Lorenzo Depau

Cancer selectivity of tetrabranched neurotensin peptides is generated by simultaneous binding to sulfated glycosaminoglycans and protein receptors.

In previous papers we demonstrated that tetrabranched peptides containing the sequence of human neurotensin, NT4, are much more selective than native monomeric analogues for binding to different human cancer cells and tissues. We show here that the much higher binding of NT4 peptides, with respect to native neurotensin, to either cancer cell lines or human cancer surgical samples is generated by a switch in selectivity toward additional membrane receptors, which are specifically expressed by different human cancers. We demonstrate that the branched structure provides NT4 with ability to bind heparin and receptors belonging to the low density lipoprotein receptor (LDLR) family, known to be involved in cancer biology. Systematic modification of neurotensin sequence in NT4 peptides led to identification of a multimeric positively charged motif, which mediates interaction with both heparin and endocytic receptors. Our findings provide the molecular basis for construction of cancer theranostics with high cancer selectivity.

Tumor-selective peptide-carrier delivery of Paclitaxel increases in vivo activity of the drug

Taxanes are highly effective chemotherapeutic drugs against proliferating cancer and an established option in the standard treatment of ovarian and breast cancer. However, treatment with paclitaxel is associated with severe side effects, including sensory axonal neuropathy, and its poor solubility in water complicates its formulation. In this paper we report the in vitro and in vivo activity of a new form of paclitaxel, modified for conjugation with a tumor-selective tetrabranched peptide carrier (NT4). NT4 selectively targets tumor cells by binding to membrane sulfated glycosaminoglycans (GAG) and to endocytic receptors, like LRP1 and LRP6, which are established tumor markers. Biological activity of NT4-paclitaxel was tested in vitro on MDA-MB 231 and SKOV-3 cell lines, representing breast and ovarian cancer, respectively, and in vivo in an orthotopic mouse model of human breast cancer. Using in vivo bioluminescence imaging, we found that conjugation of paclitaxel with the NT4 peptide led to increased therapeutic activity of the drug in vivo. NT4-paclitaxel induced tumor regression, whereas treatment with unconjugated paclitaxel only produced a reduction in tumor growth. Moreover, unlike paclitaxel, NT4-paclitaxel is very hydrophilic, which may improve its pharmacokinetic profile and allow the use of less toxic dilution buffers, further decreasing its general chemotherapic toxicity.

Neurotensin Branched Peptide as a Tumor-Targeting Agent for Human Bladder Cancer

Despite recent advances in multimodal therapy, bladder cancer still ranks ninth in worldwide cancer incidence. New molecules which might improve early diagnosis and therapeutic efficiency for tumors of such high epidemiological impact therefore have very high priority. In the present study, the tetrabranched neurotensin peptide NT4 was conjugated with functional units for cancer-cell imaging or therapy and was tested on bladder cancer cell lines and specimens from bladder cancer surgical resections, in order to evaluate its potential for targeted personalized therapy of bladder cancer. Fluorophore-conjugated NT4 distinguished healthy and cancer tissues with good statistical significance (P < 0.05). NT4 conjugated to methotrexate or gemcitabine was cytotoxic for human bladder cancer cell lines at micromolar concentrations. Their selectivity for bladder cancer tissue and capacity to carry tracers or drugs make NT4 peptides candidate tumor targeting agents for tracing cancer cells and for personalized therapy of human bladder cancer.

Insights into the role of sulfated glycans in cancer cell adhesion and migration through use of branched peptide probe

The tetra-branched peptide NT4 selectively binds to different human cancer cells and tissues. NT4 specifically binds to sulfated glycosaminoglycans on cancer cell membranes. Since sulfated glycosaminoglycans are involved in cancer cell interaction with the extracellular matrix, we evaluated the effect of NT4 on cancer cell adhesion and migration. We demonstrated here that the branched peptide NT4 binds sulfated glycosaminoglycans with high affinity and with preferential binding to heparan sulfate. NT4 inhibits cancer cell adhesion and migration on different proteins, without modifying cancer cell morphology or their ability to produce protrusions, but dramatically affecting the directionality and polarity of cell movement. Results obtained by taking advantage of the selective targeting of glycosaminoglycans chains by NT4, provide insights into the role of heparan sulfate proteoglycans in cancer cell adhesion and migration and suggest a determinant role of sulfated glycosaminoglycans in the control of cancer cell directional migration.

