Yongbaek Kim

Intraoperative detection and removal of microscopic residual sarcoma using wide-field imaging

The goal of limb‐sparing surgery for a soft tissue sarcoma of the extremity is to remove all malignant cells while preserving limb function. After initial surgery, microscopic residual disease in the tumor bed will cause a local recurrence in approximately 33% of patients with sarcoma. To help identify these patients, the authors developed an in vivo imaging system to investigate the suitability of molecular imaging for intraoperative visualization.

p53 Functions in Endothelial Cells to Prevent Radiation-Induced Myocardial Injury in Mice

Without the transcription factor p53 in cardiac endothelial cells, the heart is more susceptible to radiation-induced damage. Keeping the Heart Safe from Radiation The cells that make up the muscular part of the heart, or the myocardium, are generally not actively dividing. Therefore, one would not expect radiation therapy aimed at eliminating rapidly dividing cancer cells to cause heart disease. However, radiation-related heart disease is a clinically important long-term side effect of radiation therapy. The transcription factor p53 is activated in response to radiation and other DNA-damaging stresses; whether it promotes or attenuates radiation-related heart disease is unclear. Lee et al. generated mice lacking p53 in endothelial cells, the cells that line blood vessels, and whole-heart irradiation in these mice resulted in damage to the vasculature of the heart, triggering ischemia (disrupted blood flow) in the myocardium and eventual heart failure. p53 inhibitors that protect normal cells from the effects of radiation therapy have been proposed as an approach to improve the therapeutic ratio of radiation therapy, but these data suggest that combining radiation therapy with p53 inhibitors may actually increase the risk of cardiac injury. Radiation therapy, which is used for the treatment of some cancers, can cause delayed heart damage. In the heart, p53 influences myocardial injury that occurs after multiple types of stress. Here, we demonstrated that p53 functioned in endothelial cells to protect mice from myocardial injury after whole-heart irradiation. Mice with an endothelial cell–specific deletion of p53 succumbed to heart failure after whole-heart irradiation as a result of myocardial necrosis, systolic dysfunction, and cardiac hypertrophy. Moreover, the onset of cardiac dysfunction was preceded by alterations in myocardial vascular permeability and density, which resulted in cardiac ischemia and myocardial hypoxia. Mechanistic studies with primary cardiac endothelial cells irradiated in vitro indicated that p53 signaling caused mitotic arrest and protected cardiac endothelial cells from cell death resulting from abnormal mitosis or mitotic catastrophe. Furthermore, mice lacking the cyclin-dependent kinase inhibitor p21, which is a transcriptional target of p53, were also sensitized to myocardial injury after whole-heart irradiation. Together, our results demonstrate that the p53-p21 axis functions to prevent radiation-induced myocardial injury in mice.

Hydrogen peroxide promotes epithelial to mesenchymal transition and stemness in human malignant mesothelioma cells.

Reactive oxygen species (ROS) are known to promote mesothelial carcinogenesis that is closely associated with asbestos fibers and inflammation. Epithelial to mesenchymal cell transition (EMT) is an important process involved in the progression of tumors, providing cancer cells with aggressiveness. The present study was performed to determine if EMT is induced by H2O2 in human malignant mesothelioma (HMM) cells. Cultured HMM cells were treated with H2O2, followed by measuring expression levels of EMT-related genes and proteins. Immunohistochemically, TWIST1 expression was confined to sarcomatous cells in HMM tissues, but not in epithelioid cells. Treatment of HMM cells with H2O2 promoted EMT, as indicated by increased expression levels of vimentin, SLUG and TWIST1, and decreased E-cadherin expression. Expression of stemness genes such as OCT4, SOX2 and NANOG was also significantly increased by treatment of HMM cells with H2O2. Alteration of these genes was mediated via activation of hypoxia inducible factor 1 alpha (HIF-1α) and transforming growth factor beta 1 (TGF-β1). Considering that treatment with H2O2 results in excess ROS, the present study suggests that oxidative stress may play a critical role in HMM carcinogenesis by promoting EMT processes and enhancing the expression of stemness genes.

Identification and characterization of microRNAs in normal equine tissues by Next Generation Sequencing.

The role of microRNAs (miRNAs) as a post-transcriptional gene regulator has been elucidated in a broad range of organisms including domestic animals. Characterization of miRNAs in normal tissues is an important step to investigate the functions of miRNAs in various physiological and pathological conditions. Using Illumina Next Generation Sequencing (NGS) technology, we identified a total of 292 known and 329 novel miRNAs in normal horse tissues including skeletal muscle, colon and liver. Distinct sets of miRNAs were differentially expressed in a tissue-specific manner. The miRNA genes were distributed across all the chromosomes except chromosomes 29 and 31 in the horse reference genome. In some chromosomes, multiple miRNAs were clustered and considered to be polycistronic transcript. A base composition analysis showed that equine miRNAs had a higher frequency of A+U than G+C. Furthermore, U tended to be more frequent at the 5′ end of miRNA sequences. This is the first experimental study that identifies and characterizes the global miRNA expression profile in normal horse tissues. The present study enriches the horse miRNA database and provides useful information for further research dissecting biological functions of miRNAs in horse.

