scholarly journals CD146+CD107a+ Mesenchymal Stem/Stromal Cells with Signature Attributes Correlate to Therapeutic Potency as “First Responders” to Injury and Inflammation

2019 ◽  
Author(s):  
Annie C. Bowles ◽  
Dimitrios Kouroupis ◽  
Melissa A. Willman ◽  
Carlotta Perucca Orfei ◽  
Ashutosh Agarwal ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Immune Cell ◽  
First Responders ◽  
Infrapatellar Fat Pad ◽  
M2 Macrophage ◽  
List Type ◽  
Fat Pad ◽  
Secretory Capacity

ABSTRACTCD146+ bone marrow–derived Mesenchymal Stem/Stromal Cells (BM-MSC) play key roles in the perivascular niche, skeletogenesis and hematopoietic support, however elucidation of therapeutic potency has yet to be determined. Here, inflammatory challenge to crude BM-MSC captured a baseline of signatures including enriched expression of CD146+ with CD107a+, CXCR4+, and LepR+, transcriptional profile, enhanced secretory capacity, robust secretome and immunomodulatory function with stimulated target immune cells. These responses were significantly more pronounced in CD146+ (POS)-selected subpopulation than in the CD146- (NEG). Mechanistically, POS uniquely mediated robust immunosuppression while inducing significant frequencies of Naïve and Regulatory T cells in vitro. Moreover, POS promoted a pivotal M1-to-M2 macrophage shift in vivo, ameliorating inflammation/fibrosis of joint synovium and fat pad of the knee, failed by NEG. This study provides high-content evidence of CD146+CD107a+ BM-MSC, herein deemed ‘first responders’ to inflammation, as the underrepresented subpopulation within crude BM-MSC with innately higher secretory capacity and therapeutic potency.HIGHLIGHTSSignature phenotypic, transcriptional, and secretome profiles were identified and enriched in human CD146+ (POS)-selected subpopulation in response to inflammationInflammatory challenge consistently altered stemness (LIF) and differentiation master regulators (SOX9, RUNX2, PPARγ) in crude, POS, and NEG BM-MSC, and deduced unique expressions in POS compared to NEGPOS BM-MSC mediated the strongest immunomodulation, e.g. target immune cell suppression, Treg induction, diminished T cell differentiationPOS BM-MSC promoted the largest M1-to-M2 shift in vivo alleviating induced synovitis and infrapatellar fat pad fibrosis of the knee

871 In vitro immunoregulatory effect from cervical cancer derived mesenchymal stromal cells over molecules expression in monocyte derived macrophage polarization

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A923-A923
Author(s):  
Víctor Cortés-Morales ◽  
Juan Montesinos ◽  
Luis Chávez-Sánchez ◽  
Sandra Espíndola-Garibay ◽  
Alberto Monroy-García ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Macrophage Polarization ◽  
Induction Medium ◽  
M2 Macrophage ◽  
List Type ◽  
M2 Phenotype

