Modeling Chemotherapy Resistant Leukemia <em>In Vitro</em>

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

West Virginia University School of Medicine

Even though direct cause and effect has not been proved, clinical evidence suggests serotonin and substance P (SP) are involved in the emetic response following chemotherapy. Because of several parallels, we hypothesized that SP release, like serotonin, may be propagated by chemotherapy and both substances can be measured in biological fluids, and correlated with a particular phase of emesis.

5-Hydroxyindoleacetic acid and substance P profiles in patients receiving emetogenic chemotherapy.

Respiratory syncytial virus (RSV) is the most common respiratory pathogen in infants and young children. The pathophysiology of this infection in the respiratory system has been studied extensively, but little is known about its consequences in other systems. We studied whether RSV infects human bone marrow stromal cells (BMSCs) in vitro and in vivo, and investigated whether and how this infection affects BMSC structure and hematopoietic support function. Primary human BMSCs were infected in vitro with recombinant RSV expressing green fluorescent protein. In addition, RNA from naive BMSCs was amplified by PCR, and the products were sequenced to confirm homology with the RSV genome. The BMSC cytoskeleton was visualized by immunostaining for actin. Finally, we analyzed infected BMSCs for the expression of multiple cytokines and chemokines, evaluated their hematopoietic support capacity, and measured their chemotactic activity for both lymphoid and myeloid cells. We found that BMSCs support RSV replication in vitro with efficiency that varies among cell lines derived from different donors; furthermore, RNA sequences homologous to the RSV genome were found in naive primary human BMSCs. RSV infection disrupted cytoskeletal actin microfilaments, altered cytokine/chemokine expression patterns, decreased the ability of BMSCs to support B cell maturation, and modulated local chemotaxis. Our data indicate that RSV infects human BMSCs in vitro, and this infection has important structural and functional consequences that might affect hematopoietic and immune functions. Furthermore, we have amplified viral RNA from naive primary BMSCs, suggesting that in vivo these cells provide RSV with an extrapulmonary target.

Respiratory syncytial virus infection in human bone marrow stromal cells.

Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotropic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the responsiveness of the microenvironment to diverse stress that is important to consider in therapeutic settings.

Melphalan exposure induces an interleukin-6 deficit in bone marrow stromal cells and osteoblasts.

