Lung Cancer Onset in Wild Type Mice Following Bone Marrow Reconstitution with kras(v12) Cells.

A role for bone-marrow-derived cells (BMDCs) in tissue repair and malignancy onset has been proposed, but their contribution is still debated. We tested the ability of BMDCs containing the inducible kras(V12) oncogene to initiate lung adenocarcinoma. For our experimental strategy, we reconstituted lethally irradiated wild type mice with BMDCs carrying inducible kras(V12) and subsequently induced oncogene expression by 4-OHT administration. Epithelial lung lesions, from adenoma to adenocarcinomas, appeared at successive time points. These results show that lung tumors were derived from donor BMDCs and indicate a direct involvement of bone marrow cells in the development of epithelial cancers.

The CDC42-interacting protein 4 controls epithelial cell cohesion and tumor dissemination.

The role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-ß1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors.

A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination.

The mechanisms by which tumor cells metastasize and the role of endocytic proteins in this process are not well understood. We report that overexpression of the GTPase RAB5A, a master regulator of endocytosis, is predictive of aggressive behavior and metastatic ability in human breast cancers. RAB5A is necessary and sufficient to promote local invasion and distant dissemination of various mammary and nonmammary tumor cell lines, and this prometastatic behavior is associated with increased intratumoral cell motility. Specifically, RAB5A is necessary for the formation of invadosomes, membrane protrusions specialized in extracellular matrix (ECM) degradation. RAB5A promotes RAB4- and RABENOSYN-5-dependent endo/exocytic cycles (EECs) of critical cargos (membrane-type 1 matrix metalloprotease [MT1-MMP] and ß3 integrin) required for invadosome formation in response to motogenic stimuli. This trafficking circuitry is necessary for spatially localized hepatocyte growth factor (HGF)/MET signaling that drives invasive, proteolysis-dependent chemotaxis in vitro and for conversion of ductal carcinoma in situ to invasive ductal carcinoma in vivo. Thus, RAB5A/RAB4 EECs promote tumor dissemination by controlling a proteolytic, mesenchymal invasive program.

CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP.

Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high-density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53-dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair.

Complementary populations of human adipose CD34+ progenitor cells promote growth, angiogenesis, and metastasis of breast cancer.

Obesity is associated with an increased frequency, morbidity, and mortality of several types of neoplastic diseases, including postmenopausal breast cancer. We found that human adipose tissue contains two populations of progenitors with cooperative roles in breast cancer. CD45(-)CD34(+)CD31(+)CD13(-)CCRL2(+) endothelial cells can generate mature endothelial cells and capillaries. Their cancer-promoting effect in the breast was limited in the absence of CD45(-)CD34(+)CD31(-)CD13(+)CD140b(+) mesenchymal progenitors/adipose stromal cells (ASC), which generated pericytes and were more efficient than endothelial cells in promoting local tumor growth. Both endothelial cells and ASCs induced epithelial-to-mesenchymal transition (EMT) gene expression in luminal breast cancer cells. Endothelial cells (but not ASCs) migrated to lymph nodes and to contralateral nascent breast cancer lesions where they generated new vessels. In vitro and in vivo, endothelial cells were more efficient than ASCs in promoting tumor migration and in inducing metastases. Granulocyte colony-stimulating factor (G-CSF) effectively mobilized endothelial cells (but not ASCs), and the addition of chemotherapy and/or of CXCR4 inhibitors did not increase endothelial cell or ASC blood mobilization. Our findings suggest that adipose tissue progenitor cells cooperate in driving progression and metastatic spread of breast cancer.

Human haemato-endothelial precursors: cord blood CD34+ cells produce haemogenic endothelium.

Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144-), triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45-) capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level.

Spontaneous cell fusion of acute leukemia cells and macrophages observed in cells with leukemic potential.

