AAV-mouse DNase I sustains long-term DNase I expression in vivo and suppresses breast cancer metastasis.

Neutrophil extracellular traps (NETs) have been implicated in the pathology of various inflammatory conditions. In cancer, NETs have been demonstrated to induce systemic inflammation, impair peripheral vessel and organ function and promote metastasis. Here we show that the plasma level of NETs is significantly higher in patients with metastatic breast cancer compared to those with local disease, or those that were considered cured at a 5-year follow-up, confirming NETs as interesting therapeutic targets in metastatic breast cancer. Administration of DNase I is one strategy to eliminate NETs but long-term treatment requires repeated injections and species-specific versions of the enzyme. To enhance administration and therapeutic efficacy, we have developed an adeno-associated virus (AAV) vector system for delivery of murine DNase I and addressed its potential to counteract cancer-associated pathology in the murine MMTV-PyMT model for metastatic mammary carcinoma. The AAV vector is comprised of capsid KP1 and an expression cassette encoding hyperactive murine DNase I (AAV-mDNase I) under the control of a liver-specific promotor. This AAV-mDNase I vector could support elevated expression and serum activity of murine DNase I over at least 8 months. Neutrophil Gelatinase-Associated Lipocalin (NGAL), a biomarker for kidney hypoperfusion that is upregulated in urine from MMTV-PyMT mice, was suppressed in mice receiving AAV-mDNase I compared to an AAV-null control group. Furthermore, the proportion of mice that developed lung metastasis was reduced in the AAV-mDNase I group. Altogether, our data indicate that AAV-mDNase I has the potential to reduce cancer-associated impairment of renal function and development of metastasis. We conclude that AAV-mDNase I could represent a promising therapeutic strategy in metastatic breast cancer.

Imaging Tumor-Targeting Bacteria Using 18F-Fluorodeoxysorbitol Positron Emission Tomography.

BACKGROUND: Microbial-based cancer treatments are an emerging field, with multiple bacterial species evaluated in animal models and some advancing to clinical trials. Noninvasive bacteria-specific imaging approaches can potentially support the development and clinical translation of bacteria-based cancer treatments by assessing the tumor and off-target bacterial colonization. METHODS: 18F-Fluorodeoxysorbitol (18F-FDS) positron emission tomography (PET), a bacteria-specific imaging approach, was used to visualize an attenuated strain of Yersinia enterocolitica, currently in clinical trials as a microbial-based cancer treatment, in murine models of breast cancer. RESULTS: Y. enterocolitica demonstrated excellent 18F-FDS uptake in in vitro assays. Whole-body 18F-FDS PET demonstrated a significantly higher PET signal in tumors with Y. enterocolitica colonization compared to those not colonized, in murine models utilizing direct intratumor or intravenous administration of bacteria, which were confirmed using ex vivo gamma counting. Conversely, 18F-fluorodeoxyglucose (18F-FDG) PET signal was not different in Y. enterocolitica colonized versus uncolonized tumors. CONCLUSIONS: Given that PET is widely used for the management of cancer patients, 18F-FDS PET could be utilized as a complementary approach supporting the development and clinical translation of Y. enterocolitica-based tumor-targeting bacterial therapeutics.

Vaccination against galectin-1 promotes cytotoxic T-cell infiltration in melanoma and reduces tumor burden.

Galectin-1 (Gal1) is a glycan-binding protein that promotes tumor progression by several distinct mechanisms. Through direct binding to vascular endothelial growth factor (VEGF)-receptor 2, Gal1 is able to induce VEGF-like signaling, which contributes to tumor angiogenesis. Furthermore, several studies have demonstrated an immunosuppressive function of Gal1 through effects on both effector and regulatory T cells. Elevated Gal1 expression and secretion have been shown in many tumor types, and high Gal1 serum levels have been connected to poor prognosis in cancer patients. These findings suggest that therapeutic strategies directed against Gal1 would enable simultaneous targeting of angiogenesis, immune evasion and metastasis. In the current study, we have analyzed the potential of Gal1 as a cancer vaccine target. We show that it is possible to generate high anti-Gal1 antibody levels in mice immunized with a recombinant vaccine protein consisting of bacterial sequences fused to Gal1. Growth of Gal1 expressing melanomas was significantly impaired in the immunized mice compared to the control group. This was associated with improved perfusion of the tumor vasculature, as well as increased infiltration of macrophages and cytotoxic T cells (CTLs). The level of granzyme B, mainly originating from CTLs in our model, was significantly elevated in Gal1 vaccinated mice and correlated with a decrease in tumor burden. We conclude that vaccination against Gal1 is a promising pro-immunogenic approach for cancer therapy that could potentially enhance the effect of other immunotherapeutic strategies due to its ability to promote CTL influx in tumors.

