Preparation and Testing of Cells Expressing Fluorescent Proteins for Intravital Imaging of Tumor Microenvironment

Vodopyanov, Stepan S.; Kunin, Mikhail; Garanina, Anastasiia S.; Grinenko, N. F.; Vlasova, Kseniya Yu.; Melnikov, Pavel; Chekhonin, V. P.; Sukhinich, K. K.; Макаров, А. В.; Naumenko, Victor; Abakumov, Maxim; Majouga, Alexander G.

doi:10.1007/s10517-019-04475-3

Cited by 6 publications

(4 citation statements)

References 17 publications

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“…produced similar results; mouse EL-4 lymphoma cells expressing eGFP did not grow after subcutaneous injection to immunocompetent mice, unlike the parental cell line [13]. In another study, subcutaneous injection of GFP expressing 4T1 (murine breast cancer) and B16-F10 (murine melanoma) cells had no effect on the tumor growth rate, whereas CT26-GFP (murine colon cancer) cells were rejected after implantation [14]. When the results of our xenograft study were evaluated together with the aforementioned studies, it was clearly seen that the tumorigenic capacity of GFP-expressing cells varies regardless of the animal model or cell type used.…”

Section: Macroscopic and Microscopic Evaluation Of Xenograft Models R...mentioning

confidence: 72%

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GFP-expressing prostate cancer cells demonstrate attenuated tumorigenicity through regulation of energy metabolism, translational activity, and cytoskeletal dynamics: a proteomic study

Yanar

Sarıhan

Kasap

et al. 2023

Preprint

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Background Green Fluorescent Protein is widely used as a cellular marker tool, but its potential influence on cells has been questioned. Although the potential off-target effects of GFP on tumor cells have been studied to some extent, the findings at the molecular level are insufficient to explain the effect of GFP expression on the tumorigenic capacity of cancer cells. Here, we aimed to investigate the effect of GFP expression on the tumorigenicity of PC3 prostate cancer cells. Methods and Results Using GFP-expressing and wild-type PC3 cells, xenograft models were generated in athymic BALB/C mice. To identify differentially expressed proteins, the change in the proteome of cells was investigated by label‐free quantification with nano‐high performance liquid chromatography to tandem mass spectrometry (nHPLC‐MS/MS). The proteins that showed significantly altered expression levels were evaluated using the bioinformatics tools. The results showed that GFP-expressing cells, in contrast to the wild-type cells, failed to develop tumor. Label-free quantification revealed a total of 216 differentially regulated proteins, of which 98 were upregulated and 117 were downregulated. Conclusion Upon GFP expression, the immune system, translational machinery, energy metabolism, elements of cytoskeletal and VEGF signaling pathway were regulated. Alterations in these cellular events caused attenuated tumorigenicity. Therefore, ultimate care should be taken into account to prevent reporting deceitful mechanisms generated from studies utilizing GFP.

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Section: Macroscopic and Microscopic Evaluation Of Xenograft Models R...mentioning

confidence: 72%

“…Comparison of different mouse tumor cell lines expressing uorescent proteins resulted in varying tumorigenic capacity. Subcutaneous injection of GFP expressing 4T1 and B16-F10 cells had no effect on the tumor growth rate, while both CT26-GFP and CT26-RFP cells were rejected after implantation [14].…”

Section: Introductionmentioning

confidence: 94%

GFP-expressing prostate cancer cells demonstrate attenuated tumorigenicity through regulation of energy metabolism, translational activity, and cytoskeletal dynamics: a proteomic study

Yanar

Sarıhan

Kasap

et al. 2023

Preprint

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“…It is well-known that xenogeneic proteins expression can affect cell growth rate and their tumorigenic potential by suppression of the tumor growth via immune mechanisms [ 16 ]. For a comparative assessment of the growth dynamics of tumors obtained from 4T1 and 4T1-Qt cells, tumor volumes were measured on different days after subcutaneous tumor cells implantation.…”

