CD44 standard and CD44v10 isoform expression on leukemia cells distinctly influences niche embedding of hematopoietic stem cells
© Erb et al.; licensee BioMed Central Ltd. 2014
Received: 14 January 2014
Accepted: 25 March 2014
Published: 31 March 2014
A blockade of CD44 is considered a therapeutic option for the elimination of leukemia initiating cells. However, anti-panCD44 can interfere with hematopoiesis. Therefore we explored, whether a CD44 variant isoform (CD44v)-specific antibody can inhibit leukemia growth without attacking hematopoiesis. As a model we used CD44v10 transfected EL4 thymoma cells (EL4-v10).
The therapeutic efficacy of anti-panCD44 and anti-CD44v10 was evaluated after intravenous application of EL4/EL4-v10. Ex vivo and in vitro studies evaluated the impact of anti-panCD44 and anti-CD44v10 as well as of EL4 and EL4-v10 on hematopoietic stem cells (HSC) in cocultures with bone marrow stroma cells with a focus on adhesion, migration, cell cycle progression and apoptosis resistance.
Intravenously injected EL4-v10 grow in bone marrow and spleen. Anti-panCD44 and, more pronounced anti-CD44v10 prolong the survival time. The higher efficacy of anti-CD44v10 compared to anti-panCD44 does not rely on stronger antibody-dependent cellular cytotoxicity or on promoting EL4-v10 apoptosis. Instead, EL4 compete with HSC niche embedding. This has consequences on quiescence and apoptosis-protecting signals provided by the stroma. Anti-panCD44, too, more efficiently affected embedding of HSC than of EL4 in the bone marrow stroma. EL4-v10, by catching osteopontin, migrated on bone marrow stroma and did not or weakly interfere with HSC adhesion. Anti-CD44v10, too, did not affect the HSC – bone marrow stroma crosstalk.
The therapeutic effect of anti-panCD44 and anti-CD44v10 is based on stimulation of antibody-dependent cellular cytotoxicity. The superiority of anti-CD44v10 is partly due to blocking CD44v10-stimulated osteopontin expression that could drive HSC out of the niche. However, the main reason for the superiority of anti-CD44v10 relies on neither EL4-v10 nor anti-CD44v10 severely interfering with HSC – stroma cell interactions that, on the other hand, are affected by EL4 and anti-panCD44. Anti-panCD44 disturbing HSC embedding in the osteogenic niche weakens its therapeutic effect towards EL4. Thus, as far as leukemic cells express CD44v isoforms, the therapeutic use of anti-panCD44 should be avoided in favor of CD44v-specific antibodies.
KeywordsLeukemia Antibody therapy CD44v10 Hematopoiesis Bone marrow stroma
CD44 is important for HSC and LIC homing [12–14]. Anti-CD44 prohibits the development of cobblestone areas in murine long term bone marrow cultures (LTBMC) [15, 16]. Bone marrow stroma formation also requires CD44 that supports the process by induction of IL-6 secretion . HSC synthesize and express hyaluronic acid (HA) and HA expression correlates with selective migration of HSC to the endosteal niche . Furthermore, anti-CD44 efficiently mobilizes HSC [16, 19, 20]. HSC homing can be blocked by anti-CD44, soluble HA or hyaluronidase treatment . In addition, CXCL12 stimulates adhesion of progenitor cells via CD44, which demonstrates a cross-talk between CD44 and CXCR4 signaling and suggests a key role of HA and CD44 in CXCL12-dependent transendothelial migration of HSC and their anchorage within specific niches . Besides contributing to homing and settlement of HSC in the bone marrow niche, CD44 promotes quiescence versus proliferation [22–24].