BackgroundMacrophages are immunological cells that sense microenvironmental signals that may result in the polarized expression of either proinflammatory (M1) or anti-inflammatory (M2) phenotype.1 Macrophages M2 are present in tumoral microenvironment and their presence in patients with cervical cancer (CeCa) is related with less survival.2Mesenchymal Stromal Cells (MSCs) are also present in tumor microenvironment of cervical cancer (CeCa-MSC), which have shown immunoregulatory effects over CD8 T cells, decreasing their cytotoxic effect against tumoral cells.3 Interestingly, MSCs from bone marrow (BM-MSC) decrease M1 and increase M2 macrophage polarization in an in vitro coculture system.4 Macrophages and MSCs are present in microenvironment of cervical cancer, however it is unknown if MSCs play a role in macrophage polarization. In the present study, we have evaluated the immunoregulatory capacity of CeCa-MSCs to induce macrophage polarization.MethodsCD14 monocytes were isolated from peripheral blood and cultivated in the absence or presence of MSCs from BM, normal cervix (NCx) and CeCa. Two culture conditions were included, in the presence of induction medium to favors M1 (GM-CSF, LPS and IFNg) or M2 (M-CSF, IL-4 and IL-13) macrophage polarization. M1 (HLA-DR, CD80, CD86 and IFNg) or M2 (CD14, CD163, CD206, IDO and IL-10) macrophage molecular markers were evaluated by flow cytometry. Finally, we evaluated concentration of IL-10 and TNFa in conditioned medium form all coculture conditions.ResultsWe observed that CeCa-MSCs and BM-MSCs in presence of M1 induction medium, decreased M1 macrophage markers (HLA-II, CD80, CD86 and IFNg), and increase the expression of CD14 (M2 macrophage marker). Interestingly, in presence of M2 induction medium, BM-MSCs and CaCe-MSCs but not CxN-MSC increased CD163, CD206, IDO and IL-10 (M2 macrophage markers). We observed a decreased concentration of TNFa in the supernatant medium from all cocultures with MSCs, but only in presence of CeCa-MSCs, increased IL-10 concentration was detected in such cocultures.ConclusionsIn contrast to NCx-MSCs, CeCa-MSCs similarly to BM-MSCs have in vitro capacity to decrease M1 and increase M2 macrophage phenotype.AcknowledgementsAcknowledgments The authors are indebted to gratefully acknowledge to CONACYT (Grant No. 272793) and IMSS (Grant no. 1731) for support to Juan J. Montesinos research.ReferencesMartinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep 2014;6-13.Petrillo M, Zannoni GF, Martinelli E, et al. Polarization of tumor-associated macrophages toward M2 phenotype correlates with poor response to chemoradiation and reduced survival in patients with locally advanced cervical cancer. PLoS One 2015;10: e0136654.Montesinos JJ, Mora-García Mde L, et al. In vitro evidence of the presence of mesenchymal stromal cells in cervical cancer and their role in protecting cancer cells from cytotoxic T cell activity. Stem Cells Dev 2013;22:2508-2519.Vasandan AB, Jahnavi S, Shashank C. Human mesenchymal stem cells program macrophage plasticity by altering their metabolic status via a PGE 2-dependent mechanism. Sci Rep 2016;6:38308.

scholarly journals Perinatal Mesenchymal Stromal Cells and Their Possible Contribution to Fetal-Maternal Tolerance

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1401 ◽  
Author(s):  
Marta Magatti ◽  
Francesca Romana Stefani ◽  
Andrea Papait ◽  
Anna Cargnoni ◽  
Alice Masserdotti ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Immune Cell ◽  
Immune Cell Subsets ◽  
New Perspective ◽  
Antigen Presenting ◽  
Open Question ◽  
Maternal Tolerance

During pregnancy, a successful coexistence between the mother and the semi-allogenic fetus occurs which requires a dynamic immune system to guarantee an efficient immune protection against possible infections and tolerance toward fetal antigens. The mechanism of fetal-maternal tolerance is still an open question. There is growing in vitro and in vivo evidence that mesenchymal stromal cells (MSC) which are present in perinatal tissues have a prominent role in generating a functional microenvironment critical to a successful pregnancy. This review highlights the immunomodulatory properties of perinatal MSC and their impact on the major immune cell subsets present in the uterus during pregnancy, such as natural killer cells, antigen-presenting cells (macrophages and dendritic cells), and T cells. Here, we discuss the current understanding and the possible contribution of perinatal MSC in the establishment of fetal-maternal tolerance, providing a new perspective on the physiology of gestation.

scholarly journals Infrapatellar Fat Pad-derived Mesenchymal Stem Cell-based Spheroids Enhance Their Therapeutic Efficacy to Reverse Synovitis and Fat Pad Fibrosis

2020 ◽  
Author(s):  
Dimitrios Kouroupis ◽  
Melissa A Willman ◽  
Thomas M Best ◽  
Lee D Kaplan ◽  
Diego Correa
Keyword(s):  
Articular Cartilage ◽  
Rat Model ◽  
Cartilage Damage ◽  
Infrapatellar Fat Pad ◽  
Molecular Response ◽  
Fat Pad ◽  
Transcriptional Profiles ◽  
3D Spheroids

