1. Cancer Biology
  2. Immunology and Inflammation
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The newly-arisen Devil facial tumour disease 2 (DFT2) reveals a mechanism for the emergence of a contagious cancer

  1. Alison Caldwell
  2. Rachel Coleby
  3. Cesar Tovar
  4. Maximilian R Stammnitz
  5. Young Mi Kwon
  6. Rachel S Owen
  7. Marios Tringides
  8. Elizabeth P Murchison
  9. Karsten Skjødt
  10. Gareth J Thomas
  11. Jim Kaufman
  12. Tim Elliott
  13. Gregory M Woods
  14. Hannah VT Siddle  Is a corresponding author
  1. University of Southampton, United Kingdom
  2. University of Tasmania, Australia
  3. University of Cambridge, United Kingdom
  4. University of Southern Denmark, Denmark
  5. Faculty of Medicine, University of Southampton, United Kingdom
Research Article
Cite as: eLife 2018;7:e35314 doi: 10.7554/eLife.35314
5 figures, 2 tables, 2 data sets and 5 additional files

Figures

DFT2 cells express β2m in vitro.

Flow cytometry to compare β2m expression shows (A) DFT1_4906, (B) DFT1_4906 + IFNγ, (C) DFT2_RV, (D) DFT2_SN, (E) DFT2_TD549 and (F) Fibroblast_Salem cells stained with α-β2m (solid line) and secondary only control (shaded peak). DFT1_4906 + IFNγ (B) stained with blocked α-β2m antibody is also shown (dashed line). Fluorescence intensity for α-β2m on x-axis and cell counts on y-axis.

https://doi.org/10.7554/eLife.35314.003
Figure 2 with 1 supplement
DFT2 cells in vitro and in vivo express mRNA for β2m, Saha-UK, Saha-UA, UB and UC.

RT-qPCR of (A) β2m, (B) Saha-UA, -UB and -UC and (C) Saha-UK mRNA expression by DFT2 cell lines (DFT2_RV, DFT2_SN, DFT2_TD549), fibroblast cells (Fibroblasts_Salem) and DFT1 cells treated with IFNγ (DFT1_4906 + IFNγ) relative to DFT1_4906 cells. Gene expression levels are normalized against RPL13A as a housekeeping gene. Data are represented as mean ± S.E.M of three technical replicates. (D) An unpaired T-test was performed to test for statistical significance. (E) RT-PCR on DFT2 cell lines and DFT2 primary tumours for β2m, Saha-UA, -UB and -UC and Saha-UK. RPL13A was used as a loading control. A no DNA control (NDC) is included for each RT-PCR. A marker at 300 base pairs is indicated by an asterisk.

https://doi.org/10.7554/eLife.35314.004
Figure 2—figure supplement 1
RT-PCR amplification of RPL13A and Saha-UD from DFT1, DFT2 and Fibroblast cells.

mRNA from DFT1_4906, DFT1_4906 + IFNγ, DFT2_RV-CL, DFT2_SN-CL, DFT2_TD549-CL and Fibroblast_Salem cell lines was used. A no cDNA negative control was included in each experiment.

https://doi.org/10.7554/eLife.35314.005
Figure 3 with 3 supplements
DFT2 tumours express variable levels of classical MHC class I (Saha-UA, -UB and -UC) and non-classical MHC class I (Saha-UK) in vivo.

IHC staining of DFT2 tumours (DFT2_RVT1, DFT2_SNT2, DFT2_818T1, DFT2_547T1, DFT2_523 DFT2_812) with α-UA/UB/UC_15-25-18 against Saha-UA, -UB and -UC and α-UK_15-29-1 against Saha-UK. Arrows indicate tumour cells for each biopsy; arrow heads indicate stroma separating nests of tumour cells where present. Isotype and secondary antibody controls can be found in Figure 3—figure supplement 2. Boxed insets are at 600 x magnification and are taken from areas indicated by arrows. Scale bars represent 50 μm. Positive cells for each marker are stained brown, nuclei are stained blue.

https://doi.org/10.7554/eLife.35314.006
Figure 3—source data 1

Nucleotide sequences for the pET22B + SahaUC and pET22B + SahaUK constructs used to test the specificity of the MHC class I antibodies (a- UA/UB/UC 15-25-18 and a- UK 15-29-1) described in Figure 3.

https://doi.org/10.7554/eLife.35314.010
Figure 3—figure supplement 1
Western blots illustrating specificity of the MHC class I heavy-chain monoclonal antibodies.

