Exosomes are thought to play an important role in metastasis. Luga and colleagues have described the production of exosomes by stromal cells such as cancer-associated fibroblasts that are taken up by breast cancer cells and are then loaded with Wnt 11, which is associated with stimulation of the invasiveness and metastasis of the breast cancer cells. Previous studies have shown that exosomes produced by breast cancer cells are taken up by stromal fibroblasts and other stromal cells, suggesting that exosomes are agents of cross-talk between cancer and stromal cells to stimulate metastasis. Imaging of exosomes by labeling with fluorescent proteins will enlighten the process by which exosomes enhance metastasis, including premetastatic niche formation.
To determine whether acute acorbic acid infusions alters the effect of hyperglycemia on endothelial function in adolescents with type 1 diabetes.
Research Design and Methods
The forearm blood flow (FBF) reactive hyperemic response to 5 min of upper arm occlusion was studied in 8 adolescents with type 1 diabetes during euglycemic and hyperglycemic insulin clamp (40 mU/m2/min) with and without ascorbic acid infusion (3 mg/min).
The ratio of post-occlusion to pre-occlusion FBF decreased during hyperglycemia without ascorbic acid (p=0.013) but did not change during hyperglycemia with ascorbic acid. The changes during hyperglycemia were different between the two studies (p=0.038). Similar results were found when the percent change in forearm vascular resistance following occlusion was assessed.
These results indicate that antioxidant treatment with ascorbic acid blocks acute hyperglycemic impairment of endothelial function in adolescents with type 1 diabetes.
While the role of the macular pigment carotenoids in the prevention of age-related macular degeneration has been extensively studied in adults, comparatively little is known about the physiology and function of lutein and zeaxanthin in the developing eye. We therefore developed a protocol using a digital video fundus camera (RetCam) to measure macular pigment optical density (MPOD) and distributions in premature infants and in children.
We used blue light reflectance to image the macular pigment in premature babies at the time of retinopathy of prematurity (ROP) screening and in children aged under 7 years who were undergoing examinations under anesthesia for other reasons. We correlated the MPOD with skin carotenoid levels measured by resonance Raman spectroscopy, serum carotenoids measured by HPLC, and dietary carotenoid intake.
We enrolled 51 infants and children ranging from preterm to age 7 years. MPOD correlated significantly with age (r = 0.36; P = 0.0142), with serum lutein + zeaxanthin (r = 0.44; P = 0.0049) and with skin carotenoid levels (r = 0.42; P = 0.0106), but not with dietary lutein + zeaxanthin intake (r = 0.13; P = 0.50). All premature infants had undetectable macular pigment, and most had unusually low serum and skin carotenoid concentrations.
Our most remarkable finding is the undetectable MPOD in premature infants. This may be due in part to foveal immaturity, but the very low levels of serum and skin carotenoids suggest that these infants are carotenoid insufficient as a consequence of low dietary intake and/or severe oxidative stress. The potential value of carotenoid supplementation in the prevention of ROP and other disorders of prematurity should be a fruitful direction for further investigation.
Little is known about the physiology and function of lutein and zeaxanthin in the developing eye. We therefore developed a protocol using a digital fundus camera (RetCam) to measure macular pigment optical density (MPOD) and distributions in premature infants and in children.
macular pigment; carotenoid; imaging; lutein; zeaxanthin
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 mm3 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.
Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in two additional individuals. These recurrent mutations all occur within or in very close proximity to sites that regulate mtDNA replication, strongly implying that these variations alter the replication dynamics of the mutated mtDNA genome. These recurrent variants are all independent of each other and do not occur in the mtDNA coding regions. The most parsimonious explanation of the data is that these frequently repeated mutations experience tissue-specific positive selection, probably through replication advantage.
DNA mutations are expected to be formed randomly, thus any reproducible pattern of DNA somatic mutations across multiple individuals or even across organs within each individual is highly unexpected. Using next generation sequencing of multiple tissues from the same individuals we found several somatic mutations in mitochondrial DNA that appear in a heteroplasmic state in all individuals examined, but only in particular tissues. These mutations were only found in known regions of replication control for the mitochondrial DNA. These data imply the presence of tissue-specific positive selection for these variants.
