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BACKGROUND: Mutations in SLC37A4, which encodes the intracellular glucose transporter G6PT, cause the rare glycogen storage disease type 1b (GSD1b). A long-term consequence of GSD1b is kidney failure, which requires KRT. The main protein markers of proximal tubule function, including NaPi2A, NHE3, SGLT2, GLUT2, and AQP1, are downregulated as part of the disease phenotype.METHODS: We utilized an inducible mouse model of GSD1b, TM-G6PT-/-, to show that glycogen accumulation plays a crucial role in altering proximal tubule morphology and function. To limit glucose entry into proximal tubule cells and thus to prevent glycogen accumulation, we administered an SGLT2-inhibitor, dapagliflozin, to TM-G6PT-/- mice.RESULTS: In proximal tubule cells, G6PT suppression stimulates the upregulation and activity of hexokinase-I, which increases availability of the reabsorbed glucose for intracellular metabolism. Dapagliflozin prevented glycogen accumulation and improved kidney morphology by promoting a metabolic switch from glycogen synthesis toward lysis and by restoring expression levels of the main proximal tubule functional markers.CONCLUSION: We provide proof of concept for the efficacy of dapagliflozin in preserving kidney function in GSD1b mice. Our findings could represent the basis for repurposing this drug to treat patients with GSD1b.

Introduction: MGC018 is an ADC composed of a humanized anti-B7-H3 IgG1 monoclonal antibody conjugated via a cleavable linker to vc-seco-duocarmycin-hydroxybenzamide azaindole (DUBA), a synthetic DNA alkylating agent whose cytotoxic activity is cell-cycle independent and retained in multidrug-resistant cells. MGC018 has shown preliminary clinical activity in B7-H3-expressing tumors, including castration-resistant metastatic prostate cancer. Owing to its expression in several childhood tumors, B7-H3 is a potential target for pediatric cancers, including neuroblastoma (NB). Methods: B7-H3 expression was evaluated by flow-cytometry (FCM) in a panel of human NB cell lines. Cytotoxicity was evaluated in monolayer and in multicellular tumor spheroid (MCTS) models by MTS assay and Cell Titer Glo 3D cell viability assay, respectively. Apoptotic cell death was investigated by the Annexin V staining. Pseudometastatic, orthotopic, and resected mouse NB models were developed via tumor cells tail vein injection, implantation in the adrenal gland, or implantation followed by surgical resection of the primary tumor mass, respectively, to mimic disease conditions related to circulating tumor cells and metastases, primary tumor growth, and minimal residual disease. Results: All cell lines expressed cell surface B7-H3 in a unimodal fashion, ranging between 200 and 400 in mean ratio fluorescence intensity over unstained cells. MGC018 was cytotoxic in a dose- and time-dependent manner against all NB cell lines (IC50 range 5.1-53.9 ng/mL) and NB MCTS (IC50 range 17.8-364 ng/mL). MGC018 was inactive against a murine NB cell line (NX-S2) that did not express human B7-H3; however, NX-S2 cells were killed in the presence of MGC018 when co-cultured with human B7-H3-expressing cells, demonstrating by-stander activity. In the pseudometastatic model and in five orthotopic NB mouse models, weekly iv treatments with 1 mg/kg MGC018 for 3 weeks resulted in delayed tumor growth and increased survival rates compared to animals treated with an irrelevant (anti-CD20) duocarmycin-ADC or an anti-GD2 antibody (dinutuximab beta). A 4-week course of treatment further ameliorated MGC018 antitumor effect in both the orthotopic and resected NB models and increased the survival of NB-bearing mice co-treated with TOpotecan-TEMozolomide (TOTEM), the standard-of-care therapy for relapsed disease. In the orthotopic model, tumor relapse was temporarily or completely arrested by 2 or 3 courses of MGC018, respectively. MGC018 treatment was not associated with body weight loss, hematological toxicity, or clinical chemistry abnormalities. Conclusion: MGC018 exerts relevant antitumor activity in pre-clinical NB models and may represent a potential candidate for future NB clinical translation. Citation Format: Chiara Brignole, Veronica Bensa, Enzo Calarco, Elena Giusto, Eleonora Ciampi, Patrizia Perri, Maria Valeria Corrias, Simonetta Astigiano, Michele Cilli, Deryk Loo, Ezio Bonvini, Fabio Pastorino, Mirco Ponzoni. Anti-tumor activity of vobramitamab duocarmazine (MGC018), an investigational duocarmycin-based anti-B7-H3 antibody-drug conjugate (ADC), in preclinical neuroblastoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2981.

Tumor vasculature is an important component of the tumor microenvironment and deeply affects anticancer immune response. Eribulin is a non-taxane inhibitor of the mitotic spindle. However, off-target effects interfering with the tumor vasculature have been reported. The mechanisms responsible of this effect are still unclear.We designed an in vitro study to investigate the effect of eribulin, with or without TGF-β, on neo-angiogenesis, and on the expression of the adhesion molecules ICAM-1 and VCAM-1. We also investigated the effects of paclitaxel and vinorelbine under the same experimental conditions.Eribulin up-regulated the epithelial markers VE-cadherin and CD-31 in HUVEC and inhibited tube formation in HUVEC cells cultured in Matrigel. The drug effectively arrested tube formation even in the presence of TGF-β and counteracted the TGF-β-induced change in cell shape from the endothelial cobblestone-like morphology to an elongated spindle-shaped morphology. We also observed that eribulin was able to upregulate ICAM-1 and to counteract its down-regulation induced by TGF-β.Eribulin exerts different off-label effects: increases vascular remodeling, counteracts the endothelial-to-mesenchymal transition (EndMT) mediated by TGF-β and promotes tumor infiltration by immune cells via increasing the expression of ICAM-1 and transcription of CD31 and VE-cadherin. Moreover, eribulin was able to inhibit vasculature remodeling and the induction of EndMT mediated by TGF-β better than vinorelbine and paclitaxel. The effects observed in this study might have important therapeutic consequence if the drug is combined with immunotherapy.

