Flura-seq identifies organ-specific metabolic adaptations during early metastatic colonization
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, United States
- Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, United States
- Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program, United States
- Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, United States
- Memorial Sloan Kettering Cancer Center, United States
Figures
Figure 1 with 1 supplement
Cell-type-specific labeling and isolation of RNAs by Flura-tagging.
(A) Schematic diagram showing RNA labeling and isolation using CD and 5-FC; (B) Chemical reactions steps involved in the labeling of RNA using CD and 5-FC; (C) Enrichment of mRNAs immunopurified by …
Figure 1—figure supplement 1
Cell-type-specific labeling and isolation of RNAs by cytosine-deaminase-based 5-FU tagging.
(A) Anti-BrdU antibody detects CD-based 5-fluorouracil (5-FU) derivatives but not 5-fluorocytosine (5-FC) derivatives. CD-expressing or control 293 T cells were treated with either 5-FC or 5-FU for …
Figure 2
Flura-tagging system effectively captures signal dependent change in gene expression.
(A–D) MDA231 cells expressing CD/UPRT were treated with 5-FC for 30 min prior to TGF-β or SB-505124 (SB, a TGF-β receptor inhibitor) treatment for 150 min. (A) Change in gene expression in …
Figure 3 with 1 supplement
Flura-tagging of rare metastatic cells in situ.
(A) Schematic diagram of lung colonization xenograft assay used for evaluation of Flura-tagging in vivo. Athymic mice were injected through the tail vein with 50,000 MDA231 cells expressing CD/UPRT …
Figure 3—figure supplement 1
Flura-tagging of rare metastatic cells in situ.
(A) Representative H and E stained sections of mouse lungs harboring micrometastases (arrows) used in Flura-tagging experiments. Inset shows higher magnification. Scale bar, 2 mm (top), 100 μM …
Figure 4 with 1 supplement
Flura-seq identifies organ specific in situ transcriptomes in micrometastases.
(A) Schematic diagram of experimental design used to obtain tissue specific transcriptomes of MDA231 cells in mice; (B) Principal component analysis of genes expressed by MDA231 cells in the …
Figure 4—figure supplement 1
Flura-seq identifies organ-specific in situ transcriptomes in micrometastases.
(A) Representative H and E stained sections of mouse lungs (left) and brain (right) harboring micrometastases (arrows) used in Flura-seq experiments. Inset shows higher magnification. Scale bar, 1 …
Figure 5 with 2 supplements
Mitochondrial Complex I expression and oxidative stress in lung micrometastases.
(A) Gene Ontology (GO) analysis of biological processes (BP) of genes that were upregulated in MDA231 lung micrometastases compared to brain micrometastases or mammary tumors. The top functional …
Figure 5—figure supplement 1
Differential gene expression in brain and lung micrometastatic cells.
(A) Genes differentially expressed in lung and brain micrometastases relative to mammary fat pad tumors were identified by Flura-seq, and the overlap of the upregulated genes (left) and …
Figure 5—figure supplement 2
Oxidative stress and antioxidant programs are elevated in lung micrometastases relative to brain micrometastases in HCC1954 xenograft metastasis model.
(A–C) Oxidative stress (A), antioxidant GPX1 (B) and NRF2 (C) in lung and brain tissue sections containing HCC1954 micrometastases were examined by IHC using anti-4-HNE, anti-GPX1 and anti-NRF2 …
Figure 6
Specific oxidative stress in patient-derived lung metastasis tissues.
(A–D) Expression of nuclear Complex I and antioxidant genes in a gene expression data set of matched primary tumors and lung metastases from patients with breast cancer (Siegel et al., 2018). (A) …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-BrdU; BrdU antibody (Rat monoclonal) | Abcam | Cat#ab6326 | (1:200) |
| Antibody | anti-CD31; CD31 antibody (Rat monoclonal) | Dianova | Cat#DIA-310 | (1:100) |
| Antibody | anti-GFP; GFP antibody (Chicken monoclonal) | Aves Labs | Cat#GFP-1020 | (1:500) |
| Antibody | anti-4-Hydroxynonenal; 4-HNE antibodyl (Rabbit polyclonal) | Abcam | Cat#ab46545 | (1:75) |
| Antibody | anti-NRF2; NRF2 antibody (Rabbit polyclonal) | Abcam | Cat#ab137550 | (1:600) |
| Antibody | anti-Glutathione Peroxidase 1; GPX1 antibody (Rabbit polyclonal) | Abcam | Cat#ab22604 | (1:200) |
| Antibody | Goat polyclonal anti-chicken | Thermo Fisher | Cat#A-11039 | (1:1000) |
| Antibody | Goat polyclonal anti-rat | Thermo Fisher | Cat#A-11006 | (1:1000) |
| Antibody | Goat polyclonal anti-mouse | Abcam | Cat#ab150117 | (1:1000) |
| Biological sample (Human) | Human breast cancer lung metastases tissue microarray (TMA) | This paper (Section of lung tissue containing cancer cells was surgically removed from breast cancer patients, preserved in paraflim and a small portion of the preserved tumor was used to make the TMA) | N/A | Tissue microarray Available from Edi Brogi |
