Bispecific aptamer-decorated and light-triggered nanoparticles targeting tumor and stromal cells in breast cancer derived organoids: implications for precision phototherapies.

BACKGROUND: Based on the established role of cancer-stroma cross-talk in tumor growth, progression and chemoresistance, targeting interactions between tumor cells and their stroma provides new therapeutic approaches. Dual-targeted nanotherapeutics selectively acting on both tumor and stromal cells may overcome the limits of tumor cell-targeting single-ligand nanomedicine due to the complexity of the tumor microenvironment. METHODS: Gold-core/silica-shell nanoparticles embedding a water-soluble iridium(III) complex as photosensitizer and luminescent probe (Iren-AuSiO2_COOH) were efficiently decorated with amino-terminated EGFR (CL4) and PDGFRß (Gint4.T) aptamers (Iren-AuSiO2_Aptamer). The targeting specificity, and the synergistic photodynamic and photothermal effects of either single- and dual-aptamer-decorated nanoparticles have been assessed by confocal microscopy and cell viability assays, respectively, on different human cell types including mesenchymal subtype triple-negative breast cancer (MES-TNBC) MDA-MB-231 and BT-549 cell lines (both EGFR and PDGFRß positive), luminal/HER2-positive breast cancer BT-474 and epidermoid carcinoma A431 cells (only EGFR positive) and adipose-derived mesenchymal stromal/stem cells (MSCs) (only PDGFRß positive). Cells lacking expression of both receptors were used as negative controls. To take into account the tumor-stroma interplay, fluorescence imaging and cytotoxicity were evaluated in preclinical three-dimensional (3D) stroma-rich breast cancer models. RESULTS: We show efficient capability of Iren-AuSiO2_Aptamer nanoplatforms to selectively enter into target cells, and kill them, through EGFR and/or PDGFRß recognition. Importantly, by targeting EGFR+ tumor/PDGFRß+ stromal cells in the entire tumor bulk, the dual-aptamer-engineered nanoparticles resulted more effective than unconjugated or single-aptamer-conjugated nanoparticles in either 3D spheroids cocultures of tumor cells and MSCs, and in breast cancer organoids derived from pathologically and molecularly well-characterized tumors. CONCLUSIONS: Our study proposes smart, novel and safe multifunctional nanoplatforms simultaneously addressing cancer-stroma within the tumor microenvironment, which are: (i) actively delivered to the targeted cells through highly specific aptamers; (ii) localized by means of their luminescence, and (iii) activated via minimally invasive light, launching efficient tumor death, thus providing innovative precision therapeutics. Given the unique features, the proposed dual targeted nanoformulations may open a new door to precision cancer treatment.

Tissue Inhibitor of Metalloproteinases-1 Overexpression Mediates Chemoresistance in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is among the most aggressive breast cancer subtypes. Despite being initially responsive to chemotherapy, patients develop drug-resistant and metastatic tumors. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a secreted protein with a tumor suppressor function due to its anti-proteolytic activity. Nevertheless, evidence indicates that TIMP-1 binds to the CD63 receptor and activates noncanonical oncogenic signaling in several cancers, but its role in mediating TNBC chemoresistance is still largely unexplored. Here, we show that mesenchymal-like TNBC cells express TIMP-1, whose levels are further increased in cells generated to be resistant to cisplatin (Cis-Pt-R) and doxorubicin (Dox-R). Moreover, public dataset analyses indicate that high TIMP-1 levels are associated with a worse prognosis in TNBC subjected to chemotherapy. Knock-down of TIMP-1 in both Cis-Pt-R and Dox-R cells reverses their resistance by inhibiting AKT activation. Consistently, TNBC cells exposed to recombinant TIMP-1 or TIMP-1-enriched media from chemoresistant cells, acquire resistance to both cisplatin and doxorubicin. Importantly, released TIMP-1 reassociates with plasma membrane by binding to CD63 and, in the absence of CD63 expression, TIMP-1-mediated chemoresistance is blocked. Thus, our results identify TIMP-1 as a new biomarker of TNBC chemoresistance and lay the groundwork for evaluating whether blockade of TIMP-1 signal is a viable treatment strategy.

Aptamer-Based Strategies to Boost Immunotherapy in TNBC.

