A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins.

Aminopeptidase N/CD13, a membrane-bound enzyme upregulated in tumor vasculature and involved in angiogenesis, can be used as a receptor for the targeted delivery of drugs to tumors through ligand-directed targeting approaches. We describe a novel peptide ligand (VGCARRYCS, called "G4") that recognizes CD13 with high affinity and selectivity. Enzymological and computational studies showed that G4 is a competitive inhibitor that binds to the catalytic pocket of CD13 through its N-terminal region. Fusing the peptide C-terminus to tumor necrosis factor-alpha (TNF) or coupling it to a biotin/avidin complex causes loss of binding and inhibitory activity against different forms of CD13, including natural or recombinant ectoenzyme and a membrane form expressed by HL60 promyelocytic leukemia cells (likely due to steric hindrance), but not binding to a membrane form of CD13 expressed by endothelial cells (ECs). Furthermore, G4-TNF systemically administered to tumor-bearing mice exerted anticancer effects through a CD13-targeting mechanism, indicating the presence of a CD13 form in tumor vessels with an accessible binding site. Biochemical studies showed that most CD13 molecules expressed on the surface of ECs are catalytically inactive. Other functional assays showed that these molecules can promote endothelial cell adhesion to plates coated with G4-avidin complexes, suggesting that the endothelial form of CD13 can exert catalytically independent biological functions. In conclusion, ECs express a catalytically inactive form of CD13 characterized by an accessible conformation that can be selectively targeted by G4-protein conjugates. This form of CD13 may represent a specific target receptor for ligand-directed targeted delivery of therapeutics to tumors.

A simple and robust nanosystem for photoacoustic imaging of bladder cancer based on α5ß1-targeted gold nanorods.

BACKGROUND: Early detection and removal of bladder cancer in patients is crucial to prevent tumor recurrence and progression. Because current imaging techniques may fail to detect small lesions of in situ carcinomas, patients with bladder cancer often relapse after initial diagnosis, thereby requiring frequent follow-up and treatments. RESULTS: In an attempt to obtain a sensitive and high-resolution imaging modality for bladder cancer, we have developed a photoacoustic imaging approach based on the use of PEGylated gold nanorods (GNRs) as a contrast agent, functionalized with the peptide cyclic [CphgisoDGRG] (Iso4), a selective ligand of α5ß1 integrin expressed by bladder cancer cells. This product (called GNRs@PEG-Iso4) was produced by a simple two-step procedure based on GNRs activation with lipoic acid-polyethyleneglycol(PEG-5KDa)-maleimide and functionalization with peptide Iso4. Biochemical and biological studies showed that GNRs@PEG-Iso4 can efficiently recognize purified integrin α5ß1 and α5ß1-positive bladder cancer cells. GNRs@PEG-Iso4 was stable and did not aggregate in urine or in 5% sodium chloride, or after freeze/thaw cycles or prolonged exposure to 55 °C, and, even more importantly, do not settle after instillation into the bladder. Intravesical instillation of GNRs@PEG-Iso4 into mice bearing orthotopic MB49-Luc bladder tumors, followed by photoacoustic imaging, efficiently detected small cancer lesions. The binding to tumor lesions was competed by a neutralizing anti-α5ß1 integrin antibody; furthermore, no binding was observed to healthy bladders (α5ß1-negative), pointing to a specific targeting mechanism. CONCLUSION: GNRs@PEG-Iso4 represents a simple and robust contrast agent for photoacoustic imaging and diagnosis of small bladder cancer lesions.

CXCL10 levels at hospital admission predict COVID-19 outcome: hierarchical assessment of 53 putative inflammatory biomarkers in an observational study.

