Biology subjects

Nyström, N. N.

Publications and source records attributed to Nyström, N. N..

3 recordsLinked to original sources

bioRxiv2022

Sensitive Spatiotemporal Tracking of Spontaneous Metastasis in Deep Tissues via a Genetically-Encoded Magnetic Resonance Imaging Reporter

Metastasis remains a poorly understood aspect of cancer biology and the leading cause of cancer-related death, yet most preclinical cancer studies do not examine metastasis, focusing solely on the primary tumor. One major factor contributing to this paradox is a gap in available tools for accurate spatiotemporal measurements of metastatic spread in vivo. Our objective was to develop an imaging reporter system that offers sensitive three-dimensional detection of cancer cells at high resolutions in live mice. We utilized organic anion-transporting polypeptide lb3 (oatp1b3) as a magnetic resonance imaging (MRI) reporter gene to this end, and systematically optimized its framework for in vivo tracking of viable cancer cells in a spontaneous metastasis model. We were able to image metastasis on oatp1b3-MRI at the single lymph node level and continued to track its progression over time as cancer cells spread to multiple lymph nodes and different organ systems in single animals. While initial single lesions were successfully imaged in parallel via bioluminescence, later metastases were obscured by light scatter from the initial node. Importantly, we demonstrate and validate that 100-m isotropic resolution MR images could detect micrometastases in lung tissue estimated to contain fewer than 103 cancer cells. In summary, oatp1b3-MRI enables precise determination of lesion size and location over time and offers a path towards deep-tissue tracking of any oatp1b3-engineered cell type with combined high resolution, high sensitivity, 3D spatial information, and surrounding anatomical context.

bioengineering↗

bioRxiv2020

Molecular imaging reveals a high degree of cross-seeding of spontaneous metastases in a novel mouse model of synchronous bilateral breast cancer

Synchronous bilateral breast cancer (SBBC) patients present with cancer in both breasts at the time of diagnosis or within a short time interval. They show higher rates of metastasis and lower overall survival compared to women with unilateral breast cancer. However, the lack of a preclinical model has led to a dearth in knowledge regarding the patterns of SBBC metastasis. Here we established an SBBC model and used molecular imaging to visualize the development of spontaneous lung metastases arising from each primary tumor. We engineered human breast cancer cells to express either Akaluc or Antares2 for bioluminescence imaging (BLI), and tdTomato or zsGreen for ex vivo fluorescence microscopy. Both cell populations were implanted into contralateral mammary fat pads of mice (n=10) and BLI was performed weekly for up to day 29 (n=3), 38 (n=4), or 42 (n=3). Signal from both Antares2 and Akaluc was first detected in the lungs on day 28 and was present in 9 of 10 mice at endpoint. Ex vivo fluorescence microscopy of the lungs revealed that for mice sacrificed on day 38, a significant percentage of micrometastases were composed of cancer cells from both primary tumors (mean 37%; range 27% to 45%), while two mice sacrificed on day 42 showed percentages of 51% and 70%. These results reveal a high degree of metastatic cross-seeding which may contribute to faster metastatic growth and intratumoral heterogeneity. We posit our work will help understand treatment resistance and optimal planning of treatment for SBBC patients.

cancer biology↗

bioRxiv2020

A Safe Harbor-Targeted CRISPR/Cas9 Homology Independent Targeted Integration (HITI) System for Multi-Modality Reporter Gene-Based Cell Tracking

Imaging reporter genes can provide valuable, longitudinal information on the biodistribution, growth and survival of engineered cells in preclinical models and patients. A translational bottleneck to using reporter genes in patients is the necessity to engineer cells with randomly-integrating vectors. CRISPR/Cas9 targeted knock-in of reporter genes at a genomic safe harbor locus such as adeno-associated virus integration site 1 (AAVS1) may overcome these safety concerns. Here, we built Homology Independent Targeted Integration (HITI) CRISPR/Cas9 minicircle donors for precise AAVS1-targeted simultaneous knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency at the AAVS1 site using HITI vectors compared to homology-directed repair donor vectors. Characterization of select HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significantly increased Oatp1a1-mediated uptake of the clinically-approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. As few as 106 Oatp1a1-expressing cells in a 50 {micro}l subcutaneous injection could be detected in vivo with contrast-enhanced MRI. Contrast-enhanced MRI also improved the conspicuity of both sub-cutaneous and metastatic Oatp1a1-expressing tumours prior to them being palpable or even readily visible on pre-contrast images. Our work demonstrates the first CRISPR/Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multi-modal longitudinal in vivo imaging of cells. This work lays the foundation for safer, non-viral reporter gene tracking of multiple cell types.

bioengineering↗