Reports of XMLV strains being present in human xenograft cultures appeared in the 1970s.10,12,33
While several reports have documented this phenomenon, most reports are sporadic case reports without systematic analysis of true frequency. Two reports examined multiple xenograft cultures12,33
and found frequencies of 7 and 67% respectively. However, because of the relatively crude detection methods available at that time and failure to check for mouse cell contamination, the true frequencies cannot be determined from these reports. Because of our initial failure to culture SCLC tumors directly from tumor samples, we developed long-term cultures from xenografted tumors.34
In the mid 1980s we detected (by electron microscopy) large numbers of retroviral particles released by SCLC cell line NCI-N417. We destroyed stocks of this cell line and persuaded the American Type Culture Collection to stop distribution. However, prior to this finding, the cell line had been distributed extensively to the scientific community and we obtained a culture from Gerold Bepler, Moffitt Cancer Center, Tampa, FL. While we did not report on the viral findings, others have done so14,15
and we refer to this XMLV strain as N417 although it has also been referred to as VB3.2.21
Recent reports of squirrel monkey retroviruses (SMRV) infecting human cultures (by unknown routes or sources) and their possible large scale horizontal spread have also been published,35,36
and several human cell lines for human immunodeficiency virus research were found to release infectious XMLV.22,37
However, there are virtually no reports of contamination of xenograft cultures published during the past 15 y and most scientists appear unaware of the contamination problem (authors' observations). The purpose of our study was to determine the frequency of xenograft culture contamination by xenotropic viruses and their horizontal spread in multiple laboratories. By sequencing the viruses so isolated we could determine whether a single or multiple strains of XMLV were responsible for contamination and horizontal spread.
For our initial study we obtained 26 xenograft cultures from seven independent laboratories. Only two of the laboratory chiefs that participated in this study were previously aware of the XMLV contamination issue. We utilized three primer sets that detected virtually all XMLV stains as well as most MLV strains, covering the three major structural and functional regions of the mouse retroviral genome. Our multiple qPCR approaches had advantage to detect all possible MLV sequences (increased sensitivity) with one or two additional probe confirmation (increased specificity) as compared with a single qPCR assay. Nine of the samples were positive by either two or all three of the probes used. However, three of the nine positive samples contained varying amounts of mouse DNA, presumably as a result of survival of mouse stromal cells from the mouse xenograft. Apparently the mouse stromal cells may persist for lengthy periods in culture, occasionally in excess of one year. These three cultures were removed from further study as the mouse genome contains multiple endogenous MLV provirus sequences which make interpretation difficult. Six of the remaining 23 xenograft cultures (26%) were positive for one (or in one case, two) strains of XMLV or related viruses. These six cultures came from six independent labs indicating the widespread nature of the contamination. Of interest, our viral sequencing homology analysis indicated that multiple strains of XMLV were present in the six positive lines, demonstrating that there are multiple strains of XMLV or MLV-related viruses capable of infecting human cells individually or simultaneously after xenografting. The LAPC-4 cell line, widely used as a model for androgen independent prostate cancer, with over 120 citations in PubMed, contained two strains. Early and late passage cultures from the originator (Dr. Charles Sawyers) as well as a culture distributed to the Hsieh lab contained the same strain mixture.
Supernatant fluids were available from five of the six viral positive xenograft cell lines. Four of these released large numbers of potentially infectious viral particles, indicating possibilities of horizontal spread to other human cultures as well as posing a biohazard of unknown potential to laboratory personnel. We presume that in one of these five cell lines the XMLV virus existed in a latent form. Because of the possibility of spread to non-xenografted human cultures, we examined cultures maintained in the same culture facilities. A total of 78 cultures were obtained from five of the laboratories. Thirteen of these cultures (17%) from four of the laboratories were positive for XMLV. The viruses identified as being responsible for horizontal spread were identical to those identified in the positive xenograft cultures in the respective laboratories. By contrast, 50 cell lines from the Gazdar lab maintained in a facility free of xenograft cultures tested negative for virus. These differences were significant, indicating the potential for horizontal viral spread to other cultures maintained in the same laboratory facility as xenograft cultures. In one case XMRV virus infection to a non-xenograft colorectal carcinoma cell line RKO38
was demonstrated from an XMRV containing prostate xenograft derived cell line 22Rv1 even though the two cell lines had been maintained in the same culture facility for only a few days.