Arginine metabolism plays a significant role in regulating cell function, affecting tumor growth and metastatization. To study the effect of the arginine-catabolizing enzyme Arginase1 (ARG1) on tumor microenvironment, we generated a mouse model of mammary carcinogenesis by crossbreeding a transgenic mouse line overexpressing ARG1 in macrophages (FVBArg+/+) with the MMTV-Neu mouse line (FVBNeu+/+). This double transgenic line (FVBArg+/−;Neu+/+) showed a significant shortening in mammary tumor latency, and an increase in the number of mammary nodules. Transfer of tumor cells from FVBNeu+/+ into either FVB wild type or FVBArg+/+ mice resulted in increase regulatory T cells in the tumor infiltrate, suggestive of an impaired antitumor immune response. However, we also found increased frequency of tumor stem cells in tumors from FVBArg+/−;Neu+/+ transgenic compared with FVBNeu+/+ mice, suggesting that increased arginine metabolism in mammary tumor microenvironment may supports the cancer stem cells niche. We provide in vivo evidence of a novel, yet unexploited, mechanism through which ARG1 may contribute to tumor development.

Castration-resistant prostate cancer (CRPC) is an incurable stage of the disease. A multivariate principal component analysis on CRPC in vitro models identified aspartyl (asparaginyl) β hydrolase (ASPH) as the most relevant molecule associated with the CRPC phenotype. ASPH is overexpressed in various malignant neoplasms and catalyzes the hydroxylation of aspartyl and asparaginyl residues in the epidermal growth factor (EGF)-like domains of proteins like NOTCH receptors and ligands, enhancing cell motility, invasion and metastatic spread. Bioinformatics analyses of ASPH in prostate cancer (PCa) and CRPC datasets indicate that ASPH gene alterations have prognostic value both in PCa and CRPC patients. In CRPC cells, inhibition of ASPH expression obtained through specific small interfering RNA or culturing cells in hypoxic conditions, reduced cell proliferation, invasion and cyclin D1 expression through modulation of the NOTCH signaling. ASPH and HIF1α crosstalk, within a hydroxylation-regulated signaling pathway, might be transiently driven by the oxidative stress evidenced inside CRPC cells. In addition, increased phosphorylation of GSK3β by ASPH silencing demonstrates that ASPH regulates GSK3β activity inhibiting its interactions with upstream kinases. These findings demonstrate the critical involvement of ASPH in CRPC development and may represent an attractive molecular target for therapy.

Tumor vasculature is an important component of the tumor microenvironment and deeply affects anticancer immune response. Eribulin is a non-taxane inhibitor of the mitotic spindle. However, off-target effects interfering with the tumor vasculature have been reported. The mechanisms responsible of this effect are still unclear.We designed an in vitro study to investigate the effect of eribulin, with or without TGF-β, on neo-angiogenesis, and on the expression of the adhesion molecules ICAM-1 and VCAM-1. We also investigated the effects of paclitaxel and vinorelbine in the same experimental conditions.Eribulin up-regulated the epithelial markers VE-cadherin and CD-31 in HUVEC and inhibited tube formation in HUVEC cultured in Matrigel. The drug effectively arrested tube formation even in presence of TGF-β and counteracted the TGF-β-induced change in cell shape from the endothelial cobblestone-like morphology to an elongated spindleshaped morphology.We also observed that eribulin was able to upregulate ICAM-1 and to counteract its downregulation induced by TGF-β.Eribulin therefore exerts different off-label effects: increases vascular remodeling, counteracts the endothelial tomesenchymal transition (EndMT) mediated by TGF-β and promotes tumor infiltration by immune cells by increasing expression of ICAM-1 and transcription of CD31 and VE-cadherin.Moreover, eribulin was able to inhibit vasculature remodeling and the induction of EndMT mediated by TGF-β better than vinorelbine and paclitaxel.The effects observed in this study might have important therapeutic consequence if the drug will be administered with immunotherapy.

Tumor vasculature is an important component of the tumor microenvironment and deeply affect anticancer immune response. Eribulin is a non taxane inhibitor of the mitotic spindle. However, off-target effect interfering with the tumor vasculature have been reported. The mechanisms responsible of this effect is not clear.We designed an in vitro study to investigate the effect of eribulin on neo-angiogenesis and on the adhesion molecules ICAM-1 and VCAM-1, with or without TGF-β. We also investigated the effects of paclitaxel and vinorelbine in the same experimental conditions.Eribulin was able to up-regulate the epithelial markers VE-cadherin and CD-31 in the HUVEC and tube formation in HUVEC cultured in Matrigel. The drug effectively arrested tube formation even in presence of TGF-β. Eribulin counteracted the TGF-β induced change in cell shape from the endothelial cobblestone-like morphology to an elongated spindle-shaped morphology that is characteristic of EndMT.We also observed that eribulin is able to upregulate ICAM-1 and to counteract its downregulation induced by TGF-β.In this study, eribulin was able to inhibit the vasculature remodeling and the downregulation of ICAM-1 induced by TGF-β. These effects might have important therapeutic consequence if the drug will be administered with immunotherapy.

Glycogen Storage Disease type 1b (GSDIb) is a genetic disorder with long term severe complications. Accumulation of the glucose analog 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in neutrophils inhibits the phosphorylation of glucose in these cells, causing neutropenia and neutrophil dysfunctions. This condition leads to serious infections and inflammatory bowel disease (IBD) in GSDIb patients. We show here that dapagliflozin, an inhibitor of the renal sodium-glucose co-transporter-2 (SGLT2), improves neutrophil function in an inducible mouse model of GSDIb by reducing 1,5AG6P accumulation in myeloid cells.

