Number Of Tumor Vessels Research Articles

Exploring the Efficacy and Molecular Mechanism of Systemic Therapy-Based Split-Course Stereotactic Body Radiation Therapy for Solid Tumors

Considering the therapeutic toxicity, the question of how to administer adequate chemotherapy to synchronize Stereotactic body radiation therapy (SBRT) treatment strategy for maximizing the benefits of neoadjuvant therapy to improve prognosis is a challenging and debatable issue. This study explores the feasibility and theoretical advantages of the simultaneous implementation of split-course SBRT with systemic treatment. We evaluated the effect of three irradiation regimens (conventional radiotherapy (C-R, 15×2 Gy, (D1-5, D8-12, D15-19)), continuous SBRT (C-S, 3×7 Gy, (D1-3)), split SBRT (S-S, 3×7 Gy, (D1, D10, D19)) in combination with systemic therapy (chemotherapy (gemcitabine), PD-1 inhibitor (Anti-mouse PD-1 antibody) and angiogenesis-targeting agent (Anlotinib)) on tumor growth and survival by using the immunologically active C57BL/6 bilateral subcutaneous model with different cancer cells. At different time points after treatment, we monitored changes in the tumor microenvironment and explored the possible mechanisms. In the case of radiotherapy alone, we found similar tumor control in all groups but the best survival in the S-S group. When combined with systemic therapy, the S-S group has shown advantages in tumor control and survival. And the S-S combination group induced more CD4+ and CD8+ T lymphocyte infiltration in the tumor, which had the highest number of tumor vessels and the smallest area of hypoxia. Tumor cell PD-L1 expression was increased after radiotherapy in all but the most elevated and persistent S-S combination treatment group. CD3+ T cells, CD4+ T cells and CD8+ T cells peaked at weeks 2-3 after treatment, and all remained at the highest level for 6 weeks in the S-S combination group. Meanwhile, PD-L1 expression was significantly higher in all tumors in the third week than in other groups. The most significant reduction in the hypoxic area and increase in perfusion level was observed in the S-S group in the third week. RNA sequencing analysis revealed that immune response-related pathways were upregulated on the irradiated and non-irradiated side, tumor proliferation and invasion on the non-irradiated side, and angiogenesis-related pathways were down-regulated in the fractionated SBRT combination treatment group. In both irradiated side and non-irradiated side tumor tissues where the abscopal effect was observed, immune response-related pathways were upregulated in the S-S group relative to the C-R group, and tumor proliferation and invasion and angiogenesis-related pathways were found to be downregulated in the non-irradiated side. In preclinical models, the combination of split SBRT with systemic therapy has been shown to be more advantageous than conventional segmentation, possibly by affecting immune cell infiltration in the tumor microenvironment and reducing tumor hypoxia.

Выживаемость пациентов с увеальной меланомой малых и средних размеров в зависимости от клинико-патоморфологической картины и клеточного микроокружения

Purpose. To assess the survival rate of patients with small and mediumsized uveal melanoma, depending on the clinical and pathomorphological picture and the cellular microenvironment. Material and methods. For a prospective study, 33 patients with T1-2N0M0 stage of the tumor were selected for the period from June 2005 to July 2018 at an average age of 54±2.7 years. In all cases, the enucleation of the affected eye was carried out with a morphological study of it and a study of the composition of the cellular microenvironment of the tumor node, which became the main criterion for compliance and inclusion in the study. Results. During the study period of observation after enucleation due to eye uveal tract melanoma, 9 (27.3%) of 33 patients died from metastatic disease. Of these, 2 with small and 7 with middle size of tumor. The timing of the appearance of metastases ranged from 15 to 96 (average 41.3±34.5) months. The median follow-up was 64.5 (95% confidence interval 29.3– 99.7) month. The 5-year survival rate of patients with small and medium sized uveal melanoma was 78.7%, and the 10-year survival rate was 72.7%. A significant dependence of the mortality of patients was revealed with the epithelioid cell type of melanoma, increase of tumor basal diameter, involvement of optic disc area and ciliary body, a significant degree of pigmentation, the presence of necrosis in tumor stroma, ingrowth into sclera emissarium and large number of tumor vessels. Survival decreases in patients with a large accumulation of lymphocytes around the tumor, the absence of macrophages, mast cells, fibroblasts on the one hand, and the presence of plasmocytes, myeloid-derived suppressor cells (promyelocytes, neutrophils, eosinophils) in tumor microenvironment. Conclusion. Studies have revealed a certain relationship between the life expectancy of patients and their mortality from metastatic disease with clinical and pathological signs and a specific composition of the cell microenvironment. Key words: uveal melanoma, survival, cell microenvironment

Periostin Exon-21 Antibody Neutralization of Triple-Negative Breast Cancer Cell-Derived Periostin Regulates Tumor-Associated Macrophage Polarization and Angiogenesis.

