CCL2 blockade combined with PD-1/P-selectin immunomodulators impedes breast cancer brain metastasis.

Over the last two decades, the diagnosis and treatment of breast cancer patients have considerably improved. However, brain metastases remain a major clinical challenge and a leading cause of mortality. Thus, a better understanding of the pathways involved in the metastatic cascade is essential. To this end, we have investigated the reciprocal effects of astrocytes and breast cancer cells, employing traditional 2-dimensional cell culture and our unique 3-dimensional multicellular tumoroid models. Our findings revealed that astrocytes enhance the proliferation, migration, and invasion of breast cancer cells, suggesting a supportive role for astrocytes in breast cancer outgrowth to the brain. Elucidating the key players in astrocyte-breast cancer cells crosstalk, we found that CCL2 is highly expressed in breast cancer brain metastases tissue sections from both patients and mice. Our in vitro and in vivo models further confirmed that CCL2 has a functional role in brain metastasis. Given their aggressive nature, we sought additional immune checkpoints for rationale combination therapy. Among the promising candidates were the adhesion molecule P-selectin, which we have recently shown to play a key role in the crosstalk with microglia cells, and the co-inhibitory receptor PD-1, the main target of currently approved immunotherapies. Finally, combining CCL2 inhibition with immunomodulators targeting either PD-1/PD-L1 or P-selectin/P-Selectin Ligand-1 axes in our human 3-dimensional tumoroid models and in vivo presented more favorable outcomes than each monotherapy. Taken together, we propose that CCL2-CCR2/CCR4 is a key pathway promoting breast cancer brain metastases and a promising target for an immunotherapeutic combination approach.

Deoxyhypusine hydroxylase: A novel therapeutic target differentially expressed in short-term vs long-term survivors of glioblastoma.

Glioblastoma (GB) is the most aggressive neoplasm of the brain. Poor prognosis is mainly attributed to tumor heterogeneity, invasiveness and drug resistance. Only a small fraction of GB patients survives longer than 24 months from the time of diagnosis (ie, long-term survivors [LTS]). In our study, we aimed to identify molecular markers associated with favorable GB prognosis as a basis to develop therapeutic applications to improve patients' outcome. We have recently assembled a proteogenomic dataset of 87 GB clinical samples of varying survival rates. Following RNA-seq and mass spectrometry (MS)-based proteomics analysis, we identified several differentially expressed genes and proteins, including some known cancer-related pathways and some less established that showed higher expression in short-term (<6 months) survivors (STS) compared to LTS. One such target found was deoxyhypusine hydroxylase (DOHH), which is known to be involved in the biosynthesis of hypusine, an unusual amino acid essential for the function of the eukaryotic translation initiation factor 5A (eIF5A), which promotes tumor growth. We consequently validated DOHH overexpression in STS samples by quantitative polymerase chain reaction (qPCR) and immunohistochemistry. We further showed robust inhibition of proliferation, migration and invasion of GB cells following silencing of DOHH with short hairpin RNA (shRNA) or inhibition of its activity with small molecules, ciclopirox and deferiprone. Moreover, DOHH silencing led to significant inhibition of tumor progression and prolonged survival in GB mouse models. Searching for a potential mechanism by which DOHH promotes tumor aggressiveness, we found that it supports the transition of GB cells to a more invasive phenotype via epithelial-mesenchymal transition (EMT)-related pathways.

Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

Glioblastoma is an aggressive and invasive brain malignancy with high mortality rates despite current treatment modalities. In this study, we show that a 7-gene signature, previously found to govern the switch of glioblastomas from dormancy to aggressive tumor growth, correlates with improved overall survival of patients with glioblastoma. Using glioblastoma dormancy models, we validated the role of 2 genes from the signature, thrombospondin-1 ( TSP-1) and epidermal growth factor receptor ( EGFR), as regulators of glioblastoma dormancy and explored their therapeutic potential. EGFR up-regulation was reversed using EGFR small interfering RNA polyplex, antibody, or small-molecule inhibitor. The diminished function of TSP-1 was augmented via a peptidomimetic. The combination of EGFR inhibition and TSP-1 restoration led to enhanced therapeutic efficacy in vitro, in 3-dimensional patient-derived spheroids, and in a subcutaneous human glioblastoma model in vivo. Systemic administration of the combination therapy to mice bearing intracranial murine glioblastoma resulted in marginal therapeutic outcomes, probably due to brain delivery challenges, p53 mutation status, and the aggressive nature of the selected cell line. Nevertheless, this study provides a proof of concept for exploiting regulators of tumor dormancy for glioblastoma therapy. This therapeutic strategy can be exploited for future investigations using a variety of therapeutic entities that manipulate the expression of dormancy-associated genes in glioblastoma as well as in other cancer types.-Tiram, G., Ferber, S., Ofek, P., Eldar-Boock, A., Ben-Shushan, D., Yeini, E., Krivitsky, A., Blatt, R., Almog, N., Henkin, J., Amsalem, O., Yavin, E., Cohen, G., Lazarovici, P., Lee, J. S., Ruppin, E., Milyavsky, M., Grossman, R., Ram, Z., Calderón, M., Haag, R., Satchi-Fainaro, R. Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

Nanoparticulate vaccine inhibits tumor growth via improved T cell recruitment into melanoma and huHER2 breast cancer.

Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle-immune cell interactions and treatments regime is required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-γ cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction.

interFLOW: maximum flow framework for the identification of factors mediating the signaling convergence of multiple receptors.

Cell-cell crosstalk involves simultaneous interactions of multiple receptors and ligands, followed by downstream signaling cascades working through receptors converging at dominant transcription factors, which then integrate and propagate multiple signals into a cellular response. Single-cell RNAseq of multiple cell subsets isolated from a defined microenvironment provides us with a unique opportunity to learn about such interactions reflected in their gene expression levels. We developed the interFLOW framework to map the potential ligand-receptor interactions between different cell subsets based on a maximum flow computation in a network of protein-protein interactions (PPIs). The maximum flow approach further allows characterization of the intracellular downstream signal transduction from differentially expressed receptors towards dominant transcription factors, therefore, enabling the association between a set of receptors and their downstream activated pathways. Importantly, we were able to identify key transcription factors toward which the convergence of multiple receptor signaling pathways occurs. These identified factors have a unique role in the integration and propagation of signaling following specific cell-cell interactions.

Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker.

Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2.

Intranasal Multiepitope PD-L1-siRNA-Based Nanovaccine: The Next-Gen COVID-19 Immunotherapy.

The first approved vaccines for human use against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are nanotechnology-based. Although they are modular, rapidly produced, and can reduce disease severity, the currently available vaccines are restricted in preventing infection, stressing the global demand for novel preventive vaccine technologies. Bearing this in mind, we set out to develop a flexible nanovaccine platform for nasal administration to induce mucosal immunity, which is fundamental for optimal protection against respiratory virus infection. The next-generation multiepitope nanovaccines co-deliver immunogenic peptides, selected by an immunoinformatic workflow, along with adjuvants and regulators of the PD-L1 expression. As a case study, we focused on SARS-CoV-2 peptides as relevant antigens to validate the approach. This platform can evoke both local and systemic cellular- and humoral-specific responses against SARS-CoV-2. This led to the secretion of immunoglobulin A (IgA), capable of neutralizing SARS-CoV-2, including variants of concern, following a heterologous immunization strategy. Considering the limitations of the required cold chain distribution for current nanotechnology-based vaccines, it is shown that the lyophilized nanovaccine is stable for long-term at room temperature and retains its in vivo efficacy upon reconstitution. This makes it particularly relevant for developing countries and offers a modular system adaptable to future viral threats.

The role of P-selectin in cancer-associated thrombosis and beyond.

Cells in our body interact with their environment by a large group of diverse cell adhesion molecules (CAMs). CAMs are involved in intercellular, intracellular, and cell-extra-cellular matrix (ECM) interactions. Besides their role in cell adhesion, CAMs regulate cell growth and motility, and various signal transduction pathways as well as inflammation. P-Selectin (SELP) is an adhesion molecule that belongs to the Selectin family of proteins, which are expressed by different cell types such as platelets, endothelial and immune cells, as well as several types of cancer cells. The high expression of SELP by activated platelets makes it an important component in the pathogenesis of thrombosis, in general, and in cancer-associated thrombosis (CAT), in particular. Interestingly, the mechanisms by which SELP mediates CAT are associated with tumor-promoting processes such as inflammation and metastasis establishment. Moreover, SELP was shown to have a role in tumor-host interactions and cancer immunity. Thus, SELP has been the focus of several studies exploring its role in cancer progression. In this review, we explore the current knowledge on the role of SELP in CAT, tumor biology and immunology, in addition to recent advances in SELP-targeted therapies.

