Orthotopic Xenograft Nude Mouse Model Research Articles

Potential Value of the Stretched Exponential and Fractional Order Calculus Model in Discriminating Between Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma: an Animal Experiment of Orthotopic Xenograft Nude Mice.

In clinical practice, Preoperative differentiation between hepatocellular carcinoma and intrahepatic cholangiocarcinoma is challenging but critical for treatment decisions. This study investigated the discriminatory power of the stretched-exponential model and fractional-order calculus model parameters for hepatocellular carcinoma versus intrahepatic cholangiocarcinoma in orthotopic xenograft nude mice. Prototype orthotopic xenograft models of hepatocellular carcinoma and intrahepatic cholangiocarcinoma were developed using 20 nude mice divided into two groups and separately transplanted with MHCC97H and HUCCT1 cells. Readout-segmented diffusion-weighted imaging with multiple b-values (0-2000 s/mm2) was obtained using a 3.0-T magnetic resonance imaging scanner. The apparent diffusion coefficient was calculated using the mono-exponential model. The distributed diffusion coefficient and intravoxel water molecular diffusion heterogeneity (α) were calculated using the stretched-exponential model. The diffusion coefficient (D), fractional-order derivative in space (β), and spatial parameter (μ) were calculated using the fractional-order calculus model. The liver and tumor specimens of nude mice were immunostained after euthanasia to clarify the liver cancer type. Differences in diffusion-related parameters between the groups were evaluated using Mann-Whitney U-test and univariate logistic analysis. Receiver operating characteristic curves were used to assess the diagnostic efficacy of each parameter. P<0.05 was deemed significant. α, D, and β were significant discriminators between the groups. The area under the curve for these three variables was 0.890, 0.830, and 0.870, respectively, with cutoff values of 0.491, 0.435, and 0.782, respectively. The stretched-exponential model parameters α and the fractional-order calculus model parameters D and β showed high diagnostic efficacy in discriminating intrahepatic cholangiocarcinoma from hepatocellular carcinoma in orthotopic xenograft nude mouse models.

Oncogenes and Methionine Addiction of Cancer: Role of c-MYC.

Methionine addiction is a general and fundamental hallmark of cancer cells, termed the Hoffman effect. Previously Vanhamme and Szpirer showed that methionine addiction could be induced by transfection of the activated HRAS1 gene to a normal cell line. In the present study, we investigated the role of the c-MYC oncogene in methionine addiction of cancer, by comparison of c-Myc expression and malignancy of methionine-addicted osteosarcoma cells and rare methionine-independent revertants, derived from the methionine-addicted cells. Methionine-independent revertant 143B osteosarcoma cells (143B-R) were derived from methionine-addicted parental 143B osteosarcoma cells (143B-P), by continuous culture in medium depleted of methionine by recombinant methioninase. To compare in vitro malignancy of methionine-addicted parental cells and methionine-independent revertant cells, the following experiments were performed: for 143B-P and 143B-R cells, cell proliferation capacity was measured with a cell-counting assay, and colony-formation capacity was determined on plastic and in soft agar, all in methionine-containing Dulbecco's Modified Eagle's Medium (DMEM). Tumor growth was measured in orthotopic xenograft nude-mouse models, to compare in vivo malignancy of 143B-P and 143B-R cells. c-MYC expression was examined with western immunoblotting and compared in 143B-P and 143B-R cells. 143B-R cells had reduced cell proliferation capacity, compared to 143B-P cells, in methionine-containing medium (p=0.003). 143B-R cells had reduced colony formation capacity on plastic (p=0.003) and in soft agar, compared to 143B-P cells in methionine-containing medium. 143B-R cells had reduced tumor growth in orthotopic xenograft nude-mouse models, compared to 143B-P cells, (p=0.002). These results demonstrate that 143B-R methionine-independent revertant cells lost malignancy. Expression of c-MYC was reduced in 143B-R methionine-independent revertant osteosarcoma cells, compared to 143B-P cells, (p=0.0007). The present study demonstrated that c-MYC expression is linked to malignancy and methionine addiction of cancer cells. The present study on c-MYC, and the previous study on HRAS1, suggest that oncogenes may play a role in methionine addiction, which is a hallmark of all cancers, as well as in malignancy.

