Project description:Microarray analysis in the mouse metastatic tumor after ɣ-irradiation(ɣ-IR): non-irradiated primary tumor vs. radiated primary tumor vs. metastatic tumor after ɣ-irradiation Metastatic tumors in C6-L (rat glioma cells ) xenografted mice were studied after local treatment with fractionated γ-IR. To accurately detect the metastatic nodules after γ-IR, we observed the effect of γ-IR on distant metastatic tumor growth. Metastatic nodules after γ-IR indicated extensive colonization of C6-L cells in the lungs within 6 weeks after γ-IR. Identified and described the molecular events occurring after γ-IR through gene expression profiling to elucidate genetic changes (differentially expressed genes between the γ-IR primary tumors vs. non-γ-IR primary tumors and metastatic lung nodules vs. γ-IR primary tumors). We investigated the change of gene expression profile in the γ-IR primary tumors vs. non-γ-IR primary tumors and metastatic lung nodules vs. γ-IR primary tumors in rat glioma (C6-L cell) xenograft model.
Project description:To examine whether the local carbon ion radiotherapy affects the characteristics of the metastatic tumors, the expression profiles of the primary tumors and the lung metastases were studied in a mouse squamous cell carcinoma model by applying local radiotherapy with no irradiation (negative control), gamma-ray irradiation (reference beam), and carbon-ion irradiation. Keywords: mouse, squamous cell carcinoma, primary tumor, lung metastases, radiotherapy, carbon ion, gamma ray A highly metastatic mouse squamous cell carcinoma NR-S1 was implanted into the hind leg of synergetic C3H/HeNrs mice and irradiated with 5 Gy of carbon ion beam. 8 Gy of gamma ray was used as a reference beam. At 2 weeks after the irradiation, the lung tissue was sampled. In order to collect samples of primary tumors, the tumors were implanted in other mice and irradiated in the same manner, and the primary tumors were collected at 1 week after the irradiation. The tumor cells of the primary and metastatic tumors were collected by laser microdissection, and oligonucleotide microarray analysis of the irradiated primary tumors and the metastatic tumors were all performed in comparison to the non-irradiated primary tumor by two-color methods.
Project description:To examine whether the local carbon ion radiotherapy affects the characteristics of the metastatic tumors, the expression profiles of the primary tumors and the lung metastases were studied in a mouse squamous cell carcinoma model by applying local radiotherapy with no irradiation (negative control), gamma-ray irradiation (reference beam), and carbon-ion irradiation. Keywords: mouse, squamous cell carcinoma, primary tumor, lung metastases, radiotherapy, carbon ion, gamma ray Overall design: A highly metastatic mouse squamous cell carcinoma NR-S1 was implanted into the hind leg of synergetic C3H/HeNrs mice and irradiated with 5 Gy of carbon ion beam. 8 Gy of gamma ray was used as a reference beam. At 2 weeks after the irradiation, the lung tissue was sampled. In order to collect samples of primary tumors, the tumors were implanted in other mice and irradiated in the same manner, and the primary tumors were collected at 1 week after the irradiation. The tumor cells of the primary and metastatic tumors were collected by laser microdissection, and oligonucleotide microarray analysis of the irradiated primary tumors and the metastatic tumors were all performed in comparison to the non-irradiated primary tumor by two-color methods.
Project description:BACKGROUND:Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can preferentially accumulate at defined sites, including tumors. Analyzing the potential of EVs to target specific cells remains challenging, considering the unspecific binding of lipophilic tracers to other proteins, the limitations of fluorescence for deep tissue imaging and the effect of external labeling strategies on their natural tropism. In this work, we determined the cell-type specific tropism of B16F10-EVs towards cancer cell and metastatic tumors by using fluorescence analysis and quantitative gold labeling measurements. Surface functionalization of plasmonic gold nanoparticles was used to promote indirect labeling of EVs without affecting size distribution, polydispersity, surface charge, protein markers, cell uptake or in vivo biodistribution. Double-labeled EVs with gold and fluorescent dyes were injected into animals developing metastatic lung nodules and analyzed by fluorescence/computer tomography imaging, quantitative neutron activation analysis and gold-enhanced optical microscopy. RESULTS:We determined that B16F10 cells preferentially take up their own EVs, when compared with colon adenocarcinoma, macrophage and kidney cell-derived EVs. In addition, we were able to detect the preferential accumulation of B16F10 EVs in small metastatic tumors located in lungs when compared with the rest of the organs, as well as their precise distribution between tumor vessels, alveolus and tumor nodules by histological analysis. Finally, we observed that tumor EVs can be used as effective vectors to increase gold nanoparticle delivery towards metastatic nodules. CONCLUSIONS:Our findings provide a valuable tool to study the distribution and interaction of EVs in mice and a novel strategy to improve the targeting of gold nanoparticles to cancer cells and metastatic nodules by using the natural properties of malignant EVs.
Project description:Successful combinations of radiotherapy and immunotherapy depend on the presence of live T cells within the tumor; however, radiotherapy is believed to damage T cells. Here, based on longitudinal in vivo imaging and functional analysis, we report that a large proportion of T cells survive clinically relevant doses of radiation and show increased motility, and higher production of interferon gamma, compared with T cells from unirradiated tumors. Irradiated intratumoral T cells can mediate tumor control without newly-infiltrating T cells. Transcriptomic analysis suggests T cell reprogramming in the tumor microenvironment and similarities with tissue-resident memory T cells, which are more radio-resistant than circulating/lymphoid tissue T cells. TGF? is a key upstream regulator of T cell reprogramming and contributes to intratumoral Tcell radio-resistance. These findings have implications for the design of radio-immunotherapy trials in that local irradiation is not inherently immunosuppressive, and irradiation of multiple tumors might optimize systemic effects of radiotherapy.
