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Tracking of Individual Cell Populations by 19F MRI using a "Cargo Internalization Receptor (CIR)" System

ABSTRACT: Magnetic resonance imaging (MRI) is an important key for non-invasive visualization of immigrating cells in in vivo studies. We use Perflourcarbons (PFC) coupled to green fluorescent protein (GFP) for a 1H and 19F MRI detection. To this end, we engineered synthetic cell surface receptors, “Cargo Internalization Receptors” (CIR), which contain a GFP-nanobody and specifically bind GFP, followed by a rapid internalization of the GFP-PFC particles, but without inducing any signaling in the targeted cells. In order to explore the suitability of different uptake mechanisms we constructed three different CIRs, which contain different cytoplasmic domains that have different internalizations motifs and properties to enable the specific uptake of the GFP-PFC cargo in CIR-transfected cells. We show that the CIR approach is working in vitro, and has the potential to specifically track CIR containing cells by 1H and 19F MRI.

ORGANISM(S): Mus musculus

PROVIDER: GSE106215 | GEO | 2019/05/27

REPOSITORIES: GEO

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Project description:High-grade gliomas, the most common and aggressive primary brain tumors, are characterized by a complex and multifaceted tumor microenvironment (TME). Among the non-malignant cells of the glioma TME, tumor-associated microglia and macrophages (TAMs) constitute the major compartment. In patients, a greater abundance of TAMs is associated with high-grade and more aggressive disease. In a range of glioma mouse models, we have observed specific alterations in TAM dynamics and functions during tumor development and following different therapeutic interventions, including irradiation. The underlying TAM abundance may also modulate the subsequent response to TAM-targeted therapy, including the anti-CSF1R inhibitor BLZ945, which results in a pronounced tumor regression in different preclinical models of brain cancers, as our lab has recently shown. These observations indicate that it is crucial to evaluate TAM abundance and dynamics in gliomas. Current techniques to quantify TAMs in patients rely mainly on histological staining of tumor biopsies. While informative, these techniques require an invasive procedure to harvest the tissue sample and typically only result in a snapshot of a small region at a single point in time. Fluorine isotope 19 MRI (19F MRI) has been shown to be a powerful tool to non-invasively and longitudinally monitor myeloid cells in other pathological conditions by injecting perfluorocarbon-containing nanoparticles (PFC-NP). In this study, we demonstrated the feasibility and power of 19F MRI in preclinical glioma models and brain metastasis models and showed that the major cellular source of 19F signal consists of TAMs. Moreover, PFC-NP accumulation occurred predominantly in proximity to dysmorphic vessels, thereby enabling the identification of a TAM subpopulation with a specific oxidative metabolism. We also evaluated this imaging modality in the context of irradiation (IR) treatment and found that the 19F signal enables the tracking of TAM dynamics over time following IR. Moreover, multispectral 19F MRI with two different PFC-NP allowed us to identify spatially- and temporally-distinct TAM niches in IR-recurrent tumors. Together, we have been able to image TAMs non-invasively and longitudinally with a powerful integrated cellular, spatial and temporal resolution, while revealing new biological insights into the many functions of TAMs, including in disease recurrence.

Project description:The pir genes comprise the largest multi-gene family in Plasmodium, with members found in P. vivax, P. knowlesi and the rodent malaria species. Despite comprising up to 5% of the parasite genome, little is known about the functions of the proteins encoded by pir genes. P. chabaudi causes chronic infection in mice, which may be due antigenic variation. In this model, pir genes are called cirs and may be involved in this mechanism allowing evasion of host immune responses. We have annotated the cir repertoire and performed detailed bioinformatic characterization of the encoded CIR proteins. Two major sub-families were identified: A and B, which display different amino acid motifs, and are thus predicted to have undergone functional divergence. The expression of all cirs was analyzed via RNA sequencing and microarray. Up to 40% of cir genes were expressed in the parasite population during infection, including members of both sub-families. Dominant cir transcripts could also be identified. Finally, specific cir genes were expressed at different time points during the blood stages of infection. Together our data characterizing the cir genes and their expression throughout the intra-erythrocytic cycle of development indicate that CIR proteins are likely to be important for parasite survival in the host. P. chabaudi AS is a highly synchronous parasite for which development in the blood follows its hostM-bM-^@M-^Ys circadian rhythm. Twelve time-points were then collected; one every two hours, to cover the entire 24 h cycle of blood stage development. At the peak of parasitaemia, one mouse was sacrificed at each time point and thin blood films were made and stained with Giemsa for optical microscopy. The pan-rodent microarray was designed using the OligoRankPick program as previously described: Liew, K et al.2010, Defining species specific genome differences in malaria parasites. BMC genomics 11,128. The RNA preparation, Cy-dye coupling to cDNA, hybridization and slide scanning were performed as described by Bozdech and colleagues Bozdech, Z et al 2003, The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 1, E5.

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Tracking of Individual Cell Populations by 19F MRI using a "Cargo Internalization Receptor (CIR)" System

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