This study examines the effects of types of liver resection on the growth of liver and lung metastasis.
Materials and Methods
Experimental liver metastases were established by spleen injection of the Colon 26 murine adenocarcinoma cell line expressing GFP into transgenic nude mice expressing RFP. Experimental lung metastases were established by tail vein injection with Colon 26-GFP. Three days after cell injection, groups of mice underwent liver resection (35%+35% [repeated minor resection] vs. 70% [major resection]). Metastatic tumor growth was measured by color-coded fluorescence imaging of the GFP-expressing cancer cells and RFP-expressing stroma.
Although major and repeated minor resection removed the same volume of liver parenchyma, the two procedures had very different effects on metastatic tumor growth: major resection, stimulated liver and lung metastatic growth as well as recruitment of host-derived stroma compared to repeated minor resection. Repeated minor resection did not stimulate metastasis or stromal recruitment. There was no significant difference in liver regeneration between the two groups. Host-derived stroma density, which is stimulated by major resection compared to repeated minor resection, may stimulate growth in the liver-metastatic tumor. TGF-β is also preferentially stimulated by major resection and may play a role in stroma and metastasis stimulation.
The results of this study indicate that when liver resection is necessary, repeated minor liver resection is superior to major liver resection, since major resection, in contrast to repeated minor resection, stimulates metastasis, which should be taken into consideration in clinical situations indicating liver resection.
Nude mice; liver resection; lung metastasis; liver metastasis; stroma; green fluorescent protein; red fluorescent protein; color-coded imaging
We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells. Nestin-expressing cells were initially identified in the hair follicle bulge area (BA) using a transgenic mouse model in which the nestin promoter drives the green fluorescent protein (ND-GFP). The hair-follicle ND-GFP-expressing cells are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. Subsequently, we showed that the nestin-expressing stem cells could affect nerve and spinal cord regeneration after injection in mouse models. In the present study, we separated the mouse vibrissa hair follicle into three parts (upper, middle and lower). Each part of the follicle was cultured separately in DMEM-F12 containing B-27 and 1% methylcellulose supplemented with basic FGF. After 2 mo, the nestin-expressing cells from each of the separated parts of the hair follicle proliferated and formed spheres. Upon transfer of the spheres to RPMI 1640 medium containing 10% FBS, the nestin-expressing cells in the spheres differentiated to neurons, as well as glia, keratinocytes, smooth muscle cells and melanocytes. The differentiated cells were produced by spheres which formed from nestin-expressing cells from all segments of the hair follicle. However, the differentiation potential is greatest in the upper part of the follicle. This result is consistent with trafficking of nestin-expressing cells throughout the hair follicle from the bulge area to the dermal papilla that we previously observed. The nestin-expressing cells from the upper part of the follicle produced spheres in very large amounts, which in turn differentiated to neurons and other cell types. The results of the present study demonstrate that multipotent, nestin-expressing stem cells are present throughout the hair follicle and that the upper part of the follicle can produce the stem cells in large amounts that could be used for nerve and spinal cord repair.
hair follicle; bulge area; nestin; outer-root sheath; stem cell; green fluorescent protein; differentiation; neuron
Although alcohol use has long been a significant cause of hospital presentations, little is published regarding the long-term demographic changes that have occurred at a single hospital site. To address this deficit, we prospectively studied all acute alcohol-related presentations to Bellevue Hospital Center (New York, NY) and compared this contemporary data set with one from the same institution from 1902 to 1935. We prospectively identified all patients presenting to the emergency department because of acute alcohol use over an 8-week period in 2009. We described the basic attributes of patients presenting currently because of alcohol and compared these data to those previously described between 1902 and 1935. We also compared our census data with contemporaneous data from all patients presenting to this hospital site. During the study period, 560 patients presented because of acute alcohol use which extrapolated to an estimated 3,800 patients over the calendar year. This compares to 7,600 presentations recorded annually early in the twentieth century. Twelve percent of patients in 2009 were female as compared to 18 % of patients between 1934 and 1935. Patients with alcohol-related presentations in 2009 were more likely to be admitted than contemporaneous patients without an alcohol-related presentation (30 vs. 19 % admitted; p < 0.001). Since first measured 110 years ago at one large New York City hospital, alcohol-related presentations remain common representing 5 % of all emergency department visits. This demonstrates alcoholism's continuing toll on society's limited medical resources and on public health as a whole.
