PS02.174: THE ACTION OF A NOVEL RADIOSENSITISER WITHIN THE OESOPHAGEAL ADENOCARCINOMA TUMOUR MICROENVIROMENT

2018 ◽  
Vol 31 (Supplement_1) ◽  
pp. 171-171
Author(s):  
Amy Buckley ◽  
Niamh Lynam-Lennon ◽  
Susan Kennedy ◽  
Aoife Cannon ◽  
Dermott O’Toole ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Survival Rates ◽  
Oesophageal Adenocarcinoma ◽  
Naive Patient ◽  
Human Patient ◽  
Hypoxic Conditions ◽  
Treatment Naïve

Abstract Background Oesophageal Cancer (OAC) is an aggressive disease with survival rates of ∼15-20%. Current therapeutic regimes focus on neo-adjuvant therapy (chemo-radiation) prior to surgery. Unfortunately, only 20–30% of patients show a beneficial response, with 70–80% of patients as non-responders. This major clinical challenge of treatment resistance reinforces the need for the identification of novel treatments which can act as radio-sensitisers in the neo-adjuvant setting. Methods Through a drug screening approach in-vivo, we have identified a novel anti-angiogenic and anti-metabolic compound, 11B_CC8 with radiosensitising activity. The ability of 11B_CC8 to act as an anti-metabolic agent under hypoxia was evaluated using the XFe24 Seahorse analyser and the Don Whitley i2 workstation. The ability of 11B_CC8 to radiosensitise our isogenic OAC cells under hypoxic conditions was assessed by clonogenic assay. The effect of 11B_CC8 on inflammatory, metabolic and angiogenic protein secretions from OAC treatment naïve tumour conditioned media (TCM) was evaluated by multiplex ELISA. Fresh treatment naïve patient biopsies were screened for their metabolic activity using the XFe24 seahorse analyser at baseline and post- treatment with 11B_CC8. The elucidation of the possible mechanism of action of 11B_CC8 was evaluated by Mass-Spectrometry. Results Our novel anti-angiogenic and anti-metabolic agent can enhance radiosensitivity in our isogenic model of OAC under both normoxic and hypoxic (0.5% O 2) conditions. 11B_CC8 significantly reduces oxygen consumption rate (OCR) under normoxic but not hypoxic conditions. Ex-vivo, 11B_CC8 significantly reduced the secretion of IL1β (P = 0.0117). Real-time ex-vivo metabolic rate analysis of our treatment naïve OAC biopsies showed significantly elevated OCR, when compared to Extracellular Acidification rate, a measure of glycolysis (P = 0.0059). Treatment with 11B_CC8 produced a reduction in OCR (P = 0.0039). Conclusion Our novel anti-angiogenic and anti-metabolic agent can enhance radiosensitivity in-vitro under both normoxic and hypoxic conditions. Ex-vivo, treatment naïve OAC human patient samples, 11B_CC8 can significantly reduce the secretion of IL1β and altered metabolic programming, specifically oxidative phosphorylation in human explants. Disclosure All authors have declared no conflicts of interest.

86 BIRTH OF HEALTHY CALVES AFTER INTRAFOLLICULAR OOCYTE TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 136
Author(s):  
M. Hoelker ◽  
A. Kassens ◽  
E. Held ◽  
C. Wrenzycki ◽  
U. Besenfelder ◽  
...  
Keyword(s):  
Lipid Content ◽  
Ex Vivo ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
In Vitro Production ◽  
Negative Effects ◽  
Uterine Flushing