Anti-PD-1 or anti-PD-L1 blocking monoclonal antibodies (mAbs) have shown potent anti-tumor effects in adult cancer patients and clinical studies have recently been started in pediatric cancers, including high-risk/relapsing neuroblastoma (NB). Therefore, we studied the effects of anti-PD-1/PD-L1 mAbs in two syngeneic models of disseminated NB generated by the injection of either Neuro2a or NXS2 cells, which express PD-L1. In addition, we tested the combination of these agents with the immune-enhancing cytokine IL-21, the Ecto-NTPDase inhibitor POM-1, an anti-CD25 mAb targeting Treg cells, or an anti-CD4 mAb. We previously showed that CD4-transient depletion removes CD4+CD25+ Treg cells and other CD4+CD25− regulatory subsets. Here we show that mono-therapy with anti-PD-1/PD-L1 mAbs had no effect on systemic NB progression in vivo, and also their combination with IL-21, POM-1 or anti-CD25 mAb was ineffective. The combined use of anti-PD-1 with an anti-CD4 mAb mediated a very potent, CD8-dependent, synergistic effect leading to significant elongation of tumor-free survival of mice, complete tumor regression and durable anti-NB immunity. Similar results were obtained by combining the anti-PD-L1 and anti-CD4 mAbs. These findings indicate that both PD-1/PD-L1 and CD4+ T cell-related immune-regulatory mechanisms must be simultaneously blocked to mediate therapeutic effects in these models.

The microenvironment of solid tumours is extremely acidic and this condition arises since the precancerous stage. This acidic milieu could therefore provide a useful target for both prophylactic and therapeutic approaches. In TRAMP transgenic mice, an in vivo model of prostate adenocarcinoma (AC), oral administration of alkaline water was devoid of unwanted side effects, and when started from an early age was as effective as NaHCO3 in significantly delaying tumour progression, while when started when prostate tumours were already present, a nonstatistically significant trend in the same direction was detected. These findings indicate that the use of alkalinizing drugs should be considered for chemoprevention and, in association with standard chemotherapy, for treatment of human prostate AC.

Background: Prostate cancer (PCa) is a significant health concern throughout the world. Standard therapy for advanced disease consists of anti-androgens, however, almost all prostate tumors become castration resistant (CRPC). Progression from androgen-sensitive PCa to CRPC is promoted by inflammatory signaling through cyclooxygenase-2 (COX-2) expression and ErbB family receptors/AKT activation, compensating androgen receptor inactivity. Methods: Making use of CRPC cell lines, we investigated the effects of the anti-inflammatory drug celecoxib. Biochemical data obtained using immunoblotting, enzyme-linked immunosorbent assay (ELISA), invasion, and xenografts were further integrated by bioinformatic analyses. Results: Celecoxib reduced cell growth and induced apoptosis through AKT blockade, cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), and proteasomal degradation of the anti-apoptotic protein Mcl-1. Epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 degradation, and heterogeneous nuclear ribonucleoprotein K (hnRNP K) downregulation, further amplified the inhibition of androgen signaling. Celecoxib reduced the invasive phenotype of CRPC cells by modulating NF-&kappa, B activity and reduced tumor growth in mice xenografts when administered in association with the anti-EGFR receptor antibody cetuximab. Bioinformatic analyses on human prostate cancer datasets support the relevance of these pathways in PCa progression. Conclusions: Signaling nodes at the intersection of pathways implicated in PCa progression are simultaneously modulated by celecoxib treatment. In combination therapies with cetuximab, celecoxib could represent a novel therapeutic strategy to curb signal transduction during CRPC progression.

Background Cancer cells are characterized by chromosomal instability (CIN) and it is thought that errors in pathways involved in faithful chromosome segregation play a pivotal role in the genesis of CIN. Cohesin forms a large protein ring that binds DNA strands by encircling them. In addition to this central role in chromosome segregation, cohesin is also needed for DNA repair, gene transcription regulation and chromatin architecture. Though mutations in both cohesin and cohesin-regulator genes have been identified in many human cancers, the contribution of cohesin to cancer development is still under debate. Methods Normal mucosa, early adenoma, and carcinoma samples deriving from 16 subjects affected by colorectal cancer (CRC) were analyzed by OncoScan for scoring both chromosome gains and losses (CNVs) and loss of heterozygosity (LOH). Then the expression of SMC1A was analyzed by immunochemistry in 66 subjects affected by CRC. The effects of SMC1A overexpression and mutated SMC1A were analyzed in vivo using immunocompromised mouse models. Finally, we measured global gene expression profiles in induced-tumors by RNA-seq. Results Here we showed that SMC1A cohesin core gene was present as extra-copies, mutated, and overexpressed in human colorectal carcinomas. We then demonstrated that cohesin overexpression led to the development of aggressive cancers in immunocompromised mice through gene expression dysregulation. Conclusion Collectively, these results support a role of defective cohesin in the development of human colorectal cancer. Electronic supplementary material The online version of this article (10.1186/s13046-019-1116-0) contains supplementary material, which is available to authorized users.

