BACKGROUND
murine ct <dig>  cells were developed in  <dig> by exposing balb/c mice to n-nitroso-n-methylurethane , resulting in a rapid-growing grade iv carcinoma that is easily implanted and readily metastasizes  <cit> .
used in over  <dig> published studies, the ct <dig> colon carcinoma is one of the most commonly used cell lines in drug development.
numerous cytotoxic agents as well as therapeutics targeting specific signaling pathways have been studied with these cells .
moreover, as the ct <dig> model in balb/c mice provides a syngeneic in vivo test system, it is frequently used for developing and testing immunotherapeutic concepts.
in sharp contrast to its frequent use in drug development, there have been no comprehensive studies of the genome and transcriptome of ct <dig>  kras is mutated in ct <dig>  <cit>  but other mutations are not known.
mutations in cdkn2a, mek, braf and pi3k in combination with egfr and vegf expression, for instance, may influence the results of pre-clinical investigations of treatment modalities.
moreover, while gp <dig>  the product of the envelope gene of murine leukemia virus -related cell surface antigen, is a known model antigen for studying antigen-specific immune responses in the ct <dig> system, there is no comprehensive knowledge of potential tumor antigens in this cell line system.
further, the lack of comprehensive data on the murine ct <dig> colon cancer data sharply contrasts to the extensive molecular characterization of human colorectal cancer .
as a group, human crc is highly heterogeneous with multiple evolutionary paths, with molecular signatures classifying subtypes and steps from adenoma to carcinoma.
many human crc genomes are now known and multiple molecular signatures, classifications and biomarker concepts are published .
as comprehensive genomic and transcriptomic data of ct <dig> has not been available, it is unclear how ct <dig>  a chemically-induced tumor, molecularly correlates to human crc subtypes and to what extent it may be used as model.
to answer these questions, we utilized next-generation sequencing, bioinformatics and immuno-informatics to create an integrated mouse solid tumor mutanome, transcriptome and immunome, providing an overdue analysis of the ct <dig> cancer cell line.
RESULTS
the ct <dig> tumor genome: using the ngs reads, we assessed copy number and nucleotide variations by comparing ct <dig> to balb/cj dna.
we determined absolute dna copy number using the ratio of exome-seq reads mapping to each gene from ct <dig> versus those from balb/cj, and integrating variant allele fraction .
we found that the ploidy of ct <dig> is strikingly large with large regions of triploidy and tetraploidy, in agreement with previous karyotyping results  <cit> .
the median and mean copy number in average across all genes is  <dig> and  <dig> , respectively, with  <dig>  genes in triploid regions  and  <dig>   in tetraploid regions .
no reads map to the y chromosome , suggesting that ct <dig> cells originated from a female mouse.
only one homozygous deletion was found, which contains the tumor suppressor cdkn2a  locus on mouse chromosome  <dig> figure  <dig>
the ct <dig> genome.
a) circos diagram showing : cytogenetic bands in black, gray and white.
track 1: dna copy number.
black: deleted; green: haploid; yellow: diploid; dark purple: triploid; light purple: tetraploid; dark blue: pentaploid; bright blue: hexaploid; red: higher copy number.
track 2: high confidence point mutations, plotted based on dna allele frequency.
inner is allele frequency  <dig>  outer is allele frequency  <dig>  colors are blue , green , orange , and red .
track 3: predicted mhc binding ic <dig> scores for missense mutations.
mutations in peptides likely to bind mhc are colored orange .
track 4: insertions and deletions plotted according to allele frequency.
b) dna gene copy number.
c) single nucleotide mutation changes and the nucleotide immediately 3′ of the mutation.
d) mutation classification.
we identified  <dig>  high-confidence single nucleotide variations  and  <dig> short insertions and deletions .
indels are dominated by a/t deletions .
we selected high confidence snvs in exons , the majority of which are localized in coding regions .
of the snvs in coding regions, the majority  cause non-synonymous protein changes, including  <dig>  missense and  <dig> nonsense variants.
the ccds database identifies  <dig> million protein-encoding nucleotides in the mouse genome.
relative to a  <dig> balb/cj genome, the ct <dig> variation rate in coding regions is  <dig> non-synonymous and  <dig> silent mutations per mb.
this is significantly more than the average found in spontaneous human tumors  but still within the range observed for primary human crc tumors, which ranges from less than  <dig> per mb to over  <dig> mutations per mb  <cit> .
the identified snvs represent variations between the ct <dig> genome, derived from a balb/c mouse in  <dig>  and a balb/cj mouse in  <dig>  as such, the snvs include both somatic mutations associated with the ct <dig> onco-transformation and genetic drift in the balb/c genome.
we found  <dig>  mouse snps that distinguish the balb/cj and mm <dig>  exomes.
of these, only  <dig> % show a discrepancy between the ct <dig> and  <dig> balb/cj genomes.
thus, while this does not eliminate genetic drift or conclusively identify the substrain that gave rise to ct <dig> cells, it demonstrates that the genome of the mouse that originally created the ct <dig> cells is similar to that of the current balb/cj mouse.
