scholarly journals The Therapeutic Response of Myelodsyplastic Syndromes to Azacytidine Is Independent of Endogenous Retroelement Modulation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4349-4349
Author(s):  
Chrysoula Kordella ◽  
Anastasiya Kazachenka ◽  
Eleftheria Lamprianidou ◽  
Emmanouela Zoulia ◽  
George Vrachiolias ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Transcriptional Activation ◽  
Therapeutic Response ◽  
Conflicts Of Interest ◽  
Treatment Success ◽  
Innate Immune ◽  
Proliferating Cells ◽  
Viral Mimicry

Abstract Hypomethylating agents (HMA) such as azacytidine and decitabine are the mainstay of treatment for higher risk myelodysplastic syndromes (MDS) and are also used to treat older, unfit patients with acute myeloid leukemia (AML). Being cytidine analogues, both azacytidine and decitabine are incorporated into DNA of highly proliferating cells leading to genome-wide decrease of methylation levels (Stresemann & Lyko., 2008; Gnyszka et al., 2013), whereas azacytidine is additionally incorporated into RNA molecules. Although several putative modes of action have been suggested for HMA, the precise mechanism underlying treatment success or failure remains incompletely understood. One possible mechanism of HMA action is through 'viral mimicry' of transcriptionally repressed endogenous retroelements (EREs), which is thought to trigger innate immune pathways. EREs comprise nearly half of the human genome and their transcriptional activity is repressed by diverse mechanisms including DNA methylation. According to the 'viral mimicry' hypothesis, HMA induce unphysiological levels of ERE transcription in transformed cells, which in turn generated nucleic acid species, such as double-stranded RNAs from complementary ERE transcripts, activating innate immune sensors. Although support and a mechanistic basis for this hypothesis is provided from a number of in vitro studies, in vivo evidence from the clinical use of HMA is currently lacking. To explore the possible involvement of EREs in the HMA mode of action, we have compared the transcriptional profiles of CD34+ HSCs isolated from bone marrow samples of healthy donors (n=9) and patients diagnosed with AML (n=9), chronic myelomonocytic leukemia - II (CMML-II, n=9) or high-risk MDS (n=11). For MDS and CMML, samples were obtained before, 15 days (D15) after the initiation of azacytidine and/or after cycle 6. Our analysis revealed that ERE transcription, measured as a proportion of the total polyA-selected transcriptome, is globally repressed in untreated MDS and CMML, in line with the proposed epigenetic repression that characterizes these conditions. Treatment with azacytidine had a measureable effect in overall ERE transcription in HSCs from MDS and CMML patients, which by the 6th cycle was raised to levels equivalent to those seen in HSCs healthy controls. Comparable results were also obtained following analysis of a publicly available dataset from CD34+ HSCs isolated from MDS and CMML patients prior to and after the 6th cycle of azacytidine treatment (GSE76203). However, despite noticeable upregulation of overall ERE transcription relative to gene transcription by azacytidine, the therapeutic response was not correlated with or predicted by ERE activity. Indeed, ERE transcriptional activation was frequently observed in azacytidine-treated patients who failed to respond to treatment, whereas it was frequently low or absent in patients who attained complete remission (figures 1a & b). It remained theoretically possible that a therapeutic response to azacytidine depended on the transcriptional activation of a select few ERE loci with innate immune stimulatory properties, which might have been masked by the analysis of global ERE activity. However, few individual ERE loci differed in their activity between patients who responded or not to azacytidine treatment. Moreover, our analysis failed to detect induction of either interferon-inducible genes or interferon-inducible EREs, irrespective of treatment outcome(figures 2a & b). Together, our current results do not support a role for transcriptional activation of EREs or for innate sensing of their nucleic acid products in the therapeutic response of MDS and CMML patients to azacytidine. Investigation of alternative potential mechanisms of azacytidine is therefore warranted. Disclosures No relevant conflicts of interest to declare.