Background Prostate cancer (PCa), the second most common cancer affecting men worldwide, shows a broad spectrum of biological and clinical behaviour representing the epiphenomenon of an extreme heterogeneity. Androgen deprivation therapy is the mainstay of treatment for advanced forms but after few years the majority of patients progress to castration-resistant prostate cancer (CRPC), a lethal form that poses considerable therapeutic challenges. Methods Western blotting, immunocytochemistry, invasion and reporter assays, and in vivo studies were performed to characterize androgen resistant sublines phenotype in comparison to the parental cell line LNCaP. RNA microarray, mass spectrometry, integrative transcriptomic and proteomic differential analysis coupled with GeneOntology and multivariate analyses were applied to identify deregulated genes and proteins involved in CRPC evolution. Results Treating the androgen-responsive LNCaP cell line for over a year with 10 μM bicalutamide both in the presence and absence of 0.1 nM 5-α-dihydrotestosterone (DHT) we obtained two cell sublines, designated PDB and MDB respectively, presenting several analogies with CRPC. Molecular and functional analyses of PDB and MDB, compared to the parental cell line, showed that both resistant cell lines were PSA low/negative with comparable levels of nuclear androgen receptor devoid of activity due to altered phosphorylation; cell growth and survival were dependent on AKT and p38MAPK activation and PARP-1 overexpression; their malignant phenotype increased both in vitro and in vivo. Performing bioinformatic analyses we highlighted biological processes related to environmental and stress adaptation supporting cell survival and growth. We identified 15 proteins that could direct androgen-resistance acquisition. Eleven out of these 15 proteins were closely related to biological processes involved in PCa progression. Conclusions Our models suggest that environmental factors and epigenetic modulation can activate processes of phenotypic adaptation driving drug-resistance. The identified key proteins of these adaptive phenotypes could be eligible targets for innovative therapies as well as molecules of prognostic and predictive value. Electronic supplementary material The online version of this article (10.1186/s12964-017-0206-x) contains supplementary material, which is available to authorized users.

// Adriana Amaro 1, * , Giovanna Angelini 1, * , Valentina Mirisola 1 , Alessia Isabella Esposito 1 , Daniele Reverberi 1 , Serena Matis 1 , Massimo Maffei 1 , Walter Giaretti 1 , Maurizio Viale 2 , Rosaria Gangemi 2 , Laura Emionite 3 , Simonetta Astigiano 4 , Michele Cilli 3 , Beatrice E. Bachmeier 5 , Peter H. Killian 5 , Adriana Albini 6 , Ulrich Pfeffer 1 1 Molecular Pathology, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 2 Biotherapy, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 3 Animal Facility, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 4 Immunology, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 5 Institute of Laboratory Medicine, Ludwig-Maximilians-University, Munich, Germany 6 Scientific and Technology Park, IRCCS MultiMedica, Milan, Italy * These authors have contributed equally to the work Correspondence to: Adriana Albini, email: adriana.albini@multimedica.it Ulrich Pfeffer, email: patologia.molecolare.integrata@gmail.com Keywords: breast cancer, invasion, apocrine breast cancer, metastasis, aneuploidy Received: May 16, 2016 Accepted: August 13, 2016 Published: September 10, 2016 ABSTRACT The acquisition of an invasive phenotype is a prerequisite for metastasization, yet it is not clear whether or to which extent the invasive phenotype is linked to other features characteristic of metastatic cells. We selected an invasive subpopulation from the triple negative breast cancer cell line MDA-MB-231, performing repeated cycles of preparative assays of invasion through Matrigel covered membranes. The invasive sub-population of MDA-MB-231 cells exhibits stronger migratory capacity as compared to parental cells confirming the highly invasive potential of the selected cell line. Prolonged cultivation of these cells did not abolish the invasive phenotype. ArrayCGH, DNA index quantification and karyotype analyses confirmed a common genetic origin of the parental and invasive subpopulations and revealed discrete structural differences of the invasive subpopulation including increased ploidy and the absence of a characteristic amplification of chromosome 5p14.1-15.33. Gene expression analyses showed a drastically altered expression profile including features of apocrine breast cancers and of invasion related matrix-metalloproteases and cytokines. The invasive cells showed accelerated proliferation, increased apoptosis, and an altered pattern of chemo-sensitivity with lower IC50 values for drugs affecting the mitotic apparatus. However, the invasive cell population is significantly less tumorigenic in orthotopic mouse xenografts suggesting that the acquisition of the invasive capacity and the achievement of metastatic growth potential are distinct events.

In America and Western Europe, prostate cancer is the second leading cause of death in men. Emerging evidence suggests that chronic inflammation is a major risk factor for the development and metastatic progression of prostate cancer. We previously reported that the chemopreventive polyphenol curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast cancer metastases. In this study, we analyze the effects of curcumin on prostate carcinoma growth, apoptosis and metastasis. We show that curcumin inhibits translocation of NFκB to the nucleus through the inhibition of the IκB-kinase (IKKβ, leading to stabilization of the inhibitor of NFκB, IκBα, in PC-3 prostate carcinoma cells. Inhibition of NFκB activity reduces expression of CXCL1 and -2 and abolishes the autocrine/paracrine loop that links the two chemokines to NFκB. The combination of curcumin with the synthetic IKKβ inhibitor, SC-541, shows no additive or synergistic effects indicating that the two compounds share the target. Treatment of the cells with curcumin and siRNA-based knockdown of CXCL1 and -2 induce apoptosis, inhibit proliferation and downregulate several important metastasis-promoting factors like COX2, SPARC and EFEMP. In an orthotopic mouse model of hematogenous metastasis, treatment with curcumin inhibits statistically significantly formation of lung metastases. In conclusion, chronic inflammation can induce a metastasis prone phenotype in prostate cancer cells by maintaining a positive proinflammatory and prometastatic feedback loop between NFκB and CXCL1/-2. Curcumin disrupts this feedback loop by the inhibition of NFκB signaling leading to reduced metastasis formation in vivo.

ARG1, expressed by human PMNs, inhibits T cell proliferation by depleting extracellular l-arginine. Here, we report that ARG1, released from gelatinase granules by PMNs, is inactive at physiological pH unless activated by factor(s) stored in azurophil granules. Whereas ARG1 exocytosis was induced by TNF-α or ionomycin, only the latter mediated the release of both granules, resulting in extracellular ARG enzyme activity at physiological pH. Furthermore, after fractionation of the different classes of granules, only the mixture of gelatinase and azurophil granules resulted in ARG1 activity at physiological pH. The use of protease inhibitors indicated the involvement of a PMSF- and leupeptin-susceptible serine protease in ARG1 processing and activation. Finally, the supernatant of viable PMNs undergoing frustrated phagocytosis, which mediates gelatinase and azurophil granule release, inhibited T cell proliferation through ARG-dependent mechanisms. In vivo, high ARG1 concentrations and increased ARG enzyme activity, sufficient to inhibit T cell proliferation, were observed in synovial fluids from RA. These findings suggest that PMNs, recruited at sites of immune complex deposition, induce ARG1-dependent immune suppression through concomitant exocytosis of gelatinase and azurophil granules.

