Preclinical models of prostate cancer

Although prostate cancer is a major cause of cancer death and morbidity in Western countries, one major hindrance in the study of the biology of prostate cancer has been the limited number of laboratory models, compared with the number of models available for other neoplasms. For a long time, only three cell lines, namely LNCaP, PC3, and DU145, were routinely used to study prostate cancer in the lab. The success rate to establish cell lines from human prostate cancer tissues is low, in the 1% range. Currently, only about 10 prostate cancer cell lines are available, and many of them do not reproduce typical features of the human disease, like androgen receptor expression or prostate specific antigen (PSA) secretion. Spontaneous models in animals (including rat and dog) are practically not convenient for research purposes. Several in vivo models were artificially established by transforming prostate cells by potent oncogenes. Other models were developed by injecting prostate cancer cell lines into the prostate (orthotopic model), the vessel, or the bones of immuno-deficient mice, to mimic localized and metastatic prostate cancer. This was successfully done with the MDA PCa2b cell line. This cell line was also used to generate an in vitro model of bone metastases by a co-culture system with osteoblasts. This model allows to study the paracrine cross-talk between the two cell compartments and the resulting molecular modifications. The objective of the present article is to review the currently available model systems of prostate cancer.     

Preclinical efficacy of vascular disrupting agents in non-small-cell lung cancer

There is increasing interest in the use of vascular targeted therapy for the treatment of non-small-cell lung cancer (NSCLC). Current approaches include antiangiogenic drugs, which prevent growth of new vessels, and tumor vascular disrupting drugs, which further compromise the function of tumor vasculature and induce vascular failure. Preclinical studies have led to the development of the two main classes of tumor vascular disrupting agent-tubulin polymerization inhibitors such as combretastatin A4 phosphate (CA4P) and the flavonoid class that includes flavone acetic acid (FAA) and ASA404 (vadimezan). Each class of drug has shown antitumor activity in preclinical models of lung cancer, including both rodent tumors and human tumor xenografts. Tubulin polymerization inhibitors act primarily by disrupting the tubulin network of the endothelial cell cytoskeleton, leading to shape changes and increased vascular permeability, but these agents also affect the actin cytoskeleton and endothelial cell junctions. Flavonoid vascular disrupting agents appear to accentuate pathologic signaling by cytokines such as tumor necrosis factor and vascular endothelial growth factor, leading to changes in the actin cytoskeleton, increased vascular permeability, and endothelial apoptosis. Several approaches to the measurement of vascular disrupting effects in preclinical models have been developed. They include measurement of tumor blood flow, the induction of tumor hypoxia, and the release of serotonin from platelets, measured in plasma as the metabolite 5-hydroxyindoleacetic acid. Both classes of agent combine with standard cytotoxic drugs in the treatment of experimental tumors, but consideration of the timing of combination administration is important because tumor vascular disruption will affect delivery of a second agent.     

The histone deacetylase inhibitor PXD101 increases the efficacy of irinotecan in in vitro and in vivo colon cancer models

Purpose

Histone deacetylase inhibitors (HDACIs), such as PXD101 and suberoylanilide hydroxamic acid, inhibit proliferation and stimulate apoptosis of tumor cells. The enhanced effectiveness of chemotherapy or radiotherapy when combined with HDACIs has been observed in several cancers. In this study, we investigated the antitumor effect of PXD101 combined with irinotecan in colon cancer.

Methods

HCT116 and HT29 colon cancer cells for cell viability assay were treated with PXD101 and/or SN-38, the active form of irinotecan. Antitumor effects of HCT116 and HT29 xenografts treated with these combinations were evaluated. [¹⁸F]FLT-PET was used to detect early responses to PXD101 and irinotecan in colon cancer.

Results

PXD101 and SN38 possessed dose-dependent antiproliferative activity against HCT116 and HT29 cells and exerted a synergistic effect when used in combination. In xenografted mice, PXD101 in combination with irinotecan dramatically inhibited tumor growth without causing additive toxicity. Apoptotic effects on xenograft tumors were greater with combined treatment than with irinotecan alone. [¹⁸F]FLT-PET imaging revealed a 64% decrease in [¹⁸F]FLT uptake in tumors of HCT116 xenograft-bearing mice treated with a combination of PXD101 and irinotecan, indicating a decrease in thymidine kinase 1 (TK1) activity. These results were supported by Western blot analyses showing a decrease in tumor thymidine kinase 1 protein levels, suggesting that [¹⁸F]FLT-PET can be used to non-invasively detect early responses to these agents.

