Publications by authors named "Saigopalakrishna Yerneni"

26 Publications

  • Page 1 of 1

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

Proc Natl Acad Sci U S A 2021 Apr;118(14)

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213;

Survival in the human host requires bacteria to respond to unfavorable conditions. In the important Gram-positive pathogen , 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 Δ strain displays growth arrest and extensive lysis. Furthermore, these stress conditions compromise the efficiency with which alanyl-tRNA synthetase can avoid noncognate mischarging of tRNA 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-tRNA synthetase that enables detoxification of tRNA misacylation. Furthermore, MurM can incorporate seryl groups from mischarged Seryl-tRNA 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 Δ pneumococci is caused by LytA, and the 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-tRNA by MurM may represent a first line of defense. When this mechanism is overwhelmed or absent (Δ), the stringent response shuts down translation to avoid toxic generation of mistranslated/misfolded proteins.
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http://dx.doi.org/10.1073/pnas.2018089118DOI Listing
April 2021

Engineering exosome polymer hybrids by atom transfer radical polymerization.

Proc Natl Acad Sci U S A 2021 Jan;118(2)

Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213;

Exosomes are emerging as ideal drug delivery vehicles due to their biological origin and ability to transfer cargo between cells. However, rapid clearance of exogenous exosomes from the circulation as well as aggregation of exosomes and shedding of surface proteins during storage limit their clinical translation. Here, we demonstrate highly controlled and reversible functionalization of exosome surfaces with well-defined polymers that modulate the exosome's physiochemical and pharmacokinetic properties. Using cholesterol-modified DNA tethers and complementary DNA block copolymers, exosome surfaces were engineered with different biocompatible polymers. Additionally, polymers were directly grafted from the exosome surface using biocompatible photo-mediated atom transfer radical polymerization (ATRP). These exosome polymer hybrids (EPHs) exhibited enhanced stability under various storage conditions and in the presence of proteolytic enzymes. Tuning of the polymer length and surface loading allowed precise control over exosome surface interactions, cellular uptake, and preserved bioactivity. EPHs show fourfold higher blood circulation time without altering tissue distribution profiles. Our results highlight the potential of precise nanoengineering of exosomes toward developing advanced drug and therapeutic delivery systems using modern ATRP methods.
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http://dx.doi.org/10.1073/pnas.2020241118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7812758PMC
January 2021

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

Sci Rep 2020 Aug 17;10(1):14027. Epub 2020 Aug 17.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-70340-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431546PMC
August 2020

Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes.

Neurooncol Adv 2020 Jan-Dec;2(1):vdaa056. Epub 2020 May 6.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

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 ( < .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.
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http://dx.doi.org/10.1093/noajnl/vdaa056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262743PMC
May 2020

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

Int J Mol Sci 2020 Jun 2;21(11). Epub 2020 Jun 2.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

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.
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http://dx.doi.org/10.3390/ijms21113990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312363PMC
June 2020

A Potent Branched-Tail Lipid Nanoparticle Enables Multiplexed mRNA Delivery and Gene Editing .

Nano Lett 2020 07 9;20(7):5167-5175. Epub 2020 Jun 9.

Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States.

The clinical translation of messengerRNA (mRNA) drugs has been slowed by a shortage of delivery vehicles that potently and safely shuttle mRNA into target cells. Here, we describe the properties of a particularly potent branched-tail lipid nanoparticle that delivers mRNA to >80% of three major liver cell types. We characterize mRNA delivery spatially, temporally, and as a function of injection type. Following intravenous delivery, our lipid nanoparticle induced greater protein expression than two benchmark lipids, C12-200 and DLin-MC3-DMA, at an mRNA dose of 0.5 mg/kg. Lipid nanoparticles were sufficiently potent to codeliver three distinct mRNAs (firefly luciferase, mCherry, and erythropoietin) and, separately, Cas9 mRNA and single guide RNA (sgRNA) for proof-of-concept nonviral gene editing in mice. Furthermore, our branched-tail lipid nanoparticle was neither immunogenic nor toxic to the liver. Together, these results demonstrate the unique potential of this lipid material to improve the management of diseases rooted in liver dysfunction.
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http://dx.doi.org/10.1021/acs.nanolett.0c00596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781386PMC
July 2020

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

Angiogenesis 2020 11 18;23(4):599-610. Epub 2020 May 18.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA.

