| 1. |
- Arruda, Lucas C. M., et al.
(författare)
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A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo
- 2022
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 0390-6078 .- 1592-8721. ; 107:8, s. 1786-1795
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Tidskriftsartikel (refereegranskat)abstract
- Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34(+) blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34(+)CD38(-) phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34(+) hematopoietic stem cells from peripheral blood stem cell grafts and CD34(+) blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34(+) blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.
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| 2. |
- Benninger, R. K. P., et al.
(författare)
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Fluorescence imaging of two-photon linear dichroism : Cholesterol depletion disrupts molecular orientation in cell membranes
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 88:1, s. 609-622
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Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane of cells is an ordered environment, giving rise to anisotropic orientation and restricted motion of molecules and proteins residing in the membrane. At the same time as being an organized matrix of defined structure, the cell membrane is heterogeneous and dynamic. Here we present a method where we use fluorescence imaging of linear dichroism to measure the orientation of molecules relative to the cell membrane. By detecting linear dichroism as well as fluorescence anisotropy, the orientation parameters are separated from dynamic properties such as rotational diffusion and homo energy transfer ( energy migration). The sensitivity of the technique is enhanced by using two-photon excitation for higher photo-selection compared to single photon excitation. We show here that we can accurately image lipid organization in whole cell membranes and in delicate structures such as membrane nanotubes connecting two cells. The speed of our wide-field imaging system makes it possible to image changes in orientation and anisotropy occurring on a subsecond timescale. This is demonstrated by time-lapse studies showing that cholesterol depletion rapidly disrupts the orientation of a fluorophore located within the hydrophobic region of the cell membrane but not of a surface bound probe. This is consistent with cholesterol having an important role in stabilizing and ordering the lipid tails within the plasma membrane.
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| 3. |
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| 4. |
- Benninger, Richard K. P., et al.
(författare)
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Live Cell Linear Dichroism Imaging Reveals Extensive Membrane Ruffling within the Docking Structure of Natural Killer Cell Immune Synapses
- 2009
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 96:2, s. L13-L15
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Tidskriftsartikel (refereegranskat)abstract
- We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into ruffles at the periphery, but not in the center of a mature cytolytic NK cell IS. Time-lapse imaging showed that the membrane ruffles formed at the initial point of contact between NK cells and target cells and then spread radialy across the intercellular contact as the size of the IS increased, becoming absent from the center of the mature synapse. Understanding the role of such extensive membrane ruff ling in the assembly of cytolytic synapses is an intriguing new goal.
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| 5. |
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| 6. |
- Brandt, Ludwig
(författare)
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NK Cell Cytotoxicity at the Single Cell Level
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Natural killer (NK) cells are innate immune cells with the ability to recognize and eliminate virally infected cells and cancer cells without prior sensitization. There is a functional heterogeneity between individual NK cells, where some NK cells are more efficient at killing cancer cells than others. Methods that allow studies of single NK cells are required to understand the functional differences and how they correlate with the activation and development status of the NK cell.This thesis focuses on the development and implementation of microchip- based imaging of NK cells, which is covered in five papers. Paper I presents a microchip screening platform for assessment of the cytotoxic potential of individual NK cells, by confining single NK cells together with target cells in microwells, followed by microscopy screening over extended time periods and automated image analysis. In paper II, the microchip platform was applied to test the ability of a novel trispecific killer engager (TriKE) to mediate an NK cell-dependent immune response. The process of NK cell education was studied in paper III and for that the image analysis methods for the microchip platform was further developed, in order to reveal new insight into how the education process affects the cytotoxic function of single NK cells. In paper IV, a previously developed microchip assay was extended to study NK cell migration and cytotoxicity in a more in vivo-like 3D collagen matrix. Paper V shows how NK cells can eliminate platelets in the presence of anti-platelet antibodies.In summary, this thesis covers the development and applications of time- lapse imaging using microwells for studying important NK cell functions in different settings. Understanding NK cell heterogeneity has the potential for improving e.g. cancer cell therapies.
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| 7. |
- Carannante, Valentina, et al.
(författare)
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Generation of tumor spheroids in microwells to study NK cell cytotoxicity, infiltration and phenotype
- 2023
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Ingår i: Methods in Cell Biology. - : Elsevier BV. - 0091-679X. ; , s. 195-208
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The development of new immunotherapeutic drugs and combinatorial strategies requires the implementation of novel methods to test their efficacy in vitro. Here, we present a series of miniaturized in vitro assays to assess immune cell cytotoxic activity, infiltration, and phenotype in renal carcinoma spheroids with the use of a recently developed multichambered microwell chip. We provide protocols for tumor spheroid formation, NK cell culture, fluorescence labelling and imaging of live or fixed cells directly in the chip together with data analysis.
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| 8. |
- Carannante, Valentina, et al.
(författare)
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In vitro models to study natural killer cell dynamics in the tumor microenvironment
- 2023
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 14
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Forskningsöversikt (refereegranskat)abstract
- Immunotherapy is revolutionizing cancer therapy. The rapid development of new immunotherapeutic strategies to treat solid tumors is posing new challenges for preclinical research, demanding novel in vitro methods to test treatments. Such methods should meet specific requirements, such as enabling the evaluation of immune cell responses like cytotoxicity or cytokine release, and infiltration into the tumor microenvironment using cancer models representative of the original disease. They should allow high-throughput and high-content analysis, to evaluate the efficacy of treatments and understand immune-evasion processes to facilitate development of new therapeutic targets. Ideally, they should be suitable for personalized immunotherapy testing, providing information for patient stratification. Consequently, the application of in vitro 3-dimensional (3D) cell culture models, such as tumor spheroids and organoids, is rapidly expanding in the immunotherapeutic field, coupled with the development of novel imaging-based techniques and -omic analysis. In this paper, we review the recent advances in the development of in vitro 3D platforms applied to natural killer (NK) cell-based cancer immunotherapy studies, highlighting the benefits and limitations of the current methods, and discuss new concepts and future directions of the field.
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| 9. |
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| 10. |
- Christakou, Athanasia, et al.
(författare)
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Characterization of natural killer cell immune surveillance against solid liver tumors
- 2015
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Ingår i: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. - 9780979806483 ; , s. 915-917
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Konferensbidrag (refereegranskat)abstract
- We demonstrate a method for investigating natural killer (NK) cell aggression against ultrasound-assisted human hepatocellular carcinoma (HCC) HepG2 solid tumors in a multi-well microdevice. We quantify the activity of human primary IL-2 activated NK cells against HepG2 tumors for up to five days and we present the correlation between NK cell numbers versus average tumor volume and final tumor outcome (growth or defeat). We suggest future applications on formation of tumors originated from primary tumors cells and other tumor components as well as primary NK originating from the patient for use in personalized immunotherapy.
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