A. Zannetti

Elevated expression of the tyrosine phosphatase SHP-1 defines a subset of high-grade breast tumors

Objectives: Protein tyrosine phosphatases are key regulators of intracellular signaling that contribute to determining cancer cell growth, which thus makes them attractive targets for therapeutic and diagnostic agents. SHP-1 phosphotyrosine phosphatase is rarely expressed in epithelial tumor cells, but expression has been found in several breast cancer cell lines and tumors. To determine the potential significance of SHP-1 as a prognostic marker in the clinical setting, we examined SHP-1 protein expression in breast tumors. Methods: We analyzed SHP-1 expression by immunohistochemistry in a breast tissue microarray composed of 2,081 cores, either alone or in combination with known prognostic markers. Results: Our data showed that SHP-1 expression was confined to a well-defined subset of high-grade tumors characterized by unique biological parameters. SHP-1 expression correlated directly with expression of the tyrosine kinase receptor HER-2 and inversely with expression of the estrogen receptor, while it was weakly associated with Bcl-2 expression. Conclusions: Levels of SHP-1 were correlated with conventional pathologic parameters of tumor aggressiveness and were associated with reduced patient survival, suggesting that elevated expression of SHP-1 is a common molecular abnormality in a defined subset of breast tumors and might be used in routine diagnosis to identify patients with high-risk tumors.

A novel antagonist of CXCR4 prevents bone marrow-derived mesenchymal stem cell-mediated osteosarcoma and hepatocellular carcinoma cell migration and invasion.

Recent findings suggest that bone marrow-derived mesenchymal stem cells (BM-MSCs) are recruited into the microenvironment of developing tumors, where they contribute to metastatic processes. The aim of this study was to investigate the role of BM-MSCs in promoting osteosarcoma and hepatocellular carcinoma cell progression in vitro and the possible mechanisms involved in these processes. U2OS and SNU-398 are osteosarcoma and hepatocellular carcinoma cell lines, respectively, that can be induced to proliferate when cultured in the presence of BM-MSCs. To determine the effect of BM-MSCs on U2OS and SNU-398 cells, the AKT and ERK signaling pathways were investigated, and increases were observed in active P-Akt and P-Erk forms. Moreover, BM-MSCs caused an increase in tumor cell migration and invasion that was derived from the enhancement of CXCR4 levels. Thus, when tumor cells were treated with the CXCR4 antagonist AMD3100, a reduction in their migration and invasion was observed. Furthermore, a new CXCR4 inhibitor, Peptide R, which was recently developed as an anticancer agent, was used to inhibit BM-MSC-mediated tumor invasion and to overcome AMD3100 toxicity. Taken together, these results suggest that inhibiting CXCR4 impairs the cross-talk between tumor cells and BM-MSCs, resulting in reduced metastatic potential in osteosarcoma and hepatocellular carcinoma cells.

Tumor-educated mesenchymal stem cells promote pro-metastatic phenotype

Multipotent mesenchymal stem cells (MSCs) are recruited into tumor microenvironment in response to multiple signals produced by cancer cells. Molecules involved in their homing to tumors are the same inflammatory mediators produced by injured tissues: chemokines, cytokines and growth factors. When MSCs arrive into the tumor microenvironment these are “educated” to have pro-metastatic behaviour. Firstly, they promote cancer immunosuppression modulating both innate and adaptive immune systems. Moreover, tumor associated-MSCs trans-differentiating into cancer-associated fibroblasts can induce epithelial-mesenchymal-transition program in tumor cells. This process determinates a more aggressive phenotype of cancer cells by increasing their motility and invasiveness and favoring their dissemination to distant sites. In addition, MSCs are involved in the formation and modelling of pre-metastatic niches creating a supportive environment for colonization of circulating tumor cells. The development of novel therapeutic approaches targeting the different functions of MSCs in promoting tumor progression as well as the mechanisms underlying their activities could enhance the efficacy of conventional and immune anti-cancer therapies. Furthermore, many studies report the use of MSCs engineered to express different genes or as vehicle to specifically deliver novel drugs to tumors exploiting their strong tropism. Importantly, this approach can enhance local therapeutic efficacy and reduce the risk of systemic side effects.

PET/CT in cancer research: from preclinical to clinical applications.

The identification of genetic and biochemical mechanisms underlying tumor growth and progression along with the unraveling of human genoma provided a plethora of new targets for cancer detection, treatment and monitoring. Simultaneously, the extraordinary development of a number of imaging technologies, including hybrid systems, allowed the visualization of biochemical, molecular and physiological aberrations linked to underlying mutations in a given tumor. In vivo evaluation of complex biological processes such as proliferation, apoptosis, angiogenesis, metastasis, gene expression, receptor-ligand interactions, transport of substrates and metabolism of nutrients in human cancers is feasible using PET/CT and radiolabeled molecular probes. Some of these compounds are in preclinical phases of evaluation whereas others have been already applied in clinical settings. Here we provide prominent examples on how some biological processes and target expression can be visualized by PET/CT in animal tumor models and cancer patients for the noninvasive detection of well-known markers of tumor aggressiveness, invasiveness and resistance to treatment and for the evaluation of tumor response to therapy.

Inhibition of AQP1 Hampers Osteosarcoma and Hepatocellular Carcinoma Progression Mediated by Bone Marrow-Derived Mesenchymal Stem Cells.