Western blot on classical MHC class I protein (Saha-UC) (38 kDa) and non-classical MHC class I protein (Saha-UK) (39 kDa) with MHC class I heavy-chain specific antibodies.

https://doi.org/10.7554/eLife.35314.007
Figure 3—figure supplement 2
Isotype controls (IgG1b – anti-SahaUA/UB/UC and IgG2b – anti-SahaUK) and secondary antibody only controls for the immunohistochemistry presented in Figure 3.

All images are 100 x magnification with scale bars represent 100 μm.

https://doi.org/10.7554/eLife.35314.008
Figure 3—figure supplement 3
DFT2 tumours have different growth patterns.

H and E staining of four DFT2 tumours representing different growth patterns. Scale bars represent 100 μm.

https://doi.org/10.7554/eLife.35314.009
CD3 staining of DFT2 tumours.

CD3 staining of DFT2_RVT1, DFT2_SNT2 DFT2_818T1, DFT2_TD547T1, DFT2_523T2 and DFT2_812 tumours. CD3 positive cells are indicated by arrows. DFT2_SNT2, and DFT2_547 are CD3 negative. Scale bars represent 50 μm. Positive cells for each marker are stained brown, nuclei are stained blue.

https://doi.org/10.7554/eLife.35314.011
Figure 5 with 1 supplement
DFT2 shares classical MHC class I alleles with its hosts.

Alignment of the MHC class I sequences cloned from the mRNA from DFT2 cell lines (DFT2_CL) and host devils (TD_RV, TD_SN and TD_818 (Teu)). Nucleotide alignment in (A) and amino acid alignment in (B). Red asterisks indicate residues postulated to interact with peptides and blue asterisks indicate residues predicted to interact with TCRs (Bjorkman et al., 1987).

https://doi.org/10.7554/eLife.35314.012
Figure 5—figure supplement 1
Chromatograms of α2-domian of MHC class I from spleen samples from RV, SN and 818/Teu.

Spln_RV, Spln_SN and Spln_818/Teu host tissues share the same polymorphic sites in the MHC class I α2-domian. Chromatograms of α2-domian of MHC class I (Saha-UA, -UB and –UC) from spleen samples from RV, SN and 818/Teu from PCR amplification and sequencing using the reverse primer Saha91. Polymorphic sites are indicated by an asterisk.

https://doi.org/10.7554/eLife.35314.013

Tables

Table 1
MHC class I alleles expressed by DFT2 cell lines and host animals.

The classical and non-classical MHC class I alleles expressed by DFT1, DFT2, TD_RV, TD_818 and TD_SN. Grey boxes indicate the alleles identified in each sample. Numbers in the boxes indicate the number of clones identified for each allele. SahaI*32(UD) and SahaI*UK were amplified with gene specific primers.

https://doi.org/10.7554/eLife.35314.014
NCBI allele nameDFT1DFT2TD_RVTD_818TD_SN
SahaI*46
SahaI*272222142
SahaI*27–1132513
SahaI*74/889
SahaI*351310
SahaI*9041
SahaI*49/827
SahaI*9762
SahaI*3362
SahaI*362
SahaI*3751
SahaI*2922
SahaI*32(UD)
SahaI*UK
Table 1–source data 1

MHC class I transcripts expressed in DFT2 and host devils that were used to generate Table 1 and Figure 5.

