MRI assessment of hemodynamic effects of angiopoietin-2 overexpression in a brain tumor model

Despite treatment efforts, the median survival in patients with glioblastoma multiforme, the most aggressive form of glioma, does not extend beyond 12–15 months. One of the major pathophysiological characteristics of these tumors is their ability to induce active angiogenesis. Thus, based on the lack of efficient therapies, agents that inhibit angiogenesis are particularly attractive as a therapeutic option. However, it has been recently proposed that although specifically targeting vascular endothelial growth factor, the main angiogenic factor, certainly leads to significant tumor regression, it could also be followed by tumor relapses. In this case, angiogenesis is driven by alternate pathways that include other angiogenic factors. One possible strategy to overcome this therapeutic obstacle is to overexpress antivascular factors such as angiopoietin-2 (Ang2). Here, by using MRI and histological analysis, we studied the vascular events involved in glioma growth impairment induced by Ang2 overexpression. Our results show that an increase in Ang2 expression, during the tumor growth, leads to a significant decrease in tumor growth (~86%) along with an increase in the survival median (~70%) but does not modify the tumor vascular area or cerebral blood volume. However, tumor Ang2-derived blood vessels display an abnormal, enlarged morphology. We show that the presence of Ang2 leads to an enhancement of tumor perfusion and a decrease in tumor vessel permeability. Based on our MR image evaluations of hemodynamic tumor vessel changes, we propose that Ang2-derived tumor vessels lead to an inadequate oxygenation of the tumor tissue, leading to impairment in tumor growth.     

The emergence of tumor metastases

The appearance of metastases is an ominous sign in the natural history of any malignant tumor. Their presence implies a high tumor burden and greatly decreases the probability of a cure. Metastasis development requires the evolution of tumor cells that can survive in an environment that is normally not supportive to their growth and such cells must leave the tumor to establish tumor niches elsewhere. The interactions between the appearance of cells with metastatic ability in the primary tumor and their exit from the tumor lead to complex dynamics that can be either beneficial or detrimental to the tumor. We develop a simple mathematical model to illustrate how the interplay between mutation rate and export probability affects the intratumoral dynamics of metastasis-enabled cells and the rate of metastases formation.     

Generation of effective antitumor vaccines using photodynamic therapy

Preclinical studies have shown that photodynamic therapy (PDT) of tumors augments the host antitumor immune response. However, the role of the PDT effect on tumor cells as opposed to the host tissues has not been determined. To test the contribution of the direct effects of PDT on tumor cells to the enhanced antitumor response by the host, we examined the immunogenicity of PDT-generated murine tumor cell lysates in a preclinical vaccine model. We found that the PDT-generated tumor cell lysates were potent vaccines and that PDT-generated vaccines are more effective than other modes of creating whole tumor vaccines, i.e., UV or ionizing irradiation, and unlike other traditional vaccines, PDT vaccines do not require coadministration of an adjuvant to be effective. PDT vaccines are tumor specific and appear to induce a cytotoxic T-cell response. We have demonstrated that although both UV and PDT-generated tumor cell lysates are able to induce phenotypic DC maturation, only PDT-generated lysates are able to activate DCs to express IL-12, which is critical to the development of a cellular immune response. Our results show that PDT effects on tumor cells alone are sufficient to generate an antitumor immune response, indicating that the direct tumor effects of PDT play an important role in enhancing that host antitumor immune response. These studies also suggest that in addition to the role of PDT as a therapeutic modality, PDT-generated vaccines may have clinical potential as an adjuvant therapy.     

