Maija P. Valta

FGF-8 is involved in bone metastasis of prostate cancer.

Prostate cancer is the most common malignancy of men in Western countries. Patients with advanced prostate cancer suffer from incurable bone metastases. Recent data indicate that interactions between prostate cancer cells, osteoblasts, osteoclasts and the bone matrix are essential in the formation of bone metastases. FGF‐8 is widely overexpressed in prostate cancer. Recently, FGF‐8 has been found to affect both osteoblast and osteoclast differentiation. The aim of this study was to examine the role of FGF‐8 in bone metastasis of prostate cancer. Immunohistochemistry was used to analyse FGF‐8 expression in clinical samples of prostate cancer bone metastases. The functional significance of FGF‐8 in growth of bone metastasis and formation of bone lesions was verified by using intratibial inoculations of FGF‐8 or mock transfected PC‐3 prostate cancer cells in nude mice. Intratibial tumors and bone lesions were analysed with X‐ray, micro‐CT and detailed histomorphometry using image analysis software and with immunostaining for osteocalcin and cathepsin K. Immunohistochemical analysis of tissue microarray of bone metastases of human prostate cancer showed that 76% of human bone metastasis samples (n = 25 from 11 patients) were positive for FGF‐8. FGF‐8 increased the growth of intratibial tumors and local formation of lytic and sclerotic lesions of bone. These results demonstrate that FGF‐8 is expressed at a high frequency in bone metastases of human prostate cancer and that expression of FGF‐8 in PC‐3 prostate cancer cells increases their growth as intratibial tumors and modulates formation of bone lesions in an in vivo model of prostate cancer bone metastasis.

Phosphorylcholine-Coated Semiconducting Polymer Nanoparticles as Rapid and Efficient Labeling Agents for In Vivo Cell Tracking

Despite the pressing need to noninvasively monitor transplanted cells in vivo with fluorescence imaging, desirable fluorescent agents with rapid labeling capability, durable brightness, and ideal biocompatibility remain lacking. Here, phosphorylcholine-coated near-infrared (NIR) fluorescent semiconducting polymer nanoparticles (SPNs) are reported as a new class of rapid, efficient, and cytocompatible labeling nanoagents for in vivo cell tracking. The phosphorylcholine coating results in efficient and rapid endocytosis and allows the SPN to enter cells within 0.5 h in complete culture medium apparently independent of the cell type, while its NIR fluorescence leads to a tissue penetration depth of 0.5 cm. In comparison to quantum dots and Cy5.5, the SPN is tolerant to physiologically ubiquitous reactive oxygen species (ROS), resulting in durable fluorescence both in vitro and in vivo. These desirable physical and physiological properties of the SPN permit cell tracking of human renal cell carcinoma (RCC) cells in living mice at a lower limit of detection of 10 000 cells with no obvious alteration of cell phenotype after 12 d. SPNs thus can provide unique opportunities for optimizing cellular therapy and deciphering pathological processes as a cell tracking label.

Alendronate decreases orthotopic PC-3 prostate tumor growth and metastasis to prostate-draining lymph nodes in nude mice