Conclusions

These data show that PXD101 increases the cytotoxic activity of irinotecan in in vitro and in vivo colon cancer models and suggest these agent combinations should be explored in the treatment of colon cancer.     

Preclinical in vivo antitumor efficacy of nedaplatin with gemcitabine against human lung cancer.

The antitumor efficacy of the combination of nedaplatin (NDP) with gemcitabine (GEM) was evaluated. We also compared the antitumor activity of NDP plus GEM with that of cisplatin (CDDP) plus GEM or carboplatin (CBDCA) plus GEM. Ma44, which is a human lung cancer sensitive to GEM, and NCI-H460, which is a human lung cancer refractory to GEM, were used in this study. GEM was injected i.v. once followed by i.v. injection of NDP at an interval of approximately 30 min into tumor-bearing athymic mice. GEM was administered again 3 or 4 days thereafter. Combined dosing of NDP with GEM resulted in synergistically enhanced inhibition of tumor growth in the Ma44 tumor model. NDP plus GEM was also effective against Ma44 cells when given late in the therapy, a model for advanced disease. Potent augmentation of growth inhibition by NDP with GEM was also found with the NCI-H460 tumor model. The combination effect of NDP plus GEM appeared to be superior to that of CDDP plus GEM or CBDCA plus GEM in both tumor models. Toxicity in terms of blood cell numbers was not enhanced by the combination of NDP with GEM. These results suggest the effectiveness of combination of NDP with GEM for clinical therapy.     

Preclinical models in oncology

For oncology research, the tumor model selection is driven by the study objectives. Due to the cancer heterogeneity, different tumor models will be needed according to the development stages: target validation, proof of concept of target inhibition by a lead compound (small molecules or biotherapeutics), or candidate selection for further clinical development. In most of the cases, subcutaneous implantations of murine or human tumors are the best tools to address the preclinical questions helping to prepare the clinical development. Nevertheless, the development of more complex tumor models is also requested to answer more specific preclinical questions: either using surgical procedures to graft the tumor within a specific organ for evaluating specific tumor-stroma interactions, or using genetic engineered animals to mimic the different stages of the tumor development, or to humanize the target.     

Antitumor efficacy of a novel polymer-peptide-drug conjugate in human tumor xenograft models

We have designed a new dextran-peptide-methotrexate conjugate to achieve tumor-targeted delivery of chemotherapeutics. The dextran carrier was selected to allow passive targeting and enhanced permeation and retention (EPR). The peptide linker has also been optimized to allow drug release in the presence of matrix-metalloproteinase-2 (MMP-2) and matrix-metalloproteinase-9 (MMP-9), 2 important tumor-associated enzymes. The new conjugate was assessed for its in vivo antitumor efficacy and systemic side effects. It was compared with free methotrexate (MTX) and a similar conjugate, differing by an MMP-insensitive linker, at equivalent intraperitoneal dosages. The MMP-sensitive conjugate demonstrated tolerable in vivo side effects and effective inhibition of in vivo tumor growth by 83% in each of the 2 separate tumor models that overexpress MMP (HT-1080 and U-87). The antiproliferative effect of the drug contributed to the inhibition of tumor growth. In contrast, free MTX resulted in no significant tumor reduction in the same models. Neither free MTX nor the conjugate caused any tumor inhibition in the mice bearing RT-112, a slower growing model that does not overexpress MMP. MMP-insensitive conjugates, though able to inhibit tumor growth, caused toxicity in the small intestine and bone marrow.     

Preclinical mouse models for BRCA1-associated breast cancer

A substantial part of all hereditary breast cancer cases is caused by BRCA1 germline mutations. In this review, we will discuss the insights into BRCA1 functions that we obtained from mouse models with conventional and conditional mutations in Brca1. The most advanced models closely resemble human BRCA1-related breast cancer and may therefore be useful for addressing clinically relevant questions.     

