Multiphoton intravital microscopy of rodents

Scheele, Colinda L.G.J.; Herrmann, David; Yamashita, Erika; Celso, Cristina Lo; Jenne, Craig N.; Oktay, Maja H.; Entenberg, David; Friedl, Peter; Weigert, Roberto; Meijboom, F.L.B.; Ishii, Masaru; Timpson, Paul; Rheenen, Jacco van

doi:10.1038/s43586-022-00168-w

Cited by 34 publications

(27 citation statements)

References 373 publications

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“…Intravital imaging allows us to assess cellular behavior in live tissue within an intact cellular ecosystem, thereby facilitating high-fidelity investigations of biology in the tissue or organ of choice (22)(23)(24)55). In addition, examining the reversible nature of signaling in response to drug treatment or disease progression at the single-cell level in vivo in this way provides vital information on signaling dynamics in a spatiotemporal manner, which cannot be achieved by conventional biochemical or genomic approaches that often provide single-time point insights into disease states (22,45).…”

Section: Discussionmentioning

confidence: 99%

“…Low-level expression from the Rosa26 locus was chosen to avoid any possible dominant-negative effects of expressing the biosensor in native tissues (see fig. S1, A and B) while simultaneously allowing for a sufficiently bright fluorescent signal for robust intravital FLIM-FRET imaging, as achieved previously (20,24,32,56). We used the Akt-FRET biosensor characterized previously (4, 5), which undergoes a conformational change in response to phosphorylation by AKT via a consensus sequence, assuming a FRET conformation and thereby decreasing the lifetime of the donor mTurquoise2 (57).…”

Section: Discussionmentioning

confidence: 99%

“…This was coupled with high-throughput assessment of primary Akt-FRET cancer cells in three-dimensional (3D) organotypic invasion assays to validate the anti-invasive capacity of AKT-PI3K pathway inhibition. Furthermore, using optical window imaging ( 18 – 21 ) in breast cancer GEMMs, we mapped drug targeting in an AKT hyperactivated setting, demonstrating the utility of the Akt-FRET biosensor mouse in preclinical imaging of targeted therapies in a longitudinal manner ( 22 – 24 ).…”

Section: Introductionmentioning

confidence: 99%

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Monitoring AKT activity and targeting in live tissue and disease contexts using a real-time Akt-FRET biosensor mouse

et al. 2023

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Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Monitoring AKT activity and targeting in live tissue and disease contexts using a real-time Akt-FRET biosensor mouse

et al. 2023

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“…Immediately prior to tumor imaging, skin flap surgery exposed flank tumors. Mice were anesthetized with isoflurane, then the skin around the tumor was cut into a flap and separated from the body cavity so that the tumor laid flat on the imaging stage while still connected to the vasculature (43)(44)(45). Mice were placed on a specialized microscope stage for imaging and kept in a heating chamber (air maintained at 37°C) during imaging.…”

Section: Cd4 Mcherry Reporter Mouse Breeding Tumor Inoculation and Su...mentioning

confidence: 99%

Single cell metabolic imaging of tumor and immune cells in vivo in melanoma bearing mice

et al. 2023

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IntroductionMetabolic reprogramming of cancer and immune cells occurs during tumorigenesis and has a significant impact on cancer progression. Unfortunately, current techniques to measure tumor and immune cell metabolism require sample destruction and/or cell isolations that remove the spatial context. Two-photon fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent metabolic coenzymes nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) provides in vivo images of cell metabolism at a single cell level.MethodsHere, we report an immunocompetent mCherry reporter mouse model for immune cells that express CD4 either during differentiation or CD4 and/or CD8 in their mature state and perform in vivo imaging of immune and cancer cells within a syngeneic B78 melanoma model. We also report an algorithm for single cell segmentation of mCherry-expressing immune cells within in vivo images.ResultsWe found that immune cells within B78 tumors exhibited decreased FAD mean lifetime and an increased proportion of bound FAD compared to immune cells within spleens. Tumor infiltrating immune cell size also increased compared to immune cells from spleens. These changes are consistent with a shift towards increased activation and proliferation in tumor infiltrating immune cells compared to immune cells from spleens. Tumor infiltrating immune cells exhibited increased FAD mean lifetime and increased protein-bound FAD lifetime compared to B78 tumor cells within the same tumor. Single cell metabolic heterogeneity was observed in both immune and tumor cells in vivo.DiscussionThis approach can be used to monitor single cell metabolic heterogeneity in tumor cells and immune cells to study promising treatments for cancer in the native in vivo context.

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“…[ 23 ] Optical imaging, especially fluorescence microscopy, offers a powerful tool with an excellent spatial or temporal resolution, which enables investigations on tissue structures, dynamics of cellular components [ 24 ] as well as intravital studies. [ 25 ] However, an intrinsic limitation of optical imaging is that the tissue‐penetration depth of light is limited to several millimeters, [ 26 ] resulting in a restricted application for tracking CAR T cells in large animals and humans. Among imaging modalities widely used in the clinic, MRI exhibits the highest spatial resolution (20–100 µm) to soft tissues, [ 26 ] whereas its low sensitivity limits its application in tracking the adoptive CAR T cells in vivo.…”

Section: Introductionmentioning

confidence: 99%

Nuclear Imaging of CAR T Immunotherapy to Solid Tumors: In Terms of Biodistribution, Viability, and Cytotoxic Effect

et al. 2023

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Immunotherapy has become a mainstay of cancer therapy. Since chimeric antigen receptor (CAR) T immunotherapy achieves unprecedented success in curing hematological malignancies, the possibility of it revolutionizing the paradigm of solid tumors has aroused increasing attention. However, the restricted accessibility to tumor parenchyma, the immunosuppressive tumor microenvironment, and antigen heterogeneity of solid tumors make it difficult to replicate its success. Therefore, dynamic evaluation of CAR T cells’ tumor accessibility, intratumoral viability, and anti‐tumor cytotoxicity is necessary to facilitate its translation to solid tumors. Besides, real‐timely imaging above events in vivo can help evaluate therapeutic responses and optimize CAR T immunotherapy for solid tumors. Nuclear imaging, including positron emission tomography (PET) and single‐photon emission computed tomography (SPECT) imaging, is frequently applied for evaluating adoptive cell therapies owing to its excellent sensitivity, high tissue penetration, and great translation potential. In addition, quantitative analysis can be performed in dynamic and noninvasive patterns. This review focuses on recent advances in PET/SPECT technologies and imaging probes in monitoring CAR T cells’ migration, viability, and cytotoxicity to solid tumors post‐administration. Prospects of what should be done in the next stage to promote CAR T therapy's application in solid tumors are also discussed.

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Multiphoton intravital microscopy of rodents

Cited by 34 publications

References 373 publications

Monitoring AKT activity and targeting in live tissue and disease contexts using a real-time Akt-FRET biosensor mouse

Monitoring AKT activity and targeting in live tissue and disease contexts using a real-time Akt-FRET biosensor mouse

Single cell metabolic imaging of tumor and immune cells in vivo in melanoma bearing mice

Nuclear Imaging of CAR T Immunotherapy to Solid Tumors: In Terms of Biodistribution, Viability, and Cytotoxic Effect

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