Cathy Malcontenti-Wilson

Styrene maleic acid-pirarubicin disrupts tumor microcirculation and enhances the permeability of colorectal liver metastases.

Background: Doxorubicin is a commonly used chemotherapy limited by cardiotoxicity. Pirarubicin, derived from doxorubicin, selectively targets tumors when encapsulated in styrene maleic acid (SMA), forming the macromolecular SMA pirarubicin. Selective targeting is achieved because of the enhanced permeability and retention (EPR) effect. SMA-pirarubicin inhibits the growth of colorectal liver metastases, but tumor destruction is incomplete. The role played by the tumor microcirculation is uncertain. This study investigates the pattern of microcirculatory changes following SMA-pirarubicin treatment. Methods: Liver metastases were induced in CBA mice using a murine-derived colon cancer line. SMA-pirarubicin (100 mg/kg total dose) was administered intravenously in 3 separate doses. Twenty-four hours after chemotherapy, the tumor microvasculature was examined using CD34 immunohistochemistry and scanning electron microscopy. Tumor perfusion and permeability were assessed using confocal in vivo microscopy and the Evans blue method. Results: SMA-pirarubicin reduced the microvascular index by 40%. Vascular occlusion and necrosis were extensive following treatment. Viable cells were arranged around tumor vessels. Tumor permeability was also increased. Conclusion: SMA-pirarubicin damages tumor cells and the tumor microvasculature and enhances tumor vessel permeability. However, tumor necrosis is incomplete, and the growth of residual cells is sustained by a microvascular network. Combined therapy with a vascular targeting agent may affect residual cells, allowing more extensive destruction of tumors.

Interstitial Laser Hyperthermia for Colorectal Liver Metastases: The Effect of Thermal Sensitization and the Use of a Cylindrical Diffuser Tip on Tumor Necrosis

OBJECTIVE Our aim in this study was to investigate the characteristics of a diffuser-tipped optical fiber in producing tumor necrosis, compared to a standard bare-tipped fiber. The potential synergistic effect between thermal sensitization by metronidazole and interstitial laser hyperthermia (ILH)-induced tumor necrosis is also evaluated. BACKGROUND DATA ILH is a minimally invasive technique for the treatment of colorectal liver metastases. One of the major limitations is the size of tissue necrosis achieved by a single optical fiber. Use of cylindrical diffuser-tipped fibers and thermal sensitization of tumor cells by metronidazole may increase the size of tumor necrosis achieved by a single optical fiber. MATERIALS AND METHODS A model of colorectal cancer liver metastases in male inbred CBA mice was used. Laser hyperthermia was applied to tumor tissue using either a bare optical quartz fiber or a cylindrical diffuser-tipped fiber from a Medilas fibertom 4100 Nd:YAG surgical laser generator. Six hundred joules of energy was applied at two power settings, 5 and 10 watts, using bare- and diffuser-tipped fibers, respectively. The extent of necrosis was assessed by histological techniques. A similar study with three experimental groups was treated with 300 J of applied energy. Extent of immediate tumor necrosis was compared to that seen 24 h after ILH treatment. The third group, which had been treated with intraperitoneal metronidazole prior to ILH, was also assessed for tumor necrosis after 24 h and results compared with both the previous groups. RESULTS ILH delivered using a cylindrical diffuser-tipped fiber resulted in a significantly larger diameter of tumor necrosis when compared to a bare-tipped fiber, for a given amount of applied energy. The differences were more significant at higher power settings. Six hundred joules of energy applied by ILH using a bare-tipped fiber at 5 and 10 watts produced 6.7 +/- 1.1 mm and 5.9 +/- 0.6 mm diameter of tumor necrosis, respectively. At equivalent settings, the diffuser-tipped fiber produced 7.7 +/- 1.0 mm and 8.1 +/- 0.6 mm diameter of tumor necrosis (p = 0.02 and p < 0.001). Using a diffuser-tipped fiber and an applied energy of 300 J delivered at 5 watts power, mean diameter of tumor necrosis immediately after treatment was 6.7 +/- 1.1 mm and after 24 h 7.9 +/- 1.3 mm (p = 0.006). Mean diameter of tumor necrosis 24 h after ILH in animals treated with metronidazole was 8.3 +/- 1.9 mm (p = 0.11). CONCLUSION Diffuser-tipped optical fiber appears to significantly increase the diameter of ILH-induced tumor necrosis compared to the bare fiber. In contrast to the bare fiber, it enables the application of laser energy using higher power settings without compromising the diameter of tumor necrosis achieved. In animals treated with metronidazole, a trend towards increased tumor destruction at the tumor-host interface was seen on histolopathology. In addition, a trend towards increased diameter of tumor necrosis was also seen; however, statistical significance was not achieved.

