Thèses sur le sujet « CXCR4 axi »

La terapia CAR-T rappresenta un approccio promettente, ma ha riportato una ridotta efficacia nella leucemia mieloide acuta (AML), a causa dell’eterogeneità del tumore, dell’assenza di antigeni target AML-specifici e del ruolo del microambiente leucemico nella protezione dei blasti e delle cellule staminali leucemiche (LSC). La nicchia midollare, nella quale risiedono le LSC, è coinvolta in attività che promuovono la progressione leucemica e sopprimono l’ematopoiesi sana. Quindi ipotizziamo che bersagliare le LSC nascoste nella nicchia potesse migliorare l’efficacia delle CAR-T. Per testare la nostra ipotesi, abbiamo agito su due fronti: 1) promuovere una migrazione efficiente delle CAR-T nella nicchia midollare, 2) selezionare un antigene target ristretto ai blasti leucemici e alle LSC. Prima, abbiamo proposto una strategia per guidare le cellule CD33.CAR CIK (Cytokine-Induced Killer), una sottopopolazione di cellule T effettrici, verso la nicchia leucemica. La chemochina CXCL12, rilasciata dalle cellule mesenchimali stromali (MSC), nella nicchia midollare, e il suo recettore CXCR4, sono coinvolti nella regolazione della migrazione dei leucociti all’interno della nicchia. Quindi, abbiamo ipotizzato che sfruttare questo asse potesse migliorare la capacità di homing delle CD33.CAR-CIK nella nicchia e favorire l’eradicazione della leucemia. Tuttavia i protocolli di manipolazione ex vivo delle CD33.CAR-CIK riducono l’espressione di CXCR4, compromettendo la capacità delle cellule infuse di raggiungere la nicchia. Quindi per implementare la capacità di homing delle CD33.CAR-CIK nel microambiente midollare, abbiamo sviluppato delle CD33.CAR-CIK overesprimenti CXCR4, nella sua forma wild-type o iperattiva mutata. Le CIK ingegnerizzate con i costrutti CD33.CAR-CXCR4 hanno mostrato un consistente aumento dell’espressione di CXCR4, senza riportare alterazioni fenotipiche e nelle funzioni effettrici CAR-associate. Inoltre, rispetto alle CD33.CAR-CIK, le cellule CD33.CAR-CXCR4WT -CIK ed in particolare le CD33.CAR-CXCR4MUT-CIK hanno dimostrato non solo una superiore risposta chemotattica in vitro verso il CXCL12 ed i surnatanti delle MSC, ma anche un aumentato homing in vivo. In seguito, per promuovere lo sviluppo di un approccio CAR-T più efficace e sicuro, abbiamo proposto di re-indirizzare il CAR verso un antigene espresso selettivamente dalle cellule AML, ma assente sulle cellule staminali ematopoietiche (HSC). TIM-3 è un immune checkpoint, svolge un ruolo centrale nella regolazione delle risposte immunitarie nell’AML e costituisce un marcatore selettivo per le LSC, senza essere espresso dalle HSC. Abbiamo disegnato un CAR di terza generazione diretto contro TIM-3, utilizzando la porzione scFv derivante da un anticorpo monoclonale anti-TIM-3. In vitro, le TIM-3.CAR-CIK hanno dimostrato di eliminare sia le linee AML che i blasti primari, senza dare tossicità verso le cellule TIM-3+ sane, come le CIK attivate, i monociti e le cellule NK. Inoltre, le TIM-3.CAR-CIK hanno eliminato in maniera selettiva le LSC (CD34+ CD38-). Infine, le TIM-3.CAR-CIK hanno mantenuto le loro capacità effettrici nonostante multiple ristimolazioni in vitro, gettando le basi per lo studio di questo costrutto in vivo. Complessivamente, entrambi gli approcci, uno implementando l’homing delle CAR-CIK alla nicchia midollare e l’altro conferendo una superiore selettività, potrebbero migliorare l’efficacia della terapia CAR-T nel contesto dell’AML.
Chimeric Antigen Receptor (CAR) T-cell therapy has produced remarkable clinical responses in patients affected by acute lymphoblastic leukemia. Unfortunately, CAR T-cells have not been equally successful in acute myeloid leukemia (AML) due to tumor heterogeneity, lack of truly AML-restricted target antigens and the role of leukemia microenvironment in blasts protection and leukemia stem cells (LSCs) maintenance. Specifically, the bone marrow (BM) niche, where LSCs reside, is involved in leukemia promoting activities whilst suppressing normal hematopoiesis. Therefore, we hypothesized that targeting LSCs at their location may enhance the potency and selectivity of CAR-T cells. To address this issue, we have designed two aims: 1) promote rapid and efficient localization of CAR T-cells within the BM niche, 2) select a leukemia-restricted antigen to specifically target AML blasts and LSCs. First, we proposed to harness CD33.CAR-redirected Cytokine-Induced Killer (CIK) cells, an alternative effector T-cell population with acquired NK-like cytotoxic activity as well as minimal alloreactivity, to selectively route their activity to leukemia transformed niche. The chemokine ligand 12 (CXCL12), released by mesenchymal stromal cells (MSCs) within the medullary niche, and its chemokine receptor 4 (CXCR4) are two pivotal players regulating leukocytes trafficking to the BM. In AML, CXCL12 interacts with CXCR4 overexpressed on blasts, promoting their migration and homing in the niche. Hence, taking advantage of this axis might facilitate CD33.CAR-CIK cells homing to the BM and therefore leukemia eradication. However, ex vivo manipulation protocols of CD33.CAR-CIK cells consistently downregulate CXCR4 expression and may affect the capacity of adoptively infused cells to migrate to BM and exert their anti-leukemic action. Therefore, to improve CD33.CAR-CIKs homing in the BM microenvironment we have developed CD33.CAR-CIK cells overexpressing CXCR4, in its wild-type or hyperactive mutant form. Notably, CIK cells engineering with CD33.CAR-CXCR4 constructs led to a consistent increase in CXCR4 expression, without altering CIK cells phenotype and CAR-related effector functions. Interestingly, compared to conventional CD33.CAR-CIK cells, CD33.CAR-CXCR4WT and especially CD33.CAR-CXCR4MUT-CIK cells demonstrated significantly superior in vitro chemotactic response toward CXCL12 and MSC-derived supernatants, and greater in vivo BM homing ability and persistence. Furthermore, to develop an effective anti-AML CAR T-cell therapy, it is fundamental to identify a LSC-specific marker, sparing the normal counterpart of hematopoietic stem cells (HSCs). T-cell immunoglobulin and mucin protein 3 (TIM-3) is an immune checkpoint molecule, it plays a central role in immune responses in AML and it is an LSC-specific marker, lacking expression on HSCs. Therefore, we designed a third-generation anti-TIM-3.CAR using the single-chain fragment variable (scFv) derived from an antagonistic ligand-blocking anti-TIM-3 antibody. In vitro, TIM-3.CAR-CIK cells efficiently killed both AML cell lines and primary AML blasts, but not normal TIM-3+ activated CIK cells, monocytes and NK-cells. Notably, we observed selective elimination of primary LSC-enriched population (CD34+ CD38-). Furthermore, TIM-3.CAR-CIK cells maintained their effector functions despite multiple in vitro restimulations, setting the basis for further exploration in in vivo models. Overall, both approaches, one improving CAR-CIK cells homing to the transformed niche and the other conferring superior safety and selectivity, might improve the efficacy of anti-AML CAR-CIK therapy.