Alcohol; Demographics; Emergency department; Emergency medicine; Urban
Mesenchymal stromal cells (MSCs) are multipotent adult stem cells which are recruited to the tumor microenvironment (TME) and influence tumor progression through multiple mechanisms. In this study, we examined the effects of MSCs on the tunmorigenic capacity of 4T1 murine mammary cancer cells. It was found that MSC-conditioned medium increased the proliferation, migration, and efficiency of mammosphere formation of 4T1 cells in vitro. When co-injected with MSCs into the mouse mammary fat pad, 4T1 cells showed enhanced tumor growth and generated increased spontaneous lung metastasis. Using in vivo fluorescence color-coded imaging, the interaction between GFP-expressing MSCs and RFP-expressing 4T1 cells was monitored. As few as five 4T1 cells could give rise to tumor formation when co-injected with MSCs into the mouse mammary fat pad, but no tumor was formed when five or ten 4T1 cells were implanted alone. The elevation of tumorigenic potential was further supported by gene expression analysis, which showed that when 4T1 cells were in contact with MSCs, several oncogenes, cancer markers, and tumor promoters were upregulated. Moreover, in vivo longitudinal fluorescence imaging of tumorigenesis revealed that MSCs created a vascularized environment which enhances the ability of 4T1 cells to colonize and proliferate. In conclusion, this study demonstrates that the promotion of mammary cancer progression by MSCs was achieved through the generation of a cancer-enhancing microenvironment to increase tumorigenic potential. These findings also suggest the potential risk of enhancing tumor progression in clinical cell therapy using MSCs. Attention has to be paid to patients with high risk of breast cancer when considering cell therapy with MSCs.
The effects of bacteria on cancer patients have been observed for at least two centuries. Recent studies in animal models of cancer have demonstrated efficacy of both anaerobic bacteria such as Clostridia and Bifidobacteria and facultative anaerobes such as Salmonella. In this issue of Cancer Discovery, Flentie et al have identified five Salmonella promoters that are specifically stimulated by cancer cells as well as by acid pH, a property of most tumors. One of these promoters (STM1787) was linked to a Shiga toxin gene and inserted in a wild-type Salmonella typhimurium strain, which showed in vivo antitumor efficacy. Approaches to further improving the efficacy of S. typhimurium with the use of tumor-targeting mutations are discussed. Since the barriers to efficacy of standard therapy of cancer appear to be opportunities for bacterial cancer therapy, the future of bacterial therapy of cancer appears bright.
Negative surgical margins are vital to achieve cure and prolong survival in patients with pancreatic cancer. We inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes and reduce recurrence rates in orthotopic mouse models of human pancreatic cancer.
A randomized active-control pre-clinical trial comparing bright light surgery (BLS) to fluorescence-guided surgery (FGS) was utilized. Orthotopic mouse models of human pancreatic cancer were established using the BxPC-3 pancreatic cancer cell line expressing red fluorescent protein (RFP). Two weeks after orthotopic implantation, tumors were resected with BLS or FGS. Pre- and postoperative images were obtained with the OV-100 Small Animal Imaging System to assess completeness of surgical resection. Postoperatively, whole body imaging was done to assess recurrence and follow tumor progression. Six weeks postoperatively, mice were sacrificed to evaluate primary pancreatic and metastatic tumor burden.
