Acute Lymphoblastic Leukemia Blast Cells Stimulate the Autocrine Production of CCL2 and IL-8 in Bone Marrow Stromal Cells

Abstract

The interactions of Acute Lymphoblastic Leukemia (ALL) blasts with bone marrow (BM) stromal cells have a positive impact on leukemia cell survival and resistance to chemotherapy. ALL stimulates BM stromal cells, which reciprocally promotes leukemia cell survival. To identify molecules critically involved in leukemia–microenvironment crosstalk, we performed gene expression profiling analyses of primary BM endothelial cells (BMEC) and BM mesenchymal stem cells (BMMSC) following stimulation by primary ALL cells. Leukemia stimulation of BM stromal cells upregulates the expression of several inflammatory chemokines, including CCL2 and IL-8/CXCL8. Secretion of these molecules was confirmed by ELISA assays of in vitro co-culture experiments and in BM plasma samples from pediatric ALL patients. Most primary ALL samples were found to express mRNA for CCR2 and CXCR1/CXCR2, which are the cognate receptors for CCL2 and IL-8, respectively. Primary ALL cells expressing at least one myeloid marker (CD13, CD15 or CD33) exhibited increased mRNA expression of CCR2 (p = 0.02). Leukemia cells from most patients express CCL2 and IL-8 chemokines (ELISA test) but at lower levels than that of BMEC and BMMSC. In vitro functional studies revealed that the proliferation, survival and migration of primary ALL cells co-cultured with BM stromal cells were not affected by addition of CCL2, IL-8 or of neutralizing antibodies to these chemokines. On the other hand, both chemokines were found to enhance BMEC and BMMSC survival in serum-free medium and to increase their proliferation in serum-starved conditions. Interestingly, CCL2 and IL-8 affected endothelial morphogenesis as shown in Matrigel assays. Since CCL2 and IL-8 have suppressive effects in normal hematopoiesis but do not seem to affect primary ALL cells, it is possible that these chemokines may contribute to the establishment of survival/proliferative selective advantage for ALL cells in the leukemic BM microenvironment. In addition, CCL2 and IL-8 seems indirectly to contribute to ALL cell survival by stimulating the supporting BM stromal cells. Finally, preliminary results showed that standard risk pediatric ALL patients with BM plasma levels below 577pg/ml have better survival rates than those with higher CCL2 levels (p = 0.08). In conclusion, this work suggests a significant role for the chemokines CCL2 and IL-8 in the leukemia/microenvironment crosstalk in human ALL, and suggests that these molecules may represent valuable targets for therapeutic intervention in this cancer. Supported by: CNPq, FAPESP.