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 AR, AR and AR 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 AR-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 AR signaling (p < 0.01). In vivo, TEX stimulated the formation of defined vascular structures and macrophage infiltration. This response was absent in AR rats (p < 0.05).

Conclusion: This report provides the first evidence for adenosine production by TEX to promote angiogenesis via AR. AR antagonism emerges as a potential strategy to block TEX-induced angiogenesis.
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http://dx.doi.org/10.1007/s10456-020-09728-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529853PMC
November 2020

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

Sci Rep 2020 04 24;10(1):6948. Epub 2020 Apr 24.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA.

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.
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http://dx.doi.org/10.1038/s41598-020-63658-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181876PMC
April 2020

Inkjet Printing of Curing Agent on Thin PDMS for Local Tailoring of Mechanical Properties.

Macromol Rapid Commun 2020 Mar 29;41(5):e1900569. Epub 2020 Jan 29.

Department of Electrical and Computer Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA.

Rapid prototyping of thin, stretchable substrates with engineered stiffness gradients at desired locations has potential impact in the robustness of skin-wearable electronics, as the gradients can inhibit cracking of interconnect and delamination of embedded electronic chips. Drop-on-demand inkjetting of thinned polydimethylsiloxane (PDMS) curing agent onto a spin-cast 80 µm-thick 20:1 (base: curing agent) PDMS substrate sets the elastic modulus of the subsequently cured film with sub-millimeter accuracy. The inkjet process creates digitally defined stiffness gradient spans as small as 100 µm for single droplets. Varying the drop density results in differences in elastic modulus of up to 80%. In jetting tests of curing agent into pure base PDMS, a continuous droplet spacing of 100 µm results in smooth lines with total widths of 1 mm and a curing agent gradient span of ≈300 µm. Release of freeform mesh elastomer microstructures by removing the uncured base after selective jetting of curing agent into pure base PDMS results in structural line width resolution down to 500 µm.
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http://dx.doi.org/10.1002/marc.201900569DOI Listing
March 2020

Covalent Poly(lactic acid) Nanoparticles for the Sustained Delivery of Naloxone.

ACS Appl Bio Mater 2019 Aug 25;2(8):3418-3428. Epub 2019 Jul 25.

Neuroscience Disruptive Research Lab, Allegheny Health Network Research Institute, Allegheny General Hospital, Pittsburgh, Pennsylvania 15212, United States.

The opioid epidemic currently plaguing the United States has been exacerbated by an alarming rise in fatal overdoses as a result of the proliferated abuse of synthetic mu opioid receptor (MOR) agonists, such as fentanyl and its related analogues. Attempts to manage this crisis have focused primarily on widespread distribution of the clinically approved opioid reversal agent naloxone (Narcan); however, due to the intrinsic metabolic lability of naloxone, these measures have demonstrated limited effectiveness against synthetic opioid toxicity. This work reports a novel polymer-based strategy to create a long-acting formulation of naloxone with the potential to address this critical issue by utilizing covalent nanoparticle (NP) drug delivery technology. Covalently loaded naloxone nanoparticles (Nal-NPs) were prepared via the naloxone-initiated, ring-opening polymerization (ROP) of l-lactide in the presence of a bifunctional thiourea organocatalyst with subsequent precipitation of the resulting naloxone-poly(l-lactic acid) polymer. This protocol afforded well-defined nanoparticles possessing a drug loading of approximately 7% w/w. The resulting Nal-NPs demonstrated excellent biocompatibility, while exhibiting sustained linear release kinetics and blocking the effects of high dose (10 mg/kg) acute morphine for up to 98 h in an rodent model of neuropathic pain.
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http://dx.doi.org/10.1021/acsabm.9b00380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731033PMC
August 2019

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

ACS Nano 2019 09 27;13(9):10555-10565. Epub 2019 Aug 27.

Department of Chemistry , Carnegie Mellon University , Pittsburgh , Pennsylvania 15213 , United States.

