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Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney neoplasm; bone metastasis (BM) develops in 35% to 40% of metastatic patients and results in substantial morbidity and mortality, as well as medical costs. A key feature of ccRCC is the loss of function of the von Hippel-Lindau protein, which enhances angiogenesis via vascular endothelial growth factor release. Consequently, antiangiogenic tyrosine kinase inhibitors (TKI) emerged as a treatment for ccRCC. However, limited data about their efficacy in BM is available, and no systematic comparisons have been performed. We developed mouse models of bone and lung ccRCC tumors and compared their anticancer efficacy, impact on mouse survival, and mechanisms of action, including effects on tumor cells and both immune and nonimmune (blood vessels and osteoclasts) bone stromal components. This approach elucidates the efficacy of TKIs in ccRCC bone tumors to support rational interrogation and development of therapies., Significance: TKIs showed different efficacy in synchronous bone and lung metastases and did not eradicate tumors as single agents but induced extensive reprogramming of the BM microenvironment. This resulted in a significant decrease in neoangiogenic blood vessels, bone remodeling, and immune cell infiltration (including CD8 T cells) with altered spatial distribution., (©2024 The Authors; Published by the American Association for Cancer Research.)

Bone metastases are the most common milestone in the lethal progression of prostate cancer and prominent in a substantial portion of renal malignancies. Interactions between cancer and bone host cells have emerged as drivers of both disease progression and therapeutic resistance. To best understand these central host-epithelial cell interactions, biologically relevant preclinical models are required. To achieve this goal, we here established and characterized tissue-engineered bone mimetic environments (BME) capable of supporting the growth of patient-derived xenograft (PDX) cells, ex vivo and in vivo. The BME consisted of a polycaprolactone (PCL) scaffold colonized by human mesenchymal stem cells (hMSCs) differentiated into osteoblasts. PDX-derived cells were isolated from bone metastatic prostate or renal tumors, engineered to express GFP or luciferase and seeded onto the BMEs. BMEs supported the growth and therapy response of PDX-derived cells, ex vivo. Additionally, BMEs survived after in vivo implantation and further sustained the growth of PDX-derived cells, their serial transplant, and their application to study the response to treatment. Taken together, this demonstrates the utility of BMEs in combination with patient-derived cells, both ex vivo and in vivo. STATEMENT OF SIGNIFICANCE: Our tissue-engineered BME supported the growth of patient-derived cells and proved useful to monitor the therapy response, both ex vivo and in vivo. This approach has the potential to enable co-clinical strategies to monitor bone metastatic tumor progression and therapy response, including identification and prioritization of new targets for patient treatment., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Eleonora Dondossola reports financial support was provided by The Prostate Cancer SPORE (P50 CA140388–07). Eleonora Dondossola reports financial support and equipment, drugs, or supplies were provided by Bayer HealthCare Pharmaceuticals (57440)., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Protein tyrosine phosphatases (PTPs) play major roles in cancer and are emerging as therapeutic targets. Recent reports suggest low-molecular weight PTP (LMPTP)-encoded by the ACP1 gene-is overexpressed in prostate tumors. We found ACP1 up-regulated in human prostate tumors and ACP1 expression inversely correlated with overall survival. Using CRISPR-Cas9-generated LMPTP knockout C4-2B and MyC-CaP cells, we identified LMPTP as a critical promoter of prostate cancer (PCa) growth and bone metastasis. Through metabolomics, we found that LMPTP promotes PCa cell glutathione synthesis by dephosphorylating glutathione synthetase on inhibitory Tyr 270 . PCa cells lacking LMPTP showed reduced glutathione, enhanced activation of eukaryotic initiation factor 2-mediated stress response, and enhanced reactive oxygen species after exposure to taxane drugs. LMPTP inhibition slowed primary and bone metastatic prostate tumor growth in mice. These findings reveal a role for LMPTP as a critical promoter of PCa growth and metastasis and validate LMPTP inhibition as a therapeutic strategy for treating PCa through sensitization to oxidative stress.

Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1 . Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia in vivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress in vitro and in vivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.

