The aim of the present study was to examine the efficacy of tumor-targeting Salmonella typhimurium A1-R treatment following anti-vascular endothelial growth factor (VEGF) therapy on VEGF-positive human pancreatic cancer. A pancreatic cancer patient-derived orthotopic xenograft (PDOX) that was VEGF-positive and an orthotopic VEGF-positive human pancreatic cancer cell line (MiaPaCa-2-GFP) as well as a VEGF-negative cell line (Panc-1) were tested. Nude mice with these tumors were treated with gemcitabine (GEM), bevacizumab (BEV), and S. typhimurium A1-R. BEV/GEM followed by S. typhimurium A1-R significantly reduced tumor weight compared to BEV/GEM treatment alone in the PDOX and MiaPaCa-2 models. Neither treatment was as effective in the VEGF-negative model as in the VEGF-positive models. These results demonstrate that S. typhimurium A1-R following anti-angiogenic therapy is effective on pancreatic cancer including the PDOX model, suggesting its clinical potential.
We previously defined macrophages harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors as "tumor-educated-macrophages" (Edu) and macrophages harvested from mice without tumors as "naïve-macrophages" (Naïve), and demonstrated that Edu-macrophages promoted tumor growth and metastasis. In this study, Edu- and Naïve-macrophages were compared for their ability to enhance pancreatic cancer malignancy at the cellular level in vitro and in vivo. The inhibitory efficacy of Zoledronic acid (ZA) on Edu-macrophage-enhanced metastasis was also determined. XPA1 human pancreatic cancer cells in Gelfoam co-cultured with Edu-macrophages proliferated to a greater extent compared to XPA1 cells cultured with Naïve-macrophages (P = 0.014). XPA1 cells exposed to conditioned medium harvested from Edu culture significantly increased proliferation (P = 0.016) and had more migration stimulation capability (P<0.001) compared to cultured cancer cells treated with the conditioned medium from Naïve. The mitotic index of the XPA1 cells, expressing GFP in the nucleus and RFP in the cytoplasm, significantly increased in vivo in the presence of Edu- compared to Naïve-macrophages (P = 0.001). Zoledronic acid (ZA) killed both Edu and Naïve in vitro. Edu promoted tumor growth and metastasis in an orthotopic mouse model of the XPA1 human pancreatic cancer cell line. ZA reduced primary tumor growth (P = 0.006) and prevented metastasis (P = 0.025) promoted by Edu-macrophages. These results indicate that ZA inhibits enhanced primary tumor growth and metastasis of human pancreatic cancer induced by Edu-macrophages.
Fluorescence-guided surgery (FGS) can enable successful cancer surgery where bright-light surgery often cannot. There are three important issues for FGS going forward toward the clinic: (a) proper tumor labeling, (b) a simple portable imaging system for the operating room, and (c) patient-like mouse models in which to develop the technology. The present report addresses all three.
The aim of this study is to determine the efficacy of tumor-targeting Salmonella typhimurium A1-R (A1-R) on pancreatic cancer patient-derived orthotopic xenografts (PDOX). The PDOX model was originally established from a pancreatic cancer patient in SCID-NOD mice. The pancreatic cancer PDOX was subsequently transplanted by surgical orthotopic implantation (SOI) in transgenic nude red fluorescent protein (RFP) mice in order that the PDOX stably acquired red fluorescent protein (RFP)-expressing stroma for the purpose of imaging the tumor after passage to non-transgenic nude mice in order to visualize tumor growth and drug efficacy. The nude mice with human pancreatic PDOX were treated with A1-R or standard chemotherapy, including gemcitabine (GEM), which is first-line therapy for pancreatic cancer, for comparison of efficacy. A1-R treatment significantly reduced tumor weight, as well as tumor fluorescence area, compared to untreated control (P?=?0.011), with comparable efficacy of GEM, CDDP, and 5-FU. Histopathological response to treatment was defined according to Evans's criteria and A1-R had increased efficacy compared to standard chemotherapy. The present report is the first to show that A1-R is effective against a very low-passage patient tumor, in this case, pancreatic cancer. The data of the present report suggest A1-1 will have clinical activity in pancreatic cancer, a highly lethal and treatment-resistant disease and may be most effectively used in combination with other agents.
