[18F]EF3 is not superior to [18F]FMISO for PET-based hypoxia evaluation as measured in a rat rhabdomyosarcoma tumour model.

PURPOSE: The aim of this investigation was to quantitatively compare the novel positron emission tomography (PET) hypoxia marker 2-(2-nitroimidazol-1-yl)-N-(3[(18)F],3,3-trifluoropropyl)acetamide ([(18)F]EF3) with the reference hypoxia tracer [(18)F]fluoromisonidazole ([(18)F]FMISO). METHODS: [(18)F]EF3 or [(18)F]FMISO was injected every 2 days into two separate groups of rats bearing syngeneic rhabdomyosarcoma tumours. In vivo PET analysis was done by drawing regions of interest on the images of selected tissues. The resulting activity data were quantified by the percentage of injected radioactivity per gram tissue (%ID/g) and tumour to blood (T/B) ratio. The spatial distribution of radioactivity was defined by autoradiography on frozen tumour sections. RESULTS: The blood clearance of [(18)F]EF3 was faster than that of [(18)F]FMISO. The clearance of both tracers was slower in tumour tissue compared with other tissues. This results in increasing T/B ratios as a function of time post tracer injection (p.i.). The maximal [(18)F]EF3 tumour uptake, compared to the maximum [(18)F]FMISO uptake, was significantly lower at 2 h p.i. but reached similar levels at 4 h p.i. The tumour uptake for both tracers was independent of the tumour volume for all investigated time points. Both tracers showed heterogeneous intra-tumoural distribution. CONCLUSIONS: [(18)F]EF3 tumour uptake reached similar levels at 4 h p.i. compared with tumour retention observed after injection of [(18)F]FMISO at 2 h p.i. Although [(18)F]EF3 is a promising non-invasive tracer, it is not superior over [(18)F]FMISO for the visualisation of tumour hypoxia. No significant differences between [(18)F]EF3 and [(18)F]FMISO were observed with regard to the intra-tumoural distribution and the extra-tumoural tissue retention.

Occurrence of autoimmunity after xenothymus transplantation in T-cell-deficient mice depends on the thymus transplant technique.

Xenothymus transplantation under the kidney capsule in athymic rodents frequently leads to multiorgan autoimmunity. Herein, we explore whether this is an intrinsic risk of xenothymus grafting or whether it depends on the transplant technique. We developed a new technique of "venous pouch" thymus grafting (heart-xenothymus) and compared this with the conventional kidney subcapsular technique (kidney-xenothymus) in a rat-into-nude-mouse model. Whereas lethal autoimmunity developed in 90% of kidney-xenothymus recipients, all heart-xenothymus grafted mice remained completely healthy. Autoimmunity in heart-xenothymus recipients was absent despite a significantly improved T-cell generation and was associated with significantly higher CD4+CD25+ T-cell frequencies and CD4+CD25+ cell Foxp3 mRNA levels than those observed in kidney-xenothymus recipients. In conclusion, we describe a novel vascular pouch technique of xenothymus transplantation that prevents the development of autoimmunity in nude mice. Our data further suggest that prevention of autoimmunity is related to a superior development of regulatory T-cells.

Development of a flexible and potent hypoxia-inducible promoter for tumor-targeted gene expression in attenuated Salmonella.

To increase the potential of attenuated Salmonella as gene delivery vectors for cancer treatment, we developed a hypoxia-inducible promoter system to limit gene expression specifically to the tumor. This approach is envisaged to not only increase tumor specificity, but also to target those cells that are most resistant to conventional therapies. We demonstrate that the exponential growth of the attenuated bacteria is identical under normoxia and hypoxia. A hypoxia-inducible promoter (HIP-1) was created from a portion of the endogenous Salmonella pepT promoter and was shown to drive reporter gene expression under both acute and chronic hypoxia, but not under normoxia. Genetic engineering of the TATA- and FNR-box within HIP-1 allowed fine-tuning of gene induction, resulting in hypoxic induction factors of up to 200-fold. Finally, we demonstrate that HIP-1 can drive hypoxia-mediated gene expression in bacteria which have colonized human tumor xenografts in mouse models. Expression of both GFP and RFP under control of HIP-1 demonstrated an approximately 15-fold increase relative to a constitutive promoter when tumors were made hypoxic. Moreover, the use of a constitutive promoter resulted in reporter gene expression in both tumors and normal tissues, whereas reporter gene expressing using HIP-1 was confined to the tumor.

