Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice

INTRODUCTION: Our goal was to generate xenograft mouse models of human breast cancer based on luciferase-expressing MDA-MB-231 tumor cells that would provide rapid mammary tumor growth; produce metastasis to clinically relevant tissues such as lymph nodes, lung, and bone; and permit sensitive in vivo detection of both primary and secondary tumor sites by bioluminescent imaging. METHOD: Two clonal cell sublines of human MDA-MB-231 cells that stably expressed firefly luciferase were isolated following transfection of the parental cells with luciferase cDNA. Each subline was passaged once or twice in vivo to enhance primary tumor growth and to increase metastasis. The resulting luciferase-expressing D3H1 and D3H2LN cells were analyzed for long-term bioluminescent stability, primary tumor growth, and distal metastasis to lymph nodes, lungs, bone and soft tissues by bioluminescent imaging. Cells were injected into the mammary fat pad of nude and nude-beige mice or were delivered systemically via intracardiac injection. Metastasis was also evaluated by ex vivo imaging and histologic analysis postmortem. RESULTS: The D3H1 and D3H2LN cell lines exhibited long-term stable luciferase expression for up to 4–6 months of accumulative tumor growth time in vivo. Bioluminescent imaging quantified primary mammary fat pad tumor development and detected early spontaneous lymph node metastasis in vivo. Increased frequency of spontaneous lymph node metastasis was observed with D3H2LN tumors as compared with D3H1 tumors. With postmortem ex vivo imaging, we detected additional lung micrometastasis in mice with D3H2LN mammary tumors. Subsequent histologic evaluation of tissue sections from lymph nodes and lung lobes confirmed spontaneous tumor metastasis at these sites. Following intracardiac injection of the MDA-MB-231-luc tumor cells, early metastasis to skeletal tissues, lymph nodes, brain and various visceral organs was detected. Weekly in vivo imaging data permitted longitudinal analysis of metastasis at multiple sites simultaneously. Ex vivo imaging data from sampled tissues verified both skeletal and multiple soft tissue tumor metastasis. CONCLUSION: This study characterized two new bioluminescent MDA-MB-231-luc human breast carcinoma cell lines with enhanced tumor growth and widespread metastasis in mice. Their application to current xenograft models of breast cancer offers rapid and highly sensitive detection options for preclinical assessment of anticancer therapies in vivo

A lightweight framework for secure life-logging in smart environments

As the world becomes an interconnected network where objects and humans interact with each other, new challenges and threats appear in the ecosystem. In this interconnected world, smart objects have an important role in giving users the chance for life-logging in smart environments. However, smart devices have several limitations with regards to memory, resources and computation power, hindering the opportunity to apply well-established security algorithms and techniques for secure life-logging on the Internet of Things (IoT) domain. The need for secure and trustworthy life-logging in smart environments is vital, thus, a lightweight approach has to be considered to overcome the constraints of smart objects. The purpose of this paper is to present in details the current topics of life-logging in smart environments, while describing interconnection issues, security threats and suggesting a lightweight framework for ensuring security, privacy and trustworthy life-logging. In order to investigate the efficiency of the lightweight framework and the impact of the security attacks on energy consumption, an experimental test-bed was developed including two interconnected users and one smart attacker, who attempts to intercept transmitted messages or interfere with the communication link. Several mitigation factors, such as power control, channel assignment and AES-128 encryption were pplied for secure life-logging. Finally, research into the degradation of the consumed energy regarding the described intrusions is presented

The Role of the BMP Signaling Antagonist Noggin in the Development of Prostate Cancer Osteolytic 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

The Appearance and Modulation of Osteocyte Marker Expression during Calcification of Vascular Smooth Muscle Cells

Vascular calcification is an indicator of elevated cardiovascular risk. Vascular smooth muscle cells (VSMCs), the predominant cell type involved in medial vascular calcification, can undergo phenotypic transition to both osteoblastic and chondrocytic cells within a calcifying environment.In the present study, using in vitro VSMC calcification studies in conjunction with ex vivo analyses of a mouse model of medial calcification, we show that vascular calcification is also associated with the expression of osteocyte phenotype markers. As controls, the terminal differentiation of murine calvarial osteoblasts into osteocytes was induced in vitro in the presence of calcifying medium (containing ß-glycerophosphate and ascorbic acid), as determined by increased expression of the osteocyte markers DMP-1, E11 and sclerostin. Culture of murine aortic VSMCs under identical conditions confirmed that the calcification of these cells can also be induced in similar calcifying medium. Calcified VSMCs had increased alkaline phosphatase activity and PiT-1 expression, which are recognized markers of vascular calcification. Expression of DMP-1, E11 and sclerostin was up-regulated during VSMC calcification in vitro. Increased protein expression of E11, an early osteocyte marker, and sclerostin, expressed by more mature osteocytes was also observed in the calcified media of Enpp1(-/-) mouse aortic tissue.This study has demonstrated the up-regulation of key osteocytic molecules during the vascular calcification process. A fuller understanding of the functional role of osteocyte formation and specifically sclerostin and E11 expression in the vascular calcification process may identify novel potential therapeutic strategies for clinical intervention