Tumor-associated galectin-3 modulates the function of tumor-reactive T cells

T cells play an important role in cancer immunosurveillance and tumor destruction. However, tumor cells alter immune responses by modulating immune cells through antigen stimulation and immunoregulatory cytokines. A better understanding of the interplay between tumor cells and T cells might provide new strategies to enhance antitumor immunity. Through an antigen-screening approach using colorectal tumor-reactive T cells, we identified an HLA-DR11-restricted T-cell epitope encoded by KIAA0040 as well as MHC-unrestricted human galectin-3 (Gal-3) expressed by tumor cells. Although the biological function of KIAA0040 remains to be determined, we found that Gal-3 functioned as an immune regulator for direct T-cell activation and function. T-cell activation induced by Gal-3 resulted in T-cell apoptosis. We showed that a high level of expression of Gal-3 promoted tumor growth in vitro and in vivo. Using a mouse tumor model, we showed that delivery of high doses of Gal-3 inhibited tumor-reactive T cells and promoted tumor growth in mice receiving tumor-reactive CD8(+) T cells. These findings suggest that Gal-3 may function as an immune regulator to inhibit T-cell immune responses and promote tumor growth, thus providing a new mechanism for tumor immune tolerance.     

Cooperative autocrine and paracrine functions of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the progression of skin carcinoma cells

Tumor growth and progression are critically controlled by alterations in the microenvironment often caused by an aberrant expression of growth factors and receptors. We demonstrated previously that tumor progression in patients and in the experimental HaCaT tumor model for skin squamous cell carcinomas is associated with a constitutive neoexpression of the hematopoietic growth factors granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), causing an autocrine stimulation of tumor cell proliferation and migration in vitro. To analyze the critical contribution of both factors to tumor progression, G-CSF or GM-CSF was stably transfected in factor-negative benign tumor cells. Forced expression of GM-CSF resulted in invasive growth and enhanced tumor cell proliferation in a three-dimensional culture model in vitro, yet tumor growth in vivo remained only transient. Constitutive expression of G-CSF, however, caused a shift from benign to malignant and strongly angiogenic tumors. Moreover, cells recultured from G-CSF-transfected tumors exhibited enhanced tumor aggressiveness upon reinjection, i.e., earlier onset and faster tumor expansion. Remarkably, this further step in tumor progression was again associated with the constitutive expression of GM-CSF strongly indicating a synergistic action of both factors. Additionally, expression of GM-CSF in the transfected tumors mediated an earlier recruitment of granulocytes and macrophages to the tumor site, and expression of G-CSF induced an enhanced and persistent angiogenesis and increased the number of granulocytes and macrophages in the tumor vicinity. Thus both factors directly stimulate tumor cell growth and, by modulating the tumor stroma, induce a microenvironment that promotes tumor progression.     

Functional-characterization of developing tumor vascular system and drug delivery (review)

This review summarizes our recent experiments on the process of tumor vascularization and character of tumor vessels. By vitalscopic observation with transparent chambers in rats, we found that the sites where tumor vessels originated were usually terminal portions of terminal arterioles and that an intricate tumor vascular network was constructed from incorporated preexisting vessels and newly formed vessels by three different modes, i.e., sprouting, cross-connecting and splitting. Observation and hydrogen clearance studies showed that tumor blood flow changed remarkably during the development of the tumor vascular network. At an early stage of tumor growth, there were some regions of high flow in the tumor. At an advanced stage, however, there was a rapid increase in low-flow or no-flow areas which were resistant to access of anticancer drugs and oxygen. Angiotensin II-induced hypertension produced a several-fold increase in tumor blood flow without increasing tissue blood flow of normal tissues. These good conditions for drug delivery to tumor tissue are able to enhance therapeutic effects of chemotherapy, irradiation, antibody and photodynamic therapy.     