Simple SummaryDespite remarkable advances in breast cancer treatment, few strategies other than standard cytotoxic chemotherapy are available for patients with triple-negative breast cancer (TNBC) due to the lack of therapeutic target molecules. TNBC is still the most aggressive subtype, with a high risk of recurrence and metastasis within 2 years after initial treatment. Thus, there is an unmet medical need to develop new treatments for metastatic and recurrent TNBC patients. In this study we tested a new antibody, targeting extracellular periostin protein alternative splicing variants, which are induced by conventional chemotherapy or during the process of endothelial mesenchymal transition. This antibody reduced periostin-secreting TNBC in a mouse xenograft model, accompanied by a decrease in the number of M2 tumor-associated macrophages and tumor vessels. Periostin alternative splicing variants might be a specific and safe therapeutic target in patients with TNBC.Periostin (Pn) is involved in multiple processes of cancer progression. Previously, we reported that Pn expression is correlated with mesenchymal tumor markers and poor prognosis in triple-negative breast cancer (TNBC). In the TNBC xenograft model, chemotherapy increased expression of a Pn alternative splicing variant (ASV) with exon 21, and administration of the neutralizing antibody against Pn with exon 21 (Pn-21 Ab) overcame chemoresistance with a reduction in the mesenchymal cancer cell fraction. In the present study, the role of Pn ASV with exon 21 in TNBC progression has been addressed. We first established a stable cell line carrying a fluorescence-based splicing reporter. Pn-positive TNBC has higher expression of genes related to tumor-associated macrophage (TAM) recruitment and ECM-receptor interaction than Pn-negative cells. In a xenograft model, only Pn-positive cells initiated tumor formation, and the Pn-21 Ab suppressed tumor cell growth, accompanied by decreased M2 TAM polarization and the number of tumor vessels. These data suggest that cancer cell-derived Pn ASV educates TAMs and regulates angiogenesis, which in turn establishes a microenvironmental niche that is supportive of TNBC.

STUDY OF CHANGES IN TUMOR BLOOD FLOW FOR THE ASSESSMENT OF EARLY RESPONSE TO NEOADJUVANT CHEMOTHERAPY IN BREAST CANCER PATIENTS

Background. Over the past 20 years, there has been a change in approaches to the treatment of breast cancer, in particular, a significant increase in the role of drug therapy. Breast cancer response to neoadjuvant chemotherapy is currently considered as a surrogate biomarker, which allows evaluation of the clinical course and prognosis of the disease. To solve this problem, it is necessary to assess the functional and metabolic changes in tumor tissue during treatment. Doppler ultrasound is a non-invasive, affordable, and low-cost imaging technique that can be safely used for repeated measurements.The purpose of the study was to study vascular changes in the tumor by power Doppler ultrasound for the evaluation of the early breast cancer response to neoadjuvant chemotherapy.Material and Methods. From May 2017 to August 2019, 63 patients with breast cancer received neoadjuvant chemotherapy. Changes in the tumor blood flow were assessed before starting the treatment and prior to the second course of neoadjuvant chemotherapy using Doppler scanning. Changes in tumor blood floor after chemotherapy were compared with the pathological tumor response after surgical treatment.Results. In the vast majority of cases (78 %), there was a decrease in the number of tumor vessels after the first cycle of neoadjuvant chemotherapy independent of the grade of pathological response. In 8 cases with increased vascularization after the first cycle of neoadjuvant chemotherapy, histological examination of the removed tumor showed no response / weak response to treatment in the absence of peritumoral inflammation. In 5 cases, a sharp increase in the number of vessels around large areas of intranodular necrosis and peritumoral inflammation was observed. In general, a comparison of changes in tumor vascularization and pathological response revealed a weak, although statistically significant, negative correlation between changes in the tumor blood flow after neoadjuvant chemotherapy and pathological response.Conclusion. It was not possible to establish an unambiguous relationship between the reaction of the vascular bed and the tumor response to the cytostatic effect. An increase in the number of tumor vessels in the absence of peritumoral inflammation was the only situation when changes in tumor blood flow during chemotherapy can be unambiguously interpreted as a predictive criterion for the absence / weak response of the tumor to treatment.

Synergistic Antitumorigenic Activity of Calcitriol with Curcumin or Resveratrol is Mediated by Angiogenesis Inhibition in Triple Negative Breast Cancer Xenografts.

