Development of an Injectable Slow-Release Metformin Formulation and Evaluation of Its Potential Antitumor Effects.

Metformin is an antidiabetic drug which possesses antiproliferative activity in cancer cells when administered at high doses, due to its unfavorable pharmacokinetics. The aim of this work was to develop a pharmacological tool for the release of metformin in proximity of the tumor, allowing high local concentrations, and to demonstrate the in vivo antitumor efficacy after a prolonged metformin exposition. A 1.2% w/w metformin thermoresponsive parenteral formulation based on poloxamers P407 and P124, injectable at room temperature and undergoing a sol-gel transition at body temperature, has been developed and optimized for rheological, thermal and release control properties; the formulation is easily scalable, and proved to be stable during a 1-month storage at 5 °C. Using NOD/SCID mice pseudo-orthotopically grafted with MDA-MB-231/luc+ human breast cancer cells, we report that multiple administrations of 100 mg of the optimized metformin formulation close to the tumor site cause tissue accumulation of the drug at levels significantly higher than those observed in plasma, and enough to exert antiproliferative and pro-apoptotic activities. Our results demonstrate that this formulation is endowed with good stability, tolerability, thermal and rheological properties, representing a novel tool to be pursued in further investigations for adjuvant cancer treatment.

In vitro and in vivo characterization of stem-like cells from canine osteosarcoma and assessment of drug sensitivity.

Cancer stem cell (CSC) self-renewing and drug resistance cause treatment failure and tumor recurrence. Osteosarcoma is an aggressive bone tumor characterized by biological and molecular heterogeneity, possibly dependent on CSCs. CSC identification in osteosarcoma and their efficient targeting are still open questions. Spontaneous canine osteosarcoma shares clinical and biological features with the human tumors, representing a model for translational studies. We characterized three CSC-enriched canine osteosarcoma cultures. In serum-free conditions, these CSC cultures grow as anchorage-independent spheroids, show mesenchymal-like properties and in vivo tumorigenicity, recapitulating the heterogeneity of the original osteosarcoma. Osteosarcoma CSCs express stem-related factors (Sox2, Oct4, CD133) and chemokine receptors and ligands (CXCR4, CXCL12) involved in tumor proliferation and self-renewal. Standard drugs for osteosarcoma treatment (doxorubicin and cisplatin) affected CSC-enriched and parental primary cultures, showing different efficacy within tumors. Moreover, metformin, a type-2 diabetes drug, significantly inhibits osteosarcoma CSC viability, migration and self-renewal and, in co-treatment with doxorubicin and cisplatin, enhances drug cytotoxicity. Collectively, we demonstrate that canine osteosarcoma primary cultures contain CSCs exhibiting distinctive sensitivity to anticancer agents, as a reliable experimental model to assay drug efficacy. We also provide proof-of-principle of metformin efficacy, alone or in combination, as pharmacological strategy to target osteosarcoma CSCs.

Canine osteosarcoma cell lines contain stem-like cancer cells: biological and pharmacological characterization.

Cancer stem cells (CSCs) represent a small subpopulation of cells responsible for tumor formation and progression, drug resistance, tumor recurrence and metastasization. CSCs have been identified in many human tumors including osteosarcoma (OSA). CSC distinctive properties are the expression of stem cell markers, sustained growth, self-renewal and tumorigenicity. Here we report the isolation of stem-like cells from two canine OSA cultures, characterized by self-renewal, evaluated by sphere formation ability, differential marker expression, and in vitro proliferation when cultured in a medium containing EGF and bFGF. Current therapies for OSA increased survival time, but prognosis remains poor, due to the development of drug resistance and metastases. Chemotherapy shrinks the tumor mass but CSCs remain unaffected, leading to tumor recurrence. Metformin, a drug for type 2 diabetes, has been shown to possess antitumor properties affecting CSC survival in different human and animal cancers. Here we show that metformin has a significant antiproliferative effect on canine OSA stem-like cells, validating this in vitro model for further pre-clinical drug evaluations. In conclusion, our results demonstrate the feasibility of obtaining CSC-enriched cultures from primary canine OSA cells as a promising model for biological and pharmacological studies of canine and human OSAs.

In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors.

