High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice: New Perspectives for Onco-Pharmacology

ObjectivesWe have developed a relevant preclinical model associated with a specific imaging protocol dedicated to onco-pharmacology studies in mice.ResultsThis study demonstrates the ability of ultrasound imaging to detect and monitor the in vivo orthotopic lung tumor growth by high resolution ultrasound imaging. This approach enabled us to characterize key biological parameters such as oxygenation, perfusion status and vascularization of tumors.ConclusionSuch an experimental approach has never been reported previously and it would provide a nonradiative tool for assessment of anticancer therapeutic efficacy in mice. Considering the absence of ultrasound propagation through the lung parenchyma, this strategy requires the implantation of tumors strictly located in the superficial posterior part of the lung.     

Pyruvate Induces Transient Tumor Hypoxia by Enhancing Mitochondrial Oxygen Consumption and Potentiates the Anti-Tumor Effect of a Hypoxia-Activated Prodrug TH-302

Background

TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.

Methodology/Results

The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O₂), with minimal effect under aerobic conditions (21% O₂). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm³. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm³), significantly delayed tumor growth.

Conclusions/Significance

Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.     

Xenograft Tumors Vascularized with Murine Blood Vessels May Overestimate the Effect of Anti-Tumor Drugs: A Pilot Study

Recent evidence demonstrated that endothelial cells initiate signaling events that enhance tumor cell survival, proliferation, invasion, and tumor recurrence. Under this new paradigm for cellular crosstalk within the tumor microenvironment, the origin of endothelial cells and tumor cells may have a direct impact on the pathobiology of cancer. The purpose of this pilot study was to evaluate the effect of endothelial cell species (i.e. murine or human) on xenograft tumor growth and response to therapy. Tumor xenografts vascularized either with human or with murine microvascular endothelial cells were engineered, side-by-side, subcutaneously in the dorsum of immunodefficient mice. When tumors reached 200 mm³, mice were treated for 30 days with either 4 mg/kg cisplatin (i.p.) every 5 days or with 40 mg/kg sunitinib (p.o.) daily. Xenograft human tumors vascularized with human endothelial cells grow faster than xenograft tumors vascularized with mouse endothelial cells (P<0.05). Notably, human tumors vascularized with human endothelial cells exhibited nuclear translocation of p65 (indicative of high NF-kB activity), and were more resistant to treatment with cisplatin or sunitinib than the contralateral tumors vascularized with murine endothelial cells (P<0.05). Collectively, these studies suggest that the species of endothelial cells has a direct impact on xenograft tumor growth and response to treatment with the chemotherapeutic drug cisplatin or with the anti-angiogenic drug sunitinib.     

Epidermal Growth Factor Receptor Inhibition Modulates the Microenvironment by Vascular Normalization to Improve Chemotherapy and Radiotherapy Efficacy

Background

Epidermal growth factor receptor (EGFR) inhibitors have shown only modest clinical activity when used as single agents to treat cancers. They decrease tumor cell expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF). Hypothesizing that this might normalize tumor vasculature, we examined the effects of the EGFR inhibitor erlotinib on tumor vascular function, tumor microenvironment (TME) and chemotherapy and radiotherapy sensitivity.

Methodology/Principal Findings

Erlotinib treatment of human tumor cells in vitro and mice bearing xenografts in vivo led to decreased HIF-1α and VEGF expression. Treatment altered xenograft vessel morphology assessed by confocal microscopy (following tomato lectin injection) and decreased vessel permeability (measured by Evan''s blue extravasation), suggesting vascular normalization. Erlotinib increased tumor blood flow measured by Power Doppler ultrasound and decreased hypoxia measured by EF5 immunohistochemistry and tumor O₂ saturation measured by optical spectroscopy. Predicting that these changes would improve drug delivery and increase response to chemotherapy and radiation, we performed tumor regrowth studies in nude mice with xenografts treated with erlotinib and either radiotherapy or the chemotherapeutic agent cisplatin. Erlotinib therapy followed by cisplatin led to synergistic inhibition of tumor growth compared with either treatment by itself (p<0.001). Treatment with erlotinib before cisplatin led to greater tumor growth inhibition than did treatment with cisplatin before erlotinib (p = 0.006). Erlotinib followed by radiation inhibited tumor regrowth to a greater degree than did radiation alone, although the interaction between erlotinib and radiation was not synergistic.