Magnolol Suppresses Pancreatic Cancer Development In Vivo and In Vitro via Negatively Regulating TGF-β/Smad Signaling.

Magnolol, a hydroxylated biphenyl extracted from Magnolia officinalis, has recently drawn attention due to its anticancer potential. The present study was aimed to explore the effects of Magnolol on restraining the proliferation, migration and invasion of pancreatic cancer in vivo and in vitro. Magnolol showed significant anti-growth effect in an orthotopic xenograft nude mouse model, and immunohistochemical staining of the xenografts revealed that Magnolol suppressed vimentin expression and facilitated E-cadherin expression. The cytoactive detection using CCK-8 assay showed Magnolol inhibited PANC-1 and AsPC-1 concentration-dependently. Scratch healing assay and the Transwell invasion assay proved the inhibiting effects of Magnolol on cellular migration and invasion at a non-cytotoxic concentration. Western blot and rt-PCR showed that Magnolol suppressed epithelial-mesenchymal-transition by increasing the expression level of E-cadherin and decreasing those of N-cadherin and vimentin. Magnolol suppressed the TGF-β/Smad pathway by negatively regulating phosphorylation of Smad2/3. Moreover, TGF-β1 impaired the antitumor effects of Magnolol in vivo. These results demonstrated that Magnolol can inhibit proliferation, migration and invasion in vivo and in vitro by suppressing the TGF-β signal pathway and EMT. Magnolol could be a hopeful therapeutic drug for pancreatic malignancy.

Utilizing ICG Spectroscopical Properties for Real-Time Nanoparticle Release Quantification In vitro and In vivo in Imaging Setups.

Nanoparticle imaging and tracking the release of the loaded material from the nanoparticle system have attracted significant attention in recent years. If the release of the loaded molecules could be monitored reliably in vivo, it would speed up the development of drug delivery systems remarkably. Here, we test a system that uses indocyanine green (ICG) as a fluorescent agent for studying release kinetics in vitro and in vivo from the lipid iron nanoparticle delivery system. The ICG spectral properties like its concentration dependence, sensitivity and the fluctuation of the absorption and emission wavelengths can be utilized for gathering information about the change of the ICG surrounding. We have found that the absorption, fluorescence, and photoacoustic spectra of ICG in lipid iron nanoparticles differ from the spectra of ICG in pure water and plasma. We followed the ICG containing liposomal nanoparticle uptake into squamous carcinoma cells (SCC) by fluorescence microscopy and the in vivo uptake into SCC tumors in an orthotopic xenograft nude mouse model under a surgical microscope. Absorption and emission properties of ICG in the different solvent environment, like in plasma and human serum albumin, differ from those in aqueous solution. Photoacoustic spectral imaging confirmed a peak shift towards longer wavelengths and an intensity increase of ICG when bound to the lipids. The SCC cells showed that the ICG containing liposomes bind to the cell surface but are not internalized in the SCC-9 cells after 60 minutes of incubation. We also showed here that ICG containing liposomal nanoparticles can be traced under a surgical camera in vivo in orthotopic SCC xenografts in mice.

Abstract 1999: As4O6 suppresses triple-negative breast cancer via inhibiting EGFR/mTOR mediated EMT in vitro and in vivo

