First Cytogenomic Characterization of the Murine Testicular Tumor Cell Line I-10
Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Am Klinikum 1, Jena, Germany
Croatian Institute for Brain Research, School of Medicine University of Zagreb, Salata 12, 10000 Zagreb, Croatia
Academic Editor: Andrés Moya
Special Issue: Applications of Fluorescence in Situ Hybridization II.
Received: March 25, 2022 | Accepted: June 13, 2022 | Published: June 27, 2022
OBM Genetics 2022, Volume 6, Issue 2, doi:10.21926/obm.genet.2202159
Recommended citation: Azawi S, Barf LM, Liehr T, Kankel S, Rincic M. First Cytogenomic Characterization of the Murine Testicular Tumor Cell Line I-10. OBM Genetics 2022; 6(2): 159; doi:10.21926/obm.genet.2202159.
© 2022 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.
Leydig cell tumors (LCTs) in males are a kind of testicular mass that might lead to hormonal changes in up to a quarter of the cases [1,2]. Although LCTs are rare, they are still the most common non-germ cell testicular tumors that cause adult feminization and premature childhood masculinization [1,2]; they might cause gynecomastia or infertility. All childhood LCTs and ~90% of adult cases are benign. However, 10% of adult LCTs become malignant and often metastasize before diagnosis. Orchidectomy is the primary treatment option, but in most cases, hormonal changes remain unaffected. As identifying a malignant subset of LCTs is difficult at the early stages, cellular models are necessary for conducting more directed fundamental research to identify genetic markers and facilitate the development of treatment options [1,2].
Interestingly, there has been only one murine LCT model available since 1967, which is the lutropin receptor-deficient cell line I-10 . It was established from a testicular interstitial tumor, i.e., from the LCT-transplant H10119, which was introduced into a monolayer culture and injected into a BALB/cJ mouse. Using the single-cell plating approach, the I-10 cell line was established from the third culture passage . Although available for around 55 years , I-10 has been used in only 45 studies. This might be because I-10 has not been characterized genetically, either at the chromosome level or at the DNA level.
To provide information regarding the genetic characteristics of I-10, we performed the first comprehensive cytogenomic characterization of cell line I-10 by performing murine multicolor banding (mcb), molecular karyotyping, and in silico translation of the obtained results into the human genome (as previously described ). We found that I-10 has only ~36% similarity with the imbalance patterns of LCTs but has 68% concordance with the imbalance patterns of testicular germ cell tumors.
2. Materials and Methods
2.1 Cell Line Work up and Molecular (Cyto) Genetics
After purchasing and directly cultivating the adherent murine I-10 cells following the manufacturer’s instructions (American Type Culture Collection, ATCCR CCL83™; Wesel Germany), the cells were prepared in parallel cytogenetically to obtain the chromosomes and genetically to extract the whole genomic DNA . The cell line was not used in any other studies by the authors of this study, and ATCC confirmed the identity of the cell line.
Fluorescence in situ hybridization (FISH) was performed as described in previous studies. For multicolor-FISH (mFISH), whole chromosome paints (“SkyPaintTM DNA Kit M-10 for Mouse Chromosomes”, Applied Spectral Imaging, Edingen-Neckarhausen, Germany) were used, while for FISH-banding, murine chromosome-specific multicolor banding (mcb) probe mixes  were used. At least 30 metaphases were analyzed for each set of probes (Zeiss Axioplan microscopy) equipped with the ISIS software (MetaSystems, Altlussheim, Germany). Also, 50 metaphases were analyzed for the presence of X and Y-chromosomes using the X and Y-specific BAC probes RP23–29K3 in XA2/RP23–71G11 in A6/RP23–257N12 in XC1~2 (all labeled in Spectrum-Orange) and RP24–95K23 in YA2/RP24–14O08 in YD/RP24–209O20 in YC2 (all labeled in Spectrum-Green). Chromosome microarray studies (CMA) were performed using SurePrint G3 Mouse CGH Microarray, 4 × 180K (Agilent Technologies, Waldbronn, Germany) .
Imbalances and breakpoints of I-10, based on the mcb and CMA data, were aligned to the human homologous regions using Ensembl and the UCSC Genome Browser, as previously described . The data were compared to genetic changes known from human LCTs [6,7], seminomas, non-seminomas, and testicular germ cell tumors [8,9,10,11,12].
2.2 Ethics Statement
According to the ethics committee (medical faculty) and the Animal Experimentation Commission of the Friedrich Schiller University, there are no ethical agreements necessary for studies involving murine tumor cell lines, such as I-10.
This cell line showed a slightly hyperdiploid karyotype with many single-cell aberrations, as gains and losses of (derivative) chromosomes. The karyotype is shown below and in Figure 1. 43,der(X)t(X;16)(XA1->XE1::XE3->Xqter::16B4),-Y,idic(1)(A1),der(1)t(1;16)(C5;B4),t(3;9)(H4;F2),+del(4)(D2),der(5)t(5;17)(G2;C),+del(6)(B2),+del(6)(B2),del(7)(A3),+dic(7;17)(A;A),del(9)(A5B),+dic(10;13)(10qter->10C1::10A3->10A1::13A1->13qter),neo(12)(C1->qter),+der(12)t(12;15)(12pter->C1::15B->15D3::15E3->15qter),der(13)t(13;14)(C1;A3),-14,del(18)(A1B1).