An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2−/− γc−/− mouse

The occurrence of metastases is a critical determinant of the prognosis for breast cancer patients. Effective treatment of breast cancer metastases is hampered by a poor understanding of the mechanisms involved in the formation of these secondary tumor deposits. To study the processes of metastasis, valid in vivo tumor metastasis models are required. Here, we show that increased expression of the EGF receptor in the MTLn3 rat mammary tumor cell-line is essential for efficient lung metastasis formation in the Rag mouse model. EGFR expression resulted in delayed orthotopic tumor growth but at the same time strongly enhanced intravasation and lung metastasis. Previously, we demonstrated the critical role of NK cells in a lung metastasis model using MTLn3 cells in syngenic F344 rats. However, this model is incompatible with human EGFR. Using the highly metastatic EGFR-overexpressing MTLn3 cell-line, we report that only Rag2−/−γc−/− mice, which lack NK cells, allow efficient lung metastasis from primary tumors in the mammary gland. In contrast, in nude and SCID mice, the remaining innate immune cells reduce MTLn3 lung metastasis formation. Furthermore, we confirm this finding with the orthotopic transplantation of the 4T1 mouse mammary tumor cell-line. Thus, we have established an improved in vivo model using a Rag2−/− γc−/− mouse strain together with MTLn3 cells that have increased levels of the EGF receptor, which enables us to study EGFR-dependent tumor cell autonomous mechanisms underlying lung metastasis formation. This improved model can be used for drug target validation and development of new therapeutic strategies against breast cancer metastasis formation

The aldehyde dehydrogenase enzyme 7A1 is functionally involved in prostate cancer bone metastasis

High aldehyde dehydrogenase (ALDH) activity can be used to identify tumor-initiating and metastasis-initiating cells in various human carcinomas, including prostate cancer. To date, the functional importance of ALDH enzymes in prostate carcinogenesis, progression and metastasis has remained elusive. Previously we identified strong expression of ALDH7A1 in human prostate cancer cell lines, primary tumors and matched bone metastases. In this study, we evaluated whether ALDH7A1 is required for the acquisition of a metastatic stem/progenitor cell phenotype in human prostate cancer. Knockdown of ALDH7A1 expression resulted in a decrease of the α2hi/αvhi/CD44+ stem/progenitor cell subpopulation in the human prostate cancer cell line PC-3M-Pro4. In addition, ALDH7A1 knockdown significantly inhibited the clonogenic and migratory ability of human prostate cancer cells in vitro. Furthermore, a number of genes/factors involved in migration, invasion and metastasis were affected including transcription factors (snail, snail2, and twist) and osteopontin, an ECM molecule involved in metastasis. Knockdown of ALDH7A1 resulted in decreased intra-bone growth and inhibited experimentally induced (bone) metastasis, while intra-prostatic growth was not affected. In line with these observations, evidence is presented that TGF-β, a key player in cancer invasiveness and bone metastasis, strongly induced ALDH activity while BMP7 (an antagonist of TGF-β signaling) down-regulated ALDH activity. Our findings show, for the first time, that the ALDH7A1 enzyme is functionally involved in the formation of bone metastases and that the effect appeared dependent on the microenvironment, i.e., bone versus prostate

First international consensus on the methodology of lymphangiogenesis quantification in solid human tumours

The lymphatic system is the primary pathway of metastasis for most human cancers. Recent research efforts in studying lymphangiogenesis have suggested the existence of a relationship between lymphatic vessel density and patient survival. However, current methodology of lymphangiogenesis quantification is still characterised by high intra- and interobserver variability. For the amount of lymphatic vessels in a tumour to be a clinically useful parameter, a reliable quantification technique needs to be developed. With this consensus report, we therefore would like to initiate discussion on the standardisation of the immunohistochemical method for lymphangiogenesis assessment

The frequency of osteolytic bone metastasis is determined by conditions of the soil, not the number of seeds; evidence from in vivo models of breast and prostate cancer

Background While both preclinical and clinical studies suggest that the frequency of growing skeletal metastases is elevated in individuals with higher bone turnover, it is unclear whether this is a result of increased numbers of tumour cells arriving in active sites or of higher numbers of tumour cells being induced to divide by the bone micro-environment. Here we have investigated how the differences in bone turnover affect seeding of tumour cells and/or development of overt osteolytic bone metastasis using in vivo models of hormone-independent breast and prostate cancer. Methods Cohorts of 6 (young) and 16 (mature)-week old BALB/c nude mice were culled 1, 7 and 21 days after received intracardiac injection of luciferase expressing human prostate (PC3) or breast cancer (MDA-MB-231) cell lines labelled with a fluorescent cell membrane dye (Vybrant DiD). The presence of growing bone metastases was determined by bioluminescence using an in vivo imaging system (IVIS) and followed by anatomical confirmation of tumour metastatic sites post mortem, while the presence of individual fluorescently labelled tumour cells was evaluated using two-photon microscopy ex vivo. The bone remodelling activities were compared between young and mature naïve mice (both male and female) using micro-CT analysis, ELISA and bone histomorphometry. Results Both prostate and breast cancer cells generated higher numbers of overt skeletal lesions in young mice (~80%) than in mature mice (~20%). Although mature mice presented with fewer overt bone metastases, the number of tumour cells arriving/colonizing in the tibias was comparable between young and mature animals. Young naïve mice had lower bone volume but higher bone formation and resorption activities compared to mature animals. Conclusions Our studies suggest that higher frequencies of growing osteolytic skeletal metastases in these models are linked to increased bone turnover and not to the initial number of tumour cells entering the bone microenvironment