A rare astrocytic tumor with rhabdoid features

We report an extremely rare tumor presenting with rhabdoid features in the left temporoparietal lobe near the trigone in an 18-year-old Japanese man. This tumor mainly consisted of medium to large round cells that proliferated diffusely and incoherently with a scant extracellular matrix. These tumor cells had an eccentric nucleus and an eosinophilic cytoplasm containing inclusion bodies and bundles of intermediate filaments. The nuclei of these cells were vesicular with prominent nucleoli. This tumor had an area appearing to be diffuse astrocytoma peripherally and lacked a primitive neuroectodermal tumor component, a mesenchymal component, and epithelial differentiation. INI expression, which is not observed in atypical teratoid/ rhabdoid tumor (AT/RT), was found in this tumor. From these findings, we concluded that this tumor was not AT/RT but an astrocytic tumor with rhabdoid features. We also concluded that the tumor cells exhibiting rhabdoid features had secondarily arisen from the peripheral area presenting an appearance of diffuse astrocytoma.     

Immune control of tumors by antigen presentation improvement.

Tumor cells cannot activate T lymphocytes, since they do not usually express major histocompatibility complex (MHC) class II molecules. Thus, tumor antigens can only be presented indirectly to T cells through professional antigen-presenting cells (APC). In our laboratory, we have treated a tumor cell line (Tu1-A) - derived from an induced rat mammary sarcoma - in order to increase the expression of MHC class I and class II molecules. In our tumor model, the transference of these induced cells into normal rats generated a tumor mass that exhibited a lower tumor growth rate and an earlier regression as compared to those observed in rats inoculated with wild-type Tu1-A cells. This earlier tumor regression was associated with the development of an antigen-specific immune response. 85-87% of the rats in both groups rejected the tumor and were alive at day 60 after tumor cell inoculation. However, in rats treated with wild-type cells the rejection was delayed and took place after tumor ulceration. Rats that had rejected tumors were rechallenged with wild-type cells in order to assay the presence of a long-lived antitumor immunity. All the animals were resistant to the second tumor challenge. We conclude that the development of a specific immune response could be achieved by the superexpression of MHC molecules on tumor cells or when tumor ulceration promotes APC to take up necrotic cells and tumor antigens are presented to T lymphocytes.     

Overexpression of Notch ligand Dll1 in B16 melanoma cells leads to reduced tumor growth due to attenuated vascularization

Notch signaling plays an important role in vascular development and tumor angiogenesis. It has been shown that disruption of Dll4-triggered Notch signal activation effectively inhibits tumor growth, but this treatment also results in the formation of vascular neoplasms. In this study, we investigate the effects of over-expressing Notch ligand Dll1 in B16 melanoma cells on tumor cell proliferation and tumor growth in vitro and in vivo. Our results showed that over-expression of Dll1 could activate Notch signaling in tumor cells, and promote tumor cell proliferation in vitro. In contrast, growth of Dll1-over-expressing tumors in vivo was reduced, due to abnormal tumor vessel formation. Impaired tumor vasculature enhanced hypoxia and necrosis in tumor tissues, leading to retarded tumor growth. These results suggest that activation of Notch signaling may serve as an anti-angiogenesis strategy in the treatment of malignant tumors.     

A cytokinetic analysis of bacillus calmette-guérin-induced growth control of a murine leukemia.

The cytokinetics of an isogeneic, transplantable, lymphoid leukemia, growing as an ascitic tumor in the C57BL/6 mouse, has been investigated during normal growth and during regression induced by weekly injections i.v. of 1.0 mg Bacillus Calmette-Guérin (BCG). Survival was significantly prolonged in the BCG-treated group, and 27% of the mice were apparently cured. The tumor growth curves showed, furthermore, that BCG-rreated mice could be divided into two groups according to whether the ascitic tumor cell number was at control level or below that of the controls. By methods such as stathmokinetics, tritiated thymidine autoradiography, and cytophotometry, it was demonstrated that the proliferative activity was higher in BCG mice with a low tumor mass as compared to controls and BCG mice with a tumor mass similar to that of controls. The cytokinetic characteristics of BCG mice with a low ascitic tumor cell number were especially expressed by high mitotic activity, high initial labeling indices, short potential tumor doubling time, and a low number of G0-G1 cells. Furthermore the ascitic tumor cell loss rate was increased in these mice during the whole experimental period. It was deduced from the various parameters and especially from the cytophotometric and autoradiographic results that BCG induces a preferential kill of tumor cells in G0-G1 and the first part of the S phase. In addition, the cytological aspects of the ascitic tumor were found to be related to the cell kinetic pattern as the amount of small and large tumor cells increased and decreased, respectively, with accumulation of tumor cells in G0-G1.     