Coupling to a cancer-selective heparan-sulfate-targeted branched peptide can by-pass breast cancer cell resistance to methotrexate

Cancer-selective tetra-branched peptides, named NT4, can be coupled to different functional units for cancer cell imaging or therapy. NT4 peptides specifically bind to lipoprotein receptor-related proteins (LRP) receptors and to heparan sulfate chains on membrane proteoglycans and can be efficiently internalized by cancer cells expressing these membrane targets. Since binding and internalization of NT4 peptides is mediated by specific NT4 receptors on cancer cell membranes and this may allow drug resistance produced by drug membrane transporters to be by-passed, we tested the ability of drug-armed NT4 to by-pass drug resistance in cancer cell lines.We found that MTX-conjugated NT4 allows drug resistance to be by-passed in MTX-resistant human breast cancer cells lacking expression of folate reduced carrier. NT4 peptides appear to be extremely promising cancer-selective targeting agents that can be exploited as theranostics in personalized oncological applications.Cancer-selective tetra-branched peptides, named NT4, can be coupled to different functional units for cancer cell imaging or therapy. NT4 peptides specifically bind to lipoprotein receptor-related proteins (LRP) receptors and to heparan sulfate chains on membrane proteoglycans and can be efficiently internalized by cancer cells expressing these membrane targets. Since binding and internalization of NT4 peptides is mediated by specific NT4 receptors on cancer cell membranes and this may allow drug resistance produced by drug membrane transporters to be by-passed, we tested the ability of drug-armed NT4 to by-pass drug resistance in cancer cell lines. We found that MTX-conjugated NT4 allows drug resistance to be by-passed in MTX-resistant human breast cancer cells lacking expression of folate reduced carrier. NT4 peptides appear to be extremely promising cancer-selective targeting agents that can be exploited as theranostics in personalized oncological applications.

Near-infrared quantum dots labelled with a tumor selective tetrabranched peptide for in vivo imaging

BackgroundNear-infrared quantum dots (NIR QDs) are a new class of fluorescent labels with excellent bioimaging features, such as high fluorescence intensity, good fluorescence stability, sufficient electron density, and strong tissue-penetrating ability. For all such features, NIR QDs have great potential for early cancer diagnosis, in vivo tumor imaging and high resolution electron microscopy studies on cancer cells.ResultsIn the present study we constructed NIR QDs functionalized with the NT4 cancer-selective tetrabranched peptides (NT4-QDs). We observed specific uptake of NT4-QDs in human cancer cells in in vitro experiments and a much higher selective accumulation and retention of targeted QDs at the tumor site, compared to not targeted QDs, in a colon cancer mouse model.ConclusionsNIR QDs labelled with the tetrabranched NT4 peptide have very promising performance for selective addressing of tumor cells in vitro and in vivo, proving rising features of NT4-QDs as theranostics.

Abstract 5625: Targeting different LRP receptors and sulfated proteoglycan by branched neurotensin provide high cancer selectivity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In previous paper we reported on the much higher selectivity toward cancer cells and tissues of tetra-branched neurotensin peptides (NT4) compared to monomeric NT peptide. We also demonstrated that NT4 can be coupled to many different functional units for cancer cell tracing and drug delivery and can induce tumor growth reduction in animal studies. We then proposed NT4 as promising cancer selective theranostics for different human cancers, including CRC, pancreas adenocarcinoma and urinary bladder cancer. Nonetheless, multimeric binding of tetrabranched peptides, together with the chemical modification produced by coupling to the branched core, might have modified receptor selectivity of NT4 with respect to native monomeric NT and actually we had no conclusive indication on which receptor our branched NT4 peptides were binding to. Data reported in the present paper demonstrate that synthesis of neurotensin sequence in a tetra-branched form induce a switching of receptor selectivity, by decreasing affinity to the NT high affinity receptor NTR1 and contemporarily acquiring binding to additional receptors, which produces a much higher cancer cell selectivity of NT4 with respect to monomeric NT peptides. We demonstrate here that NT4 binds sortilin and SorLa and also acquire the ability to bind different receptors belonging to the Low Density Lipoprotein Receptor Related Protein (LRP) family as well as heparin and other Heparan Sulfate Proteoglycans (HSPG). The much higher binding of NT4 in respect to native NT to either cancel cell lines or human cancer surgical samples, as well as the higher selectivity toward human cancer tissues of NT4 is due to binding to different membrane receptors, which are very selectively expressed by many different human cancers. Moreover, our results confirm that sulfated proteoglycan can mimic the ligand binding site of different LRP receptors and indicate that targeting of multiple LRP receptors together with sulfated proteoglycans produce an extremely high selectivity towards many different human cancers. Citation Format: Luisa Bracci, Chiara Falciani, Jlenia Brunetti, Barbara Lelli, Niccolo Ravenni, Luisa Lozzi, Lorenzo Depau, Alessandro Pini. Targeting different LRP receptors and sulfated proteoglycan by branched neurotensin provide high cancer selectivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5625. doi:10.1158/1538-7445.AM2013-5625

The GAG-specific branched peptide NT4 reduces angiogenesis and invasiveness of tumor cells

Heparan sulfate proteoglycans, HSPGs, modulate major transformations of cancer cells, leading to tumor growth, invasion and metastasis. HSPGs also regulate neo-angiogenesis which prompts cancer progression and metastatic spread. A different aspect of heparin and analogs is their prominent role in the coagulation of blood. The interplay between coagulation and metastasis is being actively studied: anticoagulants such as heparin-derivatives have anticancer activity and procoagulants, such as thrombin, positively modulate proliferation, migration and invasion. The branched peptide NT4 binds to HSPGs and targets selectively cancer cells and tissues. For this, it had been extensively investigated in the last years and it proved to be efficient as chemotherapeutic and tumor tracer in in vivo models of cancer. We investigated the effects of the branched peptide in terms of modulation of angiogenesis and invasiveness of cancer cells. NT4 proved to have a major impact on endothelial cell proliferation, migration and tube formation, particularly when induced by FGF2 and thrombin. In addition, NT4 had important effects on aggressive tumor cells migration and invasion and it also had an anticoagulant profile.The peptide showed very interesting evidence of interference with tumor invasion pathways, offering a cue for its development as a tumor-targeting drug, and also for its use in the study of links between coagulation and tumor progression involving HSPGs.