The complex cross-talk between tumor cells and their surrounding stromal environment plays a key role in the pathogenesis of cancer. Among several cell types that constitute the tumor stroma, bone marrow-derived mesenchymal stem cells (BM-MSCs) selectively migrate toward the tumor microenvironment and contribute to the active formation of tumor-associated stroma. Therefore, here we elucidate the involvement of BM-MSCs to promote osteosarcoma (OS) and hepatocellular carcinoma (HCC) cells migration and invasion and deepening the role of specific pathways. We analyzed the function of aquaporin 1 (AQP1), a water channel known to promote metastasis and neoangiogenes. AQP1 protein levels were analyzed in OS (U2OS) and HCC (SNU-398) cells exposed to conditioned medium from BM-MSCs. Tumor cell migration and invasion in response to BM-MSC conditioned medium were evaluated through a wound healing assay and Boyden chamber, respectively. The results showed that the AQP1 level was increased in both tumor cell lines after treatment with BM-MSC conditioned medium. Moreover, BM-MSCs-mediated tumor cell migration and invasion were hampered after treatment with AQP1 inhibitor. These data suggest that the recruitment of human BM-MSCs into the tumor microenvironment might cause OS and HCC cell migration and invasion through involvement of AQP1.

A novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) specifically detects CXCR4 expressing tumors

C-X-C chemokine receptor 4 (CXCR4) is over-expressed in multiple human cancers and correlates with tumor aggressiveness, poor prognosis and increased risk for distant metastases. Imaging agents for CXCR4 are thus highly desirable. We developed a novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) conjugating the new developed CXCR4 peptidic antagonist Peptide R with the NIR fluorescent dye VivoTag-S750. Specific CXCR4 binding was obtained in cells overexpressing human CXCR4 (B16-hCXCR4 and human melanoma cells PES43), but not in CXCR4 low expressing cells (FB-1). Ex vivo evaluation demonstrated that PepR-NIR750 specifically detects B16-hCXCR4-derived subcutaneous tumors and lung metastases. Fluorescence Molecular Tomography (FMT) in vivo imaging was performed on mice carrying subcutaneous CHO and CHO-CXCR4 tumors. PepR-NIR750 accumulates only in CXCR4-positive expressing subcutaneous tumors. Additionally, an intense NIR fluorescence signal was detected in PES43-derived lung metastases of nude mice injected with PepR-NIR750 versus mice injected with VivoTag-S750. With a therapeutic intent, mice bearing PES43-derived lung metastases were treated with Peptide R. A the dramatic reduction in PES43-derived lung metastases was detected through a decrease of the PepR-NIR750 signal. PepR-NIR750 is a specific probe for non-invasive detection of human high CXCR4-expressing tumors and metastatic lesion and thus a valuable tool for cancer molecular imaging.

Functional imaging of multidrug resistance in breast cancer.

Intrinsic or acquired multidrug resistance is the major cause of treatment failure in many human cancers. Multiple cellular mechanisms may contribute to the development of multidrug resistance including overexpression of P-glycoprotein (Pgp). The use of 99mTc-labeled lipophilic cations, which are transport substrate of Pgp, raised the possibility to predict the tumor response to treatment and to identify patients who will become refractory to subsequent therapy. Among these agents, 99mTc-MIBI is the most widely evaluated tracer and may serve as a paradigm of this class of compounds. In particular, many studies have shown the prognostic value of 99mTc-MIBI scan in different types of malignancy including breast cancer and the correlation with the expression of Pgp. However, additional mechanisms of cell resistance, mainly involving alterations of apoptosis, may also affect 99mTc-MIBI uptake in tumors. In particular, overexpression of the anti-apoptotic protein Bcl-2 prevents tumor cells to enter apoptosis and inhibits tracer accumulation into mitochondria. Therefore, while an absent or reduced early tracer uptake in large breast carcinomas reflects the existence of a defective apoptotic program, an enhanced tracer clearance in 99mTc-MIBI positive lesions reflects the activity of drug transporters such as Pgp. The existence of two different mechanisms underlying the predictive role of 99mTc-MIBI scan may be important to establish whether individual patients may benefit from Pgp inhibitors or Bcl-2 antagonists.

Autocrine signals increase ovine mesenchymal stem cells migration through Aquaporin-1 and CXCR4 overexpression

Sheep is a relevant large animal model that is frequently used to test innovative tissue engineering (TE) approaches especially for bone reconstruction. Mesenchymal stem cells (MSCs) are used in TE applications because they represent key component of adult tissue repair. Importantly, MSCs from different species show similar characteristics, which facilitated their application in translational studies using animal models. Nowadays, many researches are focusing on the use of ovine mesenchymal stem cells (oMSCs) in orthopedic preclinical settings for regenerative medicine purposes. Therefore, there is a need to amplify our knowledge on the mechanisms underlying the behaviour of these cells. Recently, several studies have shown that MSC function is largely dependent on factors that MSCs release in the environment, as well as, in conditioned medium (CM). It has been demonstrated that MSCs through autocrine and paracrine signals are able to stimulate proliferation, migration, and differentiation of different type of cells including themselves. In this study, we investigated the effects of the CM produced by oMSCs on oMSCs themselves and we explored the signal pathways involved. We observed that CM caused an enhancement of oMSC migration. Furthermore, we found that CM increased levels of two membrane proteins involved in cell migration, Aquaporin 1 (AQP1), and C‐X‐C chemokine receptor type 4 (CXCR4), and activated Akt and Erk intracellular signal pathways. In conclusion, taken together our results suggest the high potential of autologous CM as a promising tool to modulate behaviour of MSCs thus improving their use in therapeutically approaches.