&#x9020;&#x8840;&#x91CD;&#x5EFA;&#x3001; &#x4EE5;&#x4E0B;&#x9AA8;&#x9AD3;&#x6216;&#x5E72;&#x7EC6;&#x80DE;&#x79FB;&#x690D;&#xFF0C;&#x9700;&#x8981;&#x80FD;&#x591F;&#x652F;&#x6301;&#x672A;&#x6210;&#x719F;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#x548C;&#x5E72;&#x7EC6;&#x80DE;&#x6709;&#x80FD;&#x529B;&#x5206;&#x5316;&#x548C;&#x6269;&#x5927;&#x5FAE;&#x73AF;&#x5883;&#x751F;&#x6001;&#x4F4D;&#x3002;&#x6210;&#x9AA8;&#x7EC6;&#x80DE;&#x5305;&#x62EC;&#x8FD9;&#x4E2A;&#x9886;&#x57DF;&#x7684;&#x4E00;&#x4E2A;&#x91CD;&#x8981;&#x7EC4;&#x6210;&#x90E8;&#x5206;&#x3002;&#x6211;&#x4EEC;&#x5DF2;&#x786E;&#x5B9A;&#x4EBA;&#x7C7B;&#x9AA8; (&#x6EDA;) &#x4E0E;&#x9A6C;&#x6CD5;&#x5170;&#x6216;&#x526F;&#x603B;&#x88C1; 16 &#x6CBB;&#x7597;&#x5BFC;&#x81F4;&#x589E;&#x52A0;&#x78F7; Smad2&#xFF0C;&#x7B26;&#x5408; TGF-&beta;1 &#x6D3B;&#x52A8;&#x589E;&#x52A0;&#x3002;&#x8FD9;&#x79CD;&#x589E;&#x52A0;&#x662F;&#x6EDA;&#x5200;&#x80FD;&#x529B;&#x964D;&#x4F4E;&#xFF0C;&#x4EE5;&#x652F;&#x6301;&#x672A;&#x6210;&#x719F; B &#x6CBF;&#x88AD;&#x7EC6;&#x80DE;&#x8D8B;&#x5316;&#x6027;&#x548C;&#x9075;&#x5B88;&#x4E0E;&#x91CD;&#x5408;&#x3002;&#x652F;&#x6301;&#x8D22;&#x653F;&#x8D64;&#x5B57;&#x5E76;&#x4E0D;&#x5C40;&#x9650;&#x4E8E;&#x81F4;&#x529B;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#xFF0C;&#x4F5C;&#x4E3A;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE; &#xFF08;hESC&#xFF09; &#x6216; /cd34 + &#x9AA8;&#x9AD3;&#x7EC6;&#x80DE;&#x6EDA;&#x5230;&#x9A6C;&#x6CD5;&#x5170;&#x3001; &#x526F;&#x603B;&#x88C1; 16 &#x6216; rTGF &beta;1 pre-exposed &#x5171;&#x57F9;&#x517B;&#x4E86;&#x4ECE;&#x672A;&#x7ECF;&#x5904;&#x7406;&#x7684;&#x6EDA;&#x5200;&#x5171;&#x57F9;&#x517B;&#x7684;&#x540C;&#x4E00;&#x7FA4;&#x4F53;&#x4E0D;&#x540C;&#x7684;&#x914D;&#x7F6E;&#x6587;&#x4EF6;&#x3002;&#x529F;&#x80FD;&#x652F;&#x6301;&#x8D64;&#x5B57;&#x662F;&#x4E0B;&#x6E38;&#x7684;&#x5316;&#x7597;&#x66B4;&#x9732;&#x540E;&#x7684;&#x6EDA;&#x5200;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x8C31;&#x7684;&#x53D8;&#x5316;&#x3002;&#x9A6C;&#x6CD5;&#x5170;&#x548C;&#x526F;&#x603B;&#x88C1; 16 &#x8BF1;&#x5BFC;&#x635F;&#x4F24;&#x7684;&#x6EDA;&#x5200;&#x8868;&#x660E;&#x6613;&#x5728;&#x5FAA;&#x73AF;&#x7CFB;&#x7EDF;&#x7597;&#x7A0B;&#x4E2D;&#x7ECF;&#x5E38;&#x4F7F;&#x7528;&#x7684;&#x6CBB;&#x7597;&#x5242;&#x7684;&#x8FD9;&#x4E2A;&#x5173;&#x952E;&#x9886;&#x57DF;&#xFF0C;&#x5E76;&#x5EFA;&#x8BAE;&#x8BE5;&#x5242;&#x91CF;&#x5347;&#x7EA7;&#x7684;&#x5316;&#x7597;&#x53EF;&#x80FD;&#x6709;&#x52A9;&#x4E8E;&#x79FB;&#x690D;&#x9020;&#x8840;&#x8D64;&#x5B57;&#x901A;&#x8FC7;&#x7834;&#x574F;&#x7684;&#x7ED3;&#x6784;&#x7EC4;&#x4EF6;&#x7684;&#x6B64;&#x652F;&#x6301;&#x7684;&#x5229;&#x57FA;&#x3002;

&#x9AA8;&#x9AD3;&#x6210;&#x9AA8;&#x7EC6;&#x80DE;&#x7684;&#x5316;&#x7597;&#x836F;&#x7269;&#x7684;&#x635F;&#x5BB3;&#x3002;

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-Î²1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-Î²1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.

Bone marrow osteoblast damage by chemotherapeutic agents.