The immune system (IS) may play a crucial role in preventing tumor development and progression, leading, over the last years, to the development of effective cancer immunotherapies. Nevertheless, immune evasion, the capability of tumors to circumvent destructive host immunity, remains one of the main obstacles to overcome for maximizing treatment success. In this context, promising strategies aimed at reshaping the tumor immune microenvironment and promoting antitumor immunity are rapidly emerging. Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype with poor outcomes, is highly immunogenic, suggesting immunotherapy is a viable strategy. As evidence of this, already, two immunotherapies have recently become the standard of care for patients with PD-L1 expressing tumors, which, however, represent a low percentage of patients, making more active immunotherapeutic approaches necessary. Aptamers are short, highly structured, single-stranded oligonucleotides that bind to their protein targets at high affinity and specificity. They are used for therapeutic purposes in the same way as monoclonal antibodies; thus, various aptamer-based strategies are being actively explored to stimulate the IS's response against cancer cells. The aim of this review is to discuss the potential of the recently reported aptamer-based approaches to boost the IS to fight TNBC.

Aptamer-Functionalized Nanoparticles Mediate PD-L1 siRNA Delivery for Effective Gene Silencing in Triple-Negative Breast Cancer Cells.

Small interfering RNA (siRNA) therapies require effective delivery vehicles capable of carrying the siRNA cargo into target cells. To achieve tumor-targeting, a drug delivery system would have to incorporate ligands that specifically bind to receptors expressed on cancer cells to function as portals via receptor-mediated endocytosis. Cell-targeting and internalizing aptamers are the most suitable ligands for functionalization of drug-loaded nanocarriers. Here, we designed a novel aptamer-based platform for the active delivery of siRNA targeting programmed cell death-ligand 1 (PD-L1) to triple-negative breast cancer (TNBC) cells. The generated nanovectors consist of PLGA-based polymeric nanoparticles, which were loaded with PD-L1 siRNA and conjugated on their surface with a new RNA aptamer, specific for TNBC and resistant to nucleases. In vitro results demonstrated that these aptamer-conjugated nanoparticles promote siRNA uptake specifically into TNBC MDA-MB-231 and BT-549 target cells, along with its endosomal release, without recognizing non-TNBC BT-474 breast cancer cells. Their efficiency resulted in an almost complete suppression of PD-L1 expression as early as 90 min of cell treatment. This research provides a rational strategy for optimizing siRNA delivery systems for TNBC treatments.

Optimization of Short RNA Aptamers for TNBC Cell Targeting.

Triple-negative breast cancer (TNBC) is an aggressive cancer with limited targeted therapies. RNA aptamers, suitably chemically modified, work for therapeutic purposes in the same way as antibodies. We recently generated 2'Fluoro-pyrimidines RNA-aptamers that act as effective recognition elements for functional surface signatures of TNBC cells. Here, we optimized three of them by shortening and proved the truncated aptamers as optimal candidates to enable active targeting to TNBC. By using prediction of secondary structure to guide truncation, we identified structural regions that account for the binding motifs of the full-length aptamers. Their chemical synthesis led to short aptamers with superb nuclease resistance, which specifically bind to TNBC target cells and rapidly internalize into acidic compartments. They interfere with the growth of TNBC cells as mammospheres, thus confirming their potential as anti-tumor agents. We propose sTN145, sTN58 and sTN29 aptamers as valuable tools for selective TNBC targeting and promising candidates for effective treatments, including therapeutic agents and targeted delivery nanovectors.

Selective Photo-Assisted Eradication of Triple-Negative Breast Cancer Cells through Aptamer Decoration of Doped Conjugated Polymer Nanoparticles.

Photodynamic therapy (PDT) may be an excellent alternative in the treatment of breast cancer, mainly for the most aggressive type with limited targeted therapies such as triple-negative breast cancer (TNBC). We recently generated conjugated polymer nanoparticles (CPNs) as efficient photosensitizers for the photo-eradication of different cancer cells. With the aim of improving the selectivity of PDT with CPNs, the nanoparticle surface conjugation with unique 2'-Fluoropyrimidines-RNA-aptamers that act as effective recognition elements for functional surface signatures of TNBC cells was proposed and designed. A coupling reaction with carbodiimide was used to covalently bind NH2-modified aptamers with CPNs synthetized with two polystyrene-based polymer donors of COOH groups for the amide reaction. The selectivity of recognition for TNBC membrane receptors and PDT efficacy were assayed in TNBC cells and compared with non-TNBC cells by flow cytometry and cell viability assays. Furthermore, in vitro PDT efficacy was assayed in different TNBC cells with significant improvement results using CL4, sTN29 and sTN58 aptamers compared to unconjugated CPNs and SCR non-specific aptamer. In a chemoresistance TNBC cell model, sTN58 was the candidate for improving labelling and PDT efficacy with CPNs. We proposed sTN58, sTN29 and CL4 aptamers as valuable tools for selective TNBC targeting, cell internalization and therapeutic improvements for CPNs in PDT protocols.