An aggressive angiomyxoma of the vulva

A case of an aggressive angiomyxoma of the vulva in a 38-year-old lady is reported. The tumor was gelatinous and 12 x 8 x 7 cm in size. Histologically, spindle or stellate-shaped tumor cells and blood vessels were found distributed in a myxoid stroma. It was found that the tumor cells were immunoreactive to vimentin but negative to desmin and smooth muscle specific actin. Ultrastructurally the tumor cells had an intercellular junction, an abundant, rough endemic reticulum, and intracytoplasmic filaments, but no focal densities of the filaments and the basal lamina. These findings have revealed that this tumor consisted of myofibroblastic cells resembling fibroblasts rather than myofibroblasts.     

Tumor necrosis factor alpha mediates homogeneous distribution of liposomes in murine melanoma that contributes to a better tumor response

Successful treatment of solid tumors with chemotherapeutics requires that adequate levels reach the tumor cells. Tumor vascular normalization has been proposed to enhance drug delivery and improve tumor response to chemotherapy. Differently, augmenting leakage of the tumor-associated vasculature, and as such enhance vascular abnormality, may improve tumor response as well. In the present study, we show that addition of low-dose tumor necrosis factor alpha (TNF) to systemic injections with pegylated long circulating liposomes augmented the tumor accumulation of these liposomes 5- to 6-fold, which strongly correlated with enhanced tumor response. Using intravital microscopy, we could study the liposomal distribution inside the tumor in more detail. Especially 100 nm liposomes effectively extravasate in the surrounding tumor tissue in the presence of TNF and this occurred without any effect on tumor vascular density, branching, and diameter. Next to that, we observed in living animals that tumor cells take up the liposomes intact, followed by intracellular degradation. To our knowledge, this is an unprecedented observation. Taken together, TNF renders more tumor vessels permeable, leading to a more homogeneous distribution of the liposomes throughout the tumor, which is crucial for an optimal tumor response. We conclude that delivery of nanoparticulate drug formulations to solid tumor benefits from augmenting the vascular leakage through vascular manipulation with vasoactive drugs like TNF.     

Approaches to the control of lymphogenic metastatic spread of tumors

Lymphopoiesis stimulating drugs were injected into the blood of rabbits bearing Brown-Pearce transplantable tumor. It was found that treatment with mannitol targeted on osmoregulation mechanisms and terrilytin--on blood and lymph coagulation intensified the lymphogenic migration of tumor cells. When used in an experimental series involving endolymphic transplantation of tumor cells, the said drugs promoted tumor cell passage from lymph to blood, which resulted in an increased number of metastasis-bearing animals. It was also found that the promoting effect may be counteracted by preliminary endolymphic injection of a fat-soluble antitumor preparation. Lymphopoiesis and lymph flow stimulation in conjunction with thoracic duct draining may be used for removal of tumor cells from the lymphatics.     

Presence of a growth-stimulating factor in serum following primary tumor removal in mice

