Abstract
We have performed whole transcriptome sequencing of 5-FU resistant and
5-FU sensitive tumors generated in a mouse model of de novo
carcinogenesis that closely recapitulates tumor initiation, progression
and maintenance in vivo. Tumors were generated using the DMBA/TPA model
of chemically induced carcinogenesis [29][1], tumor-bearing mice were
subsequently treated with 5-FU, and tumor growth as well as response to
treatment was monitored by measuring tumor volume twice a week. Based
on these measurements, we selected two 5-FU resistant and two 5-FU
sensitive tumors and performed whole transcriptome sequencing and in
order to identify differentially expressed transcripts between the two
sets. Data obtained is deposited and available through NCBI SRA
(reference number SRP155180 –
[30]https://www.ncbi.nlm.nih.gov/sra/?term=SRP155180).
__________________________________________________________________
Specifications table
Subject area Biology
More specific subject area Mus Musculus - Skin Tumors
Type of data Table, figures, raw sequence reads
How data was acquired Illumina HiSeq™ 2000
Data format Raw and analyzed
Experimental factors 5-FU sensitive VS 5-FU resistant tumors
Experimental features Mouse model of chemically-induced carcinogenesis.
Tumors were induced with DMBA/TPA and mice were treated
intraperitoneally with 5-fluorouracil. 5-FU-sensitive and resistant
tumors were harvested and processed for RNA extraction. Whole
transcriptome sequencing was performed on sensitive and resistant
tumors using Illumina HiSeq™ 2000.
Data source location Nicosia, Cyprus
Data accessibility Data is with this article and available at
[31]https://www.ncbi.nlm.nih.gov/sra/?term=SRP155180
Related research article [32][5] Loizides C, Iacovides D, Hadjiandreou
M. M, Rizki G, Achilleos A, Strati K, and Mitsis G. D. Model-based
tumor growth dynamics and therapy response in a mouse model of de novo
carcinogenesis, PloS One, 10(12), 2015, p. e0143840.
[33]https://doi.org/10.1371/journal.pone.0143840
[34]Open in a new tab
Value of the data
* •
Data in this article can be used to identify differentially
expressed genes between tumors that are sensitive or resistant to
treatment with 5-FU.
* •
These transcripts represent potential markers of response to
treatment with 5-FU. Thus, data presented herein could facilitate
the development of clinically meaningful biomarker signatures of
response to 5-FU and contribute towards personalized cancer
treatment.
1. Data
Eight raw sequence read data files are shared. Two 5-FU sensitive (S1
and S2) and two 5-FU resistant (NR1 and NR2) tumors were sequenced, and
each tumor is represented with two technical replicates (A and B)
([35]Fig. 1, [36]Fig. 2).
Fig. 1.
[37]Fig. 1
[38]Open in a new tab
Generation of 5-FU sensitive and resistant tumors in a mouse model of
chemically-induced carcinogenesis. Carcinogenesis was initiated with
DMBA, followed by bi-weekly treatments with TPA to promote tumor
growth. Isolated RNA from 2 pairs of 5-FU sensitive and 5-FU resistant
tumors was used to perform whole-transcriptome sequencing using
Illumina^2000, in order to identify differentially expressed genes
between sensitive and resistant tumors.
Fig. 2.
[39]Fig. 2
[40]Open in a new tab
(A) Total number of differentially expressed genes within each tumor
pair. Comparative analysis was performed as sensitive (S) vs resistant
(R) tumor. In total, 247 transcripts are commonly upregulated in
sensitive tumors compared to resistant tumors, and 141 transcripts are
commonly downregulated in sensitive tumors compared to resistant tumors
(B) S vs R DEGs in cancer-related molecular pathways.