The in vitro production (IVP) of bovine embryos is a well-established technique that has been available for nearly 20 years. However, there remain major differences between IVP-derived blastocysts and their in vivo-derived counterparts. Many studies have pointed out that most of these differences are due to the in vitro developmental environment. To circumvent these negative effects due to in vitro culture conditions, a new method – intrafollicular oocyte transfer (IFOT) – was established in the present study. Using modified ovum pick-up (OPU) equipment, in vitro-matured oocytes derived from slaughterhouse ovaries were injected into the dominant preovulatory follicle of synchronised heifers (follicular recipients) enabling subsequent ovulation, in vivo fertilization, and in vivo development. A total of 810 in vitro-matured oocytes were transferred into 14 heifers. Subsequently, 222 embryos (27.3%) were recovered after uterine flushing at Day 7. Based on the number of cleaved embryonic stages, 64.2% developed to the blastocyst stage, which did not differ from the IVP-derived embryos (58.2%). Interestingly, lipid content of IFOT-derived blastocysts did not differ from the fully in vivo-produced embryos, whereas IVP-derived blastocysts showed significantly higher lipid droplet accumulation compared with fully in vivo-derived and IFOT-derived blastocysts (P < 0.05). Accordingly, IFOT blastocysts showed significantly higher survival rates after cryopreservation than complete IVP-derived embryos (77% v. 10%), which might be attributed to a lower degree of lipid accumulation. In agreement, transfer of frozen-thawed IFOT blastocysts to synchronized recipients (uterine recipients) resulted in much higher pregnancy rates compared with transfer of IVP-derived blastocysts (42.1 v. 13.8%) but did not differ from frozen-thawed ex vivo blastocysts (52.4%). Of these presumed IFOT pregnancies, 7 went to term, and microsatellite analysis confirmed that 5 calves were indeed derived from IFOT, whereas 2 were caused by fertilization of the follicular recipient's own oocyte after AI. Taken together, IFOT-derived blastocysts closely resemble in vivo-derived blastocysts, confirming earlier suggestions that the ability to develop to the blastocyst stage is already determined in the matured oocyte, whereas the quality in terms of lipid content and survival rate after cryopreservation is affected by the environment thereafter. However, to the best of our knowledge, this is the first study reporting healthy calves after intrafollicular transfer of in vitro-matured oocytes.

scholarly journals Negative Impact of Hypoxia on Tryptophan 2,3-Dioxygenase Function

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Frank Elbers ◽  
Claudia Woite ◽  
Valentina Antoni ◽  
Sara Stein ◽  
Hiroshi Funakoshi ◽  
...  
Keyword(s):  
Essential Amino Acid ◽  
Negative Impact ◽  
Ex Vivo ◽  
T Cell Proliferation ◽  
Effector Functions ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Oxygen Conditions

Tryptophan is an essential amino acid for hosts and pathogens. The liver enzyme tryptophan 2,3-dioxygenase (TDO) provokes, by its ability to degrade tryptophan to N-formylkynurenine, the precursor of the immune-relevant kynurenines, direct and indirect antimicrobial and immunoregulatory states. Up to now these TDO-mediated broad-spectrum effector functions have never been observed under hypoxiain vitro, although physiologic oxygen concentrations in liver tissue are low, especially in case of infection. Here we analysed recombinant expressed human TDO andex vivomurine TDO functions under different oxygen conditions and show that TDO-induced restrictions of clinically relevant pathogens (bacteria, parasites) and of T cell proliferation are abrogated under hypoxic conditions. We pinpointed the loss of TDO efficiency to the reduction of TDO activity, since cell survival and TDO protein levels were unaffected. In conclusion, the potent antimicrobial as well as immunoregulatory effects of TDO were substantially impaired under hypoxic conditions that pathophysiologically occurin vivo. This might be detrimental for the appropriate host immune response towards relevant pathogens.

CD138 Regulates Myeloma Cell Survival, Proliferation, BM Engraftment and Tumor Organization

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2974-2974
Author(s):  
David R Fooksman ◽  
Amitabha Mazumder ◽  
Mark McCarron
Keyword(s):  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Early Stage ◽  
Serum Starvation ◽  
High Dose ◽  
Rapid Reduction ◽  
Myeloma Cells

