**4. Contamination**

12 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

in cell lines (Lacroix and Laclercq, 2004). This suggests that cell lines are an appropriate model of primary disease and, depending on the research focus, cell lines will faithfully

Since cell lines generally remain faithful in terms of the molecular and genetic profiles of the primary tumor from which they are derived, it is critical to consider the correct model system. While ER/PR status of primary tumors leans predominantly toward ER+ expression (55-60%), most breast cancer cell lines have been derived from ER - tumors or pleural effusions (McGuire et al., 1978)(Table 2). Therefore it is of utmost importance to select the proper model to answer the experimental question. A detailed analysis of the applicability of cell lines to accurately model primary breast tumors revealed that overall breast cancer cell lines as a whole do model primary tumors, however on an individual basis, one specific cell line does not accurately mirror a primary breast tumor, even with the same gene expression profile. Since variability in cell lines exist, it is generally thought that to more accurately predict outcomes in primary tissue, a panel of breast cancer cell lines rather than just 1 or 2 individual lines should be tested. Using panels more accurately reflects primary breast tumors and will help translate findings from *in vitro* studies to *in vivo* therapeutic

Microarray analysis clearly defined primary breast tumors and breast cancer cell lines at the genetic level. Perou and others have conducted detailed studies using microarray platforms and determined a molecular signature of gene expression changes found in primary breast cancer tumors (Alizadeh et al., 2001; Perou et al., 1999b; Perou et al., 2000b; Ross et al., 2000; Sorlie et al., 2001). These signatures are used to understand the molecular basis of breast cancer and to define different subtypes of cancer that occur naturally in humans. It was also developed as a diagnostic tool to detect breast cancer tumors earlier and to facilitate proper treatment based on a gene signature. Based on these studies, 5 molecular signatures and types of primary breast tumors have been identified. These are luminal A, luminal B, basallike, HER2+, and normal-like profiles (Perou et al., 1999a; Perou et al., 2000a; Ross et al., 2000; Sorlie et al., 2001). Prior to establishment of these molecular signatures, diagnosis was determined by receptor expression status, i.e. ER/PR/HER2, and treatment regimes assigned accordingly. Using this molecular approach, luminal A and luminal B tend to also be ER + expressing tumors, basal-like encompasses ER - tumors, HER2+ incorporate those HER2+ expressing tumors, and normal-like have similar expression patterns to noncancerous cells (Perou et al., 1999a; Perou et al., 2000a; Ross et al., 2000; Sorlie et al., 2001). Such molecular characterization will lead to providing more personalized therapy to patients. Efficacy of drugs in different subtypes will be easily determined and accurately assigned to patients expressing a similar molecular profile. While such personalized medicine may be still in the future, some current breast cancer treatment options that exist today are based on the molecular profile of the tumor. For example, tumors expressing the estrogen receptor are treated with selective estrogen receptor modulator (SERM) or other similar anti-estrogen compound whereas tumors lacking ER do not receive the same therapy. Similarly, HER2+ tumors are susceptible to trastuzumab because of HER2 expression. In the future as molecular characterization improves and new

chemotherapeutics are developed, more personalized options will be available.

Do cell lines reflect the molecular signature of primary tumors? In a direct comparison of the molecular profiles from cell lines and primary tumors, Kao et. al. found that instead of the 5 breast cancer subtypes identified in primary breast tumors, cell lines can be divided into three main groups, luminal, basal A, or basal B phenotypes (Kao et al., 2009). Luminal cells

reflect the processes of primary tissues.

options (Dairkee et al., 2004).

Overt contamination of cell lines, such as bacterial, fungal or yeast infections, is readily detectable merely by altered appearance of the culture and can be rectified without impacting the quality or reproducibility of the data. Less overt contamination, such as mycoplasma and cell line cross-contamination, can occur undetected and can seriously jeopardize experimental findings. While it is well recognized that periodic testing for mycoplasma is a necessary requirement when using cell lines, cross-contamination with other cell lines is less recognized as a problem and therefore and cell authentication practices are not routine.