spontaneous human crc tumors contain primarily c > t/g > a snvs  <cit> .
of the  <dig>  snvs in the ct <dig> genome,  <dig>   are transitions, of which most  are c > t/g > a mutations , similar to the human crc mutation profile.
based on data from over  <dig>  human tumors, g is the dominate nucleotide immediately 3′ of the mutated nucleotide in human crc tumors   <cit> .
conversely, we found that ct <dig> snvs are depleted in cg > tg and ca > ta mutations and enriched in ct > tt and cc > tc mutations.
this pattern, a c > t mutation followed by a pyrimidine, is found in tumor samples from human patients pre-treated with temozolomide, an alkylating anticancer drug  <cit> .
ct <dig> was originally induced by the alkylating agent nmu.
that temozolomide and nmu are both are associated with tumors enriched in c > t mutations at positions followed by a pyrimidine suggest a similar mutagenic pattern for these two alkylating agents.
of the  <dig>  ct <dig> snvs,  <dig>  are homozygous or heterozygous , even in amplified regions with high copy number.
homozygous variants cluster across chromosomes  <dig>   <dig>   <dig>   <dig>  and x. these regions could be the result of either a loss of heterozygosity  onco-transformation or genetic drift in a balb/c mouse followed by inbreeding.
if the result of an onco-transformation, that the regions experienced loh, followed by mutations and copy number amplification suggests that resulting individual alleles were amplified 2-fold , 3-fold , 4-fold , and 5-fold .
we further investigated chromosome x. mutations occur on chromosome x with 100% and 50% dna allele frequency, suggesting that chromosome x is diploid in ct <dig> cells.
female cells typically express xist and inactivate one x allele.
in ct <dig>  the rna-seq data show that xist is not expressed and, examining the allele expression of heterozygous mutations, that transcription occurs from both chromosome x alleles.
these findings are concordant with a scenario where the chromosome x experienced both a loss of the inactivated allele and an amplification of the non-inactivated allele .
in summary, the data imply that the ct <dig> has a complex genome of high ploidy which underwent several amplification events.
relative to a  <dig> balb/c genome, the number of mutations is higher than average, with many non-synonymous mutations.
the mutation pattern reflects the treatment with the nmu alkylating agent, a similar but distinct pattern than found in spontaneous primary crc.
ct <dig> snvs in onco-relevant genes: we investigated whether mutations associated with crc  are also prevalent in ct <dig>  apc, kras and tp <dig> are frequent drivers of the linear and uniform evolution of spontaneous human crc; of these, only kras is mutated in ct <dig>  the ct <dig> kras genomic locus is triploid and all alleles contain v8m  and g12d  mutations.
several crc subtypes are linked to syndromes based on inherited gene defects and mutations.
genes associated with familial crc  include mismatch repair genes mlh <dig>  mlh <dig>  mlh <dig>  msh <dig>  myh, pms <dig>  stk <dig>  mutyh and ctnnb <dig>  none are mutated in ct <dig>  the lack of mutations in mismatch repair genes mlh <dig> and msh <dig>  which are associated with crc microsatellite instability , agrees with the lack of mutation in braf, which is frequently associated with the msi-high phenotype  <cit> .
further, the tumor suppressor cdkn2a is homozygously deleted and the genomic mapk <dig>  and met loci are amplified in ct <dig>  crc-associated genes fbxw <dig>  pik2ca, pten, smad <dig>  smad <dig>  tcf7l <dig> are not mutated.
non-synonymous point mutations occur in other crc genes brca <dig> , pdgfra , nav <dig> , atr , cdk <dig> , and rel .
mutations in cancer-related genes include mtor , birc <dig> , casp <dig> , cenpe , esr <dig> , hdac <dig> , ins <dig> , insr , muc <dig> , pik3c <dig> , pik3cg , fgfr <dig> , ddr <dig> , notch <dig>  and rhoj .
frameshift-causing indels occur in oncogenes ewsr <dig>  and mpp <dig> .
ct <dig> gene expression: we generated gene expression profiles from ct <dig> cells.
cancer-relevant genes such nras, vegfa, trp <dig> , myc, mdm <dig>  and hif1a are expressed at high levels in ct <dig> .
egfr and flt <dig> are not expressed.
gene expression in ct <dig> relative to normal colon was used for pathway enrichment analysis in order to identify broadly enriched pathways .
not surprisingly, the identified pathways relate to cell proliferation  and increased translation .
we examined individual gene sets enriched in ct <dig> .
most enriched is “cell_cycle_rb1_targets”, a gene set curated from a study examining rb <dig> target genes involved in cell cycle regulation  <cit> , reflecting over-expression of all rb <dig> target genes .
rb <dig> mrna is itself 8-fold up-regulated.
ezh <dig>  downstream of the egfr-ras-raf pathway, impacts dna methylation, promotes emt and is associated with poor prognosis in crc  <cit> .
together with its target genes, ezh <dig> is over-expressed in ct <dig> cells.