Given the central role of the androgen receptor (AR) in prostate cancer cell biology, AR-targeted therapies have been the backbone of prostate cancer treatment for over 50 years. New data indicate that AR is expressed in additional cell types within the tumor microenvironment. Moreover, targeting AR for the treatment of prostate cancer has established side effects such as bone complications and an increased risk of developing cardiometabolic disease, indicating broader roles for AR. With the advent of novel technologies, such as single-cell approaches and advances in preclinical modeling, AR has been identified to have clinically significant functions in other cell types. In this mini-review, we describe new cancer cell-extrinsic roles for AR within the tumor microenvironment as well as systemic effects that collectively impact prostate cancer progression and patient outcomes., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The androgen receptor (AR) is one of the oldest therapeutic targets in oncology and continues to dominate the treatment landscape for advanced prostate cancer, where nearly all treatment regimens include some form of AR modulation. In this regard, AR remains the central driver of prostate cancer cell biology. Emerging preclinical and clinical data implicate key roles for AR in additional cancer types, thereby expanding the importance of this drug target beyond prostate cancer. In this mini-review, new roles for AR in other cancer types are discussed as well as their potential for treatment with AR-targeted agents. Our understanding of these additional functions for AR in oncology expand this receptor's potential as a therapeutic target and will help guide the development of new treatment approaches., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The increase in life expectancy and in the number of individuals over 60 years old brings new demands to health professionals and services based on the physiological changes that occur in this population. The aging process results in changes in body composition, increasing body fat and reducing muscle mass, in addition to a reduction in bone mass. Aim: The aim of this study was to examine the effect of whey protein and vitamin D supplementation on body composition and skeletal muscle in older adults living in long-term care facilities. Methods: This study is a double-blind randomized controlled trial. Thirty older adults (>60 years old) were randomized and allocated in three groups: group receiving resistance training and supplementation receiving resistance training, whey protein and vitamin D; group received resistance and placebo training receiving resistance training and placebo, and control group without any intervention. Body composition was measured by dual-energy X-ray absorptiometry at baseline, 12 weeks, and 24 weeks. Results: The mean age was 74.87 (± 8.14) years. A significant difference ( p  = 0.042) was observed between the group receiving resistance training and supplementation and control groups in relation to lean mass increase (kg) at 24 weeks. After 24 weeks of intervention, there was a significant increase in Relative index of muscle mass for the two groups that underwent resistance training, group received resistance and placebo training ( p  = 0.042) and group receiving resistance training and supplementation ( p  = 0.045), in relation to the control. Conclusion: Combined supplementation of whey protein and vitamin D with resistance training can significantly improve lean mass, total mass, and relative index of muscle mass in institutionalized older adults.

Aberrant metabolic functions play a crucial role in prostate cancer progression and lethality. Currently, limited knowledge is available on subtype-specific metabolic features and their implications for treatment. We therefore investigated the metabolic determinants of the two major subtypes of castration-resistant prostate cancer [androgen receptor-expressing prostate cancer (ARPC) and aggressive variant prostate cancer (AVPC)]. Transcriptomic analyses revealed enrichment of gene sets involved in oxidative phosphorylation (OXPHOS) in ARPC tumor samples compared with AVPC. Unbiased screening of metabolic signaling pathways in patient-derived xenograft models by proteomic analyses further supported an enrichment of OXPHOS in ARPC compared with AVPC, and a skewing toward glycolysis by AVPC. In vitro, ARPC C4-2B cells depended on aerobic respiration, while AVPC PC3 cells relied more heavily on glycolysis, as further confirmed by pharmacologic interference using IACS-10759, a clinical-grade inhibitor of OXPHOS. In vivo studies confirmed IACS-10759's inhibitory effects in subcutaneous and bone-localized C4-2B tumors, and no effect in subcutaneous PC3 tumors. Unexpectedly, IACS-10759 inhibited PC3 tumor growth in bone, indicating microenvironment-induced metabolic reprogramming. These results suggest that castration-resistant ARPC and AVPC exhibit different metabolic dependencies, which can further undergo metabolic reprogramming in bone., Implications: These vulnerabilities may be exploited with mechanistically novel treatments, such as those targeting OXPHOS alone or possibly in combination with existing therapies. In addition, our findings underscore the impact of the tumor microenvironment in reprogramming prostate cancer metabolism., (©2022 American Association for Cancer Research.)

