Gene expression profiling of 49 human tumor xenografts from in vitro culture through multiple in vivo passages--strategies for data mining in support of therapeutic studies.

BACKGROUND: Development of cancer therapeutics partially depends upon selection of appropriate animal models. Therefore, improvements to model selection are beneficial. RESULTS: Forty-nine human tumor xenografts at in vivo passages 1, 4 and 10 were subjected to cDNA microarray analysis yielding a dataset of 823 Affymetrix HG-U133 Plus 2.0 arrays. To illustrate mining strategies supporting therapeutic studies, transcript expression was determined: 1) relative to other models, 2) with successive in vivo passage, and 3) during the in vitro to in vivo transition. Ranking models according to relative transcript expression in vivo has the potential to improve initial model selection. For example, combining p53 tumor expression data with mutational status could guide selection of tumors for therapeutic studies of agents where p53 status purportedly affects efficacy (e.g., MK-1775). The utility of monitoring changes in gene expression with extended in vivo tumor passages was illustrated by focused studies of drug resistance mediators and receptor tyrosine kinases. Noteworthy observations included a significant decline in HCT-15 colon xenograft ABCB1 transporter expression and increased expression of the kinase KIT in A549 with serial passage. These trends predict sensitivity to agents such as paclitaxel (ABCB1 substrate) and imatinib (c-KIT inhibitor) would be altered with extended passage. Given that gene expression results indicated some models undergo profound changes with in vivo passage, a general metric of stability was generated so models could be ranked accordingly. Lastly, changes occurring during transition from in vitro to in vivo growth may have important consequences for therapeutic studies since targets identified in vitro could be over- or under-represented when tumor cells adapt to in vivo growth. A comprehensive list of mouse transcripts capable of cross-hybridizing with human probe sets on the HG-U133 Plus 2.0 array was generated. Removal of the murine artifacts followed by pairwise analysis of in vitro cells with respective passage 1 xenografts and GO analysis illustrates the complex interplay that each model has with the host microenvironment. CONCLUSIONS: This study provides strategies to aid selection of xenograft models for therapeutic studies. These data highlight the dynamic nature of xenograft models and emphasize the importance of maintaining passage consistency throughout experiments.

Exploiting embryonic niche conditions to grow Wilms tumor blastema in culture.

Introduction: Wilms Tumor (WT), or nephroblastoma, is the most common pediatric kidney cancer. Most WTs display a "favorable" triphasic histology, in which the tumor is comprised of blastemal, stromal, and epithelial cell types. Blastemal predominance after neoadjuvant chemotherapy or diffuse anaplasia ("unfavorable" histology; 5-8%) portend a worse prognosis. Blastema likely provide the putative cancer stem cells (CSCs), which retain molecular and histologic features characteristic of nephron progenitor cells (NPCs), within WTs. NPCs arise in the metanephric mesenchyme (MM) and populate the cap mesenchyme (CM) in the developing kidney. WT blastemal cells, like NPCs, similarly express markers, SIX2 and CITED1. Tumor xenotransplantation is currently the only dependable method to propagate tumor tissue for research or therapeutic screening, since efforts to culture tumors in vitro as monolayers have invariably failed. Therefore, a critical need exists to propagate WT stem cells rapidly and efficiently for high-throughput, real-time drug screening. Methods: Previously, our lab developed niche conditions that support the propagation of murine NPCs in culture. Applying similar conditions to WTs, we assessed our ability to maintain key NPC "stemness" markers, SIX2, NCAM, and YAP1, and CSC marker ALDHI in cells from five distinct untreated patient tumors. Results: Accordingly, our culture conditions maintained the expression of these markers in cultured WT cells through multiple passages of rapidly dividing cells. Discussion: These findings suggest that our culture conditions sustain the WT blastemal population, as previously shown for normal NPCs. As a result, we have developed new WT cell lines and a multi-passage in vitro model for studying the blastemal lineage/CSCs in WTs. Furthermore, this system supports growth of heterogeneous WT cells, upon which potential drug therapies could be tested for efficacy and resistance.

F-aza-T-dCyd (NSC801845), a Novel Cytidine Analog, in Comparative Cell Culture and Xenograft Studies with the Clinical Candidates T-dCyd, F-T-dCyd, and Aza-T-dCyd.

In this article, 5-aza-4'-thio-2'-ß-fluoro-2'-deoxycytidine (F-aza-T-dCyd, NSC801845), a novel cytidine analog, is first disclosed and compared with T-dCyd, F-T-dCyd, and aza-T-dCyd in cell culture and mouse xenograft studies in HCT-116 human colon carcinoma, OVCAR3 human ovarian carcinoma, NCI-H23 human NSCLC carcinoma, HL-60 human leukemia, and the PDX BL0382 bladder carcinoma. In three of five xenograft lines (HCT-116, HL-60, and BL-0382), F-aza-T-dCyd was more efficacious than aza-T-dCyd. Comparable activity was observed for these two agents against the NCI-H23 and OVCAR3 xenografts. In the HCT-116 study, F-aza-T-dCyd [10 mg/kg intraperitoneal (i.p.), QDx5 for four cycles], produced complete regression of the tumors in all mice with a response that proved durable beyond postimplant day 150 (129 days after the last dose). Similarly, complete tumor regression was observed in the HL-60 leukemia xenograft when mice were dosed with F-aza-T-dCyd (10 mg/kg i.p., QDx5 for three cycles). In the PDX BL-0382 bladder study, both oral and i.p. dosing of F-aza-T-dCyd (8 mg/kg QDx5 for three cycles) produced regressions that showed tumor regrowth beginning 13 days after dosing. These findings indicate that further development of F-aza-T-dCyd (NSC801845) is warranted. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/20/4/625/F1.large.jpg.