Abstract Multiple myeloma is the 2nd most common blood cancer in adults with a median survival time of 5 years despite high-dose chemotherapy and bone marrow transplantation interventions. Syndecan-1 or CD138, is a heparan-sulfate coated glycoprotein, which is highly expressed on the surface of plasma cells and myeloma cells, important for adhesion and accumulating survival signals. Expression of CD138 is heterogeneous in myeloma tumors, in vivo and in vitro leading some to speculate it may distinguish stem-like subpopulations. While this role is highly disputed, we investigated the effect of CD138 expression on tumor pathology in vivo. To characterize CD138neg and CD138high subpopulations, we used GFP+ Vk*myc myeloma model from Leif Bergsagel, which develops myeloma tumors in BM and spleen of C57Bl/6 mice. We found CD138high populations were more proliferative in vivo based on EdU incorporation experiments. We transferred equal numbers of sorted subpopulations into hosts and found that CD138high cells generated larger tumors in the BM than CD138neg cells after 12 weeks. Analysis of these tumor-bearing mice revealed that all tumors contained both subpopulations, indicating that these two subsets are hierarchically equivalent. We find that in mice with small tumors, the majority of cells (80% or more) are CD138high cells, while in large tumors, the level drops (to 30-50% of tumor) with higher composition of CD138neg cells. We also find lower CD138 levels on myeloma cells found in the blood compared to BM. Using intravital two-photon time-lapse imaging in the tibial BM, we find that tumor cells from smaller, early stage tumors are physically arrested within the BM parenchyma, while in larger, more advanced tumors, myeloma cells are more motile and active. CD138neg cells were more apoptotic based on ex vivo Annexin V staining following serum starvation. Interestingly, serum starvation led to rapid reduction in CD138 surface expression. Taken together, we propose a model where CD138 expression regulates localization and survival in the BM niche, but is downregulated from the plasma membrane when tumor size outgrows the necessary resources, allowing myeloma cells to migrate and metastasize to distant new locations. Disclosures No relevant conflicts of interest to declare.

PS02.239: SPECIFIC INHIBITION OF BONE MORPHOGENIC PROTEIN (BMP4) AS A POTENTIAL THERAPEUTIC STRATEGY FOR ESOPHAGEAL ADENOCARCINOMA

2018 ◽  
Vol 31 (Supplement_1) ◽  
pp. 190-190
Author(s):  
Kausilia Krishnadath ◽  
Sanne Hoefnagel ◽  
Silvia Calpe ◽  
Matthew Read ◽  
Danielle Straub ◽  
...  
Keyword(s):  
Esophageal Adenocarcinoma ◽  
Conflicts Of Interest ◽  
Bmp Signaling ◽  
In Vivo Studies ◽  
Tumor Xenograft ◽  
In Vivo Models ◽  
Treatment Naïve ◽  
Bmp4 Expression

Abstract Background In Western countries, the highly malignant Esophageal adenocarcinoma (EAC) have the most dramatically rising incidence of all malignancies. BMP4 is a growth factor important for carcinogenesis. We found that BMP4 is aberrantly expressed in Barret's esophagus, the pecursor lesion of EAC, and that together with CDX2 drives the intestinalization of epithelial metaplasia. However, its role in esophageal adenocarcinoma (EAC) remains uncertain. Methods Method: To elucidate whether BMP4 is involved in malignancy in EAC we used an RNA sequencing database of 56 EAC treatment naive endoscopic biopsies to investigate if there is a subgroup of cancers with high BMP signaling. We validated results by qPCR and immunohistochemistry in matching tumor samples. Next we used our recently developed effective and highly specific anti-BMP4 antibodies(1,2) to study the effect of inhibition of BMP4 on both in vitro as well as in vivo models of EAC. Results Using a gene set that was recently published for BMP signaling, we were able to distinguish a subgroup of EAC patients with increased BMP signaling. By IHC we confirmed that 70% of EAC tumors express BMP4 at the protein level. We found that patients with high levels of BMP4 expression tend to have a poorer recurrence-free survival compared to patients with low BMP4 expression, which suggests a more aggressive tumor behavior in BMP4 expressing EAC tumors. Most importantly, inhibition of BMP4 function in EAC cells by our recently developed anti-BMP4 antibodies lead to an increase in chemo-sensitivity and a decreased in invasive and migratory capabilities in vitro. Preclinical in vivo studies with a patient-derived tumor xenograft mouse model of an EAC tumor confirmed that anti-BMP4 antibodies can effectively reduce tumor growth and synergistically act with chemotherapy agents. Conclusion We identified a subgroup of EAC with increased BMP signaling. Our studies support a role of BMP4 in chemo-resistance and invasiveness in EAC, and indicate that inhibition of BMP4 with highly our specific llama-derived antibodies is an attractive therapy for improving outcomes of EAC. Disclosure All authors have declared no conflicts of interest.