The aim of this study is to determine if ultraviolet light (UVC) irradiation in combination with fluorescence-guided surgery (FGS) can eradicate metastatic human pancreatic cancer in orthotopic nude-mouse models. Two weeks after orthotopic implantation of human MiaPaCa-2 pancreatic cancer cells, expressing green fluorescent protein (GFP), in nude mice, bright-light surgery (BLS) was performed on all tumor-bearing mice (n?=?24). After BLS, mice were randomized into 3 treatment groups; BLS-only (n?=?8) or FGS (n?=?8) or FGS-UVC (n?=?8). The residual tumors were resected using a hand-held portable imaging system under fluorescence navigation in mice treated with FGS and FGS-UVC. The surgical resection bed was irradiated with 2700 J/m2 UVC (254 nm) in the mice treated with FGS-UVC. The average residual tumor area after FGS (n?=?16) was significantly smaller than after BLS only (n?=?24) (0.135±0.137 mm2 and 3.338±2.929 mm2, respectively; p?=?0.007). The BLS treated mice had significantly reduced survival compared to FGS- and FGS-UVC-treated mice for both relapse-free survival (RFS) (p<0.001 and p<0.001, respectively) and overall survival (OS) (p<0.001 and p<0.001, respectively). FGS-UVC-treated mice had increased RFS and OS compared to FGS-only treated mice (p?=?0.008 and p?=?0.025, respectively); with RFS lasting at least 150 days indicating the animals were cured. The results of the present study suggest that UVC irradiation in combination with FGS has clinical potential to increase survival.
In this study, we investigated the advantages of fluorescence-guided surgery (FGS) in mice of a portable hand-sized imaging system compared with a large fluorescence imaging system or a long-working-distance fluorescence microscope.
The XPA1 human pancreatic cancer cell line is dimorphic, with spindle stem-like cells and round non-stem cells. We report here the in vitro IC 50 values of stem-like and non-stem XPA1 human pancreatic cells cells for: (1) 5-fluorouracil (5-FU), (2) cisplatinum (CDDP), (3) gemcitabine (GEM), and (4) tumor-targeting Salmonella typhimurium A1-R (A1-R). IC 50 values of stem-like XPA1 cells were significantly higher than those of non-stem XPA1 cells for 5-FU (P = 0.007) and CDDP (P = 0.012). In contrast, there was no difference between the efficacy of A1-R on stem-like and non-stem XPA1 cells. In vivo, 5-FU and A1-R significantly reduced the tumor weight of non-stem XPA1 cells (5-FU; P = 0.028; A1-R; P = 0.011). In contrast, only A1-R significantly reduced tumor weight of stem-like XPA1 cells (P = 0.012). The combination A1-R with 5-FU improved the antitumor efficacy compared with 5-FU monotherapy on the stem-like cells (P = 0.004). The results of the present report indicate A1-R is a promising therapy for chemo-resistant pancreatic cancer stem-like cells.
?v Integrin is involved in various steps of cancer metastasis. In this report, we describe real-time imaging of ?v integrin molecular dynamics in human 143B osteosarcoma cells in vitro and in vivo. We first generated osteosarcoma cells expressing ?v integrin green fluorescent protein (GFP) by transfection of an ?v integrin GFP fusion vector (pCMV6-AC-ITGAV-GFP) into 143B cells. Confocal laser-scanning microscopy demonstrated that ?v integrin immunofluorescence staining co-localized with ?v integrin-GFP fluorescence in 143B cells. When ?v integrin-GFP-expressing 143B osteosarcoma cells were seeded on a dish coated with fibronectin, which is bound by ?v integrin, punctate ?v integrin-GFP was observed by confocal laser-scanning microscopy. When the 143B ?v integrin-GFP cells were seeded onto uncoated plastic, ?v integrin-GFP was diffuse within the cells. When ?v integrin-GFP 143B osteosarcoma cells (1×10(6)) were orthotopically transplanted into the tibia of nude mice, the cells aligned along the collagen fibers within the tumor and had punctuate expression of ?v integrin-GFP. In the orthotopic model, the invading osteosarcoma cells had punctate ?v integrin-GFP in the muscle tissue at the primary tumor margin. These results show that ?v integrin-GFP enables the imaging of the molecular dynamics of ?v integrin in osteosarcoma cells in vitro and in vivo.
