Branched-Tail Lipid Nanoparticles for Intravenous mRNA Delivery to Lung Immune, Endothelial, and Alveolar Cells in Mice.

Lipid nanoparticles (LNPs) are proven safe and effective delivery systems on a global scale. However, their efficacy has been limited primarily to liver and immune cell targets. To extend the applicability of mRNA drugs, 580 ionizable lipidoids are synthesized and tested for delivery to extrahepatocellular targets. Of these, over 40 enabled protein expression in mice, with the majority transfecting the liver. Beyond the liver, several LNPs containing new, branched-tail ionizable lipidoids potently delivered mRNA to the lungs, with cell-level specificity depending on helper lipid chemistry. Incorporation of the neutral helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 16 mol% enabled highly specific delivery to natural killer and dendritic cells within the lung. Although inclusion of the cationic lipid 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) improved lung tropism, it decreased cell specificity, resulting in equal transfection of endothelial and lymphoid cells. DOTAP formulations are also less favorable than DOPE formulations because they elevated liver enzyme and cytokine levels. Together, these data identify a new branched-tailed LNP with a unique ability to selectively transfect lung immune cell populations without the use of toxicity-prone cationic helper lipids. This novel vehicle may unlock RNA therapies for lung diseases associated with immune cell dysregulation, including cancer, viral infections, and autoimmune disorders.

Characterization and comparison of human and mouse milk cells.

Recent data has characterized human milk cells with unprecedented detail and provided insight into cell populations. While such analysis of freshly expressed human milk has been possible, studies of cell functionality within the infant have been limited to animal models. One commonly used animal model for milk research is the mouse; however, limited data are available describing the composition of mouse milk. In particular, the maternal cells of mouse milk have not been previously characterized in detail, in part due to the difficulty in collecting sufficient volumes of mouse milk. In this study, we have established a method to collect high volumes of mouse milk, isolate cells, and compare the cell counts and types to human milk. Surprisingly, we found that mouse milk cell density is three orders of magnitude higher than human milk. The cell types present in the milk of mice and humans are similar, broadly consisting of mammary epithelial cells and immune cells. These results provide a basis of comparison for mouse and human milk cells and will inform the most appropriate uses of mouse models for the study of human phenomena.

Blast-Derived Small Extracellular Vesicles in the Plasma of Patients with Acute Myeloid Leukemia Predict Responses to Chemotherapy.

The small extracellular vesicles (sEV) accumulating in acute myeloid leukemia (AML) patients' plasma are mixtures of vesicles produced by leukemic and non-malignant cells. sEV originating from leukemia blasts could serve as potential non-invasive biomarkers of AML response to therapy. To isolate blast-derived sEV from patients' plasma, we developed a bioprinted microarray-based immunoassay using monoclonal antibodies (mAbs) specific for leukemia-associated antigens (LAAs) and mAbs specific for a mix of tetraspanins (CD9, CD63, and CD81). We determined the proportion of LAA+ sEV relative to total plasma sEV (the LAA+/total sEV ratio) in serially collected samples of newly diagnosed AML patients prior to, during, and after chemotherapy. At AML diagnosis, the LAA+/total sEV ratio was significantly higher in patients than in healthy donors (HDs). In patients who achieved complete remission (CR) after induction chemotherapy, the LAA+/total sEV ratios significantly decreased after each chemotherapy cycle to levels seen in HDs. In contrast, the LAA+/total sEV ratios in AML patients with persistent leukemia after therapy remained elevated during and after therapy, as did the percentage of leukemic blasts in these patients' bone marrows. The LAA+/total sEV ratio emerges as a promising non-invasive biomarker of leukemia response to therapy.

TGFß carrying exosomes in plasma: potential biomarkers of cancer progression in patients with head and neck squamous cell carcinoma.