BACKGROUND: Host inflammation contributes to determine whether SARS-CoV-2 infection causes mild or life-threatening disease. Tools are needed for early risk assessment. METHODS: We studied in 111 COVID-19 patients prospectively followed at a single reference Hospital fifty-three potential biomarkers including alarmins, cytokines, adipocytokines and growth factors, humoral innate immune and neuroendocrine molecules and regulators of iron metabolism. Biomarkers at hospital admission together with age, degree of hypoxia, neutrophil to lymphocyte ratio (NLR), lactate dehydrogenase (LDH), C-reactive protein (CRP) and creatinine were analysed within a data-driven approach to classify patients with respect to survival and ICU outcomes. Classification and regression tree (CART) models were used to identify prognostic biomarkers. RESULTS: Among the fifty-three potential biomarkers, the classification tree analysis selected CXCL10 at hospital admission, in combination with NLR and time from onset, as the best predictor of ICU transfer (AUC [95% CI] = 0.8374 [0.6233-0.8435]), while it was selected alone to predict death (AUC [95% CI] = 0.7334 [0.7547-0.9201]). CXCL10 concentration abated in COVID-19 survivors after healing and discharge from the hospital. CONCLUSIONS: CXCL10 results from a data-driven analysis, that accounts for presence of confounding factors, as the most robust predictive biomarker of patient outcome in COVID-19.

R-CHOP preceded by blood-brain barrier permeabilization with engineered tumor necrosis factor-α in primary CNS lymphoma.

Patients with primary central nervous system lymphoma (PCNSL) are treated with high-dose methotrexate-based chemotherapy, which requires hospitalization and extensive expertise to manage related toxicity. The use of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) could overcome these difficulties, but blood-brain barrier (BBB) penetration of related drugs is poor. Tumor necrosis factor-α coupled with NGR (NGR-hTNF), a peptide targeting CD13+ vessels, induces endothelial permeabilization and improves tumor access of cytostatics. We tested the hypothesis that NGR-hTNF can break the BBB, thereby improving penetration and activity of R-CHOP in patients with relapsed/refractory PCNSL (NCT03536039). Patients received six R-CHOP21 courses, alone at the first course and preceded by NGR-hTNF (0.8 µg/m2) afterward. This trial included 2 phases: an "explorative phase" addressing the effect of NGR-hTNF on drug pharmacokinetic parameters and on vessel permeability, assessed by dynamic contrast-enhanced magnetic resonance imaging and 99mTc-diethylene-triamine-pentacetic acid-single-photon emission computed tomography, and the expression of CD13 on tumor tissue; and an "expansion phase" with overall response rate as the primary end point, in which the 2-stage Simon Minimax design was used. At the first stage, if ≥4 responses were observed among 12 patients, the study accrual would have continued (sample size, 28). Herein, we report results of the explorative phase and the first-stage analysis (n = 12). CD13 was expressed in tumor vessels of all cases. NGR-hTNF selectively increased vascular permeability in tumoral/peritumoral areas, without interfering with drug plasma/cerebrospinal fluid concentrations. The NGR-hTNF/R-CHOP combination was well tolerated: there were only 2 serious adverse events, and grade 4 toxicity was almost exclusively hematological, which were resolved without dose reductions or interruptions. NGR-hTNF/R-CHOP was active, with 9 confirmed responses (75%; 95% confidence interval, 51-99), 8 of which were complete. In conclusion, NGR-hTNF/R-CHOP was safe in these heavily pretreated patients. NGR-hTNF enhanced vascular permeability specifically in tumoral/peritumoral areas, which resulted in fast and sustained responses.

Enhancement of doxorubicin anti-cancer activity by vascular targeting using IsoDGR/cytokine-coated nanogold.

BACKGROUND: Gold nanospheres tagged with peptides containing isoDGR (isoAsp-Gly-Arg), an αvß3 integrin binding motif, represent efficient carriers for delivering pro-inflammatory cytokines to the tumor vasculature. We prepared bi- or trifunctional nanoparticles bearing tumor necrosis factor-α (TNF) and/or interleukin-12 (IL12) plus a peptide containing isoDGR, and we tested their anti-cancer effects, alone or in combination with doxorubicin, in tumor-bearing mice. RESULTS: In vitro biochemical studies showed that both nanodrugs were monodispersed and functional in terms of binding to TNF and IL12 receptors and to αvß3. In vivo studies performed in a murine model of fibrosarcoma showed that low doses of bifunctional nanoparticles bearing isoDGR and TNF (corresponding to few nanoparticles per cell) delayed tumor growth and increased the efficacy of doxorubicin without worsening its toxicity. Similar effects were obtained using trifunctional nanoparticles loaded with isoDGR, TNF and IL12. Mechanistic studies showed that nanoparticles bearing isoDGR and TNF could increase doxorubicin penetration in tumors a few hours after injection and caused vascular damage at later time points. CONCLUSION: IsoDGR-coated gold nanospheres can be exploited as a versatile platform for single- or multi-cytokine delivery to cells of the tumor vasculature. Extremely low doses of isoDGR-coated nanodrugs functionalized with TNF or TNF plus IL12 can enhance doxorubicin anti-tumor activity.

Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.

The clinical use of doxorubicin (Doxo), a widely used anticancer chemotherapeutic drug, is limited by dose-dependent cardiotoxicity. We have investigated whether chromogranin A (CgA), a cardioregulatory protein released in the blood by the neuroendocrine system and by the heart itself, may contribute to regulation of the cardiotoxic and antitumor activities of Doxo. The effects of a physiologic dose of full-length recombinant CgA on Doxo-induced cardiotoxicity and antitumor activity were investigated in rats using in vivo and ex vivo models and in murine models of melanoma, fibrosarcoma, lymphoma, and lung cancer, respectively. The effect of Doxo on circulating levels of CgA was also investigated. In vivo and ex vivo mechanistic studies showed that CgA can prevent Doxo-induced heart inflammation, oxidative stress, apoptosis, fibrosis, and ischemic injury. On the other hand, CgA did not impair the anticancer activity of Doxo in all the murine models investigated. Furthermore, we observed that Doxo can reduce the intracardiac expression and release of CgA in the blood (i.e., an important cardioprotective agent). These findings suggest that administration of low-dose CgA to patients with low levels of endogenous CgA might represent a novel approach to prevent Doxo-induced adverse events without impairing antitumor effects.-Rocca, C., Scavello, F., Colombo, B., Gasparri, A. M., Dallatomasina, A., Granieri, M. C., Amelio, D., Pasqua, T., Cerra, M. C., Tota, B., Corti, A., Angelone, T. Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.

NGR-TNF Engineering with an N-Terminal Serine Reduces Degradation and Post-Translational Modifications and Improves Its Tumor-Targeting Activity.

The therapeutic index of cytokines in cancer therapy can be increased by targeting strategies based on protein engineering with peptides containing the CNGRC (NGR) motif, a ligand that recognizes CD13-positive tumor vessels. We show here that the targeting domain of recombinant CNGRC-cytokine fusion proteins, such as NGR-TNF (a CNGRC-tumor necrosis factor-α (TNF) conjugate used in clinical studies) and NGR-EMAP-II, undergoes various post-translational modification and degradation reactions that lead to the formation of markedly heterogeneous products. These modifications include N-terminal cysteine acetylation or the formation of various asparagine degradation products, the latter owing to intramolecular interactions of the cysteine α-amino group with asparagine and/or its succinimide derivative. Blocking the cysteine α-amino group with a serine (SCNGRC) reduced both post-translational and degradation reactions. Furthermore, the serine residue reduced the asparagine deamidation rate to isoaspartate (another degradation product) and improved the affinity of NGR for CD13. Accordingly, genetic engineering of NGR-TNF with the N-terminal serine produced a more stable and homogeneous drug (called S-NGR-TNF) with improved antitumor activity in tumor-bearing mice, either when used alone or in combination with chemotherapy. In conclusion, the targeting domain of NGR-cytokine conjugates can undergo various untoward modification and degradation reactions, which can be markedly reduced by fusing a serine to the N-terminus. The SCNGRC peptide may represent a ligand for cytokine delivery to tumors more robust than conventional CNGRC. The S-NGR-TNF conjugate (more stable, homogeneous, and active than NGR-TNF) could be rapidly developed for clinical trials.

Tumor cell-associated immune checkpoint molecules - Drivers of malignancy and stemness.

