Authentication of M14 melanoma cell line proves misidentification of MDA-MB-435 breast cancer cell line

Abstract

A variety of analytical approaches have indicated that melanoma cell line UCLA-SO-M14 (M14) and breast carcinoma cell line MDA-MB-435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross-contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA-MB-435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA-MB-435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA-MB-435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA-MB-435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research.

Keywords: STR profiling; authentication; cross-contamination; human cell lines; misidentification.

Figure 1

M14 and MDA‐MB‐435 timeline and recent publications. (a) Timeline of events in the establishment and analysis of M14 and MDA‐MB‐435. Ref = citation from Reference list; JWCI = John Wayne Cancer Institute. (b) Usage of MDA‐MB‐435 in journal publications between January 2013 and December 2016 (looking at date of hard copy publication). A search was conducted to look for “MDA‐MB‐435” in the title or abstract, and available text was examined to classify usage as “breast” or “melanoma.” If authors described MDA‐MB‐435 more broadly as a cancer cell line, or lack of access to the full text meant that usage could not be determined, usage was classified as “neither/indeterminant.”

Figure 2

Characterization of M14 using Immunostaining and ABO Analysis. (a–b) Representative images of M14 cells in culture. Scale bars, 100 µm. (c–d). Immunostaining of M14 cells using a pan‐melanoma antibody cocktail. Antibodies are directed against melanosome (HMB45), MART‐1/Melan A (A103) and tyrosinase (T311). Cells displayed abundant cytoplasm and were multinucleated; some apoptotic cells were noted, as indicated by the arrows. Scale bar in C, 10 µm; scale bar in D, 20 µm. (e) ABO sequence demonstrating blood type O. Upper panel, sequence reported previously for A or B alleles and sequence reported for the O allele.21 Lower panel, forward and reverse sequence from M14 sample (passage 16, derived from passage 15 from December 2, 1975). Identical sequence results were obtained from ML14 and MDA‐MB‐435S samples (data not shown).

Figure 3

FISH Analysis of M14 using X‐ and Y‐specific probes. Metaphase spreads of M14 cell line, hybridized with the CEP X SpectrumOrange/Yq12 SpectrumGreen FISH probe set (Abbott Molecular). Red arrows indicate Xp11‐q11 and green arrows indicate Yq12 hybridization signals. Images (a–b) show two similar copies of der(22)t(Y;22), while (c–d) exhibit two different derivative chromosomes bearing Yq12; namely, the above der(22) and another unknown variant.

Figure 4

FISH Analysis and Karyotype of M14 and ML14. Metaphase spreads and GTL‐banded karyotype of M14 (a–c) and ML14 (d–f) cell lines, and the same cells hybridized with the CEP X SpectrumOrange/Yq12 SpectrumGreen FISH probe set (b, e). Red arrows indicate chromosomes carrying Xp11‐q11 sequences and green arrows indicate chromosomes carrying Yq12 sequences.