scholarly journals Novel in Vitro and In Vivo Drug Screening Platform for ALL.

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1189-1189
Author(s):  
Bandana Ajay Vishwakarma ◽  
Amy Wesa
Keyword(s):  
Bone Marrow ◽  
Drug Testing ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Drug Efficacy ◽  
Surface Marker ◽  
Surface Marker Expression ◽  
Marker Expression

Abstract Acute Lymphoblastic leukemia (ALL) is a malignancy of bone marrow. Accumulation of mutations in lymphoid progenitor cells give rise to either B-ALL or T-ALL. Treatment for ALL has improved in recent years, yet relapse of the disease and development of resistance are observed in patients. Lack of suitable and robust in vitro and in vivo drug testing platforms for primary ALL cells along with the lack of rapid development of novel therapeutics drugs encompassing the heterogeneity of the disease contribute to the delay in approved patient treatments. We have developed a short-term culture system that supports the survival of primary B-ALL and T-ALL cells. Our ALL bank includes patient-derived specimens with complete cytogenetics and surface marker expression information. Different culture conditions were evaluated to select conditions that support the survival and maintenance of primary B-ALL and T-ALL specimens. Cell growth/viability was assessed using the Cell Titer-Glo ® assay. Primary B-ALL cells survived in the optimized media for 3 days and a heterogenous dose dependent response was observed across the models to chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. BCR-ABl - B-ALL patient samples were found to be resistant to imatinib in contrast to BCR-ABL + samples which were sensitive to imatinib. Similarly, culture conditions optimized for T-ALL primary cells supported the survival until day 6 and displayed a diverse response to standard of care drugs like venetoclax, imatinib, vincristine, cytarabine and methotrexate, reflecting the heterogeneity of the patient derived specimens. Immunophenotypic characterization of ALL cells grown in culture displayed retention of the B and T cells surface marker expression. Further, a patient derived pre-clinical xenograft model was developed in NCG mice to study in vivo ALL drug efficacy. 100% engraftment was observed for B-ALL primary cells, with latency of engraftment (>3%) in peripheral blood varying from 15 days to 3.5 months. 30-90% of the bone marrow cells were occupied by human CD45 cells. Infiltration of human B-ALL cells were observed in the spleen causing splenomegaly. 8 out of the 14 models having high penetrance were passaged until P3. Flow analysis at each passage demonstrated surface marker expression displaying low divergence from the primary samples. Additionally, evaluation of ex vivo drug response from B-ALL PDX splenocytes was largely concordant with the primary specimen ex vivo data in three of the models evaluated. In an in vivo drug efficacy study administration of venetoclax, CHOP and R-CHOP inhibited the proliferation of B-ALL cells. Significant reduction of B-ALL cells was observed while on treatment with Venetoclax. At termination of the study, up to 80% reduction of human B-ALL cells was observed in whole blood, bone marrow, and spleen after treatment with CHOP and R-CHOP in comparison to the vehicle cohort. Similarly, patient derived T-ALL pre-clinical xenograft model development is in progress. Thus, we have developed a robust in vitro drug testing platform for B-ALL and T-ALL to evaluate drug efficacy. We also demonstrate that NCG mice support the growth and proliferation of primary B-ALL cells and have successfully developed an in vivo platform that will facilitate the testing of clinically relevant chemotherapeutic drugs for ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

Leukemia ◽  
2016 ◽  
Vol 31 (8) ◽  
pp. 1743-1751 ◽  
Author(s):  
S Hipp ◽  
Y-T Tai ◽  
D Blanset ◽  
P Deegen ◽  
J Wahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cell ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Selective Lysis ◽  
Bispecific T Cell Engager

Abstract B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

scholarly journals A Novel Infection Protocol in Zebrafish Embryo to Assess Pseudomonas aeruginosa Virulence and Validate Efficacy of a Quorum Sensing Inhibitor In Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 401
Author(s):  
Pauline Nogaret ◽  
Fatima El El Garah ◽  
Anne-Béatrice Blanc-Potard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Animal Model ◽  
Quorum Sensing ◽  
Drug Testing ◽  
Drug Efficacy ◽  
Embryo Viability ◽  
Immersion Method ◽  
Opportunistic Human Pathogen

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for a variety of acute infections and is a major cause of mortality in chronically infected cystic fibrosis patients. Due to increased resistance to antibiotics, new therapeutic strategies against P. aeruginosa are urgently needed. In this context, we aimed to develop a simple vertebrate animal model to rapidly assess in vivo drug efficacy against P. aeruginosa. Zebrafish are increasingly considered for modeling human infections caused by bacterial pathogens, which are commonly microinjected in embryos. In the present study, we established a novel protocol for zebrafish infection by P. aeruginosa based on bath immersion in 96-well plates of tail-injured embryos. The immersion method, followed by a 48-hour survey of embryo viability, was first validated to assess the virulence of P. aeruginosa wild-type PAO1 and a known attenuated mutant. We then validated its relevance for antipseudomonal drug testing by first using a clinically used antibiotic, ciprofloxacin. Secondly, we used a novel quorum sensing (QS) inhibitory molecule, N-(2-pyrimidyl)butanamide (C11), the activity of which had been validated in vitro but not previously tested in any animal model. A significant protective effect of C11 was observed on infected embryos, supporting the ability of C11 to attenuate in vivo P. aeruginosa pathogenicity. In conclusion, we present here a new and reliable method to compare the virulence of P. aeruginosa strains in vivo and to rapidly assess the efficacy of clinically relevant drugs against P. aeruginosa, including new antivirulence compounds.