Conclusions/Significance

EGFR inhibitors have shown clinical benefit when used in combination with conventional cytotoxic therapy. Our studies show that targeting tumor cells with EGFR inhibitors may modulate the TME via vascular normalization to increase response to chemotherapy and radiotherapy. These studies suggest ways to assess the response of tumors to EGFR inhibition using non-invasive imaging of the TME.     

Anti-cancer effect of marchantin C via inducing lung cancer cellular senescence associated with less secretory phenotype

BackgroundLung cancer is the leading cause of global cancer deaths. Current chemotherapeutic agents for lung cancer treatment are generally accompanied with severe side effects. Here, we report that marchantin C (Mar-C), a potential natural compound with little chemotherapeutic toxicity, exerts a well anti-tumor effect against lung cancer via inducing cellular senescence.MethodsThe antitumor activity of Mar-C was evaluated by MTT and colony formation in vitro cytotoxicity assays, and xenograft and homograft in vivo model. Antitumor mechanisms of Mar-C were investigated through SA-β-gal staining, Q-PCR, immunoblotting, immunofluorescence, protein array and siRNA knocking-down analysis.ResultsMar-C selectively induces senescence of lung cancer cells with limited cytotoxicity on normal or non-neoplastic cells. Mar-C-induced senescence was associated with the elevation of ROS and activation of DNA-damage, and largely dependent of prolonged p21^CIP1 accumulation. The senescence-associated secretory phenotype (SASP) induced by Mar-C was distinct from doxorubicin-induced. Furthermore, Mar-C exhibited an inhibitory activity on tumor growth with little toxicity in animal studies, and significantly prolonged the survival time of tumor-bearing mice than that of doxorubicin or vehicle treatments.ConclusionMar-C selectively inhibited tumor growth via the induction of cancer cell senescence and had little chemotherapeutic toxicity, suggesting the potential of Mar-C as a promising anticancer agent.General significanceThis study provided evidence to identify a novelty of Mar-C that exerted antitumor activity on lung cancer through induction of senescence with limited toxicity.     

The effect of cisplatin on cytotoxicity of anticancer cytokine TRAIL and its receptor-selective mutant variant DR5-B<Superscript>1</Superscript>

Cytokine TRAIL selectively induces apoptosis in vitro and in vivo in tumor cells without affecting normal cells, but its therapeutic application is limited, since many primary tumors are insensitive to TRAIL. To improve the efficiency of TRAIL, we have previously developed TRAIL mutant variant DR5-B, which binds the apoptosis-inducing death receptor DR5 as efficiently as wild type TRAIL, but shows almost no affinity to other receptors. In this study, we investigated the effect of the chemotherapeutic agent cisplatin on the cytotoxicity of TRAIL variants in 12 tumor cell lines of various origin. Cisplatin effectively enhances the cytotoxic activity of TRAIL preparations. The synergistic effect is most pronounced in the prostate cancer cell lines, where the combined effect exceeds the sum of the separate effects by more than 2 times. The cytotoxicity of DR5-B variant is significantly higher compared to wild-type TRAIL in combination with cisplatin in 9 of 12 tumor cell lines.     

