Comparison of the effects of adriamycin and methotrexate on orthotopic and induced heterotopic bone in rats

The effect of the two antineoplastic drugs, Adriamycin and methotrexate, on orthotopic bone, and on the induction of experimental heterotopic bone in rats was analyzed. The drugs were administered as single injections: Adriamycin in s.c. doses of 0.5 and 2 mg/kg body weight and methotrexate i.v. 100 and 250 mg/kg body weight followed by leucovorin rescue after 2 h. A passing, but significant, decrease in body weights occurred in the methotrexate-treated animals, but not in those given Adriamycin. Analysis of the amount of heterotopic bone formed 4 weeks after induction by demineralized bone matrix revealed a 30-40% decrease in the groups treated with either of the antineoplastic agents, whereas orthotopic bone was unaffected. Six weeks after the treatment the net effect on the induced bone had decreased. The present study shows that the two antineoplastic drugs Adriamycin and methotrexate inhibit heterotopic new bone formation induced by demineralized bone matrix in rats to an equal extent, although their mode of action on the cellular level is entirely different, and that the inhibitory effect of a single treatment diminishes in the presence of a continuous inductive process.     

Real-time cancer cell tracking by bioluminescence in a preclinical model of human bladder cancer growth and metastasis

Background

Bladder cancer is the fifth most common malignancy in the Western world and the second most frequently diagnosed genitourinary tumor. In the majority of cases, death from bladder cancer results from metastatic disease. Understanding the multistep process of carcinogenesis and metastasis in urothelial cancers is pivotal to the development of new therapeutic strategies. Molecular imaging of cancer growth and metastasis in preclinical models provides the essential link between cell-based experiments and clinical translation.

Objective

Develop preclinical models for sensitive bladder cancer cell tracking during tumor progression and metastasis.

Design, setting, and participants

A human transitional cell carcinoma UM-UC-3 cell line was generated that stably expresses luciferase 2 (UM-UC-3luc2), a mammalian codon-optimized firefly luciferase with superior expression. Preclinical models were developed with human UM-UC-3luc2 cells xenografted into the bladder (orthotopic model with metastases) or inoculated into the left cardiac ventricle (bone metastasis model) of immunocompromised mice.

Measurements

Noninvasive, sensitive bioluminescent imaging of human firefly luciferase 2-positive bladder cancer in mice using the IVIS100 imaging system.

Results and limitations

In the orthotopic model (intravesical inoculation), tumor growth could be followed directly after inoculation of UM-UC-3luc2 cells. Importantly, micrometastatic lesions originating from orthotopically implanted cancer cells could be detected in the locoregional lymph nodes and in distant organs. In addition, the superior bioluminescent indicator firefly luciferase 2 allows the detection and monitoring of micrometastatic lesions in real time after intracardiac inoculation of human bladder cancer cells in mice. The main disadvantage is the lack of T-cell immunity in the preclinical models.

Conclusions

The new bioluminescence-based preclinical bladder cancer models enable superior, noninvasive, and real-time tracking of cancer cells, tumor progression, and micrometastasis. Because of the significant improvement in detection of small cell numbers, the presented models are ideally suited for functional studies dealing with minimal residual disease as well as real-time imaging of drug response.     

Orthotopic Neobladder versus Ileal Conduit Urinary Diversion after Cystectomy – A Quality-of-Life Based Comparison

INTRODUCTION

Radical cystectomy remains the gold standard in treatment of muscle invasive bladder cancer. Evolution of pathological guidelines has empowered centres to offer orthotopic substitution (OBS) to patients undergoing radical cystectomy. We compared health-related quality of life (HRQoL) between patients who underwent OBS or ileal conduit urinary diversion (ICD) following radical cystectomy.

PATIENTS AND METHODS

A total of 57 patients who underwent cystectomy were assessed pre-operatively using Karnofsky performance scale (KPS). Of these, 52 patients (28 OBS and 24 ICD) who responded to a postal questionnaire consisting of SF-36 and a functional index questionnaire were included.