Cell line cross-contamination is most evident in the case of MDA-MB-435 cells. When Ross et. al. published the molecular profiles of breast cancer cell lines in 2000, the MDA-MB-435 cell line consistently fell outside the range of profiles of the other breast cancer cell lines and clustered with melanoma cell lines (Ross et al., 2000). This sparked great debate about the authenticity of the this line. Derived in 1976 from the pleural effusion of a 31 year old patient with metastatic adenocarcinoma of the breast, initial debate suggested that this was still a breast cancer cell line, but had been derived from a patient who may have also had undiagnosed melanoma (Cailleau et al., 1978). Data indicating that MDA-MB-435 cells expressed a mixture of both melanoma and epithelial markers fueled this debate, however the overwhelming belief was the these were indeed breast cancer cells (Chambers, 2009; Sellappan et al., 2004)(Figures 2 and 3). Indeed, early characterization of the cell line indicated that they were highly metastatic and secrete milk proteins, findings consistent with those of breast cancer cells (Howlett et al., 1994; Price, 1996; Price et al., 1990; Price and Zhang, 1990; Sellappan et al., 2004; Suzuki et al., 2006; Welch, 1997). Confusingly, MDA-MB-435 cells also expressed the melanocyte markers tyrosinase, melan A and S100 (Ellison et al.,

Breast Cancer Cell Line Development and Authentication 15

contamination, however this issue has rarely been adequately addressed (Alston-Roberts et al., 2010; Bartallon et al., 2010; Buehring et al., 2004; Capes-Davis et al., 2010; Freshney, 2008; MacLeod et al., 2008; MacLeod et al., 1999; SDO et al., 2010). Recent efforts have again been made to increase awareness of this problem and many calls for action have been published (Buehring et al., 2004; Capes-Davis et al., 2010; Freshney, 2008, 2010b; Lichter et al., 2010; MacLeod et al., 2008; MacLeod et al., 1999; SDO et al., 2010). A group of concerned scientists gathered and created the ATCC Standard Development Organization (ATCC SDO) to develop standards for cell authentication and with

Eliminating contamination has an easy solution. Cell line authentication using a standardized technique, Short Tandem Repeat Analysis (STR), can provide an unique DNA fingerprint of the cell line (Azari et al., 2007; Bartallon et al., 2010; Masters et al., 2001; Nims et al., 2010; Parson et al., 2005). STR is inexpensive, standardized, and provides proven methodology to produce cell line identities that is reproducible between labs. An aliquot of DNA can be analyzed and compared with known STR profiles to authenticate the cell line. STR profiles for the most commonly used cell lines are freely available and STR services are available at many universities or companies. According to the standards developed by the ATCC-SDO, cells in active use should be authenticated by STR every 2 months (SDO et al., 2010). The ATCC-SDO also recommends that such documentation of authenticity be provided with grant applications and with manuscript submission. Many funding agencies and journals agree with this idea and suggest that scientists provide such documentation prior to acceptance of a manuscript, however at

Use of breast cancer cell lines as models of breast disease will not diminish in the near future. These cell lines are an excellent resource to test novel hypotheses and to gain greater understanding about how cells work and how breast cancer can be treated. On the whole, the established cell lines are a good model for disease, however additional cell lines should be created. The addition on new lines, especially those derived from various forms of breast cancer will only strengthen the data gleaned from them. Likewise, cell authentication should become a routine part of experimental procedures. By periodically ensuring the cell lines being tested are truly the correct lines will eliminate the generation and publication of false data. Authentication will save money and potentially careers if

Ali, S., and Coombes, R.C. (2000). Estrogen receptor alpha in human breast cancer: occurence and signficance. J Mammary Gland Biol Neoplasia *5*, 271-281. Alizadeh, A., Ross, D., Perou, C.M., and van de Rijn, M. (2001). Towards a novel

classification of human malignancies based on gene expression patterns. J Pathol

maintaining databases of STR profiles.

this time, this is merely a recommendation.

**5. Future directions** 

done of a routine basis.

*195*, 41-52.

**6. References**

2002; Sellappan et al., 2004). Because of such conflicting results, these data just propagated the debate instead of satisfactorily squelching it as intended. MDA-MB-435 cells were still used and published as a breast cancer cell line (Figure 3).