Acetazolamide-based [18F]-PET tracer: In vivo validation of carbonic anhydrase IX as a sole target for imaging of CA-IX expressing hypoxic solid tumors

Carbonic anhydrase IX is overexpressed in many solid tumors including hypoxic tumors and is a potential target for cancer therapy and diagnosis. Reported imaging agents targeting CA-IX are successful mostly in clear cell renal carcinoma as SKRC-52 and no candidate was approved yet in clinical trials for imaging of CA-IX. To validate CA-IX as a valid target for imaging of hypoxic tumor, we designed and synthesized novel [¹⁸F]-PET tracer (1) based on acetazolamide which is one of the well-known CA-IX inhibitors and performed imaging study in CA-IX expressing hypoxic tumor model as 4T1 and HT-29 in vivo models other than SKRC-52. [¹⁸F]-acetazolamide (1) was found to be insufficient for the specific accumulation in CA-IX expressing tumor. This study might be useful to understand in vivo behavior of acetazolamide PET tracer and can contribute to the development of successful PET imaging agents targeting CA-IX in future. Additional study is needed to understand the mechanism of poor targeting of CA-IX, as if CA-IX is not reliable as a sole target for imaging of CA-IX expressing hypoxic solid tumors.     

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.     

[18F]-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography of LAPC4-CR Castration-Resistant Prostate Cancer Xenograft Model in Soft Tissue Compartments

Preclinical xenograft models have contributed to advancing our understanding of the molecular basis of prostate cancer and to the development of targeted therapy. However, traditional preclinical in vivo techniques using caliper measurements and survival analysis evaluate the macroscopic tumor behavior, whereas tissue sampling disrupts the microenvironment and cannot be used for longitudinal studies in the same animal. Herein, we present an in vivo study of [¹⁸F]-fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) designed to evaluate the metabolism within the microenvironment of LAPC4-CR, a unique murine model of castration-resistant prostate cancer. Mice bearing LAPC4-CR subcutaneous tumors were administered [¹⁸F]-FDG via intravenous injection. After a 60-minute distribution phase, the mice were imaged on a PET/CT scanner with submillimeter resolution; and the fused PET/CT images were analyzed to evaluate tumor size, location, and metabolism across the cohort of mice. The xenograft tumors showed [¹⁸F]-FDG uptake that was independent of tumor size and was significantly greater than uptake in skeletal muscle and liver in mice (Wilcoxon signed-rank P values of .0002 and .0002, respectively). [¹⁸F]-FDG metabolism of the LAPC4-CR tumors was 2.1 ± 0.8 ID/cm³*wt, with tumor to muscle ratio of 7.4 ± 4.7 and tumor to liver background ratio of 6.7 ± 2.3. Noninvasive molecular imaging techniques such as PET/CT can be used to probe the microenvironment of tumors in vivo. This study showed that [¹⁸F]-FDG-PET/CT could be used to image and assess glucose metabolism of LAPC4-CR xenografts in vivo. Further work can investigate the use of PET/CT to quantify the metabolic response of LAPC4-CR to novel agents and combination therapies using soft tissue and possibly bone compartment xenograft models.     

Metronomic chemotherapy with low-dose cyclophosphamide plus gemcitabine can induce anti-tumor T cell immunity in vivo

Several chemotherapeutic drugs have immune-modulating effects. For example, cyclophosphamide (CP) and gemcitabine (GEM) diminish immunosuppression by regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), respectively. Here, we show that intermittent (metronomic) chemotherapy with low-dose CP plus GEM can induce anti-tumor T cell immunity in CT26 colon carcinoma-bearing mice. Although no significant growth suppression was observed by injections of CP (100 mg/kg) at 8-day intervals or those of CP (50 mg/kg) at 4-day intervals, CP injection (100 mg/kg) increased the frequency of tumor peptide-specific T lymphocytes in draining lymph nodes, which was abolished by two injections of CP (50 mg/kg) at a 4-day interval. Alternatively, injection of GEM (50 mg/kg) was superior to that of GEM (100 mg/kg) in suppressing tumor growth in vivo, despite the smaller dose. When CT26-bearing mice were treated with low-dose (50 mg/kg) CP plus (50 mg/kg) GEM at 8-day intervals, tumor growth was suppressed without impairing T cell function; the effect was mainly T cell dependent. The metronomic combination chemotherapy cured one-third of CT26-bearing mice that acquired tumor-specific T cell immunity. The combination therapy decreased Foxp3 and arginase-1 mRNA levels but increased IFN-γ mRNA expression in tumor tissues. The percentages of tumor-infiltrating CD45⁺ cells, especially Gr-1^high CD11b⁺ MDSCs, were decreased. These results indicate that metronomic chemotherapy with low-dose CP plus GEM is a promising protocol to mitigate totally Treg- and MDSC-mediated immunosuppression and elicit anti-tumor T cell immunity in vivo.     