The effect of UVC irradiation was investigated on a model of brain cancer and a model of experimental brain metastasis. For the brain cancer model, brain cancer cells were injected stereotactically into the brain. For the brain metastasis model, lung cancer cells were injected intra-carotidally or stereotactically. The U87 human glioma cell line was used for the brain cancer model, and the Lewis lung carcinoma (LLC) was used for the experimental brain metastasis model. Both cancer cell types were labeled with GFP in the nucleus and RFP in the cytoplasm. A craniotomy open window was used to image single cancer cells in the brain. This double labeling of the cancer cells with GFP and RFP enabled apoptosis of single cells to be imaged at the subcellular level through the craniotomy open window. UVC irradiation, beamed through the craniotomy open window, induced apoptosis in the cancer cells. UVC irradiation was effective on LLC and significantly extended survival of the mice with experimental brain metastasis. In contrast, the U87 glioma was relatively resistant to UVC irradiation. The results of this study suggest the use of UVC for treatment of superficial brain cancer or metastasis.
Malignant glioma is the most common type of primary central nervous system cancer. Gliomas are very difficult to completely resect due to their invasiveness. In the present study, we compared fluorescence-guided and standard bright-light resection of a human glioma orthotopically implanted in nude mice. U87 human glioma cells, expressing red fluorescent protein (RFP), were injected stereotactically into the nude mouse brain through a craniotomy open window. Two weeks after cancer-cell implantation, gliomas were resected under fluorescence guidance or under bright light. U87-RFP tumors were clearly visualized with a long-working distance fluorescence microscope. Almost all cancer cells were removed using fluorescence-guided navigation without damage to the brain tissue. In contrast, brain tumors were difficult to visualize under bright light and many residual cancer cells remained in the brain after bright-light surgery. Fluorescence-guided surgery significantly extended the survival of the mice compared to those who underwent bright-light surgery. These results suggest that fluorescence-guided surgery has significant potential for brain cancer treatment.
Novel subcellular imaging technology has been developed in order to visualize drug efficacy on single cancer cells in the brain of mice in real time. The efficacy of temozolomide on cancer cells in the brain was determined by observation of subcellular cancer-cell dynamics over time through a craniotomy open window. Dual-color U87 human glioma and Lewis lung carcinoma (LLC) cells, expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm, were imaged through the craniotomy open window 10 days after treatment with temozolomide (100 mg/kg i.p. for five consecutive days). After treatment, dual-color cancer cells with fragmented nuclei were visualized, indicating apoptosis. GFP-expressing apoptotic bodies and the destruction of RFP-expressing cytoplasm were also visualized. In addition, the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay was used to confirm apoptosis visualized by imaging of the behavior of GFP-labeled cancer-cell nuclei. Tumor volume in the treated group was significantly smaller than in the control group (at day 19, p<0.001). The present study demonstrates technology capable of subcellular real-time imaging in the brain that reports induction of cancer-cell apoptosis by therapeutic treatment. More effective drugs for brain cancer and brain metastasis can be screened and can be identified with this technology.
We developed the tumor-targeting strain Salmonella typhimuium A1-R (A1-R) and have shown it to be active against a number of tumor types in nude mice. However, in immunocompetent mice, dosing of A1-R has to be adjusted to avoid toxicity. In the present study, we developed a strategy to maximize efficacy and minimize toxicity for A1-R tumor-targeting in immunocompetent mice implanted with the Lewis lung carcinoma. A small primer dose of A1-R was first administered (1×10(6) colony forming unit [cfu] i.v.) followed by a high dose (1×10(7) cfu i.v.) four hours later. The primer-dose strategy resulted in smaller tumors and no observable side-effects compared to treatment with high-dose-alone. The serum level of tumor necrosis factor (TNF-?) was elevated in the mice treated with primer dose compared to mice only given the high dose. Tumor vessel destruction was enhanced by primer dosing of A1-R in immuno-competent transgenic mice expressing the nestin-driven green fluorescent protein, which is selectively expressed in nascent blood vessels. The primer-dose may activate TNF-? and other cytokines in the mouse, necessary for invasion of the tumor by the bacteria, as well as enhance tumor vessel destruction, thereby allowing a subsequent therapeutic dose to be effective and safe. The results of the present study suggest effective future clinical strategies of bacterial treatment of cancer.
The Adjuvant Chemotherapy Trial of TS-1 for Gastric Cancer (ACTS-GC) demonstrated that S-1(TS-1, an oral fluoropyrimidine) was effective as adjuvant chemotherapy for patients with pathological stage II or III gastric cancer who underwent curative gastrectomy. The objective of this study was to clarify the risk factors for recurrence in patients who received S-1 adjuvant chemotherapy.
