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Safe and efficient gene delivery vectors would enhance the prospects for polynucleotide-based therapies. Herein we describe a new approach towards structurally-optimized gene vector design based on the preparation of clickable poly(allylamino-phosphazene)s that can be converted to several cationic and anionic derivatives via thiol-ene addition. Simultaneous co-incubation of alkylamine- and alkylcarboxylate-poly(phosphazenes) with polynucleotides generated binary-polyelectrolyte nanoparticles. Screening of a series of these complexes for transfection in glioblastoma cells showed that the inclusion of 6-mercaptohexanoic acid substituted poly(phosphazene)s in the complexes resulted in 6-fold and 19-fold higher luciferase expression in U87MG cells and primary GBM1 cell- line, respectively. This effect was attributed to the specific ionization properties of this materials that improved polyplex intracellular trafficking. Transfection in 3D-spheroid models and subcutaneous xenograft U87MG tumors confirmed higher transgene expression for the binary cationic/anionic poly(phosphazene) complexes compared to the related polycation-pDNA complexes and to PEI- pDNA complexes. The data also indicated a notable capacity of the mixed complexes to deliver genes to the inner cores of tumor spheroids. Extension of this approach to siRNA delivery showed that the mixed poly(phosphazene) complexes were able to silence DYRK1A, a gene implicated in tumor initiation and progression, reducing U87MG cell renewal in vitro and delaying tumor growth in vivo.

Lack of improvement in the treatment options of several types of cancer can largely be attributed to the presence of a subpopulation of cancer cells with stem cell signatures and to the tumoral niche that supports and protects these cells. This review analyses the main strategies that specifically modulate or suppress cancer stem cells (CSCs) and the tumoral niche (TN), focusing on the role of biomaterials (i.e. implants, nanomedicines, etc.) in these therapies. In the case of CSCs, we discuss differentiation therapies and the disruption of critical signaling networks. For the TN, we analyze diverse strategies to modulate tumor hypervascularization and hypoxia, tumor extracellular matrix, and the inflammatory and tumor immunosuppressive environment. Due to their capacity to control drug disposition and integrate diverse functionalities, biomaterial- based therapies can provide important benefits in these strategies. We illustrate this by providing case studies where biomaterial-based therapies either show CSC suppression and TN disruption or improved delivery of major modulators of these features. Finally, we discuss the future of these technologies in the framework of these emerging therapeutic concepts

Cancer progression is accompanied with alterations in the cell biomechanical phenotype, including changes in cell structure, morphology, and responses to microenvironmental stress. These alterations result in an increased deformability of transformed cells and reduced resistance to mechanical stimuli, enabling motility and invasion. Therefore, single cell biomechanical properties could be served as a powerful label-free biomarker for effective characterization and early detection of single cancer cells. Advances and innovations in microsystems and nanotechnology have facilitated interrogation of the biomechanical properties of single cells to predict their tumorigenicity, metastatic potential, and health state. This dissertation utilized Atomic Force Microscopy (AFM) for the cell biomechanical phenotyping for cancer diagnosis and early detection, efficacy screening of potential chemotherapeutic agents, and also cancer stem-like/tumor initiating cells (CSC/TICs) characterization as the critical topics received intensive attention in the search for effective cancer treatment. Our findings demonstrated the capability of exogenous sphingosine to revert the aberrant biomechanics of aggressive cells and showed a unique, mechanically homogeneous, and extremely soft characteristic of CSC/TICs, suitable for their targeted isolation. To make full use of cell biomechanical cues, this dissertation also considered the application of nonlinear viscoelastic models such as Fractional Zener and Generalized Maxwell models for the naturally complex, heterogeneous, and nonlinear structure of living cells. The emerging need for a high-throughput clinically relevant alternative for evaluating biomechanics of individual cells led us to the development of a microfluidic system. Therefore, a high-throughput, label-free, automated microfluidic chip was developed to investigate the biophysical (biomechanical-bioelectrical) markers of normal and malignant cells. Most importantly, this disserta

Cancer stem cells (CSCs), or tumor-initiating cells, comprise a subset of tumor cells with demonstrated ability for tumor growth, invasion, metastasis, and resistance to chemotherapy and radiation. Targeting of CSCs remains an attractive yet elusive therapeutic option, with the goal of increasing specificity and effectiveness in tumor eradication, as well as decreasing off-target or systemic toxicity. Research into further characterization and targeted therapy toward head and neck CSCs is an active and rapidly evolving field. This review discusses the current state of research into therapy against head and neck CSCs and future directions for targeted therapy.