CD44 also accounts for LIC homing . In acute myeloid leukemia (AML) CD44 is required for the transport to the stem cell-supportive osteogenic niche and anti-CD44 alters the fate of AML-IC by inducing differentiation . In a mouse model of chronic myeloid leukemia (CML), BCR-ABL1-transduced progenitors from CD44-mutant donors show defects in bone marrow homing. This results in decreased engraftment and impaired CML-like disease induction, pointing towards LIC being more dependent on CD44 for homing than HSC . However, a marker/signal transduction molecule that is shared by LIC and HSC presents a risk factor, when trying to attack the LIC [5, 27]. Our studies, confirming that leukemia cell homing and growth is retarded by a CD44-specific antibody, pointed towards an antibody blockade of CD44 during reconstitution to affect HSC embedding more severely than leukemia growth [28, 29].
Based on the finding that leukemia cells efficiently compete with HSC under the condition of myeloablation and reconstitution [28, 29], we here asked whether anti-CD44 also interferes with hematopoiesis in the non-compromised leukemia-bearing host and whether such interference could be prevented by replacing anti-panCD44 with a CD44 variant isoform-specific antibody. We choose the CD44v10 cDNA transfected thymoma EL4 (EL4-v10). CD44v10 is expressed on subpopulations of macrophages (Mϕ), activated Th1 cells and hematopoietic progenitor cells, particularly of the B cell lineage [30–34]. Upregulated CD44v10 expression correlates with Hodgkin’s disease relapse  and in multiple myeloma it supports adhesion to marrow endothelial cells . Upregulated CD44v10 expression, though not reaching statistical significance, was also observed in some patients with AML . CD44v10 appeared particularly suited as it is very weakly expressed on HSC, but is a receptor for osteopontin (OPN), that according to reports of several groups should support motility of hematopoietic stem and progenitor cells [37, 38]. Thus, we expected a blockade of CD44v10 not or hardly to affect HSC.
We report that anti-CD44v10 displays higher efficacy than anti-panCD44 in retarding EL4-v10 growth. This is an indirect effect, as anti-CD44v10, as expected, distinct to anti-panCD44, does not disturb hematopoiesis. In addition, CD44v10 being an OPN ligand, EL4-v10 migrate on bone marrow stroma (BM-Str), but do not tightly adhere, leaving space for HSC settlement.
The interaction with the osteogenic niche is important for maintenance of HSC quiescence and timely maturation . One of the molecules of central importance in these processes is CD44 [5, 10], which also becomes apparent by anti-CD44 efficiently mobilizing HSC . CD44 also is frequently overexpressed in leukemia such that LIC can be driven into differentiation and death by anti-CD44 [7, 25, 26]. Thus, we asked whether by replacing anti-panCD44 by a CD44 variant isoform-specific antibody, anti-leukemic effects can be maintained without interfering with hematopoiesis using as a model highly standard CD44 (CD44s)+ EL4, which do not express CD44v isoforms (Zöller, unpublished) and were transfected with CD44v10 (EL4-v10) (Figure 1B).
The impact of CD44v10 and anti-CD44v10 on bone marrow homing and intra-marrow growth
Mice received an intravenous (i.v.) injection of 1×107 CFSE-labeled EL4 or EL4-v10 concomitantly with control IgG, anti-panCD44 (IM7) or anti-CD44v10 (K926). Mice were sacrificed after 48 h and 72 h, hematopoietic organs were excised and the recovery of fluorescent EL4 and EL4-v10 was evaluated by flow cytometry. Both, EL4 and EL4-v10 preferentially home into bone marrow (BM) and spleen with EL4-v10 having a slight advantage in settlement in lymph nodes (LN) (Figure 1C). IM7 and K926 interfere with homing into BM and spleen, which is accompanied by a pronounced retention in the peripheral blood. IM7 and K926 displayed comparable efficacy in inhibiting EL4 and EL4-v10 homing (Figure 1D).
Mice receiving i.v. injections of 1×106 EL4/EL4-v10 became moribund after 27d to 29d. The mean survival time was prolonged to 32d and 33d, respectively, by IM7 treatment. Instead, EL4-v10-bearing mice receiving K926 had a mean survival time of 38.5d, which differed significantly from the survival time of EL4-v10 bearing mice receiving IM7 (Figure 1E and F). When mice were sacrificed after 21d, comparable numbers of EL4 and EL4v10 cells were recovered in the BM and the spleen, respectively. IM7 and K926 had a minor impact on the recovery in the spleen, but a significantly reduced number was seen in the BM. Distinct to the superior impact of K926 on the survival time, recovery of EL4-v10 in the BM of K926-treated mice did not significantly differ from that in IM7-treated mice (Figure 1G).