scholarly journals hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

2022 ◽  
Author(s):  
Homa Majd ◽  
Ryan M Samuel ◽  
Jonathan T Ramirez ◽  
Ali Kalantari ◽  
Kevin Barber ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Xenograft Model ◽  
Developmental Relationships ◽  
Drug Candidates ◽  
Effective Strategies ◽  
Wide Range ◽  
Functional Features ◽  
And Function

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.  

scholarly journals Tumor-Derived Factors Differentially Affect the Recruitment and Plasticity of Neutrophils

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5082
Author(s):  
Ludovica Arpinati ◽  
Naomi Kaisar-Iluz ◽  
Merav E. Shaul ◽  
Christopher Groth ◽  
Viktor Umansky ◽  
...  
Keyword(s):  
Cancer Biology ◽  
Ex Vivo ◽  
Surface Marker ◽  
Normal Density ◽  
Low Density ◽  
Ros Production ◽  
Surface Marker Expression ◽  
Density State ◽  
Surface Receptor

Neutrophils play a key role in cancer biology. In contrast to circulating normal-density neutrophils (NDN), the amount of low-density neutrophils (LDN) significantly increases with tumor progression. The correlation between these neutrophil subpopulations and intratumoral neutrophils (TANs) is still under debate. Using 4T1 (breast) and AB12 (mesothelioma) tumor models, we aimed to elucidate the source of TANs and to assess the mechanisms driving neutrophils’ plasticity in cancer. Both NDN and LDN were found to migrate in response to CXCL1 and CXCL2 exposure, and co-infiltrate the tumor site ex vivo and in vivo, although LDN migration into the tumor was higher than NDN. Tumor-derived factors and chemokines, particularly CXCL1, were found to drive neutrophil phenotypical plasticity, inducing NDN to transition towards a low-density state (LD-NDN). LD-NDN appeared to differ from NDN by displaying a phenotypical profile similar to LDN in terms of nuclear morphology, surface receptor markers, decreased phagocytic abilities, and increased ROS production. Interestingly, all three subpopulations displayed comparable cytotoxic abilities towards tumor cells. Our data suggest that TANs originate from both LDN and NDN, and that a portion of LDN derives from NDN undergoing phenotypical changes. NDN plasticity resulted in a change in surface marker expression and functional activity, gaining characteristics of LDN.

Ex vivo-expanded bone marrow CD34+ for acute myocardial infarction treatment: in vitro and in vivo studies

Cytotherapy ◽  
2011 ◽  
Vol 13 (9) ◽  
pp. 1140-1152 ◽  
Author(s):  
Monica Gunetti ◽  
Alessio Noghero ◽  
Fabiola Molla ◽  
Lidia Irene Staszewsky ◽  
Noeleen de Angelis ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Bone Marrow ◽  
Ex Vivo ◽  
In Vivo Studies

scholarly journals Simultaneous targeting of DNA replication and homologous recombination in glioblastoma with a polyether ionophore

2019 ◽  
Author(s):  
Yi Chieh Lim ◽  
Kathleen S Ensbey ◽  
Carolin Offenhäuser ◽  
Rochelle C J D’souza ◽  
Jason K Cullen ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
First Generation ◽  
Ex Vivo ◽  
Drug Efficacy ◽  
Tumor Formation ◽  
Dna Lesions ◽  
Dual Functions

Abstract Background Despite significant endeavor having been applied to identify effective therapies to treat glioblastoma (GBM), survival outcomes remain intractable. The greatest nonsurgical benefit arises from radiotherapy, though tumors typically recur due to robust DNA repair. Patients could therefore benefit from therapies with the potential to prevent DNA repair and synergize with radiotherapy. In this work, we investigated the potential of salinomycin to enhance radiotherapy and further uncover novel dual functions of this ionophore to induce DNA damage and prevent repair. Methods In vitro primary GBM models and ex vivo GBM patient explants were used to determine the mechanism of action of salinomycin by immunoblot, flow cytometry, immunofluorescence, immunohistochemistry, and mass spectrometry. In vivo efficacy studies were performed using orthotopic GBM animal xenograft models. Salinomycin derivatives were synthesized to increase drug efficacy and explore structure-activity relationships. Results Here we report novel dual functions of salinomycin. Salinomycin induces toxic DNA lesions and prevents subsequent recovery by targeting homologous recombination (HR) repair. Salinomycin appears to target the more radioresistant GBM stem cell–like population and synergizes with radiotherapy to significantly delay tumor formation in vivo. We further developed salinomycin derivatives which display greater efficacy in vivo while retaining the same beneficial mechanisms of action. Conclusion Our findings highlight the potential of salinomycin to induce DNA lesions and inhibit HR to greatly enhance the effect of radiotherapy. Importantly, first-generation salinomycin derivatives display greater efficacy and may pave the way for clinical testing of these agents.

