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Background: Orthotopic LNCaP xenograft mouse models closely mimic the progression of androgen-dependent prostate cancer in humans; however, orthotopic injection of LNCaP cells into the mouse prostate remains a challenge., Methods: Under the guidance of a stereoscopic microscope, the anatomy of the individual prostate lobes in male Balb/c athymic nude mice was investigated, and LNCaP cells were inoculated into the mouse dorsal prostate (DP) to generate orthotopic tumors that mimicked the pathophysiological process of prostate cancer in humans. Real-time ultrasound imaging was used to monitor orthotopic prostate tumorigenesis, contrast-enhanced ultrasonography (CEUS) was used to characterize tumor angiogenesis, and macroscopic and microscopic characteristics of tumors were described., Results: The DP had a trigonal bipyramid-shape and were located at the base of the seminal vesicles. After orthotopic inoculation, gray scale ultrasound imaging showed progressive changes in tumor echotexture, shape and location, and tumors tended to protrude into the bladder. After 8 weeks, the tumor take rate was 65% (n = 13/20 mice). On CEUS, signal intensity increased rapidly, peaked, and decreased gradually. Observations of gross specimens showed orthotopic prostate tumors were well circumscribed, round, dark brown, and soft, with a smooth outer surface and a glossy appearance. Microscopically, tumor cells were arranged in acini encircled by fibrous septa with variably thickened walls, mimicking human adenocarcinoma., Conclusions: This study describes a successful approach to establishing an orthotopic LNCaP xenograft Balb/c athymic nude mouse model. The model requires a thorough understanding of mouse prostate anatomy and proper technique. The model represents a valuable tool for the in vivo study of the biological processes involved in angiogenesis in prostate cancer and preclinical evaluations of novel anti-angiogenic therapies., (© 2022. The Author(s).)

Drug resistance is a major barrier against successful treatments of cancer patients. Various intrinsic mechanisms and adaptive responses of tumor cells to cancer drugs often lead to failure of treatments and tumor relapse. Understanding mechanisms of cancer drug resistance is critical to develop effective treatments with sustained anti-tumor effects. Three-dimensional cultures of cancer cells known as spheroids present a biologically relevant model of avascular tumors and have been increasingly incorporated in tumor biology and cancer drug discovery studies. In this review, we discuss several recent studies from our group that utilized colorectal tumor spheroids to investigate responses of cancer cells to cytotoxic and molecularly targeted drugs and uncover mechanisms of drug resistance. We highlight our findings from both short-term, one-time treatments and long-term, cyclic treatments of tumor spheroids and discuss mechanisms of adaptation of cancer cells to the treatments. Guided by mechanisms of resistance, we demonstrate the feasibility of designing specific drug combinations to effectively block growth and resistance of cancer cells in spheroid cultures. Finally, we conclude with our perspectives on the utility of three-dimensional tumor models and their shortcomings and advantages for phenotypic and mechanistic studies of cancer drug resistance.

This protocol provides the procedures for isolating differentiated tumor cells from medulloblastoma (MB) in mice. Procedures for transplantation into cerebella are also included to examine the tumorigenesis of differentiated MB cells. This protocol outlines the detailed steps required for (1) isolation of tumor cells from mouse MB, (2) purification of differentiated tumor cells by fluorescence-activated cell sorting, and (3) transplantation of tumor cells into cerebella. This protocol is useful to purify differentiated tumor cells for investigating mechanisms underlying MB progression. For complete details on the use and execution of this protocol, please refer to Cheng et. al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Pancreatic cancer is a particularly lethal malignancy that resists immunotherapy. In this study, using a preclinical pancreatic cancer murine model, we demonstrate a progressive decrease in IFN-γ and granzyme B and a concomitant increase in Tox and IL-10 in intratumoral tumor-specific T cells. Intratumoral myeloid cells produced elevated IL-27, a cytokine that correlates with poor patient outcome. Abrogating IL-27 signaling significantly decreased intratumoral Tox + T cells and delayed tumor growth yet was not curative. Agonistic αCD40 decreased intratumoral IL-27-producing myeloid cells, decreased IL-10-producing intratumoral T cells, and promoted intratumoral Klrg1 + Gzmb + short-lived effector T cells. Combination agonistic αCD40+αPD-L1 cured 63% of tumor-bearing animals, promoted rejection following tumor rechallenge, and correlated with a 2-log increase in pancreas-residing tumor-specific T cells. Interfering with Ifngr1 expression in nontumor/host cells abrogated agonistic αCD40+αPD-L1 efficacy. In contrast, interfering with nontumor/host cell Tnfrsf1a led to cure in 100% of animals following agonistic αCD40+αPD-L1 and promoted the formation of circulating central memory T cells rather than long-lived effector T cells. In summary, we identify a mechanistic basis for T cell exhaustion in pancreatic cancer and a feasible clinical strategy to overcome it., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Purpose: Diffuse intrinsic pontine glioma (DIPG) bears a dismal prognosis. A genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, Activin A receptor type I (ACVR1)-G328V (mACVR1), was developed for testing an immune-stimulatory gene therapy., Experimental Design: We utilized the Sleeping Beauty transposase system to generate an endogenous mouse model of mACVR1 brainstem glioma. Histology was used to characterize and validate the model. We performed RNA-sequencing analysis on neurospheres harboring mACVR1. mACVR1 neurospheres were implanted into the pons of immune-competent mice to test the therapeutic efficacy and toxicity of immune-stimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). mACVR1 neurospheres expressing the surrogate tumor antigen ovalbumin were generated to investigate whether TK/Flt3L treatment induces the recruitment of tumor antigen-specific T cells., Results: Histologic analysis of mACVR1 tumors indicates that they are localized in the brainstem and have increased downstream signaling of bone morphogenetic pathway as demonstrated by increased phospho-smad1/5 and Id1 levels. Transcriptome analysis of mACVR1 neurosphere identified an increase in the TGFβ signaling pathway and the regulation of cell differentiation. Adenoviral delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and increased median survival (MS)., Conclusions: This study provides insights into the phenotype and function of the tumor immune microenvironment in a mouse model of brainstem glioma harboring mACVR1. Immune-stimulatory gene therapy targeting the hosts' antitumor immune response inhibits tumor progression and increases MS of mice bearing mACVR1 tumors., (©2020 American Association for Cancer Research.)

Purpose: Aberrant Myc expression is a major factor in the pathogenesis of aggressive lymphoma, and these lymphomas, while clinically heterogeneous, often are resistant to currently available treatments and have poor survival. Myc expression can also be seen in aggressive lymphomas that are observed in the context of CLL, and we sought to develop a mouse model that could be used to study therapeutic strategies for aggressive lymphoma in the context of CLL., Experimental Design: We crossed the Eμ-TCL1 mouse model with the Eμ-Myc mouse model to investigate the clinical phenotype associated with B-cell-restricted expression of these oncogenes. The resulting malignancy was then extensively characterized, from both a clinical and biologic perspective., Results: Eμ-TCL1xMyc mice uniformly developed highly aggressive lymphoid disease with histologically, immunophenotypically, and molecularly distinct concurrent CLL and B-cell lymphoma, leading to a significantly reduced lifespan. Injection of cells from diseased Eμ-TCL1xMyc into WT mice established a disease similar to that in the double-transgenic mice. Both Eμ-TCL1xMyc mice and mice with disease after adoptive transfer failed to respond to ibrutinib. Effective and durable disease control was, however, observed by selective inhibition of nuclear export protein exportin-1 (XPO1) using a compound currently in clinical development for relapsed/refractory malignancies, including CLL and lymphoma., Conclusions: The Eμ-TCL1xMyc mouse is a new preclinical tool for testing experimental drugs for aggressive B-cell lymphoma, including in the context of CLL., (©2019 American Association for Cancer Research.)