Assessing the Radiation Response of Lung Cancer with Different Gene Mutations Using Genetically Engineered Mice

Purpose: Non-small cell lung cancers (NSCLC) are a heterogeneous group of carcinomas harboring a variety of different gene mutations. We have utilized two distinct genetically engineered mouse models of human NSCLC (adenocarcinoma) to investigate how genetic factors within tumor parenchymal cells influence the in vivo tumor growth delay after one or two fractions of radiation therapy (RT). Materials and Methods: Primary lung adenocarcinomas were generated in vivo in mice by intranasal delivery of an adenovirus expressing Cre-recombinase. Lung cancers expressed oncogenic KrasG12D and were also deficient in one of two tumor suppressor genes: p53 or Ink4a/ARF. Mice received no radiation treatment or whole lung irradiation in a single fraction (11.6 Gy) or in two 7.3 Gy fractions (14.6 Gy total) separated by 24 h. In each case, the biologically effective dose (BED) equaled 25 Gy10. Response to RT was assessed by micro-CT 2 weeks after treatment. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemical staining were performed to assess the integrity of the p53 pathway, the G1 cell-cycle checkpoint, and apoptosis. Results: Tumor growth rates prior to RT were similar for the two genetic variants of lung adenocarcinoma. Lung cancers with wild-type (WT) p53 (LSL-Kras; Ink4a/ARFFL/FL mice) responded better to two daily fractions of 7.3 Gy compared to a single fraction of 11.6 Gy (P = 0.002). There was no statistically significant difference in the response of lung cancers deficient in p53 (LSL-Kras; p53FL/FL mice) to a single fraction (11.6 Gy) compared to 7.3 Gy × 2 (P = 0.23). Expression of the p53 target genes p21 and PUMA were higher and bromodeoxyuridine uptake was lower after RT in tumors with WT p53. Conclusion: Using an in vivo model of malignant lung cancer in mice, we demonstrate that the response of primary lung cancers to one or two fractions of RT can be influenced by specific gene mutations.

Latency Locus Complements MicroRNA 155 Deficiency In Vivo

ABSTRACT MicroRNA-155 (miR-155) is expressed in many cancers. It also executes evolutionary conserved functions in normal B cell development. We show that the Kaposis sarcoma-associated herpesvirus (KSHV) latency locus, which contains an ortholog of miR-155, miR-K12-11, complements B cell deficiencies in miR-155 knockout mice. Germinal center (GC) formation was rescued in spleen, lymph node, and Peyers patches. Immunoglobulin levels were restored. This demonstrates that KSHV can complement the normal, physiological function of miR-155.

Imaging primary mouse sarcomas after radiation therapy using cathepsin-activatable fluorescent imaging agents

PURPOSE Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. METHODS AND MATERIALS A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. RESULTS RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. CONCLUSIONS In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.

Skp2 Deficiency Inhibits Chemical Skin Tumorigenesis Independent of p27Kip1 Accumulation

S-phase kinase-associated protein 2 (Skp2) functions as the receptor component of the Skp-Cullin-F-box complex and is implicated in the degradation of several cell cycle regulators, such as p21(Cip1), p27(Kip1), p57(Kip2), and cyclin E. Numerous studies in human and experimental tumors have demonstrated low p27(Kip1) levels and elevated Skp2 expression. However, a direct association between the inverse correlation of Skp2 and p27(Kip1) with tumorigenesis has not been demonstrated. Herein, we provide evidence that skin tumorigenesis is inhibited in Skp2(-/-) mice. An analysis of mouse keratinocytes indicates that increased p27(Kip1) levels in Skp2(-/-) epidermis cause reduced cell proliferation that is alleviated in the epidermis from Skp2(-/-)/p27(-/-) compound mice. In contrast, we establish that a p27(Kip1) deficiency does not overturn the reduced skin tumorigenesis experienced by Skp2(-/-) mice. In addition, Skp2(-/-) epidermis exhibits an accumulation of p53-cofactor CBP/p300 that is associated with elevated apoptosis in hair follicles and decreased skin tumorigenesis. We conclude that p27(Kip1) accumulation is responsible for the hypoplasia observed in normal tissues of Skp2(-/-) mice but does not have a preponderant function in reducing skin tumorigenesis.

Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins

Purpose To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features. Materials and Methods Tissue excised from a genetically engineered mouse model of sarcoma was imaged using a subcellular resolution microendoscope after topical application of a fluorescent anatomical contrast agent: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma. Results Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach. Conclusion The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

Disseminated visceral coccidiosis and cloacal cryptosporidiosis in a Japanese white-naped crane (Grus vipio).

A 4-mo-old male Japanese white-naped crane (Grus vipio) kept in an outdoor exhibit at the Everland Zoological Gardens in Korea became depressed and developed anorexia, weight loss, and diarrhea. Death of this bird was associated with an overwhelming systemic infection by an intracellular coccidian parasite, which resulted in necrosis and granulomatous inflammation in a number of major organs, including the intestine, liver, spleen, and kidney. Coccidian parasite–laden macrophages were commonly found in the blood vessels of these organs. Using electron microscopy and polymerase chain reaction assays, the parasite was identified as Eimeria sp. The bird was also infected with Cryptosporidium sp., which suggests an immunosuppressed state, although the cause of such suppression was not identified. Our findings suggest that an initial Eimeria sp. intestinal infection spread to other organs through the blood vessels, with the immunosuppressed state possibly contributing to a rapid hematogenous transmission. To our knowledge, this is the first report of disseminated visceral coccidiosis caused by Eimeria sp. in a captive Japanese white-naped crane.