IMMU-53. STING-ING GLIOBLASTOMA WITH SPHERICAL NUCLEIC ACIDS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi104-vi105
Author(s):  
Akanksha Mahajan ◽  
Lisa Hurley ◽  
Serena Tommasini-Ghelfi ◽  
Corey Dussold ◽  
Alexander Stegh ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Surface ◽  
Biological Properties ◽  
Innate Immune ◽  
Limiting Factors ◽  
Nucleic Acid Delivery ◽  
Sting Pathway ◽  
Spherical Nucleic Acids

Abstract The Stimulator of Interferon Genes (STING) pathway represents a major innate immune sensing mechanism for tumor-derived DNA. Modified cyclic dinucleotides (CDNs) that mimic the endogenous STING ligand cGAMP are currently being explored in patients with solid tumors that are amenable to intratumoral delivery. Inadequate bioavailability and insufficient lipophilicity are limiting factors for clinical CDN development, in particular when consideration is given to systemic administration approaches. We have shown that the formulation of oligonucleotides into Spherical Nucleic Acid (SNA) nanostructures, i.e.,the presentation of oligonucleotides at high density on the surface of nanoparticle cores, lead to biochemical and biological properties that are radically different from those of linear oligonucleotides. First-generation brain-penetrant siRNA-based SNAs (NCT03020017, recurrent GBM) have recently completed early clinical trials. Here, we report the development of a STING-agonistic immunotherapy by targeting cGAS, the sensor of cytosolic dsDNA upstream of STING, with SNAs presenting dsDNA at high surface density. The strategy of using SNAs exploits the ability of cGAS to raise STING responses by delivering dsDNA and inducing the catalytic production of endogenous CDNs. SNA nanostructures carrying a 45bp IFN-simulating dsDNA oligonucleotide, the most commonly used and widely characterized cGAS activator, potently activated the cGAS-STING pathway in vitro and in vivo. In a poorly immunogenic and highly aggressive syngeneic mouse glioma model, in which tumours were well-established, only one dose of intranasal treatment with STING-SNAs decelerated tumour growth, improved survival and importantly, was well-tolerated. Our use of SNAs addresses the challenges of nucleic acid delivery to intracranial tumor sites via intranasal route, exploits the binding of dsDNA molecules on the SNA surface to enhance the formation of a dimeric cGAS:DNA complex and establishes cGAS-agonistic SNAs as a novel class of immune-stimulatory modalities for triggering innate immune responses against tumor.

Pak A Proteins Are Essential for Hematopoietic Stem/Progenitor Cell (HSC/P) Engraftment Through Regulation of Signaling Pathways Controlling HSC/P Proliferation, Survival, Migration, and the Actin Cytoskeleton

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 917-917
Author(s):  
Adrienne M. Dorrance ◽  
Rachelle Kosoff ◽  
Meaghan McGuinness ◽  
Chad Harris ◽  
Serena De Vita ◽  
...  
Keyword(s):  
Cell Migration ◽  
Conflicts Of Interest ◽  
Effector Proteins ◽  
Proliferating Cells ◽  
Hematopoietic Stem ◽  
Time Points ◽  
Empty Vector ◽  
Group A