Calcitriol is a multitarget anticancer hormone; however, its effects on angiogenesis remain contradictory. Herein, we tested whether the antiangiogenic phytochemicals curcumin or resveratrol improved calcitriol antitumorigenic effects in vivo. Triple-negative breast cancer tumoral cells (MBCDF-T) were xenografted in nude mice, maintaining treatments for 3 weeks. Tumor onset, volume and microvessel density were significantly reduced in mice coadministered with calcitriol and curcumin (Cal+Cur). Vessel count was also reduced in mice simultaneously treated with calcitriol and resveratrol (Cal+Rsv). Cal+Cur and Cal+Rsv treatments resulted in less tumor activated endothelium, as demonstrated by decreased tumor uptake of integrin-targeted biosensors in vivo. The renal gene expression of Cyp24a1 and Cyp27b1 suggested increased calcitriol bioactivity in the combined regimens. In vitro, the phytochemicals inhibited both MBCDF-T and endothelial cells proliferation, while potentiated calcitriol’s ability to reduce MBCDF-T cell-growth and endothelial cells migration. Resveratrol induced endothelial cell death, as deduced by increased sub-G1 cells accumulation, explaining the reduced tumor vessel number in resveratrol-treated mice, which further diminished when combined with calcitriol. In conclusion, the concomitant administration of calcitriol with curcumin or resveratrol synergistically promoted anticancer effects in vitro and in vivo in human mammary tumor cells. Whereas the results suggest different mechanisms of action of the phytochemicals when coadministered with calcitriol, the converging biological effect was inhibition of tumor neoangiogenesis.

Expression and Prognostic Impact of VEGF, CD31 and αSMA in Resected Primary Lung Cancers.

One of the most important factors concerning cancer growth is angiogenesis. The purpose of this study was to clarify the relationship of maturation of tumor vessels and prognosis of lung cancer. Immunohistochemical stainings of 125 lung cancers for VEGF, CD31 and α-smooth muscle actin (αSMA) were scored by multiplying the intensity and the frequency from 0 to 12. Adenocarcinomas showed significantly higher staining scores of both VEGF and αSMA than squamous cell carcinomas did. In 42 cases of high CD31 score, five-year survival rate (87%) of patients with lung cancer showing mature tumor vessels was significantly better than that (69%) of patients with immature tumor vessels. Not the number of tumor vessels but their maturation may be a prognostic factor of patients with lung cancer. VEGF may not only stimulate proliferation of endothelial cells but also their maturation in differentiated lung cancers.

Alofanib, an allosteric FGFR2 inhibitor, has potent effects on ovarian cancer growth in preclinical studies.

Purpose Early data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased antitumor activity. We investigated antitumor activity of alofanib (RPT835), a novel allosteric FGFR2 inhibitor, in ovarian cancer in vitro and in vivo. Methods Equal amounts of ovarian cancer cell (SKOV3) lysates were analyzed for FGFR1-3 protein expression using Wes. To assess the efficacy of alofanib on FGF-mediated cell proliferation, SKOV3 cells were incubated and were treated with serially diluted alofanib. Basic FGF was added at a concentration of 25ng/ml. Control wells were left untreated. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Immunocompromised mice were used for xenotransplantation of SKOV3 cancer cells. Seventy animals with measurable tumors were selected on day 10 and randomized into control groups (no treatment or chemotherapy alone (paclitaxel + carboplatin) and treatment groups (alofanib orally or intravenously (different dose levels) in combination with chemotherapy). Measurements of tumor volume (mm3) were performed by digital calipers every 3days during 31days after tumor inoculation. Number of tumor vessels and Ki-67 index were calculated. Results SKOV3 cells express FGFR1 and FGFR2 but not FGFR3. Basic FGF increased proliferation of the ovarian cancer cells in untreated control group (P=0.001). Alofanib inhibited growth of FGFR2-expressing SKOV3 cells with GI50 value of 0.37μmol/L. Treatment with alofanib in combination with paclitaxel/carboplatin resulted in tumor growth delay phenotype in all treatment groups compared to control non-treatment groups. Compound exhibited a dose-dependent effect on tumor growth. Daily intravenous regimen of alofanib (total maximum dose per week was 350mg/kg) demonstrated significant effect (inhibiting growth by 80% and by 53% in comparison with vehicle and chemotherapy group alone, respectively (P<0.001). Alofanib decreased number of vessels in tumor (-49%; P<0.0001) and number of Ki-67-positive SKOV3 cells (-42%, P<0.05). There were tumor necrosis and cell degeneration in alofanib group. Conclusions We suggest that FGFR2 inhibition has potent effects on ovarian cancer growth in preclinical studies.

An Underlying Pathological Mechanism of Meningiomas with Intratumoral Hemorrhage: Undifferentiated Microvessels