Inhibitory or stimulatory immune checkpoint molecules are expressed on a sizeable fraction of tumor cells in different tumor types. It was thought that the main function of tumor cell-associated immune checkpoint molecules would be the modulation (down- or upregulation) of antitumor immune responses. In recent years, however, it has become clear that the expression of immune checkpoint molecules on tumor cells has important consequences on the biology of the tumor cells themselves. In particular, a causal relationship between the expression of these molecules and the acquisition of malignant traits has been demonstrated. Thus, immune checkpoint molecules have been shown to promote the epithelial-mesenchymal transition of tumor cells, the acquisition of tumor-initiating potential and resistance to apoptosis and antitumor drugs, as well as the propensity to disseminate and metastasize. Herein, we review this evidence, with a main focus on PD-L1, the most intensively investigated tumor cell-associated immune checkpoint molecule and for which most information is available. Then, we discuss more concisely other tumor cell-associated immune checkpoint molecules that have also been shown to induce the acquisition of malignant traits, such as PD-1, B7-H3, B7-H4, Tim-3, CD70, CD28, CD137, CD40 and CD47. Open questions in this field as well as some therapeutic approaches that can be derived from this knowledge, are also addressed.

Overcoming Biological Barriers in Neuroblastoma Therapy: The Vascular Targeting Approach with Liposomal Drug Nanocarriers.

Neuroblastoma is a rare pediatric cancer characterized by a wide clinical behavior and adverse outcome despite aggressive therapies. New approaches based on targeted drug delivery may improve efficacy and decrease toxicity of cancer therapy. Furthermore, nanotechnology offers additional potential developments for cancer imaging, diagnosis, and treatment. Following these lines, in the past years, innovative therapies based on the use of liposomes loaded with anticancer agents and functionalized with peptides capable of recognizing neuroblastoma cells and/or tumor-associated endothelial cells have been developed. Studies performed in experimental orthotopic models of human neuroblastoma have shown that targeted nanocarriers can be exploited for not only decreasing the systemic toxicity of the encapsulated anticancer drugs, but also increasing their tumor homing properties, enhancing tumor vascular permeability and perfusion (and, consequently, drug penetration), inducing tumor apoptosis, inhibiting angiogenesis, and reducing tumor glucose consumption. Furthermore, peptide-tagged liposomal formulations are proved to be more efficacious in inhibiting tumor growth and metastatic spreading of neuroblastoma than nontargeted liposomes. These findings, herein reviewed, pave the way for the design of novel targeted liposomal nanocarriers useful for multitargeting treatment of neuroblastoma.

Boosting Interleukin-12 Antitumor Activity and Synergism with Immunotherapy by Targeted Delivery with isoDGR-Tagged Nanogold.

The clinical use of interleukin-12 (IL12), a cytokine endowed with potent immunotherapeutic anticancer activity, is limited by systemic toxicity. The hypothesis is addressed that gold nanoparticles tagged with a tumor-homing peptide containing isoDGR, an αvß3-integrin binding motif, can be exploited for delivering IL12 to tumors and improving its therapeutic index. To this aim, gold nanospheres are functionalized with the head-to-tail cyclized-peptide CGisoDGRG (Iso1) and murine IL12. The resulting nanodrug (Iso1/Au/IL12) is monodispersed, stable, and bifunctional in terms of αvß3 and IL12-receptor recognition. Low-dose Iso1/Au/IL12, equivalent to 18-75 pg of IL12, induces antitumor effects in murine models of fibrosarcomas and mammary adenocarcinomas, with no evidence of toxicity. Equivalent doses of Au/IL12 (a nanodrug lacking Iso1) fail to delay tumor growth, whereas 15 000 pg of free IL12 is necessary to achieve similar effects. Iso1/Au/IL12 significantly increases tumor infiltration by innate immune cells, such as NK and iNKT cells, monocytes, and neutrophils. NK cell depletion completely inhibits its antitumor effects. Low-dose Iso1/Au/IL12 can also increase the therapeutic efficacy of adoptive T-cell therapy in mice with autochthonous prostate cancer. These findings indicate that coupling IL12 to isoDGR-tagged nanogold is a valid strategy for enhancing its therapeutic index and sustaining adoptive T-cell therapy.

Catestatin regulates vesicular quanta through modulation of cholinergic and peptidergic (PACAPergic) stimulation in PC12 cells.