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 and . 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.
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http://dx.doi.org/10.1021/acsnano.9b04651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800810PMC
September 2019

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

Oncoimmunology 2019;8(7):1593805. Epub 2019 Apr 24.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

: 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. : 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. : 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. : 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.
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http://dx.doi.org/10.1080/2162402X.2019.1593805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527269PMC
April 2019

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

Plast Reconstr Surg 2019 06;143(6):1215e-1223e

From the Department of Plastic Surgery, University of Pittsburgh; Biomedical Engineering and Biological Sciences and The Robotics Institute, Carnegie Mellon University; and The Ohio State University College of Medicine.

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.
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http://dx.doi.org/10.1097/PRS.0000000000005640DOI Listing
June 2019

Degradable Polymer Stars Based on Tannic Acid Cores by ATRP.

Polymers (Basel) 2019 Apr 28;11(5). Epub 2019 Apr 28.

Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.

Degradable polymers are crucial in order to reduce plastic environmental pollution and waste accumulation. In this paper, a natural product, tannic acid was modified to be used as a polymer star core. The tannic acid was modified with atom transfer radical polymerization (ATRP) initiators and characterized by H NMR, FT-IR, and XPS. Twenty-five arm polymer stars were prepared by photoinduced ATRP of poly(methyl methacrylate) (PMMA) or poly(oligo(ethylene oxide) methacrylate) (molar mass M = 300 g/mol) (P(OEOMA)). The polymer stars were degraded by cleaving the polymer star arms attached to the core by phenolic esters under mild basic conditions. The stars were analyzed before and after degradation by gel permeation chromatography (GPC). Cytotoxicity assays were performed on the P(OEOMA) stars and corresponding degraded polymers, and were found to be nontoxic at the concentrations tested.
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http://dx.doi.org/10.3390/polym11050752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571670PMC
April 2019

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

Exp Cell Res 2019 05 8;378(2):149-157. Epub 2019 Mar 8.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA; Departments of Immunology and Otolaryngology, Pittsburgh, PA 15213, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.yexcr.2019.03.014DOI Listing
May 2019

Molecular and Functional Profiles of Exosomes From HPV(+) and HPV(-) Head and Neck Cancer Cell Lines.

Front Oncol 2018 12;8:445. Epub 2018 Oct 12.

UPMC Hillman Cancer Center, Pittsburgh, PA, United States.

Exosomes produced by tumor cells have been shown to reprogram functions of human immune cells. Molecular cargos of exosomes isolated from supernatants of HPV(+) and HPV(-) head and neck cancer (HNC) cell lines or from HNC patients' plasma were compared. The exosome protein profiles resembled those of respective parent tumor cells. Only HPV(+) exosomes carried E6/E7, p16, and survivin. HPV(-) exosomes were negative for cyclin D1 and carried low p53 levels. Immunomodulatory molecules (TGF-β, FasL, OX40, OX40L, and HSP70) were carried by HPV(+) and HPV(-) exosomes. These exosomes co-incubated with human T cells induced apoptosis and suppressed T cell activation and proliferation. HPV(-) exosomes suppressed DC maturation and expression of antigen processing machinery (APM) components. In contrast, HPV(+) exosomes promoted DC maturation and did not suppress expression of APM components in mature DCs. While DCs readily internalized exosomes, T lymphocytes resisted their uptake during the initial 12 h co-culture. Thus, HPV(+) exosomes capable of sustaining DC functions may play a key role in promoting anti-tumor immune responses thereby improving outcome in patients with HPV(+) cancers.
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http://dx.doi.org/10.3389/fonc.2018.00445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194188PMC
October 2018

Reconstruction of a Calvarial Wound Complicated by Infection: Comparing the Effects of Biopatterned Bone Morphogenetic Protein 2 and Vascular Endothelial Growth Factor.

J Craniofac Surg 2019 Jan;30(1):260-264

Department of Plastic Surgery, University of Pittsburgh.