Bone marrow-derived mesenchymal stem cells (MSCs) are precursors of bone, cartilage and fat tissue. MSC can also regulate the immune response. For these properties, they are tested in clinical trials for tissue repair in combination with bioscaffolds or injected as cell suspension for immunosuppressant therapy. Experimental data, however, indicate that MSC can undergo or induce a tumorigenic process in determined circumstances. We used a modified model of ectopic bone formation in mice by subcutaneously implanting porous ceramic seeded with murine MSC. In this new model, host-derived sarcomas developed when we implanted MSC/bioscaffold constructs into syngeneic and immunodeficient recipients, but not in allogeneic hosts or when MSCs were injected as cell suspensions. The bioscaffold provided a tridimensional support for MSC to aggregate, thus producing the stimulus for triggering the process eventually leading to the transformation of surrounding cells and creating a surrogate tumor stroma. The chemical and physical characteristics of the bioscaffold did not affect tumor formation; sarcomas developed either when a stiff porous ceramic was used or when the scaffold was a smooth collagen sponge. The immunoregulatory function of MSC contributed to tumor development. Implanted MSC expanded clones of CD4+CD25+ T regulatory lymphocytes that suppressed host's antitumor immune response.

Arginase (ARG) is a metabolic enzyme present in two isoforms that hydrolyze l-arginine to urea and ornithine. In humans, ARG isoform 1 is also expressed in cells of the myeloid lineage. ARG activity promotes tumour growth and inhibits T lymphocyte activation. However, the two ARG transgenic mouse lines produced so far failed to show such effects. We have generated, in two different genetic backgrounds, transgenic mice constitutively expressing ARG1 under the control of the CD68 promoter in macrophages and monocytes. Both heterozygous and homozygous transgenic mice showed a relevant increase in mortality at early age, compared with wild-type siblings (67/267 and 48/181 versus 8/149, respectively, both P < 0.005). This increase was due to high incidence of haematologic malignancies, in particular myeloid leukaemia, myeloid dysplasia, lymphomas and disseminated intravascular coagulation (DIC), diseases that were absent in wild-type mice. Atrophy of lymphoid organs due to reduction in T-cell compartment was also detected. Our results indicate that ARG activity may participate in the pathogenesis of lymphoproliferative and myeloproliferative disorders, suggest the involvement of alterations of L-arginine metabolism in the onset of DIC and confirm a role for the enzyme in regulating T-cell homeostasis.

Embryonal carcinoma (EC) cells, stem cells of teratocarcinoma, represent an excellent model to study the developmental mechanisms that, inappropriately reactivated, can drive tumorigenesis. EC cells are very aggressive, and grow rapidly when injected into adult syngeneic mice. However, when injected into blastocysts, they revert to normality, giving rise to chimeric animals. In order to study the ability of postimplantation embryonic environment to "normalize" tumorigenic cells, and to study their homing, we transplanted F9, Nulli-SCC1, and P19 EC cells into 8 to 15-day allogenic CD1 mouse embryos, into allogenic CD1 newborns, and into syngeneic adult mice, and evaluated tumor formation, spreading, and homing. We found that, although at all embryonic stages successful transplantation occurred, the chances of developing tumors after birth increased with the time of injection of EC cells into the embryo. In addition, using enhanced green fluorescent protein-expressing F9 cells, we demonstrated that the cells not giving rise to tumors remained latent and could be tracked down in tissues during adulthood. Our data indicate that the embryonic environment retains a certain ability to "normalize" tumor cells also during post-implantation development. This could occur through yet unknown epigenetic signals triggering EC cells' differentiation.

Indoleamine 2,3-dioxygenase (IDO), a catabolizing enzyme of tryptophan, is supposed to play a role in tumor immune escape. Its expression in solid tumors has not yet been well elucidated: IDO can be expressed by the tumor cells themselves, or by ill-defined infiltrating cells, possibly depending on tumor type. We have investigated IDO expression in 25 cases of non small cell lung cancer (NSCLC). Using histochemistry and immunohistochemistry, we found that IDO was expressed not by tumor cells, but by normal cells infiltrating the peritumoral stroma. These cells were neither macrophages nor dendritic cells, and were identified as eosinophil granulocytes. The amount of IDO-positive eosinophils varied in different cases, ranging from a few cells to more than 50 per field at x200 magnification. IDO protein in NSCLC was enzymatically active. Therefore, at least in NSCLC cases displaying a large amount of these cells in the inflammatory infiltrate, IDO-positive eosinophils could exert an effective immunosuppressive action. On analyzing the 17 patients with adequate follow-up, a significant relationship was found between the amount of IDO-positive infiltrate and overall survival. This finding suggests that the degree of IDO-positive infiltrate could be a prognostic marker in NSCLC.

Integrin receptors are well-known mediators of cell adhesion that also have a fundamental role in controlling the migration of cells through tissues. Among the numerous members of a still growing family, two particular molecular complexes have turned out to be of key importance in tumor cell invasion of basement membranes, the α3β1 and α6β4 integrins. In this Review, we will focus on the role of these two receptors and the mechanisms by which they influence the invasion process.