The ability to visualize cancer cell mitosis and apoptosis in the brain in real time would be of great utility in testing novel therapies. In order to achieve this goal, the cancer cells were labeled with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm, such that mitosis and apoptosis could be clearly imaged. A craniotomy open window was made in athymic nude mice for real-time fluorescence imaging of implanted cancer cells growing in the brain. The craniotomy window was reversibly closed with a skin flap. Mitosis of the individual cancer cells were imaged dynamically in real time through the craniotomy-open window. This model can be used to evaluate brain metastasis and brain cancer at the subcellular level.
Breast cancer and melanoma cells commonly metastasize to the brain using homing mechanisms that are poorly understood. Cancer patients with brain metastases display poor prognosis and survival due to the lack of effective therapeutics and treatment strategies. Recent work using intravital microscopy and preclinical animal models indicates that metastatic cells colonize the brain, specifically in close contact with the existing brain vasculature. However, it is not known how contact with the vascular niche promotes microtumor formation. Here, we investigate the role of connexins in mediating early events in brain colonization using transparent zebrafish and chicken embryo models of brain metastasis. We provide evidence that breast cancer and melanoma cells utilize connexin gap junction proteins (Cx43, Cx26) to initiate brain metastatic lesion formation in association with the vasculature. RNAi depletion of connexins or pharmacological blocking of connexin-mediated cell-cell communication with carbenoxolone inhibited brain colonization by blocking tumor cell extravasation and blood vessel co-option. Activation of the metastatic gene twist in breast cancer cells increased Cx43 protein expression and gap junction communication, leading to increased extravasation, blood vessel co-option and brain colonization. Conversely, inhibiting twist activity reduced Cx43-mediated gap junction coupling and brain colonization. Database analyses of patient histories revealed increased expression of Cx26 and Cx43 in primary melanoma and breast cancer tumors, respectively, which correlated with increased cancer recurrence and metastasis. Together, our data indicate that Cx43 and Cx26 mediate cancer cell metastasis to the brain and suggest that connexins might be exploited therapeutically to benefit cancer patients with metastatic disease.
Currently-used rodent tumor models, including transgenic tumor models, or subcutaneously growing tumors in mice, do not sufficiently represent clinical cancer. We report here development of methods to obtain a highly clinically-accurate rectal cancer model. This model was established by intrarectal transplantation of mouse rectal cancer cells, stably expressing green fluorescent protein (GFP), followed by disrupting the epithelial cell layer of the rectal mucosa by instilling an acetic acid solution. Early-stage tumor was detected in the rectal mucosa by 6 days after transplantation. The tumor then became invasive into the submucosal tissue. The tumor incidence was 100% and mean volume (±SD) was 1232.4 ± 994.7 mm(3) at 4 weeks after transplantation detected by fluorescence imaging. Spontaneous lymph node metastasis and lung metastasis were also found approximately 4 weeks after transplantation in over 90% of mice. This rectal tumor model precisely mimics the natural history of rectal cancer and can be used to study early tumor development, metastasis, and discovery and evaluation of novel therapeutics for this treatment-resistant disease.
We have previously developed a telomerase-specific replicating adenovirus expressing GFP (OBP-401), which can selectively label tumors in vivo with GFP. Intraperitoneal (i.p.) injection of OBP-401 specifically labeled peritoneal tumors with GFP, enabling fluorescence visualization of the disseminated disease and real-time fluorescence surgical navigation. However, the technical problems with removing all cancer cells still remain, even with fluorescence-guided surgery. In this study, we report imaging of tumor recurrence after fluorescence-guided surgery of tumors labeled in vivo with the telomerase-dependent, GFP-containing adenovirus OBP-401.. Recurrent tumor nodules brightly expressed GFP, indicating that initial OBP-401-GFP labeling of peritoneal disease was genetically stable, such that proliferating residual cancer cells still express GFP. In situ tumor labeling with a genetic reporter has important advantages over antibody and other non-genetic labeling of tumors, since residual disease remains labeled during recurrence and can be further resected under fluorescence guidance.
Adrenal pseudocysts are rare cystic masses that arise from the adrenal gland and which are usually non-functional and asymptomatic. Adrenal pseudocysts consist of a fibrous wall without an epithelial or endothelial lining. We report the case of a patient with a giant adrenal pseudocyst presenting with right hypochondralgia and high fever.