Meningioma is the most frequent primary tumor of the central nervous system. The greatest percentage of meningiomas is benign tumors (WHO grade I). However, although surgical and radiotherapy techniques have significantly improved over the years, some meningiomas, independently from the grading, are refractory to multimodality therapies, and recur and/or undergo malignant transformation, representing an unsolved therapeutic challenge. Therefore, beside histopathologic benign appearance, biologically aggressive meningiomas need to be molecularly characterized, to identify novel therapeutic targets. In malignant tumors, recurrence is mainly ascribed to the presence of cancer stem cells (CSCs) which are expression of tumor cell heterogeneity, and sustain tumorigenesis, metastasization and drug resistance. CSCs are characterized by stem cell marker expression, self-renewal, and ability to differentiate into tumor-specific cell types. Recently, CSCs and their functional role have been also studied in benign tumors, including meningioma. A range of genes and proteins have been proposed to identify meningioma stem-like cells, among them CD105, a transmembrane glycoprotein, involved in angiogenesis and in the progression of a variety of tumors. Stemness, as well as cancer cell aggressive behavior, is a cell property strictly linked to tumor microenvironment: reciprocal interactions between growth factors, cytokines and chemokines released by both CSCs and other cell types forming the niche, modulate each other to sustain tumor growth. Chemokine signaling, and the CXCL11/CXCL12-CXCR4/CXCR7 system in particular, drives cell proliferation and migration in several solid tumors. On these premises, this study is focused on the isolation and characterization of stem-like cells from post-surgical samples of human meningiomas, delving deeply into the role of this subpopulation in meningioma aggressive behavior. Moreover, we analyzed the contribution of CXCR4-7 receptors in the regulation of their biological properties. Twenty-eight primary cell cultures have been obtained from 35 meningiomas, and maintained in stem cell-permissive culture conditions to enrich in CSCs. Putative meningioma stem cells rapidly grow, form meningospheres and express stem markers, such as Sox2, NANOG, CD133 and Oct-4. Conversely, CD105 was not differentially expressed between stem-like cells and their “non-stem” counterpart, cells grown in serum-containing medium. Moreover, stem-like cells displayed high migratory capacity and in vitro angiogenic activity, supporting their malignant phenotype. Meningioma stem-like cells displayed a distinct chemokinereceptor profile from “non-stem” cell population, and selectively respond to in vitro CXCL11 and CXCL12 stimulation enhancing proliferation, migration and vascular mimicry. Pharmacological inhibition of individual CXCR4 or CXCR7 significantly impaired CXCL12- and CXCL11-induced proliferation, chemotaxis and vessel-like structure formation, therefore suggesting that these activities are mediated by both receptors. We speculated that these receptors act as heterodimers, formed upon ligand activation and that the blockade of one of them results in a complete inhibition of biological effects. Overall our results, collected from a large number of meningioma cell cultures derived from different patients, allow the identification of a tumor subpopulation endowed with comm on stem cell-like features, and suggest that both CXCR4 and CXCR7 signaling sustains meningioma stem cell phenotype. Prospectively, the isolation and culture of stem-like cells directly from the meningioma tissues will allow to test new therapeutic compounds to block meningioma growth and invasiveness, in particular for those tumors showing an unpredictable aggressive behavior. In this context, we propose that the CXCR4-7 chemokinergic system might represent a relevant pharmacological target.

Les cellules souches et progéniteurs hématopoïétiques (CSPHs), incluant les progéniteurs multipotents (MPPs), sont responsables de la production des cellules immunes circulantes. Ils résident dans la moelle osseuse (MO) au sein de structures spécialisées, les niches endostéale et (péri)-vasculaire, qui régulent la spécification et l'engagement lymphoïde versus myéloïde des CSPHs. Dans la MO, le couple formé par la chimiokine CXCL12 et l’un de ses récepteurs, CXCR4, exerce un rôle clé dans la régulation de la rétention et la quiescence des CSPHs. Ces processus sont dérégulés dans le Syndrome WHIM (SW), une maladie immuno-hématologique rare liée à des mutations autosomiques dominantes du gène codant CXCR4, qui altèrent la désensibilisation du récepteur et conduisent à un gain de fonction en réponse à CXCL12. Cliniquement, le SW se caractérise notamment par une profonde leucopénie circulante qui affecte les lignages lymphoïde et myéloïde et dont les mécanismes restent à déterminer. Grâce à un modèle murin génétiquement modifié du SW et à l'accès à des prélèvements biologiques de patients atteints du SW, nous avons testé l'hypothèse que la lymphopénie circulante associée au SW résultait de défauts hématopoïétiques dans la MO. Nous avons révélé un rôle clé de la désensibilisation de CXCR4 dans la différenciation lymphoïde des CSPHs et identifié les MPPs comme étant le stade défectueux dans le SW. La divergence entre les lignages lymphoïde et myeloïde se produit précisément à ce stade au sein duquel règne une hétérogénéité : les MPP2/3 sont biaisés myéloïde et les MPP4 sont orientés lymphoïde. Notre compréhension de la façon dont les signaux extrinsèques (niches) et intrinsèques aux MPPs déterminent leur devenir lymphoïde versus myéloïde est encore parcellaire. Dans ce contexte, l’objectif de ma thèse a été de déterminer si et comment la signalisation de CXCR4 régule la dépendance énergétique des MPPs et à comprendre comment les voies métaboliques façonnent leur spécification lympho-myéloïde. Dans la MO des souris porteuses de la mutation gain de fonction de Cxcr4, nous avons observé une diminution du nombre de MPP4 qui contrastait avec l'augmentation des MPP2/3. L’analyse de prélèvements médullaires de patients a également permis de rapporter une diminution de la fréquence des progéniteurs lymphoïdes et une augmentation de celle des progéniteurs myéloïdes. Chez la souris mutantes, ce biais myéloïde du compartiment de MPPs s'avèrait associé à une expansion anormale et une reprogrammation moléculaire et métabolique des MPP4. Fait marquant, un traitement chronique par l’AMD3100, un antagoniste de CXCR4, permettait de normaliser le nombre de MPP4 dans la MO, de restaurer leurs propriétés métaboliques, et de corriger la lymphopénie des souris mutantes. Par conséquent, nos résultats suggèrent que l’axe CXCL12/CXCR4 est requis au maintien du potentiel lymphoïde des MPP4 au travers de la modulation de leur activité métabolique mitochondriale