Meningiomas usually present with a gradual onset of symptoms, and their acute presentation with a hemorrhagic event appears to be a rare condition. Although many clinical features of such a condition have been characterized, pathophysiological mechanisms underlying the bleeding remain unclear, and some contradictory results have been reported. The value of tumor vascularity as an index for the bleeding propensity of meningiomas is inconsistent. We sought to identify whether meningiomas have different types of blood vessels, and to explore the association of the different tumor vessels with intratumoral hemorrhage. Six patients with meningioma with acute onset due to intratumoral hemorrhage were identified, and 12 nonhemorrhagic meningiomas were matched according to specific clinical data. The characteristics of tumor vessels were examined through immunohistochemical staining of CD31, CD34, and smooth muscle actin (SMA). The number of stained vessels was counted and compared between the 2 groups. Two distinct types of blood vessels were determined in all meningiomas: undifferentiated (CD31+/CD34-) and differentiated (CD31+/CD34+) vessels, and most differentiated vessels were covered by pericytes marked by SMA. However, only the mean number of undifferentiated vessels in hemorrhagic meningiomas was significantly higher than that in controls (15.3 ± 4.9 vs. 6.4 ± 3.6; P < 0.01). Neither the number of differentiated vessels nor the total number of tumor vessels were significantly different between the 2 groups (P > 0.05). Our results suggest that tumor vasculature in meningiomas is heterogeneous, and that the undifferentiated vessels may play a pivotal role in the spontaneous intratumoral hemorrhage from meningiomas.

Acceleration of tumor growth due to dysfunction in M1 macrophages and enhanced angiogenesis in an animal model of autoimmune disease

Both autoimmunity and tumor immunity are immune responses against self-tissues or cells. However, the precise similarity or difference between them remains unclear. In this study, to understand a novel mechanism of tumor immunity, we performed transplantation experiments with a murine autoimmune model, C57BL/6J (B6)/lpr mice. A melanoma cell line, B16F10 cells, or granulocyte macrophage colony-stimulating factor- overexpressing B16F10 (B16F10/mGM) cells were transplanted into B6 or B6/lpr mice. Tumor growth by transplanted B16F10/mGM cells was significantly accelerated in B6/lpr mice compared with that in B6 mice. The accumulation of M1 macrophages in the tumor tissues of B6/lpr recipient mice was significantly lower compared with that in the control mice. In vitro co-culture experiment showed that impaired differentiation into M1 macrophages was observed in B6/lpr mice. The number of tumor vessels and vascular endothelial growth factor (VEGF) expression were also significantly enhanced in the tumor tissues of B6/lpr mice compared with those in the B6 mice. Moreover, VEGF expression was correlated with the increased expression of hypoxia-inducible factor-1α in the tumor tissues of B6/lpr mice. These results suggest that dysfunctional tumor immunity and enhanced angiogenesis in autoimmunity influence tumor growth.

Abstract 796: Alofanib, a novel allosteric FGFR2 inhibitor, shows potent antitumor activity in ovarian cancer with FGFR2 expression

Abstract Alofanib (formerly known as RPT835) is a novel allosteric FGFR2 inhibitor with activity in FGFR2-expressing triple-negative breast cancer [Tjulandin S, et al. San Antonio Breast Cancer Symposium 2014]. Early data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased antitumor activity [Cole C, et al. Cancer Biol Ther. 2010]. We investigated antitumor activity of alofanib in ovarian cancer in vitro and in vivo. To assess the efficacy of alofanib on FGF-mediated cell proliferation, ovarian cancer (SKOV-3) FGFR2-expressing cells were incubated in a 96-well microculture plate and were treated with serially diluted RPT835. Basic FGF was added at a concentration of 25 ng/ml. Control wells were left untreated. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Immunocompromised mice were used for xenotransplantation of FGFR2 high-expressing ovarian cancer cells (SCOV-3). Seventy animals with measurable tumors were selected on day 10 and randomized into control groups (no treatment or chemotherapy alone (paclitaxel + carboplatin) and treatment groups (alofanib orally or intravenously (different dose levels) in combination with chemotherapy). Measurements of tumor volume (mm3) were performed by digital calipers every 3 days during 30 days after tumor inoculation. Basic FGF significantly increased proliferation of the ovarian cancer cells in untreated control group (P = 0.001). Alofanib treatment resulted in growth inhibition of SKOV-3 cell line in vitro. Treatment of alofanib in combination with paclitaxel/carboplatin demonstrated significant tumor growth delay phenotype in all treatment groups compared to control non-treatment groups. Alofanib exhibited a dose-dependent effect on tumor growth. Daily intravenous regimen of alofanib (total maximum dose per week was 350 mg/kg) demonstrated dramatic effect (inhibiting growth by 80% and by 53% in comparison with vehicle and chemotherapy group alone, respectively (P&amp;lt;0.001). Alofanib decreased number of vessels in tumor on 49% compared with chemotherapy alone (P&amp;lt;0.05). There were tumor necrosis and cell degeneration in alofanib group. These results provide strong rationale for evaluation of alofanib in combination with paclitaxel and carboplatin in patients with ovarian cancer. Citation Format: Sergei Tjulandin, Mikhail Byakhov, Evgenia Stepanova, Dmitry Khochenkov, Daniel Harrison, Ilya Tsimafeyeu. Alofanib, a novel allosteric FGFR2 inhibitor, shows potent antitumor activity in ovarian cancer with FGFR2 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 796. doi:10.1158/1538-7445.AM2015-796