Taken together, EL4 and EL4-v10 preferentially home into BM and spleen. There is no evidence for increased tumorigenicity by CD44v10 expression. A blockade of CD44 retards homing and leukemia growth in BM and spleen. In EL4-v10 inoculated mice a blockade of CD44v10 with K926 is more efficient.
IM7 and K926 promote antibody-dependent cellular cytotoxicity, IM7 interferes with hematopoiesis
Taken together, antibody treatment promotes leukocyte recruitment into tumor nodules and suffices for strong ADCC, which does not differ between IM7 versus K926 treatment, excluding that effector cell activation accounts for the prolonged survival time of K926-treated EL4-v10 TB. Thus, we asked for the impact of EL4 versus EL4v10 as well as of IM7 versus K926 on hematopoiesis.
When controlling for the presence of HSC, we noted a slight decrease in cells expressing the HSC markers CD117 and SCA1 in the BM of IM7-treated early TB, which became significant in late TB. In addition, fewer HSC were recovered from late EL4 than EL4-v10 TB (Figure 2C). In late EL4-v10 TB a minor retention of Gr1+ cells in the BM and a slight reduction in serum IgG was noted (Figure 2C and D), which is in line with CD44v10 being engaged in monocyte and B cell maturation . Reduced recovery of HSC in IM7-treated late TB could be in line with IM7, distinct to K926, interfering with HSC embedding in the osteogenic niche. The higher recovery of HSC in late EL4-v10 than EL4 TB might be indicative for EL4-v10 not or less efficiently competing with HSC for stroma adhesion.
To consolidate these findings, BALB/c SCID mice were concomitantly "reconstituted" with C57BL6 HSC and EL4 or EL4-v10 and received weekly injections of IM7 or K926. According to our hypothesis, T- and B-cell recovery should be impaired in EL4 versus EL4-v10 bearing mice and should be additionally affected by IM7, but not K926. Three weeks after i.v. HSC and EL4/EL4-v10 application, fewer donor cells were recovered in spleen and lymph nodes of TB than control mice, the decrease was more pronounced in EL4 than EL4-v10 TB. IM7 and not or less pronounced K926 affected the recovery of donor cells also in control mice. Furthermore, the recovery of CD4+ and CD8+ cells was more strongly reduced in lymph nodes and spleen of EL4 than EL4-v10 TB and hardly any B cells were recovered in the spleen of EL4 TB. Only IM7 significantly affected the lymphocyte recovery, which also accounted for SCID mice only receiving HSC. Recovery of CD4+CD8+ TC also differed between TB and tumor-free mice and recovery of double-positive TC was strikingly reduced in IM7-treated mice. Likely due to the contribution of the SCID mice derived HSC, SCA1+ cells were not strikingly affected by EL4 or EL4-v10, but were reduced in IM7-treated mice. Recovery of CD34+ cells was reduced in EL4, but not EL4-v10 TB and IM7 contributed to a reduction in tumor-free and EL4-v10 TB mice (Figure 2E and F).
The strongly reduced recovery of mature lymphocyte in reconstituted EL4 TB mice confirming the pronounced impact of EL4 on hematopoiesis, we aimed to answer the question, why EL4-v10 and K926 exert a weaker effect on hematopoiesis and explored the interaction of EL4, EL4-v10 and HSC with BM-Str in dependence on the presence of IM7 or K926.