Bone morphogenetic protein 2 (BMP2) bioprinted on biological matrix induces osseous regeneration in large calvarial defects in rabbits, both uncomplicated and scarred. Healing in unfavorable defects scarred from previous infection is decreased due in part to the lack of vascularity. This impedes the access of mesenchymal stem cells, key to osseous regeneration and the efficacy of BMP2, to the wound bed. The authors hypothesized that bioprinted vascular endothelial growth factor (VEGF) would augment the osseous regeneration achieved with low dose biopatterned BMP2 alone. Thirteen New Zealand white rabbits underwent subtotal calvariectomy using a dental cutting burr. Care was taken to preserve the underlying dura. A 15 mm × 15 mm flap of bone was cut away and incubated in a 1 × 108 cfu/mL planktonic solution of S aureus before reimplantation. After 2 weeks of subsequent infection the flap was removed and the surgical wound debrided followed by 10 days of antibiotic treatment. On postoperative day 42 the calvarial defects were treated with acellular dermal matrix bioprinted with nothing (control), VEGF, BMP2, BMP2/VEGF combined. Bone growth was analyzed with serial CT and postmortem histology. Defects treated with BMP2 (BMP2 alone and BMP2/VEGF combination) showed significantly greater healing than control and VEGF treated defect (P < 0.5). Vascular endothelial growth factor treated defect demonstrated less healing than control and VEGF/BMP2 combination treatments achieved less healing than BMP2 alone though these differences were nonsignificant. Low dose BMP2-patterned acellular dermal matrix improves healing of scarred calvarial defects. Vascular endothelial growth factor at the doses applied in this study failed to increase healing.
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http://dx.doi.org/10.1097/SCS.0000000000004779DOI Listing
January 2019

Exosomes from HNSCC Promote Angiogenesis through Reprogramming of Endothelial Cells.

Mol Cancer Res 2018 11 24;16(11):1798-1808. Epub 2018 Jul 24.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.

For solid tumors, such as head and neck squamous cell carcinoma (HNSCC), an adequate blood supply is of critical importance for tumor development and metastasis. Tumor-derived exosomes (TEX) accumulate in the tumor microenvironment (TME) and serve as a communication system between tumor and normal stromal cells. This study evaluates and effects mediated by TEX that result in promotion of angiogenesis. TEX produced by PCI-13 (HPV) and UMSCC47 (HPV) cell lines or from plasma of HNSCC patients were isolated by mini size exclusion chromatography (mini-SEC). TEX morphology, size, numbers, and molecular profile were characterized, and the angiogenesis-inducing potential was measured in arrays and real-time PCR with human endothelial cells (HUVEC). Uptake of labeled TEX by HUVECs was demonstrated by confocal microscopy. Tube formation, proliferation, migration, and adherence by HUVECs in response to TEX were investigated. The 4-nitroquinoline-1-oxide (4-NQO) oral carcinogenesis mouse model was used to confirm that TEX induce the same results TEX were found to be potent inducers of angiogenesis and through functional reprogramming and phenotypic modulation of endothelial cells. TEX carried angiogenic proteins and were internalized by HUVECs within 4 hours. TEX stimulated proliferation ( < 0.001), migration ( < 0.05), and tube formation ( < 0.001) by HUVECs and promoted formation of defined vascular structures The data suggest that TEX promote angiogenesis and drive HNSCC progression. Future efforts should focus on eliminating or silencing TEX and thereby adding new options for improving existing antiangiogenic therapies. TEX appear to play an important role in tumor angiogenesis and thus may contribute to tumor growth and metastasis of HNSCC in this context. .
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0358DOI Listing
November 2018

Plasma-derived Exosomes Reverse Epithelial-to-Mesenchymal Transition after Photodynamic Therapy of Patients with Head and Neck Cancer.

Oncoscience 2018 Mar 29;5(3-4):75-87. Epub 2018 Apr 29.

Department of Pathology, University of Pittsburgh School of Medicine and UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA.