Effects of microbeam radiation therapy on normal and tumoral blood vessels

Microbeam radiation therapy (MRT) is a new form of preclinical radiotherapy using quasi-parallel arrays of synchrotron X-ray microbeams. While the deposition of several hundred Grays in the microbeam paths, the normal brain tissues presents a high tolerance which is accompanied by the permanence of apparently normal vessels. Conversely, the efficiency of MRT on tumor growth control is thought to be related to a preferential damaging of tumor blood vessels.The high resistance of the healthy vascular network was demonstrated in different animal models by in vivo biphoton microscopy, magnetic resonance imaging, and histological studies. While a transient increase in permeability was shown, the structure of the vessels remained intact. The use of a chick chorioallantoic membrane at different stages of development showed that the damages induced by microbeams depend on vessel maturation. In vivo and ultrastructural observations showed negligible effects of microbeams on the mature vasculature at late stages of development; nevertheless a complete destruction of the immature capillary plexus was found in the microbeam paths. The use of MRT in rodent models revealed a preferential effect on tumor vessels. Although no major modification was observed in the vasculature of normal brain tissue, tumors showed a denudation of capillaries accompanied by transient increased permeability followed by reduced tumor perfusion and finally, a decrease in number of tumor vessels. Thus, MRT is a very promising treatment strategy with pronounced tumor control effects most likely based on the anti-vascular effects of MRT.

Early onset and enhanced growth of autochthonous mammary carcinomas in C3-deficient Her2/neu transgenic mice

Aside from its classical role in fighting infections, complement is an important, although poorly understood, component of the tumor microenvironment. In particular, the tumor growth-regulatory activities of complement remain under debate. To assess the role of the complement system in the progression of autochthonous mammary carcinomas, we have crossed complement component 3 (C3)-deficient (C3−/−) BALB/c male mice with BALB/c females expressing the activated rat Her2/neu oncogene (neuT). Although neuT transgenic mice develop spontaneous mammary cancers with 100% penetrance, a significantly shorter tumor latency (i.e., earlier onset of the first palpable tumor), a higher frequency of multiple tumors (multiplicity), and a dramatic increase in the tumor growth rate were found in neuT-C3−/− animals. The accelerated tumor onset observed in neuT-C3−/− mice was paralleled by an earlier onset of spontaneous lung metastases and by an increase in Her2 expression levels, primarily on the surface of tumor cells. The percentage of immune cells infiltrating neuT carcinomas was similar in C3-deficient and C3-proficient mice, with the exception of a significant increase in the frequency of regulatory T cells in neuT-C3−/− tumors. Of particular interest, the enhanced immunosuppression imparted by C3 deficiency clearly influenced the immunogenic phenotype of autochthonous mammary tumors as neuT-C3−/− malignant cells transplanted into syngeneic immunocompetent hosts gave rise to lesions with a significantly delayed kinetics and reduced incidence as compared with cells obtained from neuT C3-proficient tumors. Finally, increased blood vessel permeability was evident in neuT-C3−/− tumors, although a similar number of tumor vessels was found in neuT and neuT-C3−/− lesions. Altogether, these data suggest that complement plays a crucial role in the immunosurveillance and, possibly, the immunoediting of Her2-driven autochthonous mammary tumors.

Littoral Cell Angioma of the Spleen

The purpose of this study was to evaluate the sonographic and pathologic features of littoral cell angioma of the spleen in 7 patients. The sonographic appearance in 7 cases of littoral cell angioma confirmed by surgical pathologic examination was retrospectively reviewed. All underwent color Doppler imaging. Two underwent contrast-enhanced sonography. The sonographic appearance was compared with pathologic findings. Splenic lesions were solitary in 5 cases and multiple in 2 cases. The masses ranged from 10 to 64 mm in maximum diameter. Five hypoechoic and 2 hyperechoic lesions on grayscale sonography corresponded to few and multiple blood-filled spaces on pathologic examination, respectively. Four hypovascular lesions, 1 hypervascular lesion, and the other 2 hypervascular lesions full of color flow signals on color Doppler imaging corresponded to few, several, and multiple arteries on pathologic examination. On contrast-enhanced sonography, 1 hypervascular lesion full of color flow signals showed homogeneous hyperenhancement for 8 minutes during the arterial and parenchymal phases. One hypovascular lesion showed inhomogeneous isoenhancement transiently during the arterial phase and became hypoechoic later. Littoral cell angioma is a primary vascular splenic neoplasm with variable features on grayscale sonography and color Doppler imaging as well as contrast-enhanced sonography. The sonographic appearance of littoral cell angioma mainly depends on the type and number of tumor vessels.