During the first year of this innovator award, we made significant progress toward two of our aims. We constructed a third generation RNAi library and made that available to the breast cancer community. This resource has nearly 75,000 independent, sequence verified clones targeting 18,000 human genes. A similar library for the mouse genome is nearing completion. We also scaled up our shRNA screening platform in preparation for lethality surveys of all suitable and available BC cell lines, including matched pairs of lines that have acquired resistance to herceptin in vitro. Relevant to our second aim, we have profiled microRNA from each of the identifiable epithelial cell types in the mouse mammary gland and are undertaking similar efforts in human. The goal is to develop microRNA sensor strategies that will permit visualization of each cell type in vivo and enable their isolation and manipulation in vitro. Finally, we showed that two microRNAs, let-7 and miR-93, can deplete tumor initiating cells from a number of basal breast cancer cell lines., The original document contains color images.

During the research period stated above, we have expanded our colony of SIRT6 F/F; Villin- Cre; APCmin mice and found that lack of SIRT6 dramatically increases intestinal tumorigenesis in vivo, and this phenotype is driven by an enhanced aerobic glycolysis in the absence of SIRT6. To study in more detail how SIRT6 (and aerobic glycolysis) modulates intestinal tumorigenesis, we have knocked-down SIRT6 in a panel of human CRC cell lines (HCT116, SW620, HT29, SW1116). However, no differences in glycolytic metabolism were found. Alternatively, we have taken advantage of our mouse model and found that lack of SIRT6 leads to an increase in the number of intestinal stem cells (ISC), suggesting that SIRT6 regulates intestinal tumorigenesis by controlling the number of tumor initiating cells. We have confirmed this result by using intestinal organoids derived from our mouse model. In parallel, we generated a SIRT6 F/F, Lgr5-EGFP-Cre; APCmin mouse line to precisely address the role of SIRT6 in the ISCs. Our preliminary data shows that SIRT6 F/F, Lgr5-EGFP-Cre; APCmin mice have increased numbers of ISCs compared to control animals, suggesting that SIRT6 might regulate ISC., The original document contains color images.

Breast tumors, like other solid tumors, contain a subset of cells known as tumor initiating cells (TICs). TICs are important in tumor initiation and recurrence, metastasis, and are resistant to radiation and chemotherapy. Our data and work published by others indicate that the transcription factor NF- B is likely to be important in the generation and maintenance of breast TICs. A major problem in breast cancer therapy is the issue of residual disease where therapy-resistant cancer cells remain dormant only to drive tumor recurrence years later. Here we propose to explore mechanisms whereby NF- B is activated in breast cancer TICs and recurrent experimental tumors and the roles that IKK/NF- B play in this process. Our hypothesis is that the IKK/NF- B pathway is essential for the development and/or maintenance of breast cancer TICs, potentially through the promotion of EMT and the regulation of expression of genes which confer stemness. We hypothesize that inhibition of IKK/NF- B will reduce or eliminate breast camcer TICs, blocking tumorigenesis. Furthermore, we hypothesize that the activation of NF- B is an important component in the generation of recurrent diseases derived from residual disease. The Aims are to: (i) Explore the mechanistic roles for IKK/NF- B in promoting basal-like and Her2+ tumor initiating cells. (ii) Test IKK inhibitors and mTOR inhibitors for effects on tumor growth and TIC phenotypes, (iii) Determine the requirement for the NF- B, TGF and mTOR pathways in promoting the survival and recurrence of residual cancer cells., The original document contains color images.