Abstract Abstract 917 Rho GTPases, including Rac, integrate multiple extracellular signals and play important regulatory roles in HSC/P functions such as engraftment, retention, migration, adhesion, proliferation, and survival (Gu et al. Science, 2003). Our studies now focus on identifying potential Rac downstream effector proteins important for normal HSC/P function(s). The p21-activated kinases (Pak) are serine/threonine kinases that interact with and are major downstream targets of Rac and Cdc42. There are six human Paks (Pak1–6), which are grouped based on homology into Group A (Pak 1–3) and Group B (4–6) Paks. Paks regulate cytoskeletal organization including stress fiber dissolution, lamellipodia formation and focal adhesion disassembly and mediate activation of MAPK pathways. To identify the possible role(s) of Pak proteins in engraftment, freshly isolated LSK cells from WT (CD45.1+/CD45.2+) BM were transduced with retrovirus containing the Pak Inhibitory Domain (PID), which inhibits Group A Pak protein function or empty vector control (Mieg3); both constructs co-express GFP. 1.0×105 GFP+ LSK+ cells were then isolated and co-transplanted with 5.0×105 BoyJ (CD45.1+) whole bone marrow (WBM) into lethally irradiated C57Bl/6J (CD45.2+) recipients. Percent chimerism was measured at 3- to 24- weeks post BMT. PID transduced LSK+ cells were incapable of contributing to recipient hematopoietic reconstitution (Table 1). To explore the underlying mechanism of this engraftment failure we performed in vivo homing assays and found a 4- and 16- fold decrease, respectively, in BM homing of PID transduced LSK+ vs controls at 12 and 48 hours (p<0.05, for both time points). Altered cell migration of LSK+ cells was confirmed by live imaging microscopy which showed a 4-fold decrease in overall cell displacement in SDF-1-stimulated directed migration in the PID-expressing LSK+ compared to controls and was associated with a two-fold increase in random cell migration of PID-transduced LSK+ cells in transwell migration assays. PID-expressing LSK+ cells also demonstrated abnormal lamellipodia associated with significant increases in both cell surface area and cell perimeter. Because cytoskeletal changes may be linked to alterations in cell growth, we next examined the effect of Pak inhibition on cell survival and proliferation. PID-expressing LSK+ cells had decreased proliferation (17.7% vs 36.8% of cells in S-phase, p<0.05) and increased apoptosis (48.1% vs 16.7% AnnexinV+ cells, p<0.05) when compared to controls, respectively. These phenotypic changes were associated with decreased pERK and pAKT in PID-expressing LSK+. To confirm the importance of Pak activation of these proteins in HSC/P, we performed experiments to rescue the observed engraftment defect by co-transducing PID or Mieg3 with a constitutively active-ERK (ca-MEK1) or ca-AKT. We found ca-MEK1, but not ca-AKT, was able to increase proliferation in vitro (% proliferating cells for PID + empty vector = 6.1% and PID+ ca-MEK1 = 9.5%; p<0.05) and partially but only transiently rescue Pak-deficient HSC/P engraftment (% donor cells for LSK+ transduced with: PID + empty vector =1.5%, PID + ca-MEK1 =15.8%, and PID + ca-AKT =0.5% at 3-weeks post-BMT; p<0.05 for empty vector vs ca-MEK1). Finally, to determine which PakA pathway is critical in HSC engraftment we studied Pak genetic knock-out cells. We found that Pak2Δ/Δ -but not Pak1−/− -cells resulted in a profound HSC/P engraftment defect (% Pak2Δ/Δ vs Pak2flox/flox and Pak1−/− vs Pak1wt/wt: 1.0% vs 26.5% and 35.8% vs 37.4%; p<0.05 and p=ns, respectively at 3-weeks). Taken together, these data suggest that Pak A proteins regulate multiple HSC/P functions and link Rac GTPases to actin cytoskeletal rearrangements, directed cell migration, and proliferation/survival of HSC/P during engraftment.TABLE 1:Percentage of GFP+ cells in peripheral blood of recipient mice at indicated time points post BMT3-weeks6-weeks10-weeks14-weeks24-weeksWT-Mieg331.6% (±6.69)27.1% (±8.6)34.6% (±15.0)43.2% (±16.3)22.2% (±10.7)WT-PID0.22 (±0.19)0.07% (0.06)0%0%0%**Data represent mean ± s.d., n=10 recipients per group, p<0.05 for all time points, two independent experiments. Disclosures: No relevant conflicts of interest to declare.