Crosstalk between EL4/EL4-v10 and bone marrow stroma
BM- and spleen-Str highly express CD44 CD54, CD90 and CD105, they are partly CD106 and weakly CD44v10 positive and express bFGF, SDF1 and OPN. Contamination by hematopoietic cells was excluded by the absence or very low expression of CD34, CD45, CD14, CD80, CD86 and MHC II particularly in BM-Str (Figure 3C). EL4 and EL4-v10 exosomes promoted upregulation of Rantes, IP10, SDF1 and OPN on BM-Str. EL4 and EL4-v10 exosomes promoted TNFRI upregulation, expression of other chemokine receptors was not affected (Figure 3D and data not shown). It is important to note that with the exception of OPN no significant differences were seen concerning the impact of EL4 versus EL4-v10 exosomes on BM-Str, which allowed to judge on the crosstalk between stroma and EL4 versus EL4-v10.
These findings indicate a more intense crosstalk between stroma cells and EL4 than EL4-v10, which affects adhesion, cell cycle progression and apoptosis protection. This crosstalk mostly is CD44s-dependent, as - with the exception of migration - it is efficiently blocked by IM7, but hardly by K926. According to these findings, one would expect a stronger effect of IM7 than of K926 on EL4/EL4-v10 growth in the bone marrow. As we observed the opposite, the suspicion arose that IM7 affects hematopoiesis. Alternatively, though not mutually exclusive, EL4 more efficiently compete for the endosteal niche, thereby negating the IM7 anti-leukemic effect.
The impact of EL4/EL4-v10 on the crosstalk between HSC and bone marrow stroma
Experiments controlling for the competition between HSC and EL4/EL4-v10 with BM-Str as well as for the impact of IM7 and K926 were performed with mitomycinC-treated EL4/EL4-v10 cells or exosomes. As we did not observe major differences between exosomes and cells, for clarity of presentation experiments shown were mostly performed with exosomes.
Thus, the superior effect of K926 compared to IM7 on survival of mice after i.v. application of EL4-v10 does not rely on K926 more efficiently affecting the leukemic cell, but rather on EL4-v10 not interfering with hematopoiesis that, on the other hand, is affected by EL4 as well as IM7, particularly with respect to HSC quiescence.
Taken together, a blockade of CD44v10 was more efficient than a panCD44 blockade in intra-marrow EL4-v10 growth retardation. The superior effect of K926 compared to IM7 on leukemia growth in the BM mostly is due to EL4-v10 and K926 - distinct to EL4 and IM7 - hardly disturbing the HSC interaction with the BM-Str, which partially negates the impact of anti-panCD44 on leukemia growth.
High CD44 expression in many leukemia and lymphoma has stimulated therapeutic considerations based on a blockade of CD44 that was particularly successful in AML and CML [25, 26]. Nonetheless, due to abundant CD44 expression in hematopoietic cells including HSC, anti-panCD44 has also been reported to be burdened by side effects [5, 44]. Therefore, we explored for CD44v10 transfected EL4, EL4 not expressing other CD44 variants (M.Zöller unpublished), whether side effects can be circumvented or mitigated by antibodies specific for splice variants that expression is far more restricted . We choose CD44v10 because it is hardly expressed in HSC (M.Zöller, unpublished), but expressed in some leukemia , particularly in cutaneous lymphoma , Hodgkin’s disease  and multiple myeloma  and because it is an OPN ligand [unpublished finding], where OPN is important in guiding HSC from the osteogenic to the vascular niche [47–49]. We report that anti-CD44v10 is advantageous as it did not/less severely interfere with HSC embedding and quiescence in the BM-Str niche.
In advance of discussing our findings, it should be remembered that CD44s expression is unaltered in EL4-v10 compared to EL4 cells. This implies that differences between EL4 and EL4-v10 will only be seen as a sequel of genuine functions of CD44v10. Correspondingly, anti-CD44v10 can be expected to confirm genuine CD44v10 activities and in case of inefficacy indirectly strengthens the impact of CD44s.
Anti-CD44 efficacy in interfering with intra-marrow leukemia growth
Leukemia frequently arising and expanding in the bone marrow, we controlled for the impact of a CD44v-specific antibody on leukemia growth versus hematopoiesis, which was approached by i.v. EL4/EL4-v10 application.
