Flura-seq identifies organ-specific metabolic adaptations during early metastatic colonization

  1. Harihar Basnet
  2. Lin Tian
  3. Karuna Ganesh
  4. Yun-Han Huang
  5. Danilo G Macalinao
  6. Edi Brogi
  7. Lydia WS Finley
  8. Joan Massagué  Is a corresponding author
  1. Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, United States
  2. Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, United States
  3. Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program, United States
  4. Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, United States
  5. Memorial Sloan Kettering Cancer Center, United States
6 figures, 1 table and 7 additional files

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 …

https://doi.org/10.7554/eLife.43627.003
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 …

https://doi.org/10.7554/eLife.43627.004
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 …

https://doi.org/10.7554/eLife.43627.005
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 …

https://doi.org/10.7554/eLife.43627.006
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 …

https://doi.org/10.7554/eLife.43627.007
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 …

https://doi.org/10.7554/eLife.43627.008
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 …

https://doi.org/10.7554/eLife.43627.009
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 …

https://doi.org/10.7554/eLife.43627.010
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 …

https://doi.org/10.7554/eLife.43627.011
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 …

https://doi.org/10.7554/eLife.43627.012
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) …

https://doi.org/10.7554/eLife.43627.013

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
Antibodyanti-BrdU; BrdU
antibody
(Rat monoclonal)
AbcamCat#ab6326(1:200)
Antibodyanti-CD31; CD31
antibody
(Rat monoclonal)
DianovaCat#DIA-310(1:100)
Antibodyanti-GFP; GFP
antibody (Chicken
monoclonal)
Aves LabsCat#GFP-1020(1:500)
Antibodyanti-4-Hydroxynonenal;
4-HNE antibodyl
(Rabbit polyclonal)
AbcamCat#ab46545(1:75)
Antibodyanti-NRF2; NRF2
antibody (Rabbit
polyclonal)
AbcamCat#ab137550(1:600)
Antibodyanti-Glutathione
Peroxidase 1;
GPX1 antibody
(Rabbit polyclonal)
AbcamCat#ab22604(1:200)
AntibodyGoat polyclonal
anti-chicken
Thermo FisherCat#A-11039(1:1000)
AntibodyGoat polyclonal
anti-rat
Thermo FisherCat#A-11006(1:1000)
AntibodyGoat polyclonal anti-mouseAbcamCat#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/ATissue microarray Available from
Edi Brogi
Chemical
compound, drug
DoxycyclineSigmal-AldrichCat#D9891
Chemical
compound, drug
5-Fluorocytosine; 5-FCSigma-AldrichCat#F7129
Chemical
compound, drug
5-Fluorouracil; 5-FUSigma-AldrichCat#F6627
Chemical
compound, drug
SB-505124Sigma-AldrichCat#S4696
Chemical
compound, drug
ThymineSigma-AldrichCat#T0376
Chemical
compound, drug
4-Thiouracil; TUSigma-AldrichCat#440736
OtherOligo (dT)25 magnetic
beads
New England BiolabsCat#S1419S
OtherProtein G DynabeadsThermo
Fisher Scientific
Cat#10004D
Commercial
assay or kit
Tissue digestion
C-tube
MiltenyiCat#130-096-334
Commercial
assay or kit
Mouse Tumor
Dissociation Kit
MiltenyiCat#130-096-730
Commercial
assay or kit
TruSeq RNA Library
Prep Kit v2
IlluminaRS-122–2001
Commercial
assay or kit
SMARTer PCR
cDNA synthesis kit
ClontechCat#634926
Commercial
assay or kit
Nextera XT DNA
library Preparation Kit
IlluminaFC-131–1024
Commercial
assay or kit
RNeasy MinElute
Cleanup kit
QiagenCat#74204
Commercial
assay or kit
cDNA kit-First
Strand Transcriptor
RocheCat#043790–12001
Cell line (Human)MDA231Laboratory of
Joan Massague
PMID: 19421193Expresses TGL
Cell line (Human)MDA231-CDThis paper (MDA231
cells were transduced
with rtTA3 and TRE-CD-IRES-RFP)
N/AAvailable from
Massague lab
Cell line (Human)MDA231-CD/UPRTThis paper (MDA231 cells were transduced
with rtTA3 and
TRE-UPRT-T2A-RFP-IRES-CD)
N/AAvailable from
Massague lab
Cell line (Human)293TLaboratory of
Joan Massague
N/A
Strain, strain
background (Mus
musculus)
Hsd:Athymic
Nude- Foxn1nu
EnvigoCat#069
Sequence-based
reagents
OligonucleotidesThis paperN/AOligonucleotide
sequences are provided
in Supplementary file 6
Recombinant
DNA reagent
CMV Tight RFP-IRES-CDThis paper (RFP-IRES-CD
was subcloned
into CMV Tight EGFP
Puro (Addgene:
Plasmid #26431) vector by
removing EGFP).
N/AAvailable 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/AAvailable from
Massague lab
Recombinant
DNA reagent
rtTA3AddgenePlasmid #26730
Software and
Algorithms
STAR2.5.2bPMID: 23104886https://github.com/alexdobin/STAR
Software and
Algorithms
HTSeq v0.6.1p1PMID: 20979621https://htseq.readthedocs.io/en/release_0.10.0/
Software and
Algorithms
DESeq2 v3.4PMID: 25516281https://bioconductor.org/packages/release/bioc/html/DESeq2.html
Software and
Algorithms
GSVA v3.4PMID: 23323831https://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

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