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women ^1,2,3. Serous tumors are the most widespread forms of ovarian cancer and ^4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter ⁶. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice. Objective: Although tumor imaging is possible ⁷, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses. Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm ^8,9,10. We orthotopically implanted MOV1^Kat in the ovary ^11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry. Results: Orthotopically implanted MOV1^Kat cells developed serous ovarian tumors. MOV1^Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers ¹⁵ (fig. 2). Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.Download video file.^{(63M, mov)}     

An Optimized Triple Modality Reporter for Quantitative In Vivo Tumor Imaging and Therapy Evaluation

We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.     

Rhus verniciflua Stokes prevents cisplatin-induced cytotoxicity and reactive oxygen species production in MDCK-I renal cells and intact mice

Cisplatin-induced oxidative stress can cause liver and kidney damage, thus limiting therapeutic efficacy. Thus, in the present study, since Rhus verniciflua Stokes (RVS) containing flavonoids has antioxidant effects, we investigated whether it can protect cisplatin-induced toxicity in vitro and in vivo, The in vitro effects of RVS on the cell viability and reactive oxygen species (ROS) production were investigated using cisplatin-treated Madin–Darby Canine kidney (MDCK)-I renal cells. Its in vivo effects were also studied in BALB/c mice inoculated with CT-26 colon adenocarcinoma cells and treated with cisplatin with or without RVS. Liver and renal functions were assessed together with indices of tissue oxidation. RVS prevented cisplatin-induced cytotoxicity and ROS release against MDCK-I cells. RVS alone exerted modest antitumor activity against CT-26 cells. When used concurrently with cisplatin, RVS prevented the increases in serum blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and NO, while reducing liver and kidney tissue MDA content, and increasing catalase, glutathione (GSH), and superoxide dismutase (SOD) activities. Moreover, the antitumor efficacy of cisplatin was not altered by concurrent administration of RVS. These findings demonstrate that RVS prevents cisplatin-induced toxicity in vitro and in vivo via an antioxidant activity without hurting its antitumor effectiveness, suggesting that RVS can be usefully applied to the neoplastic patients as a combined chemopreventive agent with cisplatin.     

Non-Invasive Imaging of Phosphoinositide-3-Kinase-Catalytic-Subunit-Alpha (PIK3CA) Promoter Modulation in Small Animal Models