mechanistically, that rb <dig>  ezh <dig>  lin <dig>  and e2f mrnas and their target genes are over-expressed suggests that the rb <dig>  ezh <dig>  lin <dig>  and e2f mrna levels, in addition to post-translational modifications, play a critical role controlling activation of each pathway.figure  <dig>
gene expression of onco-relevant genes, stem cell and proliferation markers, wnt signaling pathway genes, emt and epithelial markers, and differentiation markers in ct <dig> and mouse colon.
error bars represent the maximum and minimum values for the ct <dig> triplicates and the six normal colon samples .
reactome pathways over-expressed in ct <dig> versus normal colon.
enriched reactome pathways are displayed using cytoscape cluego.
ribosomal associated genes are over-expressed in ct26; while they are part of the influenza gene set, we posit their over-expression reflects increased translational activity rather than an influenza-related process.
for each gene set, cytoscape cluego calculates a false-discovery rate statistic  reflecting the likelihood of the given enrichment by chance: the pathways listed in the table those with a zero q-value.
each dot in the image represents a separate reactome gene set.
gene sets with common members are placed proximally, grouped into common themes, colored the same and labeled.
q-values for enriched pathways, cytoscape settings and gene membership are in the supplementary information.
gene sets over-expressed in ct <dig> versus normal colon.
a).
the top  <dig> over-expressed gene sets.
gene sets included those curated from literature and overexpression was determined using genepattern  <cit> .
b-f): gene set enrichment for the targets of specific genes, including the genepattern enrichment plot showing the enrichment plot  and the location of the individual gene set members in the expression ranked-ordered list .
the plot on the right  shows the expression of the targeting gene rb <dig> , e2h <dig> , lin <dig> , foxo <dig>  and e2f <dig> .
the gene set associated with genes down-regulated after foxo <dig> up-regulation was found to be up-regulated .
in agreement with this, foxo <dig> is significantly down-regulated in ct <dig> cells.
foxo <dig> expression has been identified as a potential biomarker for crc outcome  <cit> , with low foxo <dig> associated with 2-fold shorter survival.
the low foxo <dig> expression, the high ezh <dig> expression and the enrichment of the “melanoma metastasis” gene set  <cit>  are all in line with the aggressive and high metastatic activity of ct <dig> cells.
differentiation markers further corroborate that ct <dig> cells are in a highly proliferative, undifferentiated state.
the “undifferentiated cancer” gene set is highly up-regulated in the ct <dig> cells .
stem cell markers cldn <dig> and sox <dig> are highly expressed while differentiation markers muc <dig> and ms4a8a   <cit>  markers are not expressed .
whereas lgals <dig>  is over 30-fold up-regulated in ct <dig> cells, the orthologous gene lgals <dig> , a differentiation marker, is over 500-fold down-regulated in ct <dig> cells.
the proliferation markers top2a , mki <dig> and birc <dig>  are all highly expressed in ct <dig> cells.
epcam marks epithelial cells and colon crypt tops  <cit>  and is not expressed in ct <dig> cells.
cdh <dig>  marks the epithelial-mesenchymal transition  <cit>  and is highly expressed in normal colon but not expressed in ct <dig>  cd <dig> marks the crypt bottoms and is 18-fold up-regulated.
silencing of wnt targets such as ascl <dig>  axin <dig> and lgr <dig> is often accomplished through cpg promoter methylation and associated with poor prognosis and increase metastatic spread  <cit> .
in ct <dig>  wnt10a is highly up-regulated but wnt target genes, with the exception of birc <dig>  are not expressed.
these markers classify ct <dig> as cells that originated in the lower-crypt and are in an undifferentiated state prone to metastasize  <cit> .
crc cohort studies have identified markers for classifying patient crc tumors .
the three-group crc classification platform using differentiation marker krt <dig> and “top crypt” markers ca, ms4a <dig> and cd <dig>  <cit>  classifies ct <dig> as a tumor with a less mature phenotype and worse progression.
the classification platform using genes frmd <dig>  zeb <dig>  htr2b and cdx <dig>  <cit>  classifies ct <dig> as the “ccs3” sub-type, with poor prognosis, low therapy response and resistance to cetuximab.
the  <dig> gene “crcassigner-7” platform  <cit>  classifies ct <dig> cells as either “stem like” or “cr-ta” .
the ct <dig> cancer immunome: immunotherapy concepts include targeting tumor-specific antigens presented on mhc molecules.
we determined that ct <dig> cells have the same mhc types as the parental balb/cj mice: h-2dd, h-2kd and h-2ld  and h-2lad .
this is expected and a useful confirmation of the balb/c-ct <dig> linage, given on-going reports of cell line mis-identifications.
class i loci h-2dd and h-2kd are expressed at levels comparable to normal tissues , lower than lymph node and spleen but higher than non-immune tissues .
b2m, part of the mhc class i complex, is highly expressed.
both suggest that mhc class i is functional.
normal tissues show variable expression of mhc class ii .
ct <dig> cells express neither mhc class ii  nor the mhc class ii transactivator ciita, suggesting that ct <dig> cells do not have functional mhc class ii antigen presentation.figure  <dig>
mhc class i  and class ii  expression in normal mouse tissues and ct <dig> cells.
