Keywords

1 Detection of Periostin with Specific Antibodies

Periostin has been proposed as a biomarker for several diseases including tumor , fibrosis, inflammation and allergy [14, 16, 17, 23, 60, 70, 81, 85]. Elevated expressions of periostin were initially observed in microarray gene expression analyses [5, 22, 29, 33, 51, 82, 90]. Although gene expression of periostin could be evaluated with microarray or quantitative PCR with cDNA samples, these methods require total mRNA of the disordered tissues from biopsy samples of human patients . Biopsy is an invasive procedure that increases the burden on patients. Therefore, an alternative method to detect expression of periostin with minimal invasiveness on patients has been needed. As an alternative method, great attention has been paid to periostin in peripheral blood of patients with diseases [13, 24, 47, 48]. Periostin is a secretory protein mainly derived from fibroblastic cells and interacts with several extracellular matrix proteins such as fibronectin , tenascin-C , and collagens [31, 36]. On the other hands, a secreted periostin is transported into circulating blood through blood vessel, which would become permeabilized due to disease progression, and circulates in cardiovascular systems (Fig. 18.1). For example, neovessels formed in tumor tissue are permeable, and are easy to pass relatively large molecules including antibodies [45, 77, 91]. This permeabilization of blood vessels in tumor tissues is recognized as the enhanced permeability and retention (EPR) effect, by which molecules of appropriate sizes, such as liposomes, nanoparticles, and macromolecular drugs, accumulate in tumor tissue more than in normal tissues. Neovessels formed in inflamed tissue are also permeable. Thus, the circulating periostin in peripheral blood indicates existence of disordered tissue such as tumor microenvironment , fibrosis, and inflammation . The highly sensitive detection and quantification method of circulating periostin in peripheral blood of human patients has been evaluated as a minimally invasive procedure for disease diagnosis .

Fig. 18.1
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Expression and secretion of periostin into peripheral blood

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To utilize periostin as a biomarker for diseases, specific detection methods are required. Antibody has usually been used to specifically detect periostin in tissue thin section, blood, and whole body. In the earliest stage of periostin research, Kudo and colleagues developed rabbit polyclonal antibodies against the first FAS 1 domain (anti-RD1) and the carboxyl-terminal end of the CTD (anti-CT) [20, 80], and have revealed the spatiotemporal distribution of periostin in mouse and human tissues. Thereafter, a lot of polyclonal or monoclonal antibodies against periostin have been developed, and some of which are now commercially available. These antibodies against periostin were used to investigate periostin localization. Immunohistochemical analyses using these antibodies demonstrated that periostin physiologically localizes at collagen-dense areas in connective tissue, including the periodontal ligament [20, 30, 71, 84], periosteum [20, 71], cardiac valve [18, 57, 58], and alveolar wall in the lung [7, 35, 62]. Periostin has also been found to pathologically localize in infarcted myocardium [61, 80], fibrosis [22, 49, 62, 86, 92], the wound healing process [54, 56, 63, 98], and cancer-associated stroma [15, 28, 32, 33, 43, 55, 65, 68, 73, 83, 89, 94, 96]. Thus, expression of periostin is also closely associated with tissue regeneration post-injury [10]. In addition, function-blocking antibodies against periostin have been developed as rabbit polyclonal antibodies, PN1-Ab, PN21-Ab, PnAb, and mouse monoclonal antibody, MZ-1, OC-20, which were utilized in mouse disease models such as tumor growth and metastasis [39, 50, 52, 64, 88, 99]. These specific antibodies against periostin contribute to highly sensitive detection and quantification of periostin as a biomarker for disease progression.

Periostin-binding DNA aptamer has also been developed [40, 53, 93]. Nucleic acid-based aptamers comprise an emerging class of targeted therapeutic and diagnostic molecules [21, 69]. Aptamers are single-stranded DNAs or RNAs that are designed to bind to proteins with similar or better affinity and specificity, compared with antibodies or small molecules. Periostin-binding aptamers would also be useful in detection of periostin.

2 Detection of Periostin in Peripheral Blood

To detect and quantify the circulating periostin in peripheral blood samples, the sandwich enzyme-linked immunosorbent assay (ELISA) using antibodies against periostin has been developed [9]. An antibody against periostin is immobilized on wells of microtiter plate, which captures periostin protein in blood samples. The captured periostin is further labeled with the other antibody against periostin, which binds to the antigen distinct from the other one recognized by the antibody coated on the wells. The antibody on periostin was then recognized with an enzyme-conjugated secondary antibody. The captured periostin protein could be detected with the enzymatic activity as an output. As the enzymes conjugated on secondary antibody, alkaline phosphatase, horseradish peroxidase, and luciferase are utilized. Thus, ELISA enables highly sensitive and specific detection of periostin in peripheral blood samples.

