Targeting triple-negative breast cancer cells with a ß1-integrin binding aptamer.

Targeted therapies have increased the treatment options for triple-negative breast cancer patients. However, the paucity of targetable biomarkers and tumor heterogeneity have limited the ability of precision-guided interventions to live up to their full potential. As affinity-targeting ligands, aptamers show high selectivity toward target molecules. Compared with antibodies, aptamers have lower molecular weight, increased stability during transportation, reduced immunogenicity, and increased tissue uptake. Recently, we reported discovery of the GreenB1 aptamer, which is internalized in cultured triple-negative MDA-MB-231 human breast cancer cells. We show that the GreenB1 aptamer specifically targets ß1-integrin, a protein linked previously to breast cancer cell invasiveness and migration. Aptamer binds to ß1-integrin with low nanomolar affinity. Our findings suggest potential applications for GreenB1-guided precision agents for diagnosis and therapy of cancers overexpressing ß1-integrin.

Intravitreal CendR peptides target laser-induced choroidal neovascularization sites in mice.

Choroidal neovascularization (CNV) is a common ocular pathology that may be associated in a variety of eye diseases. Although intravitreal injection treatment of anti-vascular endothelial growth factor (anti-VEGF) drugs shows significant clinical benefits in CNV treatment, the limitations of the current therapy need to be addressed. The aim of our study was to investigate the potential utility of three C-end Rule (CendR) peptides (RPARPAR, PL3, iRGD) for CNV targeting and to evaluate the efficacy of peptides for treating experimental CNV in mice. We observed that the CendR peptides localize to the CNV lesion sites after intravitreal injection and were mainly found in the outer nuclear cell layer (ONL) of the mouse retina. Interestingly, experimental therapy with tenascin-C (TNC-C) and neuropilin-1 (NRP-1)-targeting PL3 peptide, reduced angiogenesis and decreased vascular leakage. The results suggest that PL3 and potentially other CendR peptides could serve as affinity targeting ligands and therapeutics for ocular diseases that involve pathological CNV.

New Tools for Streamlined In Vivo Homing Peptide Identification.

In vivo peptide-phage display is an unbiased technique for mapping of the vascular diversity and identification of homing peptides. This chapter is intended to serve as a structured practical guide to execute in vivo T7 phage biopanning and data analysis experiments. We discuss experimental designs and protocols with emphasis on application of high-throughput sequencing-based technologies for streamlined in vivo biopanning and validation of homing peptides.

P32-specific CAR T cells with dual antitumor and antiangiogenic therapeutic potential in gliomas.

Glioblastoma is considered one of the most aggressive malignancies in adult and pediatric patients. Despite decades of research no curative treatment is available and it thus remains associated with a very dismal prognosis. Although recent pre-clinical and clinical studies have demonstrated the feasibility of chimeric antigen receptors (CAR) T cell immunotherapeutic approach in glioblastoma, tumor heterogeneity and antigen loss remain among one of the most important challenges to be addressed. In this study, we identify p32/gC1qR/HABP/C1qBP to be specifically expressed on the surface of glioma cells, making it a suitable tumor associated antigen for redirected CAR T cell therapy. We generate p32 CAR T cells and find them to recognize and specifically eliminate p32 expressing glioma cells and tumor derived endothelial cells in vitro and to control tumor growth in orthotopic syngeneic and xenograft mouse models. Thus, p32 CAR T cells may serve as a therapeutic option for glioblastoma patients.

In vivo phage display: identification of organ-specific peptides using deep sequencing and differential profiling across tissues.

In vivo phage display is widely used for identification of organ- or disease-specific homing peptides. However, the current in vivo phage biopanning approaches fail to assess biodistribution of specific peptide phages across tissues during the screen, thus necessitating laborious and time-consuming post-screening validation studies on individual peptide phages. Here, we adopted bioinformatics tools used for RNA sequencing for analysis of high-throughput sequencing (HTS) data to estimate the representation of individual peptides during biopanning in vivo. The data from in vivo phage screen were analyzed using differential binding-relative representation of each peptide in the target organ versus in a panel of control organs. Application of this approach in a model study using low-diversity peptide T7 phage library with spiked-in brain homing phage demonstrated brain-specific differential binding of brain homing phage and resulted in identification of novel lung- and brain-specific homing peptides. Our study provides a broadly applicable approach to streamline in vivo peptide phage biopanning and to increase its reproducibility and success rate.