Reconstitution h&eacute;matopo&iuml;&eacute;tique, suivante la moelle osseuse ou cellules souches h&eacute;matopo&iuml;&eacute;tiques, n&eacute;cessite une niche microenvironnement capable de supporter des cellules souches immatures et cellules souches avec la capacit&eacute; de diff&eacute;rencier et d&#39;&eacute;largir. Ost&eacute;oblastes forment une composante importante de ce cr&eacute;neau. Nous avons d&eacute;termin&eacute; que le traitement des ost&eacute;oblastes primaires humaines (HOB) avec melphalan ou VP-16 conduit &agrave; phospho-Smad2 accrue, coh&eacute;rent avec l&#39;augmentation de l&#39;activit&eacute; TGF-&beta;1. Cette augmentation a &eacute;t&eacute; qui co&iuml;ncide avec la diminution de la capacit&eacute; &agrave; soutenir l&#39;adh&eacute;sion et chimiotactisme de cellules immature B lign&eacute;e HOB. Le d&eacute;ficit de soutien n&#39;&eacute;tait pas limit&eacute; aux cellules prog&eacute;nitrices engag&eacute;s, comme les cellules souches embryonnaires humaines (CSEh) ou humaines moelle osseuse cellules CD34 + co-culture avec plan de cuisson sont pr&eacute;-expos&eacute;es &agrave; melphalan, VP-16 ou rTGF-&beta;1 avaient des profils distincts des populations m&ecirc;mes co-culture avec plan de cuisson non trait&eacute;e. Support fonctionnel d&eacute;ficits &eacute;taient en aval des changements dans les profils d&#39;expression g&eacute;nique HOB apr&egrave;s l&#39;exposition de la chimioth&eacute;rapie. Melphalan et VP-16 induit des dommages de HOB sugg&egrave;re la vuln&eacute;rabilit&eacute; de ce cr&eacute;neau critique d&#39;agents th&eacute;rapeutiques, souvent utilis&eacute; en association avec pr&eacute;transplantatoire et sugg&egrave;re que la dose escalad&eacute;e chimioth&eacute;rapie peut-&ecirc;tre contribuer &agrave; des d&eacute;ficits h&eacute;matopo&iuml;&eacute;tiques post-greffe en endommageant les composantes structurales de ce cr&eacute;neau favorable.

Dommages ost&eacute;oblastes de moelle osseuse par des agents chimioth&eacute;rapeutiques.

H&auml;matopoetischen Rekonstitution, folgende Knochenmark oder Stammzellen-Transplantation, erfordert eine Mikromilieu Nische unterst&uuml;tzen sowohl unreifen Vorl&auml;uferzellen und Stammzellen mit der F&auml;higkeit zu differenzieren und zu erweitern. Osteoblasten bilden eine wichtige Komponente dieser Nische. Wir festgestellt, dass die Behandlung der menschlichen prim&auml;re Osteoblasten (HOB) mit Melphalan oder VP-16 erh&ouml;hte Phospho-Smad2, mit erh&ouml;hten TGF-&beta;1-Aktivit&auml;t gef&uuml;hrt. Dieser Anstieg war zusammenfallend mit eingeschr&auml;nkter Arbeitsf&auml;higkeit der KOCHSTELLE, unreifen B Linie Zelle Chemotaxis und Einhaltung zu unterst&uuml;tzen. Unterst&uuml;tzende betrug das Defizit nicht beschr&auml;nkt auf Foul Vorl&auml;uferzellen, als menschlicher embryonaler Stammzellen (hESC) oder menschliche CD34 + Knochenmarkzellen co-cultured mit Kochfeld pre-exposed Melphalan, VP-16 oder rTGF-&beta;1 hatte profile die gleichen Populationen mit unbehandelten HOB co-cultured unterscheiden. Funktionelle Unterst&uuml;tzung Defizite wurden flussabw&auml;rts von &Auml;nderungen in Kochfeld Gen-Ausdruck-Profilen nach Chemotherapie-Exposition. Melphalan und VP-16 verursachten Schaden Kochstelle schl&auml;gt Anf&auml;lligkeit dieser kritischen Nische f&uuml;r Therapeutika unpolar im Transplantationsprozesses Therapien und schl&auml;gt diese Dosis eskalierten Chemotherapie kann dazu beitragen, post-transplantation h&auml;matopoetischen Defizite durch strukturelle Komponenten dieser unterst&uuml;tzende Nische besch&auml;digen.

Knochenmark Osteoblasten Schaden durch Chemotherapeutika.