OBJECTIVES: Contributions of TGFß to cancer progression are well documented. However, plasma TGFß levels often do not correlate with clinicopathological data. We examine the role of TGFß carried in exosomes isolated from murine and human plasma as a contributor to disease progression in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: The 4-nitroquinoline-1-oxide (4-NQO) mouse model was used to study changes in TGFß expression levels during oral carcinogenesis. In human HNSCC, TGFß and Smad3 protein expression levels and TGFB1 gene expression were determined. Soluble TGFß levels were evaluated by ELISA and TGFß bioassays. Exosomes were isolated from plasma using size exclusion chromatography, and TGFß content was quantified using bioassays and bioprinted microarrays. RESULTS: During 4-NQO carcinogenesis, TGFß levels in tumour tissues and in serum increased as the tumour progressed. The TGFß content of circulating exosomes also increased. In HNSCC patients, TGFß, Smad3 and TGFB1 were overexpressed in tumour tissues and correlated with increased soluble TGFß levels. Neither TGFß expression in tumours nor levels of soluble TGFß correlated with clinicopathological data or survival. Only exosome-associated TGFß reflected tumour progression and correlated with tumour size. CONCLUSIONS: Circulating TGFß+ exosomes in the plasma of patients with HNSCC emerge as potential non-invasive biomarkers of disease progression in HNSCC.

Ionizable lipid nanoparticles deliver mRNA to pancreatic ß cells via macrophage-mediated gene transfer.

Systemic messenger RNA (mRNA) delivery to organs outside the liver, spleen, and lungs remains challenging. To overcome this issue, we hypothesized that altering nanoparticle chemistry and administration routes may enable mRNA-induced protein expression outside of the reticuloendothelial system. Here, we describe a strategy for delivering mRNA potently and specifically to the pancreas using lipid nanoparticles. Our results show that delivering lipid nanoparticles containing cationic helper lipids by intraperitoneal administration produces robust and specific protein expression in the pancreas. Most resultant protein expression occurred within insulin-producing ß cells. Last, we found that pancreatic mRNA delivery was dependent on horizontal gene transfer by peritoneal macrophage exosome secretion, an underappreciated mechanism that influences the delivery of mRNA lipid nanoparticles. We anticipate that this strategy will enable gene therapies for intractable pancreatic diseases such as diabetes and cancer.

TGFß+ small extracellular vesicles from head and neck squamous cell carcinoma cells reprogram macrophages towards a pro-angiogenic phenotype.

Transforming growth factor ß (TGFß) is a major component of tumor-derived small extracellular vesicles (TEX) in cancer patients. Mechanisms utilized by TGFß+ TEX to promote tumor growth and pro-tumor activities in the tumor microenvironment (TME) are largely unknown. TEX produced by head and neck squamous cell carcinoma (HNSCC) cell lines carried TGFß and angiogenesis-promoting proteins. TGFß+ TEX stimulated macrophage chemotaxis without a notable M1/M2 phenotype shift and reprogrammed primary human macrophages to a pro-angiogenic phenotype characterized by the upregulation of pro-angiogenic factors and functions. In a murine basement membrane extract plug model, TGFß+ TEX promoted macrophage infiltration and vascularization (p < 0.001), which was blocked by using the TGFß ligand trap mRER (p < 0.001). TGFß+ TEX injected into mice undergoing the 4-nitroquinoline-1-oxide (4-NQO)-driven oral carcinogenesis promoted tumor angiogenesis (p < 0.05), infiltration of M2-like macrophages in the TME (p < 0.05) and ultimately tumor progression (p < 0.05). Inhibition of TGFß signaling in TEX with mRER ameliorated these pro-tumor activities. Silencing of TGFß emerges as a critical step in suppressing pro-angiogenic functions of TEX in HNSCC.

Engineering pro-angiogenic biomaterials via chemoselective extracellular vesicle immobilization.

Nanoscale extracellular vesicles (EVs) represent a unique cellular derivative that reflect the therapeutic potential of mesenchymal stem cells (MSCs) toward tissue engineering and injury repair without the logistical and safety concerns of utilizing living cells. However, upon systemic administration in vivo,EVs undergo rapid clearance and typically lack controlled targeted delivery, thus reducing their effectiveness in therapeutic regenerative therapies. Here, we describe a strategy that enables long-term in vivo spatial EV retention by chemoselective immobilization of metabolically incoporated azido ligand-bearing EVs (azido-EVs) within a dibenzocyclooctyne-modified collagen hydrogel. MSC-derived azido-EVs exhibit comparable morphological and functional properties as their non-labeled EV counterparts and, when immobilized within collagen hydrogel implants via click chemistry, they elicited more robust host cell infiltration, angiogenic and immunoregulatory responses including vascular ingrowth and macrophage recruitment compared to ten times the higher dose required by non-immobilized EVs. We envision this technology will enable a wide range of applications to spatially promote vascularization and host integration relevant to tissue engineering and regenerative medicine applications.

