A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Torrey Pines Institute for Molecular Studies

Bone marrow hypoplasia and pancytopenia are among the most undesirable sequelae of chemotherapy for the treatment of cancer. We recently showed that hyaluronan (HA) facilitates hematopoietic recovery in tumor-free animals receiving chemotherapeutic agents. However, following a chemotherapeutic regimen in tumor-bearing animals, it is possible that residual tumor cells might respond to systemic injections of HA. Thus, in this study, we investigated the effect of HA on the regrowth of residual tumor cells following chemotherapy. As a model, we used the HCT-8 human colon carcinoma cell line, which expresses the HA receptor CD44, binds exogenous HA, and is susceptible to a chemotherapy protocol containing irinotecan and 5-fluorouracil in a human/mouse xenograft model. HCT-8 cells were implanted in severe combined immunodeficient mice, followed by irinotecan/5-fluorouracil treatment. After three rounds of chemotherapy, residual tumors were allowed to regrow in the presence or absence of HA. The dynamics of tumor regrowth in the group treated with HA was slower compared with the control group. By week 5 after tumor implantation, the difference in the size of regrown tumors was statistically significant and correlated with lower proliferation and higher apoptosis in HA-treated tumors as compared with controls. This finding provides evidence that HA treatment does not stimulate but delays the growth of residual cancer cells, which is an important parameter in establishing whether the use of HA can enhance current chemotherapeutic strategies.

Hyaluronan inhibits postchemotherapy tumor regrowth in a colon carcinoma xenograft model.

Pancytop&eacute;nie et hypoplasie de la moelle osseuse sont parmi les s&eacute;quelles plus n&eacute;fastes de la chimioth&eacute;rapie pour le traitement du cancer. Nous avons montr&eacute; r&eacute;cemment qu&#39;acide hyaluronique (HA) facilite la reprise h&eacute;matopo&iuml;&eacute;tique chez les animaux libres tumeur recevant des agents chimioth&eacute;rapeutiques. Cependant, suite &agrave; un sch&eacute;ma chimioth&eacute;rapeutique chez les animaux porteurs de tumeur, il est possible que les cellules tumorales r&eacute;siduelles peuvent r&eacute;pondre aux injections syst&eacute;miques de l&#39;Ah. Ainsi, dans cette &eacute;tude, nous avons &eacute;tudi&eacute; l&#39;effet du HA sur la repousse des cellules de tumeur r&eacute;siduelle apr&egrave;s chimioth&eacute;rapie. Comme un mod&egrave;le, nous avons utilis&eacute; la lign&eacute;e cellulaire de cancer du c&ocirc;lon humain de HCT-8, qui exprime le r&eacute;cepteur HA CD44, lie exog&egrave;ne HA et est sensible &agrave; un protocole de chimioth&eacute;rapie contenant l&#39;irinot&eacute;can et 5-fluorouracile dans un mod&egrave;le de x&eacute;nogreffe humain/souris. HCT-8 cellules ont &eacute;t&eacute; implant&eacute;es dans des souris immunod&eacute;ficientes combin&eacute;es s&eacute;v&egrave;res, suivies par un traitement de l&#39;irinot&eacute;can/5-fluorouracile. Apr&egrave;s trois cycles de chimioth&eacute;rapie, tumeurs r&eacute;siduelles pouvaient repousser en pr&eacute;sence ou en absence de l&#39;Ah. La dynamique de la tumeur repousse dans le groupe trait&eacute; par HA a &eacute;t&eacute; plus lent par rapport au groupe t&eacute;moin. Semaine 5 apr&egrave;s l&#39;implantation de la tumeur, la diff&eacute;rence dans la taille des tumeurs repousse &eacute;tait statistiquement significative et en corr&eacute;lation avec la plus faible prolif&eacute;ration et apoptose plus &eacute;lev&eacute;e dans les tumeurs trait&eacute;es HA comparativement aux t&eacute;moins. Cette constatation prouve que HA traitement ne stimule pas mais qu&#39;il retarde la croissance des cellules canc&eacute;reuses r&eacute;siduelles, qui est un param&egrave;tre important pour &eacute;tablir si l&#39;utilisation d&#39;acide hyaluronique peut am&eacute;liorer les strat&eacute;gies actuelles de chimioth&eacute;rapie.