Finally in 2007, DNA fingerprinting, or short tandem repeat (STR) analysis, in conjunction with SNP analysis, cytogenetic analysis, and comparative genomic hybridization using the earliest stocks of MDA-MB-435 cells revealed that these cells were identical to the M14 human melanoma cells and were melanoma rather than breast cancer cells (Garraway et al., 2005)(Rae et al., 2007). Rae et. al., who conducted the analysis, concluded that at some point early in passage, MDA-MB-435 cells were contaminated with M14 melanoma cells which took over the colony, leading to the establishment of a M14 melanoma cell line rather than a breast cancer line (Rae et al., 2007). This change was never detected. Stocks were unknowingly mislabeled, marked as MDA-MB-435 cells and distributed. Still, after the molecular characterization was published, debate as to whether MDA-MB-435 were really M14 melanoma cells or if M14 were really MDA-MB-435 breast cell still existed (Chambers, 2009). Ultimately, it was determined that MDA-MB-435 cells were really M14, based on the original 1974 publication that initially characterized the morphology, growth and tumorigenicity of MDA-MB-435 cells. In the original paper, MDA-MB-435 cells were reportedly non-tumorigenic in nude mice. After the initial creation in 1974, the MDA-MB-435 cells were not extensively used for testing until the 1990s when Price et. al. used these cells. At this time, MDA-MB-435 cells were characterized as a tumorigenic cell line (Cailleau et al., 1978; Price, 1996; Price et al., 1990; Price and Zhang, 1990).

While impossible to reconstruct that actually happened, this indirect evidence suggests that the MDA-MB-435 cells were contaminated with M14 melanoma cells and the original breast cancer cells died off. Subsequent frozen stocks were of the contaminating M14 cell lines, although they were labeled as MDA-MB-435 cells. No one was aware of this misidentification. Therefore, M14 cells were masquerading as MDA-MB-435 cells and used as a model of breast cancer until 2007. A total of 1803 PubMed indexed articles using MDA-MB-435 cells were published over that period (Figure 3). Since 2007, however, the number of publications using MDA-MB-435 cells has diminished, indicating that it is generally accepted that these cells are clearly not breast cancer cells and therefore should not be used as such.

#### **4.1 Authentication**

Cell line cross-contamination is hardly a new problem in tissue culture studies, although it still remains largely ignored. When HeLa were the only human cell line and few scientists studied them, cross-contamination was not a concern(Buehring et al., 2004; Skloot, 2010). Now, it is estimated that 20 - 30% of all cell lines are inadvertently contaminated (Alston-Roberts et al., 2010; Buehring et al., 2004; Gartler, 1968; Rojas and Steinsapir, 1983). Gartler et. al, was the first to highlight the problem in 1967 at the Second Decennial Review Conference on Cell, Tissue and Organ culture (Gartler, 1968). He was the first to demonstrate that many cultures from many labs were contaminated with other cell lines, primarily by HeLa cells. This meant that a significant amount of research was incorrectly interpreted because it was conducted in a different cell line and therefore the data were false. His findings were largely ignored. Over the years, others, including MacLeod, Freshney, Nardone, Alston-R, Buehring and Capes-Davis, have also documented contamination with HeLa and other cell lines, including cross-species

2002; Sellappan et al., 2004). Because of such conflicting results, these data just propagated the debate instead of satisfactorily squelching it as intended. MDA-MB-435 cells were still

Finally in 2007, DNA fingerprinting, or short tandem repeat (STR) analysis, in conjunction with SNP analysis, cytogenetic analysis, and comparative genomic hybridization using the earliest stocks of MDA-MB-435 cells revealed that these cells were identical to the M14 human melanoma cells and were melanoma rather than breast cancer cells (Garraway et al., 2005)(Rae et al., 2007). Rae et. al., who conducted the analysis, concluded that at some point early in passage, MDA-MB-435 cells were contaminated with M14 melanoma cells which took over the colony, leading to the establishment of a M14 melanoma cell line rather than a breast cancer line (Rae et al., 2007). This change was never detected. Stocks were unknowingly mislabeled, marked as MDA-MB-435 cells and distributed. Still, after the molecular characterization was published, debate as to whether MDA-MB-435 were really M14 melanoma cells or if M14 were really MDA-MB-435 breast cell still existed (Chambers, 2009). Ultimately, it was determined that MDA-MB-435 cells were really M14, based on the original 1974 publication that initially characterized the morphology, growth and tumorigenicity of MDA-MB-435 cells. In the original paper, MDA-MB-435 cells were reportedly non-tumorigenic in nude mice. After the initial creation in 1974, the MDA-MB-435 cells were not extensively used for testing until the 1990s when Price et. al. used these cells. At this time, MDA-MB-435 cells were characterized as a tumorigenic cell line (Cailleau