The effect of removal of a primary tumor on the kinetics of cells in a metastasis was evaluated using six different tumors (C3H, MXTa, MXTb, MC54, CD8, and 3LL) which varied relative to their origin, histology, and the strain of mice in which they were carried. There was an increase in the labeling index (LI) of distant tumor focus ("metastasis") associated with the removal of each of the tumor types and unrelated to operative and anesthetic trauma. Information presented supports the presence of a serum growth factor as being responsible for the phenomenon. Serum obtained from mice following removal of a tumor, when transferred to a recipient with the same type of tumor as in the donor, resulted in an increase in the LI of the tumor. Multiple injections of serum failed to add to the increase but did prolong its presence, suggesting that there is a finite population of cells, most likely in the G1-G0 phase, which are capable of responding to the stimulating factor. The transfer of serum obtained following removal of a tumor type different from that in recipients resulted in findings which indicate that tumors producing a stimulating growth factor are those capable of responding to it. Serum obtained from animals with unremoved tumors or less than 18 h after removal failed to substantially augment the LI of tumors in recipients. It is postulated that the growth factor released by a tumor is in an inactive form which becomes activated over time. Observations indicate that medium conditioned by the growth of C3H tumor contains a growth-stimulating factor which is capable of increasing the LI of a C3H tumor in a recipient in a fashion similar to that obtained following tumor removal. That finding indicates the capability of the tumor to elaborate growth-stimulating material which may be similar to that found in serum. The findings presented refute the premise that removal of a primary tumor is a local phenomenon with no other biological consequences. They indicate that, following primary tumor removal, metastatic behavior may be affected by an interplay of growth factor(s) which can influence the outcome of a host to its tumor.     

Prognostic value of tumor thickness in patients with Merkel cell carcinoma

BACKGROUND AND OBJECTIVES: Merkel cell carcinoma is an aggressive skin malignancy that often presents with tumor metastases. We hypothesized that tumor thickness might correlate with both regional and metastatic tumor spread and could, therefore, be used as an independent prognostic variable. The purpose of this study was to see if depth of tumor invasion would predict prognosis independent of tumor stage. METHODS: Data pertaining to clinical presentation, pathology, treatment, and survival were collected for patients diagnosed with Merkel cell carcinoma from 1972 to 2005. Patients were staged according to AJCC guidelines. Pathologic specimens were evaluated for tumor thickness. The relationship between tumor thickness and disease-free survival or overall survival was analyzed using Kaplan-Meier survival analyses. RESULTS: Sixty patients were identified. Five-year disease-free survivals for Stages 1, 2, and 3 patients were 20%, 33%, and 0%, respectively. Five-year overall survivals for Stages 1, 2, and 3 patients were 33.3%, 60%, and 16.7%, respectively. There was no correlation between tumor thickness and either disease-free survival or overall survival. CONCLUSIONS: This study suggests that tumor thickness is not an independent risk factor for survival. Mean tumor thickness did increase with the AJCC stages, but this most likely represents more advanced stage of disease.     

肿瘤磁感应治疗的临床应用

磁感应热疗通过特定的方法使磁介质适形分布于靶区内,在磁场的作用下靶区内快速形成高温区,杀死肿瘤细胞,并不伤及正常组织.这一治疗方法的特异性和高度靶向性是其他加温治疗方法无法比拟的.临床研究显示,肿瘤磁感应治疗技术在肿瘤临床治疗中将起到重要作用,拥有广阔的应用前景.     

The use of a uPAR-targeted probe for photothermal cancer therapy prolongs survival in a xenograft mouse model of glioblastoma

The development of tumor-targeted probes that can efficiently reach cancerous tissue is an important focus of preclinical research. Photothermal cancer therapy (PTT) relies on light-absorbing molecules, which are directed towards tumor tissue and irradiated with an external source of light. This light is transformed into heat, causing localized hyperthermia and tumor death. The fluorescent probe indocyanine green (ICG) is already used as an imaging agent both preclinically and in clinical settings, but its use for PTT is yet to be fully exploited due to its short retention time and non-specific tumor targeting. Therefore, increasing ICG tumor uptake is necessary to improve treatment outcome. The urokinase-type plasminogen activator receptor, uPAR, is overexpressed in multiple tumor types. ICG-Glu-Glu-AE105, consisting of the uPAR-targeting peptide AE105 conjugated to ICG, has shown great potential for fluorescence-guided surgery. In this study, ICG-Glu-Glu-AE105 was evaluated as photothermal agent in a subcutaneous mouse model of human glioblastoma. We observed that the photothermal abilities of ICG-Glu-Glu-AE105 triggered high temperatures in the tumor during PTT, leading to tumor death and prolonged survival. This confirms the potential of ICG-Glu-Glu-AE105 as photothermal agent and indicates that it could be used as an add-on to the application of the probe for fluorescence-guided surgery.     