the normal tissues are from both c57bl/6 j and balb/c.
the c57bl/6 j genome does not have the h-2 l locus.
genes with tumor-associated expression as well as genes with somatic mutations may act as tumor-associated antigens  .
gp <dig>  is a classical model tumor antigen frequently exploited when using ct <dig> system to investigate cd <dig> t cell immunity  <cit> .
expression of gp <dig> in normal mouse tissues has been observed in mice over 8 months old  <cit> ; however, gp <dig> levels are strikingly high levels in murine tumor cell lines including ct <dig>  <cit> .
indeed, our data show that gp <dig> has the highest expression of all ct <dig> genes.
while gp <dig> dna was not captured by the ngs exome-capture, we were able to determine the gp <dig> sequence using the rna-seq reads, averaging over  <dig> x coverage due to the high expression .
relative to the gp <dig> sequence in the mm <dig> genome, the ct <dig> gp <dig> sequence falls in a ct <dig> tetraploid region and has  <dig> non-synonymous mutations, including  <dig> homozygous and  <dig> heterozygous variants.
two variants are in dbsnp while three are found in genbank mrnas from other mouse tumor cell lines, suggesting that four could be unique to ct <dig> cells.
three variants introduce stop codons; however all are heterozygous such that a full length gp <dig> can likely be translated.table  <dig>
the ct <dig> gp <dig> snvs relative to the mm <dig> reference genome
aa, amino acid; *, stop codon.
the family of cancer testes  antigens has high tumor cell selectivity.
we found that ct antigens with the highest expression in ct <dig> cells are known colorectal ct antigens casc <dig>  cep <dig> and pbk .
these three, along with atad <dig> and ttk, have very low expression in the normal colon samples.
low expression of the human homologs of casc <dig>  ctage <dig>  pbk and spag <dig> has been observed in multiple tissues, such that these are cancer testes-selective antigens and they may be subject to tolerance  <cit> .
conversely, while expressed at 5-fold higher levels in ct <dig> cells, rqcd1is also expressed at significant levels in normal colon and is thus not an ideal immunotherapy target.table  <dig>
potential ct <dig> t-cell tumor expression antigens
known ct antigens with expression above  <dig> rpkm in ct <dig> and below  <dig> rpkm in normal mouse colon are shown.
ct <dig> and mouse colon expression values are in rpkm units.
in addition to tissue specific and over-expressed tumor antigens, somatic mutations provide tumor-specific immunotherapy t-cell targets  <cit>  that may be used for truly individualized cancer therapeutics and vaccines  <cit> .
a mutation for a cancer vaccine target must be expressed and presented on mhc molecules.
of the  <dig>  ct <dig> point mutations,  <dig>  are in expressed genes and, of these,  <dig> are in epitopes predicted to strongly bind to mhc molecules  .
<dig> occur in highly expressed genes .
table  <dig> shows eight such point mutations that meet these criteria.
for each snv, additional file 1: table s <dig> lists the mutation-containing epitope and mhc allele predicted to have the strongest mhc binding by the iedb algorithm  <cit> .
previous work by us and others  <cit>  finds that roughly 30% of these mutations are antigenic and capable of generating a t cell response when used in immunizations.
thus, these mutations provide a broad portfolio of potentially exploitable tsas for future studies.table  <dig>
eight potential ct <dig> mutation antigens that are in genes expressed in ct <dig> and in epitopes predicted to bind mhc class i molecules based on iedb consensus ranks
the bold letters signify the mutated amino acid.
dthe official names of mouse mhc alleles.
CONCLUSIONS
this is the first integrated genome, transcriptome and immunome map of a mouse epithelial tumor.
we found that the patterns of mutations in onco-relevant genes, the gene expression signatures and the regulated pathways in ct <dig> cells are in agreement with their origin in colon epithelia and share features with human primary crcs.
the mutations and expression profiles are similar to those reported for sporadic, undifferentiated, therapy-refractory, metastasis-prone human crc.
moreover, we identified non-synonymous snvs with predicted mhc class i binding capability which, together with the robust mhc class i expression of ct <dig> cells, provide a valuable resource for use of the ct <dig> model system to develop immunotherapeutic approaches.
the integrated use of mutation allele fraction and dna copy number allowed us to determine the absolute copy number and zygosity for each mutation.
the ct <dig> cells have extensive triploidy and tetraploidy and a high mutation rate .
while trp <dig>  braf, and pik3ca are not mutated, kras is mutated at g12d.
similar to human crc samples, there is a preference for c > t/g > a transitions.
however, the ct <dig> mutation pattern shows a preference for c > t mutations at sites that are followed by a pyrimidine, a pattern that is more similar to that found in tumors from patients pre-treated with temozolomide than to that found in most human crc tumors.
clinically-approved patient selection biomarkers for anti-egfr treatments cetuximab and panitumumab include assessment of egfr levels and kras g12d mutation status.
in ct <dig>  we found the kras g12d mutation and no expression of egfr.