RESULTS

Median age of patients was 70 years. Pre-operative KPS scores were similar. All eight HRQoL scales were favourable in both groups. OBS patients had significantly better physical functioning. In the cohort, 42% of men with OBS and 25% of diversions could maintain an erection to varying degrees. Of the OBS patients, 85% were continent with two patients reporting reduced QoL with pad usage. Of ICD patients, 63% felt less complete and 42% were embarrassed due to the stoma, with 58% apprehensive of stomal leakage. Of OBS patients, 96% had significant relationships and a more active life-style.

CONCLUSIONS

In a similar age-group population, there was no significant difference in most QoL indices but body image issues persist in ICD patients. OBS patients had significantly better physical function, continuing to have a more active life-style. They attained urethral voiding with good continence. A detailed discussion of long-term functional outcome would engender a realistic expectation allowing better adaptation.     

Functional imaging of multidrug resistance in an orthotopic model of osteosarcoma using 99mTc-sestamibi

Purpose The purpose of this work was the development of an orthotopic model of osteosarcoma based on luciferase-expressing tumour cells for the in vivo imaging of multidrug resistance (MDR) with ^99mTc-sestamibi. Methods Doxorubicin-sensitive (143B-luc⁺) and resistant (MNNG/HOS-luc⁺) osteosarcoma cell lines expressing different levels of P-glycoprotein and carrying a luciferase reporter gene were inoculated into the tibia of nude mice. Local tumour growth was monitored weekly by bioluminescence imaging and X-ray. After tumour growth, a ^99mTc-sestamibi dynamic study was performed. A subset of animals was pre-treated with an MDR inhibitor (PSC833). Images were analysed for calculation of ^99mTc-sestamibi washout half-life (t _1/2), percentage washout rate (%WR) and tumour/non-tumour (T/NT) ratio. Results A progressively increasing bioluminescent signal was detected in the proximal tibia after 2 weeks. The t _1/2 of ^99mTc-sestamibi was significantly shorter (p < 0.05) in drug-resistant MNNG/HOS-luc⁺ tumours (t _1/2 = 87.3 ± 15.7 min) than in drug-sensitive 143B-luc⁺ tumours (t _1/2 = 161.0 ± 47.4 min) and decreased significantly with PSC833 (t _1/2 = 173.0 ± 24.5 min, p < 0.05). No significant effects of PSC833 were observed in 143B-luc⁺ tumours. The T/NT ratio was significantly lower (p < 0.05) in MNNG/HOS-luc⁺ tumours than in 143B-luc⁺ tumours at early (1.55 ± 0.22 vs 2.14 ± 0.36) and delayed times (1.12 ± 0.11 vs 1.62 ± 0.33). PSC833 had no significant effects on the T/NT ratios of either tumour. Conclusion The orthotopic injection of tumour cells provides an animal model suitable for functional imaging of MDR. In vivo bioluminescence imaging allows the non-invasive monitoring of tumour growth. The kinetic analysis of ^99mTc-sestamibi washout provides information on the functional activity of MDR related to P-glycoprotein expression and its pharmacological inhibition in osteosarcoma.     

National survey on orthotopic neobladder

Objective A national survey was conducted among the urologists in India to find the preference for urinary diversion after radical cystectomy for muscle invasive carcinoma of the urinary bladder, percentage of neobladder reconstruction, segment of the bowel used, complication rate, need for self-intermittent catherisation on follow up and the survival. Material and methods A detailed questionnaire was mailed to all members of the urological society of India (USI) to find out their preference for urinary diversion following radical cystectomy for muscle invasive carcinoma urinary bladder. For the neobladder reconstruction, they were asked for the type of bowel segment used, complication rate, reoperation rate, need for intermittent clean catheterisation on follow up and 5-year survival. Results A total of 24 institutions responded to the mailed questionnaire. Of all institutions 12 (50%) did not prefer the orthotopic neobladder (ONB) reconstruction. Among the institutions carrying out neobladder reconstruction, majority perform ileal conduit in more than 50% of the cases. Ileum (66.66%) or ileocaecal (16.66%) segment was the choice of bowel segment for most of the urologists. Only three institutions used sigmoid colon. The complications encountered were wound infection (5–25%), burst abdomen (5%), urinary fistulas (3–25%), faecal fistulas (2–5%), bladder neck stenosis (5–15%) and ureterointestinal anastomosis stenosis (5–25%). The reoperation rate was 5–15% with a perioperative mortality of 0.5–3%. Around 10–100% (average 50%) of the patients require intermittent clean catherisation. Only seven institutions could provide 5-year survival rate data. Of these three institutions reported more than 50% and four institutes less than 50% 5-year survival. Conclusion Ileal conduit still remains the urinary diversion of choice following radical cystectomy for muscle invasive carcinoma of the bladder among most of the urologists in India. Orthotopic neobladder reconstruction is practiced only in selected centres. Wound infection, urinary leak and obstruction at ureterointestinal anastomosis are the main complications. Clean intermittent cathaterisation is required at an average of 50% of the patients to ensure complete emptying of the neobladder.     