Gadd45 Null Mice and Neutrophils/Macrophages Derived From the Bone Marrow of These Mice Exhibit Impaired Innate-Immune/Inflammatory Reponses.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1350-1350
Author(s):  
Dominic M Salerno ◽  
Barbara Hoffman ◽  
Dan A. Liebermann
Keyword(s):  
Signaling Pathways ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Innate Immune ◽  
White Pulp ◽  
Immune Functions ◽  
In Vivo Models ◽  
Cell Functions

Abstract Abstract 1350 Poster Board I-372 Stress sensor Gadd45 proteins modulate p38-NF-Kb and JNK signaling, which play major roles in leukocyte activation and innate immunity. We have previously documentedthat under conditions of hematological stress, notably acute stimulation with cytokines or inflammation, both gadd45a-deficient and gadd45b-deficient mice exhibited impaired inflammatory responses as indicated by lower percentages of Gr-1-positive cells in the BM and lower numbers of myeloid cells in peritoneal exudates (Gupta et. al Oncogene 25:5539-46, 2006). Recent evidence has implicated Gadd45 proteins in dendritic cell functions that influence effector Th1 responses to inflammation. However, whether gadd45 genes play a role in modulating the myeloid compartment, notably macrophage & granulocyte functions in response to inflammatory stress, remains largely unexplored. To this end, we have employed in vitro & in vivo models of inflammation using BM derived neutrophils and macrophages from WT, gadd45a and gadd45b null mice. The data obtained indicate that chemotaxis and transmigration to various chemo-attractants, including LPS and fMLP, as well as oxidative burst and phagocytic functions were impaired for both neutrophils and macrophages from mice lacking either gadd45a or gadd45b. Furthermore, upon stimulation with LPS, cytokine secretion, notably, but not exclusively IL-12 and TNFa, was significantly reduced in neutrophils and macrophages of gadd45a-/- and gadd455b-/- mice. Western Blot analysis of BM derived neutrophils lacking gadd45a and stimulated with LPS (500ng/mL) exhibited defects in p38 phosphorylation as compared to controls, suggesting a possible mechanism by which the innate response is impaired. P38 phosphorylation in gadd45b null granulocytes stimulated with LPS appeared comparable to what was observed in wt controls. This suggests that gadd45a and gadd45b utilize different signaling pathways to regulate innate-mmune/inflammtory responses. Interestingly, gadd45a, gadd45b & gadd45g null mice injected intraperitoneally with sublethal (25mg/kg body weight) doses of LPS were significantly more susceptible to septic shock compared to wt mice, as indicated by significantly increased morbidity through 5 days post LPS administration. Moreover, 18 hrs. post-injection, the spleens of KO mice were shown to have numerous apoptotic foci in the white pulp, confirmed to be tingible body macrophages ingesting dying cells by IH and IF for macrophage markers. These in vitro and in vivo data suggest a novel role for gadd45 family members in myeloid innate immune responses. Further elucidation of the signaling pathways involved is in progress and is expected to elucidate the molecular basis for the role Gadd45 proteins play in macrophage and granulocyte innate immune functions. Disclosures No relevant conflicts of interest to declare.

Pharmacokinetics and Biodistribution of Locked Nucleic Acid (LNA)-Mir-221 Inhibitor: A New Promising Anti-Myeloma Agent

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3025-3025 ◽  
Author(s):  
Maria Eugenia Gallo Cantafio ◽  
Annamaria Gulla ◽  
Nicola Amodio ◽  
Emanuela Leone ◽  
Eugenio Morelli ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Half Life ◽  
Conflicts Of Interest ◽  
Locked Nucleic Acid ◽  
Phase Cell ◽  
Systemic Delivery ◽  
Cynomolgus Monkeys ◽  
Mouse Tissues