consistent with this, ct <dig> cells have been shown to be refractory to the rodent egfr-targeting mabs  <cit> .
similarly, kras g12d mutations and mapk <dig>  and met amplification are published biomarkers for colorectal tumor sensitivity to both mek and met inhibitors  <cit> .
the homozygous kras g12d mutation and mapk <dig> and met amplifications in ct <dig> suggest sensitivity to mek and met inhibition.
in concordance with this, ct <dig> cells have been shown to be sensitive to mek and met inhibitors  <cit> .
further, the expression of markers such as top2a and cldn <dig> and lack of expression of muc <dig>  epcam and lgals <dig> show that ct <dig> cells are in an undifferentiated, proliferative state.
our study provides an overdue genomic and transcriptomic analysis of one of the most frequently used cell lines for drug development.
further, the results form the basis for the rationale design of pre-clinical studies using this model for drug development based on detailed molecular knowledge.

 murine ct cells were developed in by exposing balb/c mice to n-nitroso-n-methylurethane, resulting in a rapid-growing grade iv carcinoma that is easily implanted and readily metastasizes. used in over published studies, the ct colon carcinoma is one of the most commonly used cell lines in drug development. numerous cytotoxic agents as well as therapeutics targeting specific signaling pathways have been studied with these cells. moreover, as the ct model in balb/c mice provides a syngeneic in vivo test system, it is frequently used for developing and testing immunotherapeutic concepts. in sharp contrast to its frequent use in drug development, there have been no comprehensive studies of the genome and transcriptome of ct kras is mutated in ct but other mutations are not known. mutations in cdkn2a, mek, braf and pi3k in combination with egfr and vegf expression, for instance, may influence the results of pre-clinical investigations of treatment modalities. moreover, while gp the product of the envelope gene of murine leukemia virus -related cell surface antigen, is a known model antigen for studying antigen-specific immune responses in the ct system, there is no comprehensive knowledge of potential tumor antigens in this cell line system. further, the lack of comprehensive data on the murine ct colon cancer data sharply contrasts to the extensive molecular characterization of human colorectal cancer. as a group, human crc is highly heterogeneous with multiple evolutionary paths, with molecular signatures classifying subtypes and steps from adenoma to carcinoma. many human crc genomes are now known and multiple molecular signatures, classifications and biomarker concepts are published. as comprehensive genomic and transcriptomic data of ct has not been available, it is unclear how ct a chemically-induced tumor, molecularly correlates to human crc subtypes and to what extent it may be used as model. to answer these questions, we utilized next-generation sequencing, bioinformatics and immuno-informatics to create an integrated mouse solid tumor mutanome, transcriptome and immunome, providing an overdue analysis of the ct cancer cell line.  the ct tumor genome: using the ngs reads, we assessed copy number and nucleotide variations by comparing ct to balb/cj dna. we determined absolute dna copy number using the ratio of exome-seq reads mapping to each gene from ct versus those from balb/cj, and integrating variant allele fraction. we found that the ploidy of ct is strikingly large with large regions of triploidy and tetraploidy, in agreement with previous karyotyping results. the median and mean copy number in average across all genes is and, respectively, with genes in triploid regions and in tetraploid regions. no reads map to the y chromosome, suggesting that ct cells originated from a female mouse. only one homozygous deletion was found, which contains the tumor suppressor cdkn2a locus on mouse chromosome figure the ct genome. a) circos diagram showing: cytogenetic bands in black, gray and white. track 1: dna copy number. black: deleted; green: haploid; yellow: diploid; dark purple: triploid; light purple: tetraploid; dark blue: pentaploid; bright blue: hexaploid; red: higher copy number. track 2: high confidence point mutations, plotted based on dna allele frequency. inner is allele frequency outer is allele frequency colors are blue, green, orange, and red. track 3: predicted mhc binding ic scores for missense mutations. mutations in peptides likely to bind mhc are colored orange. track 4: insertions and deletions plotted according to allele frequency. b) dna gene copy number. c) single nucleotide mutation changes and the nucleotide immediately 3′ of the mutation. d) mutation classification. we identified high-confidence single nucleotide variations and short insertions and deletions. indels are dominated by a/t deletions. we selected high confidence snvs in exons, the majority of which are localized in coding regions. of the snvs in coding regions, the majority cause non-synonymous protein changes, including missense and nonsense variants. the ccds database identifies million protein-encoding nucleotides in the mouse genome. relative to a balb/cj genome, the ct variation rate in coding regions is non-synonymous and silent mutations per mb. this is significantly more than the average found in spontaneous human tumors but still within the range observed for primary human crc tumors, which ranges from less than per mb to over mutations per mb. the identified snvs represent variations between the ct genome, derived from a balb/c mouse in and a balb/cj mouse in as such, the snvs include both somatic mutations associated with the ct onco-transformation and genetic drift in the balb/c genome. we found mouse snps that distinguish the balb/cj and mm exomes. of these, only % show a discrepancy between the ct and balb/cj genomes. thus, while this does not eliminate