ARC (NSC 188491) has identical activity to Sangivamycin (NSC 65346) including inhibition of both P-TEFb and PKC.

BACKGROUND: The nucleoside analog, ARC (NSC 188491) is a recently characterized transcriptional inhibitor that selectively kills cancer cells and has the ability to perturb angiogenesis in vitro. In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines. METHODS: Structure-based homology searches were used to identify those compounds with similarity to ARC. Comparator compounds were then evaluated alongside ARC in the context of viability, cell cycle and apoptosis assays to establish any similarities. Following this, biological overlap was explored in detail using gene-expression analysis and kinase inhibition assays. RESULTS: Results demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1) cytotoxicity assays, 2) ability to induce a G2/M block, 3) inhibitory effects on RNA/DNA/protein synthesis, 4) transcriptomic response to treatment, 5) inhibition of protein kinase C, 6) inhibition of positive transcription elongation factor b (P-TEFb), 7) inhibition of VEGF secretion, and 8) activity within hollow fiber assays. Extending ARC activity to PKC inhibition provides a molecular basis for ARC cancer selectivity and anti-angiogenic effects. Furthermore, functional overlap between ARC and sangivamycin suggests that development of ARC may benefit from a retrospective of previous sangivamycin clinical trials. However, ARC was found to be inactive in several xenograft models, likely a consequence of rapid serum clearance. CONCLUSION: Overall, these data expand on the biological properties of ARC but suggest additional studies are required before it can be considered a clinical trials candidate.

Accelerated preclinical testing using transplanted tumors from genetically engineered mouse breast cancer models.

PURPOSE: The use of genetically engineered mouse (GEM) models for preclinical testing of anticancer therapies is hampered by variable tumor latency, incomplete penetrance, and complicated breeding schemes. Here, we describe and validate a transplantation strategy that circumvents some of these difficulties. EXPERIMENTAL DESIGN: Tumor fragments from tumor-bearing MMTV-PyMT or cell suspensions from MMTV-PyMT, -Her2/neu, -wnt1, -wnt1/p53(+/-), BRCA1/p53(+/-), and C3(1)T-Ag mice were transplanted into the mammary fat pad or s.c. into naïve syngeneic or immunosuppressed mice. Tumor development was monitored and tissues were processed for histopathology and gene expression profiling. Metastasis was scored 60 days after the removal of transplanted tumors. RESULTS: PyMT tumor fragments and cell suspensions from anterior glands grew faster than posterior tumors in serial passages regardless of the site of implantation. Microarray analysis revealed genetic differences between these tumors. The transplantation was reproducible using anterior tumors from multiple GEM, and tumor growth rate correlated with the number of transplanted cells. Similar morphologic appearances were observed in original and transplanted tumors. Metastasis developed in >90% of mice transplanted with PyMT, 40% with BRCA1/p53(+/-) and wnt1/p53(+/-), and 15% with Her2/neu tumors. Expansion of PyMT and wnt1 tumors by serial transplantation for two passages did not lead to significant changes in gene expression. PyMT-transplanted tumors and anterior tumors of transgenic mice showed similar sensitivities to cyclophosphamide and paclitaxel. CONCLUSIONS: Transplantation of GEM tumors can provide a large cohort of mice bearing mammary tumors at the same stage of tumor development and with defined frequency of metastasis in a well-characterized molecular and genetic background.

Development and validation of an immunoassay for quantification of topoisomerase I in solid tumor tissues.

BACKGROUND: Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors. METHODOLOGY/PRINCIPAL FINDINGS: We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998. CONCLUSIONS/SIGNIFICANCE: Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors.

Phase I study of PARP inhibitor ABT-888 in combination with topotecan in adults with refractory solid tumors and lymphomas.

A phase I trial of ABT-888 (veliparib), a PARP inhibitor, in combination with topotecan, a topoisomerase I-targeted agent, was carried out to determine maximum tolerated dose (MTD), safety, pharmacokinetics, and pharmacodynamics of the combination in patients with refractory solid tumors and lymphomas. Varying schedules and doses of intravenous topotecan in combination with ABT-888 (10 mg) administered orally twice a day (BID) were evaluated. Plasma and urine pharmacokinetics were assessed and levels of poly(ADP-ribose) (PAR) and the DNA damage marker γH2AX were measured in tumor and peripheral blood mononuclear cells (PBMC). Twenty-four patients were enrolled. Significant myelosuppression limited the ability to coadminister ABT-888 with standard doses of topotecan, necessitating dose reductions. Preclinical studies using athymic mice carrying human tumor xenografts also informed schedule changes. The MTD was established as topotecan 0.6 mg/m²/d and ABT-888 10 mg BID on days one to five of 21-day cycles. Topotecan did not alter the pharmacokinetics of ABT-888. A more than 75% reduction in PAR levels was observed in 3 paired tumor biopsy samples; a greater than 50% reduction was observed in PBMCs from 19 of 23 patients with measurable levels. Increases in γH2AX response in circulating tumor cells (CTC) and PBMCs were observed in patients receiving ABT-888 with topotecan. We show a mechanistic interaction of a PARP inhibitor, ABT-888, with a topoisomerase I inhibitor, topotecan, in PBMCs, tumor, and CTCs. Results of this trial reveal that PARP inhibition can modulate the capacity to repair topoisomerase I-mediated DNA damage in the clinic.