Anti-panCD44 and anti-CD44v10 were not curative, but sufficed for a prolonged survival time. Unexpectedly, therapeutic application of anti-CD44v10 was more efficient than anti-panCD44, although both IM7 and K926 comparably well promoted ADCC [50, 51]. In addition, there was no evidence for K926 more efficiently than IM7 driving the leukemia cells into apoptosis. Thus, there remained two alternative, not mutually exclusive explanations. IM7, distinct to K926, affects hematopoiesis and/or EL4/EL4-v10 distinctly interfere with hematopoiesis, a negative impact of the leukemic cells on hematopoiesis not becoming neutralized by IM7.
EL4/EL4-v10 and the crosstalk with the bone marrow niche
We first controlled on the impact of EL4 and EL4-v10 on BM-Str. EL4 and EL4v10 promoted upregulation of TNFRI, SDF1 and OPN, with EL4-v10 being more efficient in OPN upregulation that could have affected niche egress of EL4/EL4-v10 as well as of HSC [48, 52, 53]. Upregulated SDF1 might support recruitment of EL4/EL4-v10 as well as of HSC [54–56]. Otherwise, no EL4-/EL4-v10-induced changes in BM-Str were seen that could be expected to affect the crosstalk between EL4/EL4-v10 versus HSC with the stroma.
EL4 adhere more efficiently than EL4-v10 to BM- and spleen-Str, adhesion being significantly inhibited only by IM7, but not by K926. Distinct to adhesion, EL4-v10 had a minor advantage in migration on BM-Str that was neutralized by IM7 and K926. Adhesion and migration remained unaltered, when pretreating the BM-Str with EL4 or EL4-v10 exosomes (data not shown). We briefly want to point out that some unspecific impact of CFSE on functional activity cannot be safely excluded. However, as control settings were treated accordingly, observed differences should be relevant. In line with EL4 more tightly interacting with BM-Str, BM-Str affects EL4 cell cycle progression and proliferation, though to a minor degree and only transiently, the impact of the BM-Str on EL4 being waved in the presence of IM7. Finally, stroma-adherent EL4 and EL4-v10 were protected from a cytotoxic drug. Protection was more pronounced for EL4 than EL4-v10 and was abolished by IM7, but not K926, which findings strengthen our interpretation that EL4 interacts more tightly with the BM-Str than EL4-v10. In mouse models of AML and CML, CD44 targeting suppressed progression and induced differentiation by disrupting the interaction with the bone marrow niche , experimental evidence pointing towards apoptosis induction by caspase- or calpain-dependent pathways [58, 59] and to inhibition of proliferation through stabilization of the cyclin-dependent kinase inhibitor p27, which resulted in increased association with the cyclinE-Cdk2 complex [58, 60].
In the EL4 model, BM-Str protected EL4/EL4-v10 from drug-induced apoptosis and the protective effect was abolished by IM7, confirming a central role for the engagement of CD44. Yet, the thymoma line EL4 obviously does not essentially require the BM-Str for survival, as we observed only a minor effect on EL4/EL4-v10 proliferation. Instead, our findings suggest that EL4, rather than EL4-v10 might compete with HSC for the niche.
EL4/EL4-v10 competition with HSC for niche embedding
In line with the hypothesis that EL4 competes with HSC for the BM niche was the poorer recovery of HSC in late EL4, but not EL4-v10 TB. Furthermore, transplanted HSC maturation towards T and B cells was more severely impaired in EL4 than EL4-v10 TB and was strongly affected by IM7. Instead, K926 only affected B cell maturation, which is in line with our earlier report on CD44v10 expression in B progenitor cells . In vitro cocultures confirmed that EL4 more efficiently than EL4-v10 competed with HSC for BM-Str adhesion. Furthermore, in the presence of EL4, but less pronounced in the presence of EL4-v10, HSC were driven into cell cycle progression, rare cycling being one of the characteristics of HSC . Cell cycle progression and proliferation of HSC on BM-Str was also promoted by IM7. Corresponding to these findings, only in cocultures with EL4, β-catenin, cyclinD1 and c-myc expression [62–67], which was low in HSC seeded on BM-Str, became upregulated. EL4-v10 exerted no or weaker effects.