Thrombospondin-1 expression in melanoma is blocked by methylation and targeted reversal by 5-Aza-deoxycytidine suppresses angiogenesis

BackgroundReversibility of aberrant methylation via pharmacological means is an attractive target for therapies through epigenetic reprogramming. To establish that pharmacologic reversal of methylation could result in functional inhibition of angiogenesis, we undertook in vitro and in vivo studies of thrombospondin-1 (TSP1), a known inhibitor of angiogenesis. TSP1 is methylated in several malignancies, and can inhibit angiogenesis in melanoma xenografts. We analyzed effects of 5-Aza-deoxycytidine (5-Aza-dC) on melanoma cells in vitro to confirm reversal of promoter hypermethylation and restoration of TSP1 expression. We then investigated the effects of TSP1 expression on new blood vessel formation and tumor growth in vivo. Finally, to determine potential for clinical translation, the methylation status of TSP1 promoter regions of nevi and melanoma tissues was investigated. Results5-Aza-dC reduced DNA (cytosine-5)-methyltransferase 1 (DNMT1) protein, reversed promoter hypermethylation, and restored TSP1 expression in five melanoma cell lines, while having no effect on TSP1 protein levels in normal human melanocytes. In in vivo neovascularization studies, mice were implanted with melanoma cells (A375) either untreated or treated with 5Aza-dC. Vessels at tumor sites were counted by an observer blinded to treatments and the number of tumor vessels was significantly decreased at pretreated tumor sites. This difference occurred before a significant difference in tumor volumes was seen, yet in further studies the average tumor volume in mice treated in vivo with 5-Aza-dC was decreased by 55% compared to untreated controls. Knockdown of TSP1 expression with shRNA enhanced tumor-induced angiogenesis by 68%. Analyses of promoter methylation status of TSP1 in tumors derived from untreated and treated mice identified 67% of tumors from untreated and 17% of tumors from treated mice with partial methylation consistent with the methylation specific PCR analysis of A375 cells. Examination of methylation patterns in the promoter of TSP1 and comparison of aberrantly methylated TSP1 in melanoma with non-malignant nevi identified a significantly higher frequency of promoter methylation in tumor samples from melanoma patients. ConclusionsPharmacological reversal of methylation silenced TSP1 had functional biological consequences in enhancing angiogenesis inhibition and inducing antitumor effects to decrease murine melanoma growth. Angiogenesis inhibition is an additional mechanism by which epigenetic modulators can have antitumor effects.

Abstract 1894: Antitumor effects of everolimus, sorafenib, and axitinib in human renal cells carcinoma xenografts resistant to first line treatment by sunitinib

Abstract Background. Acquired resistance to sunitinib stands as a major clinical challenge in renal cell carcinoma. This study aimed to evaluate the antitumor effects of everolimus, sorafenib, and axitinib as second line treatment in renal cancer xenografts acquiring resistance to sunitinib. Materials and Methods: CAKI1 renal carcinoma cells (VHL+) were engrafted in nude mice and treated continuously with tumor-static doses of oral sunitinib (60 mg/kg) until progression (define as 100% increase tumor volume on 3 subsequent measurements). Mice that progressed were randomized to receive second line treatment with oral everolimus (2.5, 5, 10 and 20 mg/kg), sorafenib (60mg/kg), or axitinib (60mg/kg) for 3 weeks. Tumor responses and progression-free survival were calculated using Kaplan-Meier analysis. Tumors were analyzed by immunohistochemistry (IHC), Western blot, and q-RT-PCR. Results. First line sunitinib induced significant tumor growth delay compared with placebo (medians PFS: 50 and 17 days, respectively, p&amp;lt;0.0001) without toxicity. Tumors that progressed under sunitinib treatment showed higher mRNA levels of HIF-1α, VEGFA, CXCR4, and CA-IX as compared to tumors displaying response to sunitinib. An increase in CXCR4, vimentin, and HER3 protein expression was associated with sunitinib-resistance. No change in mouse CD31 mRNA was observed contrasting with an increased human CD31 mRNA expression in tumors developing resistance to sunitinib compared to sensitive tumors, suggesting that human cancer stem cells may dedifferentiate to gain angiogenic capacities at progression. Second line treatment with everolimus, sorafenib, and axitinib were well tolerated as defined by mouse weight, mortality, and behavior. Median PFS for 20 mg/kg everolimus was significantly higher than that of control group (13 and 5 days, respectively, p&amp;lt;0.001). Growth inhibition using 5 mg/kg/day everolimus was similar to that of axitinib. Doses &amp;gt;10mg/kg everolimus display higher effects on tumor growth than sorafenib and axitinib. Phospho-S6 expression was reduced in tumors treated with everolimus and axitinib but not with sorafenib, indicating that everolimus and axitinib inactivate the PI3k/AKT/mTOR signaling. IHC analysis of everolimus- and sorafenib-treated tumors showed no apoptosis, large hypoxic (by CA-IX staining) and necrotic areas associated, and a decreased number of tumor vessels (CD31 staining) compared to controls. Conversely, axitinib neither induce hypoxia nor necrosis in this model. Conclusions. This study shows that, using an appropriate tumor progression methodology, human tumor models can be used to evaluate first and second line treatment with targeted drugs. This setting also shows that xenograft models may allow testing molecular markers of response and resistance prior to clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1894. doi:1538-7445.AM2012-1894

