A Novel Inhibitor of Carbonic Anhydrases Prevents Hypoxia-Induced TNBC Cell Plasticity.

Cell plasticity is the ability that cells have to modify their phenotype, adapting to the environment. Cancer progression is under the strict control of the the tumor microenvironment that strongly determines its success by regulating the behavioral changes of tumor cells. The cross-talk between cancer and stromal cells and the interactions with the extracellular matrix, hypoxia and acidosis contribute to trigger a new tumor cell identity and to enhance tumor heterogeneity and metastatic spread. In highly aggressive triple-negative breast cancer, tumor cells show a significant capability to change their phenotype under the pressure of the hypoxic microenvironment. In this study, we investigated whether targeting the hypoxia-induced protein carbonic anhydrase IX (CA IX) could reduce triple-negative breast cancer (TNBC) cell phenotypic switching involved in processes associated with poor prognosis such as vascular mimicry (VM) and cancer stem cells (CSCs). The treatment of two TNBC cell lines (BT-549 and MDA-MB-231) with a specific CA IX siRNA or with a novel inhibitor of carbonic anhydrases (RC44) severely impaired their ability to form a vascular-like network and mammospheres and reduced their metastatic potential. In addition, the RC44 inhibitor was able to hamper the signal pathways involved in triggering VM and CSC formation. These results demonstrate that targeting hypoxia-induced cell plasticity through CA IX inhibition could be a new opportunity to selectively reduce VM and CSCs, thus improving the efficiency of existing therapies in TNBC.

Recruitment of stromal cells into tumour microenvironment promote the metastatic spread of breast cancer.

Currently, metastasis remains the primary cause of death of patients with breast cancer despite the important advances in the treatment of this disease. In the complex tumour microenvironment network, several malignant and non-malignant cell types as well as components of extracellular matrix cooperate in promoting the metastatic spread of breast carcinoma. Many components of the stromal compartment are recruited from distant sites to the tumour including mesenchymal stem cells, endothelial cells, macrophages and other immune cells whereas other cells such as fibroblasts are already present in both primary and secondary lesions. When these cells come into contact with cancer cells they are "educated" and acquire a pro-tumoural phenotype, which support all the steps of the metastatic cascade. In this Review, we highlight the role played by each stromal component in guiding cancer cells in their venture towards colonizing metastatic sites.

Recent Advances in Nuclear Imaging of Receptor Expression to Guide Targeted Therapies in Breast Cancer.

Breast cancer remains the most frequent cancer in women with different patterns of disease progression and response to treatments. The identification of specific biomarkers for different breast cancer subtypes has allowed the development of novel targeting agents for imaging and therapy. To date, patient management depends on immunohistochemistry analysis of receptor status on bioptic samples. This approach is too invasive, and in some cases, not entirely representative of the disease. Nuclear imaging using receptor tracers may provide whole-body information and detect any changes of receptor expression during disease progression. Therefore, imaging is useful to guide clinicians to select the best treatments for each patient and to evaluate early response thus reducing unnecessary therapies. In this review, we focused on the development of novel tracers that are ongoing in preclinical and/or clinical studies as promising tools to lead treatment decisions for breast cancer management.

Therapeutic Potential of a Novel αvß3 Antagonist to Hamper the Aggressiveness of Mesenchymal Triple Negative Breast Cancer Sub-Type.

The mesenchymal sub-type of triple negative breast cancer (MES-TNBC) has a highly aggressive behavior and worse prognosis, due to its invasive and stem-like features, that correlate with metastatic dissemination and resistance to therapies. Furthermore, MES-TNBC is characterized by the expression of molecular markers related to the epithelial-to-mesenchymal transition (EMT) program and cancer stem cells (CSCs). The altered expression of αvß3 integrin has been well established as a driver of cancer progression, stemness, and metastasis. Here, we showed that the high levels of αvß3 are associated with MES-TNBC and therefore exploited the possibility to target this integrin to reduce the aggressiveness of this carcinoma. To this aim, MES-TNBC cells were treated with a novel peptide, named ψRGDechi, that we recently developed and characterized for its ability to selectively bind and inhibit αvß3 integrin. Notably, ψRGDechi was able to hamper adhesion, migration, and invasion of MES-TNBC cells, as well as the capability of these cells to form vascular-like structures and mammospheres. In addition, this peptide reversed EMT program inhibits mesenchymal markers. These findings show that targeting αvß3 integrin by ψRGDechi, it is possible to inhibit some of the malignant properties of MES-TNBC phenotype.