Abstract Objective:The present study aimed to verify the anti-metastasis effects and its mechanisms of As4O6 on triple-negative breast cancer(TNBC)in vitro and in vivo. Methods:MTT assay was used to evaluate cell proliferation. Wound healing assay and the invasion assay were carried out to investigate cell migration and invasion. The xenograft zebrafish model and orthotopic xenograft nude mouse model derived from MDA-MB-231-Green fluorescent protein (GFP) cells was established to analysis the anti-metastasis effect of As4O6in vivo. Western blot and immunohistochemistry were performed to analysis the metastasis-related proteins expression. Result: MTT assay showed cell proliferation of TNBC cell MDA-MB-231 was inhibited in a dose-dependent manner by As4O6 treatment. Also, As4O6 suppressed cell migration and invasion by a Wound healing assay and the invasion assay, resulted in the reduction of MMP-2 and MMP-9 expression, especially for the inhibition of EGFR and mTOR phosphorylation. Meanwhile, As4O6 suppressed cancer metastasis in xenograft zebrafish model. The number of metastatic lesion in mouse lung was significantly reduced by the treatment of As4O6 in the orthotopic xenograft nude mouse model. The expression of p-mTOR, p-EGFR and EMT-related proteins such as Vimentin and E-cadherin were significantly reversed in As4O6 treated mouse lung tissues than that of non-treatment group. Furthermore, As4O6 treatment suppressed the expression of angiogenesis related proteins VEGF-a, VEGFR, CD34 and MMP2/9 in mouse lung tissues. Conclusion:Thus, the data suggests that As4O6suppresses triple-negative breast cancer metastasis via inhibiting EGFR/mTOR mediated EMT in vitro and in vivo. As4O6 might be served as a potential useful agent for TNBC metastasis in the future. Citation Format: Illju Bae, Zenglin Lian, Ziyao Liu, Zhaoyan Wu. As4O6 suppresses triple-negative breast cancer via inhibiting EGFR/mTOR mediated EMT in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1999.

Tumor-targeting Salmonella typhimurium A1-R is a highly effective general therapeutic for undifferentiated soft tissue sarcoma patient-derived orthotopic xenograft nude-mouse models

Undifferentiated soft tissue sarcoma (USTS) is a recalcitrant and heterogeneous subgroup of soft tissue sarcoma with high risk of metastasis and recurrence. Due to heterogeneity of USTS, there is no reliably effective first-line therapy. We have generated tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R), which previously showed strong efficacy on single patient-derived orthotopic xenograft (PDOX) models of Ewing's sarcoma and follicular dendritic cell sarcoma. In the present study, tumor resected from 4 patients with a biopsy-proven USTS (2 undifferentiated pleomorphic sarcoma [UPS], 1 undifferentiated sarcoma not otherwise specified [NOS] and 1 undifferentiated spindle cell sarcoma [USS]) were grown orthotopically in the biceps femoris muscle of mice to establish PDOX models. One USS model and one UPS model were doxorubicin (DOX) resistant. One UPS and the NOS model were partially sensitive to DOX. DOX is first-line therapy for these diseases. S. typhimurium A1-R arrested tumor growth all 4 models. In addition to arresting tumor growth in each case, S. typhimurium A1-R was significantly more efficacious than DOX in each case, thereby surpassing first-line therapy. These results suggest that S. typhimurium A1-R can be a general therapeutic for USTS and possibly sarcoma in general.

Recombinant methioninase in combination with doxorubicin (DOX) overcomes first-line DOX resistance in a patient-derived orthotopic xenograft nude-mouse model of undifferentiated spindle-cell sarcoma

We have previously established a patient-derived orthotopic xenograft (PDOX) model of undifferentiated spindle cell sarcoma (USCS). Recombinant methioninase (rMETase) has previously demonstrated efficacy in PDOX mouse models of human cancers. In the present study, we determined if rMETase in combination with doxorubicin (DOX) can overcome first-line DOX resistance in a PDOX models of USCS. The USCS PDOX mouse models were randomized into the following groups when tumor volume reached 100 mm3: G1, control without treatment; G2, doxorubicin (DOX) (3 mg/kg, intraperitoneal [i.p.] injection, weekly, for 2 weeks); G3, rMETase (100 units/mouse, i.p., daily, for 2 weeks); G4, DOX (3 mg/kg, i.p., weekly, for 2 weeks) combined with rMETase (100 units/mouse, i.p., daily, for 2 weeks). Tumor size and body weight were measured twice a week. On day 14 after initiation, the USCS PDOX tumor sizes were (G1): 360 ± 85 mm3; DOX (G2): 355 ± 111 mm3, p = .927; rMETase (G3): 182 ± 57 mm3, p = .0003; DOX + rMETase (G4): 134 ± 29 mm3, p = .00001. These results indicate that rMETase can overcome USCS resistance to DOX, which is first line therapy for this disease. The body weight of treated mice was not significantly different in any group. The present results demonstrate the power of the PDOX model to identify effective therapy for recalcitrant cancer and the potential of rMETase to overcome DOX resistance.