Bi-specific tenascin-C and fibronectin targeted peptide for solid tumor delivery.

Oncofetal fibronectin (FN-EDB) and tenascin-C C domain (TNC-C) are nearly absent in extracellular matrix of normal adult tissues but upregulated in malignant tissues. Both FN-EDB and TNC-C are developed as targets of antibody-based therapies. Here we used peptide phage biopanning to identify a novel targeting peptide (PL1, sequence: PPRRGLIKLKTS) that interacts with both FN-EDB and TNC-C. Systemic PL1-functionalized model nanoscale payloads [iron oxide nanoworms (NWs) and metallic silver nanoparticles] homed to glioblastoma (GBM) and prostate carcinoma xenografts, and to non-malignant angiogenic neovessels induced by VEGF-overexpression. Antibody blockage experiments demonstrated that PL1 tumor homing involved interactions with both receptor proteins. Treatment of GBM mice with PL1-targeted model therapeutic nanocarrier (NWs loaded with a proapoptotic peptide) resulted in reduced tumor growth and increased survival, whereas treatment with untargeted particles had no effect. PL1 peptide may have applications as an affinity ligand for delivery of diagnostic and therapeutic compounds to microenvironment of solid tumors.

A complex genomic locus drives mtDNA replicase POLG expression to its disease-related nervous system regions.

DNA polymerase gamma (POLG), the mtDNA replicase, is a common cause of mitochondrial neurodegeneration. Why POLG defects especially cause central nervous system (CNS) diseases is unknown. We discovered a complex genomic regulatory locus for POLG, containing three functional CNS-specific enhancers that drive expression specifically in oculomotor complex and sensory interneurons of the spinal cord, completely overlapping with the regions showing neuronal death in POLG patients. The regulatory locus also expresses two functional RNAs, LINC00925-RNA and MIR9-3, which are coexpressed with POLG The MIR9-3 targets include NR2E1, a transcription factor maintaining neural stem cells in undifferentiated state, and MTHFD2, the regulatory enzyme of mitochondrial folate cycle, linking POLG expression to stem cell differentiation and folate metabolism. Our evidence suggests that distant genomic non-coding regions contribute to regulation of genes encoding mitochondrial proteins. Such genomic arrangement of POLG locus, driving expression to CNS regions affected in POLG patients, presents a potential mechanism for CNS-specific manifestations in POLG disease.

Melanoblast development coincides with the late emerging cells from the dorsal neural tube in turtle Trachemys scripta.

Ectothermal reptiles have internal pigmentation, which is not seen in endothermal birds and mammals. Here we show that the development of the dorsal neural tube-derived melanoblasts in turtle Trachemys scripta is regulated by similar mechanisms as in other amniotes, but significantly later in development, during the second phase of turtle trunk neural crest emigration. The development of melanoblasts coincided with a morphological change in the dorsal neural tube between stages mature G15 and G16. The melanoblasts delaminated and gathered in the carapacial staging area above the neural tube at G16, and differentiated into pigment-forming melanocytes during in vitro culture. The Mitf-positive melanoblasts were not restricted to the dorsolateral pathway as in birds and mammals but were also present medially through the somites similarly to ectothermal anamniotes. This matched a lack of environmental barrier dorsal and lateral to neural tube and the somites that is normally formed by PNA-binding proteins that block entry to medial pathways. PNA-binding proteins may also participate in the patterning of the carapacial pigmentation as both the migratory neural crest cells and pigment localized only to PNA-free areas.

Gata2 and Gata3 regulate the differentiation of serotonergic and glutamatergic neuron subtypes of the dorsal raphe.