CYT997: a novel orally active tubulin polymerization inhibitor with potent cytotoxic and vascular disrupting activity in vitro and in vivo

CYT997 is a wholly synthetic compound that possesses highly potent cytotoxic activity in vitro through inhibition of microtubule polymerization. CYT997 blocks the cell cycle at the G2-M boundary, and Western blot analysis indicates an increase in phosphorylated Bcl-2, along with increased expression of cyclin B1. Caspase-3 activation is also observed in cells treated with CYT997 along with the generation of poly(ADP-ribose) polymerase. The compound possesses favorable pharmacokinetic properties, is orally bioavailable, and is efficacious per os in a range of in vivo cancer models, including some refractory to paclitaxel treatment. CYT997 exhibits vascular disrupting activity as measured in vitro by effects on the permeability of human umbilical vein endothelial cell monolayers, and in vivo by effects on tumor blood flow. CYT997 possesses a useful combination of pharmacologic and pharmacokinetic properties and has considerable potential as a novel anticancer agent. [Mol Cancer Ther 2009;8(11):3036–45]

Alterations in vascular architecture and permeability following Oxi4503 treatment

OXi4503 retards tumor growth in a dose-dependent manner and improves survival in a murine model of colorectal liver metastases. This agent causes extensive vascular shutdown by selectively altering the tubulin cytoskeleton within the endothelial cells of tumor vessels. The destruction of tumor vessels is incomplete, however, and tumor revascularization occurs after the treatment. This study evaluates the pattern of microcirculatory changes and alterations to the ultrastructural properties of the tumor vasculature that result from OXi4503 treatment. Male CBA mice were induced with liver metastases via an intrasplenic injection of a murine-derived colorectal cell line. After administering a single intraperitoneal dose of OXi4503, changes in tumor perfusion, microvascular architecture and permeability were assessed at various time points. One hour after a 100-mg/kg dose of OXi4503, a significant decrease in the percentage of tumor perfusion (63.96±1.98 in controls versus 43.77±2.71 in treated mice, P<0.001) was observed, which was still evident 5 days after the treatment. Substantial tumor microvascular damage and minimal normal liver injury were observed. Tumor vascular permeability was significantly elevated 45 min after the OXi4503 treatment (67.5±3.60 in controls versus 80.5±2.24 μg/g, P<0.05). The findings suggest that OXi4503 selectively targets tumor vessels and causes immediate microvascular destruction. Even at the maximum tolerated dose, however, residual patent tumor vessels were still present after treatment, implying incomplete tumor destruction. A combination of OXi4503 with other chemotherapeutic modalities might achieve complete tumor eradication and improve long-term survival.

The microtubule depolymerizing agent CYT997 causes extensive ablation of tumor vasculature in vivo.

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639–4642, 2009; Mol Cancer Ther 8:3036–3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC50 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

Physiological characterization of a mouse model of cachexia in colorectal liver metastases

Loss of skeletal muscle mass and function (cachexia) is severe in patients with colorectal liver metastases because of the large increase in resting energy expenditure but remains understudied because of a lack of suitable preclinical models. Our aim was to characterize a novel preclinical model of cachexia in colorectal liver metastases. We tested the hypothesis that mice with colorectal liver metastases would exhibit cachexia, as evidenced by a reduction in liver-free body mass, muscle mass, and physiological impairment. Twelve-week-old male CBA mice received an intrasplenic injection of Ringer solution (sham) or murine colorectal cancer cells (MoCR) to induce colorectal liver metastases. At end-point (20-29 days), the livers of MoCR mice were infiltrated completely with metastases, and MoCR mice had reduced liver-free body mass, muscle mass, and epididymal fat mass compared with sham controls (P < 0.03). MoCR mice exhibited impaired rotarod performance and grip strength (P < 0.03). Histochemical analyses of tibialis anterior muscles from MoCR mice revealed muscle fiber atrophy and reduced oxidative enzyme activity (P < 0.001). Adipose tissue remodeling was evident in MoCR mice, with reduced adipocyte diameter and greater infiltration of nonadipocyte tissue (P < 0.05). These findings reveal the MoCR mouse model exhibits significant cachexia and is a suitable preclinical model of cachexia in colorectal liver metastases. This model should be used for identifying effective treatments for cachexia to improve quality of life and reduce mortality in patients with colorectal liver metastases.

