"In questo periodo così difficile per il nostro paese e non solo, sono lieta di informarvi che lo studio di cui vi ho parlato alle cene da voi organizzate e nei report che vi ho scritto è stato pubblicato sulla rivista The British Journal of Haematology.
Nei ringraziamenti abbiamo naturalmente menzionato la vostra associazione per il preziosissimo supporto al progetto!
Grazie ancora di tutto."
Chiara Palmi, PhD
Centro Ricerca Tettamanti
Clinica Pediatrica Univ. Milano Bicocca
c/o Ospedale San Gerardo
Acknowledgements
L.B. designed the study, performed experiments, collected
and analyzed data and wrote the manuscript; E.D. contributed
to the experimental design, provided murine BMMSC
and BM-MSC related protocols and revised the manuscript;
C.B and P.M. provided scientific and logistical support
for lentivirus production and human UCB-CD34+ cells
infection; M.B. and D.A. performed experiments; A.F. provided
the inducible Ba/F3 model and revised the manuscript;
B.G. provided experimental support; S.B. and G.T.
performed and analysed gene expression profile experiments;
P.V. provided cord blood units; A.B. supervised the research;
G.d’A. contributed to the experimental design; C.P. designed
the study, performed experiments, collected and analysed
data and revised the manuscript; G.C. supervised the research
and revised the manuscript; C.P. and C.G. equally contributed
to the work.
The authors thank Dr Caserta Carolina for experimental
support. This study was supported by grants from: Fondazione
Tettamanti, Fondazione Cariplo (2018-0339), Associazione
Italiana Ricerca sul Cancro (AIRC, project number IG
2018 Id. 21999, to G.C., and IG 2014 Id.15494 to G.d’A.),
and the Beat Leukemia Foundation (www.beat-leukemia.org).
L.B. was supported by scholarships from Bianchi Industrial
and Societa Italiana Ematologia Sperimentale (SIES). The
laboratory work by P.M. was funded by the European
Research Council (CoG-2014-646903 and PoC-2018-811220),
the Agencia Estatal de Investigacion/European Regional
Development Fund (SAF2016-80481-R and SAF2016-75442-
R) and the Catalunya Government (SGR330 and PERIS
2017) to P.M; the laboratory work by and C.B. was funded
by the Asociacion Espa~nola Contra el Cancer, Beca FERO
and the ISCIII/FEDER (PI17/01028). P.M also acknowledges
the institutional support from the Obra Social La Caixa-Fundaci
o Josep Carreras.
Conflict of Interest
The authors declare no competing financial interests.
Authors’ disclosures of potential conflicts of
interest
There are no conflicts of interest to disclose.
Supporting Information
Additional supporting information may be found online in
the Supporting Information section at the end of the article.
Figure S1. Schematic representation of the competitive
niche assay.
Figure S2. The competitive niche assay in the presence of
several stimuli.
Figure S3. LPS stimulation does not provide advantage to
E/R+ Ba/F3 in the competitive mesenchymal niche.
Figure S4. Soluble factors, other than TGFb, mediate the
pre-leukemia selective advantage within the inflamed mesenchymal
niche.
Figure S5. A) Gene expression profiling by Gene Chip
Mouse 2.0 Arrays of control and E/R+ Ba/F3. Only those E/R
induction experiments showing >90% viability and >90%
FITC positivity were chosen for analysis (n = 3). B) E/R+ Ba/
F3 upregulated pathways involved in the immune and
inflammatory response, included myeloid cell activation
pathways. Gene ontology (GO) analysis was performed by
Fig 7. The inflamed mesenchymal niche increases DNA doublestrand
breaks and AID expression in both control and E/R+ Ba/F3
cells. A) E/R+ and control Ba/F3 cells were co-cultured (80%:20%)
for 4 days in standard liquid culture (basal) or on murine BM-MSC
monolayers in the presence or absence of IL6/TNFa/IL1b. Phosphorylated
levels of H2AX (cH2AX) in mCD45+, both V5-positive and
V5-negative, cells were measured as MFI by FACS. The MFI of control
Ba/F3 cells grown under basal condition was considered as reference
for fold increase calculation. Values are expressed as
mean SD of 6 independent experiments. One-sample t-test:
§P < 005. Paired Student’s t-test: *P < 005; **P < 001. B) Control
and E/R+ Ba/F3 cells were separately grown in standard liquid culture
(basal) or loaded into the upper chamber of 04 μm Transwell
inserts in the presence of MSC (lower chamber) and inflammatory
cytokines (+MSC + INFL.CK). RT-qPCR analysis was performed to
quantify AID expression, normalizing values on Hprt expression.
Analysis was performed on 3 independent experiments. Paired Student’s
t-test: two-sided, *P < 005; **P < 001.
Pro-inflammatory cytokines favor the emergence of ETV6-RUNX1-positive pre-leukemic cells in a model of mesenchymal niche
ª 2020 British Society for Haematology and John Wiley & Sons Ltd 9
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