Diffusion-weighted magnetic resonance imaging allows noninvasive in vivo monitoring of the effects of combretastatin a-4 phosphate after repeated administration.

The noninvasive assessment of anticancer treatment efficacy is very important for the improvement of therapeutic window. The purpose of the present study was to evaluate the antitumoral effects of the vascular targeting agent, combretastatin A-4 phosphate (CA-4-P), at selected time points after repeated intraperitoneal drug administrations (25 mg/kg), using diffusion-weighted magnetic resonance imaging (DW-MRI). The experiments were performed during an overall follow-up period of 3 weeks on WAG/Rij rats with subcutaneously growing rhabdomyosarcomas. Each animal served as its own baseline. The DW-MRI studies were quantified by calculating the apparent diffusion coefficient (ADC) for different low and high b-values to separate the effects on tumor vasculature and cellular integrity. The changes in ADC as well as the extent of necrosis development (proportional to the tumor volume), measured on the MR images, were of comparable magnitude after each treatment. All ADC values showed a significant decrease at 6 hours, followed by a significant increase at 2 days for various CA-4-P administrations. DW-MRI allowed us to monitor both reduction in perfusion and changes in the extent of tumor necrosis after CA-4-P injection. Repeated CA-4-P administration retains efficacy in rat rhabdomyosarcomas, with similar findings after each drug administration.

The prognostic value of the hypoxia markers CA IX and GLUT 1 and the cytokines VEGF and IL 6 in head and neck squamous cell carcinoma treated by radiotherapy +/- chemotherapy.

BACKGROUND: Several parameters of the tumor microenvironment, such as hypoxia, inflammation and angiogenesis, play a critical role in tumor aggressiveness and treatment response. A major question remains if these markers can be used to stratify patients to certain treatment protocols. The purpose of this study was to investigate the inter-relationship and the prognostic significance of several biological and clinicopathological parameters in patients with head and neck squamous cell carcinoma (HNSCC) treated by radiotherapy +/- chemotherapy. METHODS: We used two subgroups of a retrospective series for which CT-determined tumoral perfusion correlated with local control. In the first subgroup (n = 67), immunohistochemistry for carbonic anhydrase IX (CA IX) and glucose transporter-1 (GLUT-1) was performed on the pretreatment tumor biopsy. In the second subgroup (n = 34), enzyme linked immunosorbent assay (ELISA) was used to determine pretreatment levels of the cytokines vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in serum. Correlation was investigated between tumoral perfusion and each of these biological markers, as well as between the markers mutually. The prognostic value of these microenvironmental parameters was also evaluated. RESULTS: For CA IX and GLUT-1, the combined assessment of patients with both markers expressed above the median showed an independent correlation with local control (p = 0.02) and disease-free survival (p = 0.04) with a trend for regional control (p = 0.06). In the second subgroup, IL-6 pretreatment serum level above the median was the only independent predictor of local control (p = 0.009), disease-free survival (p = 0.02) and overall survival (p = 0.005). CONCLUSION: To our knowledge, we are the first to report a link in HNSCC between IL-6 pretreatment serum levels and radioresistance in vivo. This link is supported by the strong prognostic association of pretreatment IL-6 with local control, known to be the most important parameter to judge radiotherapy responses. Furthermore, the combined assessment of CA IX and GLUT-1 correlated independently with prognosis. This is a valuable indication that a combined approach is important in the investigation of prognostic markers.

The influence of combretastatin A-4 and vinblastine on interstitial fluid pressure in BT4An rat gliomas.

The influence of combretastatin A-4 disodium phosphate (CA-4, 50mg/kg intraperitoneally (i.p.)) and vinblastine (2mg/kg i.p.) on interstitial fluid pressure (IFP) was assessed in BT4An rat gliomas implanted subcutaneously in the neck. Furthermore the growth inhibitory effect of vinblastine and the distribution of fluorescence-conjugated vinblastine (BODIPY-vinblastine) were investigated. Tumors at different volumes were compared. Whereas CA-4 had no major influence on IFP, independent of tumor size, vinblastine increased the IFP in neoplasms above 8 cm(3) (P=0.03). Vinblastine yielded a significant tumor response only in tumors below 2.1 cm(3) (P=0.03). The distribution of BODIPY-vinblastine was heterogeneous and comparable despite tumor volume differences. We conclude that the influence of vinblastine on IFP is more pronounced than that of CA-4 in BT4An neck tumors, and that vinblastine may reduce subsequent drug delivery to solid tumors by increasing the IFP.