Project description:The prevalence of thyroid nodules increases with age, average 4-7% for the U.S.A. adult population, but it is much higher (19-67%) when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules). The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3.The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific (99m)Tc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO) and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 microCi of (99m)Tc-labeled mAb to galectin-3 (30 microg protein/in 100 microl saline solution). Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera.Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared.These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed.
Project description:Liver metastases often progress from primary cancers including uveal melanoma (UM), breast, and colon cancer. Molecular biomarker imaging is a new non-invasive approach for detecting early stage tumors. Here, we report the elevated expression of chemokine receptor 4 (CXCR4) in liver metastases in UM patients and metastatic UM mouse models, and development of a CXCR4-targeted MRI contrast agent, ProCA32.CXCR4, for sensitive MRI detection of UM liver metastases. ProCA32.CXCR4 exhibits high relaxivities (r 1 = 30.9 mM-1 s-1, r 2 = 43.2 mM-1 s-1, 1.5 T; r 1 = 23.5 mM-1 s-1, r 2 = 98.6 mM-1 s-1, 7.0 T), strong CXCR4 binding (K d = 1.10 ± 0.18 ?M), CXCR4 molecular imaging capability in metastatic and intrahepatic xenotransplantation UM mouse models. ProCA32.CXCR4 enables detecting UM liver metastases as small as 0.1 mm3. Further development of the CXCR4-targeted imaging agent should have strong translation potential for early detection, surveillance, and treatment stratification of liver metastases patients.
Project description:Rapid diagnosis of metastatic lymph nodes (mLNs) of colorectal cancer (CRC) is desirable either intraoperatively or in resected fresh specimens. We have developed a series of activatable fluorescence probes for peptidase activities that are specifically upregulated in various tumors. We aimed to discover a target enzyme for detecting mLNs of CRC. Among our probes, we found that gGlu-HMRG, a gamma-glutamyl transpeptidase (GGT)-activatable fluorescence probe, could detect mLNs. This was unexpected, because we have previously reported that gGlu-HMRG could not detect primary CRC. We confirmed that the GGT activity of mLNs was high, whereas that of non-metastatic lymph nodes and CRC cell lines was low. We investigated the reason why GGT activity was upregulated in mLNs, and found that GGT was induced under conditions of hypoxia or low nutritional status. We utilized this feature to achieve rapid detection of mLNs with gGlu-HMRG. GGT appears to be a promising candidate enzyme for fluorescence imaging of mLNs.
Project description:Metastatic dissemination continues to be a major cause of prostate cancer (PCa) mortality, creating a compelling need to understand factors that play a role in the metastatic cascade. Since hypoxia plays an important role in PCa aggressiveness, we characterized patterns of hypoxia in the primary tumor and metastatic environments of a human PCa xenograft. We previously developed and characterized an imaging strategy based on the hypoxia response element (HRE)-driven expression of long-lived enhanced green fluorescent protein (EGFP) and short-lived luciferase (luc) fused to the oxygen-dependent degradation domain in human PCa PC-3 cells. Both reporter proteins were placed under the transcriptional control of a five-tandem repeat HRE sequence. PC-3 cells also constitutively expressed the tdTomato red fluorescent protein, allowing cancer cell detection in vivo. This "timer" strategy can provide information on the temporal evolution of HIF activity and hypoxia in tumors. Here, for the first time, we performed in vivo and ex vivo imaging of this dual HIF reporter system in PC-3 metastatic tumors implanted orthotopically in the prostate and PC-3 nonmetastatic tumors implanted subcutaneously. We observed distinct patterns of EGFP and luc expression in subcutaneous and orthotopic tumors, and in metastatic nodules, that provide new insights into the presence of hypoxia at primary and metastatic tumor sites, and of the role of hypoxia in metastasis.
Project description:Modeling metastasis in vivo with animals is a priority for both revealing mechanisms of tumor dissemination and developing therapeutic methods. While conventional intravenous injection of tumor cells provides an efficient and consistent system for studying tumor cell extravasation and colonization, studying spontaneous metastasis derived from orthotopic tumor sites has the advantage of modeling more aspects of the metastatic cascade, but is challenging as it is difficult to detect small numbers of metastatic cells. In this work, we developed an approach for quantifying spontaneous metastasis in the syngeneic mouse B16 system using real time PCR. We first transduced B16 cells with lentivirus expressing firefly luciferase Luc2 gene for bioluminescence imaging. Next, we developed a real time quantitative PCR (qPCR) method for the detection of luciferase-expressing, metastatic tumor cells in mouse lungs and other organs. To illustrate the approach, we quantified lung metastasis in both spontaneous and experimental scenarios using B16F0 and B16F10 cells in C57BL/6Ncrl and NOD-Scid Gamma (NSG) mice. We tracked B16 melanoma metastasis with both bioluminescence imaging and qPCR, which were found to be self-consistent. Using this assay, we can quantitatively detect one Luc2 positive tumor cell out of 104 tissue cells, which corresponds to a metastatic burden of 1.8 × 104 metastatic cells per whole mouse lung. More importantly, the qPCR method was at least a factor of 10 more sensitive in detecting metastatic cell dissemination and should be combined with bioluminescence imaging as a high-resolution, end-point method for final metastatic cell quantitation. Given the rapid growth of primary tumors in many mouse models, assays with improved sensitivity can provide better insight into biological mechanisms that underpin tumor metastasis.