Context: In the past few years, research has suggested that molecular subtypes in muscle-invasive bladder cancer (MIBC) may be exploited to accelerate developments in clinical disease management and novel therapeutics., Objective: To review MIBC mouse models from a molecular subtype perspective, their advantages and limitations, and their applications in translational medicine, based on a PubMed search for publications from January 2000 to February 2018., Evidence Acquisition: Publications relevant to MIBC mouse models and their molecular subtypes were identified in a literature review., Evidence Synthesis: We classified the models according to the technique used for their establishment. For xenotransplant and allograft models, the inoculated cells and inoculated locations are the major determinants of molecular subtypes. Although the cell lines used in xenotransplant models can cover most of the basal-squamous and luminal subtypes, allograft models offer a more realistic environment in which to reconstruct aspects of the associated stromal and immune features. Autochthonous models, using genetic and/or chemical stimuli to induce disease progression, can also generate models with basal-squamous and luminal subtypes, but further molecular characterisation is needed since other mutational variants may be introduced in these models., Conclusions: We identified preclinical MIBC models with different subtype specifications and assessed their promise and current limitations. These models are versatile tools that can reproduce the molecular complexity of MIBC and support novel therapeutic development., Patient Summary: Understanding which models of muscle-invasive bladder cancer most accurately represent the clinical situation is important for the development of novel drugs and disease management strategies. We review the different models currently available and their relevance to different clinical subtypes., (Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Anorexia is a common manifestation of chronic diseases, including cancer. Here we investigate the contribution to cancer anorexia made by calcitonin gene-related peptide (CGRP) neurons in the parabrachial nucleus (PBN) that transmit anorexic signals. We show that CGRP PBN neurons are activated in mice implanted with Lewis lung carcinoma cells. Inactivation of CGRP PBN neurons before tumor implantation prevents anorexia and loss of lean mass, and their inhibition after symptom onset reverses anorexia. CGRP PBN neurons are also activated in Apc min/+ mice, which develop intestinal cancer and lose weight despite the absence of reduced food intake. Inactivation of CGRP PBN neurons in Apc min/+ mice permits hyperphagia that counteracts weight loss, revealing a role for these neurons in a 'nonanorexic' cancer model. We also demonstrate that inactivation of CGRP PBN neurons prevents lethargy, anxiety and malaise associated with cancer. These findings establish CGRP PBN neurons as key mediators of cancer-induced appetite suppression and associated behavioral changes.

PTPN11 mutation, a RAS signaling pathway mutation, is associated with MLL translocations in acute leukemia. A girl with MLL/AF10 AML was found to carry PTPN11 G503A . To study the impact of PTPN11 G503A cooperating with MLL/AF10 on leukemogenesis, we established a retroviral transduction/transplantation mouse model. Compared to the MLL/AF10(OM-LZ) leukemia cells harboring PTPN11 wt , the cells harboring PTPN11 G503A were hypersensitive to GM-CSF and IL3, and more resistant to death upon treatment with daunorubicin but sensitive to cytarabine. The cells harboring PTPN11 G503A autonomously differentiated into macrophages (1.8%) in the medium containing IL3. Further studies showed that the cells had an elevated (∼2.9-fold) Csf1 transcription level and secreted more (∼4.5-fold) M-CSF to the medium which can stimulate monocyte/macrophage differentiation of BM cells. Mice transplanted with the cells harboring PTPN11 G503A had a higher concentration of M-CSF in plasma. When mixed with the MLL/AF10(OM-LZ) leukemia cells harboring PTPN11 wt , the cells harboring PTPN11 G503A had an increased competitive engraftment and clonal expansion in the BM and spleen of recipient mice, although no competitive growth advantage was observed in the in vitro co-culturing assays. The mice transplanted with the MLL/AF10(OM-LZ) cells harboring PTPN11 wt developed myelomonocytic leukemia, while those transplanted with the cells harboring PTPN11 G503A -induced monocytic leukemia in a shorter latency. Our results demonstrated that addition of PTPN11 G503A to MLL/AF10 affected cell proliferation, chemo-resistance, differentiation, in vivo BM recruitment/clonal expansion and accelerated disease progression., (© 2016 UICC.)

Xenotransplantation is a widely used technique to test the tumorigenic potential of human cells in vivo using immunodeficient mice. Here we describe basic technologies and recent advances in xenotransplantation applied to study squamous cell carcinomas (SCCs) of the skin. SCC cells isolated from tumors can either be cultured to generate a cell line or injected directly into mice. Several immunodeficient mouse models are available for selection based on the experimental design and the type of tumorigenicity assay. Subcutaneous injection is the most widely used technique for xenotransplantation because it involves a simple procedure allowing the use of a large number of cells, although it may not mimic the original tumor environment. SCC cell injections at the epidermal-to-dermal junction or grafting of organotypic cultures containing human stroma have also been used to more closely resemble the tumor environment. Mixing of SCC cells with cancer-associated fibroblasts can allow the study of their interaction and reciprocal influence, which can be followed in real time by intradermal ear injection using conventional fluorescent microscopy. In this article, we will review recent advances in xenotransplantation technologies applied to study behavior of SCC cells and their interaction with the tumor environment in vivo., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Mouse models are commonly used for studying hepatocellular carcinoma (HCC) biology and exploring new therapeutic interventions. Currently three main modalities of HCC mouse models have been extensively employed in pre-clinical studies including chemically induced, transgenic and transplantation models. Among them, transplantation models are preferred for evaluating in vivo drug efficacy in pre-clinical settings given the short latency, uniformity in size and close resemblance to tumors in patients. However methods used for establishing orthotopic HCC transplantation mouse models are diverse and fragmentized without a comprehensive comparison. Here, we systemically evaluate four different approaches commonly used to establish HCC mice in preclinical studies, including intravenous, intrasplenic, intrahepatic inoculation of tumor cells and intrahepatic tissue implantation. Four parameters--the latency period, take rates, pathological features and metastatic rates--were evaluated side-by-side. 100% take rates were achieved in liver with intrahepatic, intrasplenic inoculation of tumor cells and intrahepatic tissue implantation. In contrast, no tumor in liver was observed with intravenous injection of tumor cells. Intrahepatic tissue implantation resulted in the shortest latency with 0.5 cm (longitudinal diameter) tumors found in liver two weeks after implantation, compared to 0.1cm for intrahepatic inoculation of tumor cells. Approximately 0.1cm tumors were only visible at 4 weeks after intrasplenic inoculation. Uniform, focal and solitary tumors were formed with intrahepatic tissue implantation whereas multinodular, dispersed and non-uniform tumors produced with intrahepatic and intrasplenic inoculation of tumor cells. Notably, metastasis became visible in liver, peritoneum and mesenterium at 3 weeks post-implantation, and lung metastasis was visible after 7 weeks. T cell infiltration was evident in tumors, resembling the situation in HCC patients. Our study demonstrated that orthotopic HCC mouse models established via intrahepatic tissue implantation authentically reflect clinical manifestations in HCC patients pathologically and immunologically, suggesting intrahepatic tissue implantation is a preferable approach for establishing orthotopic HCC mouse models.

