The haematopoietic stem cell (HSC) niche is formed by several cell types which contribute to the regulation of the haematopoiesis within the bone marrow (BM). In pathological conditions, alterations of the BM microenvironment have been reported, but it is still debated whether they are cause or consequence of the disease. Support to leukaemic cells, especially to leukaemic stem cells (LSC), at the expense of normal HSC and protection against chemotherapeutic agents characterise the malignant BM niche. Thus, in order to eradicate LSC, promising therapeutic strategies may consider the chemoprotection exerted by the BM microenvironment in addition to LSC intrinsic features of refractoriness to conventional therapies. In the first part of this PhD project, we investigated the role of mesenchymal stromal cells (MSC) in the pathogenesis of aplastic anaemia (AA). Patient-derived MSC (AA-MSC) were characterised in vitro and, especially, in vivo taking advantage of our recently described in vivo BM niche model, based on the subcutaneous implantation of cartilaginous pellets generated from MSC (Serafini et al., Stem Cell Research, 2014). AA-MSC did not exhibit morphological, phenotypical, proliferative and differentiation defects in vitro. Only a reduced clonogenic potential was observed. Most importantly, they were able to recreate a functional and complete BM niche in vivo, proving their unaltered ability to support haematopoiesis and excluding their involvement in AA pathogenesis. In the second part of this PhD project, we analysed the effects of L-asparaginase (ASNase) on acute myeloid leukaemia (AML) cells, especially on LSC, and the contribution of the microenvironment to chemoresistance. ASNase was equally effective against unfractionated AML cells and against CD34+CD38- and CD34+CD38+ LSC, while slightly affecting healthy haematopoietic cells. The action of the drug against AML primitive cells was confirmed by clonogenic assays and by experiments performed in LSC supportive culture conditions. However, the anti-leukaemic potential of ASNase could be in part counteracted by MSC, expressing asparagine synthetase, and by monocytes/macrophages and blasts themselves, expressing cathepsin B, generating a protective niche. Lastly, we tested a new conditioning regimen based on the addition of fludarabine to irradiation in SCID-beige mice, a poorly permissive strain for human engraftment. The administration of fludarabine after irradiation increased the levels of engraftment of a AML cell line, KG-1, as compared to irradiation only, and it allowed the engraftment in 50% of primary AML blasts transplanted, reproducing human leukaemic infiltration. Thus, we generated a novel AML xenograft model in which we would like to test promising therapeutic agents, as ASNase, and to study different features of the malignant BM niche.

La nicchia staminale ematopoietica è formata da molteplici tipi cellulari che contribuiscono alla regolazione dell’ematopoiesi all’interno del midollo osseo. In alcuni disordini ematopoietici il microambiente midollare risulta alterato, sebbene il suo contributo alla patogenesi non sia ancora chiaro, fornisce supporto alle cellule staminali leucemiche (LSC), a spese delle normali cellule staminali ematopoietiche, e protezione contro i chemioterapici. Dunque, per eradicare le LSC, le strategie terapeutiche devono considerare l’effetto protettivo contro i chemioterapici esercitato dal microambiente midollare oltre alla refrattarietà intrinseca delle LSC alle convenzionali terapie. Nella prima parte di questo progetto di dottorato, abbiamo valutato il ruolo delle cellule stromali mesenchimali (MSC) nella patogenesi dell’anemia aplastica (AA). Le MSC derivate dai pazienti (AA-MSC) sono state caratterizzate in vitro e, soprattutto in vivo, utilizzando il modello di nicchia midollare in vivo recentemente sviluppato dal nostro gruppo di ricerca, basato sull’impianto sottocutaneo di pellet cartilaginei derivati dalle MSC (Serafini et al., Stem Cell Research, 2014). In vitro, le AA-MSC mostravano solo un ridotto potenziale clonogenico senza difetti morfologici, fenotipici, proliferativi e differenziativi. Inoltre, le AA-MSC erano in grado di ricreare una nicchia midollare funzionale in vivo dimostrando un’inalterata capacità di supportare l’ematopoiesi e l’assenza di un loro coinvolgimento nella patogenesi della AA. Nella seconda parte di questo dottorato, abbiamo analizzato gli effetti dell’asparaginasi (ASNase) sulle cellule derivate da pazienti affetti da leucemia mieloide acuta (AML), soprattutto sulle LSC, ed il contributo del microambiente alla resistenza ai chemioterapici. L’ASNase era egualmente efficace contro il bulk delle cellule AML e contro le LSC (CD34+CD38- e CD34+CD38+), mentre l’effetto nei confronti delle cellule ematopoietiche sane era trascurabile. L’azione del farmaco contro le cellule primitive di AML è stato ulteriormente confermato in saggi clonogenici ed in esperimenti effettuati in condizioni di coltura specifiche per il mantenimento delle LSC in vitro. Tuttavia, una nicchia midollare protettiva formata dalle MSC, che esprimono asparagina sintetasi, dai monociti/macrofagi e dai blasti leucemici stessi, che esprimono catepsina B, potrebbe ridurre il potenziale anti-neoplastico del farmaco. Infine, abbiamo testato un nuovo regime di condizionamento (somministrazione di fludarabina in aggiunta all’irraggiamento) in topi SCID-beige, un ceppo poco permissivo per l’engraftment umano. Tale strategia di condizionamento ha incrementato i livelli di engraftment della linea cellulare AML, KG-1, rispetto al solo irraggiamento, e ha permesso di ottenere l’engraftment nel 50% dei blasti AML primari trapiantati. In futuro, vorremmo combinare questo nuovo modello di xenograft così ottenuto con il trapianto di pellet cartilaginei per generare un modello di nicchia AML da poter usare per studiare il microambiente midollare leucemico e per testare promettenti agenti terapeutici contro l’AML, come l’ASNase.