Figure 1 Pseudo-color banding depiction of 20 chromosome-specific murine multicolor banding experiments performed on the I-10 cell line. The del(9)(A5B) is highlighted by the letters ‘del’ in gray. The derivative chromosomes, consisting of different chromosomes, are highlighted by frames and displayed twice.
Aberration-induced chromosomal imbalances were relatively rare (13) relative to 16 derivative chromosomes. The main aberrations were unbalanced translocations; one such aberration led to the formation of unbalanced translocations of chromosomes 12 and 15 and a neocentric derivative of chromosome 12. Also, a more simple dicentric as dic(7;17)(A;A), and a more complex dicentric as dic(10;13)(10qter->10C1::10A3->10A1::13A1->13qter) were found to be stable aberrations in this cell line.
The results of CMA for I-10 are summarized in Figure 2A. The imbalances detected in murine cell line I-10 (Figure 2A) were translated into the human genome (Figure 2B and Table 1); this enabled us to compare I-10 imbalances to such in human LCTs; we identified a ~36% overlap of the detected alterations (Table 2). Additionally, 63% and 53% concordance of imbalances were detected with testicular germ cell tumors and human spermatocytic seminomas and non-seminomas (Table 2). No Y-chromosome was detected in 50 correspondingly analyzed metaphases using X and Y-specific BAC probes. However, one (derivative) X-chromosome was detectable in all cells (results not shown).
Figure 2 The results of the chromosome microarray (CMA) of the I-10 cell line are depicted based on a diploid basic karyotype. Gains are shown as green bars, losses are shown in red, and breaks are shown with arrows. (A) Imbalances observed in the cell line were depicted along a murine chromosome set. (B) The results were translated and projected along a human chromosome set.
Table 2 The copy number changes associated with molecular subtypes of human LCTs and seminomas, according to previous studies [6,7,8,9,10,11,12], compared to the copy number variants (CNVs) in cell line I-10. Concordances with human CNVs are highlighted in bold.
The first cytogenomic characterization of the tumor cell line I-10 revealed a slightly hyperdiploid karyotype of 43 chromosomes, with relatively few imbalances and breakpoints for a cell line that is in cell culture for around 55 years. Interestingly, I-10 is a tumor cell line with dicentric chromosomes, which are normally considered to be unstable [5,13,14,15,16,17,18,19,20,21,22,23,24,25]. It is one of the few cell lines with a stable neocentric chromosome [20,21]. Murine multicolor banding (mcb), along with CMA, enabled a detailed characterization of numerical and structural changes in the I-10 cell line. This study highlighted the importance of understanding the ploidy grade, the individual chromosome numbers, and the rearrangements involved before using a cell line in further, more sophisticated experiments. Data on these parameters might allow well-informed studies to be performed. According to this study, I-10 is not a suitable model to be used for knockout studies of genes located in regions that are present in three copies in this cell line; this refers to murine chromosome 4A-D2, 6A-B2, 7A1-A3, 10A1-A3, and 10C1-qter.
This male cell line of testicular origin lost its Y-chromosome in all cells. The loss of Y-chromosome has been previously reported in other murine cell lines but never in a testicular-derived murine cell line. However, Y-chromosome instability has been found in rat and human testicular tumors [26,27,28], while the loss of the Y-chromosome was reported in the human testicular cell lines JKT-1 and TCam-2 [29,30], as well. The reason for this tumor-associated sex-chromosome loss remains undetermined. Tumor-relevant genes might not be present on the Y-chromosome.
Here, a comparison of translated I-10-specific imbalances was performed, similar to that performed for other murine cell lines [5,13,14,15,16,17,18,19,20,21,22,23,24,25]. Sequencing analyses might also be fruitful in the future, as subtype-specific differences in somatic mutations were found in testicular germ cell tumors [31,32]. Clear differences in the mutation patterns between seminomas and non-seminomas were found [31,32]. However, chromosomal imbalances in both subtypes were similar [8,9,10,11]. Thus, only considering copy number alterations summrized in this study (Table 2), seminomas and non-seminomas could be regarded as one group in common.
Regarding the gains and losses present in I-10, the highest concordance of 63% was for human testicular germ cell tumors, while the lowest concordance of ~36% was for human LCTs. No overrepresentation of sequences homologous to the short arm of chromosome 12 could be found in I-10, which might be related to the invasive growth of human testicular seminomas and non-seminomas . This indicated that I-10 might be used as a model for early-onset LCT. However, it might also be used as a testicular germ cell tumor model.
The I-10 cell line is more suited as a model for human testicular germ cell tumors rather than for LCTs in particular. However, I-10 should not be used for studying metastasizing LCTs or testicular tumors. Moreover, studies on gene expression and approaches to introduce or remove gene copies need to consider the specific copy number alterations in this diploid tumor cell line.
The technical support from Dr Nadezda Kosyakova (Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany) is gratefully acknowledged.
TL conceived the study and obtained funding. SA, LMB and SK performed the FISH analysis. MR performed the CMA study and pre-evaluation. SA performed the overall data interpretation. TL and SA checked and approved the authenticity of the raw data and drafted and edited the manuscript. All authors read and agreed to the final draft of the manuscript.
The present study was supported by the Wilhelm Sander Stiftung (grant no. 2013.032.1).
The authors have declared that no competing interests exist.
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