Stromal cells within the bone marrow microenvironment support the survival and proliferation of multiple myeloma (MM) cells, and confer resistance against chemotherapeutic agents. Although drug combinations (such as velcade and melphalan) are used to overcome such resistance; outcomes for myeloma patients remains sub-optimal. The microenvironment therefore represents a target to sensitise cells to chemotherapy. To further investigate this, we established a co-culture platform to culture CD138–positive MM cells and stromal cells directly or indirectly (separated by a 0.4μm micropore membrane).
Co-culture with the human bone marrow stromal cell line (BMSC), HS-5, increased cell proliferation and cell viability of both MM cell lines (MMCLs) and 17 primary MM samples (Cell viability 53.6±11.2 (mean±s.d.) compared with 35.3±13.9 in media alone, (p<0.0001)). The stimulation of primary MM cell proliferation (p<0.01), confirmed the survival effects of BMSCs. Such antiapoptotic effects were markedly pronounced when the BMSCs were in direct contact with MM cells, however importantly conditioned media from BMSC cultures also demonstrated a significant increase in MM cell viability compared to control media alone (p<0.01). Exposure to standard chemotherapy (melphalan and dexamethasone) and novel therapies (bortezomib and the HDAC inhibitor UCL67022) resulted in a marked inhibition of MMCL and primary MM cell growth. However, these effects were attenuated when cells were either co-cultured in direct contact or in non-contact co-culture assays with HS-5 cells using transwell inserts. Such attenuation was accompanied by upregulation of cytokine-mediated STAT3 signaling, and PI3K/AKT, P38MAPK and ERK cell survival pathways within the MM cell. Cytokine profiling of supernatants from co-culture assays detected high levels of IL-6, IL-8, and VEGF. Levels of IL-8 correlated directly with resistance to velcade, and levels of both IL-8 and VEGF correlated with resistance to melphalan, whilst levels of IL-6 correlated with resistance to both agents in combination (Pearson's correlation coefficient p<0.05). Furthermore, neutralizing antibodies against these cytokines removed the protective effect conferred by the BMSC co-culture and restored drug sensitivity in primary MM cells. Combination of the IL-8 and VEGF antibodies gave an additive effect whereas all three antibodies in combination had the greatest sensitising effect.
In conclusion, we demonstrate that humoral factors secreted by stromal cells protect MM cells from both standard, novel chemotherapy and the highly clinically active combination of velcade and melphalan. Whilst IL-6 neutralising antibodies are being evaluated in the clinic, this data provides compelling evidence towards the further evaluation of IL-8 and VEGF antibodies both alone and in combination.
Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.
Asterisk with author names denotes non-ASH members.