Re-irradiation in conjunction with liposomal doxorubicin for the treatment of skin metastases of recurrent breast cancer: a radiobiological approach and 2 year of follow-up.

Thirty patients with local relapses after radical mastectomy and radiotherapy and undergoing infusion of liposomal doxorubicin (40 mg/m(2) monthly for 6 months) were randomized to receive re-irradiation. Radiotherapy was with either 17 fractions of 1.8 Gy, 5 days a week (N=15, group A) or 4 Gy plus two fractions of 3 Gy the 1st week and six fractions of 3 Gy given every second day (N=15, group B). Eight patients from group A (53.3%) and nine patients (60%) from group B demonstrating a clinically complete response (P=0.9). Grade I/II acute skin toxicity was monitored in 26.6% of patients in group A versus 73.3% in group B. The radiation schedule of group A seems superior for grade I/II acute (P=0.027) and late (P=0.015) skin toxicity. The linear quadratic model enabled the prediction of tumor response as well as normal skin reactions.

Potentiation of photodynamic therapy with hypericin by mitomycin C in the radiation-induced fibrosarcoma-1 mouse tumor model.

Hypericin, a polycyclic quinone obtained from plants of the genus Hypericum, has been shown to be a promising photosensitizer. We investigated the combination of hypericin-photodynamic therapy (PDT) and a bioreductive drug mitomycin C (MMC) in the present study. The radiation-induced fibrosarcoma-1 tumors were exposed to laser light (120 J/cm2 at 595 nm) 24 h after an intravenous injection of hypericin (1 mg/kg). Hypericin-PDT alone significantly decreased tumor perfusion and oxygen tension as demonstrated by India ink staining technique and OxyLite pO2 measurement, respectively. The in vivo-in vitro cell-survival assay revealed about 60% direct tumor cell killing immediately after PDT. No significant delayed tumor cell death was observed after PDT, which suggests that vascular damage does not contribute significantly to the overall tumor cell death. Injection of a 2.5 mg/kg dose of MMC 20 min before light application significantly decreased tumor cell survival and delayed tumor growth compared with PDT or MMC alone. No greater skin reaction was observed after the combination of MMC and PDT than after PDT alone. Our study demonstrates that combining hypericin-PDT with MMC can be effective in enhancing tumor response with little side effect.

Optimization of tumor-targeted gene delivery by engineered attenuated Salmonella typhimurium.

BACKGROUND: Attenuated Salmonella typhimurium has been demonstrated as a potential gene delivery vector. Previous findings induce the necessity to optimize tumor selectivity and bacterial dosing in relation to tumor volume and intratumoral therapeutic gene expression. MATERIALS AND METHODS: Attenuated Salmonella VNP20009 and VNP20047 (expressing cytosine deaminase) were systemically administered to tumor-bearing rats. The bacteria were quantified in tumor and normal organs. Conversion of 5-fluorocytosine to 5-fluorouracil was evaluated using thin layer chromatography. RESULTS: Tumor colonization efficiency was dependent on Salmonella density, administration route and tumor volume. Colonization of normal tissues gradually decreased with time, while intratumoral proliferation of bacteria remained high during the follow-up period. The Optimal Therapeutic Dose (OTD) was found to be 5.10(7) cfu/rat. Intratumoral VNP20047-expressed CDase leading to the conversion of 5-FC to 5-FU was detected in vivo. CONCLUSION: Our results indicate the need to define an OTD, probably for each species, when using genetically engineered Salmonella as a tumor- and species-selective vector in cancer therapy.

Unexpected interactions between nicotinamide and CPT-11 in a rhabdomyosarcoma tumor model.

