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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.

Members of the BMP and Wnt protein families play a relevant role in physiologic and pathologic bone turnover. Extracellular antagonists are crucial for the modulation of their activity. Lack of expression of the BMP antagonist noggin by osteoinductive, carcinoma-derived cell lines is a determinant of the osteoblast response induced by their bone metastases. In contrast, osteolytic, carcinoma-derived cell lines express noggin constitutively. We hypothesized that cancer cell-derived noggin may contribute to the pathogenesis of osteolytic bone metastasis of solid cancers by repressing bone formation. Intra-osseous xenografts of PC-3 prostate cancer cells induced osteolytic lesions characterized not only by enhanced osteoclast-mediated bone resorption, but also by decreased osteoblast-mediated bone formation. Therefore, in this model, uncoupling of the bone remodeling process contributes to osteolysis. Bone formation was preserved in the osteolytic lesions induced by noggin-silenced PC-3 cells, suggesting that cancer cell-derived noggin interferes with physiologic bone coupling. Furthermore, intra-osseous tumor growth of noggin-silenced PC-3 cells was limited, most probably as a result of the persisting osteoblast activity. This investigation provides new evidence for a model of osteolytic bone metastasis where constitutive secretion of noggin by cancer cells mediates inhibition of bone formation, thereby preventing repair of osteolytic lesions generated by an excess of osteoclast-mediated bone resorption. Therefore, noggin suppression may be a novel strategy for the treatment of osteolytic bone metastases.

Development and function of osteoblast lineage cells are regulated by a complex microenvironment consisting of the bone extracellular matrix, cells, systemic hormones and cytokines, autocrine and paracrine factors, and mechanical load. Apart from receptors that transduce extracellular signals into the cell, molecular transporters play a crucial role in the cellular response to the microenvironment. Transporter molecules are responsible for cellular uptake of nutritional components, elimination of metabolites, ion transport, and cell–cell communication. In this report, the expression of molecular transporters in osteoblast lineage cells was investigated to assess their roles in cell development and activity. Low-density arrays, covering membrane and vesicular transport molecules, were used to assess gene expression in osteoblasts representing early and late differentiation states. Receptors and transporters for the amino acid glutamate were found to be differentially expressed during osteoblast development. Glutamate is a neurotransmitter in the central nervous system, and the mechanisms of its release, signal transduction, and cellular reabsorption in the synaptic cleft are well understood. Less clear, however, is the control of equivalent processes in peripheral tissues. In primary osteoblasts, inhibition of glutamate transporters with nonselective inhibitors leads to an increase in the concentration of extracellular glutamate. This change was accompanied by a decrease in osteoblast proliferation, stimulation of alkaline phosphatase, and the expression of transcripts encoding osteocalcin. Enzymatic removal of extracellular glutamate abolished these pro-differentiation effects, as did the inhibition of PKC- and Erk1/2-signaling pathways. These findings demonstrate that glutamate signaling promotes differentiation and activation of osteoblast lineage cells. Consequently, the glutamate system may represent a putative therapeutic target to induce an anabolic response in the skeletal system. Known antagonists of glutamate transporters will serve as lead compounds in developing new and specific bioactive molecules.

The development of novel anti-cancer strategies requires more sensitive and less invasive methods to detect and monitor in vivo minimal residual disease in cancer models. Bone marrow metastases are indirectly detected by radiography as osteolytic and/or osteosclerotic lesions. Marrow micrometastases elude radiographic detection and, therefore, more sensitive methods are needed for their direct identification. Injection of cancer cells into the left cardiac ventricle of mice closely mimics micrometastatic spread. When luciferase-transfected cells are used, whole-body bioluminescent reporter imaging can detect microscopic bone marrow metastases of approximately 0.5 mm(3) volume, a size below the limit in which tumors need to induce angiogenesis for further growth. This sensitivity translates into early detection of intramedullary tumor growth, preceding the appearance of a radiologically evident osteolysis by approximately 2 weeks. Bioluminescent reporter imaging also enables continuous monitoring in the same animal of growth kinetics for each metastatic site and guides end-point analyses specifically to the bones affected by metastatic growth. This model will accelerate the understanding of the molecular events in metastasis and the evaluation of novel therapies aiming at repressing initial stages of metastatic growth.

Background Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long‐acting promoter system for gene transfer to the lung. Methods The study was designed to evaluate electroporation‐mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 µl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate‐early promoter/enhancer (CMV‐IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti‐luciferase antibody. Results Gene expression with the CMV‐IEPE promoter was highest 24 h after gene transfer (2932 ± 249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382 ± 318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989 ± 710 RLU/mg of total lung protein) with a peak at day 20 (2821 ± 2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. Conclusions These results provide evidence that in vivo electroporation is a safe and effective tool for non‐viral gene delivery to the lungs. If this method is used in combination with a long‐acting promoter system, sustained transgene expression can be achieved. Copyright © 2006 John Wiley & Sons, Ltd.