Characterization of systemic immunosuppression by IDH mutant glioma small extracellular vesicles.

BACKGROUND: Gliomas are the most common primary brain tumors and are universally fatal. Mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) define a distinct glioma subtype associated with an immunosuppressive tumor microenvironment. Mechanisms underlying systemic immunosuppression in IDH mutant (mutIDH) gliomas are largely unknown. Here, we define genotype-specific local and systemic tumor immunomodulatory functions of tumor-derived glioma small extracellular vesicles (TEX). METHODS: TEX produced by human and murine wildtype and mutant IDH glioma cells (wtIDH and mutIDH, respectively) were isolated by size exclusion chromatography (SEC). TEX morphology, size, quantity, molecular profiles and biodistribution were characterized. TEX were injected into naive and tumor-bearing mice, and the local and systemic immune microenvironment composition was characterized. RESULTS: Using in vitro and in vivo glioma models, we show that mutIDH TEX are more numerous, possess distinct morphological features and are more immunosuppressive than wtIDH TEX. mutIDH TEX cargo mimics their parental cells, and induces systemic immune suppression in naive and tumor-bearing mice. TEX derived from mutIDH gliomas and injected into wtIDH tumor-bearing mice reduce tumor-infiltrating effector lymphocytes, dendritic cells and macrophages, and increase circulating monocytes. Astonishingly, mutIDH TEX injected into brain tumor-bearing syngeneic mice accelerate tumor growth and increase mortality compared with wtIDH TEX. CONCLUSIONS: Targeting of mutIDH TEX represents a novel therapeutic approach in gliomas.

Grafting Polymer Brushes by ATRP from Functionalized Poly(ether ether ketone) Microparticles.

Poly(ether ether ketone) (PEEK) is a semi-crystalline thermoplastic with excellent mechanical and chemical properties. PEEK exhibits a high degree of resistance to thermal, chemical, and bio-degradation. PEEK is used as biomaterial in the field of orthopaedic and dental implants; however, due to its intrinsic hydrophobicity and inert surface, PEEK does not effectively support bone growth. Therefore, new methods to modify PEEK's surface to improve osseointegration are key to next generation polymer implant materials. Unfortunately, PEEK is a challenging material to both modify and subsequently characterize thus stymieing efforts to improve PEEK osseointegration. In this manuscript, we demonstrate how surface-initiated atom transfer radical polymerization (SI-ATRP) can be used to modify novel PEEK microparticles (PMP). The hard core-soft shell microparticles were synthesized and characterized by DLS, ATR-IR, XPS and TEM, indicating the grafted materials increased solubility and stability in a range of solvents. The discovered surface grafted PMP can be used as compatibilizers for the polymer-tissue interface.

Pneumococcal Extracellular Vesicles Modulate Host Immunity.

Extracellular vesicles (EVs) have recently garnered attention for their participation in host-microbe interactions in pneumococcal infections. However, the effect of EVs on the host immune system remain poorly understood. Our studies focus on EVs produced by Streptococcus pneumoniae (pEVs), and reveal that pEVs are internalized by macrophages, T cells, and epithelial cells. In vitro, pEVs induce NF-κB activation in a dosage-dependent manner and polarize human macrophages to an alternative (M2) phenotype. In addition, pEV pretreatment conditions macrophages to increase bacteria uptake and such macrophages may act as a reservoir for pneumococcal cells by increasing survival of the phagocytosed bacteria. When administered systemically in mice, pEVs induce cytokine release; when immobilized locally, they recruit lymphocytes and macrophages. Taken together, pEVs emerge as critical contributors to inflammatory responses and tissue damage in mammalian hosts. IMPORTANCE Over the last decade, pathogen-derived extracellular vesicles (EVs) have emerged as important players in several human diseases. Therefore, a thorough understanding of EV-mediated mechanisms could provide novel insights into vaccine/therapeutic development. A critical question in the field is: do pathogen-derived EVs help the pathogen evade the harsh environment in the host or do they help the host to mount a robust immune response against the pathogen? This study is a step towards answering this critical question for the Gram-positive pathogen, Streptococcus pneumoniae. Our study shows that while S. pneumoniae EVs (pEVs) induce inflammatory response both in vitro and in vivo, they may also condition the host macrophages to serve as a reservoir for the bacteria.