A Dendritic Cell Based Vaccination Strategy Geared towards Eradication of Leukemic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2046-2046
Author(s):  
Hetty J Bontkes ◽  
Jurjen Ruben ◽  
Willemijn van den Ancker ◽  
Theresia M Westers ◽  
G. Ossenkoppele ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Tumor Antigens ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Vaccination Strategy ◽  
Leukemic Stem Cells ◽  
Cell Immunity

Abstract Abstract 2046 Poster Board II-23 Introduction: In the majority of cases, initial remission of acute myeloid leukemia (AML) is reached but unfortunately relapse rates remain high and therefore novel treatments are needed. It is thought that recurrent AML originates from chemotherapy resistant quiescent leukemic stem cells (LSC). The application of immunotherapeutic approaches to eradicate LSC remaining after first line chemotherapy may contribute to improved disease outcome. Vaccination strategies have often used dendritic cells (DC) ex vivo pulsed with tumor-derived whole lysates or peptides as modalities to present a broad range of tumor antigens to T cells to stimulate effective anti-tumor T-cell immunity in vivo. It is likely that certain proteins expressed by LSC have a distinct antigenicity as compared to more mature AML blasts and thus provide targets for specific T-cells. Even without identification of specific antigens, LSC can be a useful source of tumor antigens in DC vaccination-based immunotherapy. CD34+CD38- LSC can be identified using malignant stem cell associated cell surface markers including CLL-1 and lineage markers such as CD7, CD19 and CD56. However, the low frequency of these cells precludes the use of LSC derived apoptotic cells or lysates for DC loading. Alternatively, mRNA isolated from LSC can be amplified and subsequently transfected into DC. Materials and Methods: We have made use of the CD38- AML derived cell line MUTZ-3 which contains a subpopulation of CD34+CLL1+ cells which resembles the phenotype of a putative LSC. CLL1+CD34+ and CLL1-CD34- cells were isolated by FACS sorting and total RNA was isolated. mRNA was converted to cDNA and amplified by PCR using the SMART system. Subsequently, mRNA was in vitro transcribed from the amplified cDNA. Mature monocyte derived DC (MoDC) were generated from healthy donor blood and transfected with amplified CLL1+CD34+ derived mRNA and used to stimulate autologous CD8β+ T-cells. After three weekly re-stimulations with CLL1+CD34+ mRNA transfected DC, specificity of the T-cells was analyzed by intracellular IFNγ staining upon 5 hour stimulation with autologous immature MoDC transfected with GFP mRNA, mRNA amplified from unsorted, CLL1+CD34+ or CLL1-CD34- MUTZ-3 subpopulations. Results: Amplification of CLL1 and survivin (also expressed by MUTZ-3) transcripts was confirmed by RT-PCR. After 3 weekly re-stimulations with CLL1+CD34+ amplified RNA transfected DC, 0.04% (range 0.01-0.12%) of the T-cells were positive for IFNγ upon a 5 hr re-stimulation with GFP transfected DC. 0.44% (range 0.04-0.69%) of the T-cells responded to DC transfected with unsorted MUTZ-3 amplified mRNA (p<0.00005 versus GFP control, 2-sided student's T-test), 0.51% (range 0.24-1.35%) responded to DC transfected with CLL1+CD34+ amplified mRNA (p<0.005 versus GFP control) and 0.46% (range 0.24-0.94%) responded to DC transfected with CLL1-CD34- amplified mRNA (p<0.0001 versus GFP control). Conclusion: We show that MoDC transfected with RNA amplified from one MUTZ-3 sub-population resembling the phenotype of LCS cells are capable of inducing T-cells which recognize both cells transfected with mRNA from the LSC resembling MUTZ-3 subset as well as the CLL1-CD34- subset. We are currently testing the efficacy and feasibility of this approach in an autologous setting in vitro. CD8β+ T-cells are stimulated with autologous MoDC from AML patients transfected with amplified mRNA isolated from their own LSC enriched populations. The capacity of these T-cells to kill autologous AML blasts and LSC is subsequently analysed in a 6-colour FACS based cytotoxicity assay. Disclosures: No relevant conflicts of interest to declare.

Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1919-1919
Author(s):  
Iman Hatem Fares ◽  
Jalila Chagraoui ◽  
Jana Krosl ◽  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

Assessing CD97 and CD99 As Markers of Leukaemia Initiating Cells in Paediatric ALL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1882-1882 ◽  
Author(s):  
Charlotte Victoria Cox ◽  
Paraskevi Diamanti ◽  
Allison Blair
Keyword(s):  
Functional Capacity ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Survival Rates ◽  
In Vivo Studies ◽  
Nsg Mice ◽  
Early Treatment Response ◽  
Current Therapies

Abstract Abstract 1882 Overall survival rates in paediatric acute lymphoblastic leukaemia (ALL) have dramatically improved but around 20% do not respond to current therapies and subsequently relapse. Leukaemia initiating cells (LIC) are the topic of much investigation, as these cells can self-renew and may have the potential to cause relapse. It has been shown that multiple subpopulations of ALL cells have the ability to initiate the disease in immune deficient mouse models. Therefore, treatment should be targeted at all cells with this capacity, if the disease is to be eradicated. Minimal residual disease (MRD) detection is an invaluable tracking tool to assess early treatment response and recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. CD97 and CD99 are two markers which were over expressed in paediatric ALL. Incorporating these markers into investigations of LIC may allow discrimination of leukaemia cells from normal haemopoietic stem cells (HSC). In this study we evaluated the expression of CD34 in combination with CD97 in B cell precursor (BCP) ALL cases and CD99 in T-ALL cases and subsequently assessed the functional capacity of the sorted subpopulations in vitro and in vivo. Ten ALL samples (6 B-ALL & 4 T-ALL) with a median age 7 years (range 2–15 years) were studied. One B-ALL case and 3 T-ALL cases were considered high risk by molecular assessment of MRD at day 28 of treatment. Flow cytometric analyses of the ALL samples and 8 normal haemopoietic cell samples demonstrated that both CD97 and CD99 were over expressed in ALL patients (78.9±14.8% & 76.4±32.8%, respectively) when compared to normal haemopoietic cells (14.1±25.4%; p=0.001, 47.1±10%; p=0.03, respectively). Cells were sorted for expression/lack of expression of these markers and proliferation of the sorted cells was assessed in suspension culture over a 6 week period. In the B-ALL patients the CD34+/CD97+ subpopulation represented the bulk of leukaemia cells (65.2±32.1%), the CD34−/CD97+ the smallest fraction (3.3±2.4%) with the CD34+/CD97− and CD34−/CD97− subpopulations representing 21.1±31.5% and 10.5±5.8% of cells, respectively. When the functional capacity of these subpopulations was assessed in vitro greatest expansion was observed in cells derived from CD34+/CD97− subpopulation (2–173 fold) from 9.4×103 at initiation up to 1.5×106 cells at week 6. Expansion was also observed, to a lesser extent in the CD34−/CD97− subpopulation (3.4–28 fold) from 8×103 up to 1.4×106 cells. No expansion was observed in cultures of CD34+/CD97+ and CD34−/CD97− subpopulations but cells were maintained throughout the culture period. These sorted subpopulations were also inoculated into NOD/LtSz-SCID IL-2Rγc null (NSG) mice to evaluate repopulating capacity. To date, engraftment has been achieved with 3 subpopulations; CD34+/CD97+ (3–28.8% CD45+), CD34+/CD97− (0.5–25.5% CD45+) and CD34−/CD97+ (23.8% CD45+) cells. When the functional capacity of T-ALL cases was assessed the CD34+/CD99+ subpopulation represented the bulk of cells at sorting (51.87±47.2%), the CD34+/CD99- subpopulation was the smallest (0.9±0.8%) and the CD34−/CD99+ and CD34−/CD99− subpopulations represented 32.1±38.9% and 27.2±33.4% of cells, respectively. Greatest expansion was observed in cultures of CD34+/CD99- cells (4.6–1798 fold) from 7.5×103 up to 2.6×106 cells at week 6. The other 3 subpopulations expanded to a lesser extent (1.3–216 fold) from 5×103 up to 1.8×106 cells. When the functional capacity of these cells was assessed in NSG mice, engraftment was achieved in all subpopulations; CD34+/CD99+ (87–90.5% CD45+), CD34+/CD99− (1.5–84.9% CD45+), CD34−/CD99+ (31.3–98.6% CD45+) and CD34−/CD99− (3–92.9% CD45+). In some cases, cells recovered from BM of NSG inoculated with CD99− cells had high expression of CD99, typical of the patient samples at diagnosis, indicating that the inoculated CD99− cells had differentiated in vivo. Studies are ongoing to assess the self-renewal capacity of these subpopulations by serial transplantation. The findings to date indicate that targeting CD97 and CD99, either alone or in combination with CD34 would not eliminate all cells with the capacity to initiate and maintain B-ALL and T-ALL, respectively. Further developments in therapy may require targeting leukaemogenic pathways, rather than only cell surface markers to improve survival outcome in paediatric ALL. Disclosures: No relevant conflicts of interest to declare.