Dielectrophoresis (DEP), an electrokinetic force, is the motion of a polarizable particle in a non-uniform electric field. Contactless DEP (cDEP) is a recently developed cell sorting and isolation technique that uses the DEP force by capacitavely coupling the electrodes across the channel. The cDEP platform sorts cells based on intrinsic biophysical properties, is inexpensive, maintains a sterile environment by using disposable chips, is a rapid process with minimal sample preparation, and allows for immediate downstream recovery. This platform is highly competitive compared to other cell sorting techniques and is one of the only platforms to sort cells based on phenotype, allowing for the isolation of unique cell populations not possible in other systems. The original purpose of this work was to determine differences in the bioelectrical fingerprint between several critical cancer types. Results demonstrate a difference between Tumor Initiating Cells, Multiple Drug Resistant Cells, and their bulk populations for experiments conducted on three prostate cancer cell lines and treated and untreated MOSE cells. However, three significant issues confounded these experiments and challenged the use of the cDEP platform. The purpose of this work then became the development of solutions to these barriers and presenting a more commercializable cDEP platform. An improved analysis script was first developed that performs whole cell detection and cell tracking with an accuracy of 93.5%. Second, a loading system for doing smart sample handling, specifically cell focusing, was developed using a new in-house system and validated. Experimental results validated the model and showed that cells were successfully focused into a tight band in the middle of the channel. Finally, a proof of concept for an on-chip micropump is presented and achieved 4.5% in-plane deformation. When bonded over a microchannel, fluid flow was induced and measured. These solutions present a stronger, more vers

Primary ovarian cancer (OC) represents a complex set of stem cell and cancer cell phenotypes embedded in a mixture of stromal and infiltrating immune cells. This grant develops techniques and approaches using mass cytometry that organize the heterogeneity within and between patient tumors to enlighten mechanisms and clinical opportunities in the apparent chaotic structure of the cancer. (1) A preliminary mass cytometry OC dataset by a social clustering strategy finds cell neighborhoods in high dimensional space reveals that 18 out of 20 samples had clusters of apparent stem-cell expressing deterministic combinations of stem cell markers. (2) Two high dimensional antibody panels were optimized and assembled to interrogate the tumor and immune cell compartments. (3) Indivumed (Hamburg Germany) is established as source for acquiring high quality HG-SOC primary specimens from single-cell dissociated tumors obtained within 3 hours of resection. (4) A QC procedure was established that surveys tumor samples (including epithelial, mesenchymal and immune cells) prior to their resection (using Abs against cleaved cCaspase 3, cleaved cPARP, vimentin, E-cadherin and CD45) to focus resection procedures on viable non necrotic tissue. (5) Eight key cell lines which bear remarkable genetic similarity to primary HG-SOC tumors have been obtained [1] as standards for primary tumors., The original document contains color images.

Breast tumors, like other solid tumors, contain a subset of cells known as tumor initiating cells (TICs). TICs are important in tumor initiation and recurrence, metastasis, and are resistant to radiation and chemotherapy. Our data and work published by others indicate that the transcription factor NF- B is likely to be important in the generation and maintenance of breast TICs. A major problem in breast cancer therapy is the issue of residual disease where therapy-resistant cancer cells remain dormant only to drive tumor recurrence years later. Here we propose to explore mechanisms whereby NF- B is activated in breast cancer TICs and recurrent experimental tumors and the roles that IKK/NF- B play in this process. Our hypothesis is that the IKK/NF- B pathway is essential for the development and/or maintenance of breast cancer TICs, potentially through the promotion of EMT and the regulation of expression of genes which confer stemness. We hypothesize that inhibition of IKK/NF- B will reduce or eliminate breast camcer TICs, blocking tumorigenesis. Furthermore, we hypothesize that the activation of NF- B is an important component in the generation of recurrent diseases derived from residual disease. The Aims are to: (i) Explore the mechanistic roles for IKK/NF- B in promoting basal-like and Her2+ tumor initiating cells. (ii) Test IKK inhibitors and mTOR inhibitors for effects on tumor growth and TIC phenotypes, (iii) Determine the requirement for the NF- B, TGF and mTOR pathways in promoting the survival and recurrence of residual cancer cells., The original document contains color images.