Ricostituzione ematopoietica, seguito del midollo osseo o trapianto di cellule staminali, richiede una nicchia microambiente in grado di supportare i progenitori immaturi e cellule staminali con la capacit&agrave; di differenziare ed espandere. Osteoblasti costituiscono una componente importante di questa nicchia. Abbiamo determinato che il trattamento degli osteoblasti primari umani (piano cottura) con melfalan o VP-16 ha portato a maggiore fosfo-Smad2, coerente con l&#39;aumento dell&#39;attivit&agrave; TGF-&beta;1. Questo aumento &egrave; stato coincida con ridotta capacit&agrave; di cottura di supporto aderenza e immatura chemiotassi di cellule di lignaggio di B. Il deficit di appoggio non era limitato alle cellule progenitrici impegnata, come cellule staminali embrionali umane (hESC) o umane CD34 + cellule di midollo osseo co-cultured con piano di cottura pre-exposed di melfalan, VP-16 o rTGF-&beta;1 avevano profili distinti dalle stesse popolazioni co-cultured con piano di cottura non trattata. Supporto funzionale deficit erano a valle dei cambiamenti nei profili di espressione genica di cottura dopo esposizione a chemioterapia. Melfalan e VP-16 indotta da danni di cottura suggerisce la vulnerabilit&agrave; di questa nicchia critica agli agenti terapeutici frequentemente utilizzati nei regimi pre-transplant e suggerisce che la dose escalation chemioterapia pu&ograve; contribuire al deficit post-trapianto ematopoietico di danneggiare componenti strutturali di questa nicchia solidale.

Midollo osseo danni degli osteoblasti da agenti chemioterapici.

&#x9020;&#x8840;&#x6A5F;&#x80FD;&#x518D;&#x69CB;&#x7BC9;&#x3084;&#x6B21;&#x306E;&#x9AA8;&#x9AC4;&#x5E79;&#x7D30;&#x80DE;&#x79FB;&#x690D;, &#x672A;&#x719F;&#x306A;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x3068;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5BB9;&#x91CF;&#x3068;&#x3092;&#x533A;&#x5225;&#x3057;&#x3001;&#x5C55;&#x958B;&#x3059;&#x308B;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3067;&#x304D;&#x308B;&#x5FAE;&#x5C0F;&#x74B0;&#x5883;&#x30CB;&#x30C3;&#x30C1;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x9AA8;&#x82BD;&#x7D30;&#x80DE;&#x306F;&#x3001;&#x3053;&#x306E;&#x30CB;&#x30C3;&#x30C1;&#x306E; 1 &#x3064;&#x306E;&#x91CD;&#x8981;&#x306A;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x3092;&#x69CB;&#x6210;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x4EBA;&#x9593;&#x306E;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x9AA8;&#x82BD;&#x7D30;&#x80DE; &#xFF08;&#x30DB;&#x30D6;&#xFF09; &#x30E1;&#x30EB;&#x30D5;&#x30A1;&#x30E9;&#x30F3;&#x307E;&#x305F;&#x306F; VP 16 &#x6CBB;&#x7642;&#x5897;&#x52A0;&#x30D5;&#x30A9;&#x30B9;&#x30D5;&#x30A9; Smad2, TGF-&beta; 1 &#x6D3B;&#x52D5;&#x306E;&#x5897;&#x52A0;&#x3068;&#x4E00;&#x8CAB;&#x6027;&#x306E;&#x7D50;&#x679C;&#x3092;&#x6C7A;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x5897;&#x52A0;&#x306F;&#x672A;&#x719F;&#x306A; B &#x7CFB;&#x7D71;&#x306E;&#x7D30;&#x80DE;&#x8D70;&#x5316;&#x6027;&#x3068;&#x5BC6;&#x7740;&#x6027;&#x3092;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x524A;&#x6E1B;&#x306E;&#x30DB;&#x30D6;&#x80FD;&#x529B;&#x3068;&#x4E00;&#x81F4;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x4EBA;&#x9593;&#x306E;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE; (hESC) &#x3068;&#x3057;&#x3066;&#x652F;&#x6301;&#x8D64;&#x5B57;&#x30B3;&#x30DF;&#x30C3;&#x30C8;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306B;&#x9650;&#x5B9A;&#x3055;&#x308C;&#x306A;&#x3044;&#x3067;&#x3057;&#x305F;&#x307E;&#x305F;&#x306F;&#x30E1;&#x30EB;&#x30D5;&#x30A1;&#x30E9;&#x30F3;&#x3001;VP 16 &#x307E;&#x305F;&#x306F; rTGF &beta; 1 pre-exposed &#x30DB;&#x30D6;&#x306B;&#x3088;&#x308B;&#x5171;&#x5B58;&#x57F9;&#x990A;&#x3072;&#x3068; cd34 &#x9AA8;&#x9AC4;&#x7D30;&#x80DE;&#x672A;&#x51E6;&#x7406;&#x306E;&#x30B3;&#x30F3;&#x30ED;&#x3067;&#x5171;&#x57F9;&#x990A;&#x540C;&#x3058;&#x96C6;&#x56E3;&#x304B;&#x3089;&#x500B;&#x5225;&#x306E;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x305F;&#x3002;&#x6A5F;&#x80FD;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x306E;&#x8D64;&#x5B57;&#x306F;&#x4E0B;&#x6D41;&#x306E;&#x5316;&#x5B66;&#x7642;&#x6CD5;&#x66DD;&#x9732;&#x5F8C;&#x30DB;&#x30D6;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x306E;&#x5909;&#x5316;&#x3060;&#x3063;&#x305F;&#x3002;&#x30E1;&#x30EB;&#x30D5;&#x30A1;&#x30E9;&#x30F3;&#x3068; VP 16 &#x8A98;&#x8D77;&#x30DB;&#x30D6;&#x306E;&#x640D;&#x50B7;&#x6CBB;&#x7642;&#x85AC; pre-transplant &#x306E;&#x30EC;&#x30B8;&#x30E1;&#x30F3;&#x306E;&#x6D3B;&#x7528;&#x983B;&#x5EA6;&#x306B;&#x3053;&#x306E;&#x91CD;&#x8981;&#x306A;&#x30CB;&#x30C3;&#x30C1;&#x5E02;&#x5834;&#x306E;&#x8106;&#x5F31;&#x6027;&#x3092;&#x793A;&#x5506;&#x3057;&#x3001;&#x305D;&#x306E;&#x7DDA;&#x91CF;&#x3092;&#x793A;&#x5506;&#x30A8;&#x30B9;&#x30AB;&#x30EC;&#x30FC;&#x30C8;&#x3055;&#x308C;&#x305F;&#x5316;&#x5B66;&#x7642;&#x6CD5;&#x306F;&#x3001;&#x3053;&#x306E;&#x652F;&#x6301;&#x306E;&#x30CB;&#x30C3;&#x30C1;&#x306E;&#x69CB;&#x9020;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x3092;&#x640D;&#x50B7;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;, &#x79FB;&#x690D;&#x5F8C;&#x306E;&#x9020;&#x8840;&#x969C;&#x5BB3;&#x306B;&#x8CA2;&#x732E;&#x3059;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3002;

