The selective Cox-2 inhibitor Celecoxib suppresses angiogenesis and growth of secondary bone tumors: an intravital microscopy study in mice

BACKGROUND: The inhibition of angiogenesis is a promising strategy for the treatment of malignant primary and secondary tumors in addition to established therapies such as surgery, chemotherapy, and radiation. There is strong experimental evidence in primary tumors that Cyclooxygenase-2 (Cox-2) inhibition is a potent mechanism to reduce angiogenesis. For bone metastases which occur in up to 85% of the most frequent malignant primary tumors, the effects of Cox-2 inhibition on angiogenesis and tumor growth remain still unclear. Therefore, the aim of this study was to investigate the effects of Celecoxib, a selective Cox-2 inhibitor, on angiogenesis, microcirculation and growth of secondary bone tumors. METHODS: In 10 male severe combined immunodeficient (SCID) mice, pieces of A549 lung carcinomas were implanted into a newly developed cranial window preparation where the calvaria serves as the site for orthotopic implantation of the tumors. From day 8 after tumor implantation, five animals (Celecoxib) were treated daily with Celecoxib (30 mg/kg body weight, s.c.), and five animals (Control) with the equivalent amount of the CMC-based vehicle. Angiogenesis, microcirculation, and growth of A549 tumors were analyzed by means of intravital microscopy. Apoptosis was quantified using the TUNEL assay. RESULTS: Treatment with Celecoxib reduced both microvessel density and tumor growth. TUNEL reaction showed an increase in apoptotic cell death of tumor cells after treatment with Celecoxib as compared to Controls. CONCLUSION: Celecoxib is a potent inhibitor of tumor growth of secondary bone tumors in vivo which can be explained by its anti-angiogenic and pro-apoptotic effects. The results indicate that a combination of established therapy regimes with Cox-2 inhibition represents a possible application for the treatment of bone metastases.

[Pulmonary microscopic tumor embolism syndrome]

HISTORY: A 76-year-old woman and a 62-year-old man were both referred to our clinic because of an unexplained weight loss, increasing dry cough and shortness of breath. INVESTIGATIONS: Investigations revealed an adenocarcinoma of the colon with retroperitoneal, mediastinal and supraclavicular lymph node metastasis and poorly differentiated carcinoma of the prostate with extensive bone metastases. During their hospital stay both patients developed increasing shortness of breath and clinical signs of right heart failure. Echocardiography confirmed severe pulmonary hypertension and dilatation of the right ventricle in both patients. Despite the high degree of clinical suspicion CT scans of the thorax could not demonstrate pulmonary embolism. DIAGNOSIS, TREATMENT AND COURSE: During the following days the patients condition deteriorated further and both patients' died from irreversible right heart failure. Both autopsies showed extensive metastatic adenocarcinoma with marked angiosis carcinomatosa of the lungs with numerous occlusions of small arteries and arterioles and resulting cor pulmonale. Thrombotic pulmonary embolism could not be detected. CONCLUSION: In patients with malignant neoplasms, especially adenocarcinomas, dyspnea and signs of increasing pulmonary artery pressure, the possibility of a microscopic pulmonary tumor embolism should be considered after exclusion of more usual causes especially thrombotic pulmonary embolism. In selected cases a cytologic examination of blood aspirated from a wedged pulmonary artery catheter can be performed to prove angiosis is carcinomatosa.

[Follow-up in a boy with Leydig cell tumor after selective surgery]

A 8(6/12) year-old-boy presented with precocious puberty and a slightly enlarged left testis. After a detailed examination a Leydig cell tumour was diagnosed. Surgical exploration revealed an encapsulated tumour, 2.7 cm in length, which was selectively removed without orchidectomy. Within one year the clinical signs of pubertal precocity disappeared, the bone age did not further advance and height velocity declined from 8.2 cm / year (+3.9 SDS) to 4.1 cm/year (-1.0 SDS). Physiologically, he entered puberty at the chronological age of twelve years, presenting at that age, in comparison to his peer group, a slightly decreased pubertal growth spurt. However, bearing in mind that being precocious in puberty he started in fact his pubertal growth spurt at a far earlier age, therefore, this acceleration of height before operation has to be added to the centimetres gained during pubertal development thereafter resuiting consequently in an absolute normal pubertal growth spurt. This underlines the fact that the individual growth spurt and, therefore, the total amount of centimetres gained is very much robust. Ten years later, the patient ended up well within his familial target height and remained free of disease. We report on a long-term follow-up of a prepubertal boy after testis-sparing surgery for Leydig-cell-tumour.