Optimizing cisplatin delivery to triple-negative breast cancer through novel EGFR aptamer-conjugated polymeric nanovectors.

BACKGROUND: Management of triple-negative breast cancer (TNBC) is still challenging because of its aggressive clinical behavior and limited targeted treatment options. Cisplatin represents a promising chemotherapeutic compound in neoadjuvant approaches and in the metastatic setting, but its use is limited by scarce bioavailability, severe systemic side effects and drug resistance. Novel site-directed aptamer-based nanotherapeutics have the potential to overcome obstacles of chemotherapy. In this study we investigated the tumor targeting and the anti-tumorigenic effectiveness of novel cisplatin-loaded and aptamer-decorated nanosystems in TNBC. METHODS: Nanotechnological procedures were applied to entrap cisplatin at high efficacy into polymeric nanoparticles (PNPs) that were conjugated on their surface with the epidermal growth factor receptor (EGFR) selective and cell-internalizing CL4 aptamer to improve targeted therapy. Internalization into TNBC MDA-MB-231 and BT-549 cells of aptamer-decorated PNPs, loaded with BODIPY505-515, was monitored by confocal microscopy using EGFR-depleted cells as negative control. Tumor targeting and biodistribution was evaluated by fluorescence reflectance imaging upon intravenously injection of Cyanine7-labeled nanovectors in nude mice bearing subcutaneous MDA-MB-231 tumors. Cytotoxicity of cisplatin-loaded PNPs toward TNBC cells was evaluated by MTT assay and the antitumor effect was assessed by tumor growth experiments in vivo and ex vivo analyses. RESULTS: We demonstrate specific, high and rapid uptake into EGFR-positive TNBC cells of CL4-conjugated fluorescent PNPs which, when loaded with cisplatin, resulted considerably more cytotoxic than the free drug and nanovectors either unconjugated or conjugated with a scrambled aptamer. Importantly, animal studies showed that the CL4-equipped PNPs achieve significantly higher tumor targeting efficiency and enhanced therapeutic effects, without any signs of systemic toxicity, compared with free cisplatin and untargeted PNPs. CONCLUSIONS: Our study proposes novel and safe drug-loaded targeted nanosystems for EGFR-positive TNBC with excellent potential for the application in cancer diagnosis and therapy.

Aptamers and antibodies: rivals or allies in cancer targeted therapy?

The goal of an efficacious cancer therapy is to specifically target diseased cells at high accuracy while sparing normal, healthy cells. Over the past three decades, immunotherapy, based on the use of monoclonal antibodies (mAbs) directed against tumor-associated antigens, to inhibit their oncogenic function, or against immune checkpoints, to modulate specific T cell responses against cancer, has proven to be an important strategy for cancer therapy. Nevertheless, the number of mAbs approved for clinical use is still limited because of significant drawbacks to their applicability. Oligonucleotide aptamers, similarly to antibodies, form high-affinity bonds with their specific protein targets, thus representing an effective tool for active cancer targeting. Compared to antibodies, aptamers' use as therapeutic agents benefits from their low size, low/no immunogenicity, simple synthesis and design flexibility for improving efficacy and stability. This review intends to highlight recently emerged applications of aptamers as recognition elements, from biomarker discovery to targeted drug delivery and targeted treatment, showing aptamers' potential to work in conjunction with antibodies for attacking cancer from multiple flanks.

Profiling Cancer Cells by Cell-SELEX: Use of Aptamers for Discovery of Actionable Biomarkers and Therapeutic Applications Thereof.