Chronic inflammation is a major risk factor for the development and metastatic progression of cancer. We have previously reported that the chemopreventive polyphenol Curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast and prostate cancer metastases. In the present study, we have analyzed the effects of Curcumin on miRNA expression and its correlation to the anti-tumorigenic properties of this natural occurring polyphenol. Using microarray miRNA expression analyses, we show here that Curcumin modulates the expression of a series of miRNAs, including miR181b, in metastatic breast cancer cells. Interestingly, we found that miR181b down-modulates CXCL1 and -2 through a direct binding to their 3′-UTR. Overexpression or inhibition of miR181b in metastatic breast cancer cells has a significant impact on CXCL1 and -2 and is required for the effect of Curcumin on these two cytokines. miR181b also mediates the effects of Curcumin on inhibition of proliferation and invasion as well as induction of apoptosis. Importantly, over-expression of miR181b in metastatic breast cancer cells inhibits metastasis formation in vivo in immunodeficient mice. Finally, we demonstrated that Curcumin up-regulates miR181b and down-regulates CXCL1 and -2 in cells isolated from several primary human breast cancers. Taken together, these data show that Curcumin provides a simple bridge to bring metastamir modulation into the clinic, placing it in a primary and tertiary preventive, as well as a therapeutic, setting.

In vitro differentiation of the murine embryonal carcinoma (EC) cell line F9 parallels that of the early blastocyst, where visceral (VE) and parietal endoderm (PE) diverge from a common precursor, the primitive endoderm. This differentiation pathway is induced by retinoic acid (RA) and dibutyryl cyclicAMP (dcAMP) and is accompanied by progressive and dramatic changes in cell morphology and functions. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins, becoming fully differentiated migratory cells; all these changes are likely to involve integrins expression and organization. We have investigated the changes in beta 1 integrin expression, its maturation, and organization on the cell surface in association with alpha 6, during the transition from undifferentiated F9 stem cells to migrating PE cells. By Western blotting and immunoprecipitation we showed a gradual decrease in the amount of the beta 1 subunit on the cell surface and a parallel progressive accumulation of immature protein, indicating that the control of beta 1 expression during F9 cells differentiation occurs first at post-translational level and then at the level of transcription. Moreover, the induction of differentiation produces a marked decrease of alpha 6B and its association to a high molecular weight protein, while alpha 6A level increases. By immunofluorescence we found that upon differentiation there is a relocation of the beta 1 and alpha 6B integrin subunits from cell-cell contacts to focal contacts where they colocalize with vinculin. On the contrary alpha 6A, weakly present in F9 stem cells, is present in the focal contacts of PE cells and along the stress fibers. We suggest different roles for the two alpha 6 isoforms.

International audience; The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.

Despite recent advances in understanding the biological basis of prostate cancer, management of the disease, especially in the phase resistant to androgen ablation, remains a significant challenge. The long latency and high incidence of prostate carcinogenesis provides the opportunity to intervene with chemoprevention to prevent or eradicate prostate malignancies. In this study, we have used human hormone-resistant prostate cancer cells, DU145 and PC3, as an in vitro model to assess the efficacy of xanthohumol (XN) against cell growth, motility and invasion. We observed that treatment of prostate cancer cells with low micromolar doses of XN inhibits proliferation and modulates focal adhesion kinase (FAK) and AKT phosphorylation leading to reduced cell migration and invasion. Oxidative stress by increased production of reactive oxygen species (ROS) was associated with these effects. Transgenic adenocarcinoma of the mouse prostate (TRAMP) transgenic mice were used as an in vivo model of prostate adenocarcinoma. Oral gavage of XN, three times per week, beginning at 4 wks of age, induced a decrease in the average weight of the urogenital (UG) tract, delayed advanced tumor progression and inhibited the growth of poorly differentiated prostate carcinoma. The ability of XN to inhibit prostate cancer in vitro and in vivo suggests that XN may be a novel agent for the management of prostate cancer.

Studies on oocyte transcriptome are important to understand the biological pathways involved in oogenesis, totipotence and early embryonic development. Moreover, genes regulating physiological pathways in gametes could represent potential candidates for reproductive disorders. In addition to oocyte specific transcription factors, also the members of the p53 family could be etiologically involved due to their biological functions. In fact, their role in the control of cell cycle, apoptosis, and germ-line genome stability is well known. Female reproductive aging is one of the causes of fertility reduction and it is often associated with egg aneuploidy increase. In order to verify the potential involvement of p73 in reproductive aging, we determined its expression in single mature MII oocytes from two groups of women, younger than 35 or older than 38 years, respectively. We found that TAp73 isoforms are down regulated in oocytes from women older than 38 years. We confirmed these data in pools of mouse oocytes. TAp73 down regulation in oocytes from women of advanced reproductive age could explain both the reduction of fertility and the increase of newborns with chromosomal abnormalities.

Background & Aims Several studies have shown that bone marrow-derived committed myelomonocytic cells can repopulate diseased livers by fusing with host hepatocytes and can restore normal liver function. These data suggest that myelomonocyte transplantation could be a promising approach for targeted and well-tolerated cell therapy aimed at liver regeneration. We sought to determine whether bone marrow-derived myelomonocytic cells could be effective for liver reconstitution in newborn mice knock-out for glucose-6-phosphatase-α. Methods Bone marrow-derived myelomonocytic cells obtained from adult wild type mice were transplanted in newborn knock-out mice. Tissues of control and treated mice were frozen for histochemical analysis, or paraffin-embedded and stained with hematoxylin and eosin for histological examination or analyzed by immunohistochemistry or fluorescent in situ hybridization. Results Histological sections of livers of treated knock-out mice revealed areas of regenerating tissue consisting of hepatocytes of normal appearance and partial recovery of normal architecture as early as 1 week after myelomonocytic cells transplant. FISH analysis with X and Y chromosome paints indicated fusion between infused cells and host hepatocytes. Glucose-6-phosphatase activity was detected in treated mice with improved profiles of liver functional parameters. Conclusions Our data indicate that bone marrow-derived myelomonocytic cell transplant may represent an effective way to achieve liver reconstitution of highly degenerated livers in newborn animals.