MicroRNA-410 acts as oncogene in NSCLC through downregulating SLC34A2 via activating Wnt/β-catenin pathway.

SLC34A2 had been reported to be down-regulated in human NSCLC cells and patient tissues, and played a significant role in lung cancer. However, the mechanism of its unusual expressionin NSCLC has not been fully elucidated. In present study, we identified SLC34A2 was a direct target of miR-410 and could be inhibited by miR-410 transcriptionally and post-transcriptionally. MiR-410 promoted the growth, invasion and migration of NSCLC cells in vitro. An orthotopic xenograft nude mouse model further affirmed that miR-410 promoted NSCLC cell growth and metastasis in vivo. Moreover, restoring SLC34A2 expression effectively reversed the miR-410-mediated promotion of cell growth, invasion and migration in NSCLC cells. In addition, miR-410high /SLC34A2low expression signature frequently existed in NSCLC cells and tumor tissues. MiR-410 significantly increased the expression of DVL2 and β-catenin protein while decreased that of Gsk3β protein of Wnt/β-catenin signaling pathway, while SLC34A2 partly blocked the effects of miR-410 on those protein expressions. Hence, our data for the first time delineated that unusual expression of SLC34A2 was modulated by miR-410, and miR-410 might positivelycontribute to the tumorigenesis and development of NSCLC by down-regulating SLC34A2 and activating Wnt/β-catenin signaling pathway. MiR-410 might be a new potential therapeutic target for NSCLC.

MP45-01 HIGH ALDO-KETO REDUCTASE 1C1 EXPRESSION IN METASTATIC BLADDER CANCER CELLS ASSOCIATED WITH INVASIVE POTENTIAL AND DRUG RESISTANCE

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2015MP45-01 HIGH ALDO-KETO REDUCTASE 1C1 EXPRESSION IN METASTATIC BLADDER CANCER CELLS ASSOCIATED WITH INVASIVE POTENTIAL AND DRUG RESISTANCE Ryuji Matsumoto, Masumi Tsuda, Takashige Abe, Satoru Maruyama, Kunihiko Tsuchiya, Naoto Miyajima, Nobuo Shinohara, and Shinya Tanaka Ryuji MatsumotoRyuji Matsumoto More articles by this author , Masumi TsudaMasumi Tsuda More articles by this author , Takashige AbeTakashige Abe More articles by this author , Satoru MaruyamaSatoru Maruyama More articles by this author , Kunihiko TsuchiyaKunihiko Tsuchiya More articles by this author , Naoto MiyajimaNaoto Miyajima More articles by this author , Nobuo ShinoharaNobuo Shinohara More articles by this author , and Shinya TanakaShinya Tanaka More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1499AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The objective of this study were to investigate the genetic changes responsible for bladder cancer metastasis, using orthotopic metastatic mouse model, and to find new therapeutic targets of invasive bladder cancer. METHODS Human UM-UC-3 bladder cancer cells were generated to express firefly luciferase 2 and red fluorescent protein tdTomato (UM-UC-3-tdTomato-luc2). Subsequently, we developed an orthotopic xenograft nude mouse model of bladder cancer by inoculating UM-UC-3-tdTomato-luc2 cells through a urethral catheter into the mouse bladder. We then performed bioluminescent imaging (BLI) to noninvasively monitor the growth of bladder tumors in their orthotopic sites. Two mice underwent autopsy after confirmation of lung or liver metastases using BLI. The primary and metastatic tumors were excised and plated on tissue culture dishes to generate bladder, lung, liver, and bone sublines. To examine the role of metastasis-related genes in bladder cancer, we then performed comparative microarray analyses of gene expression, and analyzed gene expression changes in bladder vs. lung, bladder vs. liver and bladder vs. bone. RESULTS Among the 8 genes commonly up-regulated in metastatic sublines compared to bladder cells by our microarray analysis, the expression of aldo-keto reductase 1C1 (AKR1C1) was especially up-regulated. Furthermore, AKR1C1 was significantly upregulated in the 3 metastatic sublines by qRT-PCR and immunoblot analysis. In clinical samples, we observed increased expression of AKR1C1 in human metastatic tissues compared with the corresponding primary bladder cancer tissues by qRT-PCR and immunohistochemistry analysis. Knockdown of AKR1C1 using small interfering RNA downregulated expression levels of Rac1, phosphorylated Src, and phosphorylated FAK, accompanied by reduced invasive ability. Overexpression of AKR1C isoforms, including AKR1C1, has been demonstrated to be associated with drug resistance in various cancers. UM-CU-3 metastatic cells were more resistant to cisplatin than were UM-UC-3 wild-type cells, and AKR1C1 inhibitor flufenamic acid reversed cisplatin resistance in these cells. CONCLUSIONS We found that AKR1C1 was up-regulated both in metastatic bladder cancer cells using an orthotopic mouse model and in metastatic sites of human surgical specimens. Our results demonstrate the role of AKR1C1 in regulating bladder cancer metastasis and drug resistance; therefore, AKR1C1 is a potential target for effective treatment of invasive bladder cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e535 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryuji Matsumoto More articles by this author Masumi Tsuda More articles by this author Takashige Abe More articles by this author Satoru Maruyama More articles by this author Kunihiko Tsuchiya More articles by this author Naoto Miyajima More articles by this author Nobuo Shinohara More articles by this author Shinya Tanaka More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