Hematopoietic stem and progenitor cells (HSPCs), including the multipotent progenitors (MPPs), are responsible for replenishing immune cells. They reside in bone marrow (BM) endosteal and (peri)-vascular niches, which provide all cellular and molecular components required for their lifelong maintenance and fate. Among them, the CXCL12 chemokine and one of its receptor, CXCR4, exert a dominant role in promoting HSPC retention and quiescence. These processes are deregulated in the WHIM Syndrome (WS), a rare immunodeficiency caused by inherited heterozygous autosomal gain-of-function CXCR4 mutations that affect homologous desensitization of the receptor. Clinically, WS is notably characterized by severe, chronic circulating lymphopenia whose mechanisms remain to be elucidated. Using a mouse model carrying a naturally occurring WS-linked Cxcr4 mutation as well as human BM and blood samples, we explored the possibility that the lymphopenia in WS originates from defects at the HSPC level in BM. We reported that Cxcr4 desensitization is required for lymphoid differentiation of HSPCs and further identified the MPP stage as defective in mutant mice. The divergence between lymphoid and myeloid lineages occurs at the MPP stage, which is composed of distinct subpopulations, i.e., MPP2 and MPP3 are reported as distinct myeloid-biased MPP subsets that operate together with lymphoid-primed MPP4 to control blood leukocyte production. Our understanding of how cell-extrinsic niche-related and cell-intrinsic cues drive the lymphoid versus myeloid fate decision of MPPs is still fragmentary. Therefore, my PhD project aimed at determining whether and how CXCR4 signaling regulates bioenergetics demands of MPPs and at understanding how these metabolic pathways shape the lympho-myeloid fate of MPPs. We unraveled a myeloid skewing of the HSPC compartment in BM of WS mice and patients. In mutant mice, this partly relied on the contraction of the MPP4 pool and on cell-autonomous molecular and metabolic changes that reprogramed MPP4 away from lymphoid differentiation. Interestingly, chronic treatment with the CXCR4 antagonist AMD3100 normalized mitochondrial metabolism and fate of MPP4, while correcting circulating lymphopenia in WS mice. This study provides evidence that CXCR4 signaling acts as an essential gatekeeper for integrity of the mitochondrial machinery, which in turn controls lymphoid potential of MPP4

Aim: The chemokine system not only coordinates leukocyte migration in immunity and inflammation, but it is also implicated in the pathogenesis of many human diseases, including cancer. The expression of chemokines and their receptors is altered in many malignancies and leads to aberrant chemokine receptor signalling. Emerging evidence indicates that the tumour microenvironment has critical roles in all aspects of cancer biology, including growth, angiogenesis, metastasis and progression. One of the important representatives of this system are the chemokine ligand CXCL12 and its receptor, CXCR4 as they are most commonly found on human and murine cancer cells. Our aims are to study and understand if there are any differences in activation of signalling molecules in the downstream signalling cascades in CXC- chemokine receptors in different cell types, and to identify the importance of different effector proteins in migration of cells; the two proteins of interest include Protein Kinase C (PKC) and arrestins. Methodology: Experimentation was undertaken in MCF-7 breast cancer cells and Jurkat leukemic T-lymphocytes which both naturally express the chemokine receptor CXCR4. Small molecule inhibition and protein overexpression was used in chemotaxis and calcium release assays to measure cellular responses. Immunocytochemistry was used to determine the effect of protein blocking and protein overexpression on receptor internalisation, protein localisation and the formation of cellular structures associated with migration. Results: Inhibition of PKC has no effect on Jurkat cell migration, but it blocks MCF-7 cell migration showing that there is a difference in the usage of PKC in different cell types. Arrestin 3 is important for migration in both suspension Jurkat cells and adherent breast cancer MCF-7 cells. Conclusion: Our study shows that CXCL12-induced migration may be arrestin 3 mediated. We have also shown that activation of signalling molecules needed for CXCL12-induced migration can differ between different cell lines. Overall, the research in this thesis has identified potential signalling molecules that can be targeted to interfere with migration of cells.

Malignant melanoma represents the most aggressive form of skin cancer. Although early stage disease is treatable through surgical excision alone, late stage tumours frequently metastasise to the liver, at which point treatment options remain limited. Migration of melanoma towards metastatic sites has been shown to be associated with the CXCR4-CXCL12 chemokine axis. The chemokine receptor CXCR4 is expressed by melanoma cells and the chemokine CXCL12 is secreted by the liver. Expression of CXCL12 has been shown to be increased in liver fibrosis and therefore it was hypothesized that cells involved in liver damage may promote melanoma metastasis to this organ. CXCR4 and CXCL12 expression in melanoma and liver cells in vitro and in vivo was examined by RT-PCR, Western blotting and immunohistochemical staining. Chemotaxis assays were performed to test the ability of AMD11070 to inhibit migration of melanoma cells. Quantitative RT-PCR and Western blotting determined the influence of different fibrosis models (Carbon tetrachloride (CCl4), Bile Duct Ligation (BDL) and Methapyrilene (MP)) on CXCL12 expression. Furthermore, the migration of melanoma was examined in animal models of liver injury. Results showed that melanoma cells and different liver cell types (myofibroblasts and biliary epithelial cells) express both CXCR4 and CXCL12. CXCR4 expression in melanoma promoted migration of tumour cells towards CXCL12 secreting liver cells and AMD11070 inhibited this. CXCR4 and CXCL12 proteins of varying sizes were observed in vivo suggesting that post translational modifications of these proteins may occur. CXCL12 expression increased in three models of chronic liver injury; CCl₄, BDL and MP. In an animal model, murine melanoma cells metastasized to the lungs and to both the fibrotic and normal liver. These findings suggest that the reduction of liver cells secreting CXCL12 may help to reduce melanoma metastasis to this organ.