The identification of tumor cell-specific surface markers is a key step towards personalized cancer medicine, allowing early assessment and accurate diagnosis, and development of efficacious targeted therapies. Despite significant efforts, currently the spectrum of cell membrane targets associated with approved treatments is still limited, causing an inability to treat a large number of cancers. What mainly limits the number of ideal clinical biomarkers is the high complexity and heterogeneity of several human cancers and still-limited methods for molecular profiling of specific cancer types. Thanks to the simplicity, versatility and effectiveness of its application, cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology is a valid complement to the present strategies for biomarkers' discovery. We and other researchers worldwide are attempting to apply cell-SELEX to the generation of oligonucleotide aptamers as tools for both identifying new cancer biomarkers and targeting them by innovative therapeutic strategies. In this review, we discuss the potential of cell-SELEX for increasing the currently limited repertoire of actionable cancer cell-surface biomarkers and focus on the use of the selected aptamers as components of innovative conjugates and nano-formulations for cancer therapy.

Aptamer targeted therapy potentiates immune checkpoint blockade in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a uniquely aggressive cancer with high rates of relapse due to resistance to chemotherapy. TNBC expresses higher levels of programmed cell death-ligand 1 (PD-L1) compared to other breast cancers, providing the rationale for the recently approved immunotherapy with anti-PD-L1 monoclonal antibodies (mAbs). A huge effort is dedicated to identify actionable biomarkers allowing for combination therapies with immune-checkpoint blockade. Platelet-derived growth factor receptor ß (PDGFRß) is highly expressed in invasive TNBC, both on tumor cells and tumor microenvironment. We recently proved that tumor growth and lung metastases are impaired in mouse models of human TNBC by a high efficacious PDGFRß aptamer. Hence, we aimed at investigating the effectiveness of a novel combination treatment with the PDGFRß aptamer and anti-PD-L1 mAbs in TNBC. METHODS: The targeting ability of the anti-human PDGFRß aptamer toward the murine receptor was verified by streptavidin-biotin assays and confocal microscopy, and its inhibitory function by transwell migration assays. The anti-proliferative effects of the PDGFRß aptamer/anti-PD-L1 mAbs combination was assessed in human MDA-MB-231 and murine 4 T1 TNBC cells, both grown as monolayer or co-cultured with lymphocytes. Tumor cell lysis and cytokines secretion by lymphocytes were analyzed by LDH quantification and ELISA, respectively. Orthotopic 4 T1 xenografts in syngeneic mice were used for dissecting the effect of aptamer/mAb combination on tumor growth, metastasis and lymphocytes infiltration. Ex vivo analyses through immunohistochemistry, RT-qPCR and immunoblotting were performed. RESULTS: We show that the PDGFRß aptamer potentiates the anti-proliferative activity of anti-PD-L1 mAbs on both human and murine TNBC cells, according to its human/mouse cross-reactivity. Further, by binding to activated human and mouse lymphocytes, the aptamer enhances the anti-PD-L1 mAb-induced cytotoxicity of lymphocytes against tumor cells. Importantly, the aptamer heightens the antibody efficacy in inhibiting tumor growth and lung metastases in mice. It acts on both tumor cells, inhibiting Akt and ERK1/2 signaling pathways, and immune populations, increasing intratumoral CD8 + T cells and reducing FOXP3 + Treg cells. CONCLUSION: Co-treatment of PDGFRß aptamer with anti-PD-L1 mAbs is a viable strategy, thus providing for the first time an evidence of the efficacy of PDGFRß/PD-L1 co-targeting combination therapy in TNBC.

Novel Aptamers Selected on Living Cells for Specific Recognition of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a high heterogeneous group of tumors with a distinctly aggressive nature and high rates of relapse. So far, the lack of any known targetable proteins has not allowed a specific anti-tumor treatment. Therefore, the identification of novel agents for specific TNBC targeting and treatment is desperately needed. Here, by integrating cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) for the specific recognition of TNBC cells with high-throughput sequencing technology, we identified a panel of 2'-fluoropyrimidine-RNA aptamers binding to TNBC cells and their cisplatin- and doxorubicin-resistant derivatives at low nanomolar affinity. These aptamers distinguish TNBC cells from both non-malignant and non-TNBC breast cancer cells and are able to differentiate TNBC histological specimens. Importantly, they inhibit TNBC cell capacity of growing in vitro as mammospheres, indicating they could also act as anti-tumor agents. Therefore, our newly identified aptamers are a valuable tool for selectively dealing with TNBC.