Thus, EL4 more strongly than EL4-v10 interfere with the crosstalk between HSC and BM-Str such that HSC are dislodged and possibly differentiate. Though IM7 exerted a similar effect on HSC cocultured with BM-Str, an additive effect of EL4 plus IM7 was not seen or was weak, which suggests that IM7 binds EL4 and HSC with equal efficacy. Alternatively, the crosstalk between both HSC and EL4 with the BM-Str may become affected by stroma-bound IM7 [68, 69]. Irrespective of this open question, in concern of the efficacy of IM7 on the survival time of EL4 TB, the benefit of IM7 promoted ADCC will be partly neutralized by the negative impact of EL4 on hematopoiesis. On the other hand, the weaker interaction between EL4-v10 and the BM-Str could explain the higher therapeutic efficacy of K926, where we speculate that weaker adhesion on BM-Str is due to CD44v10 being an OPN ligand and inducing OPN expression (M.Zöller, unpublished). This will favor EL4-v10 motility rather than BM-Str adhesion and will mitigate the impact on the crosstalk between HSC and BM-Str [48, 70]. Whether OPN capture by CD44v10 additionally supports HSC quiescence, as OPN can act as a negative regulator of HSC proliferation [48, 49, 71, 72], remains to be explored.
EL4/EL4-v10 and HSC apoptosis resistance
HA, particularly medium sized HA that was used throughout [73–75] and, far stronger BM-Str protected HSC from cytotoxic drug-induced apoptosis [40, 41, 76, 77]. HSC apoptosis protection was counterregulated by EL4 and EL4-v10 exosomes as well as IM7 and, less pronounced, K926. BM-Str-supported HSC protection was accompanied by activation of the PI3K/Akt pathway [76, 78, 79], which was not seen in the presence of EL4 or EL4-v10 and became downregulated by anti-panCD44, but not anti-CD44v10. On the other hand, neither EL4/EL4-v10 nor IM7 suffice to initiate caspase9 cleavage or caspase3 activation in the presence of BM-Str. One possible explanation could be that anti-apoptotic triggers, which proceed via distinct pathways [29, 42, 43], are not affected and suffice to prevent effector caspase activation. In line with this hypothesis was the finding that EL4/EL4-v10 did not promote Bcl2 and BclXl downregulation and that this phenomenon was CD44-independent. Instead, EL4 and EL4-v10 promoted Bcl2 and BclXl upregulation. Thus, it appears that EL4 and EL4-v10 can exert apoptosis promoting as well as protecting effects on BM-Str-embedded HSC, where we speculated that, besides protein interactions, additionally exosomal miRNA might be involved [80, 81]. Though the question on the impact of EL4/EL4-v10 and IM7 on the regulation of adult HSC apoptosis resistance, which is still disputed [82, 83], requires further elaboration, it is obvious that HSC apoptosis protection is not exclusively regulated via CD44 and also that it is not merely initiated by upregulation of the PI3K/Akt pathway.
CD44-directed therapeutic approaches have most intensely been explored in hematological malignancies. Using a CD44v10 expressing thymoma, our findings suggest that depending on the type of leukemia, a blockade of CD44v can be preferable. Distinct to CD44s, a blockade of CD44v10 hardly interfered with the crosstalk between HSC and BM-Str, particularly not affecting HSC quiescence. In concern of a concomitant cytotoxic drug therapy, apoptosis resistance of niche-embedded HSC was distorted in the presence of EL4 and, less efficiently, EL4-v10, due to the less tight crosstalk between EL4-v10 with the BM-Str. But, the protective effect of the BM-Str was much weaker for EL4-v10 than EL4. This implies that in combination with cytotoxic drugs, the application of a CD44v-specific antibody could bring about an additional therapeutic advantage.