Photodynamic therapy (PDT) is a palliative treatment option for head and neck squamous cell carcinoma (HNSCC) patients which induces local inflammation and alters tumor cell morphology. We show that exosomes in plasma of HNSCC patients undergoing PDT reprogram tumor cells towards an epithelial phenotype. Nine HNSCC patients were treated with PDT and plasma was collected prior to and at three timepoints after therapy. Exosome levels of E-Cadherin, N-Cadherin and TGF-β1 were tested by flow cytometry. Exosomes were co-incubated with cancer cells, and changes in expression of EMT markers were evaluated as were proliferation, migration, chemotaxis and invasiveness of tumor cells. Exosomes harvested pre- and 24h after PDT were enriched in N-Cadherin and TGF-β1. They induced the mesenchymal phenotype and up-regulated Vimentin and transcripts for Snail, Twist, α-SMA, Slug and ZEB1 in epithelial tumor cells. These exosomes also enhanced tumor proliferation, migration and invasion. In contrast, exosomes obtained on day 7 or 4-6 weeks after PDT carried E-cadherin, restored epithelial morphology and EpCAM expression in tumor cells, down-regulated expression of mesenchymal genes and inhibited proliferation, migration and invasion. The PDT-mediated conversion from the mesenchymal to epithelial tumor phenotype was mediated by exosomes, which also served as non-invasive biomarkers of this transition.
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http://dx.doi.org/10.18632/oncoscience.410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978437PMC
March 2018

Helicase-Driven Activation of NFκB-COX2 Pathway Mediates the Immunosuppressive Component of dsRNA-Driven Inflammation in the Human Tumor Microenvironment.

Cancer Res 2018 08 31;78(15):4292-4302. Epub 2018 May 31.

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania.

Presence of cytotoxic CD8 T cells (CTL) in tumor microenvironments (TME) is critical for the effectiveness of immune therapies and patients' outcome, whereas regulatory T(reg) cells promote cancer progression. Immune adjuvants, including double-stranded (ds)RNAs, which signal via Toll-like receptor-3 (TLR3) and helicase (RIG-I/MDA5) pathways, all induce intratumoral production of CTL-attractants, but also Treg attractants and suppressive factors, raising the question of whether induction of these opposing groups of immune mediators can be separated. Here, we use human tumor explant cultures and cell culture models to show that the (ds) RNA Sendai Virus (SeV), poly-I:C, and rintatolimod (poly-I:CU) all activate the TLR3 pathway involving TRAF3 and IRF3, and induce IFNα, ISG-60, and CXCL10 to promote CTL chemotaxis to -treated tumors. However, in contrast with SeV and poly I:C, rintatolimod did not activate the MAVS/helicase pathway, thus avoiding NFκB- and TNFα-dependent induction of COX2, COX2/PGE2-dependent induction of IDO, IL10, CCL22, and CXCL12, and eliminating Treg attraction. Induction of CTL-attractants by either poly I:C or rintatolimod was further enhanced by exogenous IFNα (enhancer of TLR3 expression), whereas COX2 inhibition enhanced the response to poly-I:C only. Our data identify the helicase/NFκB/TNFα/COX2 axis as the key suppressive pathway of dsRNA signaling in human TME and suggest that selective targeting of TLR3 or elimination of NFκB/TNFα/COX2-driven suppression may allow for selective enhancement of type-1 immunity. This study characterizes two different poly-I:C-induced signaling pathways in their induction of immunostimulatory and suppressive factors and suggests improved ways to reprogram the TME to enhance the antitumor efficacy of immunotherapies. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-3985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6636317PMC
August 2018

The CARMA3-Bcl10-MALT1 Signalosome Drives NFκB Activation and Promotes Aggressiveness in Angiotensin II Receptor-Positive Breast Cancer.

Cancer Res 2018 03 19;78(5):1225-1240. Epub 2017 Dec 19.

Departments of Pathology and Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.

The angiotensin II receptor AGTR1, which mediates vasoconstrictive and inflammatory signaling in vascular disease, is overexpressed aberrantly in some breast cancers. In this study, we established the significance of an AGTR1-responsive NFκB signaling pathway in this breast cancer subset. We documented that AGTR1 overexpression occurred in the luminal A and B subtypes of breast cancer, was mutually exclusive of HER2 expression, and correlated with aggressive features that include increased lymph node metastasis, reduced responsiveness to neoadjuvant therapy, and reduced overall survival. Mechanistically, AGTR1 overexpression directed both ligand-independent and ligand-dependent activation of NFκB, mediated by a signaling pathway that requires the triad of CARMA3, Bcl10, and MALT1 (CBM signalosome). Activation of this pathway drove cancer cell-intrinsic responses that include proliferation, migration, and invasion. In addition, CBM-dependent activation of NFκB elicited cancer cell-extrinsic effects, impacting endothelial cells of the tumor microenvironment to promote tumor angiogenesis. CBM/NFκB signaling in AGTR1 breast cancer therefore conspires to promote aggressive behavior through pleiotropic effects. Overall, our results point to the prognostic and therapeutic value of identifying AGTR1 overexpression in a subset of HER2-negative breast cancers, and they provide a mechanistic rationale to explore the repurposing of drugs that target angiotensin II-dependent NFκB signaling pathways to improve the treatment of this breast cancer subset. These findings offer a mechanistic rationale to explore the repurposing of drugs that target angiotensin action to improve the treatment of AGTR1-expressing breast cancers. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-1089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436094PMC
March 2018