TANK-binding kinase 1 is a mediator of platelet-induced EMT in mammary carcinoma cells.

Platelets can promote several stages of the metastatic process and thus contribute to malignant progression. As an example, platelets promote invasive properties of tumor cells by induction of epithelial to mesenchymal transition (EMT). In this study, we show that tumor necrosis factor receptor-associated factor (TRAF) family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) is a previously unknown mediator of platelet-induced EMT in mammary carcinoma cells. Coculture of 2 mammary carcinoma cell lines, Ep5 from mice and MCF10A(MII) from humans, with isolated platelets induced morphologic as well as molecular changes characteristic of EMT, which was paralleled with activation of TBK1. TBK1 depletion using small interfering RNA impaired platelet-induced EMT in both Ep5 and MCF10A(MII) cells. Furthermore, platelet-induced activation of the NF-κB subunit p65 was suppressed after TBK1 knockdown, demonstrating that TBK1 mediates platelet-induced NF-κB signaling and EMT. Using an in vivo metastasis assay, we found that depletion of TBK1 from mammary carcinoma cells during in vitro preconditioning with platelets subsequently suppressed the formation of lung metastases in mice. Altogether, these results suggest that TBK1 contributes to tumor invasiveness and may be a driver of metastatic spread in breast cancer.-Zhang, Y., Unnithan, R. V. M., Hamidi, A., Caja, L., Saupe, F., Moustakas, A., Cedervall, J., Olsson, A.-K. TANK-binding kinase 1 is a mediator of platelet-induced EMT in mammary carcinoma cells.

Pharmacological targeting of peptidylarginine deiminase 4 prevents cancer-associated kidney injury in mice.

Renal insufficiency is a frequent cancer-associated problem affecting more than half of all cancer patients at the time of diagnosis. To minimize nephrotoxic effects the dosage of anticancer drugs are reduced in these patients, leading to sub-optimal treatment efficacy. Despite the severity of this cancer-associated pathology, the molecular mechanisms, as well as therapeutic options, are still largely lacking. We here show that formation of intravascular tumor-induced neutrophil extracellular traps (NETs) is a cause of kidney injury in tumor-bearing mice. Analysis of clinical biomarkers for kidney function revealed impaired creatinine clearance and elevated total protein levels in urine from tumor-bearing mice. Electron microscopy analysis of the kidneys from mice with cancer showed reversible pathological signs such as mesangial hypercellularity, while permanent damage such as fibrosis or necrosis was not observed. Removal of NETs by treatment with DNase I, or pharmacological inhibition of the enzyme peptidylarginine deiminase 4 (PAD4), was sufficient to restore renal function in mice with cancer. Tumor-induced systemic inflammation and impaired perfusion of peripheral vessels could be reverted by the PAD4 inhibitor. In conclusion, the current study identifies NETosis as a previously unknown cause of cancer-associated renal dysfunction and describes a novel promising approach to prevent renal failure in individuals with cancer.

Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

With the aim to improve the efficacy of therapeutic vaccines that target self-antigens, we have developed a novel fusion protein vaccine on the basis of the C-terminal multimerizing end of the variable lymphocyte receptor B (VLRB), the Ig equivalent in jawless fishes. Recombinant vaccines were produced in Escherichia coli by fusing the VLRB sequence to 4 different cancer-associated target molecules. The anti-self-immune response generated in mice that were vaccinated with VLRB vaccines was compared with the response in mice that received vaccines that contained bacterial thioredoxin (TRX), previously identified as an efficient carrier. The anti-self-Abs were analyzed with respect to titers, binding properties, and duration of response. VLRB-vaccinated mice displayed a 2- to 10-fold increase in anti-self-Ab titers and a substantial decrease in Abs against the foreign part of the fusion protein compared with the response in TRX-vaccinated mice (P < 0.01). VLRB-generated Ab response had duration similar to the corresponding TRX-generated Abs, but displayed a higher diversity in binding characteristics. Of importance, VLRB vaccines could sustain an immune response against several targets simultaneously. VLRB vaccines fulfill several key criteria for an efficient therapeutic vaccine that targets self-antigens as a result of its small size, its multimerizing capacity, and nonexposed foreign sequences in the fusion protein.-Saupe, F., Reichel, M., Huijbers, E. J. M., Femel, J., Markgren, P.-O., Andersson, C. E., Deindl, S., Danielson, U. H., Hellman, L. T., Olsson, A.-K. Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