We have previously shown that the chromogranin A (CgA)-derived peptide catestatin (CST: hCgA352-372) inhibits nicotine-induced secretion of catecholamines from the adrenal medulla and chromaffin cells. In the present study, we seek to determine whether CST regulates dense core (DC) vesicle (DCV) quanta (catecholamine and chromogranin/secretogranin proteins) during acute (0.5-h treatment) or chronic (24-h treatment) cholinergic (nicotine) or peptidergic (PACAP, pituitary adenylyl cyclase activating polypeptide) stimulation of PC12 cells. In acute experiments, we found that both nicotine (60 µM) and PACAP (0.1 µM) decreased intracellular norepinephrine (NE) content and increased 3H-NE secretion, with both effects markedly inhibited by co-treatment with CST (2 µM). In chronic experiments, we found that nicotine and PACAP both reduced DCV and DC diameters and that this effect was likewise prevented by CST. Nicotine or CST alone increased expression of CgA protein and together elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate CgA expression. In contrast, PACAP increased expression of CgB and SgII proteins, with a further potentiation by CST. CST augmented the expression of tyrosine hydroxylase (TH) but did not increase intracellular NE levels, presumably due to its inability to cause post-translational activation of TH through serine phosphorylation. Co-treatment of CST with nicotine or PACAP increased quantal size, plausibly due to increased synthesis of CgA, CgB and SgII by CST. We conclude that CST regulates DCV quanta by acutely inhibiting catecholamine secretion and chronically increasing expression of CgA after nicotinic stimulation and CgB and SgII after PACAPergic stimulation.

The Importance of Detail: How Differences in Ligand Structures Determine Distinct Functional Responses in Integrin αv ß3.

Ligand-based control of protein functional motions can provide novel opportunities in the study of fundamental biological mechanisms and in the development of novel therapeutics. In this work we addressed the ligand-based modulation of integrin functions. Inhibitors of integrin αv ß3 are interesting anticancer agents but their molecular mechanisms are still unclear: Peptides and peptidomimetics characterized by the Arg-Gly-Asp (RGD) or isoAsp-Gly-Arg (isoDGR) binding motifs have shown controversial agonist/antagonist effects. We have investigated the differential mechanisms of integrin activation/deactivation by three distinct ligands (cyclo-RGDf(NMe)V (Cilengitide), cyclo[DKP3-RGD], cyclo[DKP3-isoDGR]; DKP=diketopiperazine) through a comparative analysis of ligand-controlled protein internal dynamics: Although RGD facilitates the onset of dynamic states leading to activation, isoDGR induces a diffuse rigidification of the complex consistent with antagonist activities. Computational predictions have been experimentally probed by showing that the antibody AP5, which is capable of recognizing the active form of integrin, binds specifically to the RGD complexes and not to the isoDGR complex, which supports opposite functional roles of the two motifs targeting the same binding site.

Association between preoperative Vasostatin-1 and pathological features of aggressiveness in localized nonfunctioning pancreatic neuroendocrine tumors (NF-PanNET).

BACKGROUND: A reliable and accessible biomarker for nonfunctioning pancreatic neuroendocrine tumors (NF-PanNET) is currently unavailable. Chromogranin A (CgA) represents the best-described neuroendocrine biomarker, but its accuracy is low. Vasostatin-1 (VS-1), a fragment derived from the cleavage of CgA, was recently investigated and found to be more accurate as tumor biomarker in a cohort of patients affected by mainly metastatic small intestinal NET. METHODS: Patients submitted to surgery for sporadic localized NF-PanNET at San Raffaele Hospital were included. Preoperative plasma samples were prospectively collected. Circulating levels of total-CgA and VS-1 were retrospectively investigated by sandwich Enzyme-Linked ImmunoSorbent Assays. RESULTS: Overall, 50 patients were included. VS-1 value (P=0.0001) was the only preoperatively retrievable factor independently associated with NF-PanNET size. No significant correlation between CgA and tumor diameter was found (P = 0.057). A VS-1 value of 0.39 nM was identified as the optimal VS-1 cut-off accurately associated with NF-PanNET larger than 4 cm. Patients with VS-1 > 0.39 nM had a significantly higher frequency of microvascular invasion (P = 0.005) and nodal metastasis (P = 0.027). Median VS-1 plasma level was significantly higher in the presence of microvascular invasion (P = 0.001) and nodal metastasis (P = 0.012). PPI assumption significantly increased total-CgA levels, but not those of VS-1 (P = 0.111). CONCLUSIONS: In localized, non-metastatic NF-PanNET, VS-1 is strongly associated to tumor dimension and its plasma levels are significantly higher in the presence of microvascular invasion and nodal metastases; moreover, VS-1 value is not affected by the PPI use.