Ipilimumab and Its Derived EGFR Aptamer-Based Conjugate Induce Efficient NK Cell Activation against Cancer Cells.

The immune checkpoint CTLA-4 (cytotoxic T-lymphocyte-antigen 4), which inhibits the co-stimulatory CD28 signal on T cells, has been recently found expressed on other cell populations, such as tumor and natural killer (NK) cells. We tested for the first time the effects of ipilimumab, the human anti-CTLA4 mAb in clinical use, on these cells and found that it inhibits the growth of tumor cells expressing CTLA-4 also in the absence of lymphocytes, and efficiently activates NK cells, thus suggesting an important unexplored role of NK cells in ipilimumab-modulated immune responses. Interestingly, the epidermal growth factor receptor (EGFR) has been shown to play a key role in tumor cell escape from immune surveillance, and in cytotoxic T lymphocyte inhibition. Thus, we tested combinatorial treatments of ipilimumab with an anti-EGFR aptamer endowed with anti-tumor activity, and constructed for the first time a novel bispecific immunoconjugate, made up of these two compounds. The novel immunoconjugate binds to the target cells, induces the activation of lymphocytes, including NK cells, and inhibits the growth of tumor target cells more efficiently than the parental compounds, by strongly enhancing the cytotoxic activity of both human peripheral blood mononuclear cells and NK cells against tumor cells.

Novel Human Bispecific Aptamer-Antibody Conjugates for Efficient Cancer Cell Killing.

Monoclonal antibodies have been approved by the Food and Drug Administration for the treatment of various human cancers. More recently, oligonucleotide aptamers have risen increasing attention for cancer therapy thanks to their low size (efficient tumor penetration) and lack of immunogenicity, even though the short half-life and lack of effector functions still hinder their clinical applications. Here, we demonstrate, for the first time, that two novel bispecific conjugates, consisting of an anti-epidermal growth factor receptor (EGFR) aptamer linked either with an anti-epidermal growth factor receptor 2 (ErbB2) compact antibody or with an immunomodulatory (anti-PD-L1) antibody, were easily and rapidly obtained. These novel aptamer-antibody conjugates retain the targeting ability of both the parental moieties and acquire a more potent cancer cell killing activity by combining their inhibitory properties. Furthermore, the conjugation of the anti-EGFR aptamer with the immunomodulatory antibody allowed for the efficient redirection and activation of T cells against cancer cells, thus dramatically enhancing the cytotoxicity of the two conjugated partners. We think that these bispecific antibody-aptamer conjugates could have optimal biological features for therapeutic applications, such as increased specificity for tumor cells expressing both targets and improved pharmacokinetic and pharmacodynamic properties due to the combined advantages of the aptamer and antibody.

Dual Oncogenic/Anti-Oncogenic Role of PATZ1 in FRTL5 Rat Thyroid Cells Transformed by the Ha-RasV12 Oncogene.

PATZ1 is a transcriptional factor downregulated in thyroid cancer whose re-expression in thyroid cancer cells leads to a partial reversion of the malignant phenotype, including the capacity to proliferate, migrate, and undergo epithelial-to-mesenchymal transition. We have recently shown that PATZ1 is specifically downregulated downstream of the Ras oncogenic signaling through miR-29b, and that restoration of PATZ1 in Ha-Ras transformed FRTL5 rat thyroid cells is able to inhibit their capacities to proliferate and migrate in vitro. Here, we analyzed the impact of PATZ1 expression on the in vivo tumorigenesis of these cells. Surprisingly, FRTL5-Ras-PATZ1 cells showed enhanced tumor initiation when engrafted in nude mice, even if their tumor growth rate was reduced compared to that of FRTL5-Ras control cells. To further investigate the cause of the enhanced tumor engraftment of FRTL5-Ras-PATZ1 cells, we analyzed the stem-like potential of these cells through their capacity to grow as thyrospheres. The results showed that restoration of PATZ1 expression in these cells increases stem cell markers' expression and self-renewal ability of the thyrospheres while limiting their growth capacity. Therefore, we suggest that PATZ1 may play a role in enhancing the stem cell potential of thyroid cancer cells, but, at the same time, it impairs the proliferation of non-stem cells.

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.

TNBC Challenge: Oligonucleotide Aptamers for New Imaging and Therapy Modalities.