The foreign body response (FBR) is a clinically relevant issue that can cause malfunction of implanted medical devices by fibrotic encapsulation. Whereas inflammatory aspects of the FBR have been established, underlying fibroblast-dependent mechanisms remain unclear. We here combine multiphoton microscopy with ad hoc reporter mice expressing α-smooth muscle actin (αSMA) protein to determine the locoregional fibroblast dynamics, activation, and fibrotic encapsulation of polymeric materials. Fibroblasts invaded as individual cells and established a multicellular network, which transited to a two-compartment fibrotic response displaying an αSMA cold external capsule and a long-lasting, inner αSMA hot environment. The recruitment of fibroblasts and extent of fibrosis were only incompletely inhibited after depletion of macrophages, implicating coexistence of macrophage-dependent and macrophage-independent mediators. Furthermore, neither altering material type or porosity modulated αSMA + cell recruitment and distribution. This identifies fibroblast activation and network formation toward a two-compartment FBR as a conserved, self-organizing process partially independent of macrophages.

223 Ra is a bone-seeking, α-particle-emitting radionuclide approved for the treatment of patients with metastatic prostate cancer and is currently being tested in a variety of clinical trials for primary and metastatic cancers to bone. Clinical evaluation of 223 Ra hematologic safety showed a significantly increased rate of neutropenia and thrombocytopenia in patients, hinting at myelosuppression as a side effect. Methods: In this study, we investigated the consequences of 223 Ra treatment on bone marrow biology by combining flow cytometry, single-cell RNA sequencing, three-dimensional multiphoton microscopy and bone marrow transplantation analyses. Results: 223 Ra accumulated in bones and induced zonal radiation damage confined to the bone interface, followed by replacement of the impaired areas with adipocyte infiltration, as monitored by 3-dimensional multiphoton microscopy ex vivo. Flow cytometry and single-cell transcriptomic analyses on bone marrow hematopoietic populations revealed transient, nonspecific 223 Ra-mediated cytotoxicity on resident populations, including stem, progenitor, and mature leukocytes. This toxicity was paralleled by a significant decrease in white blood cells and platelets in peripheral blood-an effect that was overcome within 40 d after treatment. 223 Ra exposure did not impair full hematopoietic reconstitution, suggesting that bone marrow function is not permanently hampered. Conclusion: Our results provide a comprehensive explanation of 223 Ra reversible effects on bone marrow cells and exclude long-term myelotoxicity, supporting safety for patients., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

223 Ra is an α-emitter approved for the treatment of bone metastatic prostate cancer (PCa), which exerts direct cytotoxicity toward PCa cells near the bone interface, whereas cells positioned in the core respond poorly because of short α-particle penetrance. β1 integrin (β1I) interference has been shown to increase radiosensitivity and significantly enhance external-beam radiation efficiency. We hypothesized that targeting β1I would improve 223 Ra outcome. Methods: We tested the effect of combining 223 Ra and anti-β1I antibody treatment in PC3 and C4-2B PCa cell models expressing high and low β1I levels, respectively. In vivo tumor growth was evaluated through bioluminescence. Cellular and molecular determinants of response were analyzed by ex vivo 3-dimensional imaging of bone lesions and by proteomic analysis and were further confirmed by computational modeling and in vitro functional analysis in tissue-engineered bone mimetic systems. Results: Interference with β1I combined with 223 Ra reduced PC3 cell growth in bone and significantly improved overall mouse survival, whereas no change was achieved in C4-2B tumors. Anti-β1I treatment decreased the PC3 tumor cell mitosis index and spatially expanded 223 Ra lethal effects 2-fold, in vivo and in silico. Regression was paralleled by decreased expression of radioresistance mediators. Conclusion: Targeting β1I significantly improves 223 Ra outcome and points toward combinatorial application in PCa tumors with high β1I expression., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

The Foreign body response (FBR) is a major unresolved challenge that compromises medical implant integration and function by inflammation and fibrotic encapsulation. Mice implanted with polymeric scaffolds coupled to intravital non-linear multiphoton microscopy acquisition enable multiparametric, longitudinal investigation of the FBR evolution and interference strategies. However, follow-up analyses based on visual localization and manual segmentation are extremely time-consuming, subject to human error, and do not allow for automated parameter extraction. We developed an integrated computational pipeline based on an innovative and versatile variant of the U-Net neural network to segment and quantify cellular and extracellular structures of interest, which is maintained across different objectives without impairing accuracy. This software for automatically detecting the elements of the FBR shows promise to unravel the complexity of this pathophysiological process., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sarti, Parlani, Diaz-Gomez, Mikos, Cerveri, Casarin and Dondossola.)

