Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

Hanna, Colton; Kwok, Letty W.; Finlay-Schultz, Jessica; Sartorius, Carol A.; Cittelly, Diana M.

doi:10.3791/54944

Cited by 15 publications

(13 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To assess whether GFP and luciferase labeling would allow in vivo imaging of these BM-PDXs, we dissociated cells from >1 cm 3 xenografts BM-PDXs F2-7, CSF-1, and G5-3 and transduced them with high titer viral particles of a GFP-luciferase vector as described ( 27 ). Labeled cells were regrown in the mammary fat pad of NSG mice and tumor labeling was assessed by measuring luciferase activity (IVIS) and GFP expression (Figure 3 A).…”

Section: Resultsmentioning

confidence: 99%

“…Breast cancer PDXs grown in the mammary fat pad rarely metastasize to distant organs, but dissociated cells can colonize lung, bones and brain after ic injection ( 27 ). To assess whether BM-PDXs retain their ability to colonize the brain, we induced experimental brain metastasis using dissociated cells from TN E22-1 BM-PDX.…”

Section: Resultsmentioning

confidence: 99%

“…A subset of BM-PDXs were labeled with lentiviral particles expressing EGFP-luciferase as we described previously ( 27 ). Briefly, >1 cm 3 tumors were resected from euthanized mice and digested in Accumax (Stemcell Tech) for 3 h at 30°C.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Development of Novel Patient-Derived Xenografts from Breast Cancer Brain Metastases

et al. 2017

Self Cite

View full text Add to dashboard Cite

Brain metastases are an increasing burden among breast cancer patients, particularly for those with HER2+ and triple negative (TN) subtypes. Mechanistic insight into the pathophysiology of brain metastases and preclinical validation of therapies has relied almost exclusively on intracardiac injection of brain-homing cells derived from highly aggressive TN MDA-MB-231 and HER2+ BT474 breast cancer cell lines. Yet, these well characterized models are far from representing the tumor heterogeneity observed clinically and, due to their fast progression in vivo, their suitability to validate therapies for established brain metastasis remains limited. The goal of this study was to develop and characterize novel human brain metastasis breast cancer patient-derived xenografts (BM-PDXs) to study the biology of brain metastasis and to serve as tools for testing novel therapeutic approaches. We obtained freshly resected brain metastases from consenting donors with breast cancer. Tissue was immediately implanted in the mammary fat pad of female immunocompromised mice and expanded as BM-PDXs. Brain metastases from 3/4 (75%) TN, 1/1 (100%) estrogen receptor positive (ER+), and 5/9 (55.5%) HER2+ clinical subtypes were established as transplantable BM-PDXs. To facilitate tracking of metastatic dissemination using BM-PDXs, we labeled PDX-dissociated cells with EGFP-luciferase followed by reimplantation in mice, and generated a BM-derived cell line (F2-7). Immunohistologic analyses demonstrated that parental and labeled BM-PDXs retained expression of critical clinical markers such as ER, progesterone receptor, epidermal growth factor receptor, HER2, and the basal cell marker cytokeratin 5. Similarly, RNA sequencing analysis showed clustering of parental, labeled BM-PDXs and their corresponding cell line derivative. Intracardiac injection of dissociated cells from BM-E22-1, resulted in magnetic resonance imaging-detectable macrometastases in 4/8 (50%) and micrometastases (8/8) (100%) mice, suggesting that BM-PDXs remain capable of colonizing the brain at high frequencies. Brain metastases developed 8–12 weeks after ic injection, located to the brain parenchyma, grew around blood vessels, and elicited astroglia activation characteristic of breast cancer brain metastasis. These novel BM-PDXs represent heterogeneous and clinically relevant models to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Development of Novel Patient-Derived Xenografts from Breast Cancer Brain Metastases

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…Indeed, fluorescent proteins' expression guarantees non-toxic, steady, and homogeneous labeling, fluorescence transmission to daughter cells, and its dynamic loss at death by physiological protein degradation. As such, this option most probably warrants a proper tracking of grafted cells and an accurate estimation of tumor volumes and dispersion across different time points [33][34][35].…”

Section: Choice Of a Suitable Cell Labeling Methodsmentioning

confidence: 99%

ZeOncoTest: Refining and Automating the Zebrafish Xenograft Model for Drug Discovery in Cancer

et al. 2019

View full text Add to dashboard Cite

The xenograft of human cancer cells in model animals is a powerful tool for understanding tumor progression and metastatic potential. Mice represent a validated host, but their use is limited by the elevated experimental costs and low throughput. To overcome these restrictions, zebrafish larvae might represent a valuable alternative. Their small size and transparency allow the tracking of transplanted cells. Therefore, tumor growth and early steps of metastasis, which are difficult to evaluate in mice, can be addressed. In spite of its advantages, the use of this model has been hindered by lack of experimental homogeneity and validation. Considering these facts, the aim of our work was to standardize, automate, and validate a zebrafish larvae xenograft assay with increased translatability and higher drug screening throughput. The ZeOncoTest reliability is based on the optimization of different experimental parameters, such as cell labeling, injection site, automated individual sample image acquisition, and analysis. This workflow implementation finally allows a higher precision and experimental throughput increase, when compared to previous reports. The approach was validated with the breast cancer cell line MDA-MB-231, the colorectal cancer cells HCT116, and the prostate cancer cells PC3; and known drugs, respectively RKI-1447, Docetaxel, and Mitoxantrone. The results recapitulate growth and invasion for all tested tumor cells, along with expected efficacy of the compounds. Finally, the methodology has proven useful for understanding specific drugs mode of action. The insights gained bring a step further for zebrafish larvae xenografts to enter the regulated preclinical drug discovery path.

show abstract

“…For the mouse / human experiment, NIH:3T3 and 293T cells were grown in standard media and mixed at a 1:1 ratio prior to capture. For the mouse xenograft experiment, an estrogen receptor positive patient derived xenograft primary cell line (UCD65 (Kabos et al 2012) ) was labeled with luc-eGFP using lentivirus (Hanna et al 2016) . Cells were xenografted into NOD/SCID/IL2rg −/− mice via intracardiac injection to generate disseminated metastases or injected into the mammary pad to generate a primary tumor.…”

Section: Cell Isolation and Single Cell Rna-seq Generationmentioning

confidence: 99%

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries

Riemondy

Ransom

Alderman

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1,313 to 2,002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%.Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.1 Riemondy et al .

show abstract

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

Cited by 15 publications

References 25 publications

Development of Novel Patient-Derived Xenografts from Breast Cancer Brain Metastases

Development of Novel Patient-Derived Xenografts from Breast Cancer Brain Metastases

ZeOncoTest: Refining and Automating the Zebrafish Xenograft Model for Drug Discovery in Cancer

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries

Contact Info

Product

Resources

About