Abstract Background: To investigate the in vitro and in vivo anti-inflammatory/anti-fibrotic capacity of IFP-MSC manufactured as 3D spheroids. According to our hypothesis, IFP-MSC do not require prior cell priming to acquire a robust immunomodulatory phenotype in vitro in order to efficiently reverse synovitis and IFP fibrosis and secondarily delay articular cartilage damage in vivo.Methods: Human IFP-MSC immunophenotype, tri-potentiality, and transcriptional profiles were assessed in 3D settings. Multiplex secretomes were assessed in IFP-MSC spheroids [Crude (non-immunoselected), CD146+ or CD146- immunoselected cells] and compared with 2D cultures with and without prior inflammatory/fibrotic cell priming. Functionally, immunopotency limiting human PBMCs proliferation and effect on stimulated synoviocytes with inflammation and fibrotic cues. Finally, spheroids were tested in vivo in a rat model of acute synovitis/fat pad fibrosis.Results: Spheroids enhanced IFP-MSC phenotypic, transcriptional and secretory immunomodulatory profiles compared to 2D cultures. Further, CD146+ IFP-MSC spheroids showed enhanced secretory and transcriptional profiles, however, not reflected in a superior capacity to suppress activated PBMC suggesting 3D environment sufficient to induce an immunomodulatory phenotype. Crude IFP-MSC spheroids modulated the molecular response of synoviocytes previously exposed to inflammatory cues. Therapeutically, IFP-MSC spheroids retained Substance P degradation potential in vivo, while effectively induced resolution of inflammation/fibrosis of synovium and fat pad, halting the articular cartilage degradation in a rat model of progressive synovitis, fat pad fibrosis and osteoarthritis.Conclusions: 3D spheroids confer IFP-MSC a reproducible and enhanced immunomodulatory effect in vitro and in vivo, circumventing the requirement of non-compliant cell priming or selection before administration, thus streamlining cell products manufacturing protocols.

scholarly journals Gli1+ mesenchymal stromal cells modulate epithelial metaplasia in lung fibrosis

2019 ◽  
Author(s):  
Monica Cassandras ◽  
Chaoqun Wang ◽  
Jaymin Kathiriya ◽  
Tatsuya Tsukui ◽  
Peri Matatia ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Hedgehog Signaling ◽  
List Type ◽  
Basal Cells ◽  
Human Lungs ◽  
Metaplastic Epithelium ◽  
Organ Fibrosis

AbstractOrgan fibrosis is often accompanied by aberrant epithelial reprogramming, culminating in a transformed barrier composed of scar and metaplastic epithelium. Understanding how the scar promotes an abnormal epithelial response could better inform strategies to reverse the fibrotic damage. Here we show that Gli1+ mesenchymal stromal cells (MSCs), previously shown to contribute to myofibroblasts in the scar, promote metaplastic differentiation of airway progenitors into KRT5+ basal cells in vitro and in vivo. During fibrotic repair, Gli1+ MSCs integrate hedgehog activation to promote metaplastic KRT5 differentiation by upregulating BMP antagonism in the progenitor niche. Restoring the balance towards BMP activation attenuated metaplastic KRT5+ differentiation while promoting adaptive alveolar differentiation. Finally, fibrotic human lungs demonstrate altered BMP activation in the metaplastic epithelium. These findings show that Gli1+ MSCs integrate hedgehog signaling as a rheostat to control BMP activation in the progenitor niche to determine regenerative outcome in fibrosis.HighlightsGli1+ MSCs are required for metaplastic airway progenitor differentiation into KRT5+ basal cells.Hedgehog activation of MSCs promotes KRT5 differentiation of airway progenitors by suppressing BMP activation.Restoring BMP activation attenuates metaplastic KRT5 differentiationMetaplastic KRT5+ basal cells in human fibrotic lungs demonstrate altered BMP activation.