3D bioprinted cancer models: from basic biology to drug development.

Effort invested in the development of new drugs often fails to be translated into meaningful clinical benefits for patients with cancer. The development of more effective anticancer therapeutics and accurate prediction of their clinical merit remain urgent unmet medical needs. As solid cancers have complex and heterogeneous structures composed of different cell types and extracellular matrices, three-dimensional (3D) cancer models hold great potential for advancing our understanding of cancer biology, which has been historically investigated in tumour cell cultures on rigid plastic plates. Advanced 3D bioprinted cancer models have the potential to revolutionize the way we discover therapeutic targets, develop new drugs and personalize anticancer therapies in an accurate, reproducible, clinically translatable and robust manner. These ex vivo cancer models are already replacing existing in vitro systems and could, in the future, diminish or even replace the use of animal models. Therefore, profound understanding of the differences in tumorigenesis between 2D, 3D and animal models of cancer is essential. This Review presents the state of the art of 3D bioprinted cancer modelling, focusing on the biological processes that underlie the molecular mechanisms involved in cancer progression and treatment response as well as on proteomic and genomic signatures.

MCP-1/CCR2 axis inhibition sensitizes the brain microenvironment against melanoma brain metastasis progression.

Development of resistance to chemo- and immunotherapies often occurs following treatment of melanoma brain metastasis (MBM). The brain microenvironment (BME), particularly astrocytes, cooperate toward MBM progression by upregulating secreted factors, among which we found that monocyte chemoattractant protein-1 (MCP-1) and its receptors, CCR2 and CCR4, were overexpressed in MBM compared with primary lesions. Among other sources of MCP-1 in the brain, we show that melanoma cells altered astrocyte secretome and evoked MCP-1 expression and secretion, which in turn induced CCR2 expression in melanoma cells, enhancing in vitro tumorigenic properties, such as proliferation, migration, and invasion of melanoma cells. In vivo pharmacological blockade of MCP-1 or molecular knockout of CCR2/CCR4 increased the infiltration of cytotoxic CD8+ T cells and attenuated the immunosuppressive phenotype of the BME as shown by decreased infiltration of Tregs and tumor-associated macrophages/microglia in several models of intracranially injected MBM. These in vivo strategies led to decreased MBM outgrowth and prolonged the overall survival of the mice. Our findings highlight the therapeutic potential of inhibiting interactions between BME and melanoma cells for the treatment of this disease.

Proteogenomics of glioblastoma associates molecular patterns with survival.

Glioblastoma (GBM) is the most aggressive form of glioma, with poor prognosis exhibited by most patients, and a median survival time of less than 2 years. We assemble a cohort of 87 GBM patients whose survival ranges from less than 3 months and up to 10 years and perform both high-resolution mass spectrometry proteomics and RNA sequencing (RNA-seq). Integrative analysis of protein expression, RNA expression, and patient clinical information enables us to identify specific immune, metabolic, and developmental processes associated with survival as well as determine whether they are shared between expression layers or are layer specific. Our analyses reveal a stronger association between proteomic profiles and survival and identify unique protein-based classification, distinct from the established RNA-based classification. By integrating published single-cell RNA-seq data, we find a connection between subpopulations of GBM tumors and survival. Overall, our findings establish proteomic heterogeneity in GBM as a gateway to understanding poor survival.

Sulfonated Amphiphilic Poly(α)glutamate Amine-A Potential siRNA Nanocarrier for the Treatment of Both Chemo-Sensitive and Chemo-Resistant Glioblastoma Tumors.

