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ActivinA enhances BCP-ALL cell migration
A
B
Figure 1. B-cell precursor-acute lym- phoblastic leukemia (BCP-ALL) cells reprogrammed the bone marrow (BM) stroma to produce high levels of ActivinA. (A) BM plasma levels of ActivinA were assessed by ELISA in healthy donors (HDs) (n=44) and BCP-ALL patients at the onset of the disease (n=108). Each box plot shows the median and the mean (+) and extends from the lowest to the highest value. *P<0.05: Mann-Whitney test. (B) Primary leukemic blasts were either direct- ly co-cultured or separated by a 0.4 mm Transwell insert with HD-mesenchymal stromal cells (MSCs). After 72 hours of cul- ture, supernatants were collected and ActivinA concentration was analyzed by ELISA (n=17 independent co-cultures). Each box plot shows the median and extends from the lowest to the highest value. **P<0.01; ***P<0.001; ****P<0.0001: Wilcoxon matched-pairs signed rank test.
Filamentous (F)-actin polymerization assay
F-actin polymerization was analyzed in 697 cells pretreated or not with ActivinA using AlexaFluor 647-labeled phalloidin (Invitrogen, Carlsbad, CA, USA) before and after CXCL12 stimu- lation, as reported in the Online Supplementary Appendix.
Calcium mobilization
Intracellular calcium mobilization was measured by flow cytometry using the Fluo-4NW Assay (Invitrogen), as reported in the Online Supplementary Appendix.
Statistical analyses
Differences between subgroups were compared with the Mann-Whitney test or Wilcoxon matched-pairs signed rank test in the case of matched values.
Results
Stroma-derived ActivinA increased in response to leukemia
It has been demonstrated that ActivinA could exert a pro-tumoral role in several types of cancer both through direct effects on tumoral cells and indirect effects on the
tumor microenvironment.7 Therefore, we first measured ActivinA levels in BM plasma samples from 44 HDs and 108 BCP-ALL patients at disease onset. ELISA assay revealed that ActivinA was significantly increased in the BM plasma of BCP-ALL patients compared to that of HDs (Figure 1A). The median concentration (mc) of ActivinA was 400 pg/mL (range: 62.5-7241 pg/mL) in BCP-ALL patients and 273.4 pg/mL (range: 62.5-2338 pg/mL) in HDs (P<0.05).
To determine whether ActivinA plasma levels at BCP- ALL diagnosis were related to disease outcome/severity, we analyzed 98 patients with available follow up out of the 108 tested, considering several clinical and biological parameters. In detail, 3- and 4-year event-free survival (EFS) and sensitive quantitative PCR-based minimal resid- ual disease (MRD) at days +33 and +78 and final risk were taken into account.10 ActivinA levels did not impact on EFS, on PCR-MRD risk, or on patients’ final risk stratifica- tion (data not shown).
It had previously been shown that BM-MSCs exhibit a basal level of ActivinA secretion.11 In view of the pivotal role of BM-MSCs in sustaining the leukemic niche,1 we explored the regulation of MSC-derived ActivinA in the context of BCP-ALL. We first confirmed that MSCs isolat- ed from the BM of HDs (HD-MSCs) were able to consti- tutively produce the molecule (mc: 103.2, range: 62.5- 526.7 pg/mL). Then, to test whether BCP-ALL cells could modulate MSC-derived ActivinA, we set up co-culture experiments of HD-MSCs with primary leukemic blasts and quantified ActivinA in supernatants after 72 h of co-culture. Interestingly, we found that primary leukemic cells significantly induced ActivinA in MSCs both through
B-cell acute lymphoblastic leukemia xenograft model
Female 7-9-week-old NOD-SCID-γchain-/- (NSG) mice (Charles River, Calco, Italy) were intravenously (i.v.) transplanted with 0.5x106 697 cells or 106 NALM-6 cells, either pretreated or not with ActivinA for 24 h. Details are described in the Online Supplementary Appendix. The study was approved by the Italian Ministry of Health (approval n. 64/2014).
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