Abstract miR-221/222 are highly homologous microRNAs (miRNAs) whose upregulation has been found in several malignancies, including multiple myeloma (MM). Both miRNAs are thought to promote cell proliferation via down-regulation of p27 and/or p57, two negative regulators of G1 to S phase cell cycle progression. We proved that silencing of both miRNAs results in significant anti-MM activity and in downregulation of canonical targets both in vitro and in vivo. In the aim to progress to clinical translation, we designed an original 13mer synthetic inhibitor, specific for systemic delivery, named LNA-i-miR-221, which took advantage from both locked nucleic acid (LNA) technology and phosphorothioate backbone, for increasing the seed sequence binding and nuclease resistance in vivo, respectively. Since no data are presently available, we evaluated the specificity of LNA-i-miR-221 to inhibit endogenous miR-221 and the pharmacokinetic properties (i.e. tissue distribution) in mice and Cynomolgus monkeys. We demonstrated that LNA-i-miR-221 inhibit growth and survival of MM cells bearing high miR-221 levels. Moreover, LNA-i-miR-221-mediated silencing of miR-221 triggered upregulation of p27Kip1 mRNA and protein, evaluated by q-RT-PCR and western blotting, respectively. In vivo, i.p.treatment with 25 mg/kg of LNA-i-miR-221 reduced tumor growth in SCID/NOD mice bearing MM xenografts. Tumors and vital organs (including liver, kidney, bone marrow and heart) were harvested from treated animals and evaluated for pharmacokinetics purposes using in situ hybridization (ISH) assay. After a single i.p. dose of 25 mg/kg, we detected the presence of LNA-i-miR-221 from 2 up to 7 days in tumors and mouse tissues. Interestingly, no toxicity was observed even after long-lasting presence of the inhibitors in vital organs. We also evaluated the LNA-i-miR-221 half-life in mouse plasma using HPLC-MS/MS, which confirmed the rapid uptake of LNA-i-miR-221 in tissues. To evaluate the maximum tolerated doses, in vivo dose escalation treatments was performed using doses from 10 to 100 mg/kg delivered at days 1,4,8,15,22. All treatments were well tolerated. No changes in mice behavior or organ toxicity were observed in treated mice. Finally, a pilot toxicity study was performed in Cynomolgus monkeys. PK results demonstrated that LNA-i-miR-221 has a short serum half-life with a rapid tissue uptake and minimum urinary escretion. In conclusion, our results suggest that LNA-i-miR-221 is a promising anti-MM agent associated with a safety profile in mice and in monkeys, supporting the rationale for development of this novel miR-221 inhibitor in early clinical trials. Disclosures No relevant conflicts of interest to declare.

CD38 Regulates Homing and Engraftment of CLL Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3873-3873
Author(s):  
Tiziana Vaisitti ◽  
Sara Serra ◽  
Valentina Audrito ◽  
Chris Pepper ◽  
Davide Rossi ◽  
...  
Keyword(s):  
Cell Line ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
In Vitro Assays ◽  
Wild Type ◽  
Proliferating Cells

Abstract Abstract 3873 Chronic lymphocytic leukemia (CLL) is considered the result of a dynamic balance between proliferating cells in lymphoid organs and circulating cells resisting apoptosis. Re-circulation of leukemic cells from blood to growth-permissive niches represents an essential step in the maintenance and progression of the disease. This equilibrium is finely tuned by a set of surface molecules expressed by CLL cells and modulated in response to environmental conditions. We previously reported that CD38, an enzyme and a receptor, functionally cooperates with the CXCL12/CXCR4 axis, enhancing the ability of CLL cells to home to bone marrow and lymph nodes. In addition, the use of anti-CD38 mAbs can enhance or impair the chemotactic behavior of the neoplastic cells. New evidence also indicates that CD38 synergizes with the CD49d integrin, increasing adhesion of CLL cells to VCAM-1 or the CS-1 fibronectin fragment, two known ligands of CD49d. To complete the picture, CD38 expression denotes a CLL subset with increased activity of the matrix metalloproteinases MMP-9. Ligation of CD38 with specific antibodies increases MMP-9 secretion and the invasive properties of CLL cells, using in vitro assays. The effects on chemotaxis, adhesion and invasion are obtained through modulation of a ERK1/2-dependent pathway. To further confirm the involvement of CD38 in CLL homing to specific niches, in vivo experiments have been set using NOD/SCID/γ chain−/− (NSG) mice. The CLL-like cell line Mec-1, constitutively CD38−/CD49d+, was adopted as a model and compared to transfectants stably expressing wild-type (wt) CD38, as well a mutant lacking enzyme activities. Results after i.v. injections of tumor cells indicate that de novo expression of CD38 by Mec-1 cells increases growth kinetics in vivo with a higher proliferation rate and metastatic potential, as compared to the Mec-1 mock-trasfected cells. Both these features are lost when the animals are injected with the enzyme-deficient variant of CD38, suggesting that the enzymatic activity is critical for in vivo growth and re-circulation of Mec-1 cells. Microarray data confirm that the genetic signature of the CD38-enzyme mutant overlaps with the wild-type cell line, clearly distinct from cells transfected with CD38. The latter cell line shows up-modulation of several genes involved in chemotaxis and adhesion. All together, these results support the notion that CD38 is part of a complex network of molecules and signals, that regulate homing of CLL cells to growth-permissive niches, suggesting a relationship between the expression of CD38, the ability to migrate and invade and the poor clinical outcome of the CD38+ subset of patients. Disclosures: No relevant conflicts of interest to declare.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