Le syndrome WHIM (SW) est un déficit rare caractérisé par une leuco-neutropénie (e. X. Myélokathexis) et la profusion des verrues cutanées et de carcinomes ano-génitaux due au Papillomavirus Humain (HPV). Il est associé à des dysfonctions du chimiorécepteur CXCR4 en réponse à son ligand SDF-1/CXCL12, qui sont souvent liées à des mutations hétérozygotes de CXCR4 conduisant à la troncation de l'extrémité C-terminale du récepteur impliquée dans le recrutement de l'arrestine (βarr) pour le processus de désensibilisation. Le récepteur muté (e. X. CXCR4¹º¹³) qui n'est donc plus désensibilisé et présente un gain de fonction confère aux leucocytes des patients des réponses exacerbées à CXCL12 dont nous proposons qu'elles contribuent à la pathogenèse du SW. Dans cette thèse, nous montrons que ces dysfonctions impliquent une association inattendue entre CXCR4¹º¹³ et βarr2. Cette interaction se traduit par une activation accrue et prolongée des voies de signalisation dépendantes de βarr2 en aval du récepteur et également de l' intégrité de la troisième boucle intracellulaire de CXCR4¹º¹³. Nous identifions que CXCR4¹º¹³ forme des dimères avec son homologue sauvage au sein desquels une association possible renforcée entre barr2 et CXCR4¹º¹³ pourrait contribuer aux réponses exacerbées à CXCL12. L'expression anormale de CXCL12 que nous avions identifiée dans les lésions dues à HPV provenant d'individus souffrants ou non du SW et le rôle critique de cette chimiokine dans le développement de nombreux cancers suggèrent l'implication de cet axe de signalisation dans la pathogénie virale. Dans les kératinocytes immortalisés par HPV à haut-risque, nous observons une expression anormale de CXCL12 et de ses deux récepteurs que nous caractérisons comme étant dépendante des protéines virales HPV-E6/7 et nécessaire à la prolifération et la migration des kératinocytes. Dans le contexte du SW, ce processus en coopération avec l'activation incontrôlée de CXCR4¹º¹³ pourrait contribuer à la maîignisation des lésions ano-génitales alors même que nous y avons identifié la seule présence d'HPV à faible potentiel cancérogène (bas-risque)
The WHIM syndrome (WS) is a rare immunodeficiency characterised by severe leukoneutropenia (e. G. Myelokathexis) and profuse human papillomavirus (HPV)-associated skin lesions and malignant ano-genital cohdyloma. The disease links to dysfunctions of the CXCR4 chemokine receptor in response to its ligand SDF-1/CXCL12, and associates in many cases to heterozygous mutations causing truncation in the cytoplasmic tail of the receptor that is important for the β-arrestin (βarr)-mediated receptor desensitisation process. Such truncated receptor (e. G. CXCR4¹º¹³) displays no desensitisation and thus manifests a gain of function in response to CXCL12 in leukocytes derived from WS patients, which likely contribute to the pathogenesis of the disorder. In this study, we demonstrated that such dysfunctions are in fact dependent on an unexpected interaction between βarr2 and CXCR4¹º¹³. Upon CXCL12 stimulation, the CXCR4¹º¹³receptor displays an augmented and prolonged |3arr2-dépendent signalling that relies on the integrity of the third intracellular loop of the receptor. We have also observed the existence of CXCR4wt/CXCR4¹º¹³ heterodimer from which the possible enhanced parr2/CXCR4¹º¹³ interaction may contribute to the augmented response of the receptor to CXCL12. With the abnormal expression of CXCL12 we observed in HPV-induced lesions derived from both WS and non-WS patients, and the critical role of the chemokine in tumor growth and metastasis, we speculate on the existence of an HPV/CXCL12 interplay that could be crucial for the viral-mediated pathogenesis. Using keratinocytes immortalised by the subgenomic fragment of high-risk HPV, we showed an HPV-E6/7-dependent expression of CXCL12 and its receptors and the critical role of this signalling axis in the prolifération and motility of these cells. In WS, such HPV/CXCL12-interplay may synergise with the hyperfunctioning of CXCR4, and contribute to the malignant development of ano-genital condyloma that is unusually associated with low-risk HPV - the only viral subtype we identified in these lesions

Breast cancer kills through the process of metastasis. In order to improve the prognosis of patients with breast cancer, a better understanding of the underlying factors driving the metastatic process in patients is required. One theory that helps explain the metastatic process suggests that chemokines, such as stromal cell-derived factor (SDF)-1, are overexpressed in specific distant metastatic organs, such as lung, liver, and bone, and serve to home in cancer cells that express their receptors, like CXCR4. The hypothesis of this thesis is that the SDF-1/CXCR4 axis plays an important role in the process of metastasis in breast cancer, and that this ligand/receptor axis can be exploited in the diagnostics and therapy of breast cancer. The first objective of this thesis was to determine if circulating levels of SDF-1 could predict breast cancer metastasis. We found low levels of plasma SDF-1 to be a strong independent prognostic marker, suggesting that the concentration gradient of low plasma SDF-1 and high SDF-1 expressed in the metastatic organ may be critical in driving cancer cells from the circulation to the target organ. We further determined that the levels of plasma SDF-1 were tumor-independent, identifying the first host-derived blood marker predictive of distant metastasis. The second objective was to determine if tumor expression of CXCR4 could modulate the prognostic effect of plasma SDF-1 levels. We found that patients with tumors that highly expressed the activated form of the receptor, phosphorylated-CXCR4, and low plasma SDF-1 levels had a much poorer prognosis than those patients with either risk factor alone. These results highlighted the importance of the dysfunctional relationship between the tumor and the host in the metastatic process. The third objective assessed the therapeutic potential of targeting CXCR4 with a peptide antagonist in a transgenic mouse model. In combination with a