Morphological detection of cancer cells in the rabbit VX2 allograft transplantation model is often difficult in a certain region such as serosal cavity where reactive mesothelial cells mimic cancer cells and both cells share common markers such as cytokeratins. Therefore, tagging VX2 cells with a specific and sensitive marker that easily distinguishes them from other cells would be advantageous. Thus, we tried to establish a successively transplantable, enhanced green fluorescent protein (EGFP)-expressing VX2 model. Cancer cells obtained from a conventional VX2-bearing rabbit were cultured in vitro and transfected with an EGFP-encoding vector, and then successively transplanted in Healthy Japanese White rabbits (HJWRs) (n = 8). Besides, conventional VX2 cells were transplanted in other HJWRs (n = 8). Clinicopathological comparison analyses were performed between the two groups. The success rate of transplantation was 100% for both groups. The sensitivity and specificity of EGFP for immunohistochemical detection of VX2 cells were 84.3 and 100%, respectively. No significant differences in cancer cell morphology, tumor size (P = 0.742), Ki-67 labeling index (P = 0.878), or survival rate (P = 0.592) were observed between the two. VX2 cells can be genetically altered, visualized by EGFP, and successively transplanted without significant alteration of morphological and biological properties compared to those of the conventional model.

Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an in vivo metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1α, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis.

Introduction: Evaluation of new surgical techniques in animal models is frequently challenging. This article describes the pitfalls, peculiarities and the final best applicable model for evaluating surgical techniques for glioma resection., Methods: The C6 glioma cell line and the Sprague-Dawley rat strain were selected. Fifty-thousand glioma cells were stereotactically transplanted in the left hemisphere of 137 male adult rats. Evaluation of solid tumour formation, tumour growth and scheduling of surgical resection was performed by MR scanning at 1, 2, and 4 weeks after transplantation and 3 and 6 months after tumour resection. Microsurgical tumour resection was performed with conventional techniques or with the waterjet dissector at a pressure of 6 bar. One subgroup of each surgical technique was sacrificed directly after surgery for histological analysis. The other subgroup was followed up for long-term analysis., Results: The transplantation site was of great importance. After transplantation of tumour cells posterior to the bregma, intra-ventricular tumour growth with spreading occurred. Homogenous and reproducible tumour growth was achieved after grafting cells lateral - 3 mm, anterior + 1 mm, and - 2.5 mm ventral to the bregma. After development of solid tumours on MR imaging, animals were subjected to surgery. MR and intra-operative findings corresponded well. However, MRI and intra-operative none-detectable perivascular tumour spreading was histologically observed in the majority of cases., Conclusions: The presented glioma rat model consisting of the C6 cell line and Sprague-Dawley rats as recipients is a well-suited model to investigate surgical techniques and their impact on tumour therapy. However, the site of transplantation, the preparation of cell grafts and the technique of tumour growth evaluation is of utmost importance to achieve reliable results.

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumorigenicity of murine hepatoma cells (MH22a) and their sensitivity to lysis by natural killers (NKs) have been studied after exposure to polychromatic visible and infrared light (VIS-IR, 480-3400 nm, 40 mW/cm2), similar to the terrestrial solar spectrum without its minor UV component, in order to elucidate the involvement of this important environmental and physiotherapeutic factor in regulation of the anti-tumor defense system. The MH22 cells were in vitro exposed to VIS-IR light and their sensitivity to lytic activity of NKs was evaluated. We found that sensitivity of MH22a cells to lysis by NKs after exposure to VIS-IR light at a dose of 4.8 J/cm2 increased 1.5-2 times, while it did not change after exposure to a dose of 9.6 J/cm2 at all ratios (1 : 5-1 : 50) of the number of NKs (effectors) to that of hepatoma cells (targets). The increase in the sensitivity of hepatoma cells to NKs was accompanied by structural changes of cell surface: the capability of supramembraneous glycoproteins (glycocalix) to sorb the vital dye alcian blue (AB) was significantly lower as compared with the unexposed cells of control group. However, no changes in AB sorption was revealed in hepatoma cells exposed to the light at a dose 9.6 J/cm2. Tumorigenicity of photo-irradiated MH22a cells has been studied in the in vivo experiments. Light-exposed (4.8 and 9.6 J/cm2) and intact hepatoma cells were transplanted into syngenic mice C3HA. Tumor volumes 25 days after transplantation proved to be smaller after exposure to the light at both doses than in the control group (4-4.5 times and 2.5-4 times, respectively), which correlated with the increase in the sensitivity to lisys by NKs and decrease in the AB sorption only after light exposure at dose 4.8 J/cm2. Using the flow cytometry method we could show that VIS-IR light at the applied doses did not interfere with the distribution of hepatoma cells over the cycle phases and thus deceleration of the tumor growth was not associated with cytostatic effect of VIS-IR light. To evaluate effect of polychromatic light on the growth of the preformed tumors, the 5-day course of daily light exposures of tumor bearing mice C3HA was carried out in 10 days after subcutaneous transplantation of 2 x 10(5) cells of syngene hepatoma when the tumors had developed in 100% animals. Like in the case of transplantation of the light-exposed cells, irradiation of the tumor bearing mice at doses 4.8-9.6 J/cm2 resulted in deceleration of tumor growth (2.1-2.9 and 2.2 times respectively) for 4 weeks as compared with non-irradiated mice.

SF188/V+ is a highly vascular human glioma model that is based on transfection of vascular endothelial growth factor (VEGF) cDNA into SF188/V- cells. This study aims to assess its growth and vascularity properties in vivo in a rat model. Thirty-two adult rats were inoculated with SF188/V+ tumor cells, and, for comparison, five were inoculated with SF188/V- tumor cells. Several conventional magnetic resonance imaging (MRI) sequences were acquired, and several quantitative structural (T(2) and T(1)), functional [isotropic apparent diffusion coefficient (ADC) and blood flow], and molecular [protein and peptide-based amide proton transfer (APT)] MRI parameters were mapped on a 4.7 T animal scanner. In rats inoculated with SF188/V+ tumor cells, conventional T(2)-weighted images showed a highly heterogeneous tumor mass, and post-contrast T(1)-weighted images showed a heterogeneous, strong enhancement of the mass. There were moderate increases in T(2), T(1), and ADC, and large increases in blood flow and APT in the tumor, compared to contralateral brain tissue. Microscopic examination revealed prominent vascularity and hemorrhage in the VEGF-secreting xenografts as compared to controls, and immunohistochemical staining confirmed increased expression of VEGF in tumor xenografts. Our results indicate that the SF188/V+ glioma model exhibits some MRI and histopathology features that closely resemble human glioblastoma.