Dbl is the prototype of a large family of GDP–GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho, Rac, and Cdc42 and to induce a transformed phenotype in murine fibroblasts. We previously reported that Dbl-null mice are viable and fertile but display defective dendrite elongation of distinct subpopulations of cortical neurons, suggesting a role of Dbl in controlling dendritic growth. To gain deeper insights into the role of Dbl in development and disease, we attempted a knock-in approach to create an endogenous allele that encodes a missense-mutation-mediated loss of function in the DH domain. We generated, by gene targeting technology, a mutant mouse strain by inserting a mutagenized human proto-Dbl cDNA clone expressing only the Dbl N terminus regulatory sequence at the starting codon of murine exon 1. Animals were monitored over a 21-month period, and necropsy specimens were collected for histological examination and immunohistochemistry analysis. Dbl knock-in mice are viable and did not manifest either decreased reproductive performances or gross developmental phenotype but revealed a reduced lifespan compared to wild-type (w.t.) mice and showed, with aging, a B cell lymphoproliferation that often has features of a frank diffuse large B cell lymphoma. Moreover, Dbl knock-in male mice displayed an increased incidence of lung adenoma compared to w.t. mice. These data indicate that Dbl is a tumor susceptibility gene in mice and that loss of function of Dbl DH domain by genetic missense mutations is responsible for induction of diffuse large B cell lymphoma.

Background The DLX gene family encodes for homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis. Their expression can be regulated by Endothelin1 (ET1), a peptide associated with breast cancer invasive phenotype. Deregulation of DLX gene expression was found in human solid tumors and hematologic malignancies. In particular, DLX4 overexpression represents a possible prognostic marker in ovarian cancer. We have investigated the role of DLX genes in human breast cancer progression. Methods MDA-MB-231 human breast carcinoma cells were grown in vitro or injected in nude mice, either subcutaneously, to mimic primary tumor growth, or intravenously, to mimic metastatic spreading. Expression of DLX2, DLX5 and DLX6 was assessed in cultured cells, either treated or not with ET1, tumors and metastases by RT-PCR. In situ hybridization was used to confirm DLX gene expression in primary tumors and in lung and bone metastases. The expression of DLX2 and DLX5 was evaluated in 408 primary human breast cancers examining the GSE1456 and GSE3494 microarray datasets. Kaplan-Meier estimates for disease-free survival were calculated for the patients grouped on the basis of DLX2/DLX5 expression. Results Before injection, or after subcutaneous growth, MDA-MB-231 cells expressed DLX2 but neither DLX5 nor DLX6. Instead, in bone and lung metastases resulting from intravenous injection we detected expression of DLX5/6 but not of DLX2, suggesting that DLX5/6 are activated during metastasis formation, and that their expression is alternative to that of DLX2. The in vitro treatment of MDA-MB-231 cells with ET1, resulted in switch from DLX2 to DLX5 expression. By data mining in microarray datasets we found that expression of DLX2 occurred in 21.6% of patients, and was significantly correlated with prolonged disease-free survival and reduced incidence of relapse. Instead, DLX5 was expressed in a small subset of cases, 2.2% of total, displaying reduced disease-free survival and high incidence of relapse which was, however, non-significantly different from the other groups due to the small size of the DLX+ cohort. In all cases, we found mutually exclusive expression of DLX2 and DLX5. Conclusions Our studies indicate that DLX genes are involved in human breast cancer progression, and that DLX2 and DLX5 genes might serve as prognostic markers.

International audience; The morphogenesis of the vertebrate skull results from highly dynamic integrated processes involving the exchange of signals between the ectoderm, the endoderm, and cephalic neural crest cells (CNCCs). Before migration CNCCs are not committed to form any specific skull element, molecular signals exchanged in restricted regions of tissue interaction are crucial in providing positional identity to the CNCCs mesenchyme and activate the specific morphogenetic process of different skeletal components of the head. In particular, the endothelin‐1 (Edn1)‐dependent activation of Dlx5 and Dlx6 in CNCCs that colonize the first pharyngeal arch (PA1) is necessary and sufficient to specify maxillo‐mandibular identity. Here, to better analyze the spatio‐temporal dynamics of this process, we associate quantitative gene expression analysis with detailed examination of skeletal phenotypes resulting from combined allelic reduction of Edn1, Dlx5, and Dlx6. We show that Edn1‐dependent and ‐independent regulatory pathways act at different developmental times in distinct regions of PA1. The Edn1→Dlx5/6→Hand2 pathway is already active at E9.5 during early stages of CNCCs colonization. At later stages (E10.5) the scenario is more complex: we propose a model in which PA1 is subdivided into four adjacent territories in which distinct regulations are taking place. This new developmental model may provide a conceptual framework to interpret the craniofacial malformations present in several mouse mutants and in human first arch syndromes. More in general, our findings emphasize the importance of quantitative gene expression in the fine control of morphogenetic events. genesis

Interferon gamma (IFN-gamma) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-gamma are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-gamma-mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1-expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1-expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-gamma-mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-gamma-induced antitumoral defense system.

Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.

cDNA libraries have been generated from Nulli-SCCl murine embryonal carcinoma (EC) cells untreated or treated for 24 h with all-trans retinoic acid (RA) or hexamethylenebisacetamide (HMBA), two chemically unrelated inducers of differentiation of EC cells. The libraries were screened for gene sequences whose expression was differentially regulated by one or both compounds. Of 20,000 cDNA clones screened, only 12 showed reproducible quantitative differences. One of the latter clones (pH 34) has been studied in detail. pH 34 cDNA hybridizes with a polyadenylated RNA (650 nucleotides) which is abundant in untreated Nulli-SCCl EC cells but whose steady-state levels decrease within 6 h of exposure to HMBA, reaching a minimum at 24 h. RA has a less-marked effect on this mRNA. Addition of inducers to the cells in fresh medium produces an early (15 min) transient increase in pH 34 mRNA levels. Nuclear run-on experiments are consistent with the view that the decrease in pH 34 mRNA is due to post-transcriptional events. Subclones of pH 34 in pGEM-4 were used to synthesize mRNA which could be translated in vitro into a 14-kDa protein. DNA sequencing of the pH 34 cDNA revealed that it is 607 bp in length with a single open reading frame capable of encoding a protein of 118 amino acids. Primer extension experiments revealed that the insert contains the full 5' sequence. Comparison with known sequences failed to reveal significant homology with previously sequenced proteins.

