Acute lymphoblastic leukemia cells create a leukemic niche without affecting the CXCR4/CXCL12 axis

Acute lymphoblastic leukemia (ALL) is characterized by an outgrowth of malignant lymphoblasts that occupy the bone marrow microenvironment where they reside with mesenchymal stromal cells (MSCs). Leukemic cells are able to disrupt the healthy microenvironment and create a leukemic niche that shelters malignant cells from elimination through cytostatic treatment and immune responses. Disrupting intercellular communication in this malignant microenvironment sensitizes leukemic cells to chemotherapeutic drugs. Homing of ALL cells toward the bone marrow microenvironment is thought to be similar to that of hematopoietic stem cells (HSCs). Human CD34 progenitor cells, are attracted by stromal cell-derived factor 1 (SDF1/CXCL12), a chemoattractant actively produced by MSCs in the bone marrow. CXCL12 binds to the CXCR4 receptor. Disturbance of the CXCR4/CXCL12 axis by treatment with cytokines and/or CXCR4 antagonists mobilizes HSCs to the peripheral blood and shows promising pre-clinical results in acute myeloid leukemia patients. Like HSCs, ALL cells have high CXCR4 surface expression and disruption of CXCR4/CXCL12 interaction reduces ALL engraftment in animal models. However, in mice suffering from ALL, CXCR4/CXCL12 inhibition as mono-treatment did not reduce the leukemic cell number in the bone marrow. In addition, CXCL12 expression was remarkably down-regulated in murine bone marrow regions of extensive ALL growth. Likewise, CXCL12 levels in the bone marrow of patients diagnosed with BCP-ALL were lower compared to those in the same patients at the time of remission and healthy controls. This suggests that ALL cells use additional mechanisms to retain a protective microenvironment. In this study, we used an ex vivo co-culture model in which we cultured B-cell precursor ALL (BCP-ALL) cells with bone marrow-derived primary MSCs. Several studies show that ALL cells home toward the bone marrow microenvironment in a CXCR4/CXCL12-dependent manner. We confirmed these findings in our model by assessing the migration of GFP-positive BCP-ALL cells (NALM6) toward MSCs using a transwell system. MSCs induced migration of BCP-ALL cells by 5-16-fold compared to medium controls (P<0.05) (Figure 1A and Online Supplementary Figure S1A and B). The induction of migration was correlated with the level of CXCL12 produced by the primary MSCs (Figure 1B). Inhibition of the CXCR4/CXCL12 axis with AMD3100 significantly impaired migration of BCPALL cells toward MSCs (4.5-fold reduction; P<0.01) (Figure 1C). Next, we hypothesized that BCP-ALL cells alter the chemoattractive properties of their microenvironment. To address this hypothesis, GFP-negative BCP-ALL cells (NALM6) were cultured with MSCs in the bottom compartment of a transwell system as a model of the leukemic niche. Migration of GFP-positive BCP-ALL cells (NALM6) from the upper to the bottom compartment was determined (Online Supplementary Figure S1A-C). GFP-positive BCP-ALL cells migrated significantly more toward ALLMSC co-cultures than toward MSCs in mono-culture (blue bars vs. gray bars; P<0.01) (Figure 1D and Online Supplementary Figure S1D and E). The leukemic niche induced migration of BCP-ALL cells by 2.2-fold (P<0.001) (Figure 1E). Surprisingly, CXCR4 inhibition by AMD3100 did not affect the preferential migration of BCP-ALL cells towards the leukemic niche (2.2-fold vs. 2.5-fold; P=0.52) (Figure 1E and Online Supplementary Figure S1F). In addition, we compared CXCL12 levels in the supernatant of monoand co-cultures of primary BCP-ALL cells derived from 9 different BCP-ALL cases and 4 primary MSCs. CXCL12 levels were high in mono-cultures of MSCs, and below detection level in mono-cultures of primary BCPALL cells (Online Supplementary Figure S2A and B). Co-culture of primary BCP-ALL cells and MSCs did not increase CXCL12 levels compared to the combined levels produced in both mono-cultures (Figure 1F). These results suggest that BCP-ALL cells create a leukemic niche that attracts leukemic cells in a CXCR4/CXCL12-independent manner. Next, we assessed the migration of healthy CD34 hematopoietic progenitor cells (HPCs) toward primary MSCs cultured with or without BCP-ALL cells. As expected, CD34 HPCs migrated more abundantly toward MSCs than to control medium without MSCs (1.8-fold; P<0.05) (Figure 1G). However, migration of CD34 HPCs toward ALL-MSC co-cultures was significantly reduced compared to migration toward MSCs in mono-cultures (20% reduction; P<0.05) (Figure 1G). In addition, we assessed the migratory behavior of healthy MSCs toward ALL-MSC cocultures. We performed a transwell migration assay in which primary MSCs were allowed to migrate toward BCP-ALL cells or other MSCs in the bottom compartment. We observed that MSC migration was induced toward other MSCs compared to control medium (P<0.05) (Figure 1H). Strikingly, MSCs also actively migrated to BCPALL cells illustrating that BCP-ALL cells are able to recruit MSCs (P<0.001) (Figure1H and Online Supplementary Figure S3A). However, MSC migration was significantly inhibited toward the leukemic niche (ALL-MSC co-cultures) compared to a bottom compartment containing only BCP-ALL cells (2-3-fold reduction; P<0.001) (Figure 1I and Online Supplementary Figure S3B and C). Interestingly, increasing the amount of ALL cells (up to 16-fold) in ALL-MSC co-cultures did not re-induce MSC migration (Figure 1I). These results show that ALL-MSC co-cultures efficiently attract leukemic cells, but reduce migration of healthy CD34 HPCs and primary MSCs, suggesting that ALL-MSC co-cultures produce chemokines that specifically attract BCP-ALL cells. To address which cytokines/chemokines may be altered in the leukemic niche, we quantified the levels of 64 cytokines known to be important in immunology/hematology in the supernatant of ex vivo co-cultures of patientderived BCP-ALL cells (n=10, representing major BCP-ALL subgroups) and MSCs (n=4) (Online Supplementary Table S1). Cytokine/chemokine profiles of mono-cultures of primary BCP-ALL cells and primary MSCs are shown in Online Supplementary Figure S2. Co-cultures of BCP-ALL cells with MSCs revealed the induction of unique secretion patterns per patient. These patterns were similar for different sources of MSC, i.e. MSCs obtained at the time of leukemia and those obtained from healthy controls. The unique profile per BCP-ALL patients suggests that leukemic cells govern the observed cytokine secretion (Figure 2, Online Supplementary Table S2 and Online Supplementary Figure S4A and B). These unique cytokine/chemokine profiles suggest the need for patient-specific approaches to disrupt the leukemic niche. However, we also found recurrently induced signaling pathways. All ALL-MSC co-cultures showed increased levels of CCL2/MCP-1 and/or CCL22/MDC (Figure 2A), both ligands for the CCR4 receptor. This chemokine receptor is implicated in T-cell leukemia and lymphoma, but has not yet been implicated as an important factor in BCP-ALL. In addition, analysis of previously published microarray-based gene expression data showed that CCR4 expression levels in pediatric ALL