Renal medullary carcinoma (RMC) is a lethal malignancy affecting individuals with sickle hemoglobinopathies. Currently, no modifiable risk factors are known. We aimed to determine whether high-intensity exercise is a risk factor for RMC in individuals with sickle cell trait (SCT). We used multiple approaches to triangulate our conclusion. First, a case-control study was conducted at a single tertiary-care facility. Consecutive patients with RMC were compared to matched controls with similarly advanced genitourinary malignancies in a 1:2 ratio and compared on rates of physical activity and anthropometric measures, including skeletal muscle surface area. Next, we compared the rate of military service among our RMC patients to a similarly aged population of black individuals with SCT in the U.S. Further, we used genetically engineered mouse models of SCT to study the impact of exercise on renal medullary hypoxia. Compared with matched controls, patients with RMC reported higher physical activity and had higher skeletal muscle surface area. A higher proportion of patients with RMC reported military service than expected compared to the similarly-aged population of black individuals with SCT. When exposed to high-intensity exercise, mice with SCT demonstrated significantly higher renal medulla hypoxia compared to wild-type controls. These data suggest high-intensity exercise is the first modifiable risk factor for RMC in individuals with SCT.

Castration-resistant prostate cancer (CRPC) is an advanced subtype of prostate cancer with limited therapeutic options. Here, we applied a systems-based modeling approach called kinome regularization (KiR) to identify multitargeted kinase inhibitors (KIs) that abrogate CRPC growth. Two predicted KIs, PP121 and SC-1, suppressed CRPC growth in two-dimensional in vitro experiments and in vivo subcutaneous xenografts. An ex vivo bone mimetic environment and in vivo tibia xenografts revealed resistance to these KIs in bone. Combining PP121 or SC-1 with docetaxel, standard-of-care chemotherapy for late-stage CRPC, significantly reduced tibia tumor growth in vivo, decreased growth factor signaling, and vastly extended overall survival, compared to either docetaxel monotherapy. These results highlight the utility of computational modeling in forming physiologically relevant predictions and provide evidence for the role of multitargeted KIs as chemosensitizers for late-stage, metastatic CRPC., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Bone metastasis is the most frequent complication in prostate cancer patients and associated outcome remains fatal. Radium223 (Rad223), a bone targeting radioisotope improves overall survival in patients (3.6 months vs. placebo). However, clinical response is often followed by relapse and disease progression, and associated mechanisms of efficacy and resistance are poorly understood. Research efforts to overcome this gap require a substantial investment of time and resources. Computational models, integrated with experimental data, can overcome this limitation and drive research in a more effective fashion., Methods: Accordingly, we developed a predictive agent-based model of prostate cancer bone metastasis progression and response to Rad223 as an agile platform to maximize its efficacy. The driving coefficients were calibrated on ad hoc experimental observations retrieved from intravital microscopy and the outcome further validated, in vivo., Results: In this work we offered a detailed description of our data-integrated computational infrastructure, tested its accuracy and robustness, quantified the uncertainty of its driving coefficients, and showed the role of tumor size and distance from bone on Rad223 efficacy. In silico tumor growth, which is strongly driven by its mitotic character as identified by sensitivity analysis, matched in vivo trend with 98.3% confidence. Tumor size determined efficacy of Rad223, with larger lesions insensitive to therapy, while medium- and micro-sized tumors displayed up to 5.02 and 152.28-fold size decrease compared to control-treated tumors, respectively. Eradication events occurred in 65 ± 2% of cases in micro-tumors only. In addition, Rad223 lost any therapeutic effect, also on micro-tumors, for distances bigger than 400 μm from the bone interface., Conclusions: This model has the potential to be further developed to test additional bone targeting agents such as other radiopharmaceuticals or bisphosphonates.