Development of chemo-resistance is a major challenge in glioblastoma (GB) treatment. This phenomenon is often driven by increased activation of genes associated with DNA repair, such as the alkyl-removing enzyme O6-methylguanine-DNA methyltransferase (MGMT) in combination with overexpression of canonical genes related to cell proliferation and tumor progression, such as Polo-like kinase 1 (Plk1). Hereby, we attempt to sensitize resistant GB cells using our established amphiphilic poly(α)glutamate (APA): small interfering RNA (siRNA) polyplexes, targeting Plk1. Furthermore, we improved brain-targeting by decorating our nanocarrier with sulfonate groups. Our sulfonated nanocarrier showed superior selectivity towards P-selectin (SELP), a transmembrane glycoprotein overexpressed in GB and angiogenic brain endothelial cells. Self-assembled polyplexes of sulfonated APA and siPlk1 internalized into GB cells and into our unique 3-dimensional (3D) GB spheroids inducing specific gene silencing. Moreover, our RNAi nanotherapy efficiently reduced the cell viability of both chemo-sensitive and chemo-resistant GB cells. Our developed sulfonated amphiphilic poly(α)glutamate nanocarrier has the potential to target siRNA to GB brain tumors. Our findings may strengthen the therapeutic applications of siRNA for chemo-resistant GB tumors, or as a combination therapy for chemo-sensitive GB tumors.

Microengineered perfusable 3D-bioprinted glioblastoma model for in vivo mimicry of tumor microenvironment.

Many drugs show promising results in laboratory research but eventually fail clinical trials. We hypothesize that one main reason for this translational gap is that current cancer models are inadequate. Most models lack the tumor-stroma interactions, which are essential for proper representation of cancer complexed biology. Therefore, we recapitulated the tumor heterogenic microenvironment by creating fibrin glioblastoma bioink consisting of patient-derived glioblastoma cells, astrocytes, and microglia. In addition, perfusable blood vessels were created using a sacrificial bioink coated with brain pericytes and endothelial cells. We observed similar growth curves, drug response, and genetic signature of glioblastoma cells grown in our 3D-bioink platform and in orthotopic cancer mouse models as opposed to 2D culture on rigid plastic plates. Our 3D-bioprinted model could be the basis for potentially replacing cell cultures and animal models as a powerful platform for rapid, reproducible, and robust target discovery; personalized therapy screening; and drug development.

P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression.

Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, its microenvironment plays a crucial role in its progression. Among the brain stromal cells, the microglia were shown to facilitate GB invasion and immunosuppression. However, the reciprocal mechanisms by which GB cells alter microglia/macrophages behavior are not fully understood. We propose that these mechanisms involve adhesion molecules such as the Selectins family. These proteins are involved in immune modulation and cancer immunity. We show that P-selectin mediates microglia-enhanced GB proliferation and invasion by altering microglia/macrophages activation state. We demonstrate these findings by pharmacological and molecular inhibition of P-selectin which leads to reduced tumor growth and increased survival in GB mouse models. Our work sheds light on tumor-associated microglia/macrophage function and the mechanisms by which GB cells suppress the immune system and invade the brain, paving the way to exploit P-selectin as a target for GB therapy.

Immune-mediated approaches against COVID-19.

The coronavirus disease-19 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The long incubation period of this new virus, which is mostly asymptomatic yet contagious, is a key reason for its rapid spread across the world. Currently, there is no worldwide-approved treatment for COVID-19. Therefore, the clinical and scientific communities have joint efforts to reduce the severe impact of the outbreak. Research on previous emerging infectious diseases have created valuable knowledge that is being exploited for drug repurposing and accelerated vaccine development. Nevertheless, it is important to generate knowledge on SARS-CoV-2 mechanisms of infection and its impact on host immunity, to guide the design of COVID-19 specific therapeutics and vaccines suitable for mass immunization. Nanoscale delivery systems are expected to play a paramount role in the success of these prophylactic and therapeutic approaches. This Review provides an overview of SARS-CoV-2 pathogenesis and examines immune-mediated approaches currently explored for COVID-19 treatments, with an emphasis on nanotechnological tools.

Rational Design of Polyglutamic Acid Delivering an Optimized Combination of Drugs Targeting Mutated BRAF and MEK in Melanoma.