Compared to other breast cancers, triple-negative breast cancer (TNBC) usually affects younger patients, is larger in size, of higher grade and is biologically more aggressive. To date, conventional cytotoxic chemotherapy remains the only available treatment for TNBC because it lacks expression of the estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2), and no alternative targetable molecules have been identified so far. The high biological and clinical heterogeneity adds a further challenge to TNBC management and requires the identification of new biomarkers to improve detection by imaging, thus allowing the specific treatment of each individual TNBC subtype. The Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique holds great promise to the search for novel targetable biomarkers, and aptamer-based molecular approaches have the potential to overcome obstacles of current imaging and therapy modalities. In this review, we highlight recent advances in oligonucleotide aptamers used as imaging and/or therapeutic agents in TNBC, discussing the potential options to discover, image and hit new actionable targets in TNBC.

Trabectedin modulates the senescence-associated secretory phenotype and promotes cell death in senescent tumor cells by targeting NF-κB.

Therapy-induced senescence is a major cellular response to chemotherapy in solid tumors. Senescent tumor cells acquire a secretory phenotype, or SASP, and produce pro-inflammatory factors, whose expression is largely under NF-κB transcriptional control. Secreted factors play a positive role in driving antitumor immunity, but also exert negative influences on the microenvironment, and promote tumor growth and metastasis. Moreover, subsets of cancer cells can escape the senescence arrest, driving tumor recurrence after treatments. Hence, removal the senescent tumor cells, or reprogramming of the senescent secretome, have become attractive therapeutic options. The marine drug trabectedin was shown to inhibit the production of pro-inflammatory mediators by tumor-infiltrating immune cells and by myxoid liposarcoma cells. Here, we demonstrate that trabectedin inhibits the SASP, thus limiting the pro-tumoral activities of senescent tumor cells in vitro. We show that trabectedin modulates NF-κB transcriptional activity in senescent tumor cells. This results in disruption of the balance between antiapoptotic and proapoptotic signals, and sensitization of cells to Fas-mediated apoptosis. Further, we found that trabectedin inhibits escape from therapy-induced senescence, at concentrations that do not affect the viability of bulk tumor population. Overall, our data demonstrate that trabectedin has the potential to inhibit multiple detrimental effects of therapy-induced senescence.

Oligonucleotide aptamers against tyrosine kinase receptors: Prospect for anticancer applications.

Transmembrane receptor tyrosine kinases (RTKs) play crucial roles in cancer cell proliferation, survival, migration and differentiation. Area of intense research is searching for effective anticancer therapies targeting these receptors and, to date, several monoclonal antibodies and small-molecule tyrosine kinase inhibitors have entered the clinic. However, some of these drugs show limited efficacy and give rise to acquired resistance. Emerging highly selective compounds for anticancer therapy are oligonucleotide aptamers that interact with their targets by recognizing a specific three-dimensional structure. Because of their nucleic acid nature, the rational design of advanced strategies to manipulate aptamers for both diagnostic and therapeutic applications is greatly simplified over antibodies. In this manuscript, we will provide a comprehensive overview of oligonucleotide aptamers as next generation strategies to efficiently target RTKs in human cancers.

PATZ1 is a new prognostic marker of glioblastoma associated with the stem-like phenotype and enriched in the proneural subtype.

Glioblastoma (GBM), the most malignant of the brain tumors, has been classified on the basis of molecular signature into four subtypes: classical, mesenchymal, proneural and neural, among which the mesenchymal and proneural subtypes have the shortest and longest survival, respectively. Here we show that the transcription factor PATZ1 gene is upregulated in gliomas compared to normal brain and, among GBMs, is particularly enriched in the proneural subtype and co-localize with stemness markers. Accordingly, in GBM-derived glioma-initiating stem cells (GSCs) PATZ1 is overexpressed compared to differentiated tumor cells and its expression significantly correlates with the characteristic stem cell capacity to grow as neurospheres in vitro. Interestingly, survival analysis demonstrated that PATZ1 lower levels informed poor prognosis in GBM and, specifically, in the proneural subgroup, suggesting it may serve a role as diagnostic and prognostic biomarker for intra-subtype heterogeneity of proneural GBM. We also show that PATZ1 suppresses the expression of the mesenchyme-inducer CXCR4, and that PATZ1 and CXCR4 are inversely correlated in GSC and proneural GBM. Overall these findings support a central role of PATZ1 in regulating malignancy of GBM.

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.