We investigated the potential chemosensitizing effect of nicotinamide on CPT-11, and the relationship between nicotinamide and CPT-11, intratumoral drug uptake in syngeneic rhabdomyosarcoma tumors in rats. Pretreatment with nicotinamide, known to improve tumor oxygenation, perfusion and radiotherapy effect, only caused a minor increase in tumor growth delay. To our surprise, intratumoral uptake of CPT-11 and its active metabolite SN-38 decreased significantly between 19% and 43%. This discrepancy suggests that the potential chemosensitizing effect of nicotinamide, seen in other studies, is based on a direct effect on tumor cells rather than on an increased delivery of anticancer drugs. A second finding is that plasma levels of CPT-11 and SN-38 respectively increase and decrease after nicotinamide exposure, suggesting inhibition of carboxylesterase, which is necessary for the conversion of CPT-11 to its active metabolite SN-38. Great care is required when combining nicotinamide with anticancer drugs, since unexpected pharmacokinetic and pharmacodynamic alterations might occur.

The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5.

Tumor hypoxia is a common microenvironmental factor that adversely influences tumor phenotype and treatment response. Cellular adaptation to hypoxia occurs through multiple mechanisms, including activation of the unfolded protein response (UPR). Recent reports have indicated that hypoxia activates a lysosomal degradation pathway known as autophagy, and here we show that the UPR enhances the capacity of hypoxic tumor cells to carry out autophagy, and that this promotes their survival. In several human cancer cell lines, hypoxia increased transcription of the essential autophagy genes microtubule-associated protein 1 light chain 3beta (MAP1LC3B) and autophagy-related gene 5 (ATG5) through the transcription factors ATF4 and CHOP, respectively, which are regulated by PKR-like ER kinase (PERK, also known as EIF2AK3). MAP1LC3B and ATG5 are not required for initiation of autophagy but mediate phagophore expansion and autophagosome formation. We observed that transcriptional induction of MAP1LC3B replenished MAP1LC3B protein that was turned over during extensive hypoxia-induced autophagy. Correspondingly, cells deficient in PERK signaling failed to transcriptionally induce MAP1LC3B and became rapidly depleted of MAP1LC3B protein during hypoxia. Consistent with these data, autophagy and MAP1LC3B induction occurred preferentially in hypoxic regions of human tumor xenografts. Furthermore, pharmacological inhibition of autophagy sensitized human tumor cells to hypoxia, reduced the fraction of viable hypoxic tumor cells, and sensitized xenografted human tumors to irradiation. Our data therefore demonstrate that the UPR is an important mediator of the hypoxic tumor microenvironment and that it contributes to resistance to treatment through its ability to facilitate autophagy.

18F-FLT and 18F-FDG PET to measure response to radiotherapy combined with celecoxib in two colorectal xenograft models.

PURPOSE: To determine the dependence of celecoxib on the tumour micro-environment in vitro and in vivo and to compare the use of (18)F-Fluorodeoxyglucose ((18)F-FDG) and (18)F- 3'-deoxy-3-fluorothymidine ((18)F-FLT) to measure tumour response. MATERIALS AND METHODS: In vitro, colony assays were performed on a cyclo-oxygenase 2 (COX-2) negative (HCT116) and a COX-2 positive cell line (HCA7). Xenograft models of these cell lines were treated with celecoxib and/or radiotherapy. Micro Positron Emission Tomography (microPET) scans with (18)F-FDG and (18)F-FLT were performed at different time-points. RESULTS: In vitro, no radiosensitising effect was seen in either of the cell lines. In vivo results showed a significant effect of celecoxib in the COX-2 negative tumours (HCT116) (enhancement ratio 1.5, p = 0.02) while no significant effect was observed in the COX-2 positive model (HCA7). A good correlation between (18)F-FDG and (18)F-FLT uptake was seen in both tumour models (r = 0.48, p = 0.002; r = 0.41, p = 0.005). After irradiation, a decrease in the uptake of both tracers was observed in both tumour models, which was more pronounced in the combination group, confirming the growth delay data. CONCLUSIONS: The contradicting in vitro and in vivo results suggest a major role of the tumour micro-environment. (18)F-FLT seems a good alternative for (18)F-FDG to follow tumour growth after radiation treatment.

Treatment of rodent liver tumor with combretastatin a4 phosphate: noninvasive therapeutic evaluation using multiparametric magnetic resonance imaging in correlation with microangiography and histology.