Cell fusion plays a well-recognized physiological role during development, while its function during progression is still unclear. Here, we show that acute myeloid leukemia (AML) cells spontaneously fused with murine host cells in vivo. AML cells fused in most cases with mouse macrophages. Other targets of AML cell fusion were dendritic and endothelial cells. Cytogenetic and molecular analysis revealed that successive recipients conserved detectable amounts of parental DNA. Moreover, in a mouse AML1-ETO model where female AML1-ETO-leukemic cells, expressing CD45.2, were injected in congenic CD45.1 male mice AML cells, we found hybrid cells expressing both allelic types of CD45 and XXY set of sexual chromosomes. More importantly, the fusion protein AML1-ETO was transferred in the hybrid cells. When sorted hybrid cells were reinjected in a secondary recipient, they gave rise to leukemia with 100% penetrance and similar time of onset of leukemic cells. Our data indicate that in vivo fusion of cancer cells with host cells may be a mechanism of gene transfer for cancer dissemination and suggest that fused cells may be used to identify still unrecognized leukemogenic genes that are conserved in hybrid cells and able to perpetuate leukemia in vivo.

Residual dormant cancer stem-cell foci are responsible for tumor relapse after antiangiogenic metronomic therapy in hepatocellular carcinoma xenografts.

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor and the third leading cause of cancer-related deaths. Currently available chemotherapeutic options are not curative due in part to tumor resistance to conventional therapies. We generated orthotopic HCC mouse models in immunodeficient NOD/SCID/IL2rγ null mice by injection of human alpha-feto protein (hAFP)- and/or luciferase-expressing HCC cell lines and primary cells from patients, where tumor growth and spread can be accurately monitored in a non-invasive way. In this model, low-dose metronomic administration of cyclophosphamide (LDM-CTX) caused complete regression of the tumor mass. A significant increase in survival (P<0.0001), reduced aberrant angiogenesis and hyperproliferation, and decrease in the number of circulating tumor cells were found in LDM-CTX-treated animals, in comparison with untreated mice. Co-administration of LDM-CTX with anti-VEGF therapy further improved the therapeutic efficacy. However, the presence of residual circulating hAFP levels suggested that some tumor cells were still present in livers of treated mice. Immunohistochemistry revealed that those cells had a hAFP+/CD13+/PCNA- phenotype, suggesting that they were dormant cancer stem cells (CSC). Indeed, discontinuation of therapy resulted in tumor regrowth. Moreover, in-vitro LDM-CTX treatment reduced hepatosphere formation in both number and size, and the resulting spheres were enriched in CD13+ cells indicating that these cells were particularly resistant to therapy. Co-treatment of the CD13-targeting drug, bestatin, with LDM-CTX leads to slower tumor growth and a decreased tumor volume. Therefore, combining a CD13 inhibitor, which targets the CSC-like population, with LDM-CTX chemotherapy may be used to eradicate minimal residual disease and improve the treatment of liver cancer.

The white adipose tissue used in lipotransfer procedures is a rich reservoir of CD34+ progenitors able to promote cancer progression.

Previous studies have suggested a "catalytic role" in neoplastic angiogenesis and cancer progression for bone marrow-derived endothelial progenitor cells (EPC). However, preclinical and clinical studies have shown that the quantitative role of marrow-derived EPCs in cancer vascularization is extremely variable. We have found that human and murine white adipose tissue (WAT) is a very rich reservoir of CD45-CD34(+) EPCs with endothelial differentiation potential, containing a mean of 263 times more CD45-CD34(+) cells/mL than bone marrow. Compared with marrow-derived CD34(+) cells mobilized in blood by granulocyte colony-stimulating factor, purified WAT-CD34(+) cells expressed similar levels of stemness-related genes, significantly increased levels of angiogenesis-related genes, and increased levels of FAP-α, a crucial suppressor of antitumor immunity. In vitro, WAT-CD34(+) cells generated mature endothelial cells and capillary tubes as efficiently as mature mesenchymal cells. The coinjection of human WAT-CD34(+) cells from lipotransfer procedures contributed to tumor vascularization and significantly increased tumor growth and metastases in several orthotopic models of human breast cancer in immunodeficient mice. Endothelial cells derived from human WAT-CD34(+) cells lined the lumen of cancer vessels. These data indicate that CD34(+) WAT cells can promote cancer progression and metastases. Our results highlight the importance of gaining a better understanding of the role of different WAT-derived cells used in lipotransfer for breast reconstruction in patients with breast cancer.