At the beginning of researches on evaluation of periostin in peripheral blood samples, Ben et al . [6] developed ELISA using commercially available antibodies against periostin and measured serum periostin levels in peripheral bloods of human patients with cancers. The authors demonstrated that concentration of periostin in blood samples of patients with colorectal cancer was significantly higher than those of healthy volunteers and of patients with benign colorectal polyps or adenomas. The authors also showed that cancer cells were negative for periostin and their surrounding stromal tissues were positive, indicating that the circulating periostin in peripheral bloods is derived from the surrounding cancer stromal tissues. These results suggest that serum levels of periostin detected by ELISA are of clinical value in identifying patients who may be at a high risk for malignancy of colorectal cancer.

Yamaguchi et al . [97] also evaluated serum periostin levels in peripheral blood samples from patients with progressive skin sclerosis. Skin sclerosis is one of the symptoms of systemic sclerosis that is an autoimmune disorder. Autoimmune reaction causes tissue destruction, resulting in proliferation of activated fibroblasts and accumulation of collagenous extracellular matrix proteins under the skin. Periostin was strongly expressed in the affected dermis biopsy samples from patients with systemic sclerosis. Serum levels of periostin in patients with systemic sclerosis were significantly elevated compared with healthy subjects, indicating that periostin secreted in the affected dermis is transported to blood vessels and circulated in peripheral bloods. This report suggests that an elevated periostin level in patients with systemic sclerosis is associated with severity of skin sclerosis, and that periostin is a potential biomarker for progressive skin fibrosis.

Jia et al . [27] also demonstrated that periostin concentrations in peripheral blood samples from patients with asthma were significantly higher than those from healthy subjects. Asthma is a condition in which airway narrow and swell produce extra mucus, which makes breathing difficulty and trigger coughing, wheezing and shortness of breath. In a histological view of asthmatic airway, as a result from inflammation and remodeling processes, activated fibroblasts proliferate and deposit collagenous extracellular matrix , causing thickening and increasing density of the basement membrane. In this study, the authors examined several biomarker candidates in peripheral blood samples from patients with severe asthma , and concluded that the serum periostin level was the single best predictor of airway eosinophilia. Other reports also demonstrated periostin as a biomarker for asthma [47]. Thus, periostin is a systemic biomarker of airway eosinophilia in asthmatic patients and has potential utility in patient selection for asthma therapeutics.

In addition to the reports described above, elevated expression of periostin mRNA has been demonstrated in mice with the transient middle cerebral artery occlusion model that is similar to human cerebral ischemia [78, 79]. The elevated expression of periostin promotes neural stem cell proliferation and differentiation , which would contribute to regeneration of brain injury [44]. Furthermore, serum periostin concentrations were significantly increased in peripheral blood samples from patients with traumatic brain injury, compared with those from healthy controls [12]. These reports suggest that brain injury induces expression of periostin in the traumatic region, and that secreted periostin is transported into blood vessels and then circulates in the cardiovascular system [25, 85].

In analogy to these initially reported ELISA experiments , elevated periostin levels in peripheral blood samples from patients with diseases such as cancer, fibrosis, inflammation , allergy , and ischemia have been reported. Diseases with increased periostin expression in disordered tissues should be intended for periostin liquid biopsy as peripheral blood samples with minimal invasiveness, which is useful for patient selection in therapeutics.

Although periostin liquid biopsy is an alternative way having minimal invasiveness and informs us existence of the disordered tissues such as cancer, fibrosis, inflammation and allergy , it is impossible to visualize the disordered tissues. To overcome this problem, a molecular imaging strategy targeting periostin-positive disordered tissues has been examined.

3 Molecular Imaging Targeting Periostin in Living Animals

Antibody can be utilized in diagnosis with molecular imaging techniques such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) (Fig. 18.2). PET is a nuclear medicine functional imaging method used to visualize localization of diagnosis probe molecules with radionuclide that emits positron. Positron-emitting radionuclides such as 11C (carbon-11), 18F (fluoride-18), and 64Cu (copper-64), are produced by nuclear reactions in cyclotron, in which hydrogen ions (proton) are accelerated under a high voltage in the magnetic field. These radionuclides are conjugated with probe molecules by chemical reactions. PET system detects pairs of gamma rays emitted indirectly by a positron-emitting radionuclide, which is introduced into a biologically active molecule called a radioactive tracer. For example, small molecule tracers such as 18F-fluorodeoxyglucose (FDG) that is a glucose analogue, have been frequently utilized as diagnosis in clinical oncology for staging, restaging and evaluation of tumor response to treatment, because cancer cells rather than non-malignant cells uptake a lot of glucose as an energy source to proliferate (O’Connor et al. [59]). On the other hand, SPECT is able to visualize localization of radiolabeled diagnosis probe molecules in human patients, which detects gamma ray from radionuclide conjugated on the probe molecule. As gamma-emitting radionuclides, 99mTc (technetium-99m), 123I (iodine-123), 131I (iodine-131) are utilized. These radionuclides themselves could also be used to scan bone, myocardial perfusion, brain, and tumor.