Targeted imaging and inhibition of triple-negative breast cancer metastases by a PDGFRß aptamer.

While the overall mortality for breast cancer has recently declined, management of triple-negative breast cancer (TNBC) is still challenging because of its aggressive clinical behavior and the lack of targeted therapies. Genomic profiling studies highlighted the high level of heterogeneity of this cancer, which comprises different subtypes with unique phenotypes and response to treatment. Platelet-derived growth factor receptor ß (PDGFRß) is an established mesenchymal/stem cell-specific marker in human glioblastoma and, as recently suggested, it may uniquely mark breast cancer cells with stem-like characteristics and/or that have undergone epithelial-mesenchymal transition. Methods: Immunohistochemical analysis for PDGFRß expression was performed on a human TNBC tissue microarray. Functional assays were conducted on mesenchymal-like TNBC cells to investigate the effect of a previously validated PDGFRß aptamer on invasive cell growth in three-dimensional culture conditions, migration, invasion and tube formation. The aptamer was labeled with a near-infrared (NIR) dye and its binding specificity to PDGFRß was assessed both in vitro (confocal microscopy and flow cytometry analyses) and in vivo (fluorescence molecular tomography in mice bearing TNBC xenografts). A mouse model of TNBC lung metastases formation was established and NIR-labeled PDGFRß aptamer was used to detect lung metastases in mice untreated or intravenously injected with unlabeled aptamer. Results: Here, we present novel data showing that tumor cell expression of PDGFRß identifies a subgroup of mesenchymal tumors with invasive and stem-like phenotype, and propose a previously unappreciated role for PDGFRß in driving TNBC cell invasiveness and metastases formation. We show that the PDGFRß aptamer blocked invasive growth and migration/invasion of mesenchymal TNBC cell lines and prevented TNBC lung metastases formation. Further, upon NIR-labeling, the aptamer specifically bound to TNBC xenografts and detected lung metastases. Conclusions: We propose PDGFRß as a reliable biomarker of a subgroup of mesenchymal TNBCs with invasive and stem-like phenotype as well as the use of the PDGFRß aptamer as a high efficacious tool for imaging and suppression of TNBC lung metastases. This study will allow for the significant expansion of the current repertoire of strategies for managing patients with more aggressive TNBC.

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.

Inhibition of Bone Marrow-Derived Mesenchymal Stem Cells Homing Towards Triple-Negative Breast Cancer Microenvironment Using an Anti-PDGFRß Aptamer.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are shown to participate in tumor progression by establishing a favorable tumor microenvironment (TME) that promote metastasis through a cytokine networks. However, the mechanism of homing and recruitment of BM-MSCs into tumors and their potential role in malignant tissue progression is poorly understood and controversial. Here we show that BM-MSCs increase aggressiveness of triple-negative breast cancer (TNBC) cell lines evaluated as capability to migrate, invade and acquire stemness markers. Importantly, we demonstrate that the treatment of BM-MSCs with a nuclease-resistant RNA aptamer against platelet-derived growth factor receptor ß (PDGFRß) causes the inhibition of receptor-dependent signaling pathways thus drastically hampering BM-MSC recruitment towards TNBC cell lines and BM-MSCs trans-differentiation into carcinoma-associated fibroblast (CAF)-like cells. Moreover, in vivo molecular imaging analysis demonstrated the aptamer ability to prevent BM-MSCs homing to TNBC xenografts. Collectively, our results indicate the anti-PDGFRß aptamer as a novel therapeutic tool to interfere with BM-MSCs attraction to TNBC providing the rationale to further explore the aptamer in more complex pre-clinical settings.