Material and methods
EL4 thymoma cells  were transfected with CD44v10 cDNA  (EL4-v10). Transfected cells were cloned by limiting dilution in RPMI/10% FCS supplemented with 250 μg/ml G418. EL4-v10 were maintained in RPMI/10% FSC/G418. EL4 were cultured in RPMI/10% FCS.
Tissue preparation and cell isolation
Mice were bled and sacrificed by cervical dislocation. Femur and tibia, lymph nodes (LN), spleen, thymus and tumor tissue were collected. BM cells (BMC) were flushed with PBS out of the bone, spleen cells (SC) and LN cells (LNC) were obtained by pressing through fine gauze. HSC were enriched by repeated magnetic bead selection, where after depletion of CD4+, CD8+, CD11b+, CD11c+, CD16+/CD32+ and CD19+ cells, Ter119+ and Gr1+ cells were eliminated and finally CD117+/SCA1+ cells were positively selected. Spleen stroma cells (spleen-Str) and BM-Str, collected from BMC that were depleted of HSC, were cultured for 4 wk in ISCOVES/10% FCS taking care to wash out all non-adherent and loosely adherent cells. When reaching confluence, cells were scrabbed off with a rubber policemen and were split into two flasks. Scrabbed off stroma cells were seeded in 24-well Plates 48 h in advance of starting coculture experiments.
EL4/EL4-v10 were cultured (48 h) in serum-free medium. Cleared supernatants (2×10 min, 500 g, 1×20 min, 2000 g, 1×30 min, 10000 g) were centrifuged (90 min, 100000 g) and washed (PBS, 90 min, 100000 g). The pellet was resuspended and purified by sucrose gradient centrifugation .
Cells (1-3×105) were stained according to routine procedures. For intracellular staining, cells were fixed and permeabilized in advance. Apoptosis was evaluated by AnnexinV-FITC or -APC/PI staining. Staining was evaluated using a FACS-Calibur (BD). Analysis was performed by the Cell Quest program. Experiments were repeated at least 3 times.
Snap frozen spleen sections (5 μm) were fixed (chloroform/acetone, 1:1, 4 min) and treated with levamisole to ablate tissue alkaline phosphatase activity. Nonspecific binding was blocked using an avidin-biotin-blocking kit (VectorLaboratories, Burlingame, CA, USA), and 2% normal serum derived from the same species as the secondary antibodies. Tissues were incubated with the primary antibodies, washed, exposed to biotinylated secondary antibodies and alkaline phosphatase conjugated avidin-biotin solution and were counterstained with hematoxilin. Images were taken using a Leica DMRBE microscope.
CFSE labeling was performed in medium without FCS. After washing in medium with 10% FCS, cells were rested for 30 min at 37°C to guarantee CFSE incorporation. Thereafter cells were washed an additional 3-times. CFSE-labeled cells were seeded on BSA (10 μg/ml in bicarbonate buffer, pH 9.6), OPN (2 μg/ml in bicarbonate buffer, pH 9.6), rooster comb HA (mean molecular weight 1–4 million) (100 μg/ml in PBS) or a monolayer of BM-Str in 24-well plates. Plates were coated for 24 h, washed and blocked with 1% BSA in PBS overnight. For BM-Str collection and seeding, see above. After 4 h incubation, non-adherent cells were removed by washing, triplicates of adherent cells were lysed and fluorescence was evaluated photometrically. Adhesion is presented as percent of seeded cells.
For videomicroscopy, CFSE-labeled cells (5×104) were seeded on a monolayer of BM-Str-or spleen-Str-coated 24-well plates. Plates were placed under an Olympus IX81 inverse microscope with a Hg/Xe lamp, an incubation chamber (37°C, 5% CO2), a CCD camera (Hamamatsu) and a ScanR acquisition soft ware (Olympus, Hamburg, Germany). Two pictures (20-fold magnification)/chamber (2 ms exposure) were taken every 20 min for 12 h. Migration was quantified according to Manual_tracking plugin running in the open-source software Image J. Assays were run in triplicates and tracking was evaluated for 20 individual cells in every well, the presented mean values deriving from 60 individual cells.