Antibody-drug conjugate MORAb-202 exhibits long-lasting antitumor efficacy in TNBC PDx models

The antibody-drug conjugate (ADC) MORAb-202, consisting of farletuzumab paired with a cathepsin B–cleavable linker and eribulin, targets folate receptor alpha (FRA), which is frequently overexpressed in various tumor types. MORAb-202 was highly cytotoxic to FRA-positive cells in vitro, with limited off-target killing of FRA-negative cells. Furthermore, MORAb-202 showed a clear in vitro bystander cytotoxic effect in coculture with FRA-positive/negative cells. In vivo antitumor efficacy studies of MORAb-202 were conducted with a single administration of MORAb-202 in triple-negative breast cancer (TNBC) patient–derived xenograft (PDx) models expressing low and high levels of FRA. MORAb-202 exhibited durable efficacy proportional to tumor FRA expression. Toxicology studies (Q3Wx2) in nonhuman primates suggested that the major observed toxicity of MORAb-202 is hematologic toxicity. Overall, these findings support the concept that MORAb-202 represents a promising investigational ADC for the treatment of TNBC patients.     

Dopamine increases the efficacy of anticancer drugs in breast and colon cancer preclinical models.

PURPOSE: Because neurotransmitter dopamine inhibits vascular permeability factor/vascular endothelial growth factor (VEGF)-induced angiogenesis and as anti-VEGF agents act synergistically with anticancer drugs, we therefore investigated whether dopamine can increase the efficacies of these drugs. EXPERIMENTAL DESIGN: The effect of dopamine was investigated in human breast cancer-(MCF-7) and colon (HT29) cancer-bearing mice. Experimental groups received either dopamine or doxorubicin or dopamine plus doxorubicin in MCF-7 tumor-bearing mice, and either dopamine or 5-fluorouracil or dopamine plus 5-fluorouracil in HT29-bearing mice. Thereafter, tumor growth, angiogenesis, tumor cell apoptosis, life span, and the effect of dopamine on the growth and survival of tumor cells in vitro were determined. Finally, the effects of dopamine on tumor vascular permeability; on VEGF receptor-2, mitogen-activated protein kinase, and focal adhesion kinase phosphorylation; and also on the proliferation and migration of tumor endothelial cells were investigated. RESULTS: Dopamine, in combination with anticancer drugs, significantly inhibited tumor growth and increased the life span when compared with treatment with dopamine or anticancer drugs alone. Dopamine had no direct effects on the growth and survival of tumor cells. The antiangiogenic action of dopamine was mediated by inhibiting proliferation and migration of tumor endothelial cells through suppression of VEGF receptor-2, mitogen-activated protein kinase, and focal adhesion kinase phosphorylation. CONCLUSION: Our study shows that dopamine significantly enhances the efficacies of commonly used anticancer drugs and also indicates that an inexpensive drug like dopamine, which is being extensively used in the clinics, might have a role as an antiangiogenic agent for the treatment of breast and colon cancer.     

Characterizing the efficacy of cancer therapeutics in patient-derived xenograft models of metastatic breast cancer

Purpose

Basal-like breast cancers are aggressive and often metastasize to vital organs. Treatment is largely limited to chemotherapy. This study aims to characterize the efficacy of cancer therapeutics in vitro and in vivo within the primary tumor and metastatic setting, using patient-derived xenograft (PDX) models.

Methods

We employed two basal-like, triple-negative PDX models, WHIM2 and WHIM30. PDX cells, obtained from mammary tumors grown in mice, were treated with twelve cancer therapeutics to evaluate their cytotoxicity in vitro. Four of the effective drugs—carboplatin, cyclophosphamide, bortezomib, and dacarbazine—were tested in vivo for their efficacy in treating mammary tumors, and metastases generated by intracardiac injection of tumor cells.

