Animals and culture conditions

Experiments were carried out using H. oligactis strain St. Petersburg and P. robusta strain L7. Animals of both strains were used for histological and immunohistochemical examinations and in situ hybridization experiments. Owing to the poor viability of tumour-bearing polyps and slow clonal propagation rate of H. oligactis tumour culture, we used only P. robusta tumour-bearing polyps for cell proliferation, migration and fitness assays. Conversely, the severity of the tumour phenotype in H. oligactis prioritized this strain for transcriptome sequencing and microarray analysis. Animals of all strains were maintained under constant environmental conditions, including culture medium, food and temperature (18 °C) according to standard procedures31. Oogenesis was induced by lowering the culture temperature to 10 °C and depriving animals of food13 for a period of 14 (H. oligactis) or 30 days (P. robusta). Single tumour-bearing polyps, which we discovered by careful observations, appeared spontaneously and independently in the mass cultures of both species. These founder polyps were asexually propagated by budding or by cutting longitudinally in two halves to establish clonal cultures, which have been maintained in the lab for more than 5 years. Tumour grows progressively with time, but typically does not kill the host. Affected polyps are able to feed and also to bud, thus still showing asexual mode of reproduction. In the case of P. robusta, the daughter polyp originated by budding of parental tumorous polyp is tumorous in 100% cases. Offspring develops tumour in normal laboratory conditions, without any stimulation and condition changes. In H. oligactis, very rarely apparently healthy polyps detach from the tumorous parental polyps. If these polyps did not develop any visible tumour in 1–2 months, we removed them from the culture and excluded them from further analysis.

Histological staining

For histological analysis, polyps were relaxed in 2% urethane and fixed with 4% formaldehyde, dehydrated in ethanol and embedded into LR-White resin (Ted Pella, Redding, CA, USA) according to the manufacturer’s instruction. Semi-thin sections (0.5 μm thick) were cut using Ultracut S ultratome, mounted on slides and stained with methylene blue/azur II as described previously32,33. Light microscopy images were taken on a Zeiss Axioscope microscope equipped with Axiocam digital camera (Zeiss, Jena, Germany).

Immunohistochemistry

Immunohistochemical detection of periculin protein in whole mount Hydra preparations was performed as described previously23 using polyclonal mouse antisera against periculin1a protein (1:500 diluted, produced by T. Bosch lab) and Alexa488-conjugated donkey-anti-mouse secondary antibodies (2 μg ml−1; Invitrogen, Eugene, OR, USA). Rhodamin-phalloidin and TO-PRO3 counterstaining was conducted as described previously34. Confocal laser-scanning microscopy was done using a TCS SP1 laser-scanning confocal microscope (Leica, Wetzlar, Germany).

In situ hybridization

Expression patterns of Cnnos1 and tpt1 genes were detected in whole mount Hydra preparation by in situ hybridization with digoxigenin (DIG)-labelled RNA probes32. Anti-sense RNA probes were designed to recognize specifically the sequence of Hydra Cnnos1 gene product (GenBank XM_002161814.1) and transcript of H. oligactis (contig 19403) homologous to human tpt1 gene (tpt1, p23, GenBank XM_002157314.2 in H. magnipapillata). DIG-labelled sense probes were used as a control. Signal was developed using anti-DIG antibodies conjugated to alkaline phosphatase (1:2000, Roche Diagnostics, Mannheim, Germany) and NBT/BCIP staining solution (Roche). Images of in situ preparations were collected on a Zeiss Axioscope microscope with Axiocam camera.

Cell-type and proliferation assays

Tissue maceration technique19 was used to analyse the cell composition of tumorous, normal and female gonad tissue in H. oligactis and P. robusta. Middle body-column region, containing tumorous tissue, developing female gonad (egg patch at stages 1–5 of oogenesis20) or normal tissue, was excised, macerated into single-cell suspension, fixed with 8% formaldehyde and dried on slides as described previously19. For quantitative morphological analysis we used in total 8 control P. robusta polyps, 6 female and 13 tumorous polyps. On examination of the slides using phase-contrast and differential interference contrast microscopy, we counted large and small ISCs19 separately. In this and similar experiments, a blind-control design was implemented, so that the investigator was unaware of the sample group allocation when assessing the experiment outcome. In total 7,952 cells were counted on slides (611.7±33.1; mean±s.e.m. cells per slide; n=13) made from macerated tumour tissue, 3,795 cells on slides (474.4±43.3; n=8) from control tissue and 3,161 cells on slides (526.8±24.9; n=6) made from female gonad tissue. P. robusta polyps were used for cell proliferation activity assessment using BrdU-labelling technique. Polyps were incubated in 5 mmol l−1 BrdU (Sigma, Steinheim, Germany) solution for 2–24 h (continuous labelling), with the solution being additionally injected into the polyp gastric cavity every 6 h. After 2, 12 or 24 h of incubation, the middle body-column region, containing tumorous tissue, developing female gonad (egg-patch) or normal tissue was excised and macerated into single-cell suspension19. In total we used five polyps of each sort for every time point (2, 12 and 24 h) of continuous labelling experiment. Immunodetection of BrdU on slides was done as described previously15 using monoclonal anti-BrdU antibodies (1:100, Roche), alkaline phosphatase-conjugated sheep-anti-mouse secondary antibodies (1:5,000, Millipore, Melbourne, Australia) and NBT/BCIP staining solution (Roche). In continuous labelling experiment, BrdU-labelling index was assessed for all interstitial cells together, without discriminating large and small ISCs. For all samples (tumour, control, female) at each time point (2, 12 and 24 h) we prepared five slides. In average we counted 990.3±39.1 (2 h; n=5), 825.0±28.8 (12 h; n=5) and 1,062.8±88.8 (24 h; n=5) cells on slides made from macerated tumour tissue; 827.0±63.7 (2 h; n=5), 638.8±36.4 (12 h; n=5) and 1,006.2±88.0 (24 h; n=5) cells on slides made from macerated control tissue; and 906.3±34.5 (2 h; n=5), 768.5±65.5 (12 h; n=5) and 877.3±36.4 (24 h; n=5) cells on slides made from macerated female gonad tissue. Statistical analysis of the data was carried out using one-way analysis of variance with Bonferroni post hoc test.