| Chemical compound, drug | Doxycycline | Sigmal-Aldrich | Cat#D9891 | |
| Chemical compound, drug | 5-Fluorocytosine; 5-FC | Sigma-Aldrich | Cat#F7129 | |
| Chemical compound, drug | 5-Fluorouracil; 5-FU | Sigma-Aldrich | Cat#F6627 | |
| Chemical compound, drug | SB-505124 | Sigma-Aldrich | Cat#S4696 | |
| Chemical compound, drug | Thymine | Sigma-Aldrich | Cat#T0376 | |
| Chemical compound, drug | 4-Thiouracil; TU | Sigma-Aldrich | Cat#440736 | |
| Other | Oligo (dT)25 magnetic beads | New England Biolabs | Cat#S1419S | |
| Other | Protein G Dynabeads | Thermo Fisher Scientific | Cat#10004D | |
| Commercial assay or kit | Tissue digestion C-tube | Miltenyi | Cat#130-096-334 | |
| Commercial assay or kit | Mouse Tumor Dissociation Kit | Miltenyi | Cat#130-096-730 | |
| Commercial assay or kit | TruSeq RNA Library Prep Kit v2 | Illumina | RS-122–2001 | |
| Commercial assay or kit | SMARTer PCR cDNA synthesis kit | Clontech | Cat#634926 | |
| Commercial assay or kit | Nextera XT DNA library Preparation Kit | Illumina | FC-131–1024 | |
| Commercial assay or kit | RNeasy MinElute Cleanup kit | Qiagen | Cat#74204 | |
| Commercial assay or kit | cDNA kit-First Strand Transcriptor | Roche | Cat#043790–12001 | |
| Cell line (Human) | MDA231 | Laboratory of Joan Massague | PMID: 19421193 | Expresses TGL |
| Cell line (Human) | MDA231-CD | This paper (MDA231 cells were transduced with rtTA3 and TRE-CD-IRES-RFP) | N/A | Available from Massague lab |
| Cell line (Human) | MDA231-CD/UPRT | This paper (MDA231 cells were transduced with rtTA3 and TRE-UPRT-T2A-RFP-IRES-CD) | N/A | Available from Massague lab |
| Cell line (Human) | 293T | Laboratory of Joan Massague | N/A | |
| Strain, strain background (Mus musculus) | Hsd:Athymic Nude- Foxn1nu | Envigo | Cat#069 | |
| Sequence-based reagents | Oligonucleotides | This paper | N/A | Oligonucleotide sequences are provided in Supplementary file 6 |
| Recombinant DNA reagent | CMV Tight RFP-IRES-CD | This paper (RFP-IRES-CD was subcloned into CMV Tight EGFP Puro (Addgene: Plasmid #26431) vector by removing EGFP). | N/A | Available from Massague lab |
| Recombinant DNA reagent | CMV Tight UPRT- T2A-RFP-IRES-CD | This paper (UPRT-T2A-RFP-IRES-CD was subcloned into CMV Tight EGFP Puro (Addgene: Plasmid #26431) vector by removing EGFP). | N/A | Available from Massague lab |
| Recombinant DNA reagent | rtTA3 | Addgene | Plasmid #26730 | |
| Software and Algorithms | STAR2.5.2b | PMID: 23104886 | https://github.com/alexdobin/STAR | |
| Software and Algorithms | HTSeq v0.6.1p1 | PMID: 20979621 | https://htseq.readthedocs.io/en/release_0.10.0/ | |
| Software and Algorithms | DESeq2 v3.4 | PMID: 25516281 | https://bioconductor.org/packages/release/bioc/html/DESeq2.html | |
| Software and Algorithms | GSVA v3.4 | PMID: 23323831 | https://bioconductor.org/packages/release/bioc/html/GSVA.html |
Additional files
-
Supplementary file 1
Genes that are differentially expressed in MDA231 cells expressing CD/UPRT and treated with indicated concentration of 5-FC for 4 hr compared to control cells were obtained by DESEQ2 analysis.
- https://doi.org/10.7554/eLife.43627.014
-
Supplementary file 2
Genes that are differentially expressed in TGF-β treated cells compared to SB-505124 by more than twofold are shown as identified by RNA-seq and Flura-seq.
Genes commonly identified by RNA-seq 6 hr post TGF-β, but not 2.5 hr post treatment, and Flura-seq 2.5 hr post TGF-β treatment are also shown.
- https://doi.org/10.7554/eLife.43627.015
-
Supplementary file 3
Genes that are differentially expressed in MDA231 cells in different organs in situ as determined by Flura-seq or in vitro after isolation from the organs as determined by RNA-seq are shown.
- https://doi.org/10.7554/eLife.43627.016
-
Supplementary file 4
Top 100 NRF2 target genes identified by two independent ChIP-seq experiments in Hela cells (ENCODE Project Consortium, 2012), and the genes that were common in both experiments were used as NRF2-responsive signature genes.
- https://doi.org/10.7554/eLife.43627.017
-
Supplementary file 5
Genes identified to be up-regulated by more than two-fold in lung metastases compared to the corresponding primary tumors in breast cancer patients described in Siegel et al. (2018) for each patients are shown.
Complex I genes are highlighted in red color and the total number of upregulated Complex I genes in each patient is shown.
- https://doi.org/10.7554/eLife.43627.018
-
Supplementary file 6
Oligonucleotide sequences used in the experiments described in the manuscript are shown.
- https://doi.org/10.7554/eLife.43627.019
-
Transparent reporting form
- https://doi.org/10.7554/eLife.43627.020
