Patient-Derived 3D Tissue-Engineered Bone Marrow Cultures Support Primary MM Growth

INTRODUCTION:

In vitro culturing of primary myeloma cells has been a major challenge because the lack of an in vitro technology capable of recreating the complicated bone marrow (BM) microenvironment which multiple myeloma (MM) cells depend on for its survival. While primary myeloma cells cannot grow ex vivo in SCID mice, they are able to growth in SCIDhu mice. However, animal models are expensive, time-consuming, and often have limited reproducibility. In addition, classic laboratory models cannot take into consideration the variability of disease in every patient, and the MM patient population is highly variable, both genetically and epigenetically, and the biological characteristics of patients are widely different, which demonstrates sensitivity of individual patients to different therapies. Typical two-dimensional (2D) models rely on a limited number of MM cell lines which cannot reflect the enormous heterogeneity and variations present in individual patients. The goal of this study is to create a patient derived three-dimensional tissue-engineered bone marrow (3DTEBM) culture system based on cross-linked MM-derived BM supernatant, including endogenous soluble growth factors and cytokines, and by incorporating mononuclear cells, including MM (CD138⁺ population) and accessory cells (CD138^- population), from the same patient.

METHODS:

The 3DTEBMwas formed through calcium cross-linking of BM supernatants combined with the CD138⁺ and CD138^- population from the same MM patient. We tested the growth of fresh primary CD138⁺ MM cells with CD138^- cells in 2D vs patient-derived 3DTEBM cultures based on the original BM ratio (CD138^-/CD138⁺) of the patient at 3 days by flow cytometry. We further selected a patient with not very aggressive tumor and evaluated the effect of CD138^- increasing densities on CD138⁺ MM growth in 3DTEBM cultures at day 3 by flow cytometry. In addition, the effect of patient-derived 3DTEBM and 2D cultures with and without CD138^- cells on CD markers expression of the CD138⁺ population was tested at day 3 by flow cytometry. Finally, the growth of frozen primary CD138⁺ MM cells with and without CD138^- cells in 2D vs patient-derived 3DTEBM cultures was analyzed for 14 days by flow cytometry.

RESULTS:

We found that patient-derived 3DTEBM cultures support primary MM cell growth. CD138⁺ MM cells from patients with less aggressive tumors (high BM ratio CD138^-/CD138⁺) showed better growth in 3DTEBM than in 2D systems at day 3. However, more aggressive tumors (low BM ratio CD138^-/CD138⁺), in which MM cells are less dependent of accessory cells, the 3DTEBM cultures showed similar CD138⁺ MM growth as in 2D cultures. After that, we detected in the patient-derived 3DTEBM cultures a direct correlation between positive and negative fractions, with increased CD138^- densities the CD138⁺ MM growth increased.

Next, we analyzed the effect of 3D cultures in CD markers expression and we showed that CD138⁺ MM cells expressed loss of the plasma cells markers (CD38, CD56, and CD138), reduction of B cells markers (CD19, CD20 and CD22), and increased of the stem cell marker CD34 in 3DTEBM compared to 2D cultures.

Finally, patient-derived 3DTEBM supported the growth of frozen primary CD138⁺ MM cells better than 2D cultures, and the addition of CD138^- cells enhanced even more CD138⁺ MM growth after 14 days in culture.

CONCLUSIONS:

Patient-derived 3DTEBM cultures support primary fresh and frozen CD138⁺ MM growth better than classic systems, and induced de-differentiation of MM cells while increased a stem-cell like phenotype. These results highlight the importance of the BM microenvironment (BM supernatant, including endogenous soluble growth factors and cytokines, and the CD138- population from the same patient) to facilitate primary MM cell growth in vitro. Therefore, patient-derived 3DTEBM cultures allowed long-term culture of primary fresh and frozen myeloma cells ex vivo, and these findings indicate that patient-derived 3DTEBM can be utilized for studying multiple myeloma biology and for testing patient-targeted therapy.