Clinical Significance of PD-L1 Exosomes in Plasma of Head and Neck Cancer Patients.

Clin Cancer Res 2018 02 12;24(4):896-905. Epub 2017 Dec 12.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.

The microenvironment of head and neck squamous cell carcinomas (HNSCC) is highly immunosuppressive. HNSCCs expressing elevated levels of PD-L1 have especially poor outcome. Exosomes that carry PD-L1 and suppress T-cell functions have been isolated from plasma of patients with HNSCC. The potential contributions of PD-L1 exosomes to immune suppression and disease activity are evaluated. Exosomes isolated from plasma of 40 HNSCC patients by size exclusion chromatography were captured on beads using anti-CD63 Abs, stained for PD-1 and PD-L1 and analyzed by flow cytometry. The percentages and mean fluorescence intensities (MFI) of PD-L1 and PD-1 exosome/bead complexes were correlated with the patients' clinicopathologic data. PD-L1 or PD-L1 exosomes were incubated with activated CD69 human CD8 T cells ± PD-1 inhibitor. Changes in CD69 expression levels on T cells were measured. Patients' plasma was tested for soluble PD-L1 (sPD-L1) by ELISA. Levels of PD-L1 carried by exosomes correlated with patients' disease activity, the UICC stage and the lymph node status ( = 0.0008-0.013). In contrast, plasma levels of sPD-L1 or exosome PD-1 levels did not correlate with any clinicopathologic parameters. CD69 expression levels were inhibited ( < 0.03) by coincubation with PD-L1 but not by PD-L1 exosomes. Blocking of PD-L1 exosome signaling to PD-1 T cells attenuated immune suppression. PD-L1 levels on exosomes, but not levels of sPD-L1, associated with disease progression in HNSCC patients. Circulating PD-L1 exosomes emerge as useful metrics of disease and immune activity in HNSCC patients.

Significance: Circulating PD-L1 exosomes in HNC patients' plasma but not soluble PD-L1 levels associate with disease progression. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-2664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126905PMC
February 2018

Circulating exosomes carrying an immunosuppressive cargo interfere with cellular immunotherapy in acute myeloid leukemia.

Sci Rep 2017 10 31;7(1):14684. Epub 2017 Oct 31.

UPMC Hillman Cancer Center, Pittsburgh, PA, USA.

Exosomes, small (30-150 nm) extracellular vesicles (EVs) isolated from plasma of patients with acute myeloid leukemia (AML) carry leukemia-associated antigens and multiple inhibitory molecules. Circulating exosomes can deliver suppressive cargos to immune recipient cells, inhibiting anti-tumor activities. Pre-therapy plasma of refractory/relapsed AML patients contains elevated levels of immunosuppressive exosomes which interfere with anti-leukemia functions of activated immune cells. We show that exosomes isolated from pre-therapy plasma of the AML patients receiving adoptive NK-92 cell therapy block anti-leukemia cytotoxicity of NK-92 cells and other NK-92 cell functions. NK-92 cells do not internalize AML exosomes. Instead, signaling via surface receptors expressed on NK-92 cells, AML exosomes simultaneously deliver multiple inhibitory ligands to the cognate receptors. The signals are processed downstream and activate multiple suppressive pathways in NK-92 cells. AML exosomes reprogram NK-92 cells, interfering with their anti-leukemia functions and reducing the therapeutic potential of adoptive cell transfers. Plasma-derived exosomes interfere with immune cells used for adoptive cell therapy and may limit expected therapeutic benefits of adoptive cell therapy.
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http://dx.doi.org/10.1038/s41598-017-14661-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666018PMC
October 2017

Perivascular extracellular matrix hydrogels mimic native matrix microarchitecture and promote angiogenesis via basic fibroblast growth factor.