Epithelial ovarian cancers (EOCs) often exhibit morphologic features of embryonic Müllerian duct-derived tissue lineages and colonize peritoneal surfaces that overlie connective and adipose tissues. However, the mechanisms that enable EOC cells to readily adapt to the peritoneal environment are poorly understood. In this study, we show that expression of HOXA9, a Müllerian-patterning gene, is strongly associated with poor outcomes in patients with EOC and in mouse xenograft models of EOC. Whereas HOXA9 promoted EOC growth in vivo, HOXA9 did not stimulate autonomous tumor cell growth in vitro. On the other hand, expression of HOXA9 in EOC cells induced normal peritoneal fibroblasts to express markers of cancer-associated fibroblasts (CAFs) and to stimulate growth of EOC and endothelial cells. Similarly, expression of HOXA9 in EOC cells induced normal adipose- and bone marrow-derived mesenchymal stem cells (MSCs) to acquire features of CAFs. These effects of HOXA9 were due in substantial part to its transcriptional activation of the gene encoding TGF-β2 that acted in a paracrine manner on peritoneal fibroblasts and MSCs to induce CXCL12, IL-6, and VEGF-A expression. These results indicate that HOXA9 expression in EOC cells promotes a microenvironment that is permissive for tumor growth.

We studied the content of tissue inhibitors of matrix metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) and activities of matrix metalloproteinases (MMP) in the serum and lungs of mice with Lewis lung carcinoma metastasizing into the lung. Metastasizing was associated with increased serum content of TIMP-1 and TIMP-2 (only on day 20 at the terminal stage of the tumor process). These data confirm the hypothesis on pro-tumorigenic role of TIMP-1 in the serum. Locally, the development of metastases was associated with a decrease in TIPM-1 concentration (day 7), an increase in TIMP-2 concentration (days 7 and 20), and elevated activity of MMP at all terms of the study (days 7, 15, and 20). Increased concentration of TIMP-2 in the lungs (but not in the serum) can be regarded as an indicator of Lewis lung carcinoma metastasizing.

Background: The aim of this study was to improve fluorescence laparoscopy of pancreatic cancer in an orthotopic mouse model with the use of a light-emitting diode (LED) light source and optimal fluorophore combinations., Study Design: Human pancreatic cancer models were established with fluorescent FG-RFP, MiaPaca2-GFP, BxPC-3-RFP, and BxPC-3 cancer cells implanted in 6-week-old female athymic mice. Two weeks postimplantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source or a Stryker X8000 xenon light source 24 hours after tail-vein injection of CEA antibodies conjugated with Alexa 488 or Alexa 555. Cancer lesions were detected and localized under each light mode. Intravital images were also obtained with the OV-100 Olympus and Maestro CRI Small Animal Imaging Systems, serving as a positive control. Tumors were collected for histologic analysis., Results: Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor deposits in the peritoneal cavity. The simultaneous use of different fluorophores (Alexa 488 and Alexa 555), conjugated to antibodies, brightened the fluorescence signal, enhancing detection of submillimeter lesions without compromising background illumination. Adjustments to the LED light source permitted simultaneous detection of tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence., Conclusions: Using an LED light source with adjustments to the red, blue, and green wavelengths, it is possible to simultaneously identify tumor metastases expressing fluorescent proteins of different wavelengths, which greatly enhanced the signal without compromising background illumination. Development of this fluorescence laparoscopy technology for clinical use can improve staging and resection of pancreatic cancer., (Copyright © 2012 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Malignant glioma is an incurable disease with a relatively short median survival. Several clinical trials have demonstrated that immunotherapy with vaccination is a safe and possibly effective way of prolonging survival. Antibody-based blockade of cytotoxic T-lymphocyte antigen 4 (CTLA-4) ligation on T lymphocytes is associated with enhanced antitumor immunity in animal models of cancer and in patients with advanced melanoma. We hypothesized that sequential therapy with granulocyte-macrophage-colony-stimulating factor (GM-CSF)-expressing whole-glioma-cell vaccination and CTLA-4 blockade is an effective strategy for treating established intracranial gliomas. GL261 glioma cells were injected into the right frontal lobes of syngeneic C57/BL6 mice. At days 3, 6, and 9 after tumor implantation, mice were treated with subcutaneous injection of irradiated GM-CSF-expressing GL261 cells. Mice were also treated with intraperitoneal injection of anti-CTLA-4 monoclonal antibodies (mAbs), either at days 3, 6, and 9 or days 12, 15, and 18. Animals were followed for survival. Splenocytes were harvested at day 22 for use in enzyme-linked immunosorbent spot assays. Early treatment of established intracranial gliomas with high-dose CTLA-4 blockade was associated with increased survival in GL261-bearing mice. Later treatment with anti-CTLA-4 monoclonal antibodies did not significantly improve survival compared with control-treated mice. Early vaccination followed by subsequent CTLA-4 blockade was associated with significantly improved survival versus either treatment alone and intensified tumor-specific immunity as measured by interferon-γ enzyme-linked immunosorbent spot assay. Sequential immunotherapy with GM-CSF-expressing irradiated glioma cells and CTLA-4 blockade synergistically prolongs survival in mice bearing established intracranial gliomas.

Cancer stem cells represent the most important target cells for antitumor therapy. TRAIL (TNF-related apoptosis-inducing ligand) is a potential anticancer agent that induces apoptosis in a wide variety of tumor cells, but its ability to target cancer stem cells is currently unknown. Here we investigated whether TRAIL targets leukemia-initiating cells. Limiting dilution transplantation assays were performed on xenografts from pediatric patients with precursor B-cell acute lymphoblastic leukemia (pre-B ALL) in NSG mice. In vitro treatment of xenograft cells with TRAIL significantly reduced and delayed their engraftment and procrastinated animal death from leukemia. Systemic TRAIL treatment of mice injected with patient-derived pre-B ALL xenograft cells abrogated leukemia in 3 of 5 mice in 1 sample. In conclusion, our data suggest that TRAIL targets leukemia-initiating cells derived from pre-B ALL xenografts in vitro and in vivo, and hence constitutes an attractive candidate drug for treatment of ALL.

Background: Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo., Methods and Results: Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of vegfc and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth., Conclusion: Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development.

