carriers with high affinity for TAMs. METHODS: Poly(lactic-co-glycolic acid) nanoparticles were coated with M2pep, a peptide ligand selectively binding to M2-polarized macrophages, via a simple surface modification method based on tannic acid-iron complex. The interactions of M2pep-coated nanoparticles with macrophages of different phenotypes were tested in vitro and in vivo. PLX3397, an inhibitor of the colony stimulating factor-1 (CSF-1)/CSF-1 receptor (CSF-1R) pathway and macrophage survival, was delivered to B16F10 tumors via M2pep-modified PLGA nanoparticles. RESULTS: In bone marrow-derived macrophages polarized to M2 phenotype, M2pep-coated nanoparticles showed greater cellular uptake than those without M2pep. Consistently, M2pep-coated nanoparticles showed relatively high localization of CD206+ macrophages in B16F10 tumors. PLX3397 encapsulated in M2pep-coated nanoparticles attenuated tumor growth better than the free drug counterpart. CONCLUSION: These results support that M2pep-coating can help nanoparticles to interact with M2-like TAMs and facilitate the delivery of drugs that control the tumor-supportive functions of TAMs.

Modulating the Inflammatory Reflex in Rats Using Low-Intensity Focused Ultrasound Stimulation of the Vagus Nerve.

Tumor necrosis factor α (TNF-α) is linked to several chronic inflammatory diseases. Electrical vagus nerve stimulation reduces serum TNF-α levels but may cause chronic nerve damage and requires surgery. Alternatively, we proposed focused ultrasound stimulation of the vagus nerve (uVNS), which can be applied non-invasively. In this study, we induced an inflammatory response in rats using lipopolysaccharides (LPS) and collected blood to analyze the effects of uVNS on cytokine concentrations. We applied one or three 5-min pulsed focused ultrasound stimulation treatments to the vagus nerve (250 kHz, ISPPA = 3 W/cm2). Animals receiving a single ultrasound application had an average reduction in TNF-α levels of 19%, similar to the 16% reduction observed in electrically stimulated animals. With multiple applications, uVNS therapy statistically reduced serum TNF-α levels by 73% compared with control animals without any observed damage to the nerve. These findings suggest that uVNS is a suitable way to attenuate TNF-α levels.

Surface modification of polymer nanoparticles with native albumin for enhancing drug delivery to solid tumors.

Albumin is a promising surface modifier of nanoparticulate drug delivery systems. Serving as a dysopsonin, albumin can protect circulating nanoparticles (NPs) from the recognition and clearance by the mononuclear phagocytic system (MPS). Albumin may also help transport the NPs to solid tumors based on the increased consumption by cancer cells and interactions with the tumor microenvironment. Several studies have explored the benefits of surface-bound albumin to enhance NP delivery to tumors. However, it remains unknown how the surface modification process affects the conformation of albumin and the performance of the albumin-modified NPs. We use three different surface modification methods including two prevalent approaches (physisorption and interfacial embedding) and a new method based on dopamine polymerization to modify the surface of poly(lactic-co-glycolic acid) NPs with albumin and compare the extent of albumin binding, conformation of the surface-bound albumin, and biological performances of the albumin-coated NPs. We find that the dopamine polymerization method preserves the albumin structure, forming a surface layer that facilitates NP transport and drug delivery into tumors via the interaction with albumin-binding proteins. In contrast, the interfacial embedding method creates NPs with denatured albumin that offers no particular benefit to the interaction with cancer cells but rather promotes the MPS uptake via direct and indirect interactions with scavenger receptor A. This study demonstrates that the surface-bound albumin can bring distinct effects according to the way they interact with NP surface and thus needs to be controlled in order to achieve favorable therapeutic outcomes.

Limited fibrosis accompanies triple-negative breast cancer metastasis in multiple model systems and is not a preventive target.

The lysophosphatidic acid receptor 1 (LPAR1) is mechanistically implicated in both tumor metastasis and tissue fibrosis. Previously, metastasis was increased when fulminant fibrosis was first induced in mice, suggesting a direct connection between these processes. The current report examined the extent of metastasis-induced fibrosis in breast cancer model systems, and tested the metastasis preventive efficacy and fibrosis attenuation of antagonists for LPAR1 and Idiopathic Pulmonary Fibrosis (IPF) in breast and ovarian cancer models. Staining analysis demonstrated only focal, low-moderate levels of fibrosis in lungs from eleven metastasis model systems. Two orally available LPAR1 antagonists, SAR100842 and EPGN9878, significantly inhibited breast cancer motility to LPA in vitro. Both compounds were negative for metastasis prevention and failed to reduce fibrosis in the experimental MDA-MB-231T and spontaneous murine 4T1 in vivo breast cancer metastasis models. SAR100842 demonstrated only occasional reductions in invasive metastases in the SKOV3 and OVCAR5 ovarian cancer experimental metastasis models. Two approved drugs for IPF, nintedanib and pirfenidone, were investigated. Both were ineffective at preventing MDA-MB-231T metastasis, with no attenuation of fibrosis. In summary, metastasis-induced fibrosis is only a minor component of metastasis in untreated progressive breast cancer. LPAR1 antagonists, despite in vitro evidence of specificity and efficacy, were ineffective in vivo as oral agents, as were approved IPF drugs. The data argue against LPAR1 and fibrosis as monotherapy targets for metastasis prevention in triple-negative breast cancer and ovarian cancer.