Novel TGFß Inhibitors Ameliorate Oral Squamous Cell Carcinoma Progression and Improve the Antitumor Immune Response of Anti-PD-L1 Immunotherapy.

TGFß is a key regulator of oral squamous cell carcinoma (OSCC) progression, and its potential role as a therapeutic target has been investigated with a limited success. This study evaluates two novel TGFß inhibitors as mono or combinatorial therapy with anti-PD-L1 antibodies (α-PD-L1 Ab) in a murine OSCC model. Immunocompetent C57BL/6 mice bearing malignant oral lesions induced by 4-nitroquinoline 1-oxide (4-NQO) were treated for 4 weeks with TGFß inhibitors mRER (i.p., 50 µg/d) or mmTGFß2-7m (10 µg/d delivered by osmotic pumps) alone or in combination with α-PD-L1 Abs (7× i.p. of 100 µg/72 h). Tumor progression and body weight were monitored. Levels of bioactive TGFß in serum were quantified using a TGFß bioassay. Tissues were analyzed by immunohistology and flow cytometry. Therapy with mRER or mmTGFß2-7m reduced tumor burden (P < 0.05) and decreased body weight loss compared with controls. In inhibitor-treated mice, levels of TGFß in tumor tissue and serum were reduced (P < 0.05), whereas they increased with tumor progression in controls. Both inhibitors enhanced CD8+ T-cell infiltration into tumors and mRER reduced levels of myeloid-derived suppressor cells (P < 0.001). In combination with α-PD-L1 Abs, tumor burden was not further reduced; however, mmTGFß2-7m further reduced weight loss (P < 0.05). The collagen-rich stroma was reduced by using combinatorial TGFß/PD-L1 therapies (P < 0.05), enabling an accelerated lymphocyte infiltration into tumor tissues. The blockade of TGFß signaling by mRER or mmTGFß2-7m ameliorated in vivo progression of established murine OSCC. The inhibitors promoted antitumor immune responses, alone and in combination with α-PD-L1 Abs.

A molecular link between cell wall biosynthesis, translation fidelity, and stringent response in Streptococcus pneumoniae.

Survival in the human host requires bacteria to respond to unfavorable conditions. In the important Gram-positive pathogen Streptococcus pneumoniae, cell wall biosynthesis proteins MurM and MurN are tRNA-dependent amino acyl transferases which lead to the production of branched muropeptides. We demonstrate that wild-type cells experience optimal growth under mildly acidic stressed conditions, but ΔmurMN strain displays growth arrest and extensive lysis. Furthermore, these stress conditions compromise the efficiency with which alanyl-tRNAAla synthetase can avoid noncognate mischarging of tRNAAla with serine, which is toxic to cells. The observed growth defects are rescued by inhibition of the stringent response pathway or by overexpression of the editing domain of alanyl-tRNAAla synthetase that enables detoxification of tRNA misacylation. Furthermore, MurM can incorporate seryl groups from mischarged Seryl-tRNAAlaUGC into cell wall precursors with exquisite specificity. We conclude that MurM contributes to the fidelity of translation control and modulates the stress response by decreasing the pool of mischarged tRNAs. Finally, we show that enhanced lysis of ΔmurMN pneumococci is caused by LytA, and the murMN operon influences macrophage phagocytosis in a LytA-dependent manner. Thus, MurMN attenuates stress responses with consequences for host-pathogen interactions. Our data suggest a causal link between misaminoacylated tRNA accumulation and activation of the stringent response. In order to prevent potential corruption of translation, consumption of seryl-tRNAAla by MurM may represent a first line of defense. When this mechanism is overwhelmed or absent (ΔmurMN), the stringent response shuts down translation to avoid toxic generation of mistranslated/misfolded proteins.

Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes.

BACKGROUND: Glioblastoma is one of the most immunosuppressive human tumors. Emerging data suggest that glioblastoma-derived exosomes (GBex) reprogram the tumor microenvironment into a tumor-promoting milieu by mechanisms that not yet understood. METHODS: Exosomes were isolated from supernatants of glioblastoma cell lines by size exclusion chromatography. The GBex endosomal origin, size, protein cargos, and ex vivo effects on immune cell functions were determined. GBex were injected intravenously into mice to evaluate their ability to in vivo modulate normal immune cell subsets. RESULTS: GBex carried immunosuppressive proteins, including FasL, TRAIL, CTLA-4, CD39, and CD73, but contained few immunostimulatory proteins. GBex co-incubated with primary human immune cells induced simultaneous activation of multiple molecular pathways. In CD8+ T cells, GBex suppressed TNF-α and INF-γ release and mediated apoptosis. GBex suppressed natural killer (NK) and CD4+ T-cell activation. GBex activated the NF-κB pathway in macrophages and promoted their differentiation into M2 cells. Inhibition of the NF-κB pathway in macrophages reversed the GBex-mediated effects. GBex-driven reprogramming of macrophages involved the release of soluble factors that promoted tumor proliferation in vitro. In mice injected with GBex, the frequency of splenic CD8+ T cells, NK cells, and M1-like macrophages was reduced, while that of naïve and M2-like macrophages increased (P < .05). CONCLUSIONS: GBex reprogrammed functions of all types of immune cells in vitro and altered their frequency in vivo. By creating and sustaining a highly immunosuppressive environment, GBex play a key role in promoting tumor progression.

Arginase-1+ Exosomes from Reprogrammed Macrophages Promote Glioblastoma Progression.

Interactions between tumor cells and tumor-associated macrophages (TAMs) are critical for glioblastoma progression. The TAMs represent up to 30% of the glioblastoma mass. The role of TAMs in tumor progression and in the mechanisms underlying tumor growth remain unclear. Using an in vitro model resembling the crosstalk between macrophages and glioblastoma cells, we show that glioblastoma-derived exosomes (GBex) reprogram M1 (mediate pro-inflammatory function) and M2 (mediate anti-inflammatory function) macrophages, converting M1 into TAMs and augmenting pro-tumor functions of M2 macrophages. In turn, these GBex-reprogrammed TAMs, produce exosomes decorated by immunosuppressive and tumor-growth promoting proteins. TAM-derived exosomes disseminate these proteins in the tumor microenvironment (TME) promoting tumor cell migration and proliferation. Mechanisms underlying the promotion of glioblastoma growth involved Arginase-1+ exosomes produced by the reprogrammed TAMs. A selective Arginase-1 inhibitor, nor-NOHA reversed growth-promoting effects of Arginase-1 carried by TAM-derived exosomes. The data suggest that GBex-reprogrammed Arginase-1+ TAMs emerge as a major source of exosomes promoting tumor growth and as a potential therapeutic target in glioblastoma.

Tumor-derived exosomes promote angiogenesis via adenosine A2B receptor signaling.

RATIONALE: One hallmark of tumor-derived exosomes (TEX) is the promotion of cancer progression by stimulating angiogenesis. This study was performed to evaluate the role of adenosine receptors in TEX-induced angiogenesis. METHODS: TEX produced by UMSCC47 head and neck cancer cell line were isolated by mini size exclusion chromatography (mini-SEC). Enzymatic activity of ectonucleotidases CD39/CD73 carried by TEX was measured by HPLC. Adenosine content of TEX was measured by UPLC-MS/MS. Primary human macrophages were co-incubated with TEX or exosomes derived from the plasma of head and neck cancer patients and their marker expression profile was analyzed by flow cytometry. The macrophage secretome was analyzed by angiogenesis arrays. The in vitro angiogenic potential of TEX was evaluated in endothelial growth studies. Results were validated in vivo using basement membrane extract plug assays in A1R-/-, A2AR-/- and A2BR-/- rats. Vascularization was analyzed by hemoglobin quantification and immunohistology with vessel and macrophage markers. RESULTS: TEX carried enzymatically active CD39/CD73 and adenosine. TEX promoted A2BR-mediated polarization of macrophages toward an M2-like phenotype (p < 0.05) and enhanced their secretion of angiogenic factors. Growth of endothelial cells was stimulated directly by TEX and indirectly via macrophage-reprogramming dependent on A2BR signaling (p < 0.01). In vivo, TEX stimulated the formation of defined vascular structures and macrophage infiltration. This response was absent in A2BR-/- rats (p < 0.05). CONCLUSION: This report provides the first evidence for adenosine production by TEX to promote angiogenesis via A2BR. A2BR antagonism emerges as a potential strategy to block TEX-induced angiogenesis.