Background: Bone-targeting radiotherapy with Radium-223 (Rad-223), a radioisotope emitting genotoxic alpha-radiation with limited tissue penetrance (∼100 µm), prolongs the survival of patients with metastatic prostate cancer (PCa). Confoundingly, the clinical response to Rad-223 is often followed by detrimental relapse and progression, and whether Rad-223 causes tumor-cell directed cytotoxicity in vivo remains unclear. We hypothesized that limited radiation penetrance in situ defines outcome., Methods: We tested Rad-223 overall response by PC3 and C4-2B human PCa cell lines in mouse bones (n = 5-18 tibiae per group). Rad-223 efficacy at subcellular resolution was determined by intravital microscopy analysis of dual-color fluorescent PC3 cells (n = 3-4 mice per group) in tissue-engineered bone constructs. In vivo data were fed into an in silico model to predict Rad-223 effectiveness in lesions of different sizes (1-27, 306 initial cells; n = 10-100 simulations) and the predictions validated in vivo by treating PCa tumors of varying sizes in bones (n = 10-14 tibiae per group). Statistical tests were performed by two-sided Student t test or by one-way ANOVA followed by Tukey's post-hoc test., Results: Rad-223 (385 kBq/kg) delayed the growth (means [SD]; comparison with control-treated mice) of PC3 (6.7 × 105[4.2 × 105] vs 2.8 × 106 [2.2 × 106], P = .01) and C4-2B tumors in bone (7.7 × 105 [4.0 × 105] vs 3.5 × 106 [1.3 × 106], P < .001). Cancer cell lethality in response to Rad-223 (385 kBq/kg) was profound but zonally confined along the bone interface compared with the more distant tumor core, which remained unperturbed (day 4; 13.1 [2.3%] apoptotic cells, 0-100 µm distance from bone vs 3.6 [0.2%], >300 µm distance; P = .01).In silico simulations predicted greater efficacy of Rad-223 on single-cell lesions (eradication rate: 88.0%) and minimal effects on larger tumors (no eradication, 16.2% growth reduction in tumors of 27 306 cells), as further confirmed in vivo for PC3 and C4-2B tumors., Conclusions: Micro-tumors showed severe growth delay or eradication in response to Rad-223, whereas macro-tumors persisted and expanded. The relative inefficacy in controlling large tumors points to application of Rad-223 in secondary prevention of early bone-metastatic disease and regimens co-targeting the tumor core., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mechanistic analysis of metastatic prostate cancer (PCa) biology and therapy response critically depends upon clinically relevant three-dimensional (3D) bone-like, organotypic culture. We here combine an engineered bone-mimetic environment (BME) with longitudinal microscopy to test the growth and therapy response of 3D PCa tumoroids. Besides promoting both tumor-cell autonomous and microenvironment-dependent growth in PCa cell lines and patient-derived xenograft cells, the BME enables in vivo-like tumor cell response to therapy, and reveals bone stroma dependent resistance to chemotherapy and BME-targeted localization and induction of cytoxicity by Radium-223. The BME platform will allow the propagation, compound screening and mechanistic dissection of patient-derived bone tumor isolates and applications toward personalized medicine., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Intravital multiphoton microscopy (iMPM) in mice provides access to cellular and molecular mechanisms of metastatic progression of cancers and the underlying interactions with the tumor stroma. Whereas iMPM of malignant disease has been performed for soft tissues, noninvasive iMPM of solid tumor in the bone is lacking. We combined miniaturized tissue-engineered bone constructs in nude mice with a skin window to noninvasively and repetitively monitor prostate cancer lesions by three-dimensional iMPM. In vivo ossicles developed large central cavities containing mature bone marrow surrounded by a thin cortex and enabled tumor implantation and longitudinal iMPM over weeks. Tumors grew inside the bone cavity and along the cortical bone interface and induced niches of osteoclast activation (focal osteolysis). Interventional bisphosphonate therapy reduced osteoclast kinetics and osteolysis without perturbing tumor growth, indicating dissociation of the tumor-stroma axis. The ossicle window, with its high cavity-to-cortex ratio and long-term functionality, thus allows for the mechanistic dissection of reciprocal epithelial tumor-bone interactions and therapy response., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

A systematic review with meta-analysis was performed to estimate the accuracy of IgM ELISA for Leptospirosis diagnosis. A search of Medline, Lilacs, Embase, Cochrane Central Register of Controlled Trials and Grey literature (Google Scholar and British Library) was conducted. The medical subject headings (MeSHs) and the words "leptospirosis", "human leptospirosis" and "IgM ELISA" were used. Fifty-two studies were analyzed, which included 10,775 samples. The pooled sensitivity of all the studies was 86% (CI 95%, 85%-87%) and specificity was 90% (CI 95%, 89%-91%). In studies of the acute phase, the sensitivity and specificity were 84% (CI 95%, 82%-85%) and 91% (CI 95%, 90%-91%), respectively. In conclusion, IgM ELISA is sensitive for use as an initial screen for leptospiral infections.