genetic drift or conclusively identify the substrain that gave rise to ct cells, it demonstrates that the genome of the mouse that originally created the ct cells is similar to that of the current balb/cj mouse. spontaneous human crc tumors contain primarily c > t/g > a snvs. of the snvs in the ct genome, are transitions, of which most are c > t/g > a mutations, similar to the human crc mutation profile. based on data from over human tumors, g is the dominate nucleotide immediately 3′ of the mutated nucleotide in human crc tumors. conversely, we found that ct snvs are depleted in cg > tg and ca > ta mutations and enriched in ct > tt and cc > tc mutations. this pattern, a c > t mutation followed by a pyrimidine, is found in tumor samples from human patients pre-treated with temozolomide, an alkylating anticancer drug. ct was originally induced by the alkylating agent nmu. that temozolomide and nmu are both are associated with tumors enriched in c > t mutations at positions followed by a pyrimidine suggest a similar mutagenic pattern for these two alkylating agents. of the ct snvs, are homozygous or heterozygous, even in amplified regions with high copy number. homozygous variants cluster across chromosomes and x. these regions could be the result of either a loss of heterozygosity onco-transformation or genetic drift in a balb/c mouse followed by inbreeding. if the result of an onco-transformation, that the regions experienced loh, followed by mutations and copy number amplification suggests that resulting individual alleles were amplified 2-fold, 3-fold, 4-fold, and 5-fold. we further investigated chromosome x. mutations occur on chromosome x with 100% and 50% dna allele frequency, suggesting that chromosome x is diploid in ct cells. female cells typically express xist and inactivate one x allele. in ct the rna-seq data show that xist is not expressed and, examining the allele expression of heterozygous mutations, that transcription occurs from both chromosome x alleles. these findings are concordant with a scenario where the chromosome x experienced both a loss of the inactivated allele and an amplification of the non-inactivated allele. in summary, the data imply that the ct has a complex genome of high ploidy which underwent several amplification events. relative to a balb/c genome, the number of mutations is higher than average, with many non-synonymous mutations. the mutation pattern reflects the treatment with the nmu alkylating agent, a similar but distinct pattern than found in spontaneous primary crc. ct snvs in onco-relevant genes: we investigated whether mutations associated with crc are also prevalent in ct apc, kras and tp are frequent drivers of the linear and uniform evolution of spontaneous human crc; of these, only kras is mutated in ct the ct kras genomic locus is triploid and all alleles contain v8m and g12d mutations. several crc subtypes are linked to syndromes based on inherited gene defects and mutations. genes associated with familial crc include mismatch repair genes mlh mlh mlh msh myh, pms stk mutyh and ctnnb none are mutated in ct the lack of mutations in mismatch repair genes mlh and msh which are associated with crc microsatellite instability, agrees with the lack of mutation in braf, which is frequently associated with the msi-high phenotype. further, the tumor suppressor cdkn2a is homozygously deleted and the genomic mapk and met loci are amplified in ct crc-associated genes fbxw pik2ca, pten, smad smad tcf7l are not mutated. non-synonymous point mutations occur in other crc genes brca, pdgfra, nav, atr, cdk, and rel. mutations in cancer-related genes include mtor, birc, casp, cenpe, esr, hdac, ins, insr, muc, pik3c, pik3cg, fgfr, ddr, notch and rhoj. frameshift-causing indels occur in oncogenes ewsr and mpp. ct gene expression: we generated gene expression profiles from ct cells. cancer-relevant genes such nras, vegfa, trp, myc, mdm and hif1a are expressed at high levels in ct. egfr and flt are not expressed. gene expression in ct relative to normal colon was used for pathway enrichment analysis in order to identify broadly enriched pathways. not surprisingly, the identified pathways relate to cell proliferation and increased translation. we examined individual gene sets enriched in ct. most enriched is “cell_cycle_rb1_targets”, a gene set curated from a study examining rb target genes involved in cell cycle regulation, reflecting over-expression of all rb target genes. rb mrna is itself 8-fold up-regulated. ezh downstream of the egfr-ras-raf pathway, impacts dna methylation, promotes emt and is associated with poor prognosis in crc. together with its target genes, ezh is over-expressed in ct cells. mechanistically, that rb ezh lin and e2f mrnas and their target genes are over-expressed suggests that the rb ezh lin and e2f mrna levels, in addition to post-translational modifications, play a critical role controlling activation of each pathway.figure gene expression of onco-relevant genes, stem cell and proliferation markers, wnt signaling pathway genes, emt and epithelial markers, and differentiation markers in ct and mouse colon. error bars represent the maximum and minimum values for the ct triplicates and the six normal colon samples. reactome pathways over-expressed in ct versus normal colon. enriched reactome pathways are displayed using cytoscape cluego. ribosomal associated genes are over-expressed in ct26; while they are part of the influenza gene set, we posit their over-expression reflects increased translational activity rather than an influenza-related process. for each gene set, cytoscape cluego calculates a false-discovery rate statistic reflecting the likelihood of the given enrichment by chance: the pathways listed in the table those with a zero q-value. each dot in the image represents a separate reactome gene set. gene sets with common members are placed proximally, grouped into common