&#x5316;&#x5B66;&#x7642;&#x6CD5;&#x306E;&#x30A8;&#x30FC;&#x30B8;&#x30A7;&#x30F3;&#x30C8;&#x306B;&#x3088;&#x308B;&#x9AA8;&#x9AC4;&#x9AA8;&#x82BD;&#x7D30;&#x80DE;&#x306E;&#x640D;&#x50B7;&#x3002;

Hematopoietic &#xC7AC;&#xAD6C;&#xC131;, &#xB2E4;&#xC74C; &#xACE8; &#xC218; &#xB610;&#xB294; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xAC00; &#xC2DD;&#xC740;, microenvironment &#xD2C8;&#xC0C8; &#xC2DC;&#xC7A5;&#xC744; &#xC9C0; &#xC6D0;&#xD558;&#xB294; &#xBBF8; &#xC219; progenitors&#xC640; &#xC6A9;&#xB7C9;&#xC744; &#xAC00;&#xC9C4; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xD654; &#xBC0F; &#xD655;&#xC7A5;&#xC744; &#xC694;&#xAD6C; &#xD55C;&#xB2E4;. Osteoblasts&#xC774;&#xC774; &#xD2C8;&#xC0C8;&#xC758; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xB97C; &#xAD6C;&#xC131; &#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xC99D;&#xAC00; &#xC778;-Smad2, TGF-&beta;1 &#xD65C;&#xB3D9; &#xC99D;&#xAC00;&#xC640; &#xC77C;&#xCE58; &#xADC0;&#xCC29;&#xB418; melphalan &#xB610;&#xB294; &#xBD80;&#xC0AC;&#xC7A5; 16 &#xC778;&#xAC04;&#xC758; &#xAE30;&#xBCF8; osteoblasts (&#xD649;)&#xC758; &#xCE58;&#xB8CC; &#xACB0;&#xC815;. &#xC774; &#xC99D;&#xAC00; &#xBBF8; &#xC219; B &#xD608;&#xD1B5; &#xC140; chemotaxis &#xBC0F; &#xC900;&#xC218; &#xC9C0;&#xC6D0; &#xD558;&#xB3C4;&#xB85D; &#xAC10;&#xC18C; &#xD649; &#xC6A9;&#xB7C9; &#xC77C;&#xCE58; &#xD569;&#xB2C8;&#xB2E4;. &#xC9C0;&#xC6D0; &#xC801;&#xC790;&#xB294; &#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; (hESC)&#xB85C; &#xC11C; &#xCD5C;&#xC120;&#xC744; &#xB2E4;&#xD558;&#xACE0; &#xC2DC; &#xC870; &#xC140;&#xC73C;&#xB85C; &#xC81C;&#xD55C; &#xB610;&#xB294; &#xC778;&#xAC04; &#xACE8; &#xC218; &#xC138;&#xD3EC; CD34 + &#xD649; melphalan, &#xBD80;&#xC0AC;&#xC7A5;-16 &#xB610;&#xB294; rTGF &beta;1 pre-exposed&#xC640; co-cultured &#xD504;&#xB85C;&#xD544; &#xCE58;&#xB8CC; &#xD638;&#xACFC; co-cultured &#xAC19;&#xC740; &#xC778;&#xAD6C; &#xAD6C;&#xBD84; &#xD588;&#xB2E4;. &#xAE30;&#xB2A5; &#xC9C0;&#xC6D0; &#xC801;&#xC790; &#xB2E4;&#xC6B4;&#xC2A4;&#xD2B8;&#xB9BC; &#xD654;&#xD559;&#xC694;&#xBC95; &#xB178;&#xCD9C; &#xB2E4;&#xC74C; &#xD649; &#xC9C4; &#xC2DD; &#xD504;&#xB85C;&#xD544;&#xC5D0; &#xBCC0;&#xD654; &#xD588;&#xB2E4;. Melphalan&#xC640; &#xBD80;&#xC0AC;&#xC7A5; 16 &#xC720;&#xB3C4; &#xD649;&#xC758; &#xC190;&#xC0C1; &#xCE58;&#xB8CC;&#xC81C; pre-transplant regimens&#xC5D0; &#xC790;&#xC8FC; &#xD65C;&#xC6A9;&#xC774; &#xC911;&#xC694; &#xD55C; &#xD2C8;&#xC0C8;&#xC758; &#xCDE8;&#xC57D;&#xC810;&#xC744; &#xC81C;&#xC548; &#xD558; &#xACE0; &#xBCF5;&#xC6A9;&#xB7C9;&#xC744; &#xC81C;&#xC548; &#xC5D0;&#xC2A4;&#xCEEC;&#xB808;&#xC774;&#xC158; &#xB41C; &#xD654;&#xD559;&#xC694;&#xBC95;&#xC774; &#xC9C0;&#xC9C0; &#xD2C8;&#xC0C8;&#xC758; &#xAD6C;&#xC870;&#xC801; &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xB97C; &#xC190;&#xC0C1;&#xC5D0; &#xC758;&#xD574; post-transplantation &#xC870; &#xD608; &#xC801;&#xC790;&#xC5D0; &#xAE30;&#xC5EC;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xD654;&#xD559;&#xC694;&#xBC95; &#xC5D0;&#xC774;&#xC804;&#xD2B8;&#xC5D0; &#xC758;&#xD574; &#xACE8; &#xC218; osteoblast &#xC190;&#xC0C1;&#xC785;&#xB2C8;&#xB2E4;.