Acide hyaluronique inhibe la repousse de la tumeur postchemotherapy dans un mod&egrave;le de x&eacute;nogreffe de cancer du c&ocirc;lon.

Pancitopenia e ipoplasia del midollo osseo sono tra le sequele pi&ugrave; indesiderate della chemioterapia per il trattamento del cancro. Abbiamo recentemente mostrato che acido ialuronico (HA) facilita il recupero ematopoietico in animali senza tumore ricevendo agenti chemioterapici. Tuttavia, a seguito di un regime chemioterapico negli animali del tumore-cuscinetto, &egrave; possibile che le cellule tumorali residue potrebbero rispondere alle iniezioni sistemiche di HA. Cos&igrave;, in questo studio, abbiamo studiato l&#39;effetto di HA sulla ricrescita di cellule tumorali residue dopo la chemioterapia. Come un modello, abbiamo utilizzato l&#39;HCT-8 umano del colon carcinoma a cell line, che esprime il recettore HA CD44, lega HA esogeno ed &egrave; suscettibile di un protocollo di chemioterapia contenente irinotecan e 5-fluorouracile in un modello di xenoinnesto umani e mouse. HCT-8 celle furono impiantate in gravi combinati topi immunodeficienti, seguiti da un trattamento di irinotecan/5-fluorouracile. Dopo tre cicli di chemioterapia, tumori residui erano autorizzati a ricrescere in presenza o assenza di HA. La dinamica della ricrescita del tumore nel gruppo trattato con HA &egrave; stato pi&ugrave; lento rispetto al gruppo di controllo. Settimana 5 dopo l&#39;impianto del tumore, la differenza delle dimensioni dei tumori regrown era statisticamente significativa e correlato con proliferazione inferiore e superiore apoptosi in tumori HA trattati rispetto ai controlli. Questa scoperta fornisce la prova che HA trattamento non stimola ma ritarda la crescita di cellule tumorali residue, che &egrave; un parametro importante nello stabilire se l&#39;uso di HA pu&ograve; migliorare strategie attuali chemioterapici.

Acido ialuronico inibisce la ricrescita del tumore sviluppatasi in un modello di xenoinnesto carcinoma del colon.

The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2(flox/flox);Has1(-/-);Has3(-/-) triple knock-out (tKO) mice as compared with wild type (WT) and Has1(-/-);Has3(-/-) double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. Collectively, the results suggest that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment.

Hyaluronan expressed by the hematopoietic microenvironment is required for bone marrow hematopoiesis.