In human prostate cancer, Arginase 2 (ARG2) and nitric oxide synthase (NOS) are concomitantly expressed by tumor cells, and induce tumor immune escape via peroxynitrite-dependent Tyrosine nitrosylation. Since there were no data regarding this immune suppressive mechanism in other tumor types, and an evaluation of its clinical relevance in human tumors had still to be provided, we have investigated presence and clinical relevance of ARG2 and NOS expression in lung cancer. No evidence of NOS expression was found, no significant NOS enzymatic activity was detected. Instead, ARG2 protein was expressed by tumor cells. In a cohort of 120 patients, the amount of ARG2-positive tumor cells was significantly higher in small cell lung cancers (SCLC) than in non-small cell lung cancers (NSCLC). Large cell undifferentiated carcinomas had twice ARG2 than the other NSCLC subtypes. ARG2 expression was increased in Grade 3 tumors, as compared to Grades 1 and 2. However, no relationship was found with tumor size and stage, and with patient survival. Indeed, the enzyme was active, since the Arginine catabolite Ornithine was produced, but Arginine depletion was not attained. In addition, nitrotyrosine was not found in tumor tissue. Accordingly, when tumor cells isolated from lung cancer were incubated with activated autologous T cells, no inhibition of proliferation was detected. Our results indicate that ARG2 is expressed in lung cancer, but it does not induce tumor immune escape and does not affect disease progression, most probably due to the lack of concomitant NOS expression.

Tumors derived from rat LA7 cancer stem cells (CSCs) contain a hierarchy of cells with different capacities to generate self-renewing spheres and tubules serially ex vivo and to evoke tumors in vivo . We isolated two morphologically distinct cell types with distinct tumorigenic potential from LA7-evoked tumors: cells with polygonal morphology that are characterized by expression of p21/ WAF1 and p63 and display hallmarks of CSCs and elongated epithelial cells, which generate tumors with far less heterogeneity than LA7 CSCs. Serial transplantation of elongated epithelial cells results in progressive loss of tumorigenic potential; tumor heterogeneity; CD44, E-cadherin, and epithelial cytokeratin expression and increased α-smooth muscle actin I and vimentin expression. In contrast, serial transplantation of LA7 CSCs can be performed indefinitely and results in tumors that maintain their heterogeneity, consistent with self-renewal and multilineage differentiation potential. Collectively, our data show that polygonal cells are CSCs, whereas epithelial elongated cells are lineage-committed progenitors with tumorigenic potential, and suggest that tumor progenitors, although lacking indefinite self-renewal potential, nevertheless may make a substantial contribution to tumor development. Because LA7 cells can switch between conditions that favor maintenance of pure CSCs vs. differentiation into other tumor cell types, this cell system provides the opportunity to study factors that influence CSC self-renewal and differentiation. One factor, p63 , was identified as a key gene regulating the transition between CSCs and early progenitor cells.

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing cancer-initiating cells with stem cell properties at the single cell level has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell, derived from rat mammary adenocarcinoma has: the ability to serially re-generate mammospheres in long-term non-adherent cultures, the differentiation potential to generate all the cell lineages of the mammary gland and branched duct-like structures that recapitulate morphologically and functionally the ductal–alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multi-lineage differentiation and the tubular-like structure formation potential suggest that LA7 cells is a cancer stem model system to study the dynamics of tumor formation at the single cell level.

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing a single cancer-initiating cell with stem cell properties has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell derived from rat mammary adenocarcinoma has the following properties: the differentiation potential to generate all of the cell lineages of the mammary gland; the ability to generate branched duct-like structures that recapitulate morphologically and functionally the ductal–alveolar-like architecture of the mammary tree; and the capacity to initiate heterogeneous tumors in nonobese diabetic-SCID mice. In addition, we show that cultured cells derived from tumors generated by a single LA7 cell-injection have properties similar to LA7 cells, can generate all of the cell lineages of the mammary gland, and recapitulate the ductal–alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multilineage differentiation potential, and single-cell tumor-initiation potential suggest that LA7 cells are cancer stem cells and can be used as a model system to study the dynamics of tumor formation at the single-cell level.

The homeopathic compound of resonance FMS*Calciumfluor (FMS*) reportedly promotes osteogenic differentiation of rat pre-osteoblasts in vitro. Here, we show that the continuous exposure of differentiating rat osteogenic cells (ROB) to FMS* modulates the level of expression of mRNAs for 7 of the 8 osteogenic markers tested. Alkaline phosphatase (AP), osteocalcin (OC), metalloproteinases (MMP-2 and -14), procollagenase C (BMP-1), biglycan (BG) and integrin 1 are expressed at higher levels in FMS*-treated osteoblasts than in control cultures. MMP-2 and -14 mRNA are not down-modulated at mineralization. Also, the pattern of expression induced by FMS* for some of these genes (BMP-1, BG and integrin 1) is changed, but collagen type I (Coll I) mRNA levels are not affected by treatment with FMS*. This suggests that FMS* modulates mRNA levels and that this is not generalized, but gene(s) specific. We also report that exposure to FMS* rapidly and transiently induces activation of mitogen-activated protein kinases (MAPKs) 42,44 in populations of early osteoblasts, but not in pre-osteoblasts, with a cell differentiation stage-dependent and pertussis toxin (PTX)-sensitive response. Subsequent to FMS* MAPK signaling activation, an increase in AP and MMP-14 mRNA is detected, which is also inhibited by PTX, suggesting that FMS* activation of MAPK signaling could be an early event required for the induction of these genes. Exposure to FMS* does not cause changes in the activity of p125 (FAK)-mediated signaling.