223 High aldo-keto reductase 1C1 expression in metastatic bladder cancer cells associated with invasive potential and drug resistance

INTRODUCTION AND OBJECTIVES: The objective of this study were to investigate the genetic changes responsible for bladder cancer metastasis, using orthotopic metastatic mouse model, and to find new therapeutic targets of invasive bladder cancer. METHODS: Human UM-UC-3 bladder cancer cells were generated to express firefly luciferase 2 and red fluorescent protein tdTomato (UM-UC-3-tdTomato-luc2). Subsequently, we developed an orthotopic xenograft nude mouse model of bladder cancer by inoculating UM-UC-3-tdTomato-luc2 cells through a urethral catheter into the mouse bladder. We then performed bioluminescent imaging (BLI) to noninvasively monitor the growth of bladder tumors in their orthotopic sites. Two mice underwent autopsy after confirmation of lung or liver metastases using BLI. The primary and metastatic tumors were excised and plated on tissue culture dishes to generate bladder, lung, liver, and bone sublines. To examine the role of metastasis-related genes in bladder cancer, we then performed comparative microarray analyses of gene expression, and analyzed gene expression changes in bladder vs. lung, bladder vs. liver and bladder vs. bone. RESULTS: Among the 8 genes commonly up-regulated in metastatic sublines compared to bladder cells by our microarray analysis, the expression of aldo-keto reductase 1C1 (AKR1C1) was especially up-regulated. Furthermore, AKR1C1 was significantly upregulated in the 3 metastatic sublines by qRT-PCR and immunoblot analysis. In clinical samples, we observed increased expression of AKR1C1 in human metastatic tissues compared with the corresponding primary bladder cancer tissues by qRT-PCR and immunohistochemistry analysis. Knockdown of AKR1C1 using small interfering RNA downregulated expression levels of Rac1, phosphorylated Src, and phosphorylated FAK, accompanied by reduced invasive ability. Overexpression of AKR1C isoforms, including AKR1C1, has been demonstrated to be associated with drug resistance in various cancers. UM-CU-3 metastatic cells were more resistant to cisplatin than were UM-UC-3 wild-type cells, and AKR1C1 inhibitor flufenamic acid reversed cisplatin resistance in these cells. CONCLUSIONS: We found that AKR1C1 was up-regulated both in metastatic bladder cancer cells using an orthotopic mouse model and in metastatic sites of human surgical specimens. Our results demonstrate the role of AKR1C1 in regulating bladder cancer metastasis and drug resistance; therefore, AKR1C1 is a potential target for effective treatment of invasive bladder cancer.

Role of aldolase A in osteosarcoma progression and metastasis: in vitro and in vivo evidence.