Effects of the novel vascular targeting agent MDS-11P on tumor vascularity and its antitumor activity

Vascular disrupting agents show selective effects on tumor established vasculature, and achieve encouraging results in both pre-clinical and clinical experiments. In the present study, we investigated the effects of a new CA4 derivative MDS-11 and its prodrug MDS-11P on vascular disrupting activity in vitro and in vivo. Surface plasmon resonance (SPR) and tubulin polymerization assay showed that MDS-11 interacted with tubulin directly and inhibited tubulin polymerization in a cell free system, and western blot assay further confirmed the action in the cellular level. MDS-11 was found to significantly disrupt the microtubulin skeleton in proliferating HUVECs than quiescent ones determined by confocal microscopy. Furthermore, MDS-11 was found to damage the HUVEC-formed tube quickly, but did not influence structures of microvessels from aortic ring possessing pericytes and smooth muscle cells until 3h treatment. In A549 xenograft mice, immunohistochemistry staining of tumor sections revealed that a single dose of MDS-11P led to large areas of necrosis within tumor and reduced the number of tumor vessels, which was consolidated by perfused vascular volume assay. Pharmacokinetic studies of MDS-11P indicated that MDS-11P rapidly converted to the active form, MDS-11, and exhibited a much faster elimination in mice. The antitumor analysis using H22 and A549 mice xenograft models revealed that the growth inhibition rates of MDS-11P at 50mg/kg (twice a day for three weeks) reached 59.4%, 60.5% respectively without obvious weight loss. Taken together, these results suggest that MDS-11 is a potential vascular disrupting agent for further development of antitumor drug.

3D-CTA and 4D-CE-MRA for dynamic monitoring tumor angiogenesis in a rabbit VX2 tumor

To study possibility of dynamic monitoring tumor angiogenesis in vivo by 3D-CTA on a 64-row multidetector CT and 4D-CE-MRA on a 3T MR scanner; compare the advantages and faults of these two methods; and analyze the relationship between tumor angiogenesis and tumor growth. This study had been approved by the institutional animal care and use committee. Thirty New Zealand white rabbits with implanted VX2 tumors in the right thigh muscle were randomly divided into five groups according to survive time (n = 6). The rabbits were scanned by a 64-row multidetector CT and a 3T MR at 4, 7, 10, 13, 16 days after tumor implantation respectively, and then sacrificed to extract the tumor. The tumors' long diameters, short diameters and volume measurements derived from CT and MR imaging were compared with pathological data. The minimum tumor diameter and the number of new tumor blood vessels detectable by 3D-CTA and 4D-CE-MRA were also compared. The morphologic process of tumor angiogenesis was monitored and described. (1) The volume of tumors measured by CT imaging, MR imaging and pathological data had no significant difference (P > 0.05). (2) The minimum diameter of tumor vessels displayed on 3D-CTA and 4D-CE-MRA images was 0.68 mm +/- 0.07 mm and 0.85 mm +/- 0.12 mm respectively. The minimum diameter of tumor vessels displayed on 3D-CTA imaging was significantly smaller than 4D-CE-MRA imaging (t = -6.5075, P = 0.005). (3) The number of tumor vessels on 3D-CTA imaging and 4D-CE-MRA imaging had no significant difference at 4, 7, 10 days after tumor implantation. The number of tumor vessels on 3D-CTA images were significantly more than that on 4D-CE-MRA images at 13, 16 days after tumor implantation (all P < 0.01). (4) The morphologic process of tumor angiogenesis was demonstrated as sprouting from pre-existing blood vessel, forming lots of new vessels around tumor, and forming the immature vessels web with lots of tortuous, dilated, irregular vessels penetrating into the tumor at last. Volume CT and high magnet field MR can monitor tumor growth in vivo. 3D-CTA and 4D-CE-MRA can dynamicly monitor morphological changes of tumor angiogenesis in vivo. Of the two methods, 3D-CTA has better spatial resolution, but 4D-CE-MRA allows better temporal resolution of tumor angiogenesis monitoring.

A comparison of 3D-CTA and 4D-CE-MRA for the dynamic monitoring of angiogenesis in a rabbit VX2 tumor