Regulatory-compliant conditions during cell product manufacturing enhance in vitro immunomodulatory properties of infrapatellar fat pad-derived mesenchymal stem/stromal cells

Cytotherapy ◽  
2020 ◽  
Vol 22 (11) ◽  
pp. 677-689
Author(s):  
Dimitrios Kouroupis ◽  
Annie C. Bowles ◽  
Dylan N. Greif ◽  
Clarissa Leñero ◽  
Thomas M. Best ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Infrapatellar Fat Pad ◽  
Fat Pad ◽  
Cell Product ◽  
Product Manufacturing ◽  
Immunomodulatory Properties

CD10/Neprilysin Enrichment in Infrapatellar Fat Pad–Derived Mesenchymal Stem Cells Under Regulatory-Compliant Conditions: Implications for Efficient Synovitis and Fat Pad Fibrosis Reversal

2020 ◽  
Vol 48 (8) ◽  
pp. 2013-2027 ◽  
Author(s):  
Dimitrios Kouroupis ◽  
Annie C. Bowles ◽  
Thomas M. Best ◽  
Lee D. Kaplan ◽  
Diego Correa
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Infrapatellar Fat Pad ◽  
Therapeutic Effects ◽  
Fat Pad ◽  
In Vitro Proliferation ◽  
Differentiation Potential

Background: Synovitis and infrapatellar fat pad (IFP) fibrosis participate in various conditions of the knee. Substance P (SP), a neurotransmitter secreted within those structures and historically associated with nociception, also modulates local neurogenic inflammatory and fibrotic responses. Exposure of IFP mesenchymal stem cells (IFP-MSCs) to a proinflammatory/profibrotic environment (ex vivo priming with TNFα, IFNγ, and CTGF) induces their expression of CD10/neprilysin, effectively degrading SP in vitro and in vivo. Purpose/Hypothesis: The purpose was to test the therapeutic effects of IFP-MSCs processed under regulatory-compliant protocols, comparing them side-by-side with standard fetal bovine serum (FBS)–grown cells. The hypothesis was that when processed under such protocols, IFP-MSCs do not require ex vivo priming to acquire a CD10-rich phenotype efficiently degrading SP and reversing synovitis and IFP fibrosis. Study Design: Controlled laboratory study. Methods: Human IFP-MSCs were processed in FBS or either of 2 alternative conditions—regulatory-compliant pooled human platelet lysate (hPL) and chemically reinforced medium (Ch-R)—and then subjected to proinflammatory/profibrotic priming with TNFα, IFNγ, and CTGF. Cells were assessed for in vitro proliferation, stemness, immunophenotype, differentiation potential, transcriptional and secretory profiles, and SP degradation. Based on a rat model of acute synovitis and IFP fibrosis, the in vivo efficacy of cells degrading SP plus reversing structural signs of inflammation and fibrosis was assessed. Results: When compared with FBS, IFP-MSCs processed with either hPL or Ch-R exhibited a CD10High phenotype and showed enhanced proliferation, differentiation, and immunomodulatory transcriptional and secretory profiles (amplified by priming). Both methods recapitulated and augmented the secretion of growth factors seen with FBS plus priming, with some differences between them. Functionally, in vitro SP degradation was more efficient in hPL and Ch-R, confirmed upon intra-articular injection in vivo where CD10-rich IFP-MSCs also dramatically reversed signs of synovitis and IFP fibrosis even without priming or at significantly lower cell doses. Conclusion: hPL and Ch-R formulations can effectively replace FBS plus priming to induce specific therapeutic attributes in IFP-MSCs. The resulting fine-tuned, regulatory-compliant, cell-based product has potential future utilization as a novel minimally invasive cell therapy for the treatment of synovitis and IFP fibrosis. Clinical Relevance: The therapeutic enhancement of IFP-MSCs manufactured under regulatory-compliant conditions suggests that such a strategy could accelerate the time from preclinical to clinical phases. The therapeutic efficacy obtained at lower MSC numbers than currently needed and the avoidance of cell priming for efficient results could have a significant effect on the design of clinical protocols to potentially treat conditions involving synovitis and IFP fibrosis.