Cell migration assays

Migratory activity of the interstitial cells was examined in P. robusta strain. Donor polyps were labelled with BrdU for 24 h, and a 4–6 mm3 piece of tumour tissue, normal control tissue or developing female gonad (oogenesis stage 2–3) was laterally grafted into the middle body part of healthy unlabelled polyp according to the transplantation procedure described by Takano and Sugiyama35. We excised, pooled and macerated oral and aboral part of the recipient body column at 24 and 72 h after transplantation. Immunodetection of BrdU on slides was done as described above. On examination of the slides using phase-contrast microscopy, we counted BrdU-positive ISCs and non-labelled epithelial cells. To calculate migration index, we normalized the number of BrdU-positive ISCs by the number of non-labelled epithelial cells. To control for the occasional contamination of the recipient tissue with the donor epithelial tissue, we discarded from the analysis the slides that showed >1% of the BrdU-positive epithelial cells. In total, 20 grafts were analysed at 24 h (controls: n=5; female gonads: n=7; tumours: n=8) and 21 grafts at 72 h after transplantation (controls: n=5; female gonads: n=9; tumours: n=7). In total 17,731 cells were counted (432.5±7.2 cells per slide). The data were analysed using one-way analysis of variance with Bonferroni post hoc test.

To check whether the tumorous cells invading normal host tissue are able to induce de novo tumour formation, we grafted small pieces of unlabelled tumour tissue into the healthy hosts. We fed the host polyps ad libitum daily, and 35 days after transplantation we screened the clonal progeny generated by the budding for the presence of tumours. In total, we analysed seven clonal lines with grafted tumours and three control lines, where healthy tissue was transplanted.

Apoptosis analysis

Apoptosis activity in H. oligactis tumour-bearing, control polyps and female polyps at different oogenesis phases (stage 1–7) was analysed by TUNEL assay and acridine orange staining as described previously27. In TUNEL experiment, polyps were relaxed in 2% urethane, fixed with 4% formaldehyde and treated with 150 U ml-1 TdT enzyme (Fermentas) in the presence of 5 μmol l−1 DIG-dUTP for 2 h at 37 °C. Detection of incorporated DIG was performed as in in situ hybridization experiments. To detect apoptotic cells in vivo, polyps were stained 10 min in 1 μg ml−1 acridine orange (Sigma) solution, relaxed in 2% urethane and monitored under Zeiss Axioscope microscope with Axiocam camera and fluorescein isothiocyanate filter set. Confocal laser-scanning microscopy was done using a TCS SP1 laser scanning confocal microscope (Leica, Wetzlar, Germany).

Fitness assays

To assess the growth rate we established P. robusta clonal lines of tumour-bearing (n=12) and normal polyps (n=12) by putting one founder polyp per well into a 24-well plate. The polyps were fed daily ad libitum. The number of clonal progeny propagated by budding within each clonal line was counted daily for a period of 4 weeks. To estimate fecundity, tumour-bearing (n=41) and normal (n=39) polyps were kept at 10 °C and deprived of food for 30 days. After this period we counted the number of tumour-bearing and normal polyps that produced eggs. These data were used to generate four-field contingency table and to calculate further odds ratio value for egg production by tumorous polyps compared with control polyps. The statistical significance of fecundity was analysed by Fisher’s exact test.