Serotonergic and glutamatergic neurons of the dorsal raphe regulate many brain functions and are important for mental health. Their functional diversity is based on molecularly distinct subtypes; however, the development of this heterogeneity is poorly understood. We show that the ventral neuroepithelium of mouse anterior hindbrain is divided into specific subdomains giving rise to serotonergic neurons as well as other types of neurons and glia. The newly born serotonergic precursors are segregated into distinct subpopulations expressing vesicular glutamate transporter 3 (Vglut3) or serotonin transporter (Sert). These populations differ in their requirements for transcription factors Gata2 and Gata3, which are activated in the post-mitotic precursors. Gata2 operates upstream of Gata3 as a cell fate selector in both populations, whereas Gata3 is important for the differentiation of the Sert+ precursors and for the serotonergic identity of the Vglut3+ precursors. Similar to the serotonergic neurons, the Vglut3-expressing glutamatergic neurons, located in the central dorsal raphe, are derived from neural progenitors in the ventral hindbrain and express Pet1 Furthermore, both Gata2 and Gata3 are redundantly required for their differentiation. Our study demonstrates lineage relationships of the dorsal raphe neurons and suggests that functionally significant heterogeneity of these neurons is established early during their differentiation.

Differentiation and molecular heterogeneity of inhibitory and excitatory neurons associated with midbrain dopaminergic nuclei.

Local inhibitory GABAergic and excitatory glutamatergic neurons are important for midbrain dopaminergic and hindbrain serotonergic pathways controlling motivation, mood, and voluntary movements. Such neurons reside both within the dopaminergic nuclei, and in adjacent brain structures, including the rostromedial and laterodorsal tegmental nuclei. Compared with the monoaminergic neurons, the development, heterogeneity, and molecular characteristics of these regulatory neurons are poorly understood. We show here that different GABAergic and glutamatergic subgroups associated with the monoaminergic nuclei express specific transcription factors. These neurons share common origins in the ventrolateral rhombomere 1, where the postmitotic selector genes Tal1, Gata2 and Gata3 control the balance between the generation of inhibitory and excitatory neurons. In the absence of Tal1, or both Gata2 and Gata3, the GABAergic precursors adopt glutamatergic fates and populate the glutamatergic nuclei in excessive numbers. Together, our results uncover developmental regulatory mechanisms, molecular characteristics, and heterogeneity of central regulators of monoaminergic circuits.

Defects in sensory organ morphogenesis and generation of cochlear hair cells in Gata3-deficient mouse embryos.

The development of the inner ear sensory epithelia involves a complex network of transcription factors and signaling pathways and the whole process is not yet entirely understood. GATA3 is a DNA-binding factor that is necessary for otic morphogenesis and without GATA3 variable defects have been observed already at early stages in mouse embryos. In the less severe phenotypes, one small oval shaped vesicle is formed whereas in the more severe cases, the otic epithelium becomes disrupted and the endolymphatic domain becomes separated from the rest of the otic epithelium. Despite these defects, the early sensory fate specification occurs in Gata3-/- otic epithelium. However, due to the early lethality of Gata3-deficient embryos, the later morphogenesis and sensory development have remained unclear. To gain information of these later processes we produced drug-rescued Gata3-/- embryos that survived up to late gestation. In these older Gata3-/- embryos, a similar variability was observed as earlier. In the more severely affected ears, the development of the separate endolymphatic domain arrested completely whereas the remaining vesicle formed an empty cavity with variable forms, but without any distinguishable otic compartments or morphologically distinct sensory organs. However, the dorsal part of this vesicle was able to adopt a sensory fate and to produce some hair cells. In the less severe cases of Gata3-/- ears, distinct utricular, saccular and cochlear compartments were present and hair cells could be detected in the vestibular sensory epithelia. Although clear cristae and maculae formed, the morphology and size of these sensory areas were abnormal and they remained often un-separated. In contrast to the vestibule, the cochlear sensory compartment remained more immature and no hair or supporting cells could be detected. Our results suggest that GATA3 is critical for normal vestibular and cochlear morphogenesis and that it is especially important for cochlear sensory differentiation.

Gata2 is required for the development of inner ear semicircular ducts and the surrounding perilymphatic space.

Gata2 has essential roles in the development of many organs. During mouse inner ear morphogenesis, it is expressed in otic vesicle and the surrounding periotic mesenchyme from early on, but no defects in the ear development of Gata2 null mice have been observed before lethality at embryonic day (E) 10.5. Here, we used conditional gene targeting to reveal the role of Gata2 at later stages of inner ear development. We show that Gata2 is critically required from E14.5-E15.5 onward for vestibular morphogenesis. Without Gata2 the semicircular ducts fail to grow to their normal size and the surrounding mesenchymal cells are not removed properly to generate the perilymphatic space. Gata2 is the first factor known to control the clearing of the vestibular perilymphatic mesenchyme, but interestingly, it is not required for the formation of the cochlear perilymphatic areas, suggesting distinct molecular control for these processes.