A more complete resection of pancreatic cancer was achieved using FGS compared to BLS: 98.9% vs. 77.1%, p=0.005. The majority of mice undergoing BLS (63.2%) had evidence of gross disease with no complete resections, whereas 20% of mice undergoing FGS had complete resection and an additional 75% had only minimal residual disease (p=0.0001). The mean postoperative tumor burden was significantly less with FGS compared to BLS: 0.08 ± 0. 06 mm2 vs. 2.64 ± 0.63 mm2, p=0.001. The primary tumor burden at termination was significantly less with FGS compared to BLS: 19.3 ± 5.3 mm2 vs. 6.2 ± 3.6 mm2, p=0.048. FGS resulted in significantly longer disease-free survival than BLS (p=0.02, HR=0.39, 95% CI: (0.17, 0.88)).
Surgical outcomes were improved in pancreatic cancer using fluorescence-guidance. This novel approach has significant potential to improve surgical treatment of cancer.
pancreatic cancer; orthotopic mouse models; surgery; fluorescence; resection; recurrence; survival
Drug overdose is a leading cause of cardiac arrest and is currently the second leading cause of overall injury-related fatality in the United States. Despite these statistics, the incidence of adverse cardiovascular events (ACVEs) in emergency department (ED) patients following acute drug overdose is unknown. With this study, we address the 2010 American Heart Association Emergency Cardiovascular Care update calling for research to characterize the incidence of in-hospital ACVE following drug overdose.
This was a prospective cohort study at two tertiary care hospitals over 12 months. Consecutive adult ED patients with acute drug overdose were prospectively followed to hospital discharge. The main outcome was occurrence of in-hospital ACVE, defined as the occurrence of one or more of the following: myocardial injury, shock, ventricular dysrhythmia, and cardiac arrest.
There were 459 ED patients with suspected drug overdose, of whom 274 acute drug overdose qualified and were included for analysis (mean [±SE] age = 40.3 [±1.0] years; 63% male). Hospital course was complicated by ACVE in 16 patients (some had more than one): 12 myocardial injury, three shock, two dysrhythmia, and three cardiac arrest. The incidence of ACVE was 5.8% overall (95% confidence interval [CI] = 3.6% to 9.3%) and 10.7% (95% CI = 6.6% to 16.9%) among inpatient admissions, with all-cause mortality at 0.7% (95% CI = 0.2% to 2.6%).
Based on this study of adult patients with acute drug overdose, ACVE may occur in up to 9.3% overall and up to 16.9% of hospital admissions. Implications for the evaluation and triage of ED patients with acute drug overdose require further study with regard to optimizing interventions to prevent adverse events.
In psoriasis, inflammation and epidermal hyperplasia are thought to be controlled by T cell-derived cytokines. Evidence suggests that the Th17 cell cytokine interleukin-17 (IL-17) may play a role in disease pathogenesis.
To understand the impact that neutralization of IL-17 has on the clinical features of psoriasis and to understand the role that IL-17 has in inflammatory pathways underlying psoriasis in human subjects.
We examined skin lesions obtained from 40 subjects participating in a phase 1, randomized, double-blind, placebo-controlled trial of an anti-IL-17 monoclonal antibody, ixekizumab (previously LY2439821), in which subjects received subcutaneous 5mg, 15mg, 50mg or 150mg ixekizumab or placebo at weeks 0, 2, and 4.
There were significant, dose-dependent reductions from baseline in keratinocyte proliferation, hyperplasia, epidermal thickness, infiltration into the dermis and epidermis by T cells and dendritic cells and keratinocyte expression of innate defense peptides at 2 weeks. By week 6, the skin appeared normal. Quantitative reverse transcriptase polymerase chain reaction and microarrays revealed an ablation of the disease-defining mRNA expression profile by 2 weeks after the first dose of study drug. The effect of IL-17 blockade on expression of genes synergistically regulated by IL-17 and Tumor necrosis factor (TNF) was of higher magnitude at 2 weeks than in prior studies with TNF antagonism.
Our data suggest that IL-17 is a key “driver” cytokine in psoriasis that activates pathogenic inflammation. Neutralizing IL-17 with ixekizumab may be a successful therapeutic strategy.