Results

RNA sequencing showed that global gene expression of PDX cells grown in the mammary gland was similar to those tested in culture. In vitro, carboplatin was cytotoxic to WHIM30 but not WHIM2, whereas bortezomib, dacarbazine, and cyclophosphamide were cytotoxic to both lines. Yet, these drugs were ineffective in treating both primary and metastatic WHIM2 tumors in vivo. Carboplatin and cyclophosphamide were effective in treating WHIM30 mammary tumors and reducing metastatic burden in the brain, liver, and lungs. WHIM2 and WHIM30 metastases showed distinct patterns of cytokeratin and vimentin expression, regardless of treatment, suggesting that different tumor cell subpopulations may preferentially seed in different organs.

Conclusions

This study highlights the utility of PDX models for studying the efficacy of therapeutics in reducing metastatic burden in specific organs. The differential treatment responses between two PDX models of the same intrinsic subtype, in both the primary and metastatic setting, recapitulates the challenges faced in treating cancer patients and highlights the need for combination therapies and predictive biomarkers.

     

Preclinical evaluation of EC145, a folate-vinca alkaloid conjugate

We recently developed a new group of folate-conjugated Vinca alkaloids, one of which, EC145, emerged as a candidate for clinical development. Brief treatment of nude mice bearing approximately 100 mm(3) folate receptor-positive human xenografts led to complete response (CR) in 5/5 mice and cures (i.e., remission without a relapse for >90 days post-tumor implantation) in 4/5 mice. Multiple CRs and cures were also noted when EC145 was used to treat mice initially bearing tumors as large as 750 mm(3). Likewise, complete cures (5/5) resulted following the treatment of an aggressive folate receptor-positive J6456 lymphoma model. The activity of EC145 was not accompanied by noticeable weight loss or major organ tissue degeneration. Furthermore, no significant antitumor activity (0/5 CR) was observed in EC145-treated animals that were co-dosed with an excess of a benign folate ligand, thus demonstrating the target-specific activity of EC145. The enhanced therapeutic index due to folate conjugation was also evidenced by the fact that the unconjugated drug (desacetylvinblastine monohydrazide) was found to be completely inactive when administered at nontoxic dose levels and only marginally active when given at highly toxic dose levels. Subsequent dose regimen studies confirmed that EC145 given on a more frequent, qdx5 schedule resulted in the most effective antitumor response as compared with an equivalent total dose given on thrice- or single-injection-per-week schedule. Taken together, these studies show that EC145 has significant antiproliferative activity and tolerability, thus lending support to an ongoing phase 1 trial for the treatment of advanced malignancies.     

Preclinical safety and antitumor efficacy of insulin combined with irradiation.

BACKGROUND AND PURPOSE: We have previously reported that insulin significantly enhances tumor oxygenation (pO(2)) and increases radiation-induced tumor regrowth delay in experimental models. Considering the large radiosensitizing effect, clinical trials might be envisioned. The aim of the present pre-clinical study was to obtain a more complete set of safety and efficacy data which would further justify the commencement of such clinical trials. MATERIAL AND METHODS: Toxicity on normal (early and late-responding) tissues was measured by the intestinal crypt regeneration assay and the late leg contracture assay. Efficacy in terms of enhancement of pO(2) (measured by in vivo EPR oximetry) and increase in radiation-induced tumor regrowth delay was evaluated with a dose-response study on mice bearing FSaII fibrosarcoma. RESULTS: The effect on regrowth delay was directly correlated with the effect on the tumor pO(2), with a maximal effect using 400 mU kg(-1) insulin. Importantly, there was no increase in the radiation toxicity for normal tissues. Finally, we found that the hypoglycaemia induced by insulin can be corrected by simultaneous glucose infusion without modification of efficacy. CONCLUSION: Insulin here demonstrated a therapeutic gain and a lack of toxicity to normal tissues. The results of this study fully justify further larger preclinical assays such as the use of fractionated irradiation and a tumor control dose assay, before determining the utility of insulin as a radiosensitizer for human patients in the clinic.     