IMMU-20. HYDROGEL-CXCL9 VACCINE RESULTS IN MRNA DELIVERY TO DENDRITIC CELLS AND POTENT ANTI-TUMOR RESPONSES IN GBM

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi96-vi96
Author(s):  
Farhad Dastmalchi ◽  
Aida Karachi ◽  
Yusuf Mehkri ◽  
Ashley O’Malley ◽  
Vignesh Subramaniam ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Single Dose ◽  
Nk Cells ◽  
Immune Cell ◽  
Control Group ◽  
Cell Trafficking ◽  
Fat Pad ◽  
Tumor Bearing

Abstract INTRODUCTION Our group and others have shown mRNA-vaccines have significant anti-tumor efficacy in the treatment of brain tumors and are currently being tested in first-in-human trials. To further enhance mRNA delivery, a hydrogel platform was developed with the addition of CXCL9 to promote immune cell trafficking. METHODS We generated the vaccine by utilizing a hydrogel platform. CXCL9 and Nano-mRNA were loaded into the hydrogel. In vitro recruitment of DCs and NK was evaluated by fluorescence microscopy. In vivo recruitment of immune cells was analyzed by flowcytometry after collecting the fat pad, spleen, and tumor samples from KR158b-luc and Gl261-gp100 tumor-bearing animals 3, 5 and 10 days after vaccine delivery. The efficacy of the vaccine was evaluated by measuring overall survival, and tumor growth was measured by IVIS live-imaging. RESULTS Dendritic cells(DCs) and natural killer(NK) cells were able to efficiently migrate within the hydrogel-CXCL9 platform and uptake and express mRNA in vivo. In vitro, the hydrogel-CXCL9 was combined with nanoparticles loaded with total tumor RNA, and the vaccine was delivered to KR-158-luc and GL261-gp100 tumor-bearing animals via mammary fat pad SQ injection. Flow cytometry of the fat pad and draining lymph nodes demonstrated showed significant recruitment of endogenous DCs including inflammatory-DC(P= 0.0035), conventional-DC1(P= 0.0076), pDC(P=0.0028), NK(p= 0.0025 compared to the control group. In two different tumor models, a single dose of the vaccine resulted in significant survival benefits compared to control animals (n=10,P< 0.0001). SQ injection was superior to intracranial injection of the vaccine. Vaccinated animals showed an increased number of antigen-specific CD8 T cells in spleen(P= 0.0001) and tumor-microenvironment(P= 0.0070). CONCLUSION The hydrogel-CXCL9 platform results in efficient delivery of mRNA loaded nanoparticles to endogenous DCs and also causes an upregulation of NK cells with resultant improved survival in murine GBM models including the highly resistant KR158 model with a single dose. Further studies are ongoing.

scholarly journals 888 An integrative approach to optimize a synthetic EphA2/CD137 agonist: balancing potency, physiochemical properties, and pharmacokinetics to achieve robust anti-tumor activity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A931-A931
Author(s):  
Punit Upadhyaya ◽  
Gemma Mudd ◽  
Kristen Hurov ◽  
Johanna Lahdenranta ◽  
Elizabeth Repash ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
Immune Cell ◽  
List Type ◽  
Continuous Exposure ◽  
Target Engagement ◽  
In Vivo Models ◽  
Anti Tumor Activity

