Myofibroblasts enable invasion of endothelial cells into three-dimensional tumor cell clusters: a novel in vitro tumor model

Purpose In an effort to study the importance of stromal involvement in angiogenesis, we developed a novel, multicellular model that utilizes three of the primary cell types involved in tumor angiogenesis.Methods Fluorescently labeled human microvascular endothelial cells (HMVECs), 10T1/2 cells and myofibroblasts were incubated in the presence of a three-dimensional tumor cell cluster resuspended in collagen and embedded in Matrigel.Results HMVECs cultured in the presence of a human SKOV-3 ovarian carcinoma tumor cell cluster, surrounded the tumor cell cluster, while myofibroblasts invaded the cluster, localizing within the tumor cell mass. In contrast, 10T1/2 cells, a pluripotent mouse mesenchymal cell line with pericyte-like properties, did not demonstrate the same invasive phenotype. HMVECs cultured in the presence of myofibroblasts invaded the tumor cell cluster and colocalized with the myofibroblasts as demonstrated by fluorescent microscopy and immunohistochemistry. The angiogenesis inhibitors SU6668 and paclitaxel inhibited stromal invasion, while a broad-spectrum matrix metalloproteinase inhibitor did not.Conclusions This model emphasizes the critical interaction between endothelial cells and myofibroblasts and provides a more complete in vitro model for studying angiogenesis and tumor progression.     

ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, induces the formation of inactive EGFR/HER2 and EGFR/HER3 heterodimers and prevents heregulin signaling in HER2-overexpressing breast cancer cells.

PURPOSE: ZD1839 is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) that has shown clinical activity against EGFR-expressing tumors. Our aim was to explore the effects of ZD1839 in breast cancer cell lines expressing different levels of EGFR and the closely related HER2 receptor. EXPERIMENTAL DESIGN: We studied the growth-inhibitory effects of ZD1839 in a series of breast carcinoma cell lines. In HER2-overexpressing BT-474 breast cancer cells, we studied the effects of ZD1839 on cell growth and heterodimerization of receptors under basal and ligand-stimulated conditions. RESULTS: ZD1839 was an equally potent inhibitor of growth in breast cancer cells expressing high levels of EGFR and HER2. In BT-474 breast cancer cells, ZD1839 abolished EGF- and heregulin-induced activation of ErbB receptors and downstream signaling molecules. Because ZD1839 does not inhibit the HER2 tyrosine kinase in vitro, and because heregulin is a ligand that activates HER2 by binding to HER3 and HER4 but does not bind to the EGFR, our findings suggested that ZD1839 interfered with HER2 function in intact cells. Searching for mechanisms, we report that ZD1839 induces the formation of inactive unphosphorylated EGFR/HER2 and EGFR/HER3 heterodimers. Furthermore, ZD1839 completely abolishes basal and heregulin-induced formation of active phosphorylated HER2/HER3 heterodimers. CONCLUSIONS: ZD1839 inhibits the growth of HER2-overexpressing breast cancer cells, possibly by sequestration of HER2 and HER3 receptors in an inactive heterodimer configuration with the EGFR. Our findings suggest that there is a strong rationale to conduct clinical trials of ZD1839 in patients with HER2-overexpressing breast tumors.     

Postoperative low-pelvic irradiation for stage I-IIA cervical cancer patients with risk factors other than pelvic lymph node metastasis

: To retrospectively investigate whether postoperative low-pelvic radiotherapy (RT) is an appropriate treatment for node-negative, high-risk Stage I–IIA cervical cancer patients.

: A total of 228 Stage I–IIA cervical cancer patients treated by radical surgery and postoperative RT were included in this study. All patients had histopathologically negative pelvic node metastasis, but at least one of the following risk factors: parametrial involvement, positive or close resection margins, invasion depth two-thirds or greater cervical stromal thickness. Seventy-nine patients (35%) received 30–50 Gy (median 44) to whole pelvis and a boost dose to the low pelvis (whole-pelvic RT group); the other 149 patients (65%) received low-pelvic RT only (low-pelvic RT group). For both groups, the total external RT dose to the low pelvis ranged from 40 to 60 Gy (median 50). The potential factors associated with survival, small bowel (gastrointestinal) complications, and leg lymphedema were analyzed, and patients who had a relapse in the upper pelvis were identified.