BackgroundMetastatic prostate cancer is associated with a high morbidity and mortality but the spreading mechanisms are still poorly understood. The aminobisphosphonate alendronate, used to reduce bone loss, has also been shown to inhibit the invasion and migration of prostate cancer cells in vitro. We used a modified orthotopic PC-3 nude mouse tumor model of human prostate cancer to study whether alendronate affects prostate tumor growth and metastasis.MethodsPC-3 cells (5 × 105) were implanted in the prostates of nude mice and the mice were treated with alendronate (0.5 mg/kg/day in PBS, s.c.) or vehicle for 4 weeks. After sacrifice, the sizes of tumor-bearing prostates were measured and the tumors and prostate-draining regional iliac and sacral lymph nodes were excised for studies on markers of proliferation, apoptosis, angiogenesis and lymphangiogenesis, using histomorphometry and immunohistochemistry.ResultsTumor occurrence in the prostate was 73% in the alendronate-treated group and 81% in the control group. Mean tumor size (218 mm3, range: 96–485 mm3, n = 11) in the alendronate-treated mice was 41% of that in the control mice (513 mm3, range: 209–1350 mm3, n = 13) (p < 0.05). In the iliac and sacral lymph nodes of alendronate-treated mice, the proportion of metastatic area was only about 10% of that in control mice (p < 0.001). Immunohistochemical staining of tumor sections showed that alendronate treatment caused a marked decrease in the number of CD34-positive endothelial cells in tumors (p < 0.001) and an increase in that of ISEL positive apoptotic cells in tumors as well as in lymph node metastases (p < 0.05) compared with those in the vehicle-treated mice. The density of m-LYVE-1-stained lymphatic capillaries was not changed.ConclusionOur results demonstrate that alendronate treatment opposes growth of orthotopic PC-3 tumors and decreases tumor metastasis to prostate-draining lymph nodes. This effect could be at least partly explained by decreased angiogenesis and increased apoptosis. The results suggest that bisphosphonates have anti-tumoral and anti-invasive effects on primary prostate cancer.

FGF-8b Induces Growth and Rich Vascularization in an Orthotopic PC-3 Model of Prostate Cancer

Fibroblast growth factor 8 (FGF‐8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF‐8b on proliferation of PC‐3 prostate cancer cells and growth of PC‐3 tumors, and to identify FGF‐8b‐associated molecular targets. Expression of ectopic FGF‐8b in PC‐3 cells caused a 1.5‐fold increase in cell proliferation in vitro and a four‐ to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF‐8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT‐qPCR of FGF‐8b mRNA. Microarray analyses revealed significantly altered, up‐ and downregulated, genes in PC‐3 cell cultures (169 genes) and in orthotopic PC‐3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell‐to‐cell signaling and energy production, and the downregulated genes associated with differentiation (DKK‐1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT‐qPCR. In conclusion, our results demonstrate that FGF‐8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF‐8b actions on prostate cancer progression and metastasis. J. Cell. Biochem. 107: 769–784, 2009.

Preclinical trial of a new dual mTOR inhibitor, MLN0128, using renal cell carcinoma tumorgrafts

mTOR is a rational target in renal cell carcinoma (RCC) because of its role in disease progression. However, the effects of temsirolimus, the only first‐generation mTOR inhibitor approved by the FDA for first‐line treatment of metastatic RCC, on tumor reduction and progression‐free survival are minimal. Second‐generation mTOR inhibitors have not been evaluated on RCC. We compared the effects of temsirolimus and MLN0128, a potent second‐generation mTOR inhibitor, on RCC growth and metastasis using a realistic patient‐derived tissue slice graft (TSG) model. TSGs were derived from three fresh primary RCC specimens by subrenal implantation of precision‐cut tissue slices into immunodeficient mice that were randomized and treated with MLN0128, temsirolimus, or placebo. MLN0128 consistently suppressed primary RCC growth, monitored by magnetic resonance imaging (MRI), in three TSG cohorts for up to 2 months. Temsirolimus, in contrast, only transiently inhibited the growth of TSGs in one of two cohorts before resistance developed. In addition, MLN0128 reduced liver metastases, determined by human‐specific quantitative polymerase chain reaction, in two TSG cohorts, whereas temsirolimus failed to have any significant impact. Moreover, MLN0128 decreased levels of key components of the two mTOR subpathways including TORC1 targets 4EBP1, p‐S6K1, HIF1α and MTA1 and the TORC2 target c‐Myc, consistent with dual inhibition. Our results demonstrated that MLN0128 is superior to temsirolimus in inhibiting primary RCC growth as well as metastases, lending strong support for further clinical development of dual mTOR inhibitors for RCC treatment.