Techniques for experimental heterotopic and orthotopic tracheal transplantations - When to use which model?

BACKGROUND: Different animal models have been developed to study the pathogenesis and treatment of obliterative airway disease (OAD). Here we describe the techniques of heterotopic and orthotopic tracheal transplantations in the rat, comparing the kinetics of systemic host immune response and of histopathologic OAD development. METHODS: Heterotopic and orthotopic tracheal transplantations were performed in both allogeneic (Brown Norway-to-Lewis) and syngeneic (Lewis-to-Lewis) models. Grafts were harvested after 7, 30, and 60 days post-transplant for histologic evaluation and analysis of host cellular and humoral response. RESULTS: Syngeneic tracheal grafts did not develop luminal obliteration and were morphologically indistinguishable from native tracheas. In heterotopic allografts, airway epithelium was rapidly destroyed and OAD progressed with complete luminal occlusion by 30 days. Orthotopic allografts showed enhanced early infiltration (1298+/-45 vs. 674+/-75 cells/high power field, p<0.001) with concomitant greater day 7 luminal narrowing (45+/-6% vs. 14+/-3%, p<0.001). In this model, donor-type BN epithelium (62+/-17%, 21+/-19%, and 1+/-1% on days 7, 30, and 60) was gradually replaced by recipient-type epithelial cells (2+/-4%, 70+/-22%, and 98+/-2%). OAD developed with circular orientation of cells and connective tissue fibers to 45+/-6% obliteration by day 60. Cellular host response, as determined by IFN-gamma-ELISPOT assay (548+/-132 vs. 402+/-197 spots, p=0.046) and anti-donor alloreactive IgM antibody production (2827+/-148 vs. 1565+/-393 mean channel fluorescence, p<0.001) were significantly stronger in rats bearing orthotopic vs. heterotopic allografts. CONCLUSIONS: The orthotopic tracheal transplantation model may be more representative of OAD found in human lung transplant recipients and we therefore encourage the wider use of this model.     

Health related quality of life after radical cystectomy: Comparison of ileal conduit to continent orthotopic neobladder

Aims

To compare health related quality of life (HRQOL) between patients with two different types of urinary diversion, ileal conduit and orthotopic neobladder, and between them and an age-matched population of healthy subjects.

Materials and methods

Eighty eight patients treated with radical cystectomy for bladder cancer at our institutions between 2002 and 2007 were contacted for this survey. All of them had a follow-up of more than 12 months after surgery and were recurrence free. The SF-36 questionnaire was provided to each patient during a follow-up visit at outpatient clinics. Overall, 79 patients (90%) returned the questionnaire and were included in this analysis. They were divided into two groups: group 1 comprised 44 patients with an ileal conduit diversion, and group 2 included 35 patients with a neobladder. As a control, normative values of an age-matched healthy Italian population were considered.

Results

No significant difference was found in scale scores between the neobladder and ileal conduit groups. Scale scores for role-physical functioning, social functioning and role-emotional functioning in both the neobladder and ileal conduit groups were significantly below the Italian population norm. Patients with a neobladder 65 years old or older (n = 18) had significantly lower scores for role-physical functioning and role-emotional functioning than those younger than 65 years (n = 17; p < 0.05).

Conclusion

Few differences between ileal conduit and orthotopic bladder substitution have been detected. Thus, the assumption that continent reconstruction provides better HRQOL than ileal conduit diversion cannot be supported. Patient education and active participation in treatment decisions seem to be the key to postoperative satisfaction.     