Hypoxia-Inducible Factor (HIF)-1alpha Inhibitionin Myeloma Cells Significantly Increases the Anti-Myeloma Effect of Lenalidomide in Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3012-3012
Author(s):  
Paola Storti ◽  
Valentina Marchica ◽  
Denise Toscani ◽  
Irma Airoldi ◽  
Sophie Maiga ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Direct Action ◽  
Severe Combined Immunodeficiency ◽  
Hypoxia Inducible Factor ◽  
Hexokinase Ii ◽  
Inhibition Of Proliferation ◽  
Vitro Effect

Abstract The immunomodulatory drugs (IMiDs®) exert an anti-myeloma effect by cereblon-dependent destruction of IKZF proteinseither through a direct action on multiple myeloma (MM) cells or through indirect immunomodulatory and anti-angiogenic effects.Previous data indicated that MM cells overexpress hypoxia inducible factor (HIF)-1α and that HIF-1α suppression significantly blocks MM-induced angiogenesis and reduces in vivo tumoral burden in MM mouse models. Interestingly, it has been recently reported that HIF-1α knock-down in MM cells potentiates the in vitro effect of Lenalidomide (LEN) on cell proliferation without changing cell viability and that LEN is not able to suppress HIF-1α expression in MM cells. These evidences give the rationale design to investigate the in vivo effect of HIF-1α stable suppression in MM cells on LEN sensitivity. Thus, in this study, we assessed the effect of LEN in vivo in combination with HIF-1α inhibition in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) subcutaneous mouse model using JJN3, a cell line known to be resistant to the cytotoxic effect of LEN. Different groups of animals were injected with JJN3-pLKO.1 (empty vector) or JJN3-anti-HIF1α, obtained by anti-HIF1α lentiviral shRNA pool. When tumors became palpable, mice were treated with LEN (5mg/kg), using the intraperitoneal route. After three weeks, we evaluated tumor volume and weight. Moreover, by immunohistochemistry on ex vivo plasmacytomas, we evaluated the expression of p27 and the microvascular density (MVD), checked by CD34 immunostaining. In addition, the expression of a p27 inhibitor, the S-phase kinase-associated protein 2 (SKP2), the expression of the HIF-1α target key mediator of glycolysis and tumoral growth, Hexokinase II (HK2), and the levels of pERK 1/2, and total Caspase-3 (Casp-3) were evaluated in the ex vivo plasmacytomas lysates by western blot. We found that LEN treatment induced a significant weight and volume reduction of the tumor burden in mice injected with JJN3 anti-HIF1α as compared to JJN3-pLKO.1. The p27 nuclear expression was significantly increased by LEN treatment in JJN3-anti-HIF1α as compared to JJN3 pLKO.1 mice and compared to JJN3-anti-HIF1α mice treated with vehicle. In addition, we demonstrated that LEN in combination with HIF-1α inhibition significantly reduced in vivo the expression of p-ERK1/2, total Casp-3, HK2 and the p27 inhibitor, SKP2. Because it is known that LEN exerts its anti-MM effect targeting the IKZF proteins, we further checked in vitro whether the effect of HIF-1α suppression and LEN treatment combination could be mediated by IKZF proteins modulation. Interestingly, after LEN (2-10µM) treatment we found that both IKZF1 and IKZF3 were not differentially expressed, whereas IRF4 was down regulated, in JJN3-anti-HIF1α as compared to JJN3 pLKO.1. Finally, regarding a possible combinatory effect on the in vivo angiogenesis, we found that both the number of CD34 positive vessels and the MVD were reduced in mice colonized by JJN3-anti-HIF1α as compared to JJN3-pLKO.1. On the other hand, LEN treatment did not further significantly reduce the number of CD34 positive vessels and the MVD. Accordingly, we did not find any significant inhibitory effect by LEN treatment on the main pro-angiogenic molecules in JJN3 anti-HIF1α as compared to JJN3 pLKO.1 even after 72 hours. Overall, our data indicate that HIF-1α suppression in MM cells significantly increases the anti-MM effect of LEN in vivo, mainly through the inhibition of proliferation signaling including the modulation of p27 pathway and the IKZF target protein IRF4, rather than to an anti-angiogenic effect. These data suggest that the combination of LEN and HIF-1α inhibition has a therapeutic rationale in MM. Disclosures No relevant conflicts of interest to declare.