Aldolase A (ALDOA) has been reported to be negative survival marker of osteosarcoma (OS) and may be implicated in OS development and progression. In the present study, we assessed for the first time the functional role of ALDOA in OS cell invasion and survival invitro and invivo, using human OS cell lines and an orthotopic xenograft nude mouse model. Overexpression and knockdown of ALDOA were respectively performed in MG-63 and U-2 OS cells, which showed relatively low and high constitutive ALDOA expression levels, respectively. Overexpression of ALDOA in MG-63 cells significantly increased invitro cell invasion, matrix metalloproteinase (MMP)-2 expression, and cell survival against cisplatin-induced apoptosis. On the other hand, knockdown of ALDOA in U-2 cells markedly decreased invitro cell invasion, MMP-2 expression, and cell survival against cisplatin-induced apoptosis. In an orthotopic xenograft nude mouse model, intra-tibial injection of MG-63 cells overexpressing ALDOA led to significantly increased primary tumor volume and pulmonary metastasis as well as decreased cell apoptosis in the primary tumors, compared with the controls. In contrast, intra-tibial injection of U-2 cells with knockdown of ALDOA led to markedly decreased primary tumor volume and pulmonary metastasis as well as increased cell apoptosis in the primary tumors, compared with the controls. In conclusion, our invitro data indicate that ALDOA promotes OS cell invasion and survival, and our in vivo data demonstrate an important role of ALDOA in promoting OS tumor growth and metastasis. The present study provides the first invitro and invivo evidence supporting a critical functional role of ALDOA in OS progression and metastasis, suggesting that ALDOA could serve as a novel therapeutic target in OS. Additionally, our results suggest that ALDOA is involved in the development of OS chemoresistance.

Nitric oxide synthase II suppresses the growth and metastasis of human cancer regardless of its up-regulation of protumor factors

Inducible nitric oxide (NO) synthase (NOS) II has been implicated in macrophage-mediated antitumor activity. However, use of the NOS II gene in cancer therapy is problematic because of the double-edged nature of NO action. Herein we show that adenoviral vectors mediated effective NOS II gene transfer into various human tumors. Production of NO significantly up-regulated multiple angiogenic molecules. However, the NO-producing tumor cells did not form tumors or metastases in ectopic or orthotopic xenograft nude mouse models. The dramatic loss of malignancy was due to NO-mediated apoptosis. We also generated a series of adenoviral vectors harboring mutant NOS II genes that expressed mutant NOS II proteins with defined levels of enzymatic activity. Tumor cells transduced with these NOS II genes produced NO at different levels, which directly correlated with the antitumor activity in vitro and in vivo. This demonstration using a relevant biological system shows that NO produces dose-dependent antitumor activity in vitro and in vivo, regardless of its up-regulation of protumor factors.

Contortrostatin, a dimeric disintegrin from Agkistrodon contortrix contortrix, inhibits breast cancer progression.

We report the results of a multidisciplinary study on the inhibitory effect of a snake venom disintegrin, contortrostatin, a 13.5 kDa homodimeric protein isolated from Agkistrodon contortrix contortrix (southern copperhead) venom, on breast cancer progression. We demonstrate that contortrostatin binds to integrins and blocks the adhesion of human breast cancer cells (MDA-MB-435) to extracellular matrix (ECM) proteins including fibronectin and vitronectin, but it has no effect on adhesion of the cells to laminin and Matrigel. Contortrostatin also prevents invasion of MDA-MB-435 cells through an artificial Matrigel basement membrane. Daily local injection of contortrostatin (5 microg per mouse per day) into MDA-MB-435 tumor masses in an orthotopic xenograft nude mouse model inhibits growth of the tumor by 74% (p = 0.0164). More importantly, it reduces the number of pulmonary macro-metastasis of the breast cancer by 68% (p < 0.001), and micro-metastasis by 62.4% (p < 0.001). Contortrostatin is not cytotoxic to cancer cells, and does not inhibit proliferation of the breast cancer cells in vitro. However, contortrostatin inhibits angiogenesis induced by the breast cancer, as shown by immunohistochemical quantitation of the vascular endothelial cells in tumor tissue removed from the nude mice. We have identified alpha(v)beta3, an important integrin mediating cell motility and tumor invasion, as one of the binding sites of contortrostatin on MDA-MB-435 cells. We conclude that contortrostatin blocks alpha(v)beta3, and perhaps other integrins, and thus inhibits in vivo progression.