OBJECTIVES: To document tumoricidal events after intravenous administration of a vascular targeting agent combretastatin A-4-phosphate (CA4P) in rodent liver tumors by using multiparametric magnetic resonance imaging (MRI) in correlation with microangiography and histopathology. MATERIALS AND METHODS: Thirty rhabdomyosarcomas of 8 to 14 mm in diameter were obtained 16 days after implantation in liver lobes of 15 rats. Using a 1.5T magnet and a 4-channel wrist coil, T2-weighted imaging (T2WI), pre- and postcontrast T1-weighted imaging (T1WI), diffusion-weighted imaging (DWI), and dynamic susceptibility imaging (DSI) with relative blood volume (rBV) and flow (rBF) maps were acquired at baseline, 1 hour, 6 hours, and 48 hours after iv injection of CA4P at 10 mg/kg and vehicle in 9 treated and 6 control rats, respectively. In vivo data including signal intensity (SI), tumor volume, apparent diffusion coefficient (ADC), rBV, and rBF were correlated with ex vivo microangiographic and histopathologic findings. RESULTS: CA4P-treated tumors (n = 18) grew slower than those (n = 12) of controls (P < 0.05), with vascular shutdown evident on CE-T1WI at 1 hour but more prominent at 6 hours. However, enhanced rim occurred in the periphery 48 hours after treatment, indicating neovascularization. ADC map enabled distinction between necrotic and viable tumors. DSI-derived tumoral rBV and rBF decreased significantly at 1 hour through 6 hours and partly recovered at 48 hours. SI-time curve reflected diverse therapeutic responses between tumor and liver. MRI findings were verified by ex vivo techniques. CONCLUSIONS: Clinical MRI allowed monitoring of CA4P-related vascular shutdown, necrosis, and neovascularization of liver tumors in rats. Single dose of CA4P seemed insufficient for tumor eradication because of evident peripheral residue and recurrence.

Inhibition of 4E-BP1 sensitizes U87 glioblastoma xenograft tumors to irradiation by decreasing hypoxia tolerance.

PURPOSE: Eukaryotic initiation factor 4E (eIF4E) is an essential rate-limiting factor for cap-dependent translation in eukaryotic cells. Elevated eIF4E activity is common in many human tumors and is associated with disease progression. The growth-promoting effects of eIF4E are in turn negatively regulated by 4E-BP1. However, although 4E-BP1 harbors anti-growth activity, its expression is paradoxically elevated in some tumors. The aim of this study was to investigate the functional role of 4E-BP1 in the context of solid tumors. METHODS AND MATERIALS: In vitro and in vivo growth properties, hypoxia tolerance, and response to radiation were assessed for HeLa and U87 cells, after stable expression of shRNA specific for 4E-BP1. RESULTS: We found that loss of 4E-BP1 expression did not significantly alter in vitro growth but did accelerate the growth of U87 tumor xenografts, consistent with the growth-promoting function of deregulated eIF4E. However, cells lacking 4E-BP1 were significantly more sensitive to hypoxia-induced cell death in vitro. Furthermore, 4E-BP1 knockdown cells produced tumors more sensitive to radiation because of a reduction in the viable fraction of radioresistant hypoxic cells. Decreased hypoxia tolerance in the 4E-BP1 knockdown tumors was evident by increased cleaved caspase-3 levels and was associated with a reduction in adenosine triphosphate (ATP). CONCLUSIONS: Our results suggest that although tumors often demonstrate increases in cap-dependent translation, regulation of this activity is required to facilitate energy conservation, hypoxia tolerance, and tumor radioresistance. Furthermore, we suggest that targeting translational control may be an effective way to target hypoxic cells and radioresistance in metabolically hyperactive tumors.

Diffusion weighted imaging in small rodents using clinical MRI scanners.

Diffusion weighted imaging (DWI) has emerged as a unique and powerful non-invasive magnetic resonance imaging (MRI) technique with a major potential impact on imaging-based diagnosis in a variety of clinical applications including oncology and tissue viability assessment. In light of increasing demand for applying this technique in preclinical investigations using small animals, we have explored the potentials of a clinical magnet for acquiring the DWI in rats and mice with either cerebral ischemia or solid tumors. Through technical adaptation and optimization, we have been able to perform a series of clinically relevant animal studies with conclusions based on DWI quantification. Focusing more on practical aspects and cross-referencing with the current literature, this paper is aimed to summarize our ongoing DWI studies on small rodents with stroke and tumors, and to provide protocols for researchers to replicate similar techniques in their own preclinical and clinical studies.

