Mesenchymal Stromal Cells Derived From Chronic Lymphocytic Leukemic Patients Express Different Genes and Produce Different Cytokines Compared to MSC Derived From Normal Subjects,

Mesenchymal stromal cells are one of the components in the CLL microenvironment known to support leukemic cells survival and growth. Crosstalk between CLL B-cells and marrow-derived mesenchymal stromal cells (MSC) can modulate the activation of both cell types. Indeed we have shown that soluble factors secreted from CLL B cells can activate MSC with increased production of angiogenic cytokines (Blood. 2010. 116:2984). We hypothesize that MSC derived from CLL patients are modified by their interaction with leukemic cells in their gene expression profiles (GEP) and cytokine production (CP) capabilities. Given this, intrinsic differences should be present amongst MSC derived from CLL patients versus normal subjects.

MSC collected from CLL patients with Rai stage 1–4 before any therapy or who have not had therapies for at least 2 years as well as normal MSC from healthy controls were cultured and expanded one time in vitro. Subsequently, MSC after one in vitro passage were sorted with CD45 negative selection to eliminate any contaminating non-stromal cells. Approximately 100,000 to 300,000 cells were then stored at −70C and used for RNA purification. Nine CLL MSC and five normal MSC cell samples had RNA purified from each sample and then the RNA preparations were used for comparative analysis of GEP using affymetrix U133A plus B chip. Data were transformed using two different methods: MAS5 or Plier and only probes that were present in at least 50% of cases were included in the analysis. We have recently observed that MSC derived from bone marrow of CLL patients promotes Th17 cell differentiation (Blood. 2010. 116: Abstract 2414). Since IL-17 is the signature cytokine produced by TH17 cells, we decided to test if IL-17 could alter the cytokine production from CLL and normal MSC. Cytokine production from both CLL MSC and normal MSC as secreted into culture medium (CM) spontaneously or with recombinant IL-17 during a 72h-culture were tested using a commercial cytokine array (R&D).

Both MAS5 and Plier method of transformation yielded similar results for GEPs of the MSC. The preliminary data from these analyses showed that multiple transcription factors including PITX2, HOXB7, SIM1, ZFY, EMX2, HOXB3, HOXB4, and IRF6 were significantly up-regulated (more than 2 fold) in CLL MSC compared to normal MSC. In contrast, the genes up-regulated in normal MSC compared to CLL MSC include genes involved in the immune response (IGH, IGK, IGL, BST2, PPBP, RGS1, etc.), antigen processing and presentation (HLA genes, IFI30, etc.) and inflammatory response (CD163, LYZ, TNFAIP6, etc.). We are currently in the process of performing further analysis using additional MSC samples to validate these findings with further GEP assays and also by validating the dysregulated genes detected by our GEP studies using real-time PCR analysis. We performed the analysis of multiple cytokines produced by CLL (n=16) and normal MSC (n=12) with and without stimulation with recombinant IL-17 (8 ng/ml) over a 72 hour culture period. Without stimulation, there is approximately two-fold increase of MCP-1 production in CLL MSC compared to normal MSC (CLL vs. normal, 164.2 ± 26.1 vs. 82.7 ± 15.4 pg/ml, p = 0.02). After IL-17 stimulation, an approximately four-fold increase of MCP-1 production (CLL vs. normal, 1200.1 ± 258.6 vs. 334.1 ± 68.2 pg/ml, p = 0.005), three-fold increase of IL-8 production (CLL vs. normal, 2073.1 ± 504.1 vs. 708.7 ± 218.9 pg/ml, p = 0.02), two-fold increase of IL-6 production (CLL vs. normal, 1400.6 ± 214.0 vs. 705.6 ± 98.2 pg/ml, p = 0.008) were detected in CLL MSC compared to normal MSC.

We have detected the presence of intrinsic mRNA expression differences for both transcription factors and immune response genes between CLL and normal MSC. The expression differences of these genes could be facilitating the differential cytokine responses we observed in CLL MSC compared to normal MSC. Given these findings it is possible that the dynamic interactions between the CLL leukemic cell and MSC influence the transcription profiles and cytokine production from MSC. This is of biologic and clinical interest in that the GEP dysregulation in CLL MSC may be a primary mechanism for the differential cytokine production seen in CLL MSC. This outcome in turn has the potential to modify the immune and non-immune environment in favor of tumor proliferation and progression.

Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.