Chromogranin A (CgA), a neuroendocrine secretory protein, and its fragments are present in variable amounts in the blood of normal subjects and cancer patients. We investigated whether circulating CgA has a regulatory function in tumor biology and progression. Systemic administration of full-length CgA, but not of fragments lacking the C-terminal region, could reduce tumor growth in murine models of fibrosarcoma, mammary adenocarcinoma, Lewis lung carcinoma, and primary and metastatic melanoma, with U-shaped dose-response curves. Tumor growth inhibition was associated with reduction of microvessel density and blood flow in neoplastic tissues. Neutralization of endogenous CgA with antibodies against its C-terminal region (residues 410-439) promoted tumor growth. Structure-function studies showed that the C-terminal region of CgA contains a bioactive site and that cleavage of this region causes a marked loss of anti-angiogenic and anti-tumor potency. Mechanistic studies showed that full-length CgA could induce, with a U-shaped dose-response curve, the production of protease nexin-1 in endothelial cells, a serine protease inhibitor endowed of anti-angiogenic activity. Gene silencing or neutralization of protease nexin-1 with specific antibodies abolished both anti-angiogenic and anti-tumor effects of CgA. These results suggest that circulating full-length CgA is an important inhibitor of angiogenesis and tumor growth, and that cleavage of its C-terminal region markedly reduces its activity. Pathophysiological changes in CgA blood levels and/or its fragmentation might regulate disease progression in cancer patients.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of leukemic B cells in peripheral blood, bone marrow (BM) and lymphoid tissues, and by their recirculation between these compartments. We observed that circulating chromogranin A (CgA) and its N-terminal fragment (called vasostatin-1, CgA1-76), two neuroendocrine secretory polypeptides that enhance the endothelial barrier function, are present in variable amounts in the blood of CLL patients. Studies in animal models showed that daily administration of full-length human CgA1-439 (0.3 μg, i.v., or 1.5 μg/mouse, i.p.) can reduce the BM/blood ratio of leukemic cells in Eμ-TCL1 mice, a transgenic model, and decrease BM, lung and kidney infiltration in Rag2-/-γc-/- mice engrafted with human MEC1 CLL cells, a xenograft model. This treatment also reduced the loss of body weight and improved animal motility. In vitro, CgA enhanced the endothelial barrier integrity and the trans-endothelial migration of MEC1 cells, with a bimodal dose-response curve. Vasostatin-1, but not a larger fragment consisting of N-terminal and central regions of CgA (CgA1-373), inhibited CLL progression in the xenograft model, suggesting that the C-terminal region is crucial for CgA activity and that the N-terminal domain contains a site that is activated by proteolytic cleavage. These findings suggest that circulating full-length CgA and its fragments may contribute to regulate leukemic cell trafficking and reduce tissue infiltration in CLL., Competing Interests: No competing financial interest.

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.

Implanted biomaterials often fail because they elicit a foreign body response (FBR) and concomitant fibrotic encapsulation. To design clinically relevant interference approaches, it is crucial to first examine the FBR mechanisms. Here, we report the development and validation of infrared-excited nonlinear microscopy to resolve the three-dimensional (3D) organization and fate of 3D-electrospun scaffolds implanted deep into the skin of mice, and the following step-wise FBR process. We observed that immigrating myeloid cells (predominantly macrophages of the M1 type) engaged and became immobilized along the scaffold/tissue interface, before forming multinucleated giant cells. Both macrophages and giant cells locally produced vascular endothelial growth factor (VEGF), which initiated and maintained an immature neovessel network, followed by formation of a dense collagen capsule 2-4 weeks post-implantation. Elimination of the macrophage/giant-cell compartment by clodronate and/or neutralization of VEGF by VEGF Trap significantly diminished giant-cell accumulation, neovascularization and fibrosis. Our findings identify macrophages and giant cells as incendiaries of the fibrotic encapsulation of engrafted biomaterials via VEGF release and neovascularization, and therefore as targets for therapy., Competing Interests: Competing Financial Interests: the authors do not have competing financial interest to declare.

In response to an urgent need for improved diagnostic and predictive serum biomarkers for management of metastatic prostate cancer, we used phage display fingerprinting to analyze sequentially acquired serum samples from a patient with advancing prostate cancer. We identified a peptide ligand, CTFAGSSC, demonstrating an increased recovery frequency over time. Serum antibody reactivity to this peptide epitope increased in the index patient, in parallel with development of deteriorating symptoms. The antigen mimicking the peptide epitope was identified as alpha-2-Heremans-Schmid glycoprotein, also known as fetuin-A. Metastatic prostate cancer cell lines and bone metastasis samples displayed robust fetuin-A expression, and we demonstrated serum immune reactivity to fetuin-A with concomitant development of metastatic castrate-resistant disease in a large cohort of prostate cancer patients. Whereas fetuin-A is an established tumor antigen in several types of cancer, including breast cancer, glioblastoma, and pancreas cancer, this report is to our knowledge the first study implicating fetuin-A in prostate cancer and indicating that autoantibodies specific for fetuin-A show utility as a prognostic indicator for prostate cancer patients prone to progress to metastatic disease.