Section: Resultsmentioning

confidence: 99%

New Approach to Non-Invasive Tumor Model Monitoring via Self-Assemble Iron Containing Protein Nanocompartments

et al. 2022

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According to the World Health Organization, breast cancer is the most common oncological disease worldwide. There are multiple animal models for different types of breast carcinoma, allowing the research of tumor growth, metastasis, and angiogenesis. When studying these processes, it is crucial to visualize cancer cells for a prolonged time via a non-invasive method, for example, magnetic resonance imaging (MRI). In this study, we establish a new genetically encoded material based on Quasibacillus thermotolerans (Q.thermotolerans, Qt) encapsulin, stably expressed in mouse 4T1 breast carcinoma cells. The label consists of a protein shell containing an enzyme called ferroxidase. When adding Fe2+, a ferroxidase oxidizes Fe2+ to Fe3+, followed by iron oxide nanoparticles formation. Additionally, genes encoding mZip14 metal transporter, enhancing the iron transport, were inserted into the cells via lentiviral transduction. The expression of transgenic sequences does not affect cell viability, and the presence of magnetic nanoparticles formed inside encapsulins results in an increase in T2 relaxivity.

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“…4T1 (mouse breast cancer cells) were purchased from the American Type Culture CollectionF (ATCC, Manassas, VA, USA). A GFP-expressing tumor cell line was obtained via lentivirus transduction and had previously been characterized for its in vitro and in vivo growth kinetics [55]. The cells were cultured in RPMI-1640 medium (gibco, New York, NY, USA) with 10% fetal bovine serum (FBS, gibco) and 2 mM L-glutamine (gibco).…”

Section: Animals and Tumor Modelmentioning

confidence: 99%

Neutrophil as a Carrier for Cancer Nanotherapeutics: A Comparative Study of Liposome, PLGA, and Magnetic Nanoparticles Delivery to Tumors

Garanina,

Vishnevskiy,

Chernysheva

et al. 2023

Pharmaceuticals

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Insufficient drug accumulation in tumors is still a major concern for using cancer nanotherapeutics. Here, the neutrophil-based delivery of three nanoparticle types—liposomes, PLGA, and magnetite nanoparticles—was assessed both in vitro and in vivo. Confocal microscopy and a flow cytometry analysis demonstrated that all the studied nanoparticles interacted with neutrophils from the peripheral blood of mice with 4T1 mammary adenocarcinoma without a significant impact on neutrophil viability or activation state. Intravital microscopy of the tumor microenvironment showed that the neutrophils did not engulf the liposomes after intravenous administration, but facilitated nanoparticle extravasation in tumors through micro- and macroleakages. PLGA accumulated along the vessel walls in the form of local clusters. Later, PLGA nanoparticle-loaded neutrophils were found to cross the vascular barrier and migrate towards the tumor core. The magnetite nanoparticles extravasated in tumors both via spontaneous macroleakages and on neutrophils. Overall, the specific type of nanoparticles largely determined their behavior in blood vessels and their neutrophil-mediated delivery to the tumor. Since neutrophils are the first to migrate to the site of inflammation, they can increase nanodrug delivery effectiveness for nanomedicine application.

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Preparation and Testing of Cells Expressing Fluorescent Proteins for Intravital Imaging of Tumor Microenvironment

Cited by 6 publications

References 17 publications

GFP-expressing prostate cancer cells demonstrate attenuated tumorigenicity through regulation of energy metabolism, translational activity, and cytoskeletal dynamics: a proteomic study

GFP-expressing prostate cancer cells demonstrate attenuated tumorigenicity through regulation of energy metabolism, translational activity, and cytoskeletal dynamics: a proteomic study

New Approach to Non-Invasive Tumor Model Monitoring via Self-Assemble Iron Containing Protein Nanocompartments

Neutrophil as a Carrier for Cancer Nanotherapeutics: A Comparative Study of Liposome, PLGA, and Magnetic Nanoparticles Delivery to Tumors

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