Self-targeting of TNF-releasing cancer cells in preclinical models of primary and metastatic tumors.

Circulating cancer cells can putatively colonize distant organs to form metastases or to reinfiltrate primary tumors themselves through a process termed "tumor self-seeding." Here we exploit this biological attribute to deliver tumor necrosis factor alpha (TNF), a potent antitumor cytokine, directly to primary and metastatic tumors in a mechanism that we have defined as "tumor self-targeting." For this purpose, we genetically engineered mouse mammary adenocarcinoma (TSA), melanoma (B16-F10), and Lewis lung carcinoma cells to produce and release murine TNF. In a series of intervention trials, systemic administration of TNF-expressing tumor cells was associated with reduced growth of both primary tumors and metastatic colonies in immunocompetent mice. We show that these malignant cells home to tumors, locally release TNF, damage neovascular endothelium, and induce massive cancer cell apoptosis. We also demonstrate that such tumor-cell-mediated delivery avoids or minimizes common side effects often associated with TNF-based therapy, such as acute inflammation and weight loss. Our study provides proof of concept that genetically modified circulating tumor cells may serve as targeted vectors to deliver anticancer agents. In a clinical context, this unique paradigm represents a personalized approach to be translated into applications potentially using patient-derived circulating tumor cells as self-targeted vectors for drug delivery.

Circulating chromogranin A and its fragments as diagnostic and prognostic disease markers.

Chromogranin A (CgA), a secretory protein released in the blood by neuroendocrine cells and neurons, is the precursor of various bioactive fragments involved in the regulation of the cardiovascular system, metabolism, innate immunity, angiogenesis, and tissue repair. After the original demonstration that circulating CgA can serve as a biomarker for a wide range of neuroendocrine tumors, several studies have shown that increased levels of CgA can be present also in the blood of patients with cardiovascular, gastrointestinal, and inflammatory diseases with, in certain cases, important diagnostic and prognostic implications. Considering the high structural and functional heterogeneity of the CgA system, comprising precursor and fragments, it is not surprising that the different immunoassays used in these studies led, in some cases, to discrepant results. Here, we review these notions and we discuss the importance of measuring total-CgA, full-length CgA, specific fragments, and their relative levels for a more thorough assessment of the pathophysiological function and diagnostic/prognostic value of the CgA system.

Enhancement of Tumor Homing by Chemotherapy-Loaded Nanoparticles.

Targeted delivery of anticancer drugs with nanocarriers can reduce side effects and ameliorate therapeutic efficacy. However, poorly perfused and dysfunctional tumor vessels limit the transport of the payload into solid tumors. The use of tumor-penetrating nanocarriers might enhance tumor uptake and antitumor effects. A peptide containing a tissue-penetrating (TP) consensus motif, capable of recognizing neuropilin-1, is here fused to a neuroblastoma-targeting peptide (pep) previously developed. Neuroblastoma cell lines and cells derived from both xenografts and high-risk neuroblastoma patients show overexpression of neuropilin-1. In vitro studies reveal that TP-pep binds cell lines and cells derived from neuroblastoma patients more efficiently than pep. TP-pep, after coupling to doxorubicin-containing stealth liposomes (TP-pep-SL[doxorubicin]), enhances their uptake by cells and cytotoxic effects in vitro, while increasing tumor-binding capability and homing in vivo. TP-pep-SL[doxorubicin] treatment enhances the Evans Blue dye accumulation in tumors but not in nontumor tissues, pointing to selective increase of vascular permeability in tumor tissues. Compared to pep-SL[doxorubicin], TP-pep-SL[doxorubicin] shows an increased antineuroblastoma activity in three neuroblastoma animal models mimicking the growth of neuroblastoma in humans. The enhancement of drug penetration in tumors by TP-pep-targeted nanoparticles may represent an innovative strategy for neuroblastoma.