Le cancer du sein tue par le processus des métastases. Dans le but d'améliorer le pronostic des patients atteints du cancer du sein, une meilleure compréhension des facteurs sous-jacents qui conduisent à la transformation métastatique est nécessaire. Une théorie qui explique la transformation métastatique propose que les chimiokines, telles que le stromal cell-derived factor (SDF)-1, sont surexprimées dans des organes métastatiques distants spécifiques, tels que le poumon, le foie, et les os et servent à attirer les cellules cancéreuses qui expriment leurs récepteurs, tels CXCR4. L'hypothèse de cette t hèse est donc que l'axe SDF-1/CXCR4 joue un rôle important dans la transformation métastatique dans le cancer du sein, et que cet axe ligand/récepteur peut être exploité dans le diagnostic et la thérapie du cancer du sein. Le premier objectif de cette thèse était de déterminer si les niveaux circulants de SDF-1 peuvent prédire la présence de métastases du cancer du sein. Nous avons découvert que des niveaux peu élevés de SDF-1 dans le plasma représentent un bon déterminant pronostique indépendant, suggérant que le gradient de concentration de faible niveaux de SDF-1 dans le plasma et de niveaux élevés de SDF-1 dans l'organe métastasé peut être un événement critique dans le transfert des cellules cancéreuses de la circulation sanguine jusqu'à l'organe-cible. Nous avons de plus déterminé que les niveaux plasmatiques de SDF-1 sont indépendants des tumeurs, identifiant le premier marqueur sanguin, dérivé de l'hôte, de prédiction de métastases éloignées. Le second objectif était de déterminer si l'expression tumorale de CXCR4 pourrait moduler l'effet pronostique des niveaux plasmatiques de SDF-1. Nous avons découvert que les patients dont les tumeurs expriment de façon élevée la forme activée du récepteur, CXCR4 phosphorylé, et des niveaux plasmatiq

O tráfego de leucócitos é um processo complexo, dependente da ação de inúmeras substâncias químicas, além da perfeita interação celular. Desta forma, este estudo teve como objetivo avaliar a ação dos GCe e da ANXA1 sobre o eixo SDF-1α/CXCR4 e IL-17/IL-23/G-CSF e sobre a expressão de moléculas de adesão CD18, CD49d e CD62L. Foram utilizados camundongos machos Balb/C selvagens (WT) ou ANXA1-/-. As avaliações foram realizadas em condições basais, na presença de altas concentrações de GCe e na vigência de processo inflamatório, induzidos pela administração de ACTH (5 µg/animal, i.p.) ou pela injeção de LPS (100 µg/kg, i.p.), respectivamente, ou na ausência da ação dos GCe, pela ação do RU 38486 (RU, 10 mg/kg, i.p.). A participação da ANXA1 e do receptor FPR2 foi avaliada pelo pré-tratamento com Ac2-26 (1 mg/Kg, i.p.) ou com BOC2 (10 µg/animal, i.p.) durante 4 dias, 1 vez ao dia. A quantificação total e diferencial das células foi realizada em câmara de Neubauer e em esfregaços corados por May-Grunwald ou citometria de fluxo. As quantificações de CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L e maturação granulocítica (CD11b/Ly6G) em células da medula e da circulação foram realizadas por citometria de fluxo. A expressão de ANXA1 nos tecidos do estomago e do baço foi realizada por western blotting e nas células da medula óssea e sangue circulante foi realizada por imunofluorescência. As quantificações de IL-17, IL-23, G-CSF, SDF-1α e corticosterona foram realizadas por ELISA. A quimiotaxia de neutrófilos da medula óssea e sangue periférico foi ensaiada na placa de quimiotaxia com filtro de poro de 8 µm. A fagocitose de neutrófilos apoptóticos por macrófagos da medula óssea foi avaliada por ensaio in vitro. Para verificar os efeitos do ACTH na migração de neutrófilos no processo inflamatório, foi empregado o modelo de bolsa de ar (100 µg/mL; LPS); e o comportamento dos leucócitos circulantes de animais tratados com ACTH foi avaliado pela técnica de microscopia intravital. Os resultados obtidos, que estão apresentados em quatro temáticas, mostraram que: 1) neutrófilos da medula óssea e sangue periférico expressam ANXA1 no citoplasma e membrana, bem como o receptor FPR2, constitutivamente, e a expressão de ambos é regulada pelos GCe. A ANXA1, via receptor FPR2 expresso em células da medula óssea, controlam a maturação neutrofílica e o tráfego destas células da medula óssea para o sangue. A ANXA1, via interação ao FPR2, controla o clearance de neutrófilos do sangue para a medula óssea, modulando o eixo SDF-1α/CXCR4; 2) A administração do ACTH causa neutrofilia e os neutrófilos circulantes são ANXA1+, CD18+, CD49d+, CD62L+, mostrando que injeção do ACTH in vivo altera o fenótipo destas células na circulação. Estas modificações alteram o comportamento dos neutrófilos na circulação, bem como a migração para a bolsa de ar na vigência de inflamação e para os tecidos de clearance. Estes efeitos podem ser dependentes, pelo menos em parte, da inibição de migração orientada, já que quimiotaxia frente ao fMLP ou ao SDF-1α estavam reduzidas. Ainda, o clearance de neutrófilos é reduzido em animais tratados com o ACTH pela menor atividade fagocítica e secretora dos macrófagos medulares; 3) Animais tratados com RU 38486 e ANXA1-/- mobilizam granulócitos da medula óssea para o sangue circulante e, deste compartimento para o foco de inflamação com maior intensidade que o observado em animais controles. O eixo IL-17/IL-23/G-CSF parece estar envolvido na granulopoiese e na mobilização de neutrófilos para o sangue durante a inflamação, mas não é alvo de ação da ANXA1 e o GCe nesta etapa do processo inflamatório. Adicionalmente, foi observado que na vigência de peritonite, as moléculas de adesão, CD49d e CD62L estão envolvidas no processo de migração de neutrófilos da medula óssea para o sangue. Os resultados aqui obtidos permitem concluir que os GCe e a ANXA1 são relevantes para granulopoiese e tráfego dos neutrófilos da medula óssea em condições fisiológicas e na vigência de processo inflamatório. Ainda, em conjunto com os dados da literatura, os nossos resultados podem sugerir a participação da ANXA1 dos GCe na plasticidade fenotípica dos neutrófilos de acordo com os estímulos a que são submetidos, e podem auxiliar na compreensão dos novos conceitos sobre a produção, tempo de vida, localização e funções de neutrófilos.