Biomaterials 2017 04 30;123:142-154. Epub 2017 Jan 30.

Department of Cardiothoracic Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA.

Extracellular matrix (ECM)-derived bioscaffolds have been shown to elicit tissue repair through retention of bioactive signals. Given that the adventitia of large blood vessels is a richly vascularized microenvironment, we hypothesized that perivascular ECM contains bioactive signals that influence cells of blood vessel lineages. ECM bioscaffolds were derived from decellularized human and porcine aortic adventitia (hAdv and pAdv, respectively) and then shown have minimal DNA content and retain elastin and collagen proteins. Hydrogel formulations of hAdv and pAdv ECM bioscaffolds exhibited gelation kinetics similar to ECM hydrogels derived from porcine small intestinal submucosa (pSIS). hAdv and pAdv ECM hydrogels displayed thinner, less undulated, and fibrous microarchitecture reminiscent of native adventitia, with slight differences in ultrastructure visible in comparison to pSIS ECM hydrogels. Pepsin-digested pAdv and pSIS ECM bioscaffolds increased proliferation of human adventitia-derived endothelial cells and this effect was mediated in part by basic fibroblast growth factor (FGF2). Human endothelial cells cultured on Matrigel substrates formed more numerous and longer tube-like structures when supplemented with pAdv ECM bioscaffolds, and FGF2 mediated this matrix signaling. ECM bioscaffolds derived from pAdv promoted FGF2-dependent in vivo angiogenesis in the chick chorioallantoic membrane model. Using an angiogenesis-focused protein array, we detected 55 angiogenesis-related proteins, including FGF2 in hAdv, pAdv and pSIS ECMs. Interestingly, 19 of these factors were less abundant in ECMs bioscaffolds derived from aneurysmal specimens of human aorta when compared with non-aneurysmal (normal) specimens. This study reveals that Adv ECM hydrogels recapitulate matrix fiber microarchitecture of native adventitia, and retain angiogenesis-related actors and bioactive properties such as FGF2 signaling capable of influencing processes important for angiogenesis. This work supports the use of Adv ECM bioscaffolds for both discovery biology and potential translation towards microvascular regeneration in clinical applications.
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http://dx.doi.org/10.1016/j.biomaterials.2017.01.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5319845PMC
April 2017

Controlled Release of Small Molecules from Elastomers for Reducing Epidermal Downgrowth in Percutaneous Devices.

ACS Biomater Sci Eng 2016 Sep 18;2(9):1464-1470. Epub 2016 Aug 18.

Department of Biomedical Engineering, Institute for Complex Engineered Systems, and Department of Materials Science and Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213, United States.

The elevated infection rise associated with indwelling devices can compromise the performance of percutaneous devices and increase the risk of complications. High infection rates are associated with both the high bacterial load on the skin and epidermal downgrowth at the interface of the indwelling material. Here, we propose a drug-eluting material that promotes local dermal regeneration to reduce epidermal downgrowth. Mesoporous elastomeric matrices composed of naturally occurring monomers were prepared by a combination of photo- and thermal-crosslinking. Elastomeric devices loaded with conjugated linoleic acids (CLA), a class of small molecules that promote local anti-inflammatory responses, can deliver these compounds for 7 d ( = 3.94 × 10 cm/s, 95% CI [3.12 × 10, 4.61 × 10]). In a mouse model, CLA-eluting elastomeric matrices increase the M2 population (5.0 × 10 ± 1.4 × 10 cells/cm), compared to blank devices (3.8 × 10 ± 2.2 × 10 cells/cm), and also reduce skin contraction (98.9 ± 6.4%), compared to blank devices (70.9 ± 9.3%) at 7 d. Dermal downgrowth is also attenuated at 14 d (60.4 ± 32.4 μm) compared to blank devices (171.7 ± 93.8 μm). CLA-eluting elastomers are therefore a viable strategy to reduce epidermal downgrowth in percutaneous devices.
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http://dx.doi.org/10.1021/acsbiomaterials.6b00192DOI Listing
September 2016