Our results indicated the frequent presence of XMLV viruses in cultures initiated from human xenografted tumors, with integration of the viral genomes and production of abundant virions. We also found evidence of widespread contamination of non-xenografted cultures maintained in the same tissue culture facilities with xenografted cultures. However, to rigorously prove that the virions released by the xenografted cultures were infectious for human cells, we collected supernatant fluids from three XMLV-positive xenografted cultures including LAPC-4 and used them to infect five other human cell lines. All three viruses readily infected two SCLC lines, one NSCLC line and one virus infected a non-tumorigenic immortalized bronchial epithelial cell culture. The fifth cell line, NSCLC line NCI-H460, appeared to undergo an abortive infection, as monitored by reverse transcriptase activity. However genomic analyses demonstrated the presence of relatively low amounts of integrated forms of two of three infected viruses, indicating latent infection. Thus human cells demonstrate heterogeneity for XMLV viral sensitivity and the XMLV viruses demonstrate variable infective potential.
Several reports, have described the finding, usually of partial XMRV or XMLV related sequences at low abundance, in human tumors and tissues.19,39,40
These methods often utilize nested PCR techniques for detection, and multiple other labs have failed to confirm the findings. Considerable recent evidence indicates contamination as the probable cause of these sequences in human cells.41,42
Because of sequence similarities, the presence of XMRV virus in human tumors and cells has been attributed to contamination, especially from the frequently used 22Rv1 prostate cancer cell line41
but it has not been confirmed in any cell lines, to our knowledge.23
Hue et al. screened human tumor 411 cell lines from the COSMIC collection and found XMLV sequences positive in nine cell lines (2.2%), in which five are closely related to DG-75 strains but none of these cell lines are confirmed to be infected with XMRV and the verification of mouse DNA contamination was not addressed.23
Our findings demonstrated that the XMLV virus in colorectal cell line RKO from the Maitra Lab is an XMRV isolate contaminated from the virus containing xenograft derived 22Rv1 prostate cancer culture. The window during which both cell lines were maintained together in the same culture facility was only a few days, indicating that horizontal spread may occur rapidly. This XMRV-positive RKO culture probably represents the first report of horizontal spread of the XMRV virus.
Our results indicate that human tumor cells frequently become infected with MLV virus after xenografting and subsequent culture. We have observed that mouse stromal cells may persist in culture for lengthy periods. Mouse stromal cells, while they contain abundant provirus forms of MLV, including ecotropic, polytropic and xenotropic strains, seldom spontaneously release large amounts of infectious virus (authors' unpublished findings). Virus infection of xenografted cells may require activation of XMLV virus by chemical or immunological induction in mouse and by prolonged mouse and human cell contact. Viral transfer may occur in the mouse host or during subsequent xenograft culture. Our findings of infectivity of XMLV-positive supernatant fluids demonstrated that XMLV can readily infect other human cultures without presence of mouse cells or other aiding factors, indicating that these viruses are highly infectious.
In conclusion, our studies demonstrated that several MLV strains were present in over one fourth of xenograft cell lines. Infected cell lines were identified in most laboratories working with or establishing xenograft cultures, indicating that such contamination was widespread. Infected cultures usually release large numbers of infectious virions, and intra-laboratory spread of MLV virus to other cell lines maintained in the same facilities may occur, confirming the highly infectious nature of MLV virus. Retroviruses have been associated with multiple diseases including solid and hematologic malignancies, AIDS as well as with non-malignant diseases. The high susceptibility of human cells to infection with XMLV, the high levels of reverse transcriptase activity present in culture supernatant fluids and the demonstrated infectivity of the shed virions suggest that such viruses may present potential biohazards to laboratory personnel involved in cell culture facilities or to those handling human xenografts. In addition, the effects of the integrated provirus or the released virions on the biology of infected tumor cells are unknown. Provirus integration into the genome is not random, and occurs preferentially at transcription start sites, CpG islands, DNase-hypersensitive sites and gene-dense regions, suggesting that provirus integration may influence transcription in the host cell.43
Thus laboratories handling or culturing human xenografts should monitor for the presence of MLV, and should consider monitoring personnel for viral antigens or antibodies to them. Laboratories working with xenograft cultures should have full knowledge and understanding of the potential biological and biohazardous risks and should not distribute or publish their findings without full disclosure of the virus status of their xenograft-derived materials.