BACKGROUND: Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. METHODS: Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. RESULTS: We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. CONCLUSIONS: Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals.

CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis.

BACKGROUND: Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation. RESULTS: A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours. CONCLUSIONS: These results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis.

Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential.

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs.

Receptor tyrosine kinase inhibitors and cytotoxic drugs affect pleural mesothelioma cell proliferation: insight into EGFR and ERK1/2 as antitumor targets.

Malignant pleural mesothelioma (MPM) is an aggressive chemotherapy-resistant cancer. Up-regulation of epidermal growth factor receptor (EGFR) plays an important role in MPM development and EGFR-tyrosine kinase inhibitors (TKIs) may represent novel therapeutic options. We tested the effects of the EGFR TKIs gefitinib and erlotinib and TKIs targeted to other growth factors (VEGFR and PDGFR), in comparison to standard antineoplastic agents, in two human MPM cell lines, IST-Mes2 and ZL55. All drugs showed IC(50) values in the micromolar range: TKIs induced cytostatic effects at concentrations up to the IC(50,) while conventional drug growth-inhibitory activity was mainly cytotoxic. Moreover, the treatment of IST-Mes2 with TKIs (gefitinib and imatinib mesylate) in combination with cisplatin and gemcitabine did not show additivity. Focusing on the molecular mechanisms underlying the antiproliferative and pro-apoptotic effects of EGFR-TKIs, we observed that gefitinib induced the formation and stabilization of inactive EGFR homodimers, even in absence of EGF, as demonstrated by EGFR B(max) and number of sites/cell. The analysis of downstream effectors of EGFR signaling demonstrated that EGF-induced proliferation, reverted by gefitinib, involved ERK1/2 activation, independently from Akt pathway. Gefitinib inhibits MPM cell growth and survival, preventing EGF-dependent activation of ERK1/2 pathway by blocking EGFR-TK phosphorylation and stabilizing inactive EGFR dimers. Along with the molecular definition of TKIs pharmacological efficacy in vitro, these results may contribute to delve deep into the promising but still controversial role for targeted and conventional drugs in the therapy of MPM.

Paraneoplastic leukocytosis in a dog with a renal carcinoma.

A 7-year-old male German Shepherd dog in poor body condition had a 3-month history of intermittent hematuria. Nonregenerative anemia, mild leukocytosis, marked hypoalbuminemia, and hematuria were observed. Subsequently, marked neutrophilia and moderate monocytosis were noted; anemia, hypoalbuminemia, and hematuria persisted; and the dog developed disseminated intravascular coagulation. Ultrasonographic examination of the abdomen revealed the presence of an enlarged and irregularly shaped right kidney with a large area of cavitation, and a nephrectomy was performed 30 days after initial examination. Cytologic examination of fine-needle aspirates and imprints of the right kidney revealed a neoplastic cell population suggestive of renal carcinoma. The histopathologic diagnosis was chromophobic cystic-papillary renal carcinoma. The tumor cells expressed granulocytic/macrophage-colony-stimulating factor (GM-CSF), detected by immunohistochemical staining, and elaboration of GM-CSF by the tumor may have mediated the leukocytosis in this dog. Following excision of the tumor, neutrophil and monocyte counts were only mildly increased. The dog died 135 days after initial presentation, and a necropsy was not permitted. Paraneoplastic neutrophilic leukocytosis is an uncommon finding and may be caused by elaboration of CSF by neoplastic cells.

An intra-abdominal malignant mesenchymoma associated with nonabsorbable sutures in a ferret (Mustela putorius furo).

A 6-year-old ferret (Mustela putorius furo) was presented with abdominal enlargement. Clinical examination revealed an intra-abdominal mass measuring approximately 5 cm in diameter. Abdominal survey radiographs revealed a sharply marginated mass with multifocal radiodense foci, suggestive of pathologic calcification. A complete blood cell count revealed a moderate, normocytic, normochromic, nonregenerative anemia. The mass was surgically removed en bloc, fixed in 10% neutral buffered formalin solution, and routinely processed for histologic and immunohistochemical investigation. The neoplastic tissue consisted of a mixed neoplastic cell population, including osteosarcoma and fibrosarcoma components. Immunohistochemistry revealed that both neoplastic cell populations were positive for vimentin and negative for actin (smooth and striated), desmin, and myoglobin. Nonabsorbable suture material was admixed with the neoplastic tissue in the histologic section. This material was birefringent when viewed microscopically under polarized light.