BackgroundCD137 (4-1BB) is a resurging target in immunotherapy after the first generation of monoclonal antibodies were limited by hepatotoxicity1 or lack of efficacy.2 A new generation of CD137 agonists are now in clinical development but they exclusively utilize large molecules derived from recombinant technology and are associated with long circulating terminal half-lives.3–6 Unlike checkpoint inhibition where complete saturation of the receptors drives the reversal of immunosuppression, intermittent target engagement that reflects the physiological context of T cell co-stimulation may be more appropriate for a CD137 agonist.7 Bicyclic peptides or Bicycles are a class of small (MW~2kDa), highly constrained peptides characterized by formation of two loops cyclized around a symmetric scaffold. To develop a differentiated tumor antigen dependent CD137 agonist for treating EphA2 expressing solid tumors, we integrated structure activity relationship (SAR) data from biochemical binding studies and in-vitro and in-vivo models to understand the relationship between exposure, target engagement and preclinical efficacy.MethodsOver 150 different EphA2/CD137 tumor-targeted immune cell agonists (Bicycle TICAs) were synthesized by linking Bicycle® binders to EphA2 to those binding CD137.8 The molecules were assessed in vitro using a CD137 reporter assay and by measuring cytokine production from primary human PBMC in tumor cell co-cultures. The pharmacokinetics were evaluated in rodents using Phoenix WinNonlin. The in vivo activity was determined in syngeneic mouse tumor models by measuring tumor growth kinetics and using tumor immune cell and transcriptional profiling by IHC and NanoString.ResultsEvaluation of the Bicycle TICAs in co-culture assays with EphA2-expressing tumor cell lines and Jurkat reporter cells overexpressing CD137 or human PBMCs demonstrated that constructs bearing two CD137 binding Bicycles to one EphA2 binding Bicycle (1:2 format) were more potent than the 1:1 format.8 Several Bicycle TICAs with amino acid substitutions to the EphA2 binding Bicycle maintained sub-nanomolar potency in-vitro and exhibited a plasma terminal half-life (t1/2) in rodents ranging from 0.4 and 4.0 h. Modifications that conferred aqueous solubility of greater than 10 mg/mL were considered suitable for further development. Treatment of MC38 tumors in immunocompetent mice with this series of molecules demonstrated that low MW Bicycle TICAs with sub-nanomolar potency and a t½ of ~1 h in mouse maintained target coverages necessary to produce robust modulation of the tumor immune microenvironment and tumor regression.ConclusionsA differentiated EphA2-dependent CD137 agonist was developed that exploits intermittent rather than continuous exposure for robust anti-tumor activity.ReferencesSegal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res 2017;23(8):1929–1936.Segal NH, Aiwu RH, Toshihiko D, et al. Phase I study of single-agent utomilumab (PF-05082566), a 4-1BB/CD137 agonist, in patients with advanced cancer. Clin Cancer Res 2018;24(8):1816–1823.Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1):49–57.Hinner MJ, Aiba RSB, Jaquin TJ, et al. Tumor-localized costimulatory T-cell engagement by the 4-1BB/HER2 bispecific antibody-anticalin fusion PRS-343. Clin Cancer Res 2019;25(19):5878–5889.Claus C, Ferrara, C, Xu W, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med 2019;11(496):eaav5989.Eskiocak U, Guzman W, Wolf B, et al. Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity. JCI Insight 2020;5(5):e133647.Mayes PA, Hance KW, Hoos A. The promise and challenges of immune agonist antibody development in cancer. Nat Rev Drug Discov 2018;17:509–27.Upadhyaya P, Lahdenranta J, Hurov K, et al. Anticancer immunity induced by a synthetic tumor-targeted CD137 agonist. J Immunother Cancer 2021;9:e001762.

scholarly journals IL-33 Mediates Lung Inflammation by the IL-6-Type Cytokine Oncostatin M

2020 ◽  
Vol 2020 ◽  
pp. 1-17
Author(s):  
Fernando Botelho ◽  
Anisha Dubey ◽  
Ehab A. Ayaub ◽  
Rex Park ◽  
Ashley Yip ◽  
...  
Keyword(s):  
Lung Inflammation ◽  
Immune Cell ◽  
Interleukin 1 ◽  
Oncostatin M ◽  
Mouse Lung ◽  
M2 Macrophage ◽  
Protein Levels ◽  
Arginase 1