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors in vitro and their recovery in transplanted recipients in vivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The treatment of cancer by antisense anti-IGF-I cellular therapy inducing immune response has evoked interest among many promising strategies. Here, we reported some results obtained from patients with cancer, mainly glioblastoma treated by this strategy, which was also extended to patients with colon carcinoma, ovary cystadenocarcinoma and prostate adenocarcinoma. It was shown that, in the phase I of clinical trial, patients vaccinated with their own tumour cells treated by antisense IGF-I presented a slight increase of temperature. Their peripheral blood lymphocytes showed a shift in the percentage of CD8 effector cells as judged by expression of cell surface markers CD8+ CD28+. Particularly, in two treated patients with glioblastoma, the survival time was 19 and 24 months respectively in comparison to the range of 12 to 15 months observed in the case of classical treatment such as surgery, radiation or chemotherapy. These results, although preliminary, gave indication that the reported strategy could deserve consideration owing to its safety. Furthermore, the increase in the percentage of peripheral blood monomorphonucleated cells (PBMNCs) with effector phenotype, i.e., CD8+ CD28+ in vaccinated patients might explain their prolonged survival time., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Technetium coordination chemistry has been a subject of interest in the development of radiopharmaceuticals, especially imaging radiotracers. Due to the extensive work done on developing chelates for (99m)Tc, various chelators have been investigated and applied to radiopharmceuticals. Previous studies on the coordination chemistry of the [(99m)Tc=O] core have established peptide-derived sequences as effective chelating ligands. These observations led to the design of tetradentate ligands derived from amino acid sequences. Such amino acid sequences provide a tetradentate coordination site for chelation to the radionuclide and an effective functional group for conjugation to biomolecules using conventional solid-phase synthetic routes. A derivative of a novel tripeptide chelating sequence, Pro-Gly-Cys (PGC) has been developed where it is possible to form stable technetium complexes with the [(99m)Tc=O] via N(3)S(1) tetradentate coordination core that serves this function and can be readily incorporated into biomolecules using solid-phase synthesis techniques. As a model system, the RGD peptide was selected which has been well known to target the integrin receptor for angiogenesis and tumor imaging agents. The results of in vivo studies with these novel radiolabeled compounds in tumor xenografts demonstrated a distribution in tumor targeting and other organs, such as kidney, liver and intestines., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: Chondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. An animal model of chondrosarcoma has been established--namely, the Swarm Rat Chondrosarcoma (SRC)--and shown to resemble the human disease. Previous studies with this model revealed that tumor microenvironment could significantly influence chondrosarcoma malignancy., Methods: To examine the effect of the microenvironment, SRC tumors were initiated at different transplantation sites. Pyrosequencing assays were utilized to assess the DNA methylation of the tumors, and SAGE libraries were constructed and sequenced to determine the gene expression profiles of the tumors. Based on the gene expression analysis, subsequent functional assays were designed to determine the relevancy of the specific genes in the development and progression of the SRC., Results: The site of transplantation had a significant impact on the epigenetic and gene expression profiles of SRC tumors. Our analyses revealed that SRC tumors were hypomethylated compared to control tissue, and that tumors at each transplantation site had a unique expression profile. Subsequent functional analysis of differentially expressed genes, albeit preliminary, provided some insight into the role that thymosin-β4, c-fos, and CTGF may play in chondrosarcoma development and progression., Conclusion: This report describes the first global molecular characterization of the SRC model, and it demonstrates that the tumor microenvironment can induce epigenetic alterations and changes in gene expression in the SRC tumors. We documented changes in gene expression that accompany changes in tumor phenotype, and these gene expression changes provide insight into the pathways that may play a role in the development and progression of chondrosarcoma. Furthermore, specific functional analysis indicates that thymosin-β4 may have a role in chondrosarcoma metastasis.

Natural killer T (NKT) cells are potent modulators of antitumor immunity. Their protective effects can be achieved upon their activation by glycolipid ligands presented in the context of the CD1d molecule. These CD1d-binding glycolipid antigens have been described as potent therapeutic agents against tumors, infections, as well as autoimmune diseases. Immunoregulatory and therapeutic effects of glycolipid ligands depend on their structure and modes of administration. Therefore, more studies are needed for optimization of the particular therapeutic settings. This study was focused on the tumor-inhibitory effects of 12 carbon acyl chain beta-galactosyl ceramide (C12 beta-D-Galactosyl Ceramide; beta-GalCer(C12)) on the growth of human papillomavirus type 16 (HPV16)-associated neoplasms transplanted in syngeneic mice. Treatment of tumor-bearing mice with beta-GalCer(C12) 3-14 days after tumor cell transplantation significantly inhibited the growth of the major histocompatibility complex (MHC) Class I-positive (TC-1), as well as MHC Class I-deficient (TC-1/A9) HPV16-associated tumors. Moreover, administration of beta-GalCer(C12) after surgical removal of TC-1 tumors inhibited the growth of tumor recurrences. Similar results were obtained in the treatment of tumors after chemotherapy. beta-GalCer(C12) treatment turned out to be also synergistic with immunotherapy based on administration of IL-12-producing cellular vaccines. These results suggest that beta-GalCer(C12), whose antitumor effects have so far not been studied in detail, can be effective for the treatment of minimal residual tumor disease as well as an adjuvant for cancer immunotherapy.

Purpose: Uveal melanoma is the most common primary intraocular malignant tumor in adults and is defined by a poor natural outcome, as 50% of patients die from metastases. The aim of this study was to develop and characterize a panel of human uveal melanoma xenografts transplanted into immunodeficient mice., Experimental Design: Ninety tumor specimens were grafted into severe combined immunodeficient mice, and 25 transplantable xenografts were then established (28%). Relationship between tumor graft and clinical, biological, and therapeutic features of the patients included were investigated. Characterization of 16 xenografts included histology, molecular analyses by immunohistochemistry, genetic alteration analysis (single-nucleotide polymorphism), and specific tumor antigen expression by quantitative reverse transcription-PCR. Pharmacologic characterization (chemosensitivity) was also done in four models using two drugs, temozolomide and fotemustine, currently used in the clinical management of uveal melanoma., Results: Take rate of human uveal melanoma was 28% (25 of 90). Tumor take was independent of size, histologic parameters, or chromosome 3 monosomy but was significantly higher in metastatic tumors. Interestingly, in vivo tumor growth was prognostic for a lower metastasis-free survival in patients with primary tumors. A high concordance between the patients' tumors and their corresponding xenografts was found for all parameters tested (histology, genetic profile, and tumor antigen expression). Finally, the four xenografts studied displayed different response profiles to chemotherapeutic agents., Conclusions: Based on these results, this panel of 16 uveal melanoma xenografts represents a useful preclinical tool for both pharmacologic and biological assessments.

Background/aim: We attempted to develop a humanized mouse model for prostate cancer to study immune recognition and responses to human prostate-tumor antigens in mice., Materials and Methods: Our study was based on cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) tumors. TRAMP cells were modified to express a chimeric MHC I molecule comprising the extracellular domains of human HLA-A2.1 with the transmembrane and intracellular domain of K(b)., Results: These modified TRAMP cells were immunologically rejected following recognition of human tumor epitopes known to be immunodominant in the context of HLA-A2.1. Immune-compromised SCID-beige mice did not reject these tumors., Conclusion: We conclude that epitopes derived from endogenous murine homologs were being presented by the chimeric MHC class I molecules due to a lack of central tolerance to these epitopes in the mice.