: The 5-year overall and disease-specific survival rate was 84% and 86%, respectively. After multivariate analysis, only bulky tumor (≥4 cm) and non-squamous cell carcinoma were significantly associated with survival. Parametrial involvement, lymph-vascular invasion, ≤50.4 Gy to the low pelvis, positive or close margins, and low-pelvic RT alone did not significantly affect survival. Grade I–V small bowel complications occurred in 33 patients (15%). Whole pelvic RT and >50.4 Gy to the low pelvis, but not old age and treatment technique (AP-PA vs. box), were significantly associated with gastrointestinal complications. Three patients (2%) in the low-pelvic RT group and 6 patients (8%) in the whole-pelvic RT group were found to have Grade III or higher small bowel complications (p = 0.023). Thirty-one percent of patients developed lymphedema of the leg. A dose to the low pelvis >50.4 Gy and an AP-PA field, but not whole-pelvic RT, old age, or the number of sampled lymph nodes, were associated with lymphedema of the leg. Five patients (3.6%) of the low-pelvic RT group and none of the whole-pelvic RT group developed upper pelvis relapse. Three of these 5 patients had upper pelvic relapse alone.

: Compared with whole-pelvic RT plus low-pelvic boost, low-pelvic RT alone significantly reduces the small bowel complications in node-negative, high-risk, Stage I–IIA cervical cancer patients. Although low-pelvic RT alone increases the incidence of upper pelvic relapse, its effect on survival is not substantial. Low-pelvic RT alone appears to be an appropriate treatment method for this group of patients.     

Ultrasonography and fluoroscopic fusion for prostate brachytherapy dosimetry

: To investigate the feasibility of performing postimplant and intraoperative dosimetry for prostate brachytherapy by fusing transrectal ultrasound (TRUS) and fluoroscopic data.

: Registration of ultrasound (prostate boundary) and fluoroscopic (seed) data requires spatial markers that are detectable by both imaging modalities. In this study, the needle tips were considered as such fiducials. Prostate phantoms were implanted with the seeds, and four localization needles were inserted. In the TRUS frame of reference, the longitudinal coordinate of the needle tip was determined by advancing the needle until the echo from its tip just registered at a known probe depth. The tip’s transverse coordinates were determined from the associated TRUS slice. The three-dimensional needle tip positions were also calculated in the fluoroscopic coordinate system using a seed reconstruction method. The transformation between the TRUS and fluoroscopy coordinate systems was established by the least-squares solution using the singular value decomposition.

: With three of four needle tips as fiducials and the one remaining needle as a test target, the mean fiducial registration error was 0.8 mm and the test target registration error was 2.5 mm. When all four points were used for registration, the errors decreased to 1.1 mm. A comparison between the proposed method and CT-based dosimetry yielded a percentage of prostate volume receiving 100% and 150% of the prescribed minimal peripheral dose and minimal dose received by 90% of the prostate gland that agreed within 0.4%, 2.7%, and 4.2%, respectively.

: The combination of TRUS and fluoroscopy is a feasible alternative to the currently used CT-based postimplant dosimetry. Furthermore, because of online imaging capability, the method lends itself to real-time intraoperative applications.     

Frontotemporal white matter changes in amyotrophic lateral sclerosis

Cognitive dysfunction can occur in some patients with amyotrophic lateral sclerosis (ALS) who are not suffering from dementia. The most striking and consistent cognitive deficit has been found using tests of verbal fluency. ALS patients with verbal fluency deficits have shown functional imaging abnormalities predominantly in frontotemporal regions using positron emission tomography (PET). This study used automated volumetric voxel-based analysis of grey and white matter densities of structural magnetic resonance imaging (MRI) scans to explore the underlying pattern of structural cerebral change in nondemented ALS patients with verbal fluency deficits. Two groups of ALS patients, defined by the presence or absence of cognitive impairment on the basis of the Written Verbal Fluency Test (ALSi, cognitively impaired, n=11; ALSu, cognitively unimpaired n=12) were compared with healthy age matched controls (n=12). A comparison of the ALSi group with controls revealed significantly (p<0.002) reduced white matter volume in extensive motor and non-motor regions, including regions corresponding to frontotemporal association fibres. These patients demonstrated a corresponding cognitive profile of executive and memory dysfunction. Less extensive white matter reductions were revealed in the comparison of the ALSu and control groups in regions corresponding to frontal association fibres. White matter volumes were also found to correlate with performance on memory tests. There were no significant reductions in grey matter volume in the comparison of either patient group with controls. The structural white matter abnormalities in frontal and temporal regions revealed here may underlie the cognitive and functional imaging abnormalities previously reported in non-demented ALS patients. The results also suggest that extra-motor structural abnormalities may be present in ALS patients with no evidence of cognitive change. The findings support the hypothesis of a continuum of extra-motor cerebral and cognitive change in this disorder.     

