Prolonged Maintenance of Ex Vivo Functional Vk*MYC Multiple Myeloma Cells Allows for in Vitro Manipulation and Subsequent In Vivo Analysis

Human multiple myeloma (MM) cell lines are easy to culture but only partially reflect the heterogeneity of primary MM samples as most cell lines are derived from primary human plasma-cell leukemia samples and have often lost their dependence on the marrow microenvironment. In contrast, maintaining primary human MM samples ex vivo and in vivo is very challenging. To bridge this gap, the immunocompetent Vk*MYC transgenic MM model was developed. It has high biological fidelity to the human disease characteristics and features easy in vivo engraftment and serial transplantation. However, Vk*MYC cells are considered notoriously difficult to grow and manipulate in vitro, limiting their usage for functional studies. Aim

To overcome these limitations, we aimed to develop a robust culture system which promotes survival and function of primary Vk*MYC MM cells in culture as a research tool for in vitro manipulation and subsequent in vivo transplantation. Method

Splenocytes from tVk*MYC (Vk12598 clone) engrafted mice were routinely maintained ex vivo and conditioned media collected for M-spike quantification. Analysis of cultures by flow cytometry allowed for Vk*MYC population quantification over time. Our novel plating method includes the following components to support Vk*MYC cell survival: 3D plating as hanging drops, low (6.5%) oxygen culture, high density cultures and a cytokine cocktail. Results

The culture method described here routinely results in maintenance or slight enrichment of Vk*MYC (CD138+B220-) splenocyte fraction. Monoclonal immunoglobulin (M spike) accumulation in conditioned culture media was assessed after 7 days culture as a clinically relevant, functional output marker. Culture media displayed the same immunoglobulin isotype as parent Vk*MYC (Vk12598 clone). Figure 1 shows previously frozen donor Vk*MYC splenocytes (day 0) assessed by flow cytometry and either injected directly, or maintained 7 days ex vivo, prior to IV injection into naive C57BL/6 recipient mice. Culture-recipient mice developed Vk*MYC MM disease with the same serum M-spike isotype band, similar engraftment ratio, similar time to evidence of biochemical disease and similar overall survival time as mice transplanted with freshly defrosted Vk*MYC splenocytes. To exemplify in vitro manipulation, Vk*MYC splenocytes were cultured as described and treated with Cy3-labelled liposomal nanoparticles (LNP), a non-toxic way to deliver treatment loads e.g. siRNAs, then maintained in culture for up to 7 days after treatment. Flow analysis showed >95% of viable CD138+ cells were Cy3 positive as a marker of LNP uptake. In addition, Vk*MYC splenocytes were transduced with GFP-encoding lentivirus, then maintained in culture for up to 7 days. Flow analysis showed viable, CD138+B220- cells expressing GFP as a marker of lentiviral transduction, highlighting the potential of this culture method to allow manipulation of Vk*MYC cells in vitro. Discussion

This innovative culture method allows for survival and enrichment, but not expansion of Vk*MYC splenocytes in culture. Our seven-day culture protocol allows in vitro manipulation with liposomal nanoparticles as well as viral vectors and subsequent in vivo investigations.

Figure 1. Experimental setup. A) (Day 0) donor tVk*MYC spleen was defrosted and assessed for MM burden by flow (CD138+ B220-). Splenocytes were maintained for 7 days ex vivo in 3D hanging drop cultures. At day 7, conditioned media was assessed by modified SPEP method for evidence of biochemical disease, and cultured cells assessed by flow or injected into naive C57BL/6 recipient mice. B) After 4 weeks, evidence of biochemical disease (M-spike) was confirmed by SPEP and mouse spleen Vk*MYC burden assessed by flow cytometry.

Hay:Janssen: Research Funding; Bristol-Myers-Squibb: Honoraria; Kite/Gilead: Honoraria; Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria. Chesi:Abcuro, Palleon Pharmaceuticals, Pi Therapeutics.: Patents & Royalties: Genetically engineered mouse model of myeloma.; Pfizer, Novartis.: Consultancy.