Allogeneic bone marrow transplantation in models of experimental autoimmune encephalomyelitis: evidence for a graft-versus-autoimmunity effect.

Autologous hematopoietic stem cell transplantation (HSCT) is being explored in the treatment of severe multiple sclerosis (MS), and is based on the concept of "resetting" the immune system. The use of allogeneic HSCT may offer additional advantages, such as the replacement of the autoreactive immune compartment by healthy allogeneic cells and development of a graft-versus-autoimmunity (GVA) effect. However, in clinical practice, the genetic susceptibility to MS of allogeneic stem cell donors is generally unknown, and GVA may therefore be an important mechanism of action. Experimental autoimmune encephalomyelitis (EAE)-susceptible and -resistant mouse strains were used to determine the roles of genetic susceptibility, level of donor-chimerism, and alloreactivity in the therapeutic potential of syngeneic versus allogeneic bone marrow transplant (BMT) for EAE. After transplantation and EAE induction, animals were evaluated for clinical EAE and ex vivo myelin oligodendrocyte glycoprotein-specific proliferation. Early after BMT, both syngeneic and allogeneic chimeras were protected from EAE development. On the longer term, allogeneic but not syngeneic BMT conferred protection, but this required high-level donor-chimerism from EAE-resistant donors. Importantly, when EAE-susceptible donors were used, robust protection from EAE was obtained when active alloreactivity, induced by donor lymphocyte infusions, was provided. Our findings indicate the requirement of a sufficient level of donor-chimerism from a nonsusceptible donor in the therapeutic effect of allogeneic BMT. Importantly, the data indicate that, independently of genetic susceptibility, active alloreactivity is associated with a GVA effect, thereby providing new evidence to support the potential role of allogeneic BMT in the treatment of MS.

Magnetic resonance imaging after radiofrequency ablation in a rodent model of liver tumor: tissue characterization using a novel necrosis-avid contrast agent.

We exploited a necrosis-avid contrast agent ECIV-7 for magnetic resonance imaging (MRI) in rodent liver tumors after radiofrequency ablation (RFA). Rats bearing liver rhabdomyosarcoma (R1) were randomly allocated to three groups: group I, complete RFA, group II, incomplete RFA, and group III, sham ablation. Within 24 h after RFA, T1-weighted (T1-w) MRI was performed before and after injection of ECIV-7 at 0.05 mmol/kg and followed up from 6-24 h. Signal intensities (SIs) were measured with relative enhancement (RE) and contrast ratio (CR) calculated. The MRI findings were verified histomorphologically. On plain T1-w MRI the contrasts between normal liver, RFA lesion, residual and/or intact tumor were vague. Early after administration of ECIV-7, the liver SI was strongly enhanced (RE=40-50%), leaving the RFA lesion as a hypointense region in groups I and II. At delayed phase, two striking peri-ablational enhancement patterns appeared (RE=90% and CR=1.89%), i.e., "O" type of hyperintense rim in group I and "C" type of incomplete rim in group II. These MRI manifestations could be proven histologically. In this study, tissue components after RFA could be characterized with discernable contrasts by necrosis-avid contrast agent (NACA)-enhanced MRI, especially at delayed phase. This approach may prove useful for defining the ablated area and identifying residual tumor after RFA.

Expression of carbonic anhydrase IX (CA IX), a hypoxia-related protein, rather than vascular-endothelial growth factor (VEGF), a pro-angiogenic factor, correlates with an extremely poor prognosis in esophageal and gastric adenocarcinomas.