A case of interscapular fibrosarcoma in a dwarf rabbit (Oryctolagus cuniculus).

A 1-year-old, intact, male dwarf rabbit (Oryctolagus cuniculus) was vaccinated against myxomatosis and rabbit viral hemorrhagic disease in February 1999, and a localized reaction appeared in the same anatomic site within a few days. No regression was observed after subcutaneous antibiotic treatment. The rabbit was kept under observation, and the swelling apparently disappeared in 3 months. The owner then decided to avoid any further subcutaneous drug administration. The referring veterinarian examined the animal on July 2006 for the sudden appearance of a nodular, 4.5 cm x 3.5 cm x 2.0 cm, subcutaneous mass located over the interscapular space. Fine-needle aspiration was performed, and a population of neoplastic spindle cells, rare pleomorphic multinucleated cells, and rare leukocytes were observed. The mass was surgically removed, fixed in 10% neutral buffered formalin, and routinely processed for histologic, histochemical, and immunohistochemical diagnostic investigation. The neoplastic tissue exhibited fascicles composed of malignant spindle-shaped cells with elongated to oval hyperchromatic nuclei and scant cytoplasm. Occasional multinucleated cells were also observed. The neoplastic cells were immunoreactive for vimentin but did not stain for smooth muscle actin, desmin, myoglobin, and cytokeratins (AE1/AE3). Moreover, the histochemical stain for aluminum was positive. The diagnosis was fibrosarcoma based on morphologic and immunohistochemical results. The histologic features of this neoplasm were remarkably similar to feline injection-site sarcoma.

17beta-estradiol promotes breast cancer cell proliferation-inducing stromal cell-derived factor-1-mediated epidermal growth factor receptor transactivation: reversal by gefitinib pretreatment.

The coordinated activity of estrogens and epidermal growth factor receptor (EGFR) family agonists represents the main determinant of breast cancer cell proliferation. Stromal cell-derived factor-1 (SDF-1) enhances extracellular signal-regulated kinases 1 and 2 (ERK1/2) activity via the transactivation of EGFR and 17beta-estradiol (E2) induces SDF-1 production to exert autocrine proliferative effects. On this basis, we evaluated whether the inhibition of the tyrosine kinase (TK) activity of EGFR may control different mitogenic stimuli in breast tumors using the EGFR-TK inhibitor gefitinib to antagonize the proliferation induced by E2 in T47D human breast cancer cells. EGF, E2, and SDF-1 induced a dose-dependent T47D cell proliferation, that being nonadditive suggested the activation of common intracellular pathways. Gefitinib treatment inhibited not only the EGF-dependent proliferation and ERK1/2 activation but also the effects of SDF-1 and E2, suggesting that these activities were mediated by EGFR transactivation. Indeed, both SDF-1 and E2 caused EGFR tyrosine phosphorylation. The molecular link between E2 and SDF-1 proliferative effects was identified because 1,1'-(1,4-phenylenebis(methylene))-bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist, inhibited SDF-1- and E2-dependent proliferation and EGFR and ERK1/2 phosphorylation. EGFR transactivation was dependent on c-Src activation. E2 treatment caused a powerful SDF-1 release from T47D cells. Finally, in SKBR3, E2-resistant cells, EGFR was constitutively activated, and AMD3100 reduced EGFR phosphorylation and cell proliferation, whereas HER2-neu was transactivated by SDF-1 in SKBR3 but not in T47D cells. In conclusion, we show that activation of CXCR4 transduces proliferative signals from the E2 receptor to EGFR, whose inhibition is able to revert breast cancer cell proliferation induced by multiple receptor activation.

Immunohistochemical evaluation of STAT3-p-tyr705 expression in feline mammary gland tumours and correlation with histologic grade.