We demonstrate the development of a long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular. To observe cytoplasmic and nuclear dynamics of cancer cells in the living mouse, 143B human osteosarcoma cells are labeled with green fluorescent protein in the nucleus and red fluorescent protein in the cytoplasm. These dual-color cells are injected by a vascular route in an abdominal skin flap in nude mice. The mice are then imaged with the Olympus MVX10 macroview fluorescence microscope. With the MVX10, the nuclear and cytoplasmic behavior of cancer cells trafficking in blood vessels of live mice is observed. We also image lung metastases in live mice from the macro- to the subcellular level by opening the chest wall and imaging the exposed lung in live mice. Injected splenocytes, expressing cyan fluorescent protein, could also be imaged on the lung of live mice. We demonstrate that the MVX10 microscope offers the possibility of full-range in vivo fluorescence imaging from macro- to subcellular and should enable widespread use of powerful imaging technologies enabled by genetic reporters and other fluorophores.
Cancer cells, with and without fluorescent protein expression, were irradiated with various doses of UVC (100, 400, and 600 J/m(2)). Dual-color Lewis lung carcinoma cells (LLC) and U87 human glioma cells, expressing GFP in the nucleus and RFP in the cytoplasm and non-colored LLC and U87 cells were cultured in 96-well plates. Eight hours after seeding, the cells were irradiated with the various doses of UVC. The resulting cell number was determined after 24 hours. Compared to non-colored LLC cells, the number of dual-color LLC cells decreased significantly due to UVC irradiation with 100 J/m(2) (p=0.003). Although there was no significant difference in the number of dual-color and non-colored U87 cells after 100 J/m(2) UVC irradiation (p=0.852), the number of dual-color U87 cells decreased significantly with respect to non-colored cells due to UVC irradiation with 400 J/m(2) and 600 J/m(2) (p=0.011 and p=0.009, respectively). Thus, both dual-color LLC and dual-color U87 cells were more sensitive to UVC light than non-colored LLC and U87 cells. These results suggest that the expression of fluorescent proteins in cancer cells can enhance photodynamic therapy (PDT) using UVC and possibly with other wavelengths of light as well.
Integrins play a role in tumor growth and metastasis. However, the effect of integrin inhibition has not been visualized on single cancer cells in vivo. In this study, we used a powerful subcellular in vivo imaging model to demonstrate how an anti-integrin antibody affects seeding and growth of osteosarcoma cells on the lung. The 143B human osteosarcoma cell line, expressing red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus, was established. Such double-labeled cells enable imaging of apoptosis and mitosis and other nuclear-cytoplasmic dynamics. Using the double-labeled osteosarcoma cells, single cancer-cell seeding in the lung after i.v. injection of osteosarcoma cells was imaged. The anti-?1 integrin monoclonal antibody, AIIB2, greatly inhibited the seeding of cancer cells on the lung (experimental metastasis) while a control antibody had no effect. To image the efficacy of the anti-integrin antibody on spontaneous metastasis, mice with orthotopically-growing 143B-RFP cells in the tibia were also treated with AIIB2 or control anti-rat IgG1 antibody. After 3 weeks treatment, mice were sacrificed and primary tumors and lung metastases were evaluated with fluorescence imaging. AIIB2 significantly inhibited spontaneous lung metastasis but not primary tumor growth, possibly due to inhibition of lung seeding of the cancer cells as imaged in the experimental metastasis study. AIIB2 treatment also increased survival of mice with orthotopically growing 143B-RFP.
Malignant glioma tumors are the most common primary central nervous system tumors. Despite the multidisciplinary approach to treatment, prognosis remains poor. In this study, we demonstrated that the Salmonella typhimurium A1-R tumor-targeting strain can inhibit and eradicate human glioma in an orthotopic nude-mouse model. S. typhimurium A1-R was administered by injection through a craniotomy open-window or intravenously in nude mice. To establish the model, 2x10(5) U87-RFP human glioma cells were injected stereotactically into the mouse brain through the craniotomy open window. Two weeks after glioma-cell implantation, mice were treated with S. typhimurium A1-R [2x10(7) CFU/200 ?l intravenous injection (i.v.) or 1x10(6) CFU/1 ?l intracranial injection (i.c.)] once a week for 3 weeks. Brain tumors were observed by fluorescence imaging through the craniotomy open window over time. S. typhimurium A1-R, administered i.c., inhibited brain tumor growth 7.6-fold compared with untreated mice (p=0.009) and improved survival 73% (p=0.001). Two of ten mice appeared to have their tumors eradicated. Intravenous administration of S. typhimurium A1-R was not effective. The craniotomy open window enabled observation of tumor growth in the brain in real time in both treated and untreated mice. The results of the present study demonstrate that bacterial therapy of brain cancer is a novel, effective and safe treatment strategy in a highly treatment-resistance cancer.
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