themes, colored the same and labeled. q-values for enriched pathways, cytoscape settings and gene membership are in the supplementary information. gene sets over-expressed in ct versus normal colon. a). the top over-expressed gene sets. gene sets included those curated from literature and overexpression was determined using genepattern. b-f): gene set enrichment for the targets of specific genes, including the genepattern enrichment plot showing the enrichment plot and the location of the individual gene set members in the expression ranked-ordered list. the plot on the right shows the expression of the targeting gene rb, e2h, lin, foxo and e2f. the gene set associated with genes down-regulated after foxo up-regulation was found to be up-regulated. in agreement with this, foxo is significantly down-regulated in ct cells. foxo expression has been identified as a potential biomarker for crc outcome, with low foxo associated with 2-fold shorter survival. the low foxo expression, the high ezh expression and the enrichment of the “melanoma metastasis” gene set are all in line with the aggressive and high metastatic activity of ct cells. differentiation markers further corroborate that ct cells are in a highly proliferative, undifferentiated state. the “undifferentiated cancer” gene set is highly up-regulated in the ct cells. stem cell markers cldn and sox are highly expressed while differentiation markers muc and ms4a8a markers are not expressed. whereas lgals is over 30-fold up-regulated in ct cells, the orthologous gene lgals, a differentiation marker, is over 500-fold down-regulated in ct cells. the proliferation markers top2a, mki and birc are all highly expressed in ct cells. epcam marks epithelial cells and colon crypt tops and is not expressed in ct cells. cdh marks the epithelial-mesenchymal transition and is highly expressed in normal colon but not expressed in ct cd marks the crypt bottoms and is 18-fold up-regulated. silencing of wnt targets such as ascl axin and lgr is often accomplished through cpg promoter methylation and associated with poor prognosis and increase metastatic spread. in ct wnt10a is highly up-regulated but wnt target genes, with the exception of birc are not expressed. these markers classify ct as cells that originated in the lower-crypt and are in an undifferentiated state prone to metastasize. crc cohort studies have identified markers for classifying patient crc tumors. the three-group crc classification platform using differentiation marker krt and “top crypt” markers ca, ms4a and cd classifies ct as a tumor with a less mature phenotype and worse progression. the classification platform using genes frmd zeb htr2b and cdx classifies ct as the “ccs3” sub-type, with poor prognosis, low therapy response and resistance to cetuximab. the gene “crcassigner-7” platform classifies ct cells as either “stem like” or “cr-ta”. the ct cancer immunome: immunotherapy concepts include targeting tumor-specific antigens presented on mhc molecules. we determined that ct cells have the same mhc types as the parental balb/cj mice: h-2dd, h-2kd and h-2ld and h-2lad. this is expected and a useful confirmation of the balb/c-ct linage, given on-going reports of cell line mis-identifications. class i loci h-2dd and h-2kd are expressed at levels comparable to normal tissues, lower than lymph node and spleen but higher than non-immune tissues. b2m, part of the mhc class i complex, is highly expressed. both suggest that mhc class i is functional. normal tissues show variable expression of mhc class ii. ct cells express neither mhc class ii nor the mhc class ii transactivator ciita, suggesting that ct cells do not have functional mhc class ii antigen presentation.figure mhc class i and class ii expression in normal mouse tissues and ct cells. the normal tissues are from both c57bl/6 j and balb/c. the c57bl/6 j genome does not have the h-2 l locus. genes with tumor-associated expression as well as genes with somatic mutations may act as tumor-associated antigens. gp is a classical model tumor antigen frequently exploited when using ct system to investigate cd t cell immunity. expression of gp in normal mouse tissues has been observed in mice over 8 months old; however, gp levels are strikingly high levels in murine tumor cell lines including ct. indeed, our data show that gp has the highest expression of all ct genes. while gp dna was not captured by the ngs exome-capture, we were able to determine the gp sequence using the rna-seq reads, averaging over x coverage due to the high expression. relative to the gp sequence in the mm genome, the ct gp sequence falls in a ct tetraploid region and has non-synonymous mutations, including homozygous and heterozygous variants. two variants are in dbsnp while three are found in genbank mrnas from other mouse tumor cell lines, suggesting that four could be unique to ct cells. three variants introduce stop codons; however all are heterozygous such that a full length gp can likely be translated.table the ct gp snvs relative to the mm reference genome aa, amino acid; *, stop codon. the family of cancer testes antigens has high tumor cell selectivity. we found that ct antigens with the highest expression in ct cells are known colorectal ct antigens casc cep and pbk. these three, along with atad and ttk, have very low expression in the normal colon samples. low expression of the human homologs of casc ctage pbk and spag has been observed in multiple tissues, such that these are cancer testes-selective antigens and they may be subject to tolerance. conversely, while expressed at 5-fold higher levels in ct cells, rqcd1is also expressed at significant levels in normal colon and is thus not an ideal immunotherapy target.table potential ct t-cell tumor expression antigens known ct antigens with expression above rpkm in ct and below rpkm in normal mouse colon are shown. ct and mouse colon expression values are in rpkm units. in