Ectopic growth hormone-releasing hormone secretion by a metastatic bronchial carcinoid tumor: a case with a non hypophysial intracranial tumor that shrank during long acting octreotide treatment

Ectopic acromegaly represents less than 1% of the reported cases of acromegaly. Although clinical improvement is common after treatment with somatostatin (SMS) analogs, the biochemical response and tumor size of the growth hormone-releasing hormone (GHRH)-producing tumor and its metastases are less predictable. Subject A 36-year-old male was referred because of a 3-year history of acromegaly related symptoms. He had undergone lung surgery in 1987 for a "benign" carcinoid tumor. Endocrine evaluation confirmed acromegaly Plasma IGF-1: 984 ng/ml (63-380), GH: 49.8 ng/ml (<5). MRI showed a large mass in the left cerebellopontine angle and diffuse pituitary hyperplasia. Pulmonary, liver and bone metastases were shown by chest and abdominal CT scans. Ectopic GHRH secretion was suspected. Methods Measurement of circulating GHRH levels by fluorescence immunoassay levels and immunohistochemical study of the primary lung tumor and metastatic tissue with anti-GHRH and anti-somatostatin receptor type 2 (sst2A) antibodies. Results Basal plasma GHRH: 4654 pg/ml (<100). Pathological study of liver and bone biopsy material and lung tissue removed 19 years earlier was consistent with an atypical carcinoid producing GHRH and exhibiting sst2A receptor expression. Treatment with octreotide LAR 20-40 mg q. month resulted in normalization of plasma IGF-1 levels. Circulating GHRH levels decreased dramatically. The size of the left prepontine cistern mass, with SMS receptors shown by a radiolabeled pentetreotide scan, decreased by 80% after 18 months of therapy. Total regression of pituitary enlargement was also observed. No changes were observed in lung and liver metastases. After 24 months of therapy the patient is asymptomatic and living a full and active life.

Reconstruction of Skull Base and Fronto-orbital Defects following Tumor Resection

Reconstruction of the anterior skull base and fronto-orbital framework following extensive tumor resection is both challenging and controversial. Dural defects are covered with multiple sheets of fascia lata that provide sufficient support and avoid herniation. Plating along the skull base is contraindicated. After resection of orbital walls, grafting is necessary if the periosteum or parts of the periorbital tissue had to be removed, to avoid enophthalmus or strabism. Free bone grafts exposed to the sinonasal or pharyngeal cavity are vulnerable to infection or necrosis: therefore, covering the grafts with vascularized tissue, such as the Bichat fat-pad or pedicled temporalis flaps, should reduce these complications. Alloplastic materials are indispensable in cranial defects, whereas microsurgical free tissue transfer is indicated in cases of orbital exenteration and skin defects. The authors review their experience and follow-up of 122 skull base reconstructions following extensive subcranial tumor resection. Most significant complications were pneumocranium in 4.9%, CSF leaks in 3.2%, and partial bone resorption in 8.1%.

Regulation of Id1 expression by SRC: implications for targeting of the bone morphogenetic protein pathway in cancer

Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Src-blocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Src-responsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)-induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1.

Evaluation of 15 mandibular reconstructions with Dumbach Titan Mesh-System and particulate cancellous bone and marrow harvested from bilateral posterior ilia

This study reports on 15 mandibular reconstructions using the Dumbach Titan Mesh-System and particulate cancellous bone and marrow harvested from bilateral posterior ilia. All cases showed segmental defects. Eleven cases involved patients with malignant tumor. Six patients had received irradiation of 40-50 Gy. Reconstructions were performed immediately in 1 patient and secondarily in the remaining 14 patients. In 13 cases, mandibles were successfully reconstructed. Of these 13 patients, 9 reconstructions were completed without complications, whereas the other 4 cases showed complications. In 2 cases, reconstruction failed completely. Overall success rate was 87%. Statistical analysis revealed the extent of mandibular defect, but not malignancy of the original disease or radiotherapy of

Cholesteatoma triggering squamous cell carcinoma: case report and literature review of a rare tumor

BACKGROUND: The aim of this study was to report a case of squamous cell carcinoma of the petrous part of the temporal bone associated with a long history of secondary acquired cholesteatoma in a 71-year-old man. PATIENTS AND METHODS: We present the case of a 71-year-old man diagnosed with secondary acquired cholesteatoma in 1950. Treatments consisted of repetitive surgery owing to several relapses. In 2004, he presented with progressive fetid otorrhea. Clinical and computed tomography findings were indicative for relapsing cholesteatoma and a subtotal petrosectomy was performed. RESULTS: Histologic work-up demonstrated a moderately differentiated squamous cell carcinoma. The staging revealed stadium pT3 cN0 cM0. Postoperative treatment consisted of local radiation therapy with intensity-modulated beam geometry with a total of 64.2 Gy in 30 fractions using a simultaneous integrated boost. CONCLUSION: Middle ear carcinoma can arise from acquired cholesteatoma. The pathogenesis of squamous cell carcinoma associated with cholesteatoma has not been elucidated satisfactorily. Due to the complex anatomic features, intensity-modulated radiation therapy is the technique of choice for postoperative radiotherapy.

Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.