scholarly journals Detecting apoptosis as a clinical endpoint for proof of a clinical principle

2020 ◽  
Author(s):  
Piero Zollet ◽  
Timothy E.Yap ◽  
M Francesca Cordeiro
Keyword(s):  
Drug Development ◽  
Therapeutic Response ◽  
Imaging Techniques ◽  
Brain Diseases ◽  
Promising Strategy ◽  
Sight Loss ◽  
Apoptosis Detection ◽  
Novel Therapeutic Strategies

The transparent eye media represent a window through which to observe changes occurring in the retina during pathological processes. In contrast to visualising the extent of neurodegenerative damage that has already occurred, imaging an active process such as apoptosis has the potential to report on disease progression and therefore the threat of irreversible functional loss in various eye and brain diseases. Early diagnosis in these conditions is an important unmet clinical need to avoid or delay irreversible sight loss. In this setting, apoptosis detection is a promising strategy with which to diagnose, provide prognosis, and monitor therapeutic response. Additionally, monitoring apoptosis in vitro and in vivo has been shown to be valuable for drug development in order to assess the efficacy of novel therapeutic strategies both in the pre-clinical and clinical setting. Detection of Apoptosing Retinal Cells (DARC) technology is to date the only tool of its kind to have been tested in clinical trials, with other new imaging techniques under investigation in the fields of neuroscience, ophthalmology and drug development. We summarize the transitioning of techniques detecting apoptosis from bench to bedside, along with the future possibilities they encase.

scholarly journals Long-Term Severe In Vitro Hypoxia Exposure Enhances the Vascularization Potential of Human Adipose Tissue-Derived Stromal Vascular Fraction Cell Engineered Tissues

2021 ◽  
Vol 22 (15) ◽  
pp. 7920
Author(s):  
Myroslava Mytsyk ◽  
Giulia Cerino ◽  
Gregory Reid ◽  
Laia Gili Sole ◽  
Friedrich S. Eckstein ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Therapeutic Potential ◽  
Stromal Vascular Fraction ◽  
Human Adipose Tissue ◽  
Bioreactor Culture ◽  
Proliferating Cells ◽  
Angiogenic Potential ◽  
Engineered Tissues

The therapeutic potential of mesenchymal stromal/stem cells (MSC) for treating cardiac ischemia strongly depends on their paracrine-mediated effects and their engraftment capacity in a hostile environment such as the infarcted myocardium. Adipose tissue-derived stromal vascular fraction (SVF) cells are a mixed population composed mainly of MSC and vascular cells, well known for their high angiogenic potential. A previous study showed that the angiogenic potential of SVF cells was further increased following their in vitro organization in an engineered tissue (patch) after perfusion-based bioreactor culture. This study aimed to investigate the possible changes in the cellular SVF composition, in vivo angiogenic potential, as well as engraftment capability upon in vitro culture in harsh hypoxia conditions. This mimics the possible delayed vascularization of the patch upon implantation in a low perfused myocardium. To this purpose, human SVF cells were seeded on a collagen sponge, cultured for 5 days in a perfusion-based bioreactor under normoxia or hypoxia (21% and <1% of oxygen tension, respectively) and subcutaneously implanted in nude rats for 3 and 28 days. Compared to ambient condition culture, hypoxic tension did not alter the SVF composition in vitro, showing similar numbers of MSC as well as endothelial and mural cells. Nevertheless, in vitro hypoxic culture significantly increased the release of vascular endothelial growth factor (p < 0.001) and the number of proliferating cells (p < 0.00001). Moreover, compared to ambient oxygen culture, exposure to hypoxia significantly enhanced the vessel length density in the engineered tissues following 28 days of implantation. The number of human cells and human proliferating cells in hypoxia-cultured constructs was also significantly increased after 3 and 28 days in vivo, compared to normoxia. These findings show that a possible in vivo delay in oxygen supply might not impair the vascularization potential of SVF- patches, which qualifies them for evaluation in a myocardial ischemia model.