(2019). The role of the bone marrow microenvironment in aplastic anaemia and acute myeloid leukaemia: from pathogenesis to chemoresistance. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).

The role of the bone marrow microenvironment in aplastic anaemia and acute myeloid leukaemia: from pathogenesis to chemoresistance

MICHELOZZI, ILARIA MARINA
2019

Abstract

The haematopoietic stem cell (HSC) niche is formed by several cell types which contribute to the regulation of the haematopoiesis within the bone marrow (BM). In pathological conditions, alterations of the BM microenvironment have been reported, but it is still debated whether they are cause or consequence of the disease. Support to leukaemic cells, especially to leukaemic stem cells (LSC), at the expense of normal HSC and protection against chemotherapeutic agents characterise the malignant BM niche. Thus, in order to eradicate LSC, promising therapeutic strategies may consider the chemoprotection exerted by the BM microenvironment in addition to LSC intrinsic features of refractoriness to conventional therapies. In the first part of this PhD project, we investigated the role of mesenchymal stromal cells (MSC) in the pathogenesis of aplastic anaemia (AA). Patient-derived MSC (AA-MSC) were characterised in vitro and, especially, in vivo taking advantage of our recently described in vivo BM niche model, based on the subcutaneous implantation of cartilaginous pellets generated from MSC (Serafini et al., Stem Cell Research, 2014). AA-MSC did not exhibit morphological, phenotypical, proliferative and differentiation defects in vitro. Only a reduced clonogenic potential was observed. Most importantly, they were able to recreate a functional and complete BM niche in vivo, proving their unaltered ability to support haematopoiesis and excluding their involvement in AA pathogenesis. In the second part of this PhD project, we analysed the effects of L-asparaginase (ASNase) on acute myeloid leukaemia (AML) cells, especially on LSC, and the contribution of the microenvironment to chemoresistance. ASNase was equally effective against unfractionated AML cells and against CD34+CD38- and CD34+CD38+ LSC, while slightly affecting healthy haematopoietic cells. The action of the drug against AML primitive cells was confirmed by clonogenic assays and by experiments performed in LSC supportive culture conditions. However, the anti-leukaemic potential of ASNase could be in part counteracted by MSC, expressing asparagine synthetase, and by monocytes/macrophages and blasts themselves, expressing cathepsin B, generating a protective niche. Lastly, we tested a new conditioning regimen based on the addition of fludarabine to irradiation in SCID-beige mice, a poorly permissive strain for human engraftment. The administration of fludarabine after irradiation increased the levels of engraftment of a AML cell line, KG-1, as compared to irradiation only, and it allowed the engraftment in 50% of primary AML blasts transplanted, reproducing human leukaemic infiltration. Thus, we generated a novel AML xenograft model in which we would like to test promising therapeutic agents, as ASNase, and to study different features of the malignant BM niche.
SERAFINI, MARTA
nicchia midollare; MSC; anemia aplastica; LAM; asparaginasi
BM niche; MSC; aplastic anaemia; AML; asparaginasi
MED/38 - PEDIATRIA GENERALE E SPECIALISTICA
English
12-feb-2019
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
31
2017/2018
open
(2019). The role of the bone marrow microenvironment in aplastic anaemia and acute myeloid leukaemia: from pathogenesis to chemoresistance. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/241079
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