2. Experimental design, materials and methods
2.1. Breeding and genotyping
In order to accelerate tumor formation, we used transgenic mice
expressing the HPV E6/E7 viral oncogenes under the keratin-14 promoter
[41][2], [42][3]. These mice were obtained by crossing K14E6H females
with K14E7h heterozygous females (K14E6hK14E7h). To confirm presence of
the E6 and E7 transgenes, we extracted DNA for mouse tail and performed
PCR genotyping, using Sigma DNA extraction Kit (Sigma-Aldrich, cat. #
G1N10) and KAPATaq (Kappa Biosystems, cat. # KK1015). (K14709-4/E7TTL)
and (Oligo2/E6TTL) primers were used to detect E6 and E7 respectively,
as previously described [43][4].
2.2. DMBA/TPA treatment
Treatment with 200 μl of 0.03 μmol/μl DMBA (Sigma-Aldrich, cat. #
D3254) was administered once on the back of 7–8 weeks old mice
previously shaved at the area. Two weeks after DMBA administration, we
initiated treatment with 2.5 μg TPA diluted in 200 μl acetone twice a
week (Sigma-Aldrich, cat. #P8139) until a mouse was sacrificed.
2.3. Treatment with 5-Fluorouracil
Treatment with 50 mg/kg of 5-FU was initiated when the first tumor on a
mouse reached 3–4 mm in size. The drug was administered
intraperitonially (5-FU; Sigma-Aldrich, cat. # F6627) once a week,
until any tumor on the animal reached ~1 cm in diameter, at which point
the mouse was sacrificed.
2.4. Tumor measurements
Measurements of tumor volume on a mouse were recorded twice a week
during treatment with 5-FU, as previously described [44][5]. As tumors
generated here had an ellipsoid shape, tumor volume (V) was calculated
using the formula:
[MATH:
V=π6(xy)3
2 :MATH]
where x and y denote the length and width of the tumor respectively
[45][6].
In order to determine sensitivity and resistance to 5-FU treatment, we
compared tumor volume measurements obtained right before commencing
treatment with 5-FU with volumes of the same tumors just right before
sacrificing the animal. Sensitive tumors were selected based on at
least 50% reduction in tumor size, and resistant tumors were selected
as those with at least 2-fold growth in volume despite treatment with
5-FU. One sensitive and one resistant tumor were selected from each
animal for sequencing.
2.5. Transcriptomic profiling
Total RNA was extracted from 5-FU sensitive and resistant mouse tumors
using the RNeasy kit (Qiagen, cat. # 74104), DNase-treated with TURBO
DNA-free kit (Ambion,cat. # AM1907) and quantified using NanoDrop. RNA
purity was assessed by A260/A280 and A260/230 ratios (>1.8) using
Nanodrop.
Magnetic beads with Oligo (dT) were subsequently used to isolate mRNA
which was then fragmented into short fragments, purified and resolved
for end reparation and single nucleotide A (adenine) addition. The
short mRNA fragments were connected with adapters, subjected to agarose
gel electrophoresis, and cDNA was subsequently synthesized using the
mRNA fragments as templates through PCR amplification. Agilent 2100
Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used for
quantification and qualification of the sample library, and the library
was then sequenced using Illumina HiSeq™ 2000. Sequence alignment and
quantification analysis of gene expression was done using
SOAPaligner/SOAP2 [46][7]. Transcript reads were assembled using
Cufflink [47][8]. KEGG [48][9] was used to perform pathway enrichment
analysis of differentially expressed genes (DEGs), and expression
pattern analysis was performed using Cluster [49][10] and JavaTreeView
[50][11]. Gene expression levels were measured and normalized using the
RPKM method [51][12]. For each pair of sensitive and resistant tumor
samples, we then calculated the log2-ratio of each gene׳s corresponding
RPKM value, and subsequently calculated the p-value for the test that
the expression levels between the two samples for a specific gene are
different. Since multiple comparisons were performed, we corrected
these p-values using the Benjamini and Yekutieli method [52][13]. We
consider significant DEGs those with adjusted p values less than 1e^−4.
Acknowledgements