Establishment and exploitation of hyperdiploid and non-hyperdiploid human myeloma cell lines

Abstract

The establishment of clinically relevant human myeloma cell lines is central for our understanding of myeloma pathogenesis and development of novel therapies for the disease. Unfortunately, most available lines were generated from extramedullary sites, harbored multiple genetic abnormalities and categorized as non-hyperdiploid. In contrast, hyperdiploid myeloma cell lines, which represent more than 50% of patients, are rare. We established procedures for establishment of stroma-dependent myeloma lines by passaging primary myeloma cells, in severe combined immunodeficient-human (SCID-hu) or SCID-rab mice followed by maintenance in co-culture with stromal cells. We described the establishment and characterization of two hyperdiploid (LD and CF) and two non-hyperdiploid (JB and BN) cell lines. Using our animal models, we also established bortezomib-sensitive and -resistant BN lines. These cell lines were cellularly, phenotypically and molecularly characterized using flow cytometry immunophenotyping, DNA content, G-band and multicolor spectral karyotyping (SKY) and global gene expression profiling. All four cell lines were infected with lentiviral-expressing luciferase for detection of tumour cells at high sensitivity level and for monitoring myeloma growth in co-cultures and in vivo by live animal imaging. These myeloma cell lines and the procedures used for their establishment provide essential tools for studying myeloma biology and therapy.

Fig 1

Immunophenotypic analysis of four myeloma cell lines. LD, CF, JB and BN cells were stained with CD45/CD38 and CD138/CD45 and analysed by flow cytometry. Note the high expression of CD38 and CD138, and various expression level of CD45 and CD56 by these cells.

Fig 2

Representative G-band (top panel) and spectral karyotyping (SKY; bottom panel) karyotypes demonstrating different translocations and chromosome aneuploidies in LD (A), CF (B), JB (C) and BN (D) myeloma cell lines. The most common structural aberrations in the cell lines were duplications and translocations of 1q. Note that each cell line harbors basically the same karyotype aberrations found in the original tumour specimen of the patients.

Fig 3

Bioluminescent imaging of myeloma growth in vivo. Our myeloma cell lines were infected with luciferase-containing lentivirus as a marker for tracing myeloma growth in live SCID-hu and SCID-rab mice. Luciferase activity was detected using IVIS 200 imaging system. (A) BN myeloma cells were injected into mice and immediately analysed for bioluminescence intensity (photons/s/cm²/steradian). Note detection of 5000 or more BN cells by this system. (B, C) Bioluminescence intensity of BN cells (100 000 cells) was increased with time (B and C) and was highly correlated increased circulating hIg (B). (D) Treatment of BN- but not CF-bearing hosts with bortezomib (BOR, subcutaneously, 0.5 mg/kg/body weight, twice a week) for 5 weeks resulted in eradication of myeloma growth.

Fig 4

Establishment of a bortezomib-resistant myeloma cell line in vivo. SCID-hu mice engrafted with BN cells were treated with bortezomib (BOR) or left untreated. Note that hosts initially responded to BOR; however, when myeloma recurred, BN cells were resistant to the drug (A). Bortezomib-resistant BN cells (BN-R) recovered from mice (and were not continuously exposed to the drug), remained resistant to bortezomib following passage into newly constructed SCID-rab mice (B) and in co-culture with stromal cells (C), while bortezomib-sensitive BN cells (BN-S) responded to BOR in a dose–response manner (C).

Fig 5

Establishment of in vitro growth assay of myeloma cells in co-culture with stromal cells. (A) Luciferase (Luc) activity was highly correlated with number of tumour cells in co-cultures (r = 0.997). (B) A study demonstrating the effect of various doses of bortezomib, dexamethasone and melphalan (72 h, triplicates) on growth of BN cells in co-culture with stromal cells. Results are expressed as mean ± SEM.