Preclinical efficacy spectrum and pharmacokinetics of ixabepilone

Purpose  Ixabepilone, a semisynthetic analog of natural epothilone B, was developed for use in cancer treatment. This study extends previous findings regarding the efficacy of ixabepilone and its low susceptibility to tumor resistance mechanisms and describes the pharmacokinetics of this new antineoplastic agent. Methods  The cytotoxicity of ixabepilone was assessed in vitro in breast, lung, and colon tumor cell lines and in vivo in human xenografts in mice. Antitumor activities of ixabepilone and taxanes were compared in multidrug-resistant models in vivo. Differential drug uptake of ixabepilone and paclitaxel was assessed in a P-glycoprotein (P-gp)-resistant colon cancer model in vitro. The pharmacokinetic profile of ixabepilone was established in mice and humans. Results  Ixabepilone demonstrated potent cytotoxicity in a broad range of human cancer cell lines in vitro and in a wide range of xenografts in vivo. Ixabepilone was ~3-fold more potent than docetaxel in the paclitaxel-resistant Pat-21 xenograft model (resistant due to overexpression of βIII-tubulin and a lack of βII-tubulin). Ixabepilone activity against P-gp-overexpressing breast and colon cancer was confirmed in in vivo models. Cellular uptake of ixabepilone, but not paclitaxel, was established in a P-gp-overexpressing model. The pharmacokinetics of ixabepilone was characterized by rapid tissue distribution and extensive tissue binding. Conclusions  Cytotoxicity studies against a range of tumor types in vitro and in vivo demonstrate that ixabepilone has potent and broad-spectrum antineoplastic activity. This is accompanied by favorable pharmacokinetics. Ixabepilone has reduced susceptibility to resistance due to P-gp overexpression, tubulin mutations, and alterations in β-tubulin isotype expression.     

Preclinical analysis of the antitumor efficacy of TS-1 using human uterine cervical cancer tumor xenografts

We investigated the antitumor activity of TS-1 in comparison with that of UFT and cisplatin (CDDP) against cervical cancer using xenografts of a human uterine cervical squamous cell cancer cell line, CaSki, transplanted into female Balb/cA JcL-nu mice. CaSki cell xenografts were prepared by subcutaneous (s.c.) implantation of 3x10(6) cells/animal into the right dorsal region of the mice. The tumor volume was measured twice a week and the relative tumor volume (RTV) was calculated. We divided the animals into four groups according to the treatment administered; TS-1 (10 mg/kg orally, once daily for 14 consecutive days), UFT (24 mg/kg orally, once daily for 14 consecutive days), CDDP (7.6 mg/kg injected intravenously once on the 1st day) and control (no treatment) groups. The antitumor effects of the drugs were measured. On the 35th day after the completion of treatment, the mean tumor volume in the mice treated with TS-1 or CDDP changed from 132.873+/-11.783 mm(3) to 706.401+/-613.122 mm(3) and 133.809+/-19.366 mm(3) to 722.630+/-855.509 mm(3), respectively. The mean tumor volume in the groups treated with TS-1 or CDDP was significantly lower compared to that in the control group (p<0.001; one-tailed Student's t-test). The relative inhibition of the tumor growth was 65.31 in the TS-1 group, 48.31 in the UFT group and 64.51 in the CDDP group. We conclude that TS-1 administered orally for 14 consecutive days showed the highest antitumor activity.     

Activity of the histone deacetylase inhibitor belinostat (PXD101) in preclinical models of prostate cancer

Histone deacetylase inhibitors (HDACi) represent a promising new class of anticancer agents. In the current investigation, we examined the activity of the HDACi belinostat in preclinical models of prostate cancer. In vitro proliferation assays demonstrated that belinostat potently inhibited the growth of prostate cancer cell lines (IC(50) < 1.0 microM) and was cytotoxic to these cells. Washout experiments indicated that exposure to belinostat for relatively short periods of time (<12 hr) induced suboptimal growth-inhibition and that cells exposed to 1.0 microM belinostat for 48 hr retained the capacity for regrowth following drug withdrawal, while cells exposed to 4.0 microM belinostat were irreversibly growth-inhibited. Cell cycle analyses demonstrated that belinostat induced G2/M arrest and increased the percentage of cells with subG1 DNA content, thus confirming the growth-inhibitory and cytotoxic effects of this compound. Normal prostate epithelial cells were generally less susceptible to the effects of belinostat than were prostate cancer cells. In an orthotopic prostate cancer tumor model, belinostat inhibited tumor growth by up to 43%. Moreover, metastatic lung lesions were present in 47% of vehicle-treated animals but in none of the animals administered belinostat. Consistent with its observed antimetastatic activity, belinostat inhibited the migration of prostate tumor cells and increased the production of tissue inhibitor of metalloproteinase-1 (TIMP-1) by these cells, the latter effect being replicated by siRNA knockdown of HDAC3. Belinostat also increased the expression of p21 and decreased the expression of potentially oncogenic proteins (mutant p53 and ERG). These results support the clinical evaluation of belinostat for the treatment of prostate cancer.     