Styrene maleic acid copolymer-pirarubicin induces tumor-selective oxidative stress and decreases tumor hypoxia as possible treatment of colorectal cancer liver metastases

BACKGROUND Pirarubicin, a derivative of doxorubicin, induces tumor destruction via the production of reactive oxygen species (ROS) but is associated with cardiotoxicity. As a macromolecule (conjugated to styrene-maleic acid [SMA]), SMA-pirarubicin is selective to tumors resulting in improved survival with decreased systemic toxicity. Tumor destruction is, however incomplete, and resistant cells at the periphery of the tumor contribute to recurrence. Tumor hypoxia is a major factor in tumor resistance. Understanding the effect of oxidative stress induced by SMA-pirarubicin on the tumor microenvironment may be key to overcoming resistance. This study investigated the pattern of ROS production and tumor hypoxia after treatment with SMA-pirarubicin in a murine model of colorectal liver metastases. METHODS Liver metastases were induced in male, CBA mice using a murine-derived colon cancer cell line. SMA-pirarubicin (maximum tolerated dose, 100 mg/kg) or pirarubicin, (maximum tolerated dose, 10 mg/kg) were administered intravenously 14 days after tumor induction. Systemic oxidative stress in serum, liver, and cardiac tissue was quantified using the thiobarbituric acid reactive substances assay. Flow cytometry and fluorescence microscopy were used to assess ROS production for 48 hours after treatment. Tumor hypoxia was quantified using immunohistochemistry for pimonidazole adducts. RESULTS SMA-pirarubicin (100 mg/kg) induced ROS exclusively in tumors with minimal levels in serum and cardiac tissue. ROS levels were induced in a time-dependent and dose-dependent manner optimal between 4 and 24 hours after drug administration. Although tumor hypoxia was decreased overall, residual tumor cells adjacent to patent vessels were hypoxic. CONCLUSION This study provides insight into the tumor microenvironment after chemotherapy. SMA-pirarubicin inhibits the growth of colorectal liver metastases by inducing ROS, which seems to be largely tumor selective. The temporal pattern of ROS production can be used to improve future dosing regimens. Furthermore, the observation that residual tumor cells are hypoxic clarifies the need for a multimodal approach with agents that can alter the hypoxic state to effect complete tumor destruction.

Abstract 3707: Captopril inhibits colorectal cancer liver metastases following 70% partial hepatectomy in a mouse model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Blockade of the renin-angiotensin system (RAS) with the angiotensin-converting enzyme inhibitor, Captopril, has been shown to inhibit CRCLM. Partial hepatectomy (PH) for colorectal cancer liver metastases (CRCLM) can stimulate tumour recurrence. Aim: This study investigated the effects of Captopril on liver regeneration (LR), as well as on CRCLM in the regenerating liver. Methods: Male CBA mice were used for 70% PH as well as 70% PH following CRCLM induction. Mice were randomly assigned to control or Captopril-treated groups. Liver and tumour samples were collected on days 1, 2, 4, 6 and 8, 16 and 21 post-surgery. The rate of LR was measured by liver-to-body weight (LBW) ratio. The percentage of liver metastases was quantified using quantitative stereology and immunohistochemistry was performed to quantify hepatocyte and tumour cell proliferation (anti-Ki67) and apoptosis (anti-caspase-3). Results: Captopril increased LBW (0.57 ± 0.02 Captopril, 0.49 ± 0.02 control, p = 0.027) compared to controls on day 2 following 70% PH. At day 6, Captopril had decreased LBW (0.5 ± 0.03, 0.73 ± 0.06, p = 0.006). However, by day 8, LBW was not different between the control and Captopril groups. At day 21, Captopril decreased the percentage of liver metastases compared to controls (24.4 ± 6.2%; 48.7 ± 4.7%, p = 0.008) in the regenerating liver. Tumour volume (388.3 ± 150.4; 1046.2 ± 200.2mm3, p = 0.02) and tumour nodule count per image field (68 ± 17.6; 181.1 ± 28.5, p = 0.005) were also decreased by Captopril. In contrary, Captopril did not significantly alter liver volume compared to controls (886.1 ± 65.2; 1044.1 ± 115.8, p = 0.254). Furthermore, LBW during LR in the presence of CRCLM at day 2 (0.53 ± 0.01; 0.51 ± 0.02, p = 0.449) and day 6 (0.71 ± 0.01; 0.73 ± 0.03, p = 0.51) were not different between Captopril and control groups. Conclusion: Captopril enhanced the early stage of LR following 70% PH. Captopril inhibits CRCLM in the regenerating liver without inhibiting LR. Understanding the mechanisms of actions of Captopril is required to improve CRCLM patient outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3707. doi:1538-7445.AM2012-3707

Gastrin is not required for liver regeneration.

Background:  Although several growth factors are known to be essential for liver regeneration, the role of gastrin remains controversial.