Direct and Continuous Inhibition of ADAM17 Using a Novel Selective Inhibitor Restores Functional Platelet Yield From Human Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2323-2323
Author(s):  
Shinji Hirata ◽  
Ryoko Jono-Ohnishi ◽  
Satoshi Nishimura ◽  
Naoya Takayama ◽  
Sou Nakamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Imaging System ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
P38 Map Kinase

Abstract Abstract 2323 Platelet transfusion is therapeutically important for patients with thrombocytopenia and/or bleeding disorders. Problems associated with a lack of donors and unknown infections in the blood have not been fully resolved, however. In that context, human induced pluripotent stem cells (hiPSCs) are a potentially abundant source of infection-free platelets. The pluripotent state of hiPSCs and their differentiation depend upon appropriate culture conditions defined in part by oxygen and temperature. We therefore initially examined whether temperatures at or below 24°C, which are required for preservation of platelet concentrates ex vivo, allow hiPSC differentiation to generate platelets. We found that only at 37°C were platelets generated. But at 37°C in vitro, platelets are subject to degradation exemplified by the shedding of GPIbα, a receptor for von Willebrand factor (vWF), which is caused by a disintegrin and metalloprotease (ADAM) 17. We therefore developed KP-457, a novel ADAM17 inhibitor that has a reverse hydroxamic acid structure and has been found safe in rats and dogs. Although inhibition of p38 MAP kinase, putatively upstream of ADAM17, reportedly inhibits GPIbα shedding in stored human platelets, even at 37°C, administration of the p38 inhibitor SB203580 induces cytotoxicity during differentiation, leading to a loss of platelet yield from hiPSCs. By contrast, KP-457 significantly protected GPIbα expression in platelets from hiPSCs and in aged human platelets in culture at 37°C. Moreover, iPSC-derived platelets generated in the presence of KP-457 displayed improved hemostatic function when studied using an imaging system that enables characterization of single-platelet kinetics during thrombus formation after laser-induced injury in vivo. We propose this new drug could markedly improve the maintenance of functional platelets generated in culture, particularly those derived from hiPSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Fracture Healing Research—Shift towards In Vitro Modeling?

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 748
Author(s):  
Moritz Pfeiffenberger ◽  
Alexandra Damerau ◽  
Annemarie Lang ◽  
Frank Buttgereit ◽  
Paula Hoff ◽  
...  
Keyword(s):  
Fracture Healing ◽  
Ex Vivo ◽  
3D Models ◽  
In Vitro Models ◽  
Human Patient ◽  
In Vivo Models ◽  
Underlying Mechanisms ◽  
Biological Situation

Fractures are one of the most frequently occurring traumatic events worldwide. Approximately 10% of fractures lead to bone healing disorders, resulting in strain for affected patients and enormous costs for society. In order to shed light into underlying mechanisms of bone regeneration (habitual or disturbed), and to develop new therapeutic strategies, various in vivo, ex vivo and in vitro models can be applied. Undeniably, in vivo models include the systemic and biological situation. However, transferability towards the human patient along with ethical concerns regarding in vivo models have to be considered. Fostered by enormous technical improvements, such as bioreactors, on-a-chip-technologies and bone tissue engineering, sophisticated in vitro models are of rising interest. These models offer the possibility to use human cells from individual donors, complex cell systems and 3D models, therefore bridging the transferability gap, providing a platform for the introduction of personalized precision medicine and finally sparing animals. Facing diverse processes during fracture healing and thus various scientific opportunities, the reliability of results oftentimes depends on the choice of an appropriate model. Hence, we here focus on categorizing available models with respect to the requirements of the scientific approach.

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