The traffic leukocytes is a complex process dependent on the action of severals chemical mediators, in addition to perfect cell interaction. Therefore, this study aimed to evaluate the effect of GCe and ANXA1 on SDF-1α/CXCR4 and IL-17/IL-23/G-CSF and on the expression of adhesion molecules CD18, CD49d and CD62L. Balb/C wild type and ANXA1-/- male mice were employed. The analysis were performed at physiological conditions, in the presence of high concentrations of GCe and during of inflammatory process induced by ACTH administration (5 µg/animal, i.p.) or LPS injection (100 µg/kg, i.p.), respectively or in the absence of GCe action, by the action of RU 38486 (RU, 10 mg/kg , i.p.). The involvement of the receptor FPR2 and ANXA1 was assessed by pre-treatment with Ac2-26 (1 mg/kg, i.p.) or BOC2 (10 µg/animal, i.p.) for 4 days, once a day. The quantification of total and differential cell was performed in a Neubauer chamber and stained smears by May-Grunwald and flow cytometry. Quantification of expression of CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L and granulocytic maturation (CD11b/Ly6G) in the bone marrow and circulation were performed by flow cytometry. The expression of ANXA1 on tissues was performed by western blotting and on cells from bone marrow and blood by immunocytochemistry. Quantification of IL-17, IL-23, G-CSF, SDF-1α and corticosterone were performed by ELISA. The chemotaxis of neutrophils from the bone marrow and blood was tested in the chemotaxis chamber with filter pore of 8 microns. The phagocytosis of apoptotic neutrophils by bone marrow macrophages was assessed by in vitro assay. To investigate the effects of ACTH in the migration of neutrophils in the inflammatory process, the model employed was air pouch (100 µg/ ml, LPS), and the behavior of circulating leukocytes from animals treated with ACTH were evaluated by intravital microscopy. The results obtained, which are presented in three sections, showed that: 1) neutrophils from the bone marrow and blood expressed ANXA1 in the cytoplasm and membrane, as well as FPR2, constitutively and the expression of both is regulated by GCe. The ANXA1 via FPR2 receptor expressed in bone marrow cells, controls the neutrophilic maturation and traffic of these cells from the bone marrow into the blood. The ANXA1 via interaction to FPR2 controls the clearance of neutrophils from the blood to the bone marrow by modulating the SDF-1α/CXCR4 axis; 2) the administration of ACTH induces neutrophilia and the circulating neutrophils are ANXA1+, CD18+, CD49d+ and CD62L+, showing that the injection of ACTH in vivo alters the phenotype of these cells in the blood. These modifications alter the behavior of neutrophils in the blood, as well as the migration to the air pouch in the presence of inflammation and to the tissue clearance, and these effects may be dependent, at least in part, on inhibition of migration oriented events, as chemotaxis in response to fMLP or SDF-1α were reduced. Further, the clearance of neutrophils is reduced in animals treated with ACTH due to the lower phagocytic and secretory activity of medullary macrophages; 3) Animals treated with RU 38486 and ANXA1-/- mobilize granulocytes from bone marrow into the blood, and from this compartment to the focus of inflammation with higher intensity than that observed in the control group. The axis IL-17/IL-23/G-CSF seems to be involved in granulopoiesis and mobilization of neutrophils into the blood during inflammation, but it is not the target of action of ANXA1 and GCe at this step of inflammatory process. Additionally, it was observed that in the presence of peritonitis, the adhesion molecules, CD49d and CD62L are involved in the migration of neutrophils from the bone marrow into the blood. The results obtained allow concluding that the GCe and ANXA1 are relevant to the granulopoiesis and the traffic of neutrophils from bone marrow under physiological conditions and in the presence of inflammation. Furthermore, together with literature data, the data presented here may suggest the involvement of ANXA1 the GCe in phenotypic plasticity of neutrophils according to the stimuli that are submitted, and may support to understand the new concepts of production, half-life, location and function of neutrophils.

Finalidad: El eje CXCL12/CXCR4 es un sistema de quimioquinas (ligando) y su receptor, que funcionan normalmente y son necesarios para la homeostasis de diversos sistemas de nuestro cuerpo. Se sospecha que algunos carcinomas de cabeza y cuello utilizan este eje para diseminarse hacia ganglios regionales del tumor primario. Existen pocos trabajos que sustancien esta vía como posible marcador que prediga la aparición de recidivas ganglionares. Estudiamos la capacidad pronóstica del eje CXCL12/CXCR4 sobre la posible recidiva ganglionar en pacientes tratados de CECC. Diseño experimental y resultados: Se analizaron muestras tumorales y sanas de 111 pacientes afectos de CECC (cavidad oral, orofaringe, laringe e hipofaringe) tratados con intención radical con seguimiento mínimo de 3 años. Se valoró la aparición de recidiva ganglionar, la supervivencia libre de recidiva regional, local y a distancia, y la supervivencia ajustada en función de la expresión del eje CXCL12/CXCR4 y la expresión de otras vías inflamatorias relacionadas con la respuesta al tratamiento y la capacidad de diseminación conocidas (COX, NFκB, IL-1, CD-45, MPO, MPC-1,MMP- 2 y 9, Hsp-90 y SOD). Pudimos comprobar que la expresión de CXCL12 fue menor y las de CXCR4 mayor en el tumor que en la mucosa sana. Los pacientes fueron distribuidos, a través de un método de análisis de partición recursiva, en grupos según la expresión de CXCL12 y CXCR4. Los pacientes con expresión baja de CXCR4 y, alta de CXCR4 y alta de CXCL12, tuvieron un riesgo bajo de recidiva tumoral a nivel ganglionar, 2.6% y 4.5% respectivamente. El grupo de pacientes formado por una alta expresión de CXCR4 y baja de CXCL12 contaron con un riesgo alto de recidiva regional, un 34.0%. El análisis multivariante calculó un riesgo 10.7 veces mayor de aparición de recidiva regional en éstos pacientes respecto a los del grupo anterior. Todo ello independientemente de la categoría de extensión inicial del tumor a nivel regional, del tipo de tratamiento realizado sobre las áreas ganglionares o del control local de la enfermedad. Adicionalmente se demostró una correlación significativa entre la expresión transcripcional de CXCL12 y de CXCR4 con PGIS (PGI2), SOD-2 y COX-2, PGIS, VEFG y MMP-2 respectivamente. Conclusiones: La expresión de CXCL12 fue inferior y las de CXCR4 superior de forma significativa en las muestras de tumor que la correspondiente a las muestras de mucosa sana. Los niveles de expresión de CXCL12/CXCR4 no difirieron en función de la localización del tumor ni del estatus del HPV. La expresión baja de CXCL12 y elevada de CXCR4 en pacientes con CECC demuestra un claro aumento en el riesgo a padecer una recidiva ganglionar durante la evolución del tratamiento de estos pacientes, disminuyendo la supervivencia de forma significativa con respecto a los pacientes con expresión CXCL12 y CXCR4 alta o con expresión CXCR4 baja. Existe una correlación entre CXCL12/CXCR4 y la expresión de diversos genes relacionados con la respuesta al tratamiento y la capacidad de diseminación de los CECC como PGIS, COX-2 , VEFG y MMP-2.