Targeted therapies against cancer can relieve symptoms and induce remission, however, they often present limited duration of disease control, cause side effects and often induce acquired resistance. Therefore, there is a great motivation to develop a unique delivery system, targeted to the tumor, in which we can combine several active entities, increase the therapeutic index by reducing systemic exposure, and enhance their synergistic activity. To meet these goals, we chose the biocompatible and biodegradable poly(α,L-glutamic acid) (PGA) as a nanocarrier that facilitates extravasation-dependent tumor targeting delivery. The RAS/RAF/MEK/ERK pathway when aberrantly activated in melanoma, can lead to uncontrolled cell proliferation, induced invasion, and reduced apoptosis. Here, we selected two drugs targeting this pathway; a MEK1/2 inhibitor (selumetinib; SLM) and a modified BRAF inhibitor (modified dabrafenib; mDBF), that exhibited synergism in vitro. We synthesized and characterized our nanomedicine of PGA conjugated to SLM and mDBF (PGA-SLM-mDBF). PGA-SLM-mDBF inhibited the proliferation of melanoma cells and decreased their migratory and sprouting abilities without inducing a hemolytic effect. Moreover, the polymer-2-drugs conjugate exhibited superior anti-tumor activity in comparison with the two separate polymer-drug conjugates in vitro and with free drugs in a mouse model of primary melanoma and prolonged survival at a lower dose.

Immunization with mannosylated nanovaccines and inhibition of the immune-suppressing microenvironment sensitizes melanoma to immune checkpoint modulators.

A low response rate, acquired resistance and severe side effects have limited the clinical outcomes of immune checkpoint therapy. Here, we show that combining cancer nanovaccines with an anti-PD-1 antibody (αPD-1) for immunosuppression blockade and an anti-OX40 antibody (αOX40) for effector T-cell stimulation, expansion and survival can potentiate the efficacy of melanoma therapy. Prophylactic and therapeutic combination regimens of dendritic cell-targeted mannosylated nanovaccines with αPD-1/αOX40 demonstrate a synergism that stimulates T-cell infiltration into tumours at early treatment stages. However, this treatment at the therapeutic regimen does not result in an enhanced inhibition of tumour growth compared to αPD-1/αOX40 alone and is accompanied by an increased infiltration of myeloid-derived suppressor cells in tumours. Combining the double therapy with ibrutinib, a myeloid-derived suppressor cell inhibitor, leads to a remarkable tumour remission and prolonged survival in melanoma-bearing mice. The synergy between the mannosylated nanovaccines, ibrutinib and αPD-1/αOX40 provides essential insights to devise alternative regimens to improve the efficacy of immune checkpoint modulators in solid tumours by regulating the endogenous immune response.

Molecular Weight-Dependent Activity of Aminated Poly(α)glutamates as siRNA Nanocarriers.

RNA interference (RNAi) can contribute immensely to the area of personalized medicine by its ability to target any gene of interest. Nevertheless, its clinical use is limited by lack of efficient delivery systems. Polymer therapeutics can address many of the challenges encountered by the systemic delivery of RNAi, but suffer from inherent drawbacks such as polydispersity and batch to batch heterogeneity. These characteristics may have far-reaching consequences when dealing with therapeutic applications, as both the activity and the toxicity may be dependent on the length of the polymer chain. To investigate the consequences of polymers' heterogeneity, we have synthesized two batches of aminated poly(α)glutamate polymers (PGAamine), differing in their degree of polymerization, but not in the monomer units or their conjugation. Isothermal titration calorimetry study was conducted to define the binding affinity of these polymers with siRNA. Molecular dynamics simulation revealed that Short PGAamine:siRNA polyplexes exposed a higher amount of amine moieties to the surroundings compared to Long PGAamine. This resulted in a higher zeta potential, leading to faster degradation and diminished gene silencing. Altogether, our study highlights the importance of an adequate physico-chemical characterization to elucidate the structure⁻function-activity relationship, for further development of tailor-designed RNAi delivery vehicles.

Successful intracranial delivery of trastuzumab by gene-therapy for treatment of HER2-positive breast cancer brain metastases.