Psoriasis; interleukin -17; Th17 cells; TNF
Pancreatic-cancer-patient tumor specimens were initially established subcutaneously in SCID-NOD mice immediately after surgery. The patient tumors were then harvested from SCID-NOD mice and passaged orthotopically in transgenic nude mice ubiquitously expressing RFP. The primary patient tumors acquired RFP-expressing stroma. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Further passage to transgenic nude mice ubiquitously expressing GFP resulted in tumors and metastasis that acquired GFP stroma in addition to their RFP stroma, including CAFs and TAMs and blood vessels. The RFP stroma persisted in the tumors growing in the GFP mouse. Further passage to transgenic nude mice ubiquitously expressing CFP resulted in tumors and metastasis acquiring CFP stroma in addition to persisting RFP and GFP stroma including RFP- and GFP-expressing CAFs and TAMs and blood vessels. This model can be used to image primary and metastatic progression of patient pancreatic tumors to visually target stroma as well as cancer cells and individualize therapy.
human-patient pancreas cancer; imaging; tumor microenvironment; GFP; RFP; CFP
We have previously reported that hair follicles contain multipotent stem cells which express nestin. The nestin-expressing cells form the hair follicle sensory nerve. In vitro, the nestin-expressing hair follicle cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, the sciatic nerve was excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP mice). The ND-GFP cells of the sciatic nerve were also found to be multipotent as the ND-GFP cells in the hair follicle. When the ND-GFP cells in the mouse sciatic nerve cultured on Gelfoam® and were imaged by confocal microscopy, they were observed forming fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker β-III tubulin, the immature Schwann-cell marker p75NTR and TrkB which is associated with neurons. The fibers also contain nestin-negative spherical cells expressing GFAP, a Schwann-cell marker. The β-III tubulin-positive fibers had growth cones on their tips expressing F-actin, indicating they are growing axons. When the sciatic nerve from mice ubiquitously expressing red fluorescent protein (RFP) was co-cultured on Gelfoam® with the sciatic nerve from ND-GFP transgenic mice, the interaction of nerves was observed. Proliferating nestin-expressing cells in the injured sciatic nerve were also observed in vivo. Nestin-expressing cells were also observed in posterior nerves but not in the spinal cord itself, when placed in 3-D Gelfoam® culture. The results of the present report suggest a critical function of nestin-expressing cells in peripheral nerve growth and regeneration.
Stickler syndrome is a heterogeneous condition due to mutations in COL2A1, COL11A1, COL11A2, and COL9A1. To our knowledge, neither non-penetrance nor mosaicism for COL2A1 mutations has been reported for Stickler syndrome. We report on a family with two clinically affected sibs with Stickler syndrome who have clinically unaffected parents. Both sibs have a novel heterozygous mutation in exon 26 of COL2A1 (c.1525delT); this results in a premature termination codon downstream of the mutation site. One parent was found to have low level mosaicism in DNA extracted from whole blood. This scenario encourages consideration of molecular testing in seemingly unaffected parents for recurrence risks and potential screening for mild age-related manifestations.
Stickler syndrome; Mosaicism; COL2A1; Recurrence risk
The aim of this study was to improve fluorescence laparoscopy of pancreatic cancer in an orthotopic mouse model with the use of an LED light source and an optimal fluorophore combination.
Human pancreatic cancer models were established with fluorescent FG-RFP, MiaPaca2-GFP, BxPC-3-RFP, and BxPC-3 cancer cells implanted in 6-week-old female athymic mice. Two weeks post-implantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source or a Stryker X8000 xenon light source 24 hours after tail vein injection of CEA antibodies conjugated with Alexa 488 or Alexa 555. Cancer lesions were detected and localized under each light mode. Intravital images were obtained with the OV-100 Olympus Small Animal Imaging System and Maestro CRI Small Animal Imaging System, serving as a positive control. Tumors were collected for histologic review.
Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor deposits in the peritoneal cavity. The simultaneous use of different fluorophores (Alexa 488 and Alexa 555) conjugated to antibodies brightened the fluorescence signal, enhancing detection of sub-millimeter lesions without compromising background illumination. Adjustments to the LED light source permitted simultaneous detection of tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence.