Purpose: The CXCL12 / CXCR4 axis is a chemokine system (ligand) and its receptor, which function normally and are necessary for the homeostasis of various body systems. It is suspected that some head and neck carcinomas using this axis to spread to regional lymph from the primary tumor. Few studies have substantiated this pathway as a possible marker to predict the occurrence of lymph node recurrences. We studied the prognostic axis CXCL12 / CXCR4 on possible nodal recurrence in patients treated for HNSCC. Experimental design and results: Healthy and tumor samples from 111 patients with HNSCC (oral cavity, oropharynx, larynx and hypopharynx) and treated with radical intent with minimum follow-up of 3 years were analyzed. The appearance of lymph node recurrence, regional recurrence free survival, local or remote and survival adjusted depending on the expression of CXCL12/CXCR4 axis expression and other inflammatory pathways associated with response to treatment and was rated capacity known spread (COX, NFkB, IL-1, CD-45, MPO, MPC-1, MMP-2 and 9, Hsp-90 and SOD). We found that the expression of CXCL12 was lower and CXCR4 higher in the tumor than in healthy mucosa. Patients were distributed through an analysis method of recursive partitioning, in groups according to the expression of CXCL12 and CXCR4. Patients with low expression of CXCR4 and high CXCR4 and CXCL12 high, had a low risk of tumor recurrence at nodal level, 2.6% and 4.5% respectively. The patient group consisting of high expression of CXCR4 CXCL12 and counted down with a high risk of local relapse, 34.0%. Multivariate analysis calculated a 10.7 times greater risk of regional recurrence in these patients compared to the previous group. All this regardless of the category of initial tumor extension at regional level, the type of treatment performed on nodal areas or local control of the disease. Additionally, a significant correlation between the transcriptional expression of CXCL12 and CXCR4 with PGIS (PGI2), SOD-2 and COX-2, PGIS, VEGF and MMP-2 respectively is shown. Conclusions: CXCL12 expression was significantly lower and upper CXCR4 in tumor samples corresponding to samples of healthy mucosa. Expression levels of CXCL12/CXCR4 not differ depending on the location of the tumor nor HPV status. The low expression of CXCL12 and CXCR4 elevated in patients with HNSCC shows a clear increase in the risk of suffering a relapse during evolution nodal treatment of these patients, decreasing significantly with survival compared to patients with high expression of CXCL12 and high CXCR4 or CXCR4 expression low. There is a correlation between CXCL12/CXCR4 and the expression of various genes related to response to treatment and the ability to spread the HNSCC as PGIS, COX-2, VEGF and MMP-2.

Dissertação de mestrado em Ciências da Saúde
The crosstalk between acute myeloid leukemia (AML) cells and their neighboring stromal cells is often key for cancer cells survival, resistance to treatment and disease relapse. Importantly, altered metabolic conditions of the bone marrow (BM) niche can influence stem cell behavior. Type 2 diabetes mellitus (T2DM) induces persistent changes in the BM microenvironment, disturbing stem cell niches and consequently hematopoiesis and BM cells function. T2DM and AML share the average age of onset, both disorders more prevalent in the elderly. Moreover, T2DM is often associated with poorer AML outcomes. Yet the association between these two disorders has not been clarified or dissected. The CXCL12/CXCR4 chemoattraction axis is a possible mechanism by which diabetes affects AML progression. The CXCL12/CXCR4 axis altered function is known to impair hematopoietic stem cells mobilization in a diabetic context, and to enhance survival and chemoresistance of AML cells. Thus, the aim of this project was to dissect the role of the CXCL12/CXCR4 axis on the AML poorer outcomes associated with diabetes‐induced damage. Specifically, we addressed the activity of the CXCL12/CXCR4 chemoattraction axis on the crosstalk between BM stromal cells and AML cells in a diabetes context. Using primary human samples, a deregulation of the CXCL12/CXCR4 patterns within the BM diabetic microenvironment was observed. Therefore, we next evaluated the effect of such alteration on the crosstalk between the AML and stromal cells. Submitting two AML cell lines (HL‐60 and KG‐1) to either BMSCs‐conditioned media or a BMSCs‐AML direct contact co‐culture system, upon normal and diabetic conditions, a strong association between CXCR4 activation and AML cells tumorigenicity was demonstrated in a diabetic context. In fact, BM stromal cells from diabetic patients were shown to activate CXCR4 receptor in HL‐60 cells, which led to activation of the Akt/MEK survival pathways, and increase of the cancer cells’ tumorigenicity. Briefly, our results support the hypothesis that the CXCL12/CXCR4 axis may play a critical role on the prognostic of AML patients that also suffer from metabolic disorders, as diabetes. This project unveils the very “tip of the iceberg” on the diabetes facilitating‐role on AML progression and tumorigenicity, within BM microenvironment.
A comunicação entre as células de leucemia mielóide aguda (LMA) e as células estromais vizinhas é, muitas vezes, crucial para a sobrevivência das células cancerígenas, resistência ao tratamento e recaída. Mais importante, condições metabólicas alteradas do nicho da medula óssea (MO) podem influenciar o comportamento das células estaminais. Diabetes mellitus tipo 2 (T2DM) induz alterações persistentes no microambiente da MO, perturbando os nichos das células estaminais e, consequentemente, a hematopoiese e as funções das células da MO. T2DM e LMA partilham a idade média de diagnóstico, sendo ambas doenças prevalentes na população idosa. Além disso, T2DM é frequentemente associada com maus prognósticos da LMA. No entanto, a associação entre as duas doenças não foi ainda estudada. O eixo de quimioatração CXCL12/CXCR4 é um mecanismo possível pelo qual diabetes afeta a progressão da LMA. A função alterada do eixo CXCL12/CXCR4 prejudica a mobilização das células estaminais hematopoiéticas num contexto de diabetes, e aumenta a sobrevivência e quimioresistência das células de LMA. Assim sendo, o objetivo deste projeto era avaliar os possíveis efeitos que o dano induzido pela diabetes na MO teria na progressão da LMA, tendo em conta o eixo de quimioatração CXCL12/CXCR4. Usando amostras humanas primárias, foi observada uma desregulação dos padrões de CXCL12/CXCR4 no microambiente diabético da MO. Portanto, de seguida avaliamos o efeito de tal alteração na comunicação entre células de LMA e células estromais. Submetendo duas linhas de AML (HL‐60 e KG‐1) a meio condicionado de células estromais derivadas da MO, ou a um sistema de co‐cultura direta, em condições normal e diabética, uma forte associação entre a ativação do CXCR4 e a tumorigenicidade das células de LMA foi demonstrada no contexto diabético. Na verdade, as células estromais da MO de pacientes diabéticos são capazes de ativar o receptor CXCR4 nas células HL‐60, levando à ativação das cascatas de sobrevivência Akt/MEK, e consequente aumento da tumorigenicidade das células de AML. Em resumo, os resultados deste projeto sugerem a hipótese de que o eixo de quimioatração CXCL12/CXCR4 tem um papel importante no prognóstico de pacientes de LMA que também sofrem de doenças metabólicas, como a diabetes. Este projeto desvenda a “ponta do iceberg” do papel facilitador da diabetes na progressão e tumorigenicidade da LMA, no microambiente da MO.