Metastasis is the most lethal step of cancer progression in patients with invasive melanoma. In most human cancers, including melanoma, tumor dissemination through the lymphatic vasculature provides a major route for tumor metastasis. Unfortunately, molecular mechanisms that facilitate interactions between melanoma cells and lymphatic vessels are unknown. Here, we developed an unbiased approach based on molecular mimicry to identify specific receptors that mediate lymphatic endothelial-melanoma cell interactions and metastasis. By screening combinatorial peptide libraries directly on afferent lymphatic vessels resected from melanoma patients during sentinel lymphatic mapping and lymph node biopsies, we identified a significant cohort of melanoma and lymphatic surface binding peptide sequences. The screening approach was designed so that lymphatic endothelium binding peptides mimic cell surface proteins on tumor cells. Therefore, relevant metastasis and lymphatic markers were biochemically identified, and a comprehensive molecular profile of the lymphatic endothelium during melanoma metastasis was generated. Our results identified expression of the phosphatase 2 regulatory subunit A, α-isoform (PPP2R1A) on the cell surfaces of both melanoma cells and lymphatic endothelial cells. Validation experiments showed that PPP2R1A is expressed on the cell surfaces of both melanoma and lymphatic endothelial cells in vitro as well as independent melanoma patient samples. More importantly, PPP2R1A-PPP2R1A homodimers occur at the cellular level to mediate cell-cell interactions at the lymphatic-tumor interface. Our results revealed that PPP2R1A is a new biomarker for melanoma metastasis and show, for the first time to our knowledge, an active interaction between the lymphatic vasculature and melanoma cells during tumor progression.

Non-malignant cells found within neoplastic lesions express alanyl (membrane) aminopeptidase (ANPEP, best known as CD13), and CD13-null mice exhibit limited tumor growth and angiogenesis. We have recently demonstrated that a subset of bone marrow-derived CD11b + CD13 + myeloid cells accumulate within neoplastic lesions in several murine models of transplantable cancer to promote angiogenesis. If these findings were confirmed in clinical settings, CD11b + CD13 + myeloid cells could become a non-malignant target for the development of novel anticancer regimens.

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

Cancer progression involves the seeding of malignant cells in circulation and the colonization of distant organs. However, circulating neoplastic cells can also reinfiltrate the tumor of origin. This process, called "tumor-self seeding," can select more aggressive cells that may contribute to cancer progression. Here, using mouse mammary adenocarcinoma models, we observed that both tumor self-seeding and organ colonization were inhibited by chromogranin A (CgA), a protein present in variable amounts in the blood of cancer patients. Mechanism studies showed that CgA inhibited the shedding of cancer cells in circulation from primary tumors, as well as the reinfiltration of tumors and the colonization of lungs by circulating tumor cells. CgA reduced gap formation induced by tumor cell-derived factors in endothelial cells, decreased vascular leakage in tumors, and inhibited the transendothelial migration of cancer cells. Together, our findings point to a role for circulating CgA in the regulation of tumor cell trafficking from tumor-to-blood and from blood-to-tumor/normal tissues. Inhibition of the multidirectional trafficking of cancer cells in normal and neoplastic tissues may represent a novel strategy to reduce cancer progression.

The angiogenic switch is a fundamental process for many diseases and for tumor growth. The main proangiogenic stimulus is hypoxia, through activation of the hypoxia-inducible factor (HIF)-1α pathway in endothelial cells (ECs). We have previously shown that the vasostatin-1 (VS-1) fragment of chromogranin A inhibits TNF-α-induced vessel permeability and VEGF-induced EC proliferation, together with migration and matrix invasion, which are all critical steps in angiogenesis. The present study was undertaken to investigate the effect of VS-1 on tumor angiogenesis. We found mouse mammary adenocarcinomas (TS/A), genetically engineered to secrete VS-1 (TS/A 1B8), to be characterized by reduced vascular density and more regular vessels, compared with nontransfected tumors [TS/A wild type (WT)]. Supernatants from TS/A WT cells, but not those from TS/A 1B8, generated tip cells and promoted the permeability of primary human umbilical vein ECs, via VE-cadherin redistribution and cytoskeletal disorganization. These effects were abrogated by mAb 5A8, a VS-1-blocking antibody. Furthermore, VS-1 inhibited hypoxia-driven EC morphological changes, VE-cadherin redistribution, intercellular gap formation, tube morphogenesis, and HIF-1α nuclear translocation in vitro. Our findings highlight a previously undescribed function of VS-1 as a regulator of tumor vascularization.