BACKGROUND: Trastuzumab is a monoclonal antibody which demonstrates efficacy for HER2 positive breast cancer patients. Recently, an increased incidence of brain metastasis in trastuzumab-treated patients has been reported. The reason for this may be the effectiveness of systemic trastuzumab allowing patients to survive longer thus providing time for brain metastases to develop, along with the lack of penetration of systemic therapies through the blood brain barrier. In recent years, several administration routes to the brain have been evaluated. Albeit advances in the field, there is still a need for improved delivery of therapeutic antibodies to the brain. To address this challenge, we have developed two gene therapy-based methods enabling continuous secretion of active trastuzumab in the brain. METHODS: We have developed two gene therapy approaches for the delivery of the therapeutic anti-HER2 monoclonal antibody, trastuzumab, to the brain. We utilized the helper dependent adenovirus vector, containing trastuzumab light and heavy chains coding sequences (HDAd-trastuzumab). In the first approach, we used the Transduced Autologous Restorative Gene Therapy (TARGT) platform, in which dermal fibroblasts of human and mouse origin, are ex-vivo transduced with HDAd-trastuzumab vector, rendering continuous secretion of active trastuzumab from the cells locally. These genetically engineered cells were subsequently implanted intracranially to mice, contralateral to HER2 positive breast carcinoma cells inoculation site, enabling continuous secretion of trastuzumab in the brain. In the second approach, we used the same HDAd-trastuzumab viral vector, directly injected intracranially, contralateral to the HER2 positive breast carcinoma cells inoculation site. Both methods enabled therapeutic concentrations of local in-vivo production of active trastuzumab in a mouse model of brain metastatic breast cancer. RESULTS: Trastuzumab secreted from the TARGT platform demonstrated in-vitro affinity and immune recruitment activity (ADCC) similar to recombinant trastuzumab (Herceptin, Genentech). When implanted in the brain of HER2 positive tumor-bearing mice, both the TARGT platform of dermal fibroblasts engineered to secrete trastuzumab and direct injection of HDAd-trastuzumab demonstrated remarkable intracranial tumor growth inhibitory effect. CONCLUSIONS: This work presents two gene therapy approaches for the administration of therapeutic antibodies to the brain. The TARGT platform of dermal fibroblasts engineered to secrete active trastuzumab, and the direct injection of HDAd-trastuzumab viral vector, both rendered continuous in-vivo secretion of active trastuzumab in the brain and demonstrated high efficacy. These two approaches present a proof of concept for promising gene therapy based administration methods for intracranial tumors as well as other brain diseases.

Image-guided surgery using near-infrared Turn-ON fluorescent nanoprobes for precise detection of tumor margins.

Complete tumor removal during surgery has a great impact on patient survival. To that end, the surgeon should detect the tumor, remove it and validate that there are no residual cancer cells left behind. Residual cells at the incision margin of the tissue removed during surgery are associated with tumor recurrence and poor prognosis for the patient. In order to remove the tumor tissue completely with minimal collateral damage to healthy tissue, there is a need for diagnostic tools that will differentiate between the tumor and its normal surroundings. Methods: We designed, synthesized and characterized three novel polymeric Turn-ON probes that will be activated at the tumor site by cysteine cathepsins that are highly expressed in multiple tumor types. Utilizing orthotopic breast cancer and melanoma models, which spontaneously metastasize to the brain, we studied the kinetics of our polymeric Turn-ON nano-probes. Results: To date, numerous low molecular weight cathepsin-sensitive substrates have been reported, however, most of them suffer from rapid clearance and reduced signal shortly after administration. Here, we show an improved tumor-to-background ratio upon activation of our Turn-ON probes by cathepsins. The signal obtained from the tumor was stable and delineated the tumor boundaries during the whole surgical procedure, enabling accurate resection. Conclusions: Our findings show that the control groups of tumor-bearing mice, which underwent either standard surgery under white light only or under the fluorescence guidance of the commercially-available imaging agents ProSense® 680 or 5-aminolevulinic acid (5-ALA), survived for less time and suffered from tumor recurrence earlier than the group that underwent image-guided surgery (IGS) using our Turn-ON probes. Our "smart" polymeric probes can potentially assist surgeons' decision in real-time during surgery regarding the tumor margins needed to be removed, leading to improved patient outcome.