Vaccines targeting self-antigens: mechanisms and efficacy-determining parameters.

We recently showed that it is possible to compromise tumor vessel function and, as a consequence, suppress growth of aggressive preclinical tumors by immunizing against the tumor vascular markers extra domain-A (ED-A) or -B (ED-B) of fibronectin, using a fusion protein consisting of the ED-A or ED-B peptide fused to bacterial thioredoxin. To address the mechanism behind fusion protein-induced immunization and the specific contribution of the different vaccine constituents to elicit an anti-self-antibody response, we immunized mice with modified or unmodified self-antigens, combined with different adjuvant components, and analyzed antibody responses by ELISA in sera. Several essential requirements to circumvent tolerance were identified: (1) a potent pattern recognition receptor agonist like an oligonucleotide containing unmethylated cytosine and guanine dinucleotides (CpG); (2) a depot adjuvant to keep the CpG at the site of injection; and (3) the presence of foreign sequences in the vaccine protein. Lack of either of these factors abolished the anti-self-response (P = 0.008). In mice genetically deficient for type I IFN signaling, there was a 60% reduction in the anti-self-response compared with wild-type (P = 0.011), demonstrating a key role of this pathway in CpG-induced circumvention of self-tolerance. Identification of these mechanistic requirements to generate a potent anti-self-immune response should significantly aid the design of efficient, specific, and safe therapeutic cancer vaccines.

Spatial organization of the tenascin-C microenvironment in experimental and human cancer.

The extracellular matrix (ECM) molecule tenascin-C (TNC) promotes tumor progression. This has recently been demonstrated in the stochastic murine RIP1-Tag2 insulinoma model, engineered to either express TNC abundantly or to be devoid of TNC. However, our knowledge about organization of the TNC microenvironment is scant. Here we determined the spatial distribution of TNC together with other ECM molecules in murine RIP1-Tag2 insulinoma and human cancer tissue (insulinoma and colorectal carcinoma). We found that TNC is organized in matrix tracks together with other ECM molecules of the AngioMatrix signature, a previously described gene expression profile that characterizes the angiogenic switch. Moreover, stromal cells including endothelial cells, fibroblasts and leukocytes were enriched in the TNC tracks. Thus, TNC tracks may provide niches for stromal cells and regulate their behavior. Given similarities of TNC rich niches for stromal cells in human insulinoma and colon cancer, we propose that the RIP1-Tag2 model may be useful for providing insights into the contribution of the tumor stroma specific ECM as promoter of cancer progression.

Tenascin-C: Exploitation and collateral damage in cancer management.

Despite an increasing knowledge about the causes of cancer, this disease is difficult to cure and still causes far too high a death rate. Based on advances in our understanding of disease pathogenesis, novel treatment concepts, including targeting the tumor microenvironment, have been developed and are being combined with established treatment regimens such as surgical removal and radiotherapy. Yet it is obvious that we need additional strategies to prevent tumor relapse and metastasis. Given its exceptional high expression in most cancers with low abundance in normal tissues, tenascin-C appears an ideal candidate for tumor treatment. Here, we will summarize the current applications of targeting tenascin-C as a treatment for different tumors, and highlight the potential of this therapeutic approach.

Therapeutic vaccination against fibronectin ED-A attenuates progression of metastatic breast cancer.