Basal-like breast cancers (BLBCs) generally become resistant to cytotoxic agents and resistance has been attributed to the presence of tumor initiating cancer stem cells (CSCs). Furthermore, LRP6/Wnt appears to play a crucial role in BLBC and CSC progression, and may represent an excellent therapeutic target. We have previously described that TRA-8, a monoclonal antibody specific to death receptor 5, kills both the CSCs and non-CSC population of BLBCs. This study examined two questions: whether niclosamide (an FDA approved antihelminthic, that inhibits Wnt/ -catenin signaling) is cytotoxic to BLBCs and its CSC population; and whether niclosamide in combination with TRA-8 produces synergistic cytotoxicity. We characterized non-adherent ALDH enriched (NAAE) cells as a CSC enriched population from BLBC cell lines. Both Adherent and NAAE cells from 2LMP, SUM159, HCC1187, HCC1143 cell lines and patient pleural effusion samples showed that niclosamide inhibited Wnt/ -catenin pathway activation, down regulated LRP6, and decreased downstream -catenin signaling. The combination of TRA-8 and niclosamide showed additive to synergistic cytotoxicity and further reduced Wnt/ -catenin activity. In vivo studies also showed that intraperitoneal administration of niclosamide in combination with TRA-8 suppressed growth of established 2LMP orthotopic tumor xenografts. Treatment with niclosamide in combination with TRA-8 may be an effective therapy against BLBC., The original document contains color images.

Basal-like breast cancers (BLBCs) generally become resistant to cytotoxic agents and resistance has been attributed to the presence of tumor initiating cancer stem cells (CSCs). Furthermore, LRP6/Wnt appears to play a crucial role in BLBC and CSC progression, and may represent an excellent therapeutic target. We have previously described that TRA-8, a monoclonal antibody specific to death receptor 5, kills both the CSCs and non-CSC population of BLBCs. This study examined two questions: whether niclosamide (an FDA approved antihelminthic, that inhibits Wnt/ -catenin signaling) is cytotoxic to BLBCs and its CSC population; and whether niclosamide in combination with TRA-8 produces synergistic cytotoxicity. We characterized non-adherent tumorsphere enriched (NATE) cells as a CSC enriched population from BLBC cell lines. Both Adherent and NATE cells from 2LMP, SUM159, HCC1187, HCC1143 cell lines and patient pleural effusion samples showed that niclosamide inhibited Wnt/ -catenin pathway activation, down regulated LRP6, and decreased downstream - catenin signaling. The combination of TRA-8 and niclosamide showed additive to synergistic cytotoxicity and further reduced Wnt/ -catenin activity. In vivo studies also showed that intraperitoneal administration of niclosamide in combination with TRA-8 suppressed growth of established 2LMP orthotopic tumor xenografts. Treatment with niclosamide in combination with TRA-8 may be an effective therapy against BLBC., The original document contains color images.

Prostate cancer (CaP) is the second leading cause of cancer related deaths in men in the United States. Currently, androgen depletion is an essential strategy for CaP combined with surgery, chemotherapy and radiation. Hormone independent cancer stem cells escaping conventional therapy present a major therapeutic challenge. The available treatment regimens for hormone resistant CaP are only palliative and marginally increase survival. Therefore, novel strategies to eradicate CaP including stem cells are imperative. Oncolytic virus (OV) therapy is a novel approach that overcomes the limitations posed by radiation and chemotherapy. Oncolytic virotherapy of cancer is based on the use of replication competent, tumor selective viruses with limited toxicity. Newcastle Disease Virus (NDV), an avian paramyxovirus, is a safe and promising OV successfully used in many clinical trials. NDV is inherently tumor selective and cytotoxic but replication restricted in normal cells. But, systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity and extracellular matrix. Overcoming these hurdles is paramount to realize the exceptional oncolytic efficacy of NDV. Therefore, we engineered the fusion (F) glycoprotein of NDV and generated a recombinant NDV (rNDV) cleavable exclusively by prostate specific antigen (PSA). The rNDV replicated efficiently and specifically only in prostate cancer (CaP) cells but failed to replicate in the absence of PSA. Further, PSA-cleavable rNDV caused specific lysis of androgen independent and dependent/responsive CaP cells with a mean effective concentration (EC50) ranging from 0.01 to 0.1 multiplicity of infection (MOI). PSA retargeted rNDV efficiently lysed three-dimensional prostaspheres, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating absence of pathogenicity to its natural hos

Prostate cancer (CaP) is the second leading cause of cancer related deaths in men in the United States. Currently, androgen depletion is an essential strategy for CaP combined with surgery, chemotherapy and radiation. Hormone independent cancer stem cells escaping conventional therapy present a major therapeutic challenge. The available treatment regimens for hormone resistant CaP are only palliative and marginally increase survival. Therefore, novel strategies to eradicate CaP including stem cells are imperative. Oncolytic virus (OV) therapy is a novel approach that overcomes the limitations posed by radiation and chemotherapy. Oncolytic virotherapy of cancer is based on the use of replication competent, tumor selective viruses with limited toxicity. Newcastle Disease Virus (NDV), an avian paramyxovirus, is a safe and promising OV successfully used in many clinical trials. NDV is inherently tumor selective and cytotoxic but replication restricted in normal cells. But, systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity and extracellular matrix. Overcoming these hurdles is paramount to realize the exceptional oncolytic efficacy of NDV. Therefore, we engineered the fusion (F) glycoprotein of NDV and generated a recombinant NDV (rNDV) cleavable exclusively by prostate specific antigen (PSA). The rNDV replicated efficiently and specifically only in prostate cancer (CaP) cells but failed to replicate in the absence of PSA. Further, PSA-cleavable rNDV caused specific lysis of androgen independent and dependent/responsive CaP cells with a mean effective concentration (EC50) ranging from 0.01 to 0.1 multiplicity of infection (MOI). PSA retargeted rNDV efficiently lysed three-dimensional prostaspheres, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating absence of pathogenicity to its natural hos

Gene delivery vectors that are safe, efficient and affordable could significantly enhance the prospects for genetic-based therapies. Here we describe an approach to such vectors, using new variants of polyphosphazene materials and describe the synthesis of a series of degradable polyphosphazenes with both cationic and anionic side-chains, and report their use as mixed polyelectrolyte complexes for DNA and RNA delivery to glioblastoma cells in vitro and in vivo. Precursor poly(allylamino-phosphazene)s were converted to cationic and anionic derivatives via a,w-thiolated alkylamines and alkylcarboxylates, respectively. Simultaneous co-incubation of alkylamine- and alkylcarboxylate-poly(phosphazenes) with nucleic acids generated polyelectrolyte complexes which were more compact than poly(alkylamino-phosphazene):DNA analogues but with similar positive surface charges. Screening of a series of these complexes for transfection of U87MG glioblastoma cells, showed that 6-mercaptohexanoic acid substituted poly(phosphazene)s mixed in the polycation/DNA complexes resulted in the highest luciferase expression in the cells. These data were consistent with an increased buffering capability of the 6-mercaptohexanoic acid substituted polymer across the early endosomal pH range in comparison with other anionic side-chain substituted polymers. Transfection assays in 3D spheroid models and in subcutaneous xenograft U87MG tumours confirmed higher transgene expression for these mixed cationic and anionic poly(phosphazene)s compared to the related poly(alkylamino-phosphazene)-DNA complexes, and also to PEI-DNA complexes. Extension of the approach to siRNA delivery showed that the mixed cationic and anionic poly(phosphazene)s were able to silence a gene encoding for a kinase implicated in tumour progression (DYRK1A), resulting in a reduced renewal ability of U87MG cells in vitro and in delay of tumour growth in a xenograft model.