STAT3 (signal transducer and activator of transcription 3) is a cytoplasmic transcription factor that plays a role in the G1 to S phase cell-cycle transition and is induced by cytokines and growth factors. The expression of STAT3 phosphorylated on tyrosine 705 (STAT3-p-tyr705) in normal, hyperplastic and neoplastic feline mammary gland tissue was assessed by immunohistochemistry in 45 cats. The samples included 4 normal mammary non-lactating tissues, 9 hyperplastic tissues (5 fibroepithelial hyperplasia and 4 lobular epithelial hyperplasia), 2 benign tumours (1 complex adenoma, and 1 simple adenoma), and 30 carcinomas (18 simple tubular papillary, 6 simple tubular, 2 simple solid, 3 cribriform, and 1 adenosquamous carcinoma). For immunohistochemistry, tissue sections were incubated with an anti-STAT3-p-tyr705 monoclonal antibody and visualized with EnVision-DAB polymer. STAT3-p-tyr705 positivity was quantified in a semi-quantitative manner. All positive samples showed cytoplasmic and/or nuclear positivity. Normal non-lactating mammary tissue showed a low number of positive cells, similar to hyperplastic tissue. In neoplastic tissues, a high number of positive cells with a moderate to intense reaction was observed. Moreover, a correlation was observed between nuclear positivity for STAT3-p-tyr705 and histologic grade (P=0.013; r=0.447), tubular formation (P=0.043; r=0.820), and mitotic activity (P<0.0001; r=0.689). In contrast, no such correlations were observed for cytoplasmic reactivity of STAT3-p-tyr705. A significant difference was observed between malignant lesions and hyperplasia with regards to expression of STAT3-p-tyr 705 in the cytoplasm (P=0.008; U=59.00) and nuclei (P=0.002; U=47.00). These results confirm previous our data and reinforce the potential role of STAT3 in malignancy as reported for human breast cancer and other sporadic tumours.

Undifferentiated tumor in the ovary of a corn snake (Elaphe guttata guttata).

A 6-year-old intact female corn snake (Elaphe guttata guttata) was presented with a 3-week history of anorexia. Coelomic radiographs revealed a 9 x 4 cm soft tissue opacity suggestive of a right ovarian enlargement. The mass (9 x 5 x 4 cm) was surgically removed, and multiple smears from tissue sections were stained with Diff-Quik. Multiple tissue samples from the mass were collected and fixed in formalin. Cytologic specimens were moderately cellular and contained light pink amorphous background material. The cells were primarily spindle-shaped with moderate to marked anisocytosis and anisokaryosis. Cells sometimes were round to polygonal, and rarely were arranged in small clusters. Macrophages occasionally were observed. Histologic specimens consisted of a highly cellular mass composed of pleomorphic, spindle-shaped cells and, occasionally, round to polygonal cells arranged in irregular fascicles. The neoplastic cells were immunoreactive for cytokeratin (AE1/AE3), smooth muscle actin, and skeletal muscle actin, but did not stain for vimentin or desmin. On the basis of the morphologic and immunohistochemical results, a diagnosis of ovarian undifferentiated carcinoma was made. In this report, we describe the challenges of using immunohistochemistry to diagnose this uncommon type of tumor in reptiles.

Bilateral nodular lymphocytic conjunctivitis in a horse.

A Russian jumper horse presented because of an ocular perilimbal conjunctival mass and, on clinical examination, two bilateral conjunctival masses were found, of different size and conformation. Attempts at complete excision of the left eye mass and excisional biopsy of the right eye mass were performed. The left eye mass recurred rapidly, but resolved completely after topical corticosteroid therapy. The two lesions had similar histopathologic features, characterized by focal, chronic, primarily lymphocytic conjunctivitis with follicular lymphoid hyperplasia. Special histopathologic staining techniques (Gomori methenamine silver and acid fast stains) and immunohistochemistry (for CD3, BLA36 and lysozyme) failed to reveal any etiologic agents and indicated an inflammatory lesion composed of a heterogeneous population of lymphocytes and macrophages (nodular lymphocytic conjunctivitis). The lesions were indistinguishable, clinically and behaviorally, from what has been reported as 'conjunctival pseudotumor' in the horse. Equine conjunctival pseudotumor/nodular lymphocytic conjunctivitis has been reported to be unilateral and have a good prognosis after partial or complete surgical excision. This is the first reported case of bilateral nodular lymphocytic conjunctivitis in a horse and for which surgical excision alone was not curative.