The overall purpose of this study was to establish human head and neck squamous cell carcinoma (HNSCC) xenografts in mice by transplantation of surgical tumor tissue and to characterize the growth, histologic and vascular properties of these xenografts. Primary surgical specimens of HNSCC were xenografted into eight-to-twelve week old severe combined immunodeficiency (SCID) mice. Histologic features of primary HNSCC specimens, initial and established xenografts were compared for tumors established from three different head and neck subsites, namely, oral cavity, larynx and base of tongue (one tumor per site). Growth rates of xenografts were compared along with magnetic resonance imaging (MRI) measures of tumor vascularity and correlative CD31-immunostaining. Initial and established xenografts from all three sites demonstrated a squamous phenotype similar to the original patient tumor histology. Established xenografts of oral cavity and larynx exhibited increased keratinization (H&E) compared to initial xenografts and the primary tumor. No differences in tumor growth rates were observed between established xenografts from the different subsites. Xenografts established from SCC of the larynx exhibited increased microvessel density and lumen area (CD31 staining) along with enhanced permeability to the MR contrast agent compared to oral cavity and base of tongue tumors. Our results show that the combination of non-invasive imaging along with histologic evaluation of patient tumor xenografts offers a valuable platform for preclinical investigations in head and neck cancer. However, it is important to recognize the influence of tumor-host interactions on the histologic phenotype of transplanted tumors.

The CXCR4-SDF-1 axis plays a central role in the trafficking and retention of normal and malignant stem cells in the bone marrow (BM) microenvironment. Here, we used a mouse model of acute promyelocytic leukemia (APL) and a small molecule competitive antagonist of CXCR4, AMD3100, to examine the interaction of mouse APL cells with the BM microenvironment. APL cells from a murine cathepsin G-PML-RARalpha knockin mouse were genetically modified with firefly luciferase (APL(luc)) to allow tracking by bioluminescence imaging. Coculture of APL(luc) cells with M2-10B4 stromal cells protected the leukemia cells from chemotherapy-induced apoptosis in vitro. Upon injection into syngeneic recipients, APL(luc) cells rapidly migrated to the BM followed by egress to the spleen then to the peripheral blood with death due to leukostasis by day 15. Administration of AMD3100 to leukemic mice induced a 1.6-fold increase in total leukocytes and a 9-fold increase of circulating APL blast counts, which peak at 3 hours and return to baseline by 12 hours. Treatment of leukemic mice with chemotherapy plus AMD3100 resulted in decreased tumor burden and improved overall survival compared with mice treated with chemotherapy alone. These studies provide a proof-of-principle for directing therapy to the critical tethers that promote AML-niche interactions.

Liposomes modified with a high concentration of polyethylene glycol (PEG) could significantly prolong the retention time of the carried drug in the circulation, thus improving the drug accumulation in the tumor. In this study, 6 mol% rather than 0.9 mol% PEGylated liposomes (100 nm in diameter) encapsulated with indium-111 were used in a human colorectal carcinoma HT-29/luc tumor-bearing mouse model for comparing the PEGylation effect. Pharmacokinetics, biodistribution, passive-targeted assay, bioluminescence imaging (BLI) and tumor growth measurements were used for the spatial and temporal distribution, tumor localization and therapeutic evaluation of the drug. Pharmacokinetic studies indicated that the terminal half-life (T((1/2))lambdaz) and C(max) of 6 mol% PEG (111)In liposomes were similar to those of 0.9 mol% PEG (111)In liposomes. In the blood, the total body clearance (Cl) of 6 mol% PEG (111)In liposomes was about 1.7-fold lower and the area under the curve (AUC) was 1.7-fold higher than those of 0.9 mol% PEG (111)In liposomes. These results showed that the long-term circulation and localization of 6 mol% PEGylated liposomes was more appropriate for use in the tumor-bearing animal model. In addition, the biodistribution of 6 mol% PEG (111)In liposomes showed significantly lower uptake in the liver, spleen, kidneys, small intestine and bone marrow than those of 0.9 mol% PEG (111)In liposomes. The clearance rate of both drugs from the blood decreased with time, with the maximum at 24 h post intravenous (i.v.) injection. Prominent tumor uptake and the highest tumor/muscle ratios were found at 48 h post injection. Both AUC and relative ratio of the AUCs (RR-AUC) also showed that 6 mol% PEGylated liposomes significantly reduced the uptake of drugs in the reticuloendothelial system (RES), yet enhanced the uptake in the tumor. Gamma scintigraphy at 48 h post injection also demonstrated more distinct tumor uptake with 6 mol% PEG (111)In liposomes as compared to that of 0.9 mol% PEGylated liposomes (p<0.01). BLI and in vivo tumor growth tracing showed that growth in tumor volume could largely be inhibited by 6 mol% PEG (111)In liposomes. The results suggest that 6 mol% PEGylated liposomes might be a more suitable liposomal carrier for drug delivery than 0.9 mol% PEGylated liposomes, not only by reducing the drug accumulation in the RES or its related organs, but by prolonging drug circulation and eventually enhancing the targeting efficiency in the tumor to reach a better therapeutic index.

This study evaluated the effects of cohabitation with a B16F10 melanoma-bearer cage mate on behavior and immune functions in mice. Five different experiments were conducted. In each of them, the female mice were divided into two groups: control and experimental. One mouse of each control pair was kept undisturbed and called "companion of health partner" (CHP). One mouse of each experimental pair was inoculated with B16F10 cells and the other, the subject of this study, was called "companion sick partner" (CSP). On Day 20 of cohabitation, behavior and immune parameters from CHP and CSP mice were analyzed. In comparison to the CHP, the CSP mice: (1) presented an increased general locomotion in the open field and a decreased exploration time and number of entries in the plus-maze open arms; (2) had an enhanced expression of the CD80 costimulatory molecule on Iab(+)CD11c(+) spleen cells, but no differences were found on lymph nodes cells; (3) presented an altered differentiation of bone marrow cells in the presence of GM-CSF, IL-4, and LPS in vitro, resulting in a lower percentage of Iab(+)CD80(+) cells; (4) had a deficit in the establishment of a Delayed Type of Hypersensitivity to ovalbumin, which was associated to an in vitro proliferation of an IL-10-producing lymphocyte subpopulation after ovalbumin stimulation. Corticosterone levels detected on Day 20 of cohabitation were similar in CHP and CSP mice. It is shown here that DCs phenotype in mice is affected by conditions associated with behavioral alterations indicative of an anxiety-like state induced by the cohabitation with a tumor-bearer conspecific. This phenomenon occurred probably through a nondependent corticosterone mechanism.

HKH40A (RTA 502), an optimized 8-methoxy analog of the unsymmetrical bifunctional antitumor agent WMC79, was found to be potently active against liver cancer cell growth in vitro and in vivo. Studies on selected human hepatocellular carcinoma (HCC) cell lines with differing p53 status (HepG2, Hep3B, and PLC/PRF/5), revealed that drug-mediated growth inhibition was independent of p53 status. FACS analysis showed an accumulation of cells in S-phase within 24 h of treatment with 100 nM HKH40A. Subsequent incubation of cells, either in the presence of drug or without, caused cell cycle block at the S and G2/M checkpoints, which was consistent with the observed up-regulation of p21, cyclin A, cyclin B1, sustained phosphorylation of Cdk1, and down-regulation of Cdc6, Cdc7, and RRM2. This irreversible growth arrest eventually led to apoptosis. HKH40A completely suppressed growth of the rat transplantable HCC cell line (JM-1) in an orthotopic model in Fisher 344 rats in vivo, without evidence of toxicity. HKH40A may be a useful agent for new therapeutic strategies focusing on inhibition of HCC cell proliferation.