Fig. 18.2
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Immuno-PET imaging

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In case of antibody , the small molecule metal chelator, such as DOTA, NOTA, and NODAGA, is covalently conjugated to antibody, with amine coupling reaction for example, and radionuclide metal ion such as 64Cu produced in cyclotron is then introduced to the chelators conjugated on antibody [72]. Radionuclide-labeled antibody is administrated into patients with disease such as cancer, who is thereafter scanned with PET camera and CT (computed tomography). For example, PET scan of patients with aggressive breast cancer were performed with 64Cu-labeled antibody against HER2 (epidermal growth factor receptor 2) (64Cu-trastuzumab) [37, 38, 74, 87]. 64Cu-trastuzumab was administrated to patients with metastatic HER2-positive breast cancer, and scanned with medical PET/CT camera, clearly visualizing its accumulation not only in the primary lesion of tumors but also in brain metastasis. Thus, PET imaging technique with radiolabeled antibody (Immuno-PET) would be useful for diagnosis of diseases [46].

Heidari et al . [19] have utilized an antibody against periostin in PET imaging with mice bearing the esophageal squamous cell carcinoma cell line expressing periostin. The authors used a commercially available anti-periostin monoclonal antibody, and prepared a F(ab’)2 fragment by enzymatic digestion. The F(ab’)2 fragment was then labeled with DOTA chelator and subsequently with 64Cu. PET imaging clearly showed specific accumulation of the radiolabeled antibody to tumor tissue derived from the inoculated cell line. Although this study demonstrated that periostin immuno-PET imaging is a powerful method to visualize periostin-positive tissues, it remains elusive whether periostin immuno-PET imaging could visualize the fibrotic region of dense connective tissues in tumor microenvironment , fibrosis, inflammation and allergy . Further studies on periostin immuno-PET imaging are required in order to visualize periostin-related diseases.

4 Clinical Imaging of Tenascin-C and Extracellular Matrix

Tenascin-C , which is one of the interacting proteins with periostin, has been targeted in PET or SPECT imaging studies [1, 3, 4, 34, 66, 67, 75, 76]. Akabani et al . [2] demonstrated that 131I-labeled anti-tenascin 81C6 murine monoclonal antibody accumulated lesion of patients with malignant gliomas in MRI/SPECT imaging. Jacobson et al . [26] developed a radiolabeled single-stranded DNA aptamer that targets tenascin-C and utilized it in PET imaging of mice bearing tenascin-C-positive tumor . Thus, molecular imaging techniques could target extracellular matrix proteins accumulated in the disordered tissues. It should be possible to visualize periostin in the extracellular matrix and matricellular space of the disordered tissues such as tumor microenvironment .

In addition to periostin and tenascin-C , the other extracellular matrix proteins and related transmembrane proteins have also been targeted in molecular imaging. Radiolabeled probes that recognize fibrin, fibronectin , collagens, MMPs, and integrins have been developed to visualize fibrosis and fibrogenesis [11]. These molecular probes including antibodies against periostin could be useful not only for diagnosis but also for the drug delivery system, which enables targeted delivery of drug only to the disordered tissues and reduces the adverse side effect of drug. For example, tumor microenvironment , which is rich in stromal fibroblasts and extracellular matrix proteins, is one of the crucial factors that cause tumor drug-resistance [8, 41, 42, 95]. Remodeling of tumor microenvironment by the drug delivery system targeting the extracellular matrix or transmembrane proteins described above would improve anti-tumor efficacy in drug treatment. Thus, targeting the fibrotic lesion in the disordered tissues is of importance in therapeutics as well as diagnosis .

5 Conclusion

Periostin should be a promising biomarker for diseases such as tumor, fibrosis, inflammation , and allergy . The advantage of periostin is to detect and quantify it in peripheral blood samples of human patients. This liquid biopsy could be performed with minimal invasiveness, which does not burden patients. Periostin in peripheral blood indicates existence of the disordered tissues, in which abnormally activated fibroblastic cells highly express and secret periostin that is transported into circulating blood through blood vessels. Basic researches on periostin in peripheral bloods have utilized ELISA in detection, whereas rapid detection methods, such as the quantitative immuno-chromatography, are required to expand its utility in routine clinical practice.

Antibodies against periostin could be also utilized in clinical immuno-PET diagnosis . Specific and high affinity antibodies against periostin have been developed and utilized for immunological detection, functional blocking, and molecular imaging. Immuno-PET diagnosis could visualize distribution of the disordered tissues in living patients with minimally invasiveness, which contributes to accurate operative treatment as well as evaluation of outcome from therapy. Because clinical molecular imaging such as immuno-PET is an emerging method, periostin in the disordered tissues would be targeted as a useful biomarker for disease diagnosis .