To compare three-dimensional computed tomography angiography (3D-CTA) and four-dimensional contrast-enhanced magnetic resonance angiography (4D-CE-MRA) for the in vivo monitoring of tumor angiogenesis. VX2 tumors were implanted into the right thigh muscle of 30 New Zealand white rabbits. The animals were randomly assigned to 5 groups, which, respectively, were scanned by 3D-CTA and 4D-CE-MRA on day 4, 7, 10, 13, or 16 after tumor implantation. After scanning, tumors were resected and processed for conventional histology and CD-31 immunohistochemistry. Tumor volume measurements derived from CT and MR imaging were compared with histopathological data. The minimum tumor diameter and the number of new tumor blood vessels detectable by 3D-CTA and 4D-CE-MRA were also compared. There were no significant differences in the tumor volume measurements derived from CT, MR, and histological analysis. The minimum diameter of tumor vessels detectable by 3D-CTA (0.68 ± 0.07 mm) was significantly less than that by 4D-CE-MRA (0.85 ± 0.12 mm) (P=0.005). The number of tumor vessels detected by each imaging method was not significantly different until day 13 after implantation, when 3D-CTA detected a greater number (P<0.001). The morphologic process of tumor angiogenesis was demonstrated dynamically by 3D-CTA and 4D-CE-MRA in vivo. Tumor angiogenesis can be dynamically monitored in vivo by 3D-CTA and 4D-CE-MRA. Of the two methods, 3D-CTA has better spatial resolution, but 4D-CE-MRA allows temporal resolution of tumor angiogenesis.

Abstract 1367: The dual selective VEGFR-FGFR kinases inhibitor E-3810 decreases tumor perfusion and inhibits tumor growth: an analysis using dynamic contrast-enhanced magnetic resonance imaging

Abstract Background: E-3810 is a potent VEGFR1, 2 and 3 and FGFR 1 kinases inhibitor showing potent anti-angiogenic and antitumor activity in several in vitro and in vivo mouse models (Proceedings AACR-EORTC-NCI Meeting, Boston, November 2009; Abstracts No. 252 and 257). The aim of this study was to investigate, in a rat model, the antitumor activity of E-3810 and its anti-angiogenic effects by using DCE-MRI and to correlate the response with a panel of biological markers. Methods: Nude rats were subcutaneously xenografted with human Calu-6 lung cancer cells and treated orally once daily for 14 consecutive days, either with E-3810 − 10 mpk (dose chosen on the basis of a preliminary study in the same model) or Sorafenib − 100 mpk. Tumor growth and parameters reflecting tumor vascular permeability/perfusion (Ktrans) were evaluated by T2-weighted MRI and by T1-weighted DCE-MRI using Gd-DTPA (Magnevist®) as contrast agent at D-1, D1, D3, D7 and D14. The number, maturity and functionality of tumor vessels were assessed using CD31/α-SMA colocalisation by IHC and injection of Hoechst fluorescent dye on D1, D3, D7 and D14. On the same days, the level of circulating endothelial cells (CEC) and collagen IV were measured by FACS and ELISA assays. Results: In comparison to vehicle-treated animals, a marked tumor growth inhibition was observed in E-3810-treated rats (max T/C 21%) with no remarkable signs of toxicity. In the same animals, Ktrans value in the tumor rim was significantly decreased (up to 49% on D14). This DCE-MRI finding was supported by the significant decrease in the Hoechst stained area observed at D1 (−97%), D7 (−96%) and D14 (−75%). In addition, the number of tumor vessels (both CD31 and colocalized CD31/α-SMA) was significantly decreased at D3 (−72%), D7 (−68%) and D14 (−86%) in E-3810-treated rats. Always in comparison to vehicle group, no significant changes in CEC were observed after treatment with E-3810, whereas collagen IV levels significantly increased at D14 (+38). Similar effects on all the above mentioned parameters were observed in Sorafenib treated animals. Conclusions: E-3810 displayed a marked antitumor activity in the model of Calu-6 tumor bearing nude rats. The antitumor effect is coupled with a strong antiangiogenic activity, as indicated by decrease in vascular permeability/perfusion and decrease of microvessels density that occur early on and are maintained throughout the treatment period. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1367.

Metronomic treatment of malignant glioma xenografts with irinotecan (CPT-11) inhibits angiogenesis and tumor growth

Irinotecan (CPT-11) has shown emerging promise in the treatment of malignant gliomas. It is believed the mechanism of action of irinotecan is to sensitize glioma cells to the cytotoxic action of radiation therapy and alkylating agents. However, clinical trials using weekly or three-weekly doses of CPT-11 have demonstrated imaging responses in only 10-15% of patients. In this study, we evaluated another mechanism of action, angiosuppression by CPT-11 of ACNU-resistant gliomas, using a metronomic administration schedule. Two different types of treatment, (1) conventional and (2) metronomic, were applied to the subcutaneous U87 model. We found that metronomic administration of CPT-11 significantly inhibited malignant glioma growth by inhibiting angiogenesis; this treatment procedure reduced the number of tumor vessels and the area of hypoxic lesions and reduced expression of VEGF and HIF-1alpha, the most important angiogenic factors in gliomas. Metronomic treatment was superior to conventional treatment with regard to the severe systemic side effect of body weight loss. The growth inhibitory effect was very similar for both low and high doses of CPT-11. These angiosuppressive effects of CPT-11 show promise for another use of CPT-11 in metronomic and scheduled angiosuppressive chemotherapy with low dose and long-term administration for malignant gliomas without systemic side effects.