OBJECTIVE: To evaluate the expression of carbonic anhydrase IX (CA IX) and vascular-endothelial growth factor (VEGF) in esophageal and gastric adenocarcinomas and in turn with the histologic subtype. SUMMARY BACKGROUND DATA: Tumor hypoxia is an important factor in therapy resistance. A low oxygen concentration in tumors stimulates a.o. the expression of CA IX, a marker of hypoxia, and VEGF, a pro-angiogenic factor. METHODS: We evaluated the immunohistochemical expression of CA IX and VEGF on paraffin-embedded material of 154 resection specimens: 39 esophageal, 73 cardiac, and 42 distal gastric adenocarcinomas (UICC classification). The adenocarcinomas were subtyped according to the Lauren classification (intestinal- and diffuse-type). STATISTICAL ANALYSIS: chi test, Kaplan-Meier survival analysis, log-rank test, and Cox proportional hazards model. RESULTS: CA IX and VEGF expression were independent of the localization of the tumor. However, intestinal-type adenocarcinomas showed a significantly higher expression of CA IX as well as VEGF than diffuse-type tumors. VEGF expression was associated with a high microvessel density. Although survival analysis showed that CA IX expression (P = 0.008) as well as the coexpression of CA IX and VEGF (P = 0.008) correlate with a poor outcome, only CA IX expression is an independent prognostic factor for overall survival and metastasis-free survival. CONCLUSION: The difference in expression of CA IX and VEGF between intestinal- and diffuse-type adenocarcinomas may possibly explain the different clinical behavior of these tumors. CA IX expression, rather than VEGF positivity in tumors, enables the identification of a subpopulation, characterized by a more aggressive behavior and a poorer prognosis.

Liver tumor model with implanted rhabdomyosarcoma in rats: MR imaging, microangiography, and histopathologic analysis.

In compliance with institutional regulations for care and use of laboratory animals, the aim of this study was to establish and characterize a rodent liver tumor model to provide a platform for preclinical assessment of new diagnostic and therapeutic strategies. A rhabdomyosarcoma tumor was implanted in the right and left liver lobes of 20 rats, for a total of 40 tumors. T1- and T2-weighted magnetic resonance (MR) images, diffusion-weighted images, and dynamic susceptibility contrast agent-enhanced perfusion-weighted images were obtained up to 16 days after tumor implantation and were compared with postmortem three-dimensional computed tomographic (CT) images, digital microangiograms, and histopathologic findings. Fifteen tumors were examined with proton ((1)H) MR spectroscopy. All tumors grew, with a mean volume doubling time of 2.2 days +/- 0.9 (standard deviation) and a final size of 591 mm(3)+/- 124. The rhabdomyosarcoma tumor showed hypervascularity at MR imaging, three-dimensional CT, microangiography, and histologic analysis. On dynamic susceptibility contrast-enhanced perfusion-weighted images, the maximum signal intensity decrease differed in time and extent between the tumor and the liver, with a significantly (P < .001) higher relative blood volume, relative blood flow, and permeability value in the tumor than in the liver. With (1)H MR spectroscopy, the rhabdomyosarcoma tumor and the liver featured significant (P < .001) choline and lipid peaks, respectively. Implantation of a rhabdomyosarcoma tumor in the livers of rats is feasible and reproducible, and this animal model seems promising for future testing of new diagnostic and therapeutic strategies.

CD4+ T lymphocytes expressing CD40 ligand help the IgM antibody response to soluble pneumococcal polysaccharides via an intermediate cell type.

Streptococcus pneumoniae causes serious infections in children, the elderly, and immunocompromised patients. Protection against infections with S. pneumoniae is mediated through Abs against the capsular polysaccharides (caps-PS). We previously showed that the murine Ab response to caps-PS is dependent on CD40-CD40L interaction. In the present paper, we addressed the question of whether the CD40-CD40L-mediated modulation of the anti-caps-PS immune reaction is the result of a direct interaction between B lymphocytes and T lymphocytes or of an indirect interaction. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice did not mount anti-caps-PS Abs. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice and CD4+ T lymphocytes from wild-type mice but not CD4+ T lymphocytes from CD40L knockout mice stimulated the anti-caps-PS Ab response. This indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in a CD40L-dependent manner. SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice and CD4+ T lymphocytes from wild-type mice generated an anti-caps-PS Ab response that could be inhibited by MR1, a blocking anti-CD40L Ab. These data indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in an indirect way. Finally, lethally irradiated CD40 knockout mice reconstituted with bone marrow from wild-type mice mounted an anti-caps-PS Ab response that was comparable to the Ab response in wild-type mice, revealing that the required CD40 was on hemopoietic cells. In conclusion, we provide evidence that CD4+ T lymphocytes expressing CD40L stimulate the Ab response to soluble caps-PS by interacting with CD40-expressing non-B cells.