Background and Purpose: Paclitaxel is highly efficacious in the treatment of breast, head and neck, non-small cell lung cancers and ovarian carcinoma. For malignant gliomas, paclitaxel is prevented from reaching its target by the presence of the efflux pump P-glycoprotein (P-gp) at the blood-brain barrier. We investigated the utilization of a new drug delivery system to increase brain delivery of paclitaxel., Experimental Approach: Paclitaxel molecules were conjugated to a brain peptide vector, Angiopep-2, to provide a paclitaxel-Angiopep-2 conjugate named ANG1005. We determined the brain uptake capacity, intracellular effects and antitumour properties of ANG1005 in vitro against human tumour cell lines and in vivo in human xenografts. We then determined ANG1005 activity on brain tumours with intracerebral human tumour models in nude mice., Key Results: We show by in situ brain perfusion that ANG1005 enters the brain to a greater extent than paclitaxel and bypasses the P-gp. ANG1005 has an antineoplastic potency similar to that of paclitaxel against human cancer cell lines. We also demonstrate that ANG1005 caused a more potent inhibition of human tumour xenografts than paclitaxel. Finally, ANG1005 administration led to a significant increase in the survival of mice with intracerebral implantation of U87 MG glioblastoma cells or NCI-H460 lung carcinoma cells., Conclusions and Implications: These results demonstrate the antitumour potential of a new drug, ANG1005, and establish that conjugation of anticancer agents with the Angiopep-2 peptide vector could increase their efficacy in the treatment of brain cancer.

Melanoma cells originate from the neural crest and are characterized by high migratory potential and invasive growth. After transplantation into the neural tube of the chick embryo, melanoma cells spontaneously emigrate along the neural crest pathways without tumor formation or malignant growth. This emigration depends on the constitutive over-expression of bone morphogenetic protein-2 (BMP-2) and can be ablated by the BMP-antagonist noggin. When transplanted into the embryonic optic cup, melanoma cells invade the host tissue and form malignant tumors. Here, we asked if the invasive growth of melanoma cells in the optic cup could be influenced by BMP-2 or noggin. Mouse B16-F1 cells were grown as aggregates, treated with BMP-2 or noggin during aggregation and transplanted into the optic cup of 3-day chick embryos. After 3 days of subsequent incubation, embryos were evaluated for melanoma cell invasiveness. Immunohistochemical examination revealed that untreated and BMP-2-treated melanoma cells had grown malignantly into the host tissue. However, noggin pretreatment of the aggregates had blocked melanoma cell invasiveness and tumor formation. We conclude that invasive growth of melanoma cells in vivo is BMP-dependent and can be ablated by noggin, thus rendering noggin a promising agent for the treatment of BMP-over-expressing melanoma., ((c) 2007 Wiley-Liss, Inc.)

Human tumors are dependent on angiogenesis for growth, and the vascular endothelial growth factor (VEGF) is a major regulator of this process. Bevacizumab (Avastin), a monoclonal antibody directed against VEGF, has shown promise in treating a variety of cancers. In this study, we first examined the anti-tumor effects of bevacizumab on head and neck squamous cell carcinoma (HNSCC). Then we examined the effects of bevacizumab combined with paclitaxel, a chemotherapeutic agent, in HNSCC. This is the first demonstration of the anti-tumor effects of bevacizumab on HNSCC. In vitro, bevacizumab did not show any antiproliferative effects against the HNSCC cell lines. However, in vivo, bevacizumab showed dramatic anti-tumor effects against HNSCC tumor xenografts in mice. In addition, treatment with a bevacizumab-paclitaxel combination resulted in a remarkable inhibition of the HNSCC tumor xenografts, compared to the effects of each agent separately. A decreased blood vessel density and an increased apoptotic index were seen in the shrunken tumors. These results suggest that bevacizumab in combination with paclitaxel could have useful clinical application in HNSCC.

Object: Current treatments for malignant gliomas produce only a modest increase in survival time. New therapeutic approaches are desperately needed. Suberoylanilide hydroxamic acid (SAHA) is an effective inhibitor of the growth of many solid and hematological malignancies. Nevertheless, very few studies have investigated the effects of SAHA on glial tumors. The present study was designed to investigate the therapeutic effects of the intracranial local delivery of SAHA in an orthotopic glioma model., Methods: The antiproliferative effect of SAHA was examined in six glioblastoma and one endothelial cell lines in vitro. In addition, one glioblastoma cell line (U87MG) used in in vivo short term (14 days) and survival studies in an orthotopic human glioma athymic mice model. Tumor volume, apoptosis rate, microvessel density, and proliferation index were determined by immunohistochemistry., Results: SAHA treatment inhibited the growth of all cell lines in concentrations ranging from 1 microM to 30 microM. For short-term studies, histological analysis showed an 80% reduction of tumor volume in the treatment group (P < 0.001). This reduction in tumor volume was associated with a significant increase in the apoptosis rate (31.9%, P < 0.001), a significant decrease in the proliferation (36.8%, P < 0.001) and angiogenesis rates (30%, P < 0.05). For survival studies, the mean survival time was 22 days in the control group, whereas it was 42 days in the treatment group., Conclusions: These results suggest that local delivery with SAHA inhibits intracranial glioma growth in vitro and in vivo. SAHA is a promising candidate for further preclinical and clinical studies on glial tumors.

To confirm the cytotoxic effect of instant curry containing combined spices on cancer cells in vivo, cancer was induced by transplanting cancer cells to mice, and the development of cancer upon feeding pure curry were examined. The concentration of lipid peroxide in the groups transplanted with cancer cells which were fed with normal feed was 19.6 nM, and it was increased as the amount of pure curry was increased. The concentration of cytochrome P-450 was decreased in the group transplanted with cancer cells which were fed with pure curry and the group without the transplant which were fed with pure curry when compared with the groups which were fed with normal feed. The activity of cytochrome P-450 was decreased as the concentration of cytochrome P-450 was decreased in the groups transplanted with cancer cells. However, it was increased in the groups without cancer cell transplant when over 2% of pure curry was fed. The amount of glutathione was increased in the groups transplanted with cancer cells when over 2% of pure curry was fed. The activities of glutathione peroxidase and glutathione S-transferase were decreased in the groups transplanted with cancer cells which were fed with over 1% of pure curry, and were restored to the level of the group without cancer cell transplant which were fed with normal feed. The superoxide dismutase activity in the groups transplanted with cancer cells was restored to the level of the group without cancer cell transplant which was fed with normal feed when over 1% of pure curry was fed.