Reconstituci&oacute;n hematopoy&eacute;tica, m&eacute;dula siguiente &oacute;sea o trasplante de c&eacute;lulas madre, necesita un nicho de microambiente capaz de soportar tanto progenitores inmaduras y c&eacute;lulas con la capacidad para diferenciar y ampliar. Los osteoblastos forman un componente importante de este nicho. Determinamos que el tratamiento de osteoblastos humanos primarios (HOB) con melfal&aacute;n o VP-16 dio lugar a mayor phospho-Smad2, consistente con una mayor actividad de TGF-&beta;1. Este aumento fue coincidente con la capacidad reducida de la encimera para apoyar la adherencia y Quimiotaxis de c&eacute;lulas de linaje de B inmaduro. El d&eacute;ficit apoyo no se limita a las c&eacute;lulas progenitoras comprometidas, como c&eacute;lulas madre embrionarias humanas (hESC) o humanas c&eacute;lulas CD34 + m&eacute;dula &oacute;sea co-cultured con placa pre-exposed melfal&aacute;n, VP-16 u rTGF-&beta;1 ten&iacute;an perfiles distintos de las mismas poblaciones co-cultured con encimera sin tratar. D&eacute;ficit de apoyo funcional fueron aguas abajo de los cambios en los perfiles de expresi&oacute;n g&eacute;nica encimera despu&eacute;s de la exposici&oacute;n de la quimioterapia. Melfal&aacute;n y VP-16 inducida por da&ntilde;o de encimera sugiere vulnerabilidad de este nicho cr&iacute;tica a agentes terap&eacute;uticos utilizados con frecuencia en reg&iacute;menes pre-trasplante y sugiere que la dosis escalada quimioterapia puede contribuir al d&eacute;ficit hematopoy&eacute;ticos post-transplante por da&ntilde;ar componentes estructurales de este nicho apoyo.

Da&ntilde;os por agentes quimioterap&eacute;uticos en m&eacute;dula &oacute;sea osteobl&aacute;stica.

Osteoblasts are a major component of the bone marrow microenvironment, which provide support for hematopoietic cell development. Functional disruption of any element of the bone marrow niche, including osteoblasts, can potentially impair hematopoiesis. We have studied the effect of two widely used drugs with different mechanisms of action, etoposide (VP16) and melphalan, on murine osteoblasts at distinct stages of maturation. VP16 and melphalan delayed maturation of preosteoblasts and altered CXCL12 protein levels, a key regulator of hematopoietic cell homing to the bone marrow. Sublethal concentrations of VP16 and melphalan also decreased the levels of several transcripts which contribute to the composition of the extracellular matrix (ECM) including osteopontin (OPN), osteocalcin (OCN), and collagen 1A1 (Col1a1). The impact of chemotherapy on message and protein levels for some targets was not always aligned, suggesting differential responses at the transcription and translation or protein stability levels. As one of the main functions of a mature osteoblast is to synthesize ECM of a defined composition, disruption of the ratio of its components may be one mechanism by which chemotherapy affects the ability of osteoblasts to support hematopoietic recovery coincident with altered marrow architecture. Collectively, these observations suggest that the osteoblast compartment of the marrow hematopoietic niche is vulnerable to functional dysregulation by damage imposed by agents frequently used in clinical settings. Understanding the mechanistic underpinning of chemotherapy-induced changes on the hematopoietic support capacity of the marrow microenvironment may contribute to improved strategies to optimize patient recovery post-transplantation.

Bone marrow osteoblast vulnerability to chemotherapy.

Acute lymphoblastic leukemia (ALL) treatment regimens have dramatically improved the survival of ALL patients. However, chemoresistant minimal residual disease that persists following cessation of therapy contributes to aggressive relapse. The bone marrow microenvironment (BMM) is an established "site of sanctuary" for ALL, as well as myeloid-lineage hematopoietic disease, with signals in this unique anatomic location contributing to drug resistance. Several models have been developed to recapitulate the interactions between the BMM and ALL cells. However, many in vitro models fail to accurately reflect the level of protection afforded to the most resistant subset of leukemic cells during coculture with BMM elements. Preclinical in vivo models have advantages, but can be costly, and are often not fully informed by optimal in vitro studies. We describe an innovative extension of 2-D coculture wherein ALL cells uniquely interact with bone marrow-derived stromal cells. Tumor cells in this model bury beneath primary human bone marrow-derived stromal cells or osteoblasts, termed "phase dim" ALL, and exhibit a unique phenotype characterized by altered metabolism, distinct protein expression profiles, increased quiescence, and pronounced chemotherapy resistance. Investigation focused on the phase dim subpopulation may more efficiently inform preclinical design and investigation of the minimal residual disease and relapse that arise from BMM-supported leukemic tumor cells.

Debbie Piktel

West Virginia University School of Medicine

Modeling Chemotherapy Resistant Leukemia In Vitro