addition to tissue specific and over-expressed tumor antigens, somatic mutations provide tumor-specific immunotherapy t-cell targets that may be used for truly individualized cancer therapeutics and vaccines. a mutation for a cancer vaccine target must be expressed and presented on mhc molecules. of the ct point mutations, are in expressed genes and, of these, are in epitopes predicted to strongly bind to mhc molecules. occur in highly expressed genes. table  shows eight such point mutations that meet these criteria. for each snv, additional file 1: table s lists the mutation-containing epitope and mhc allele predicted to have the strongest mhc binding by the iedb algorithm. previous work by us and others finds that roughly 30% of these mutations are antigenic and capable of generating a t cell response when used in immunizations. thus, these mutations provide a broad portfolio of potentially exploitable tsas for future studies.table eight potential ct mutation antigens that are in genes expressed in ct and in epitopes predicted to bind mhc class i molecules based on iedb consensus ranks the bold letters signify the mutated amino acid. dthe official names of mouse mhc alleles.  this is the first integrated genome, transcriptome and immunome map of a mouse epithelial tumor. we found that the patterns of mutations in onco-relevant genes, the gene expression signatures and the regulated pathways in ct cells are in agreement with their origin in colon epithelia and share features with human primary crcs. the mutations and expression profiles are similar to those reported for sporadic, undifferentiated, therapy-refractory, metastasis-prone human crc. moreover, we identified non-synonymous snvs with predicted mhc class i binding capability which, together with the robust mhc class i expression of ct cells, provide a valuable resource for use of the ct model system to develop immunotherapeutic approaches. the integrated use of mutation allele fraction and dna copy number allowed us to determine the absolute copy number and zygosity for each mutation. the ct cells have extensive triploidy and tetraploidy and a high mutation rate. while trp braf, and pik3ca are not mutated, kras is mutated at g12d. similar to human crc samples, there is a preference for c > t/g > a transitions. however, the ct mutation pattern shows a preference for c > t mutations at sites that are followed by a pyrimidine, a pattern that is more similar to that found in tumors from patients pre-treated with temozolomide than to that found in most human crc tumors. clinically-approved patient selection biomarkers for anti-egfr treatments cetuximab and panitumumab include assessment of egfr levels and kras g12d mutation status. in ct we found the kras g12d mutation and no expression of egfr. consistent with this, ct cells have been shown to be refractory to the rodent egfr-targeting mabs. similarly, kras g12d mutations and mapk and met amplification are published biomarkers for colorectal tumor sensitivity to both mek and met inhibitors. the homozygous kras g12d mutation and mapk and met amplifications in ct suggest sensitivity to mek and met inhibition. in concordance with this, ct cells have been shown to be sensitive to mek and met inhibitors. further, the expression of markers such as top2a and cldn and lack of expression of muc epcam and lgals show that ct cells are in an undifferentiated, proliferative state. our study provides an overdue genomic and transcriptomic analysis of one of the most frequently used cell lines for drug development. further, the results form the basis for the rationale design of pre-clinical studies using this model for drug development based on detailed molecular knowledge. 
mhc class i but not mhc class ii is expressed. the highest expressed gene is a mutated form of the mouse tumor antigen gp of the non-synonymous point variations, are both in expressed genes and in peptides predicted to bind mhc and thus potential targets for immunotherapy development. as ct is one of the most extensively used syngeneic mouse tumor models, our data provide a map for the rationale design of mode-of-action studies for pre-clinical evaluation of targeted- and immunotherapies. tumor models are critical for our understanding of cancer and the development of cancer therapeutics.
 tumor models are critical for our understanding of cancer and the development of cancer therapeutics. here, we present an integrated map of the genome, transcriptome and immunome of an epithelial mouse tumor, the ct colon carcinoma cell line.  we found that kras is homozygously mutated at p.g12d, apc and tp are not mutated, and cdkn2a is homozygously deleted. proliferation and stem-cell markers, including top2a, birc, cldn and mki are highly expressed while differentiation and top-crypt markers muc ms4a8a and epcam are not. myc, trp, mdm hif1a, and nras are highly expressed while egfr and flt are not. mhc class i but not mhc class ii is expressed. several known cancer-testis antigens are expressed, including atad cep and pbk. the highest expressed gene is a mutated form of the mouse tumor antigen gp of the non-synonymous point variations, are both in expressed genes and in peptides predicted to bind mhc and thus potential targets for immunotherapy development. based on its molecular signature, we predicted that ct is refractory to anti-egfr mabs and sensitive to mek and met inhibitors, as have been previously reported.  ct cells share molecular features with aggressive, undifferentiated, refractory human colorectal carcinoma cells. as ct is one of the most extensively used syngeneic mouse tumor models, our data provide a map for the rationale design of mode-of-action studies for pre-clinical evaluation of targeted- and immunotherapies. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords immunotherapycancer modelscomputational immunologycolorectal cancerissue-copyright-statement© the author 2014 