Anti-VEGF and beyond: shaping a new generation of anti-angiogenic therapies for cancer.

The anti-angiogenic class of drugs is one of the few where representatives have gained international approval for clinical use in oncology during the past decade. Most of the biological and clinical activity of the currently available generation of anti-angiogenic drugs targets vascular endothelial growth factor (VEGF) and its related pathways. However, the clinical benefits associated with the use of these drugs have, so far, been limited. There is, therefore, an unmet need for biomarkers that can be used to identify patients who are most likely to benefit therapeutically and also to predict the best schedule and dosage for these drugs. Here, we discuss some of the emerging new combination strategies involving the approved anti-angiogenic drugs, some of the emerging targets associated with neoplastic angiogenesis and some novel agents used as a paradigm of the next generation of anti-angiogenic drugs.

Therapeutic effect of lenalidomide in a novel xenograft mouse model of human blastic NK cell lymphoma/blastic plasmacytoid dendritic cell neoplasm.

PURPOSE: Blastic natural killer (NK) cell lymphoma/blastic plasmacytoid dendritic cell neoplasm (BNKL) is a rare and aggressive neoplasia characterized by infiltration of blast CD4(+)/CD56(+) cells in the skin, the bone marrow, and peripheral blood. Currently, more efforts are required to better define molecular and biological mechanisms associated with this pathology. To the best of our knowledge, no mouse model recapitulated human BNKL so far. EXPERIMENTAL DESIGN: Primary bone marrow cells from a BNKL patient were injected in nonobese diabetes/severe combined immunodeficient interleukin (IL) 2rγ(-/-) mice with the intent to generate the first BNKL orthotopic mouse model. Moreover, because of the lack of efficient treatments for BNKL, we treated mice with lenalidomide, an immunomodulatory and antiangiogenic drug. RESULTS: We generated in mice a fatal disease resembling human BNKL. After lenalidomide treatment, we observed a significant reduction in the number of peripheral blood, bone marrow, and spleen BNKL cells. Tumor reduction parallels with a significant decrease in the number of circulating endothelial and progenitor cells and CD31(+) murine endothelial cells. In mice treated with lenalidomide, BNKL levels of active caspase-3 were significantly augmented, thus showing proapoptotic and cytotoxic effects of this drug in vivo. An opposite result was found for proliferating cell nuclear antigen, a proliferation marker. CONCLUSIONS: Our BNKL model might better define the cellular and molecular mechanisms involved in this disease, and lenalidomide might be considered for the future therapy of BNKL patients.

Evidence of distinct tumour-propagating cell populations with different properties in primary human hepatocellular carcinoma.

BACKGROUND AND AIMS: Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC). METHODS: After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice. RESULTS: The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features. CONCLUSIONS: Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution.

Circulating perivascular progenitors: a target of PDGFR inhibition.