T1 alpha is the first marker gene known to be expressed in the adult lung solely by the alveolar type I epithelial cell. Previous studies showed that T1 alpha transcripts are abundant in early rat embryos where they are found in the nervous system and in the foregut and certain of its derivatives including the primitive lung. By mid- to late gestation T1 alpha messenger RNA (mRNA) expression is lost from neural tissues but appears to increase in the lung throughout fetal life. To determine whether the T1 alpha transcripts are translated into protein, especially in early embryos which sometimes express transcripts that are translationally silent, we performed immunohistochemistry on embryos and fetal tissues and analyzed certain tissues by western blotting using a monoclonal antibody against T1 alpha protein. T1 alpha protein is present at all sites that have previously been shown to express the mRNA and at similar developmental stages. As estimated from western blots, T1 alpha protein abundance peaks at about fetal day 16 in the brain and decreases thereafter to a relative level in the adult that is lower than that of the neural tube of the day 13 embryo. Relative protein abundance in the lung is very low, although detectable, on embryonic day 13 but increases slowly until fetal day 20 when there is a dramatic increase. At the time of birth, restriction to the type I cell is not complete and therefore must occur during postnatal lung development. Immunostaining reveals additional sites of expression in fetal and adult rats that had not been clearly visualized in previous in situ hybridization studies. T1 alpha is present in mesonephric tubules and apparently in primitive germ cells but is not detectable in specific cells in the adult kidney, ovary, or testis. However, cells of the choroid plexus of the central nervous system and the ciliary epithelium of the eye express T1 alpha in both fetuses and adults. The well-known functions of these epithelia are to elaborate cerebrospinal fluid and aqueous humor respectively by processes of active ion transport and water fluxes, probably through the aquaporin 1 (channel-forming integral membrane protein [CHIP] 28). We speculate therefore that T1 alpha protein may modulate or participate in these types of cellular functions in the lung.

HER-2/neu, a tumor-associated antigen (TAAg), plays a critical role in oncogenesis of various tumor types, and its selective overexpression by malignant tumor cells makes it an ideal target for immunotherapy. A prerequisite for clinical vaccines is the construction of safe and highly immunogenic reagents able to generate efficient immune responses against TAAg. Previous protein vaccines, consisting of the extracellular domain of HER-2/neu (pNeuECD), were shown to elicit an immune response that did not provide protection from transplantable tumors expressing HER-2/neu. Here we showed that virosomes, which consist of reconstituted viral envelopes without viral genetic material, can act as a carrier and an adjuvant for a truncated protein pNeuECD. Mice vaccinated with pNeuECD either encapsulated in virosomes or bound to the virosomal membrane (Vir-pNeuECD), generated rNeu-specific humoral and cytotoxic immune responses. In addition, Vir-p(NeuECD) induced significant tumor rejection and additionally did not lead to delayed tumor formation when compared with free pNeuECD in complete Freund's adjuvant. There was no difference between the virosomal constructs. Taken together these results suggest that virosomes, as clinically approved safe vaccines, can be used to elicit both humoral and cell-mediated responses against TAAg and induce tumor rejection. Our model is providing important preclinical data to design human vaccination trials for patients with tumors overexpressing HER-2/neu, either as a primary vaccination or as a boost in combination with other vaccines in a context of an adjuvant treatment plan.

The development of novel anti-cancer strategies requires more sensitive and less invasive methods to detect and monitor in vivo minimal residual disease in cancer models. Bone marrow metastases are indirectly detected by radiography as osteolytic and/or osteosclerotic lesions. Marrow micrometastases elude radiographic detection and, therefore, more sensitive methods are needed for their direct identification. Injection of cancer cells into the left cardiac ventricle of mice closely mimics micrometastatic spread. When luciferase-transfected cells are used, whole-body bioluminescent reporter imaging can detect microscopic bone marrow metastases of ≈0.5 mm 3 volume, a size below the limit in which tumors need to induce angiogenesis for further growth. This sensitivity translates into early detection of intramedullary tumor growth, preceding the appearance of a radiologically evident osteolysis by ≈2 weeks. Bioluminescent reporter imaging also enables continuous monitoring in the same animal of growth kinetics for each metastatic site and guides end-point analyses specifically to the bones affected by metastatic growth. This model will accelerate the understanding of the molecular events in metastasis and the evaluation of novel therapies aiming at repressing initial stages of metastatic growth.

T1 alpha is the first marker gene known to be expressed in the adult lung solely by the alveolar type I epithelial cell. Previous studies showed that T1 alpha transcripts are abundant in early rat embryos where they are found in the nervous system and in the foregut and certain of its derivatives including the primitive lung. By mid- to late gestation T1 alpha messenger RNA (mRNA) expression is lost from neural tissues but appears to increase in the lung throughout fetal life. To determine whether the T1 alpha transcripts are translated into protein, especially in early embryos which sometimes express transcripts that are translationally silent, we performed immunohistochemistry on embryos and fetal tissues and analyzed certain tissues by western blotting using a monoclonal antibody against T1 alpha protein. T1 alpha protein is present at all sites that have previously been shown to express the mRNA and at similar developmental stages. As estimated from western blots, T1 alpha protein abundance peaks at about fetal day 16 in the brain and decreases thereafter to a relative level in the adult that is lower than that of the neural tube of the day 13 embryo. Relative protein abundance in the lung is very low, although detectable, on embryonic day 13 but increases slowly until fetal day 20 when there is a dramatic increase. At the time of birth, restriction to the type I cell is not complete and therefore must occur during postnatal lung development. Immunostaining reveals additional sites of expression in fetal and adult rats that had not been clearly visualized in previous in situ hybridization studies. T1 alpha is present in mesonephric tubules and apparently in primitive germ cells but is not detectable in specific cells in the adult kidney, ovary, or testis. However, cells of the choroid plexus of the central nervous system and the ciliary epithelium of the eye express T1 alpha in both fetuses and adults. The well-known functions of these epithelia are to elaborate cerebrospinal fluid and aqueous humor respectively by processes of active ion transport and water fluxes, probably through the aquaporin 1 (channel-forming integral membrane protein [CHIP] 28). We speculate therefore that T1 alpha protein may modulate or participate in these types of cellular functions in the lung.