博士
長庚大學
臨床醫學研究所
99
We have divided this PhD thesis into three parts. While each is independent, these three parts of study are closely related in the subjects of cancer metastasis, tumor microenvironment and cancer stem cells. In the first part, since the understanding of immunobiology of bone marrow metastases (designated BM-NPC) versus primary tumors (P-NPC) of the nasopharynx is far from complete, the goal of this part of study was to determine if there would be differences between cultured P-NPCs and BM-NPCs with respect to (i) the expression levels of constitutive IL-6 and the gp80 subunit of IL-6 receptor (IL-6Rα) in the spent media of nontransduced P-NPC and BM-NPC cells, and (ii) the expression levels of IL-6 and IL-6Rα in the spent media of P-NPC and BM-NPC cells transduced with a retroviral vector containing the IFN–γ gene. Our results showed that in day 3 culture supernatants, low levels of soluble IL-6 were detected in 5/5 cultured tumor cell lines derived from P-NPCs, while much higher constitutive levels of IL-6 were detected in 3/3 metastasis-derived NPC cell lines including one originated from ascites; the difference was significant (p = 0.025). An inverse relationship was found between IL-6Rα and IL-6 in their release levels in cultured P-NPCs and metastasis-derived NPCs. In IFN-γ-transduced-P-NPCs, IL-6 production increased and yet IL-6Rα decreased substantially, as compared to nontransduced counterparts. At variance with P-NPC cells, the respective ongoing IL-6 and IL-6Rα release patterns of BM-NPC cells were not impeded as much following IFN-γ transduction. These observations were confirmed by extended kinetic studies with representative NPC cell lines and clonal sublines. The latter observation with the clonal sublines also indicates that selection for high IL-6 or low IL-6Rα producing subpopulations did not occur as a result of IFN-γ-transduction process. P-NPCs, which secreted constitutively only marginal levels of IFN-γ (8.4 ~ 10.5 pg/ml), could be enhanced to produce higher levels of IFN-γ (6.8- to 10.3-fold increase) after IFN-γ gene transduction. Unlike P-NPCs, BM-NPCs spontaneously released IFN-γ at moderate levels (83.8 ~ 100.7 pg/ml), which were enhanced by 1.3 to 2.2-fold in the spent media of their IFN-γ-transduced counterparts. In conclusion, our results clearly showed that cultured P-NPCs and BM-NPCs could be distinguished from one another on the basis of their differential baseline secretion pattern of IFN-γ, IL-6 and IL-6Rα, and their differential response profiles to IFN-γ gene transfer of the production of these three soluble molecules. These results suggest that the IL-6 and IFN-γ pathways in a background of genetic instability be involved in the acquisition of metastatic behavior in BM-NPCs. In the second part of the study, we looked into the mechanisms underlying tumor dormancy in human P-NPCs and BM-NPCs. The specific aim of this part of study was to determine differences in the fates of P-NPC cells, IFN-γ-transduced primary NPC (IFN-γ-P-NPC), bone marrow metastatic NPC (BM-NPC) and IFN-γ-transduced BM-NPC (IFN-γ-BM-NPC) cells following xenotransplantation into 4 different groups of SCID mice through subcutaneous injection of 5 x 106 cells/site/animal (4 animals/group). The injected mice were monitored for tumor development at the sites of injection. In only the group injected with IFN-γ-P-NPC cells, the resulting nodules remained small throughout the 60-days observation period after injection, but gradually became palpably prickly. Histopathological examination revealed that these lesions invariably consisted of mostly structures of horny pearls and keratin bridges with occasional apoptotic and degenerative cells. In contrast, animals injected with non-transduced-P-NPC cells developed tumors progressively with occasional central necroses. In the two groups injected with IFN-γ-NPC-BM and NPC-BM cells, progressive growths of tumors were noted with the latter being at slightly faster rates; while the xenografts of both groups showed a poorly differentiated phenotype with abundant vascularity. Our results highlight the high susceptibility of P-NPC, but not BM-NPC, cells, following IFN-γ gene transfer to the induction of tumor dormancy which is mediated via induced cell differentiation. Thus induced cell differentiation by agents such as IFN-γ could provide a new mechanism by which tumor dormancy is initiated and established. In the last part of the study, we looked into how the metastatic cancer stem cells (mCSCs) were converted from cancer stem cells (CSCs). As an advanced status of CSCs, mCSC shave been proposed to be the essential seeds that initiate tumor metastasis. In this study, we used a lymph node metastatic CEA-producing carcinoma cell line, UP-LN1, characterized by the persistent appearance of adherent (A) and floating (F) cells in culture, to determine the distribution of CSCs and mechanisms for the induction of mCSCs. F and A cells displayed distinct phenotypes, CD44high/CD24low and CD44low/CD24high, respectively. The CSC-rich nature of F cells was typified by stronger expression of multiple drug resistance genes and a 7.8-fold higher frequency of tumor-initiating cells in NOD/SCID mice when compared to A cells. F cells exhibited a greater depression in HLA class I expression and an extreme resistance to NK/LAK-mediated cytolysis. Moreover, the NK/LAK-resistant F cells were highly susceptible to IFN-γ-mediated induction of surface CXCR4 with concomitant down-regulation of cytoplasmic CXCL12 expression, whereas these two parameters remained essentially unchanged in NK/LAK-sensitive A cells. Following the induction of surface CXCR4, enhanced migratory/invasive potential of F cells was demonstrated by in vitro assays. Confocal immunofluorescence microscopy showed the two distinct phenotypes of F and A cells could be correspondingly identified in monodispersed and compact tumor cell areas within the patient’s LN tumor lesion. In response to IFN-γ or activated NK/LAK cells, the CXCR4+ mCSCs could be only induced from the CSCs which were harbored in the highly tumorigenic CD44high/CD24low F subset. Collectively, these results revealed the complexity and heterogeneity of the CSC of this cell line/tumor, localization of the major niche of CSCs/mCSCs in F subsets, and the differential immunomodulatory functions of F and A subsets.