Infectivity-enhanced adenoviruses deliver efficacy in clinical samples and orthotopic models of disseminated gastric cancer.

PURPOSE: Metastatic gastric cancer remains a common and devastating disease without curative treatment. Recent proof-of-concept clinical trials have validated gene therapy with adenoviruses as an effective and safe modality for the treatment of cancer. However, expression of the primary coxsackie-adenovirus receptor is variable in advanced cancers, and therefore, the use of heterologous receptors could be advantageous. EXPERIMENTAL DESIGN: Here, we used capsid-modified adenoviruses for increasing the transduction and subsequent antitumor efficacy. 5/3 chimeric viruses have a serotype 3 knob which allows binding to a receptor distinct from coxsackie-adenovirus receptor. The fiber of Ad5lucRGD is modified with an integrin-targeted motif. Polylysine motifs, pK7 and pK21, bind to heparan sulfates. Oncolytic adenoviruses replicate in and kill tumor cells selectively. Gastric cancer cell lines and fresh clinical samples from patients were infected with transductionally targeted viruses. Capsid-modified oncolytic adenoviruses were used in cell killing experiments. To test viral transduction and therapeutic efficacy in vivo, we developed orthotopic mouse models featuring i.p. disseminated human gastric cancer, which allowed the evaluation of biodistribution and antitumor efficacy in a system similar to humans. RESULTS: Capsid modifications benefited gene transfer efficiency and cell killing in gastric cancer cell lines and clinical samples in vitro and in vivo. Modified oncolytic adenoviruses significantly increased the survival of mice with orthotopic gastric cancer. CONCLUSIONS: These preclinical data set the stage for the clinical evaluation of safety and efficacy in patients with disease refractory to current modalities.     

Preclinical antitumor efficacy of selective exportin 1 inhibitors in glioblastoma

Background

Glioblastoma (GBM) is poorly responsive to current chemotherapy. The nuclear transporter exportin 1 (XPO1, CRM1) is often highly expressed in GBM, which may portend a poor prognosis. Here, we determine the efficacy of novel selective inhibitors of nuclear export (SINE) specific to XPO1 in preclinical models of GBM.

Methods

Seven patient-derived GBM lines were treated with 3 SINE compounds (KPT-251, KPT-276, and Selinexor) in neurosphere culture conditions. KPT-276 and Selinexor were also evaluated in a murine orthotopic patient-derived xenograft (PDX) model of GBM. Cell cycle effects were assayed by flow cytometry in vitro and immunohistochemistry in vivo. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase 3/7 activity assays.

Results

Treatment of GBM neurosphere cultures with KPT-276, Selinexor, and KPT-251 revealed dose-responsive growth inhibition in all 7 GBM lines [range of half-maximal inhibitory concentration (IC₅₀), 6–354 nM]. In an orthotopic PDX model, treatment with KPT-276 and Selinexor demonstrated pharmacodynamic efficacy, significantly suppressed tumor growth, and prolonged animal survival. Cellular proliferation was not altered with SINE treatment. Instead, induction of apoptosis was apparent both in vitro and in vivo with SINE treatment, without overt evidence of neurotoxicity.

Conclusions

SINE compounds show preclinical efficacy utilizing in vitro and in vivo models of GBM, with induction of apoptosis as the mechanism of action. Selinexor is now in early clinical trials in solid and hematological malignancies. Based on these preclinical data and excellent brain penetration, we have initiated clinical trials of Selinexor in patients with relapsed GBM.