NGR-TNF is a derivative of TNF-α that targets tumor blood vessels and enhances penetration of chemotherapeutic drugs. Because of this property, NGR-TNF is being tested in combination with chemotherapy in various phase II and III clinical trials. Here we report that chromogranin A (CgA), a protein present in variable amounts in the blood of normal subjects and cancer patients, inhibits the synergism of NGR-TNF with doxorubicin and melphalan in mouse models of lymphoma and melanoma. Pathophysiologically relevant levels of circulating CgA blocked NGR-TNF-induced drug penetration by enhancing endothelial barrier function and reducing drug extravasation in tumors. Mechanistic investigations done in endothelial cell monolayers in vitro showed that CgA inhibited phosphorylation of p38 MAP kinase, disassembly of VE-cadherin-dependent adherence junctions, paracellular macromolecule transport, and NGR-TNF-induced drug permeability. In this system, the N-terminal fragment of CgA known as vasostatin-1 also inhibited drug penetration and NGR-TNF synergism. Together, our results suggest that increased levels of circulating CgA and its fragments, as it may occur in certain cancer patients with nonneuroendocrine tumors, may reduce drug delivery to tumor cells particularly as induced by NGR-TNF. Measuring CgA and its fragments may assist the selection of patients that can respond better to NGR-TNF/chemotherapy combinations in clinical trials., (©2011 AACR.)

This study is the first to report on vascular effect of the chromogranin A derived Vasostatin-I (CgA(1-76)) in vivo. Cardiovascular parameters were recorded in 29 rabbits with sympathetically decentralized right carotid vascular bed. The recombinant human STA CgA(1-78) (VS-1) was infused at 480 μg/kg over 25 min. Group I was kept awake while groups II-V were anesthetized with Ketamine-xylazine. VS-1 was given alone in groups I-II while in presence of either phentolamine, phentolamine plus propranolol or hexamethonium in groups III-V. Serum VS-1 peaked at 2 μg/ml (200 nM) before onset of vascular effects and declined rapidly to ~200 ng/ml within 30 min. In all groups but III and IV VS-1 induced a brief vasoconstriction, being larger in intact than in sympathetically decentralized beds. The VS-1 induced vasoconstriction was not altered by hexamethonium but was abolished by phentolamine. In presence of the α-adrenergic blocker a long lasting vasodilatation, unaffected by propranolol, was apparent on both innervated and decentralized sides. In conclusion, VS-1 induced an α-adrenoceptor-mediated vasoconstriction presumably brought about by noradrenaline release from sympathetic nerves when infused at a dose giving an initial serum concentration of ~200 nM. This initial vasoconstriction masked a persistent adrenoceptor-independent vasodilatation, consistent with previous reports from in vitro models., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Fibroblast adhesion can be modulated by proteins released by neuroendocrine cells and neurons, such as chromogranin A (CgA) and its N-terminal fragment vasostatin-1 (VS-1, CgA(1-78)). We have investigated the mechanisms of the interaction of VS-1 with fibroblasts and of its pro-adhesive activity and have found that the proadhesive activity of VS-1 relies on its interaction with the fibroblast membrane via a phospholipid-binding amphipathic alpha-helix located within residues 47-66, as well as on the interaction of the adjacent C-terminal region 67-78, which is structurally similar to ezrin-radixin-moesin-binding phosphoprotein 50 (a membrane-cytoskeleton adapter protein), with other cellular components critical for the regulation of cell cytoskeleton.

Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degradation reaction occurring in vivo during tissue aging. For instance, non-enzymatic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymatically "repaired" to RGD by protein-l-isoAsp-O-methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an alpha(v)beta3-integrin binding site containing the L-isoDGR sequence, which is enzymatically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGR-to-isoDGR sequence transition in fibronectin fragments generates alpha(v)beta3 antagonists (named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of molecular clock for activating latent integrin binding sites in proteins.