Activation of the PI3K/Akt pathway, a critical step for survival in cancer cells is often associated with decreased sensitivity to several chemotherapeutic drugs. PIK3CA gene amplification is observed in 16–24% of epithelial ovarian cancer (EOC) patients in conjunction with p53 mutations. A 900 bp long PIK3CA promoter is shown to be negatively regulated by p53 in ovarian surface epithelial cells but the consequence of chemotherapeutic drug treatments on this promoter in ovarian cancer cells is largely unknown. We aim to study the modulation of this promoter by cisplatin using an improved fusion reporter in ovarian cancer cells and tumor xenografts by non-invasive imaging approach. A PIK3CA sensor was developed using a bi-fusion reporter from a newly constructed library of bi- and tri-fusion vectors comprising of two mutant far red fluorescent proteins (mcherry/mch and tdTomato/tdt), a mutant firefly luciferase (fluc2), and a PET reporter protein (ttk). In vivo imaging of mice implanted with 293T cells transiently expressing these bi- and tri-fusion reporters along with respective controls revealed comparable activity of each reporter in the fusion background and fluc2-tdt as the most sensitive one. Repression of the PIK3CA sensor by drugs was inversely proportional to cellular p53 level in a germline (PA1) and in an EOC (A2780) cell line but not in a p53 deficient EOC (SKOV3) cell line. Bioluminescence imaging of tumor xenografts stably expressing the PIK3CA sensor in PA1 and A2780 cells exhibited attenuating activity without any change in SKOV3 tumors expressing the PIK3CA sensor after cisplatin treatment. Sequential mutation at p53 binding sites showed gradual increase in promoter activity and decreased effects of the drugs. These newly developed PIK3CA-fluc2-tdt and the mutant reporter sensors thus would be extremely useful for screening new drugs and for functional assessment of PIK3CA expression from intact cells to living subjects.     

Oral Metronomic Topotecan Sensitizes Crizotinib Antitumor Activity in ALKF1174L Drug-Resistant Neuroblastoma Preclinical Models

BACKGROUND: Anaplastic lymphoma kinase (ALK) inhibitor crizotinib has proven to be effective in the treatment of ALK-mutated neuroblastoma, but crizotinib resistance was commonly observed in patients. We aimed to overcome crizotinib resistance by combining with the MEK inhibitor trametinib or low-dose metronomic (LDM) topotecan in preclinical neuroblastoma models. METHODS: We selected a panel of neuroblastoma cell lines carrying various ALK genetic aberrations to assess the therapeutic efficacy on cell proliferation in vitro. Downstream signals of ALK activation, including phosphorylation of ERK1/2, Akt as well as HIF-1α expression were evaluated under normoxic and hypoxic conditions. Tumor growth inhibition was further assessed in NOD/SCID xenograft mouse models. RESULTS: All NBL cell lines responded to crizotinib treatment but at variable ED50 levels, ranging from 0.25 to 5.58 μM. ALK-mutated cell lines SH-SY5Y, KELLY, LAN-5, and CHLA-20 are more sensitive than ALK wild-type cell lines. In addition, we demonstrated that under hypoxic conditions, all NBL cell lines showed marked decrease of ED50s when compared to normoxia except for KELLY cells. Taking into consideration the hypoxia sensitivity to crizotinib, combined treatment with crizotinib and LDM topotecan demonstrated a synergistic effect in ALK^F1174L-mutated SH-SY5Y cells. In vivo, single-agent crizotinib showed limited antitumor activity in ALK^F1174L-mutated SH-SY5Y and KELLY xenograft models; however, when combined with topotecan, significantly delayed tumor development was achieved in both SH-SY5Y and KELLY tumor models. CONCLUSIONS: Oral metronomic topotecan reversed crizotinib drug resistance in the ALK^F1174L-mutated neuroblastoma preclinical model.     

Mapping Tumor Hypoxia In Vivo Using Pattern Recognition of Dynamic Contrast-enhanced MRI Data

In solid tumors, hypoxia contributes significantly to radiation and chemotherapy resistance and to poor outcomes. The “gold standard” pO₂ electrode measurements of hypoxia in vivo are unsatisfactory because they are invasive and have limited spatial coverage. Here, we present an approach to identify areas of tumor hypoxia using the signal versus time curves of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) data as a surrogate marker of hypoxia. We apply an unsupervised pattern recognition (PR) technique to determine the differential signal versus time curves associated with different tumor microenvironmental characteristics in DCE-MRI data of a preclinical cancer model. Well-perfused tumor areas are identified by rapid contrast uptake followed by rapid washout; hypoxic areas, which are regions of reduced vascularization, are identified by delayed contrast signal buildup and washout; and necrotic areas exhibit slow or no contrast uptake and no discernible washout over the experimental observation. The strength of the PR concept is that it captures the pixel-enhancing behavior in its entirety—during both contrast agent uptake and washout—and thus, subtleties in the temporal behavior of contrast enhancement related to features of the tumor microenvironment (driven by vascular changes) may be detected. The assignment of the tumor compartments/microenvironment to well vascularized, hypoxic, and necrotic is validated by comparison to data previously obtained using complementary imaging modalities. The proposed novel analysis approach has the advantage that it can be readily translated to the clinic, as DCE-MRI is used routinely for the identification of tumors in patients, is widely available, and easily implemented on any clinical magnet.     

