Cancer Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 1531 Views Jun 5, 2023

Exosomes are lipid bilayer–enclosed vesicles, actively secreted by cells, containing proteins, lipids, nucleic acids, and other substances with multiple biological functions after entering target cells. Exosomes derived from NK cells have been shown to have certain anti-tumor effects and potential applications as chemotherapy drug carriers. These developments have resulted in high demand for exosomes. Although there has been large-scale industrial preparation of exosomes, they are only for generally engineered cells such as HEK 293T. The large-scale preparation of specific cellular exosomes is still a major problem in laboratory studies. Therefore, in this study, we used tangential flow filtration (TFF) to concentrate the culture supernatants isolated from NK cells and isolated NK cell–derived exosomes (NK-Exo) by ultracentrifugation. Through a series of characterization and functional verification of NK-Exo, the characterization, phenotype, and anti-tumor activity of NK-Exo were verified. Our study provides a considerably time- and labor-saving protocol for the isolation of NK-Exo.

0 Q&A 1885 Views Jan 20, 2022

Nanomaterials are increasingly used for the diagnosis and treatment of cancer, including lung cancer. For the clinical translation of nano-based theranostics, it is vital to detect and monitor their accumulation in the tumor, as well as their interaction with tumor, immune cells, and the tumor microenvironment (TME). While high resolution microscopy of fixed tumor specimens can provide some of this information from individual thin slices, it cannot capture cellular events over time and lacks 3D information of the tumor tissue. On the other hand, in vivo optical procedures either fall short of providing the necessary cellular resolution, as in the case of epifluorescence optical imaging, or are very demanding, as for instance intravital lung microscopy. We describe an alternative approach to investigate nanoparticle-cell interactions in entire mouse lung lobes, by longitudinal live cell confocal microscopy at nanometer resolution. By filling the lung ex vivo with 1% agarose, we were able to stabilize the lung lobes and visualize the interaction of fluorescent cells and nanoparticles for at least 4 hours post mortem. This high resolution ex vivo live cell imaging approach is an easy 4D tool for assessing several dynamic processes in tumor tissue, such as the traffic of cells, shedding of extracellular vesicles (EVs), and the accumulation of nanoparticles in tumor tissue.

Graphic abstract:

Schematic of the workflow for live cell imaging in the mouse lung.

0 Q&A 6133 Views Apr 5, 2020
The binding interactions of PD-1 and PD-L1 have been studied by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) over the past few years, but these investigations resulted in controversy regarding the values of the dissociation constant (Kd) (Freeman et al., 2000). MST is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. The technique is based on the movement of molecules along microscopic temperature gradients, and it detects changes in their hydration shell, charge or size. One binding partner is fluorescently labeled, while the other binding partner remains label-free. We used a protocol that allows the determination of the binding affinity by MST without purification of the target protein from the cell lysate. The application of this MST method to PD-1-eGFP and PD-L1-eGFP expressed in CHO-K1 cells allowed us, for the first time, to determine the affinity of the complex formed between PD-1 and its ligand PD-L1 during tumor escape. The protocol has a variety of potential applications for studying the interactions of proteins with small molecules.

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