Simultaneous Inhibition of Glycolysis and Oxidative Phosphorylation Triggers a Multi-Fold Increase in Secretion of Exosomes: Possible Role of 2'3'-cAMP.

Exosome secretion by cells is a complex, poorly understood process. Studies of exosomes would be facilitated by a method for increasing their production and release. Here, we present a method for stimulating the secretion of exosomes. Cultured cells were treated or not with sodium iodoacetate (IAA; glycolysis inhibitor) plus 2,4-dinitrophenol (DNP; oxidative phosphorylation inhibitor). Exosomes were isolated by size-exclusion chromatography and their morphology, size, concentration, cargo components and functional activity were compared. IAA/DNP treatment (up to 10 µM each) was non-toxic and resulted in a 3 to 16-fold increase in exosome secretion. Exosomes from IAA/DNP-treated or untreated cells had similar biological properties and functional effects on endothelial cells (SVEC4-10). IAA/DNP increased exosome secretion from mouse organ cultures, and in vivo injections enhanced the levels of circulating exosomes. IAA/DNP decreased ATP levels (p < 0.05) in cells. A cell membrane-permeable form of 2',3'-cAMP and 3'-AMP mimicked the potentiating effects of IAA/DNP on exosome secretion. In cells lacking 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase; an enzyme that metabolizes 2',3'-cAMP into 2'- and 3'-AMP), effects of IAA/DNP on exosome secretion were enhanced. The IAA/DNP combination is a powerful stimulator of exosome secretion, and these stimulatory effects are, in part, mediated by intracellular 2',3'-cAMP.

Rapid On-Demand Extracellular Vesicle Augmentation with Versatile Oligonucleotide Tethers.

Exosomes show potential as ideal vehicles for drug delivery because of their natural role in transferring biological cargo between cells. However, current methods to engineer exosomes without negatively impacting their function remain challenging. Manipulating exosome-secreting cells is complex and time-consuming, while direct functionalization of exosome surface proteins suffers from low specificity and low efficiency. We demonstrate a rapid, versatile, and scalable method with oligonucleotide tethers to enable diverse surface functionalization on both human and murine exosomes. These exosome surface modifiers, which range from reactive functional groups and small molecules to aptamers and large proteins, can readily and efficiently enhance native exosome properties. We show that cellular uptake of exosomes can be specifically altered with a tethered AS1411 aptamer, and targeting specificity can be altered with a tethered protein. We functionalize exosomes with an immunomodulatory protein, FasL, and demonstrate their biological activity both in vitro and in vivo. FasL-functionalized exosomes, when bioprinted on a collagen matrix, allows spatial induction of apoptosis in tumor cells and, when injected in mice, suppresses proliferation of alloreactive T cells. This oligonucleotide tethering strategy is independent of the exosome source and further circumvents the need to genetically modify exosome-secreting cells.

Circulating exosomes measure responses to therapy in head and neck cancer patients treated with cetuximab, ipilimumab, and IMRT.

