RESUMO
Apoptosis is the programmed cell death pathway that is critical for maintaining homeostasis, in which cancer cells can evade to ensure survival. For pharmaceutical drug discovery, it is important to characterize and compare different cancer therapeutics (i.e., small molecules, antibody drugs, cell therapies) that can initiate the process of apoptosis, enabling the identification of potential therapeutic candidates. In this work, we developed and demonstrated a multiplex detection method for monitoring apoptosis and necrosis with Annexin V, Caspase-3, and Propidium Iodide (PI) using the Cellaca® PLX Image Cytometer (Revvity Health Sciences, Inc., Lawrence, MA). First, apoptosis was induced in Jurkat and K562 cell lines with staurosporine over the course of 24 h, where apoptosis and necrosis were assessed at 0, 1, 1.5, 2, 4, 20, and 24 h timepoints. Samples were stained with Hoechst 33342 (total dye), Annexin V-APC (early-stage apoptosis), Caspase-3 488 (late-stage apoptosis), and PI (necrosis) at each timepoint and evaluated using image cytometry. Results showed that apoptotic factors and cascades were successfully detected along the pathway from early- to late-stage apoptosis, and ultimately necrosis. A clear trend was observed analyzing apoptotic and necrotic populations during the first 1.5 h, showing differences of up to ~15% in single Annexin V+ and Caspase-3+ populations in treated Jurkat cells, however, a significant increase in double positive apoptotic/necrotic cells for Annexin V+PI+ and Capase-3+PI+ was not observed until 20 h. Upon further analysis between apoptotic populations only, Annexin V+ only populations were higher than Caspase-3+ only populations by up to ~20% between 0 and 1.5 h. Conversely, K562 cells did not exhibit a notable change in apoptotic and necrotic populations due to low sensitivity to staurosporine. The proposed image cytometric detection method may provide an effective and efficient tool for rapid and reliable simultaneous detection of early- late-stage apoptosis, and necrosis. Therefore, allowing researchers to better characterize and screen potential cancer therapeutic drug candidates for their treatment efficacy in a higher throughput manner.
RESUMO
Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field.
Assuntos
Apoptose , Humanos , Imunofenotipagem , Transfecção , Linhagem Celular , Células Jurkat , Citometria de Fluxo/métodosRESUMO
Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation - arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.
Assuntos
Carcinogênese/genética , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Receptores ErbB/genética , Receptores ErbB/metabolismo , Mutação , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
Inflammation in the diabetic retina is mediated by leukocyte adhesion to the retinal vasculature and alteration of the blood-retinal barrier (BRB). We investigated the role of chemokines in the alteration of the BRB in diabetes. Animals were made diabetic by streptozotocin injection and analyzed for gene expression and monocyte/macrophage infiltration. The expression of CCL2 (chemokine ligand 2) was significantly up-regulated in the retinas of rats with 4 and 8 weeks of diabetes and also in human retinal endothelial cells treated with high glucose and glucose flux. Additionally, diabetes or intraocular injection of recombinant CCL2 resulted in increased expression of the macrophage marker, F4/80. Cell culture impedance sensing studies showed that purified CCL2 was unable to alter the integrity of the human retinal endothelial cell barrier, whereas monocyte conditioned medium resulted in significant reduction in cell resistance, suggesting the relevance of CCL2 in early immune cell recruitment for subsequent barrier alterations. Further, using Cx3cr1-GFP mice, we found that intraocular injection of CCL2 increased retinal GFP+ monocyte/macrophage infiltration. When these mice were made diabetic, increased infiltration of monocytes/macrophages was also present in retinal tissues. Diabetes and CCL2 injection also induced activation of retinal microglia in these animals. Quantification by flow cytometry demonstrated a two-fold increase of CX3CR1+/CD11b+ (monocyte/macrophage and microglia) cells in retinas of wildtype diabetic animals in comparison to control non-diabetic ones. Using CCL2 knockout (Ccl2-/-) mice, we show a significant reduction in retinal vascular leakage and monocyte infiltration following induction of diabetes indicating the importance of this chemokine in alteration of the BRB. Thus, CCL2 may be an important therapeutic target for the treatment of diabetic macular edema.