Abstract 1153: Heparan sulfate proteoglycans as novel target in cancer precise therapy

Heparan sulfate proteoglycans (HSPGs) have crucial regulatory roles in tumor onset and progression. HSPGs are composed of a core protein and glycan chains characterized by repeated disaccharide units which can be sulfated at different amount and position. HSPG have enormous structural diversity due to the different possible modifications of the single saccharide units within the polysaccharide, such as position, sulphation and acetylation. As a result HSPG can bind and modulate their binding to signaling molecules such as growth factors, morphogens and chemokines1 . HSPG proved to be important in mediating cancer development and progression by enhancing the binding of growth factors, morphogens and cytokines to their cognate receptors, thus activating signaling pathways that give rise to angiogensis, cell growth and proliferation, together with invasion and metastasis 2-3. NT4 is a branched peptides that targets HSPGs. NT4 specifically binds to sulfated glycosaminoglycans on cancer cells and tissues. NT4 can be conjugated to many different cytotoxic units and tracers. NT4 conjugated to paclitaxel produced tumor regression in a breast cancer orthotopic mouse model 4. NT4 conjugated to tracers can discriminate between tumor and healthy tissue in different human cancer specimen5. We will show the ability of NT4 to drive tracers onto tumor lesions by means of Qdots and in vivo imaging, proving their promising features as theranostics. We will also show NT4 ability to interfere with HSPG- modulated activities such as: tumor cell proliferation, migration and invasion of matrix; as well as endothelial cells proliferation, migration and tube formation. NT4-HSPG interactions and consequent modulation of signaling pathways will prove the importance of this versatile tool, NT4, in addressing tumor cells and interfering in their cell-cell and cell-matrix communications. Bibliography 1. Gharbaran R. et al. Tumour Biol. 2016; 37:11573-11588. 2. Purushothaman A et al. Blood. 2010; 115:2449-57. 3. Lee JH et al. J Biol Chem. 2009; 284:27167-75. 4. Brunetti, J. et al. Scientific Reports. DOI:10.1038/srep17736 5. Falciani C. et al. J Med Chem. 2013; 56:5009-18. Citation Format: Chiara Falciani, Jlenia Brunetti, Lorenzo Depau, Alessandro Pini, Giulia Riolo, Elisabetta Mandarini, Luisa Bracci. Heparan sulfate proteoglycans as novel target in cancer precise therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1153. doi:10.1158/1538-7445.AM2017-1153

Abstract 3899: Preclinical development of tetra-branched NT4 peptide theranostics

The tetra-branched peptide NT4 is a potential cancer theranostic, which very selectively binds to human cancer tissues in different malignancies and can efficiently and selectively deliver drugs or liposomes for cancer cell imaging or therapy, in vitro and in vivo. By using NT4 conjugated to methotrexate or 5FdU we obtained significant reduction of tumor growth in xenografted nude mice. Very recently we reported that conjugation of paclitaxel to NT4 leads to increased therapeutic activity of the drug in an orthotopic model of breast cancer in mice and produces tumor regression which is not achieved with unconjugated paclitaxel in identical experimental conditions. We demonstrated that NT4 specifically binds to sulfated glycosaminoglycans and LRP receptors on cancer cells and tissues. Considering the role of sulfated glycosaminoglycans in cancer cell interaction with the extracellular matrix, we have analyzed the effect of NT4 in cancer cell adhesion and migration on different supports. NT4 inhibits adhesion and migration of different human cancer cell lines, strongly affecting directionality of cell movement. We have also constructed and validated a novel theranostics nanodevices, by conjugation of NT4 to quantum dots, for selective diagnosis and imaging of different human carcinomas. Thanks to their high cancer selectivity and versatile chemical conformation, NT4 peptides can be exploited for constructing cancer theranostics, which may also reduce tumor aggressiveness and metastatic potential by inhibiting cancer cell migration. References: Falciani, C. et al. Cancer selectivity of tetrabranched neurotensin peptides is generated by simultaneous binding to sulfated glycosaminoglycans and protein receptors. J Med Chem. 2013, 56, 5009-18. Brunetti, J. et al. Tumor-selective peptide-carrier delivery of Paclitaxel increases in vivo activity of the drug. Scientific Reports. DOI:10.1038/srep17736. Citation Format: Jlenia Brunetti, Lorenzo Depau, Chiara Falciani, Giulia Riolo, Elisabetta Mandarini, Alessandro Pini, Luisa Bracci. Preclinical development of tetra-branched NT4 peptide theranostics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3899.