Glycine N-methylation in NGR-Tagged Nanocarriers Prevents Isoaspartate formation and Integrin Binding without Impairing CD13 Recognition and Tumor Homing.

NGR (asparagine-glycine-arginine) is a tumor vasculature-homing peptide motif widely used for the functionalization of drugs, nanomaterials and imaging compounds for cancer treatment and diagnosis. Unfortunately, this motif has a strong propensity to undergo rapid deamidation. This reaction, which converts NGR into isoDGR, is associated with receptor switching from CD13 to integrins, with potentially important manufacturing, pharmacological and toxicological implications. It is found that glycine N-methylation of NGR-tagged nanocarriers completely prevents asparagine deamidation without impairing CD13 recognition. Studies in animal models have shown that the methylated NGR motif can be exploited for delivering radiolabeled compounds and nanocarriers, such as tumor necrosis factor-α (TNF)-bearing nanogold and liposomal doxorubicin, to tumors with improved selectivity. These findings suggest that this NGR derivative is a stable and efficient tumor-homing ligand that can be used for delivering functional nanomaterials to tumor vasculature.

Chromogranin A regulates vesicle storage and mitochondrial dynamics to influence insulin secretion.

Chromogranin A (CgA) is a prohormone and a granulogenic factor that regulates secretory pathways in neuroendocrine tissues. In ß-cells of the endocrine pancreas, CgA is a major cargo in insulin secretory vesicles. The impact of CgA deficiency on the formation and exocytosis of insulin vesicles is yet to be investigated. In addition, no literature exists on the impact of CgA on mitochondrial function in ß-cells. Using three different antibodies, we demonstrate that CgA is processed to vasostatin- and catestatin-containing fragments in pancreatic islet cells. CgA deficiency in Chga-KO islets leads to compensatory overexpression of chromogranin B, secretogranin II, SNARE proteins and insulin genes, as well as increased insulin protein content. Ultrastructural studies of pancreatic islets revealed that Chga-KO ß-cells contain fewer immature secretory granules than wild-type (WT) control but increased numbers of mature secretory granules and plasma membrane-docked vesicles. Compared to WT control, CgA-deficient ß-cells exhibited increases in mitochondrial volume, numerical densities and fusion, as well as increased expression of nuclear encoded genes (Ndufa9, Ndufs8, Cyc1 and Atp5o). These changes in secretory vesicles and the mitochondria likely contribute to the increased glucose-stimulated insulin secretion observed in Chga-KO mice. We conclude that CgA is an important regulator for coordination of mitochondrial dynamics, secretory vesicular quanta and GSIS for optimal secretory functioning of ß-cells, suggesting a strong, CgA-dependent positive link between mitochondrial fusion and GSIS.

Angiopoietin-2 in Bone Marrow milieu promotes Multiple Myeloma-associated angiogenesis.

Angiopoietin-2 (Ang-2) is involved in angiogenesis in both solid and hematological malignancies. In Multiple Myeloma (MM), serum Ang-2 correlates with disease progression and response to therapy. To address the patho-physiologic role of Ang-2 in MM associated angiogenesis, we used sera from patients with active MM, which contained significantly higher levels of the molecule, compared to those from patients with smoldering MM and Monoclonal Gammopathy of Undetermined Significance. MM Bone Marrow (BM) sera with high Ang-2 concentration specifically contributed to endothelial cell (EC) activation, while Ang-1 containing sera maintained EC stabilization. The functional dichotomy of Ang-1 and Ang-2 was confirmed by the triggering of distinctive signaling pathways down-stream the common Tie-2 receptor, i.e., the Akt or the ERK- phosphorylation pathway. Notably, Ang-2 but not VEGF serum levels correlated with BM micro-vessel density, further underscoring the key role of Ang-2 in angiogenesis. Western Blot, RT-PCR and immunocytochemistry identified MMEC as the major source of Ang-2, at variance with MM cells and CD14(+) BM monocytes. These data suggest that Ang-2 produced in the BM milieu may contribute to MM angiogenesis and suggest that the molecule can be further exploited both as angiogenesis biomarker and as a potential therapeutic target.