Gata2 is a tissue-specific post-mitotic selector gene for midbrain GABAergic neurons.

Midbrain GABAergic neurons control several aspects of behavior, but regulation of their development and diversity is poorly understood. Here, we further refine the midbrain regions active in GABAergic neurogenesis and show their correlation with the expression of the transcription factor Gata2. Using tissue-specific inactivation and ectopic expression, we show that Gata2 regulates GABAergic neuron development in the mouse midbrain, but not in rhombomere 1, where it is needed in the serotonergic lineage. Without Gata2, all the precursors in the embryonic midbrain fail to activate GABAergic neuron-specific gene expression and instead switch to a glutamatergic phenotype. Surprisingly, this fate switch is also observed throughout the neonatal midbrain, except for the GABAergic neurons located in the ventral dopaminergic nuclei, suggesting a distinct developmental pathway for these neurons. These studies identify Gata2 as an essential post-mitotic selector gene of the GABAergic neurotransmitter identity and demonstrate developmental heterogeneity of GABAergic neurons in the midbrain.

Analysis of Netrin 1 receptors during inner ear development.

Netrin 1 plays key roles in axon guidance and neuronal migration during central nervous system (CNS) development. Outside the CNS, Netrin 1 has been shown to be involved in epithelial morphogenesis of various organs. We have shown that Netrin 1 is essential for inner ear semicircular duct formation, but the involvement of Netrin 1 receptors in this process has remained unknown. Netrin 1 receptors include members of the Deleted in colorectal cancer (Dcc), Unc5-homologue and integrin families. Here we have analysed the expression of these receptor genes during inner ear development and verified the inner ear phenotypes of several receptor mutant mice. Special interest was directed to receptors that could cooperate with Netrin 1 during semicircular duct formation. We show that Neogenin (Neo1), Unc5c as well as integrin b1 (Itgb1) are expressed in periotic mesenchyme, while Dcc, Unc5b, Unc5c, Itga3, Itga6 and Itgb1 are expressed in different parts of the otic epithelium. In spite of the broad and strong expression of several receptors in ear region, none of the analysed receptor mutant embryos showed any defects in inner ear development.

Comparative analysis of Gata3 and Gata2 expression during chicken inner ear development.

The inner ear is a complex sensory organ with hearing and balance functions. Gata3 and Gata2 are expressed in the inner ear, and to gain more insight into their roles in otic development, we made a detailed expression analysis in chicken embryos. At early stages, their expression was highly overlapping. At later stages, Gata2 expression became prominent in vestibular and cochlear nonsensory epithelia. In contrast to Gata2, Gata3 was mainly expressed in the developing sensory epithelia, reflecting the importance of this factor in the sensory-neural development of the inner ear. While the later expression patterns of both Gata3 and Gata2 were highly conserved between chicken and mouse, important differences were observed especially with Gata3 during early otic development, providing indications of divergent molecular control during placode invagination in mice and chickens. We also found indications that the regulatory hierarchy observed in mouse, where Gata3 is upstream of Gata2 and Fgf10, could be conserved in chicken.

Gata3 is required for early morphogenesis and Fgf10 expression during otic development.

Inner ear develops from an induced surface ectoderm placode that invaginates and closes to form the otic vesicle, which then undergoes a complex morphogenetic process to form the membranous labyrinth. Inner ear morphogenesis is severely affected in Gata3 deficient mouse embryos, but the onset and basis of the phenotype has not been known. We show here that Gata3 deficiency leads to severe and unique abnormalities during otic placode invagination. The invagination problems are accompanied often by the formation of a morphological boundary between the dorsal and ventral otic cup and by the precocious appearance of dorsal endolymphatic characteristics. In addition, the endolymphatic domain often detaches from the rest of the otic epithelium during epithelial closure. The expression of several cell adhesion mediating genes is altered in Gata3 deficient ears suggesting that Gata3 controls adhesion and morphogenetic movements in early otic epithelium. Inactivation of Gata3 leads also to a loss of Fgf10 expression in otic epithelium and auditory ganglion demonstrating that Gata3 is an important regulator of Fgf-signalling during otic development.