Cancer blood vessels consist of two interacting types of cells: inner lining endothelial cells (ECs) and surrounding perivascular cells (pericytes, vascular smooth muscle cells or mural cells). PDGFRbeta(CD140b)+ progenitor perivascular cells (PPC) can differentiate into pericytes and regulate vessel stability and vascular survival in tumors. Similarly to what we have done with circulating ECs and progenitors, we developed a flow cytometry procedure for the enumeration of circulating PPCs and the study of their viability in murine models of cancer and in cancer patients. DNA+CD45-CD31-CD140b+ cells were enumerated by six-colour flow cytometry, their morphology was studied by electron microscopy, PPC specificity confirmed by reverse trascription-PCR (RT-PCR) expression of CD140b mRNA, and viability assessed by Syto16 and 7AAD. In preclinical marrow transplantation studies, 9 ± 4% of circulating PPCs were derived from the marrow donor. PPCs were increased in cancer-bearing mice and in patients affected by some types of cancer. At variance with the kinetic of circulating endothelial progenitors, high-dose cyclophosphamide reduced the number of viable PPCs. The administration of sunitinib, a drug known to inhibit PDGFR, was associated in murine models and in cancer patients with an increase of apoptotic/necrotic circulating PPC, suggesting a direct targeting of these cells. PPC enumeration might be studied as a tool for the definition of the optimal biologic dose of anti-PDGFR drugs and investigated clinically as a possible predictive/prognostic tool in patients receiving anti-PDGFR drugs.

Cellular and soluble markers of tumor angiogenesis: from patient selection to the identification of the most appropriate postresistance therapy.

Antiangiogenic drugs are now intensively used in clinical oncology, but some drawbacks still hamper their development. First, it is frequently unclear what patient subpopulation is likely to gain clinical benefit from these expensive therapies; second, there is evidence of (sometimes rapid) development of drug resistance in many patients; third, the results of some preclinical and clinical studies have suggested acceleration of malignant cell aggressiveness when some antiangiogenic therapies are terminated. Here we discuss the role of soluble molecules and cellular markers of neoplastic angiogenesis for patient selection and follow-up during treatment. These markers should help clinicians to decide the right therapy, advise them of the generation of mechanisms of drug resistance during antiangiogenic treatment, and finally suggest the most appropriate next line of therapy according to the new patterns of cancer vascularization induced by antiangiogenic therapies.

Molecular and functional analysis of the stem cell compartment of chronic myelogenous leukemia reveals the presence of a CD34- cell population with intrinsic resistance to imatinib.

We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin(-)CD34(-)) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that approximately one-third of CD34(-) cells are leukemic. CML Lin(-)CD34(-) cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells and cell cycling, and increased clonogenic activity and expression of BCR-ABL transcript. Lin(-)CD34(-) cells showed hematopoietic cell engraftment rate in 2 immunodeficient mouse strains similar to Lin-CD34(+) cells, whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell-cycle arrest genes and genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated compared with normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin(-)CD34(-) cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity, and clonogenic efficiency of CML Lin(-)CD34(-) cells in vitro. Moreover, leukemic CD34(-) cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34(-) leukemic stem cell subset in CML with peculiar molecular and functional characteristics.

Human acute leukemia cells injected in NOD/LtSz-scid/IL-2Rgamma null mice generate a faster and more efficient disease compared to other NOD/scid-related strains.

Transplantation of human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) primary cells and cell lines in different strains of immunodeficient mice has led to preclinical models extensively used to investigate acute leukemia stem cells, biology and drug sensitivity. We studied the engraftment kinetics of AML and ALL cell lines and primary cells in 3 strains of NOD.CB17-Prkdc(scid) (NOD/scid, NS)-related mice (NOD.Cg-Prkdc(scid)B2m(tm1Unc)/J, abbreviated NOD/scid/beta2 null, NSB; and NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ, abbreviated NOD/scid/IL-2Rgamma null, NSG). The engraftment of human malignant cells was investigated by means of clinicopathological criteria, flow cytometry, PCR and immunohistochemistry. In NSG mice, we observed a significantly faster development of leukemia-related symptoms and a higher percentage of leukemia cells in the blood, in the marrow and in the spleen. The leukemia-related angiogenic switch (measured as the number of circulating endothelial cells and progenitors) was faster in NSG compared to NS and NSB mice. These models will be instrumental to studies on leukemia-initiating stem cells, leukemia biology, preclinical treatment studies, and to obtain patient-specific preclinical models to design and investigate patient-tailored therapies.