While impossible to reconstruct that actually happened, this indirect evidence suggests that the MDA-MB-435 cells were contaminated with M14 melanoma cells and the original breast cancer cells died off. Subsequent frozen stocks were of the contaminating M14 cell lines, although they were labeled as MDA-MB-435 cells. No one was aware of this misidentification. Therefore, M14 cells were masquerading as MDA-MB-435 cells and used as a model of breast cancer until 2007. A total of 1803 PubMed indexed articles using MDA-MB-435 cells were published over that period (Figure 3). Since 2007, however, the number of publications using MDA-MB-435 cells has diminished, indicating that it is generally accepted that these cells are clearly not breast cancer cells and therefore should not be used

Cell line cross-contamination is hardly a new problem in tissue culture studies, although it still remains largely ignored. When HeLa were the only human cell line and few scientists studied them, cross-contamination was not a concern(Buehring et al., 2004; Skloot, 2010). Now, it is estimated that 20 - 30% of all cell lines are inadvertently contaminated (Alston-Roberts et al., 2010; Buehring et al., 2004; Gartler, 1968; Rojas and Steinsapir, 1983). Gartler et. al, was the first to highlight the problem in 1967 at the Second Decennial Review Conference on Cell, Tissue and Organ culture (Gartler, 1968). He was the first to demonstrate that many cultures from many labs were contaminated with other cell lines, primarily by HeLa cells. This meant that a significant amount of research was incorrectly interpreted because it was conducted in a different cell line and therefore the data were false. His findings were largely ignored. Over the years, others, including MacLeod, Freshney, Nardone, Alston-R, Buehring and Capes-Davis, have also documented contamination with HeLa and other cell lines, including cross-species

used and published as a breast cancer cell line (Figure 3).

et al., 1978; Price, 1996; Price et al., 1990; Price and Zhang, 1990).

as such.

**4.1 Authentication** 

contamination, however this issue has rarely been adequately addressed (Alston-Roberts et al., 2010; Bartallon et al., 2010; Buehring et al., 2004; Capes-Davis et al., 2010; Freshney, 2008; MacLeod et al., 2008; MacLeod et al., 1999; SDO et al., 2010). Recent efforts have again been made to increase awareness of this problem and many calls for action have been published (Buehring et al., 2004; Capes-Davis et al., 2010; Freshney, 2008, 2010b; Lichter et al., 2010; MacLeod et al., 2008; MacLeod et al., 1999; SDO et al., 2010). A group of concerned scientists gathered and created the ATCC Standard Development Organization (ATCC SDO) to develop standards for cell authentication and with maintaining databases of STR profiles.

Eliminating contamination has an easy solution. Cell line authentication using a standardized technique, Short Tandem Repeat Analysis (STR), can provide an unique DNA fingerprint of the cell line (Azari et al., 2007; Bartallon et al., 2010; Masters et al., 2001; Nims et al., 2010; Parson et al., 2005). STR is inexpensive, standardized, and provides proven methodology to produce cell line identities that is reproducible between labs. An aliquot of DNA can be analyzed and compared with known STR profiles to authenticate the cell line. STR profiles for the most commonly used cell lines are freely available and STR services are available at many universities or companies. According to the standards developed by the ATCC-SDO, cells in active use should be authenticated by STR every 2 months (SDO et al., 2010). The ATCC-SDO also recommends that such documentation of authenticity be provided with grant applications and with manuscript submission. Many funding agencies and journals agree with this idea and suggest that scientists provide such documentation prior to acceptance of a manuscript, however at this time, this is merely a recommendation.