Cell cycle, proliferation and apoptosis
Cells were seeded on BSA-coated plates or on a monolayer of BM-Str. Cell cycle progression was evaluated after starving EL4/EL4-v10 for 48 h and HSC for 24 h, adding RPMI/10% FCS (EL4/EL4-v10) or Iscove'sMEM/10% FCS (HSC) for 2 h. Cells were washed with ice cold PBS and resuspended in 70% cold ethanol and incubated overnight at 4°C. Cells were washed with cold PBS and resuspended in PI-RNAse solution (250U RNAse type I-A, 100 μg/ml, 10 μg/ml PI, =.1% TritonX-100 in PBS). After 15 min incubation at room temperature in the dark, PI staining was evaluated by flow cytometry. For determining cell division, CFSE-labeled cells were starved overnight and cultured for 24 h-72 h, determining CFSE-dilution by flow-cytometry. Apoptosis was determined by AnnV (1 μg/ml)/PI (0.3 μg/ml) staining. Where indicated, cultures contained 2.5-20 μg/ml cisplatin. Mean values ± SD of triplicates are presented.
ADCC activity was evaluated after in vitro restimulation in the presence of 100 U/ml IL2 for 48 h. Where indicated, cultures contained 10 μg/ml anti-CD44 (IM7) or anti-CD44v10 (K926). 3H-thymidine labeled (12 h, 10 μCi/ml 3H-thymidine) target cells (104/well) were seeded on titrated numbers (10×105 - 6×104) of effector cells in 96 well plates. After 6 h at 37°C, plates were harvested, and radioactivity was determined in a β-counter. Cytotoxicity is presented as % cytotoxicity = 100 × (counts in control wells - counts in test wells)/(counts in control wells). The spontaneous release [100 × (total counts – counts in control wells)/total counts)] of the target cells ranged between 6%-10%. Mean values ± SD of triplicates are presented. SD were in the range of 3%-5%.
C57BL6 mice (5/group) received an intravenous (i.v.) injection of 1×106 EL4 or EL4-v10 and 2×/week 100 μg/mouse IM7 or K926 in 200 μl 0.9% NaCl solution. Mice were sacrificed according to weight loss and fatigue. To evaluate tumor cell homing, mice (3 mice/group) received 1×107 CFSE-labeled tumor cells, which were preincubated (30 min, 4°C) with 200 μg IM7 or K296 in 200 μl 0.9% NaCl solution (i.v.). Mice were sacrificed after 48 h and 72 h. Organs were excised and single cell suspensions were prepared. The presence of CFSE-labeled tumor cells was evaluated by flow cytometry. For evaluating the impact of EL4/EL4-v10 and IM7/K926 on HSC maturation BALB/c SCID mice (5/group) received an i.v. injection of 2×106 C57BL6 HSC. Where indicated, mice received concomitantly 1×106 EL4 or EL4-v10, i.v. and 1 day later IM7 or K926 (100 μg / mouse in 100 μl 0.9% NaCl solution). The antibody application was repeated every 5th day. Mice were sacrificed after 3 wk. BM, thymus, LN and spleen were excised and the recovery of donor cells (H-2b), B cells CD4+, CD8+ and CD4+CD8+ T cells as well as of SCA1+ and CD34+ was evaluated by flow cytometry. The mean percent of marker-positive cells per organ from 5 mice is presented. Animal experiments were approved by the local Government.
Significance was evaluated by the two tailed Student’s t-test (in vitro assays) or the Kruskal-Wallis test (in vivo assays). P values <0.05 were considered significant and are indicated by an asterisk or s.
Antibody-dependent cellular cytotoxicity
CD44 standard isoform
CD44 variant isoform
CD44v10 transfected EL4 cells
Hematopoietic stem cells
Leukemia initiating cells
This investigation was supported by the Deutsche Krebshilfe (MZ). We cordially thank Christine Niesik and Angela Frank for help with animal experiments, flow cytometry and immunohistology.
1 Table listing antibodies and other reagents.
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