建立结肠癌SW480细胞原位移植瘤模型的实验研究

目的建立接近于临床表现的结肠癌动物模型,以便了解结肠癌生长及形态学变化。方法选SPF级Balb／c品系裸鼠,皮下接种SW480细胞,4周后取出瘤体,种植于裸鼠结肠系膜根部,建立结肠癌外科原位手术移植动物模型,4周后取出结肠原位肿瘤,应用病理学方法对结肠癌原位移植动物模型进行评价和鉴定。结果5只裸鼠皮下接种SW480细胞后4周,皮下成瘤3只;10只裸鼠结肠原位移植肿瘤,8只结肠成瘤,显微镜下可见肿瘤浸润肠壁。结论实验成功建立了结肠癌SW480细胞动物结肠原位移植瘤模型,为结肠癌相关实验提供临床研究动物模型。     

Induction of M2-macrophages by tumour cells and tumour growth promotion by M2-macrophages: A quid pro quo in pancreatic cancer

IntroductionMore effective therapies are required to improve survival of pancreatic cancer. Possible immunologic targets include tumour associated macrophages (TAMs), generally consisting of M1- and M2-macrophages. We have analysed the impact of TAMS on pancreatic cancer in a syngeneic orthotopic murine model.Methods6606PDA murine pancreatic cancer cells were orthotopically injected into C57BL6 mice. Tumour growth was monitored using MRI. Macrophages were depleted by clodronate liposomes. Tumours including microvessel density were evaluated using immunohistochemistry, immunofluorescence and/or cytometric beads assays. Naïve macrophages were generated employing peritoneal macrophages. In vitro experiments included culturing of macrophages in tumour supernatants as well as tumour cells cultured in macrophage supernatants using arginase as well as Griess assays.ResultsClodronate treatment depleted macrophages by 80% in livers (p = 0.0051) and by 60% in pancreatic tumours (p = 0.0169). MRI revealed tumour growth inhibition from 221.8 mm³ to 92.3 mm³ (p = 0.0216). Micro vessel densities were decreased by 44% (p = 0.0315). Yet, MCP-1-, IL-4- and IL-10-levels within pancreatic tumours were unchanged. 6606PDA culture supernatants led to a shift from naïve macrophages towards an M2-phenotype after a 36 h treatment (p < 0.0001), reducing M1-macrophages at the same time (p < 0.037). In vivo, M2-macrophages represented 85% of all TAMs (p < 0.0001). Finally, culture supernatants of M2-macrophages induced tumour growth in vitro by 63.2% (p = 0.0034).ConclusionsThis quid pro quo of tumour cells and M2-macrophages could serve as a new target for future immunotherapies that interrupt tumour promoting activities of TAMs and change the iNOS-arginase balance towards their tumoricidal capacities.     

Metabolic syndrome after liver transplantation: Short-term prevalence and pre- and post-operative risk factors

Background

The metabolic syndrome is a common condition among liver transplanted patients and contributes to morbidity and mortality by favouring the development of cardiovascular diseases.

Aims

This prospective study assessed the prevalence of metabolic syndrome in the first year after orthotopic liver transplantation, the associated pre-operative and post-operative risk factors and the influence of nutritional factors.

Methods

84 cirrhotic patients (75% male, mean age 53.9 ± 9.3 years) were evaluated at baseline and after liver transplantation. Metabolic syndrome was defined according to 2004 Adult Treatment Panel-III criteria. Nutritional habits were assessed using 3-day food records.

Results

Prevalence of metabolic syndrome before orthotopic liver transplantation was 14/84 (16.6%); at 3, 6 and 12 months post-orthotopic liver transplantation it was 27/84 (32.1%), 30/84 (35.7%), and 32/81 (39.5%), respectively. Diabetes, family history of diabetes, and excess body weight at baseline independently correlated with incidence of metabolic syndrome. After orthotopic liver transplantation, patients with metabolic syndrome showed a higher increase in the intake of total energy and saturated fats and a higher prevalence of complications, especially cardiovascular events, than subjects without metabolic syndrome.

Conclusion

Occurrence of metabolic syndrome is an early phenomenon after liver transplantation. Pre-operative and post-operative factors predispose patients to metabolic syndrome, which may be reduced by controlling modifiable risk factors, such as body weight and dietary intake.