Using an LED light source with adjustments to the red, blue and green wavelengths, we can simultaneously identify tumor metastases expressing fluorescent proteins of different wavelengths, which greatly enhanced the signal without compromising background illumination. Development of this technology for clinical use can improve staging and treatment of pancreatic cancer.
pancreatic cancer; orthotopic mouse models; fluorescence; laparoscopy; LED light source
Optic pathway gliomas (OPGs) occur in 15%–20% of children with neurofibromatosis type 1 (NF1); up to half become symptomatic. There is little information regarding ophthalmologic outcomes after chemotherapy. A retrospective multicenter study was undertaken to evaluate visual outcomes following chemotherapy for NF1-associated OPG, to identify risks for visual loss, and to ascertain indications for treatment. Subjects included children undergoing initial treatment for OPGs with chemotherapy between January 1997 and December 2007. Of 115 subjects, visual acuity (VA) decline and tumor progression were the primary reasons to initiate treatment, although there were significant differences in the pattern of indications cited among the institutions. Eighty-eight subjects and 168 eyes were evaluable for VA outcome. At completion of chemotherapy, VA improved (32% of subjects), remained stable (40%), or declined (28%). Tumor location was the most consistent prognostic factor for poor VA outcome. There was poor correlation between radiographic and VA outcomes. Although visual outcomes for NF1-associated OPG are not optimal, approximately one-third of children regain some vision with treatment. Since radiographic outcomes do not predict visual outcomes, their use as the primary measure of treatment success is in question. The lack of consensus regarding the indications for treatment underlines the need for better standardization of care. Future clinical trials for OPG require standardized visual assessment methods and clear definitions of visual outcomes.
neurofibromatosis; optic glioma; outcomes; visual acuity
Early biomarkers and effective therapeutic strategies are desperately needed to treat pancreatic ductal adenocarcinoma (PDAC), which has a dismal 5-year patient survival rate. Here, we report that the novel tyrosine kinase PEAK1 is upregulated in human malignancies, including human PDACs and pancreatic intraepithelial neoplasia (PanIN). Oncogenic KRas induced a PEAK1-dependent kinase amplification loop between Src, PEAK1, and ErbB2 to drive PDAC tumor growth and metastasis in vivo. Surprisingly, blockade of ErbB2 expression increased Src-dependent PEAK1 expression, PEAK1-dependent Src activation, and tumor growth in vivo, suggesting a mechanism for the observed resistance of patients with PDACs to therapeutic intervention. Importantly, PEAK1 inactivation sensitized PDAC cells to trastuzumab and gemcitabine therapy. Our findings, therefore, suggest that PEAK1 is a novel biomarker, critical signaling hub, and new therapeutic target in PDACs.
There are many challenges that face surgeons when attempting curative resection for gastrointestinal cancers. The ability to properly delineate tumor margins for complete resection is of utmost importance in achieving cure and giving the patient the best chance of prolonged survival. Targeted tumor imaging techniques have gained significant interest in recent years to enable better identification of tumor lesions to improve diagnosis and treatment of cancer from preoperative staging modalities to optimizing the surgeon's ability to visualize tumor margins at the initial operation. Using unique characteristics of the tumor to fluorescently label the tissue can delineate tumor margins from normal surrounding tissue, allowing improved precision of surgical resection. In this paper, different methods of fluorescently labeling native tumor are discussed as well as the development of fluorescence laparoscopy and the potential role for fluorescence-guided surgery in the treatment of gastrointestinal cancers.
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, 2 × 105 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 [2 × 107 CFU/200 µl intravenous injection (i.v.) or 1 × 106 CFU/1 µl 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.