https://doi.org/10.7554/eLife.35314.015
Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Gene
(Sarcophilus harrisii)
Saha-UKMurchison et al. (2012)ensembl:
ENSSHAG00000002942
Devil_ref v7.0
Gene (S. harrisii)Saha-UCMurchison et al. (2012)ensembl:
ENSSHAG00000000117
Devil_ref v7.0
Gene (S. harrisii)Saha-UDMurchison et al. (2012)ensembl:
ENSSHAG00000010776
Devil_ref v7.0
Cell line (S. harrisii)DFT1_4906Siddle et al. (2013) and
Murchison et al. (2012)
RRID:CVCL_LB78;
DFTD 4906; 86T
Devil Facial Tumour 1;
Supplementary file 1
Cell line (S. harrisii)DFT2_RVPye et al. (2016b)DFT2_202T1Devil Facial Tumour 2;
Supplementary file 1
Cell line (S. harrisii)DFT2_SNPye et al. (2016b)DFT2_203T3Devil Facial Tumour 2;
Supplementary file 1
Cell line (S. harrisii)DFT2_549This paperDevil Facial Tumour 2;
Supplementary file 1
Cell line
(S. harrisii) (Female)
Fibroblasts_SalemMurchison et al. (2012)91 HTasmanian devil fibroblasts
Cell line
(Cricetulus griseus)
CHO_SahaIFNySiddle et al. (2013)Chinese Hamster Ovary (CHO)
cell line transfected with
pcDNA3_SahaIFNy
Biological sample
(S. harrisii)
DFT2_RVPye et al. (2016b)DFT2_202T1Devil Facial Tumour 2;
Supplementary file 1
Biological sample
(S. harrisii)
DFT2_SNPye et al. (2016b)DFT2_203T2Devil Facial Tumour 2;
Supplementary file 1
Biological sample
(S. harrisii)
DFT2_818Stammnitz et al. (2018)Devil Facial Tumour 2;
Supplementary file 1
Biological sample
(S. harrisii)
DFT2_547Stammnitz et al. (2018)807T1Devil Facial Tumour 2;
Supplementary file 1
Biological sample
(S. harrisii)
DFT2_523Stammnitz et al. (2018)638T1Devil Facial Tumour 2;
Supplementary file 1
Biological sample
(S. harrisii)
DFT2_812Stammnitz et al. (2018)Devil Facial Tumour 2;
Supplementary file 1
Biological sample
(S. harrisii)
TD_RVStammnitz et al. (2018)202H1Tasmanian devil spleen
biopsy
Biological sample
(S. harrisii)
TD_SNStammnitz et al. (2018)203 HTasmanian devil kidney
biopsy
Biological sample
(S. harrisii)
TD_818Stammnitz et al. (2018)818Tasmanian deil spleen
biopsy
Antibodyα-UA/UB/UC_15-25-18;
Classical MHC class I Saha-UA,
-UB and -UC
This paperUA/UB/UC_15-25-18Antibody recognising MHC
class I molecules,SahaUA,
UB and UC. Generated using
a peptide immunogen
(WMEKVQDVDPGYWE).
Supernatant from hybridoma
used neat.
Antibodyα-UK_15-29-1; Non-classical
MHC class I Saha-UK
This paperα-UK_15-29-1Antibody recognising MHC
class I molecule,Saha-UK.
Generated using a peptide
immunogen (RITHRTHPDGKVTL).
Supernatant from hybridoma
used neat.
AntibodyIgG1 Isotype controlSigma Aldrichclone: MOPC-21;
cat number: M5284
0.5 mg/ml
AntibodyIgG2b Isotype controlSigma Aldrichclone: MOPC-141;
cat number: M5534
0.5 mg/ml
Antibodya-B2mSiddle et al. (2013)SahaB2m-13-34-48supernatant used neat;
B2-microglogulin
Antibodya-CD3Dako/Agilentcat number: A04521:50
Recombinant DNA
reagent
pET22B+-SahaUCThis paperSahaI*UC (SahaI*27) amplified
using primer Saha349 and Saha350
(Supplementary file 2).
Recombinant DNA
reagent
pET22B+-SahaUKThis paperSaha-UK in Pet22B + using primer
Saha335 and Saha351
(Supplementary file 2).

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Table 1, Figure 5 and Figure 3-figure supplement 1.

The following previously published data sets were used
  1. 1
    Devil_ref v7.0 (GCA_000189315.1)
    1. Murchison
    (2012)
    Publicly available at the European Nucleotide Archive (accession no: GCA_000189315.1).
  2. 2
    Genomes of Tasmanian devil transmissible cancers DFT1, DFT2 and normal animals
    1. Stammnitz
    (2018)
    Publicly available at the European Nucleotide Archive (accession no: ENA: PRJEB21902).

Additional files

Supplementary file 1

Biological samples used in this study.

Details of the tumour and host tissue samples, the date the samples were collected from the animal and the location of the animal when it was trapped. *As described previously (Pye et al., 2016b).

https://doi.org/10.7554/eLife.35314.016
Supplementary file 2

Primers used in this study with amplicon size and optimised annealing temperature.

https://doi.org/10.7554/eLife.35314.017
Supplementary file 3

Antibodies used in this study.

https://doi.org/10.7554/eLife.35314.018
Supplementary file 4

PCR conditions for the primers used in this study.

https://doi.org/10.7554/eLife.35314.019
Transparent reporting form
https://doi.org/10.7554/eLife.35314.020

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