Linkage of doxorubicin (Dox) to a water-soluble synthetic N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) eliminates most of the systemic toxicity of the free drug. In EL-4 lymphoma-bearing C57BL/6 mice, a complete regression of pre-established tumours has been achieved upon treatment with Dox-PHPMA-HuIg conjugate. The treatment was effective using a range of regimens and dosages, ranging from 62.5 to 100% cured mice treated with a single dose of 10-20 mg of Dox eq./kg, respectively. Fractionated dosages producing lower levels of the conjugate for a prolonged time period had substantial curative capacity as well. The cured mice developed anti-tumour protection as they rejected subsequently re-transplanted original tumour. The proportion of tumour-protected mice inversely reflected the effectiveness of the primary treatment. The treatment protocol leading to 50% of cured mice produced only protected mice, while no mice treated with early treatment regimen (i.e. starting on day 1 after tumour transplantation) rejected the re-transplanted tumour. Exposure of the host to the cancer cells was a prerequisite for developing protection. The anti-tumour memory was long lasting and specific against the original tumour, as the cured mice did not reject another syngeneic tumour, melanoma B16-F10. The immunity was transferable to naïve recipients in in vivo neutralization assay by spleen cells or CD8(+) lymphocytes derived from cured animals. We propose an effective treatment strategy which eradicates tumours without harming the protective immune anti-cancer responses.

Immunotherapy of cancer includes both active and adoptive, or passive, forms of immunization to target and eradicate malignant B cells in the host. Advances in the understanding of immunology and tumor-cell evasion of the host immune system, coupled with improved technologies to manipulate immune effectors and tumors, have led to a wide array of novel therapies for B-cell malignancies. As a result, investigators have proposed and tested numerous vaccine strategies able to elicit immune responses to tumor antigens. Furthermore, novel approaches to B-cell-targeted antibody therapies hold promise in advancing this line of treatment, and efficient gene transfer technologies have enabled investigators to manipulate immune effector cells to enhance antitumor activity. Significantly, an increasing number of these novel immune-based therapies are being applied to the clinical setting. Whether findings from these clinical trials, in combination with further preclinical studies, will ultimately translate into improved survival of patients with B-cell malignancies remains to be seen.

Clinical application of immunotherapy for acute myeloid leukemia (AML) requires the efficient induction of dendritic cells (DCs) from AML blast cells using in vitro culture. We examined the effect of autologous serum on the properties of leukemic DCs derived from leukemic cells of AML patients by culture in AIM-V medium with GM-CSF, IL-4, TNF-alpha, and 0, 2, 5, or 10% human autologous serum. The expressions of CD80, CD83, CD86, and HLA-DR were upregulated under all culture conditions; however, 10% autologous serum induced the highest expression levels of several molecules. The capacity of leukemic DCs to stimulate allogeneic T cells increased with increasing serum concentration. Stimulation of autologous CD3(+) T cells with leukemic DCs grown in the presence of various concentrations of autologous serum resulted in induction of more IFN-gamma-secreting cells than was the case for unprimed CD3(+) T cells. Leukemic DCs cultured with 10% autologous serum induced the highest numbers of IFN-gamma-secreting cells and CD8(+)CD56(+) T cells from autologous T cells. These results suggest that culture of AML blast cells in the presence of autologous serum could be used to generate leukemic DCs for immunotherapy against AML. The highest serum concentration appeared optimal for generating the most potent leukemic DCs.

Caulobacter crescentus is a gram negative, non-pathogenic bacterium, common in aquatic and soil environments. One feature of note is a protein surface layer (S-layer) composed of a single protein, organized as a self-assembled crystalline array that coats the bacterium. In the course of efforts to express cancer-associated peptides as genetic insertions into the S-layer, we noted a tumor suppressive effect of the unmodified bacterium. C. crescentus was examined for anti-tumor activity against three transplantable tumor mouse models: Lewis lung carcinoma cells transfected with the MUC1 gene in C57BL/6, murine mammary carcinoma (EMT-6) in BALB/c (both in prophylactic and therapeutic mode) and murine leukemia cells (L1210) in DBA2. Mice were immunized three times i.p. with C. crescentus (2 x 10(7) cells/mouse). In prophylactic mode, the mice were challenged with tumor cells two weeks after the last immunization. Immunization with live C. crescentus resulted in anti-tumor activity in all three transplantable tumor models, as measured by prolonged survival, reduced tumor mass or reduced number of lung nodules, compared to saline control groups. In the Lewis lung and the EMT-6 mammary carcinoma murine models the number of lung nodules as well as the tumor weight was lower in mice treated with C. crescentus, compared to the control group; for EMT-6, this was observed in prophylactic and therapeutic modes. In the murine leukemia and Lewis lung carcinoma models prolonged survival was observed in the groups of mice immunized with Caulobacters. In most cases the live C. crescentus cells were markedly more efficacious than heat killed or formalin fixed cells, despite the fact that they do not grow or persist in mice. The results suggest that C. crescentus may be a safe, bacterial immunomodulator for the treatment of tumors.

We investigated the regulatory effect of tamoxifen (TAM) on fibronectin (FN) expression in estrogen-dependent MCF-7 breast carcinoma cells both in vitro and in vivo. in vitro, MCF-7 cells were cultured with 17beta-estradiol (E2) and/or TAM. In the animal experiment in vivo, MCF-7 tumors were grown in ovariectomized athymic mice by implanting a sustained release E2 pellet. The E2 pellets were removed after 3 weeks of E2 treatment. Animals were then divided into four groups: 1) an E2 (0.72 mg/pellet) pellet [E2(+)]; 2) an E2 and a TAM (5 mg/pellet) pellet [E2(+)TAM]; 3) no treatment [E2(-)] and 4) a TAM pellet [E2(-)TAM]. Following each treatment for 4 weeks, all animals were sacrificed and tumors were removed. Specimens, cells (in vitro) or tumors (in vivo), were homogenized and assayed for FN by Western blots. In the in vitro experiment, FN expression in MCF-7 cells decreased by incubating with 10(-9) M E2 and increased with 10(-6) M TAM. The effect of TAM increasing FN expression was inhibited by incubation accompanied with 10(-9) M E2 or 1 microg/ml transforming growth factor-beta (TGF-beta) neutralizing antibody. In the in vivo animal experiment FN expression in the tumors of E2(+) mice was lower than that of E2(-) mice. However, TAM increased FN expression in the tumors regardless of E2 pellet. These results suggest that TAM increases FN expression of MCF-7 breast carcinoma cells and that these regulatory effects of TAM on FN expression are partly mediated by TAM-induced TGF-beta.

CDX2 is a Drosophila caudal-related homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. CDX2 is a marker of colon cancer, with strong staining in up to 90% of colonic adenocarcinomas. CDX2 heterozygous-null mice develop colonic neoplasms, which have suggested that CDX2 is a tumor suppressor. However, CDX2 has not been reported to affect xenograft growth. Furthermore, CDX2 is rarely mutated in colon cancer, which has led to suggestions that it may play only a minor role as a tumor suppressor in colon cancer. To understand the functional contributions of CDX2 to colon cancer, we disrupted CDX2 in LOVO and SW48 human colon cancer cell lines by targeted homologous recombination. Consistent with the literature, disruption of CDX2 enhanced anchorage-dependent cell proliferation. However, homozygous loss of CDX2 led to significant inhibition of anchorage-independent growth in LOVO cells, and cell lethality in SW48 cells. Further analyses revealed that disruption of CDX2 led to anchorage-independent G1 to S growth arrest and anoikis. In vivo xenograft studies confirmed that disruption of CDX2 inhibited LOVO tumor growth. These data demonstrate that CDX2 mediates anchorage-independent growth and survival. Thus, CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48.