Transcriptome sequencing and assembly

For transcriptome sequencing, mRNA was isolated from a pool of normal, tumorous and oogenesis-induced (stage 1–7 (ref. 20)) H. oligactis polyps using Illustra QuickPrep Micro mRNA purification kit (GE Healthcare, Buckinghamshire, UK). A double-stranded complementary DNA library was constructed using SMARTer PCR cDNA Synthesis Kit (Clontech Laboratories, Mountain View, CA, USA). The cDNA library, representing the complete transcriptome of H. oligactis, was pyrosequenced on Roche FLX 454 sequencer as explained elsewhere16. After removal of adaptor sequences, 690,540 reads (~

224 Mb) were assembled into contigs using the Newbler 2.5.5 assembly pipeline. After filtering, this yielded 31,473 contigs (~

20 Mb) of 100–11,823 bp in size, with an average contig length of 662 bp and N50 of 852 bp.

Microarray hybridization and analysis

Custom Agilent Gene Expression Microarray was designed using the H. oligactis transcriptome (31,473 contigs, 4 × 44 k platform). For the microarray analysis of gene expression, total RNA was isolated from H. oligactis healthy control (n=6 replicates), tumour-bearing polyps (n=4) and oogenesis-induced polyps (pooled equal ratios of stages 1–7 (ref. 20) of oogenesis, n=3) by TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. After chloroform extraction, the aqueous phase was purified on the RNA-binding cartridge of PureLink RNA Mini Kit (Ambion, Life Technologies, Darmstadt, Germany) according to the protocol of the manufacturer. The RNA samples were additionally purified by glycogen–potassium acetate precipitation followed by washing with ice-cold 80% ethanol. About 400 ng of total RNA were labelled with Cy3 using the one-colour Quick-Amp Labeling Kit (Agilent Technologies, Waldbronn, Germany). Labelled cRNA samples (n=13 in total) were hybridized to custom Agilent Gene Expression Microarray 4 × 44 k slides for 17 h at 65 °C and treated according to the Agilent protocol. The microarrays were scanned using the Agilent High Resolution G2565CA Microarray Scanner System.

Raw microarray image files were processed and quality checked by Agilent Feature Extraction 10.7.3 software. The microarray and custom-design data were deposited at the NCBI Gene Expression Omnibus repository under the accession number GSE56287. Background-subtracted signal intensity values that contain correction for multiplicative surface trends (gProcessedSignal) generated by Feature Extraction Software were used for further data analysis. Using GeneSpring microarray data analysis software, we filtered probes that were flagged as non-uniform or as population outliers. We additionally filtered data set for expression levels of contigs that lack evidence to code for proteins. The potential coding regions were initially predicted by ESTscan36 and then double checked by BLASTp analysis against H. magnipapillata gene predictions28. This procedure reduced the expression sample to 22,719 contigs that were used in the subsequent analyses.

This data set was normalized by 75th percentile normalization procedure as recommended by Agilent. To reveal the global changes in gene expression patterns, we performed principal component analysis using NIPALS algorithm. To assess the putative functional properties of the contigs that most probably code for proteins, we performed sequence similarity searches against the NCBI nr database and the predicted human genes. Significance of differences in the pairwise comparisons between tumours and controls were conducted by Student’s t-test with false discovery rate correction. By setting a threshold of >3.0-fold change in relation to healthy control and oogenesis-induced females, we obtained a set of 417 significantly differentially regulated contigs (P≤0.05). This set was further reduced by keeping only contigs that had the most significant e-value in the situations where several contigs from the full data set (22,719 contigs) had the same H. magnipapillata gene as the best BLAST match. This procedure yielded 196 contigs that were significantly differentially expressed above the threshold of 3.0-fold change and have the best correspondence to the H. magnipapillata gene models. Finally, by manual screen of the literature and sequence databases, we were able to link 44 of these genes to mammalian homologues that are implicated in tumours.

Quantitative real-time PCR gene expression analysis

To validate the results of microarray hybridization analysis and to find most promising genes for expression analysis by in situ hybridization, we performed quantitative real-time PCR. We used the same total RNA samples that we isolated in microarray experiment: RNA from H. oligactis healthy controls (n=2), tumour-bearing polyps (n=2) and oogenesis-induced polyps (n=2). cDNA samples were produced using First Strand cDNA Synthesis Kit (Thermo Scientific, Schwerte, Germany). Real-time PCR was performed using GoTaq qPCR Master Mix (Promega, Madison, USA) and oligonucleotide primers specifically designed to recognize H. oligactis genes (Supplementary Table 1). In total, we assessed expression level of 27 genes, including house-keeping genes, oogenesis-related and tumour-specific genes. The data were collected by ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, USA) and analysed by conventional ddCt method, using expression level of EF1a (translation elongation factor 1 alpha) gene as equilibration reference. The experiments were repeated twice, resulting in two biological with two technical replicates each. These data were used further to calculate mean fold change factors of gene expression in tumour related to healthy asexual polyps (Tumour versus Control, Supplementary Data 2) or related to oogenesis-induced animals (Tumour versus Female, Supplementary Data 2).

To compare expression level of 27 genes in H. oligactis tumorous tissue with female gonad tissue at different oogenesis stages, we excised tumorous tissue (n=10), developing female gonads at early (stage 1–3; n=10), and late (stage 4–7; n=10) oogenesis and normal tissue (n=10) from 10 polyps of each type. These samples were further used for RNA extraction, cDNA synthesis and PCR amplification as described above. The experiments were repeated twice, resulting in two biological replicates with two technical replicates each. These data were used further to calculate mean fold change factors of gene expression in tumorous tissue, early and late female gonad related to healthy asexual tissue (Supplementary Fig. 6).