An Altered Microenvironment Within The Spleens Of Patients With Myelofibrosis Affects CD34⁺ Cell Trafficking

We have demonstrated that the homing of peripheral blood (PB) myelofibrosis (MF) CD34⁺ cells to the marrow but not the spleen is altered (Wang X, et al. Cancer Res. 2009;69:7612-8). The defective homing to the marrow was further found to be associated with both the down-regulation of CXCR4 expression by PB MF CD34+ cells (Wang X, et al. Cancer Res. 2009;69:7612-8) and the proteolytic degradation of CXCL12 in the plasma of MF patients (Cho SY, et al. Cancer Res. 2010;70:3402-10). However, the mechanism underlying the preferential homing, retention and engraftment of the MF HSCs/HPCs to extramedullary sites has not been clarified. Cancer stem cell behavior is thought to be determined by both intrinsic properties and by regulatory signals provided by the microenvironment. The effect of alterations within the splenic microenvironment on the trafficking of MF-stem cells (MF-SC) was, therefore, investigated.

The homing of spleen and PB MF CD34+ cells to the marrow and spleens of sublethally irradiated NOD/SCID mice was first evaluated. Following the infusion of paired spleen or PB MF CD34+ cells (n=5) or normal G-CSF mobilized PB CD34⁺ cells (mPB: n=6, 5×10⁵/mouse), reduced numbers of spleen MF CD34+ cells were detected in the marrows of these mice as compared with mPB CD34+ cells (No. of CD34⁺ Cells/10⁶ BMCs: PB MF:74±6; mPB: 196±32; P=0.007). However, the number of spleen MF CD34+ cells that homed to the marrow of recipient mice were even lower than PB MF CD34+ cells (No. of CD34⁺ Cells/10⁶ BMCs: spleen MF: 74±6; PB MF: 102±13; P=0.08). By contrast, similar numbers of spleen MF, PB MF and mPB CD34+ cells were detected in the spleens of these mice. These findings suggest that both spleen MF and PB MF CD34+ cells preferentially migrate towards spleen rather than to the marrow. To determine if the expression of chemokine receptors and adhesion molecules could account for the homing and location of MF CD34⁺ cells to the spleen, the expression of CXCR4, CD47, CD44 and CD49d by spleen and PB MF CD34+ cells was evaluated and was shown to be similar. These findings suggest that the greater number of MF-SCs that were observed in the spleen as compared to PB of MF patients (Wang X, et al. J Clin Invest. 2012;122:3888-99) is not due to aberrant expression of adhesion molecules or chemokine receptors by MF-SCs and that microenvironmental conditions within the MF spleen might be responsible for MF-SC and progenitor cell homing, retention and engraftment.

Unlike intact CXCL12, truncated forms of CXCL12 either lack the ability to act as a chemo-attractant for CD34⁺ cells or act as an antagonist to the action of CXCL12. The intact and truncated forms (loss of 2 aa, 3 aa, 4 aa, and 5 aa, aa = amino acid) of CXCL12 in paired spleen and PB MF plasma (n=5) were quantified using mass spectrometry. The concentration of intact CXCL12 in spleen MF plasma was 39.6 ±13.9ng/ml, which was higher than that detected in PB MF plasma (5.5 ± 2.6ng/ml, P=0.08). However, the concentrations of the 4 truncated forms of CXC12 were comparable in spleen and PB MF plasma. The concentrations of CXCL12 were further normalized based on the albumin level of the corresponding plasma to exclude the influence of different methods used to prepare these two sources of plasmas. The normalized concentration of intact CXCL12 in spleen MF plasma was significantly higher than that of PB MF plasma (P=0.03), while the normalized concentrations of the 4 truncated forms of CXCR4 were found again similar in both spleen and PB MF plasma. However, comparable levels of serine proteases, including active MMP-9 and NE, each of which are responsible for the degradation of a number of marrow matrix proteins such as CXCL12, were detected in spleen and PB MF plasma, suggesting that the splenic microenvironment acted to blunt their proteolytic actions. The migratory behavior of spleen MF CD34+ cells towards spleen and PB MF plasma was also determined in vitro using transwell plates. A greater number of spleen MF CD34+ cells from each individual patient migrated towards spleen plasma than PB plasma. Moreover, the percentage of JAK2V617F⁺ colonies cloned from patient CD34+ cells (JAK2V617F allele burden: 90%) that had migrated towards spleen plasma was similar to that observed with PB plasma. These findings suggest that the MF spleen is characterized by increased levels of intact CXCL12 which contributes to the preferential homing of spleen MF CD34+ cells to the spleen and the retention of such cells within the spleens of MF patients.