scholarly journals The Revolutionary Roads to Study Cell–Cell Interactions in 3D In Vitro Pancreatic Cancer Models

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 930
Author(s):  
Donatella Delle Cave ◽  
Riccardo Rizzo ◽  
Bruno Sainz ◽  
Giuseppe Gigli ◽  
Loretta L. del Mercato ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Therapeutic Response ◽  
Three Dimensional ◽  
Cellular Models ◽  
Common Cancer ◽  
Cancer Models ◽  
Anti Cancer ◽  
Tumor Environment

Pancreatic cancer, the fourth most common cancer worldwide, shows a highly unsuccessful therapeutic response. In the last 10 years, neither important advancements nor new therapeutic strategies have significantly impacted patient survival, highlighting the need to pursue new avenues for drug development discovery and design. Advanced cellular models, resembling as much as possible the original in vivo tumor environment, may be more successful in predicting the efficacy of future anti-cancer candidates in clinical trials. In this review, we discuss novel bioengineered platforms for anticancer drug discovery in pancreatic cancer, from traditional two-dimensional models to innovative three-dimensional ones.

scholarly journals CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jin-Chun Qi ◽  
Zhan Yang ◽  
Tao Lin ◽  
Long Ma ◽  
Ya-Xuan Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcriptional Activation ◽  
Positive Feedback ◽  
Feedback Loop ◽  
Biological Effects ◽  
Therapeutic Benefit ◽  
Loss Of Function ◽  
Positive Feedback Loop

Abstract Background Both E2F transcription factor and cyclin-dependent kinases (CDKs), which increase or decrease E2F activity by phosphorylating E2F or its partner, are involved in the control of cell proliferation, and some circRNAs and miRNAs regulate the expression of E2F and CDKs. However, little is known about whether dysregulation among E2Fs, CDKs, circRNAs and miRNAs occurs in human PCa. Methods The expression levels of CDK13 in PCa tissues and different cell lines were determined by quantitative real-time PCR and Western blot analysis. In vitro and in vivo assays were preformed to explore the biological effects of CDK13 in PCa cells. Co-immunoprecipitation anlysis coupled with mass spectrometry was used to identify E2F5 interaction with CDK13. A CRISPR-Cas9 complex was used to activate endogenous CDK13 and circCDK13 expression. Furthermore, the mechanism of circCDK13 was investigated by using loss-of-function and gain-of-function assays in vitro and in vivo. Results Here we show that CDK13 is significantly upregulated in human PCa tissues. CDK13 depletion and overexpression in PCa cells decrease and increase, respectively, cell proliferation, and the pro-proliferation effect of CDK13 is strengthened by its interaction with E2F5. Mechanistically, transcriptional activation of endogenous CDK13, but not the forced expression of CDK13 by its expression vector, remarkably promotes E2F5 protein expression by facilitating circCDK13 formation. Further, the upregulation of E2F5 enhances CDK13 transcription and promotes circCDK13 biogenesis, which in turn sponges miR-212-5p/449a and thus relieves their repression of the E2F5 expression, subsequently leading to the upregulation of E2F5 expression and PCa cell proliferation. Conclusions These findings suggest that CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 is responsible for PCa development. Targeting this newly identified regulatory axis may provide therapeutic benefit against PCa progression and drug resistance.

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