L&#39;apport d&#39;acide hyaluronique (HA) au r&eacute;seau r&eacute;glementaire du microenvironnement h&eacute;matopo&iuml;&eacute;tique a &eacute;t&eacute; &eacute;tudi&eacute;e &agrave; l&#39;aide de la souris knock out de trois g&egrave;nes de synthase d&#39;acide hyaluronique (Has1, Has2 et Has3). Le nombre de prog&eacute;niteurs h&eacute;matopo&iuml;&eacute;tiques &eacute;tait diminu&eacute; dans la moelle osseuse et augmente dans les sites extram&eacute;dullaire de Prx1-Cre ;Has2(Flox/Flox) ;Has1(-/-) ;Souris Has3(-/-) (tKO) knock out &agrave; triples par rapport au type sauvage (WT) et Has1(-/-) ;Has3(-/-) souris de double knock out (dKO). Conform&eacute;ment &agrave; cette observation, diminution de l&#39;activit&eacute; h&eacute;matopo&iuml;&eacute;tique a &eacute;t&eacute; observ&eacute;e &agrave; long terme la moelle osseuse cultures (LTBMC) chez des souris de tKO, consid&eacute;rant que la formation de la couche adh&eacute;rente et cr&eacute;ation de cellules h&eacute;matopo&iuml;&eacute;tiques en cultures WT et dKO n&#39;&eacute;taient pas diff&eacute;rents. 4-M&eacute;thylumbellif&eacute;rone (4MU) servait &agrave; pharmacologiquement inhibent la production de HA en LTBMC. Traitement avec 4MU inhibe la synth&egrave;se des HA, a diminu&eacute; l&#39;expression de HAS2 et HAS3 et &eacute;limin&eacute; l&#39;h&eacute;matopo&iuml;&egrave;se en LTBMC, et cet effet a &eacute;t&eacute; att&eacute;nu&eacute; par l&#39;addition d&#39;acide hyaluronique exog&egrave;ne. HA exog&egrave;ne a &eacute;galement renforc&eacute; la motilit&eacute; cellulaire dans LTBMC, qui en corr&eacute;lation avec la production stimul&eacute;e par HA de chimiokines et facteurs de croissance. Les milieux conditionn&eacute;s de LTBMC induite par l&#39;HA am&eacute;lior&eacute; le chimiotactisme des cellules souches/prog&eacute;nitrices h&eacute;matopo&iuml;&eacute;tiques (CSPH) en r&eacute;ponse aux SDF-1. L&#39;exposition des cellules endoth&eacute;liales &agrave; 4MU diminu&eacute; leur capacit&eacute; &agrave; soutenir HSPC laminage et adh&eacute;rence. En outre, la migration du HSPC transplant&eacute;e dans la moelle de souris pr&eacute;trait&eacute;es 4MU &eacute;tait plus faible que chez les souris non trait&eacute;es. Collectivement, ces r&eacute;sultats sugg&egrave;rent que l&#39;appauvrissement HA r&eacute;duit la capacit&eacute; du microenvironnement de soutenir HSPC et confirmer un r&ocirc;le pour HA comme un &eacute;l&eacute;ment r&eacute;gulateur n&eacute;cessaire dans la structure du microenvironnement h&eacute;matopo&iuml;&eacute;tique.

Acide hyaluronique exprim&eacute;e par le microenvironnement h&eacute;matopo&iuml;&eacute;tique est requis pour la moelle osseuse h&eacute;matopo&iuml;&egrave;se.

Il contributo di acido ialuronico (HA) alla rete regolamentare del microambiente ematopoietico &egrave; stato studiato utilizzando topi knock-out di tre geni synthase hyaluronan (Has1, Has2 e Has3). Il numero dei progenitori ematopoietici &egrave; stato diminuito nel midollo osseo e aumentato in siti extramidollare del Cre-Prx1;Has2(flox/flox);Has1(-/-);Has3(-/-) tripli topi knock-out il (tKO) rispetto al wild type (WT) e Has1(-/-);Has3(-/-) topi knock-out double (dKO). In linea con questa osservazione, una ridotta attivit&agrave; ematopoietiche &egrave; stata osservata nel lungo termine del midollo osseo culture (LTBMC) da topi tKO, mentre la formazione dello strato aderente e la generazione di cellule ematopoietiche in WT e dKO culture non era diversi. 4-Methylumbelliferone (4MU) &egrave; stato utilizzato per inibire farmacologicamente la produzione di HA in LTBMC. Trattamento con 4MU inibita sintesi HA, diminuita espressione di HAS2 e HAS3 ed eliminato ematopoiesi nel LTBMC, e questo effetto &egrave; stato alleviato mediante l&#39;aggiunta di esogeno HA. Esogeno HA anche aumentato la motilit&agrave; delle cellule in LTBMC, che &egrave; correlata con la produzione di chemochine e fattori di crescita HA stimolato. Media condizionata da LTBMC HA indotto rafforzata la chemiotassi di cellule staminali emopoietiche/progenitrici (HSPC) in risposta a SDF-1. Esposizione delle cellule endoteliali a 4MU diminuito la loro capacit&agrave; di sostenere HSPC rotolamento e adesione. Inoltre, migrazione di HSPC trapiantato nel midollo osseo di topi pretrattati 4MU era inferiore rispetto a topi non trattati. Collettivamente, i risultati suggeriscono che HA deplezione riduce la capacit&agrave; del microambiente per supportare HSPC, confermano un ruolo per HA come elemento normativo necessario nella struttura del microambiente ematopoietico.

Valentina Goncharova

Torrey Pines Institute for Molecular Studies

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions