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).1 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.32 Disrupting intercellular communication in this malignant microenvironment sensitizes leukemic cells to chemotherapeutic drugs.4 Homing of ALL cells toward the bone marrow microenvironment is thought to be similar to that of hematopoietic stem cells (HSCs).5 Human CD34 progenitor cells, are attracted by stromal cell-derived factor 1 (SDF-1/CXCL12), a chemoattractant actively produced by MSCs in the bone marrow.65 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 blood7 and shows promising pre-clinical results in acute myeloid leukemia patients.8 Like HSCs, ALL cells have high CXCR4 surface expression and disruption of CXCR4/CXCL12 interaction reduces ALL engraftment in animal models.109 However, in mice suffering from ALL, CXCR4/CXCL12 inhibition as mono-treatment did not reduce the leukemic cell number in the bone marrow.119 In addition, CXCL12 expression was remarkably down-regulated in murine bone marrow regions of extensive ALL growth.1 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.6 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.1091 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 BCP-ALL 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 ALL-MSC 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 mono- and 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 BCP-ALL 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 co-cultures. 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 BCP-ALL cells illustrating that BCP-ALL cells are able to recruit MSCs (P<0.001) (Figure 1H 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 patient-derived 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.12 In addition, analysis of previously published microarray-based gene expression data showed that CCR4 expression levels in pediatric ALL cells were significantly higher compared to healthy hematopoietic cells (Online Supplementary Figure S4C).
In addition, we observed recurrent induction of CXCL8/IL-8 and CXCL1/GRO secretion, ligands of the CXCR1 and CXCR2 receptor. CXCR1/2 ligands were induced by BCP-ALL cells from 6 out of 10 patients (Figure 2C). In contrast to CCR4 expression, CXCR1 and CXCR2 expression levels were lower in pediatric ALL compared to healthy hematopoietic cells (Online Supplementary Figure S3C).
We validated the cytokine/chemokine patterns identified in our co-culture model using serum derived from bone marrow aspirates from 10 ALL patients at diagnosis. We compared the cytokine/chemokine patterns in these samples with serum obtained from bone marrow aspirates of the same patients taken at the end of induction chemotherapy. At this stage of treatment, the patients were in remission. Furthermore, we used bone marrow aspirates of 7 healthy controls to analyze cytokine/chemokine patterns of the healthy niche. Levels of the CCR4 ligand CCL2/MCP-1 were significantly higher in the bone marrow of untreated, newly diagnosed BCP-ALL patients compared to healthy controls (2.0-fold increase; P<0.01) (Figure 2B). Interestingly, after induction therapy, the CCL2/MCP-1 levels in the bone marrow of ALL patients decreased to levels observed in healthy controls (2.2-fold decrease; P<0.01) (Figure 2B). This suggests that the induced CCL2/MCP-1 levels at diagnosis are causally linked to the presence of leukemic cells. CCL22/MDC levels were similar between BCP-ALL patients and healthy controls (Figure 2B). Like CCL2/MCP-1, CXCL1/GRO levels were also increased in the bone marrow of BCP-ALL patients (2.3-fold increase; P<0.05) (Figure 2D) and CXCL8/IL8 levels showed a trend towards upregulation (2.5-fold; P=0.052) (Figure 2D). Similar to CCL2/MCP-1 levels, the secreted levels of these CXCR1/2 ligands were significantly reduced after induction chemotherapy (2.3-fold and 3.1-fold, respectively; P<0.05) (Figure 2D). These data implicate that CCR4 and CXCR1/2 signaling are important in the bone marrow of BCP-ALL patients and suggest that CCR4 inhibition (e.g. by mogamulizumab1)13 or CXCR1/2 inhibition might be an alternative or additional way to disrupt the BCP-ALL niche.
In conclusion, our data indicate that the BCP-ALL niche is highly potent in attracting BCP-ALL cells and repelling the influx of healthy hematopoietic cells and MSCs in a CXCL12-independent manner. Instead, we show the recurrent induction of CCR4 and CXCR1/2 ligands by BCP-ALL cells in the leukemic niche (Figure 3). Our data suggest CCR4 and CXCR1/2 to be valuable future therapeutic targets to interfere with the leukemic niche in ALL.
- Colmone A, Amorim M, Pontier AL. Leukemic cells create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells. Science. 2008; 322(5909):1861-1865. PubMedhttps://doi.org/10.1126/science.1164390Google Scholar
- Fujisaki J, Wu J, Carlson AL. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature. 2011; 474(7350):216-219. PubMedhttps://doi.org/10.1038/nature10160Google Scholar
- Nakasone ES, Askautrud HA, Kees T. Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance. Cancer Cell. 2012; 21(4):488-503. PubMedhttps://doi.org/10.1016/j.ccr.2012.02.017Google Scholar
- Polak R, de Rooij B, Pieters R. B-cell precursor acute lymphoblastic leukemia cells use tunneling nanotubes to orchestrate their microenvironment. Blood. 2015; 126(21):2404-2414. PubMedhttps://doi.org/10.1182/blood-2015-03-634238Google Scholar
- Peled A, Petit I, Kollet O. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science. 1999; 283(5403):845-848. PubMedhttps://doi.org/10.1126/science.283.5403.845Google Scholar
- van den Berk LC, van der Veer A, Willemse ME. Disturbed CXCR4/CXCL12 axis in paediatric precursor B-cell acute lymphoblastic leukaemia. Br J Haematol. 2014; 166(2):240-249. PubMedhttps://doi.org/10.1111/bjh.12883Google Scholar
- Flomenberg N, Devine SM, Dipersio JF. The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone. Blood. 2005; 106(5):1867-1874. PubMedhttps://doi.org/10.1182/blood-2005-02-0468Google Scholar
- Burger JA, Peled A. CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers. Leukemia. 2009; 23(1):43-52. PubMedhttps://doi.org/10.1038/leu.2008.299Google Scholar
- Juarez J, Dela Pena A, Baraz R. CXCR4 antagonists mobilize childhood acute lymphoblastic leukemia cells into the peripheral blood and inhibit engraftment. Leukemia. 2007; 21(6):1249-1257. PubMedhttps://doi.org/10.1038/sj.leu.2404684Google Scholar
- Sipkins DA, Wei X, Wu JW. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature. 2005; 435(7044):969-973. PubMedhttps://doi.org/10.1038/nature03703Google Scholar
- Parameswaran R, Yu M, Lim M. Combination of drug therapy in acute lymphoblastic leukemia with a CXCR4 antagonist. Leukemia. 2011; 25(8):1314-1323. PubMedhttps://doi.org/10.1038/leu.2011.76Google Scholar
- Nakagawa M, Schmitz R, Xiao W. Gain-of-function CCR4 mutations in adult T cell leukemia/lymphoma. J Exp Med. 2014; 211(13):2497-2505. PubMedhttps://doi.org/10.1084/jem.20140987Google Scholar
- Duvic M, Pinter-Brown LC, Foss FM. Phase 1/2 study of mogamulizumab, a defucosylated anti-CCR4 antibody, in previously treated patients with cutaneous T-cell lymphoma. Blood. 2015; 125(12):1883-1889. PubMedhttps://doi.org/10.1182/blood-2014-09-600924Google Scholar