CD8+-dependent CNS demyelination following ocular infection of mice with a recombinant HSV-1 expressing murine IL-2

Demyelinating diseases comprise a spectrum of immunopathologic syndromes in which myelin, the fatty covering of nerve cell fibers in the brain and spinal cord, is destroyed. In this study, we have shown for the first time that ocular infection of BALB/c mice with a recombinant herpes simplex virus type 1 (HSV-1) expressing IL-2 (HSV-IL-2) results in CNS demyelination as determined by light microscopy and EM. The demyelinated lesions involve periventricular white matter, brain stem, and spinal cord white matter. Demyelination was detected in the CNS of infected mice up to 75 days (the longest time point tested) post HSV-IL-2 infection. In contrast, mice infected with HSV-IFN-gamma or HSV-IL-4, which are identical to HSV-IL-2 but express IFN-gamma or IL-4 instead of IL-2, did not exhibit demyelination. Control mice infected with wild-type HSV-1 or parental virus also remained free of these symptoms. During early times (days 3-7), post-infection with HSV-IL-2 virus, a T(H)1 + T(H)2 pattern of cytokines was produced by lymphocytes of infected mice while mice infected with HSV-IFN-gamma or control viruses produced a T(H)1 pattern of cytokine. By day 21 post-infection, all infected groups exhibited a T(H)1 pattern of response. Immunohistochemistry and FACS analyses of infiltrates in the brains and spinal cords of HSV-IL-2-infected mice showed elevations in CD4+ and CD8+ T cells and macrophages. However, T cell depletion studies suggest that only central memory CD8+ T cells are directly involved in the demyelination process, with macrophages being involved through a bystander effect.     

Ultrastructure of cholinergic synaptic terminals in the thalamic anteroventral,ventroposterior, and dorsal lateral geniculate nuclei of the rat

The principal relay nuclei of the thalamus receive their cholinergic innervation from two midbrain cholinergic groups: the pedunculopontine tegmental nucleus and the laterodorsal tegmental nucleus. The different thalamic nuclei exhibit populations of cholinergic axons which vary in density and morphology when examined at the light microscopic level. However, the ultrastructure of the cholinergic terminals in different thalamic nuclei has not been described. This study was undertaken to confirm that synaptic contacts are formed by cholinergic axons in several principal thalamic relay nuclei, to describe their ultrastructural morphology, and to identify the types of postsynaptic elements contacted by cholinergic synaptic terminals. The thalamic nuclei examined in this study are the dorsal lateral geniculate nucleus, ventroposteromedial nucleus, ventroposterolateral nucleus, and anteroventral nucleus. Our results confirm that cholinergic axons form synaptic terminals in these thalamic nuclei. Cholinergic synaptic terminals contact structures outside the characteristic synaptic glomeruli, are never postsynaptic, and have morphologies and postsynaptic targets which differ among the thalamic nuclei. In the ventroposterior nuclei, cholinergic terminals form asymmetric synaptic contacts onto larger dendrites in the extraglomerular neuropil. In the anteroventral nucleus, cholinergic terminals form both symmetric and asymmetric synaptic contacts onto dendrites and somata. Cholinergic terminals in the anteroventral nucleus are larger than those in other nuclei. In the dorsal lateral geniculate nucleus, cholinergic terminals contact both somata and dendrites in the extraglomerular neuropil, but the synaptic contacts in this nucleus are symmetric in morphology.     

Seizure remission and antiepileptic drug discontinuation in children with tuberous sclerosis complex

BACKGROUND: Epilepsy is a common neurologic complication of tuberous sclerosis complex (TSC) and it is often refractory to treatment. Therefore, treating physicians are often reluctant to discontinue antiepileptic drugs (AEDs) in individuals with TSC who have attained seizure remission. To our knowledge, seizure remission and AED discontinuation in children with TSC has not been studied. OBJECTIVE: To characterize seizure remission and AED discontinuation in children with TSC. METHODS: Retrospective medical record and neuroimaging analysis of 15 children with TSC and epilepsy who had seizure remission, with a subsequent trial of discontinuation of AED treatment. RESULTS: The seizure remission rate for the group of patients with TSC and epilepsy was 14.2%. From the group of 15 patients who had a remission, the absolute relapse rate was 26.7% after a mean follow-up of 5 years 7 months. Patients with sustained remission were more likely to have normal intelligence and only a few cortical or subcortical lesions on neuroimaging. CONCLUSIONS: The proportion of children with TSC and epilepsy who achieve seizure remission is small. Nevertheless, some do attain seizure remission, and AEDs may be successfully discontinued. Mild cerebral involvement is a general clinical marker for seizure remission. The relapse rate in those who have undergone a trial of discontinuation of AED therapy is comparable with the rate in the general pediatric population with epilepsy.