MLN9708 Elicits Pharmacodynamic Response in the Bone Marrow Compartment and Has Strong Antitumor Activity in a Preclinical Intraosseous Model of Plasma Cell Malignancy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1834-1834 ◽  
Author(s):  
Edmund Lee ◽  
Bret Bannerman ◽  
Michael Fitzgerald ◽  
Jennifer Terkelsen ◽  
Daniel Bradley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Antitumor Activity ◽  
Plasma Cell ◽  
Research Funding ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Tumor Burden ◽  
Human Multiple Myeloma ◽  
Cell Malignancy

Abstract Abstract 1834 Poster Board I-860 Introduction The clinical success of VELCADE® (bortezomib) for Injection has validated the proteasome as a therapeutic target for the treatment of human cancer. The novel proteasome inhibitor MLN9708 is a potent, reversible, and specific inhibitor of the b5 site of the 20S proteasome identified in preclinical studies. MLN9708 is currently in human clinical development for both hematological and non-hematological malignancies. Here we describe the pharmacodynamic (PD) response of MLN9708 in the murine bone marrow compartment and its strong antitumor activity in an intraosseous xenograft model of plasma cell malignancy. Materials MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Methods It has been previously shown that double transgenic iMycCa/Bcl-XL mice develop de novo plasma cell malignancies (J. Clin. Invest. 113:1763-1773, 2004) in which neoplastic plasma cell development is driven by the targeted expression of the transgene Myc (c-myc; myelocytomatosis oncogene) and Bcl-x (Bcl2l1; encodes the oncoprotein Bcl-XL). DP54 is a plasma cell tumor cell line derived from the bone marrow of a syngeneic mouse previously inoculated with an iMycCa/Bcl-XL tumor (Cancer Res. 67:4069-4078, 2007). In vitro, DP54 cells express both the Myc and Bcl-XL transgenes, various plasma cell and B-cell markers including CD38, CD138 and B220, and has gene expression profile very similar to human multiple myeloma. To establish a preclinical intraosseous model of plasma cell malignancy for efficacy studies, freshly dissociated DP54-Luc cells (constitutively expressing firefly luciferase under a mouse Ig-k promoter) were aseptically injected into the bone marrow space of the upper shaft of the right tibia of NOD-SCID mice. Once tumor growth has been established, mice were randomized into treatment groups and then treated intravenously (IV) with vehicle, bortezomib (at 0.8 mg/kg twice weekly [BIW]) or MLN2238 (at 11 mg/kg BIW) for 3 consecutive weeks. Tumor burden was measured by bioluminescent imaging. Results MLN2238 strongly inhibited proteasome activity in the blood and bone marrow compartments of mice (maximum b5 inhibition of 84% and 83%, respectively). In vivo, when DP54 cells were aseptically injected into the bone marrow space of the mouse tibia, signs of bone erosion in the tibia, femur and cranial sagittal sultures (as determined by ex-vivo mCT imaging) were observed which resembled osteolytic lesions frequently seen in human multiple myeloma. Dissemination of DP54-Luc cells after intratibia inoculations were detected by in vivo bioluminescent and confirmed by ex vivo imaging where luminescent tumor nodules were detected in the spleen, kidneys, intestine, lymph nodes and bones including right tibia, spine and cranium. To assess the antitumor activity of MLN2238 in the bone marrow compartment, an efficacy study was performed using the DP54-Luc intraosseous xenograft model of plasma cell malignancy. Tumor burden (bioluminescence), osteolytic lesions (mCT) and overall survival after treatment with bortezomib and MLN2238 will be presented. Conclusion The novel proteasome inhibitor MLN9708 demonstrates strong activity in the bone marrow compartment in vivo. MLN9708 is currently in human clinical development for both hematological and solid tumor indications. Disclosures Lee: Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Terkelsen:Milllennium: Employment. Bradley:Milllennium: Employment, Equity Ownership, Research Funding. Li:Milllennium: Employment. Li:Milllennium: Employment. Janz:Milllennium: Research Funding. Van Ness:Milllennium: Research Funding. Manfredi:Milllennium: Employment. Kupperman:Milllennium: Employment.

The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3090-3090 ◽  
Author(s):  
Folashade Otegbeye ◽  
Nathan Mackowski ◽  
Evelyn Ojo ◽  
Marcos De Lima ◽  
David N. Wald
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Leukemia Cells ◽  
Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

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