Overexpression of vascular endothelial growth factor C increases growth and alters the metastatic pattern of orthotopic PC-3 prostate tumors

BackgroundProstate cancer metastasizes to regional lymph nodes and distant sites but the roles of lymphatic and hematogenous pathways in metastasis are not fully understood.MethodsWe studied the roles of VEGF-C and VEGFR3 in prostate cancer metastasis by blocking VEGFR3 using intravenous adenovirus-delivered VEGFR3-Ig fusion protein (VEGFR3-Ig) and by ectopic expression of VEGF-C in PC-3 prostate tumors in nude mice.ResultsVEGFR3-Ig decreased the density of lymphatic capillaries in orthotopic PC-3 tumors (p < 0.05) and inhibited metastasis to iliac and sacral lymph nodes. In addition, tumor volumes were smaller in the VEGFR3-Ig-treated group compared with the control group (p < 0.05). Transfection of PC-3 cells with the VEGF-C gene led to a high level of 29/31 kD VEGF-C expression in PC-3 cells. The size of orthotopic and subcutaneous PC-3/VEGF-C tumors was significantly greater than that of PC-3/mock tumors (both p < 0.001). Interestingly, while most orthotopic PC-3 and PC-3/mock tumors grown for 4 weeks metastasized to prostate-draining lymph nodes, orthotopic PC-3/VEGF-C tumors primarily metastasized to the lungs. PC-3/VEGF-C tumors showed highly angiogenic morphology with an increased density of blood capillaries compared with PC-3/mock tumors (p < 0.001).ConclusionThe data suggest that even though VEGF-C/VEGFR3 pathway is primarily required for lymphangiogenesis and lymphatic metastasis, an increased level of VEGF-C can also stimulate angiogenesis, which is associated with growth of orthotopic prostate tumors and a switch from a primary pattern of lymph node metastasis to an increased proportion of metastases at distant sites.

Fast growth associated with aberrant vasculature and hypoxia in fibroblast growth factor 8b (FGF8b) over-expressing PC-3 prostate tumour xenografts

BackgroundProstate tumours are commonly poorly oxygenated which is associated with tumour progression and development of resistance to chemotherapeutic drugs and radiotherapy. Fibroblast growth factor 8b (FGF8b) is a mitogenic and angiogenic factor, which is expressed at an increased level in human prostate tumours and is associated with a poor prognosis. We studied the effect of FGF8b on tumour oxygenation and growth parameters in xenografts in comparison with vascular endothelial growth factor (VEGF)-expressing xenografts, representing another fast growing and angiogenic tumour model.MethodsSubcutaneous tumours of PC-3 cells transfected with FGF8b, VEGF or empty (mock) vectors were produced and studied for vascularity, cell proliferation, glucose metabolism and oxygenation. Tumours were evaluated by immunohistochemistry (IHC), flow cytometry, use of radiolabelled markers of energy metabolism ([18F]FDG) and hypoxia ([18F]EF5), and intratumoral polarographic measurements of pO2.ResultsBoth FGF8b and VEGF tumours grew rapidly in nude mice and showed highly vascularised morphology. Perfusion studies, pO2 measurements, [18F]EF5 and [18F]FDG uptake as well as IHC staining for glucose transport protein (GLUT1) and hypoxia inducible factor (HIF) 1 showed that VEGF xenografts were well-perfused and oxygenised, as expected, whereas FGF8b tumours were as hypoxic as mock tumours. These results suggest that FGF8b-induced tumour capillaries are defective. Nevertheless, the growth rate of hypoxic FGF8b tumours was highly increased, as that of well-oxygenised VEGF tumours, when compared with hypoxic mock tumour controls.ConclusionFGF8b is able to induce fast growth in strongly hypoxic tumour microenvironment whereas VEGF-stimulated growth advantage is associated with improved perfusion and oxygenation of prostate tumour xenografts.

Effect of legislative changes in drug promotion on medical students: questionnaire survey.

Objective  The aim of this follow‐up study was to examine whether the legislative changes that took place in Finland in 2004 had an impact on the interactions between pharmaceutical companies and medical students. According to a previous survey, information provided by pharmaceutical companies represented one of the most important sources of information on pharmaceutical products for medical students and students frequently attended promotional events.