Purpose: Exosomes, small extracellular vesicles (EVs) derived from the endocytic compartment of their parent cells, are present in plasma of cancer patients and may serve as non-invasive biomarkers of disease outcome. Here, we asked whether tumor-derived (TEX) and/or T-cell derived exosomes can predict outcome in head and neck squamous cell carcinoma (HNSCC) patients treated with oncological therapy. Materials and Methods: 18 HNSCC patients enrolled in phase I clinical trial and receiving a combination of cetuximab, ipilimumab and radiation therapy were serially monitored for TEX and T cell-derived exosomes. Exosomes isolated from plasma by size exclusion chromatography were fractionated into TEX and CD3 + T cell-derived exosomes by immunocapture. Exosome-associated proteins were quantified by on-bead flow cytometry. Exosome molecular cargos of patients whose tumors recurred within 2 years (N = 5) were compared to cargos of patients who remained disease free at 2 years (N = 13) after therapy. Results: The predictive value of the exosome molecular cargo for disease recurrence was evaluated pre-, during and post therapy. In patients whose disease recurred, total exosome proteins, TEX/total exosome ratios, total CD3+, CD3(-)PD-L1+ and CD3 + 15s+ (Treg-derived) exosomes increased from the baseline levels. In patients who remained disease free, total exosome protein and TEX levels decreased, CD3+ and CD3+ CD15s+ exosomes stabilized and CD3+ CTLA4+ exosomes declined after ipilimumab therapy. Conclusion: TEX and T cell-derived circulating exosomes instead of immune cells were used for monitoring of patients' responses to oncological therapy. The results support the potential role of exosomes as a non-invasive tumor and immune cell biomarkers in cancer.

Interleukin-10 Does Not Augment Osseous Regeneration in the Scarred Calvarial Defect Achieved with Low-Dose Biopatterned BMP2.

BACKGROUND: Large calvarial defects represent a major reconstructive challenge, as they do not heal spontaneously. Infection causes inflammation and scarring, further reducing the healing capacity of the calvaria. Bone morphogenetic protein-2 (BMP2) has been shown to stimulate osteogenesis but has significant side effects in high doses. BMP2 has not been tested in combination with antiinflammatory cytokines such as interleukin-10. METHODS: Sixteen New Zealand White rabbits underwent 15 × 15-mm flap calvarectomies. The flap was incubated in Staphylococcus aureus and replaced, and infection and scarring were allowed to develop. The flap was subsequently removed and the wound débrided. A 15 × 15-mm square of acellular dermal matrix biopatterned with low-dose BMP2, interleukin-10, or a combination was implanted. Computed tomographic scans were taken over 42 days. Rabbits were then killed and histology was performed. RESULTS: Defects treated with BMP2 showed significantly (p < 0.05) greater osseous regeneration than untreated controls. Interleukin-10 did not significantly augment the healing achieved with BMP2, and interleukin-10 alone did not significantly increase healing compared with controls. Histology showed evidence of bone formation in defects treated with BMP2. Untreated controls and defects treated with interleukin-10 alone showed only fibrous tissue in the defect site. CONCLUSIONS: Low-dose BMP2 delivered directly to the scarred calvarial defect augments bony healing. Interleukin-10 at the dose applied did not significantly augment healing alone or in combination with BMP2. Healing had not finished at 42 days and analysis at later time points or the use of higher doses of BMP2 may yield greater healing.

Optimization of cell culture conditions for exosome isolation using mini-size exclusion chromatography (mini-SEC).

Extracellular vesicles (EVs) are emerging as a major intercellular communication system engaged in a variety of physiological and pathophysiological processes. Tumor-derived exosomes (TEX) are a subset of EVs of special interest as potential cancer biomarkers. Supernatants of tumor cell lines are widely used as the source of pure TEX for molecular/genetic studies. To optimize TEX isolation and characterization for these studies, we evaluated culture conditions for different tumor cell lines and used mini size exclusion chromatography (mini-SEC) for TEX isolation. Each tumor cell line showed unique culture requirements that determined the recovery, purity and total yield of TEX. Culture conditions for optimal TEX purity and recovery by mini-SEC could be modified by altering the media composition and numbers of seeded cells. TEX recovered from mini-SEC fraction #4 under optimized conditions were biologically active, were sized from 30 to 150 nm in diameter, had a typical vesicular morphology and carried endocytic markers. The most critical requirement for reproducible exosome recovery was re-seeding of tumor cells in numbers adjusted to reflect the optimized culture conditions for each tumor cell line. This study provides insights into a cell culture technique, which can be optimized for exosome production by various human or mouse tumor cell lines for isolation by mini-SEC.