The interleukin-1 family member IL-33 participates in both innate and adaptive T helper-2 immune cell responses in models of lung disease. The IL-6-type cytokine Oncostatin M (OSM) elevates lung inflammation, Th2-skewed cytokines, alternatively activated (M2) macrophages, and eosinophils in C57Bl/6 mice in vivo. Since OSM induces IL-33 expression, we here test the IL-33 function in OSM-mediated lung inflammation using IL-33-/- mice. Adenoviral OSM (AdOSM) markedly induced IL-33 mRNA and protein levels in wild-type animals while IL-33 was undetectable in IL-33-/- animals. AdOSM treatment showed recruitment of neutrophils, eosinophils, and elevated inflammatory chemokines (KC, eotaxin-1, MIP1a, and MIP1b), Th2 cytokines (IL-4/IL-5), and arginase-1 (M2 macrophage marker) whereas these responses were markedly diminished in IL-33-/- mice. AdOSM-induced IL-33 was unaffected by IL-6-/- deficiency. AdOSM also induced IL-33R+ ILC2 cells in the lung, while IL-6 (AdIL-6) overexpression did not. Flow-sorted ILC2 responded in vitro to IL-33 (but not OSM or IL-6 stimulation). Matrix remodelling genes col3A1, MMP-13, and TIMP-1 were also decreased in IL-33-/- mice. In vitro, IL-33 upregulated expression of OSM in the RAW264.7 macrophage cell line and in bone marrow-derived macrophages. Taken together, IL-33 is a critical mediator of OSM-driven, Th2-skewed, and M2-like responses in mouse lung inflammation and contributes in part through activation of ILC2 cells.

scholarly journals Methylthioadenosine is Not Dramatically Elevated in MTAP-Homozygous Deleted Primary Glioblastomas

2019 ◽  
Author(s):  
Yasaman Barekatain ◽  
Victoria C. Yan ◽  
Jeffrey J. Ackroyd ◽  
Anton H. Poral ◽  
Theresa Tran ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Stromal Cells ◽  
Homozygous Deletion ◽  
Human Tumors ◽  
Normal Brain ◽  
List Type ◽  
Human Glioblastoma ◽  
Frequent Event

In BriefThe co-deletion of MTAP in the CDKN2A locus is a frequent event in diverse cancers including glioblastoma. Recent publications report that significant accumulations of the MTAP substrate, methylthioadenosine (MTA), can sensitize MTAP-deleted cancer cells to novel inhibitors of PRMT5 and MAT2A for targeted therapy against tumors with this particular genetic alteration. In this work, using comprehensive metabolomic profiling, we show that MTA is primarily secreted, resulting in exceedingly high levels of extracellular MTA in vitro. We further show that primary human glioblastoma tumors minimally accumulate MTA in vivo, which is likely explained by the metabolism of MTA by MTAP-competent stromal cells. Together, these data challenge whether the metabolic conditions required for therapies to exploit vulnerabilities associated MTAP deletions are present in primary human tumors, questioning their translational efficacy in the clinic.HighlightsMethylthioadenosine (MTA) is elevated in MTAP-deleted cancer cells in vitro, which provides a selective vulnerability to PRMT5 and MAT2A inhibitorsAccumulation of MTA in MTAP-deleted cancer cells is predominately extracellular, suggesting active secretion of MTA.MTAP-deleted primary human glioblastoma tumors show minimal intratumoral elevations of MTA, which is likely explained by secretion and metabolism by MTAP-competent stromal cells.SUMMARYHomozygous deletion of the CDK2NA locus frequently results in co-deletion of methylthioadenosine phosphorylase (MTAP) in many fatal cancers such as glioblastoma multiforme (GBM), resulting in elevations of the substrate metabolite, methylthioadenosine (MTA). To capitalize on such accumulations, therapeutic targeting of protein arginine methyltransferase 5 (PRMT5) and methionine adenosyl transferase (MAT2A) are ongoing. While extensively corroborated in vitro, the clinical efficacy of these strategies ultimately relies on equally significant accumulations of MTA in human tumors. Here, we show that in vitro accumulation of MTA is a predominately extracellular phenomenon, indicating secretion of MTA from MTAP-deleted cells. In primary human GBMs, we find that MTA levels are not significantly higher in MTAP-deleted compared to MTAP-intact tumors or normal brain tissue. Together, these findings highlight the metabolic discrepancies between in vitro models and primary human tumors and should thus be carefully considered in the development of the precision therapies targeting MTAP-homozygous deleted GBM.

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