Abstract 4011: Validation of novel continuous live-cell assays for immune cell activation and killing of blood cell cancers
| Autor: | Derek J. Trezise, Clare Szybut, Dan Appledorn, Nicola Bevan, Hinnah Campwala, Tim Dale, Kalpana Patel |
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| Rok vydání: | 2017 |
| Předmět: | |
| Zdroj: | Cancer Research. 77:4011-4011 |
| ISSN: | 1538-7445 0008-5472 |
| Popis: | The blood cancers leukaemia, lymphoma and myeloma are expected to cause the deaths of > 55,000 people in the US in 2016. New immunological approaches afford great promise for improved therapies. Here, we describe novel high throughput live-cell image-based assays for immune cell activation and killing of target cells that are geared toward screening for new treatments for these malignancies. Myeloid and lymphoid cells (Jurkats, Raji, Ramos, WIL2-NS, THP-1, PBMCs) were plated on poly-L-ornithine (PLO) or fibronectin coated 96-well flat plates and monitored over time (h to days) using non-invasive live-cell analysis (IncuCyte). The dynamics of proliferation were quantified via phase-contrast image analysis (% confluence), and validated as a robust measure of cell number by correlating to direct cell counts (Scepter, Millipore) and ATP assays (PerkinElmer). Anti-CD3/IL-2 (0.1-100/10 ng/mL) or anti-CD28 activation (1-100 ng/mL) of PBMCs evoked time-dependent proliferation (0-5d) that was sensitive to the initial cell density and concentration of stimulus. L-Kynurenine (4.69 - 300 µM), a metabolite of the amino acid L-tryptophan caused concentration and time-dependent inhibition of proliferation of PBMCs. To quantify immune-cell killing in co-cultures, WIL2Ns and Ramos B-cell myelomas were first transduced with nuclear-targeted RFP (NucLight Red) to enable direct cell counting. PBMCs, either pre-activated or activated in situ (IL-2/CD3), were then added and the time-course of killing quantified through live (RFP) and dead/apoptotic (annexin-V) cell counting. Together, these protocol developments and validation data illustrate non-invasive continuous measurement of proliferation, activation, clustering and immune-cell killing of non-adherent tumour cells at industrial scale. Unlike flow cytometry, this approach follows the full time-course of the biology without perturbing the cells and allows cell-cell interactions to be visualised. These assays are amenable to testing new therapeutic antibodies, small molecules and genetic T-cell modulation such as CAR-T. Citation Format: Nicola Bevan, Hinnah Campwala, Clare Szybut, Kalpana Patel, Dan Appledorn, Tim Dale, Derek Trezise. Validation of novel continuous live-cell assays for immune cell activation and killing of blood cell cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4011. doi:10.1158/1538-7445.AM2017-4011 |
| Databáze: | OpenAIRE |
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