Therapeutic vaccination targeting self-molecules is an attractive alternative to monoclonal antibody-based therapies for cancer and various inflammatory diseases. However, development of cancer vaccines targeting self-molecules has proven difficult. One complicating factor is that tumor cells have developed strategies to escape recognition by the immune system. Antigens specifically expressed by the tumor vasculature can therefore provide alternative targets. The alternatively spliced extra domain-A and B (ED-A and ED-B) of fibronectin are expressed during vasculogenesis in the embryo, but essentially undetectable under normal conditions in the adult. However, these domains are re-expressed during tumor angiogenesis and matrix remodeling, which renders them highly interesting for targeted cancer therapies. Using the MMTV-PyMT transgenic model of metastatic mammary carcinoma, we show that tumor burden can be significantly decreased by immunization against ED-A in a therapeutic setting. Furthermore, we found that in mice carrying anti-ED-A antibodies the number of metastases was reduced. ED-A immunization increased infiltration of macrophages and compromised tumor blood vessel function. These findings implicate an attack of the tumor vasculature by the immune system, through a polyclonal antibody response. We conclude that tumor vascular antigens are promising candidates for development of therapeutic vaccines targeting growth of primary tumors as well as disseminated disease.

AngioMatrix, a signature of the tumor angiogenic switch-specific matrisome, correlates with poor prognosis for glioma and colorectal cancer patients.

Angiogenesis represents a rate-limiting step during tumor progression. Targeting angiogenesis is already applied in cancer treatment, yet limits of anti-angiogenic therapies have emerged, notably because tumors adapt and recur after treatment. Therefore, there is a strong need to better understand the molecular and cellular mechanisms underlying tumor angiogenesis. Using the RIP1-Tag2 transgenic murine model, we identified 298 genes that are deregulated during the angiogenic switch, revealing an ingression/expansion of specific stromal cell types including endothelial cells and pericytes, but also macrophages and perivascular mesenchymal cells. Canonical TGF-ß signaling is up-regulated during the angiogenic switch, especially in tumor-associated macrophages and fibroblasts. The matrisome, comprising extracellular matrix (ECM) and ECM-associated molecules, is significantly enriched, which allowed us to define the AngioMatrix signature as the 110 matrisomal genes induced during the RIP1-Tag2 angiogenic switch. Several AngioMatrix molecules were validated at expression level. Ablation of tenascin-C, one of the most highly induced ECM molecules during the switch, resulted in reduced angiogenesis confirming its important role. In human glioma and colorectal samples, the AngioMatrix signature correlates with the expression of endothelial cell markers, is increased with tumor progression and finally correlates with poor prognosis demonstrating its diagnostic and therapeutic potential.

Phosphonated chelates for nuclear imaging.

A series of bis-, tris- and tetra-phosphonated pyridine ligands is presented. In view of their potential use as chelates for radiopharmaceutical applications, the physico-chemical properties of the ligands and of their Co(II), Ni(II), Cu(II), and Zn(II) complexes were studied by means of potentiometry and UV-Vis absorption spectroscopy. The pKa values of the ligands and of the complexes, as well as the stability constants for the formation of the complexes, are presented. The kinetic aspects of the formation of Cu(II) complexes and of their dissociation in acidic media were studied by means of stopped flow experiments, and the stability of the Cu(II) complex toward reduction to Cu(I) was investigated by cyclic voltammetry and by titration with different reducing agents. The different thermodynamic and kinetic aspects of the polyphosphonated ligands were compared with regard to the impact of the number of phosphonic acid functions. Considering the very promising properties for complexation, preliminary SPECT/CT imaging experiments were carried out on mice with (99m)Tc using the bis- and tetra-phosphonated ligands L(2) and L(1). Finally, a bifunctional version of chelate L(1), L*, was used to label MTn12, a rat monoclonal antibody with both specificity and relatively high affinity for murine tenascin-C. The labeling was monitored by MALDI/MS spectrometry and the affinity of the labeled antibody was checked by immunostaining experiments. After chelation with (99m)Tc, the (99m)Tc-L*-MTn12 antibody was injected into a transgenic mouse with breast cancer and the biodistribution of the labeled antibody was followed by SPECT/CT imaging.

Tenascin-C downregulates wnt inhibitor dickkopf-1, promoting tumorigenesis in a neuroendocrine tumor model.