Salmonella typhimurium A1-R; fluorescent proteins; brain cancer; mouse model; in vivo imaging
Salmonella typhimurium double leu-arg auxotrophs have been shown to be highly effective as antitumor agents in nude mouse models of human metastatic cancer. In order to proceed to clinical development of the S. typhimurium double auxotroph, termed A1-R, it is necessary to evaluate antitumor efficacy in immunocompetent mice. In the present study, we have observed the efficacy of A1-R on the Lewis lung (LLC) carcinoma in vitro as well as in C57BL/6 (C57) immunocompetent mice. In vitro, A1-R treatment of LLC began to induce cell death within one hour. Various doses and schedules of A1-R were administered to C57 mice implanted with LLC, including bolus single intravenous injection; medium dose with weekly intravenous administration and metronomic treatment with small intravenous doses twice a week. Bolus treatment was toxic to the immunocompetent host in contrast to nude mice. Lower-dose weekly doses and metronomic doses were well-tolerated by the immunocompetent host. Weekly intravenous injection with 2 × 107 bacteria and twice a week intravenous injection with 107 bacteria significantly inhibited metastasis formation, while bolus injection was toxic. Intrathoracic administration was performed with 108 A1-R bacteria injected into Lewis lung-bearing C57 mice weekly for three weeks. Lung metastasis was significantly inhibited by intrathoracic bacterial administration without toxicity. The results in this report, demonstrating the anti-metastatic efficacy of S. typhimurium A1-R in immunocompetent mice, indicate the clinical potential of bacterial therapy of cancer.
Salmonella typhimurium; amino acid auxotroph; selective tumor targeting; lung; metastasis; RFP; GFP; fluorescence imaging; confocal microscopy
Ovarian cancer relapse is often characterized by metastatic spread throughout the peritoneal cavity with tumors attached to multiple organs. In this study, interaction of ovarian cancer cells with the peritoneal tumor microenvironment was evaluated in a xenograft model based on intraperitoneal injection of fluorescent SKOV3.ip1 ovarian cancer cells. Intra-vital microscopy of mixed GFP-red fluorescent protein (RFP) cell populations injected into the peritoneum demonstrated that cancer cells aggregate and attach as mixed spheroids, emphasizing the importance of homotypic adhesion in tumor formation. Electron microscopy provided high resolution structural information about local attachment sites. Experimental measurements from the mouse model were used to build a three-dimensional cellular Potts ovarian tumor model (OvTM) that examines ovarian cancer cell attachment, chemotaxis, growth, and vascularization. OvTM simulations provide insight into the relative influence of cancer cell–cell adhesion, oxygen availability, and local architecture on tumor growth and morphology. Notably, tumors on the mesentery, omentum, or spleen readily invade the “open” architecture, while tumors attached to the gut encounter barriers that restrict invasion and instead rapidly expand into the peritoneal space. Simulations suggest that rapid neovascularization of SKOV3.ip1 tumors is triggered by constitutive release of angiogenic factors in the absence of hypoxia. This research highlights the importance of cellular adhesion and tumor microenvironment in the seeding of secondary ovarian tumors on diverse organs within the peritoneal cavity. Results of the OvTM simulations indicate that invasion is strongly influenced by features underlying the mesothelial lining at different sites, but is also affected by local production of chemotactic factors. The integrated in vivo mouse model and computer simulations provide a unique platform for evaluating targeted therapies for ovarian cancer relapse.
ovarian cancer; tumor modeling; tumor microenvironment; metastasis; cellular Potts model; cell adhesion; angiogenesis; chemotaxis
To address the role of β1 integrins in pancreatic cancer progression, we stably knocked down β1 integrin subunit expression in human FG-RFP pancreatic cancer cells using lentiviral-based RNA interference. We then examined the effects of β1 integrin subunit knockdown on pancreatic cancer cell adhesion, migration, and proliferation on tumor microenvironment-specific ECM proteins in vitro, and on tumor progression in vivo using a clinically-relevant fluorescent orthotopic mouse model of pancreatic cancer. Knockdown of the β1 integrin subunit inhibited cell adhesion, migration, and proliferation on types I and IV collagen, fibronectin, and laminin in vitro. In vivo, knockdown of the β1 integrin subunit reduced primary tumor growth by 50% and completely inhibited spontaneously occurring metastasis. These observations indicate a critical role for the β1 integrin subunit in pancreatic cancer progression, and metastasis in particular. Our results suggest the β1 integrin subunit as a therapeutic target for the treatment of pancreatic cancer, especially in the adjuvant setting to prevent metastasis of this highly aggressive cancer.
integrins; extracellular matrix; pancreatic cancer; orthotopic mouse models; RNA interference