Spheroid culture of LuCaP 136 patient-derived xenograft enables versatile preclinical models of prostate cancer

LuCaP serially transplantable patient-derived xenografts (PDXs) are valuable preclinical models of locally advanced or metastatic prostate cancer. Using spheroid culture methodology, we recently established cell lines from several LuCaP PDXs. Here, we characterized in depth the features of xenografts derived from LuCaP 136 spheroid cultures and found faithful retention of the phenotype of the original PDX. In vitro culture enabled luciferase transfection into LuCaP 136 spheroids, facilitating in vivo imaging. We showed that LuCaP 136 spheroids formed intratibial, orthotopic, and subcutaneous tumors when re-introduced into mice. Intratibial tumors responded to castration and were highly osteosclerotic. LuCaP 136 is a realistic in vitro–in vivo preclinical model of a subtype of bone metastatic prostate cancer.

Abstract B9: Mouse model of primary human renal cell carcinoma metastasis to bone

More than a third of patients with renal cell carcinoma (RCC) will die from disease and thus renal cell carcinoma has the highest mortality rate of the genitourinary cancers. Localized RCC can be cured by surgery but life expectancy is short for the metastatic disease. Thirty % of RCC metastasizes to bone and these metastases are especially debilitating and resistant to therapy. As the incidence of RCC is increasing, bone metastatic renal cancer merits greater focus. However, the development of effective therapies for RCC bone metastasis is hampered by lack of realistic pre-clinical models. Recent studies have shown that tumorgrafts are the most realistic models of solid tumors that are highly predictive of therapeutic response. We have improved this methodology by implanting precision-cut tumor tissue slices rather than minced tissue chunks into immunodeficient mice. Our aim is to develop a bone metastasis model using tissue slice tumorgrafts (TSGs) derived from fresh RCC specimens rather than established RCC cell lines that do not mimic natural history of metastases. Methods: Cores (8-mm diameter) of fresh renal cancer were obtained from nephrectomy specimens and precision-cut at 300-μm using a tissue slicer. Intact tissue slices were then implanted under the renal capsules of immunodeficient mice. The presence of disseminated cells in the bone marrow of graft-bearing mice was screened by qPCR (polymerase chain reaction) for human-specific markers. Primary cells were then obtained by enzymatical digestion of TSGs and injected into the tibiae of mice to mimic a RCC bone metastasis. Mice were followed up with x-ray and micro-CT. At the end of the experiment the mice bearing intratibial tumors were sacrificed and the tibiae were fixed and longitudinally sectioned at 5-μm. The phenotype of the metastases and interactions of cancer cells with the bone marrow cells/stem cell niche was analyzed by means of immunohistology. Results: By using human specific qPCR, we detected disseminated tumor cells in bone of mice bearing TSGs derived from a clear cell carcinoma patient, who subsequently progressed to metastatic disease, 1 month after implantation. The analysis for 9 other RCC cases is currently under way. We then confirmed the presence of metastatic lesions in bone by histology. These metastases retained many phenotypic characteristics of the primary tumor (as shown by histology and immunohistochemistry for CA-IX or CD-31). Primary cells derived from the TSGs produced x-ray visible tumors/metastases already at 3 weeks after cancer cell inoculation intratibially at a 100 % engraftment rate. Micro-CT analysis revealed an osteolytic nature of these lesions, which is considered typical for renal cancer bone metastases. Conclusions: We showed that orthotopic RCC TSGs have potential to develop micrometastases and metastases to bone. We demonstrated the feasibility of an intratibial model of RCC metastasis to bone that resembles the actual disease in many ways. These are the first reported models of primary RCC metastasis to bone. Our translational research project allows realistic settings to test therapeutics to prevent or treat micro-metastases and metastases in RCC and has potential for applications in personalized medicine Citation Format: Maija Valta, Hongjuan Zhao, Alexandre Ingels, Alan Thong, Rosalie Nolley, Matthias Saar, Donna Peehl. Mouse model of primary human renal cell carcinoma metastasis to bone. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B9.