Inhibition of angiopoietin-1 expression in tumor cells by an antisense RNA approach inhibited xenograft tumor growth in immunodeficient mice.

Angiopoietin-1 (Ang1) is an angiogenic growth factor that functions through activation of its endothelium-specific tyrosine kinase receptor Tie2; it mediates the interaction between endothelial and surrounding cells to promote the remodeling, maturation and stabilization of blood vessels. Although Ang1 is expressed constitutively in many adult tissues, its role in tumor growth and metastasis is not clear. Here we describe experiments in which Ang1 expression was inhibited in HeLa cells by an antisense RNA approach. The modified HeLa cells produced significantly less Ang1 protein both in cultured cells and in tumors formed when these cells were injected into immunodeficient mice. The Ang1 antisense tumors grew much more slowly, with significantly reduced tumor angiogenesis compared with control tumors. Furthermore, they also had substantially increased tumor cell apoptosis and decreased tumor necrosis. Our results indicate that the perturbation of Ang1 expression in tumors could be an effective method to control tumor growth by inhibiting tumor angiogenesis and that antisense RNA is an efficient way to inhibit Ang1 protein production in tumor cells.     

Carcinosarcoma of the Ureter with Unusual Histologic Features

We report a rare case of carcinosarcoma of the ureter presentedas a retroperitoneal tumor. The tumor originated in the ureteras a polypoid protrusion and spread around peri-ureteral, retroperitonealtissues. The polypoid ureteral tumor was composed of an intra-epithelialcarcinoma and a submucosal mesenchymal tumor; the histologyof the retroperitoneal tumor in the original site was that ofa carcinosarcoma. The bulk of the tumor in the retroperitoneumwas composed of blastematous, epithelial and sarcomatous neoplasticcells with foci resembling an incomplete glomeruloid formation,thus mimicking a Wilms' tumor. The lesion appeared to originatefrom multipotential cells in the mucosal layer of the ureter.Whether or not this tumor has a true nephrogenic property iscurrently unknown. When a retroperitoneal tumor of the adultresembling a Wilms' tumor is found, one should suspect a possiblecarcinosarcomatous origin in the ureter.     

Tumor cell-derived nitric oxide is involved in the immune-rejection of an immunogenic murine lymphoma

The roles played by host-derived nitric oxide (NO) in the growth and subsequent immune rejection of a immunogenic murine lymphoma were investigated by growing the tumor in mice in which the gene for either inducible NO synthase (iNOS) or endothelial NOS (eNOS) had been ablated. This showed that NO from tumor-infiltrating host cells had no significant effect on either tumor growth or immune rejection, although measurements of tumor nitrite levels and protein nitration showed that there had been significant NO production in the rejected tumors, in both the eNOS and iNOS knockout mice. Inhibition of both tumor and host NOS activities, with an iNOS-selective inhibitor (1400W), a nonselective NOS inhibitor [Nomega-nitro-L-arginine methyl ester (L-NAME)], or scavenging NO with a ruthenium-based scavenger, significantly delayed tumor rejection, while having no appreciable effect on tumor growth. Incubation of tumor cells with medium taken from cultured splenocytes, that had been isolated from immunized animals and activated by incubating them with irradiated tumor cells, resulted in an increase in tumor cell NOS activity and an increase in tumor cell apoptosis, which could be inhibited using L-NAME. We propose that, during the immune rejection of this tumor model, there is induction of tumor NOS activity by cytokines secreted by activated lymphocytes within the tumor and that this results in increased levels of tumor NO that induce tumor cell apoptosis and facilitate immune rejection of the tumor.