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

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

The Strong In Vivo Anti-Tumor Effect of the UIC2 Monoclonal Antibody Is the Combined Result of Pgp Inhibition and Antibody Dependent Cell-Mediated Cytotoxicity

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10–40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC₅₀ of doxorubicin (DOX) in KB-V1 (Pgp⁺) cells in vitro almost to the level of KB-3-1 (Pgp^-) cells. At the same time, UIC2 alone did not affect the EC₅₀ value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp⁺ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp⁺ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased ¹⁸FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).     

Evaluation of oxygen dependence on in vitro and in vivo cytotoxicity of photoimmunotherapy using IR-700–antibody conjugates

Photoimmunotherapy (PIT) using the near-infrared-absorbing photosensitizing phthalocyanine dye, IRDye 700DX (IR-700), conjugated with a tumor-targeting antibody such as panitumumab (Pan) has shown efficacy in in vitro studies and several preclinical models in mice with promise for clinical translation. PIT results in rapid necrotic cell death in vitro and tumor shrinkage in vivo. Photochemical studies with the Pan-IR-700 conjugate showed that this agent can support generation of singlet oxygen and also generate reactive oxygen species after exposure to near-infrared (NIR) light. Moreover, in vitro studies using A431 cells, singlet oxygen scavengers abrogated the efficacy of PIT with Pan-IR-700, while oxygen depletion to undetectable levels in the exposure chamber almost completely inhibited the cellular cytotoxicity of PIT. Survival of tumor bearing mice was prolonged in PIT-treated animals but mice whose tumors were made transiently hypoxic prior to PIT had no benefit from the treatment. The results from this study support a central role for molecular oxygen-derived species in cell death caused by PIT.     

Poly(methylmetacrylate) (PMMA) core-shell nanospheres act as efficient pharmacophores for the antiproliferative [PtCl3(NH3)] complex by forming ionic couples

Polymer-based, covalently linked drug-delivery systems have been shown to improve the therapeutic index of effective but toxic anticancer agents, such as cisplatin, by exploiting the peculiar features of tumour blood and lymphatic circulation. In the present study, positively charged poly(methylmetacrylate) core-shell nanospheres (ZN2) were used as pharmacophores for anionic platinum-containing moieties (PtA). The antitumor effect of the resulting non-covalent adduct (PtA-ZN2) was assessed in C57BL/6 mice bearing B16 murine melanoma. PtA-ZN2 was significantly more effective than cisplatin in inhibiting B16 tumour growth when both agents were used at their respective maximum tolerated doses; importantly, mice receiving PtA-ZN2 exhibited no signs of significant general toxicity. Tumour growth in mice treated with unconjugated PtA did not significantly differ from that observed in control animals. As expected, the in vivo efficacy of PtA-ZN2, cisplatin and PtA was found to correlate with Pt intratumour accumulation. In vitro cytotoxicity assays on cultured B16 cells also evidenced a superior efficacy for PtA-ZN2 as compared with unconjugated PtA. This was associated with higher intracellular levels of Pt in cells treated with PtA-ZN2, suggesting that the pharmacophore may facilitate Pt accumulation in tumour cells, even when blood circulation and lymphatic drainage are not an issue.