The extracellular matrix molecule tenascin-C (TNC) is a major component of the cancer-specific matrix, and high TNC expression is linked to poor prognosis in several cancers. To provide a comprehensive understanding of TNC's functions in cancer, we established an immune-competent transgenic mouse model of pancreatic ß-cell carcinogenesis with varying levels of TNC expression and compared stochastic neuroendocrine tumor formation in abundance or absence of TNC. We show that TNC promotes tumor cell survival, the angiogenic switch, more and leaky vessels, carcinoma progression, and lung micrometastasis. TNC downregulates Dickkopf-1 (DKK1) promoter activity through the blocking of actin stress fiber formation, activates Wnt signaling, and induces Wnt target genes in tumor and endothelial cells. Our results implicate DKK1 downregulation as an important mechanism underlying TNC-enhanced tumor progression through the provision of a proangiogenic tumor microenvironment.

Synthesis of an activated phosphonated bifunctional chelate with potential for PET imaging and radiotherapy.

The synthesis of a phosphonated acyclic bifunctional chelate L* for the labeling of biomaterial is described. L* is based on a pyridine backbone, functionalized in ortho positions by aminomethyl-bis-methylphosphonic acids, and, in the para position, by a side chain containing a reactive NHS carbamate function. The stability of L* in aqueous solutions at different pH values was studied by mass spectrometry, showing the activated function to be sensitive to hydrolysis above neutral pH. The reactivity of L* towards amine functions was tested using ethylamine under different conditions of pH and concentrations, and by the labeling of two reference peptides containing both an N-terminal amino function and a ε-amino group of a lysine residue in the backbone, and a supplementary thiol group of a cysteine residue for one of these two peptides. The results showed the coupling to be efficient at pH 8.0, with a total selectivity for the terminal amine function with respect to lysine and cysteine. The labeling was further performed on B28-13, a mouse monoclonal antibody specifically recognizing tenascin-C protein in human cancer. The labeled antibody was characterized by means of mass spectrometry and spectrofluorimetry, unraveling a labeling ratio of one chelate per antibody. Finally, the affinity of the labeled antibody towards its target was controlled by immunofluorescence staining experiments on human colon cancer biopsies, confirming the affinity of the labeled peptide for tenascin-C.

Fibronectin and tenascin-C: accomplices in vascular morphogenesis during development and tumor growth.

In addition to soluble factors, the extracellular matrix (ECM) also plays a vital role in normal vasculogenesis and in the pathological angiogenesis of many disease states. Here we will review what is known about the role of the ECM molecules fibronectin and tenascin-C in the vasculature and highlight a potential collaborative interplay between these molecules in developmental and tumorigenic angiogenesis. We will address the evolution of these modular proteins, their cellular interactions and how they become assembled into an insoluble matrix that impacts the assembly of other ECM proteins and the bioavailability of pro-angiogenic factors. The role of fibronectin and tenascin-C networks in tumor angiogenesis and metastasis will be described. We will elaborate on lessons learned about their role in vessel function from the functional ablation or the ectopic expression of both molecules. We will also elaborate on potential mechanisms of how fibronectin and tenascin-C affect cell adhesion and signaling that are relevant to angiogenesis.

Combined lysophosphatidic acid/platelet-derived growth factor signaling triggers glioma cell migration in a tenascin-C microenvironment.

The antiadhesive extracellular matrix molecule tenascin-C abrogates cell spreading on fibronectin through competitive inhibition of syndecan-4, thereby preventing focal adhesion kinase (FAK) activation and triggering enhanced proteolytic degradation of both RhoA and tropomyosin 1 (TM1). Here, we show that simultaneous signaling by lysophosphatidic acid (LPA) and platelet-derived growth factor (PDGF) initiates glioma cell spreading and migration through syndecan-4-independent activation of paxillin and FAK and by stabilizing expression of RhoA, TM1, TM2, and TM3. By using gene silencing methods, we show that paxillin, TM1, TM2, and TM3 are essential for LPA/PDGF-induced cell spreading on a fibronectin/tenascin-C (FN/TN) substratum. LPA/PDGF-induced cell spreading and migration on FN/TN depends on phosphatidylinositol 3-kinase, RhoKinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 but is independent of phospholipase C and Jun kinase. RNA microarray data reveal expression of tenascin-C, PDGFs, LPA, and the respective receptors in several types of cancer, suggesting that the TN/LPA/PDGF axis exists in malignant tumors. These findings may in turn be relevant for diagnostic or therapeutic applications targeting cancer.