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.

Real-time PCR-based assay to quantify the relative amount of human and mouse tissue present in tumor xenografts.

BACKGROUND: Xenograft samples used to test anti-cancer drug efficacies and toxicities in vivo contain an unknown mix of mouse and human cells. Evaluation of drug activity can be confounded by samples containing large amounts of contaminating mouse tissue. We have developed a real-time quantitative polymerase chain reaction (qPCR) assay using TaqMan technology to quantify the amount of mouse tissue that is incorporated into human xenograft samples. RESULTS: The forward and reverse primers bind to the same DNA sequence in the human and the mouse genome. Using a set of specially designed fluorescent probes provides species specificity. The linearity and sensitivity of the assay is evaluated using serial dilutions of single species and heterogeneous DNA mixtures. We examined many xenograft samples at various in vivo passages, finding a wide variety of human:mouse DNA ratios. This variation may be influenced by tumor type, number of serial passages in vivo, and even which part of the tumor was collected and used in the assay. CONCLUSIONS: This novel assay provides an accurate quantitative assessment of human and mouse content in xenograft tumors. This assay can be performed on aberrantly behaving human xenografts, samples used in bioinformatics studies, and periodically for tumor tissue frequently grown by serial passage in vivo.

ASPS-1, a novel cell line manifesting key features of alveolar soft part sarcoma.

In vitro growth of alveolar soft part sarcoma (ASPS) and establishment of an ASPS cell line, ASPS-1, are described in this study. Using a recently developed xenograft model of ASPS derived from a lymph node metastasis, organoid nests consisting of 15 to 25 ASPS cells were isolated from ASPS xenograft tumors by capture on 70 µm filters and plated in vitro. After attachment to the substratum, these nests deposited small aggregates of ASPS cells. These cells grew slowly and were expanded over a period of 3 years and have maintained characteristics consistent with those of both the original ASPS tumor from the patient and the xenograft tumor including (1) presence of the alveolar soft part locus-transcription factor E3 type 1 fusion transcript and nuclear expression of the alveolar soft part locus-transcription factor E3 type 1 fusion protein; (2) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS; and (3) expression of upregulated ASPS transcripts involved in angiogenesis (ANGPTL2, HIF-1-α, MDK, c-MET, VEGF, and TIMP-2), cell proliferation (PRL, PCSK1), metastasis (ADAM9), as well as the transcription factor BHLHB3 and the muscle-specific transcripts TRIM63 and ITGß1BP3. This ASPS cell line forms colonies in soft agar and retains the ability to produce highly vascularized ASPS tumors in NOD.SCID/NCr mice. Immunohistochemistry of selected ASPS markers on these tumors indicated similarity to those of the original patient tumor as well as to the xenografted ASPS tumor. We anticipate that this ASPS cell line will accelerate investigations into the biology of ASPS including identification of new therapeutic approaches for treatment of this slow growing soft tissue sarcoma.

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.

Sodium dichloroacetate selectively targets cells with defects in the mitochondrial ETC.

The "Warburg effect," also termed aerobic glycolysis, describes the increased reliance of cancer cells on glycolysis for ATP production, even in the presence of oxygen. Consequently, there is continued interest in inhibitors of glycolysis as cancer therapeutics. One example is dichloroacetate (DCA), a pyruvate mimetic that stimulates oxidative phosphorylation through inhibition of pyruvate dehydrogenase kinase. In this study, the mechanistic basis for DCA anti-cancer activity was re-evaluated in vitro using biochemical, cellular and proteomic approaches. Results demonstrated that DCA is relatively inactive (IC(50) ≥ 17 mM, 48 hr), induces apoptosis only at high concentrations (≥ 25 mM, 48 hr) and is not cancer cell selective. Subsequent 2D-PAGE proteomic analysis confirmed DCA-induced growth suppression without apoptosis induction. Furthermore, DCA depolarizes mitochondria and promotes reactive oxygen species (ROS) generation in all cell types. However, DCA was found to have selective activity against rho(0) cells [mitochondrial DNA (mtDNA) deficient] and to synergize with 2-deoxyglucose in complex IV deficient HCT116 p53(-/-) cells. DCA also synergized in vitro with cisplatin and topotecan, two antineoplastic agents known to damage mitochondrial DNA. These data suggest that in cells "hardwired" to selectively utilize glycolysis for ATP generation (e.g., through mtDNA mutations), the ability of DCA to force oxidative phosphorylation confers selective toxicity. In conclusion, although we provide a mechanism distinct from that reported previously, the ability of DCA to target cell lines with defects in the electron transport chain and to synergize with existing chemotherapeutics supports further preclinical development.

Clinical Evolution of Epithelial-Mesenchymal Transition in Human Carcinomas.

The significance of the phenotypic plasticity afforded by epithelial-mesenchymal transition (EMT) for cancer progression and drug resistance remains to be fully elucidated in the clinic. We evaluated epithelial-mesenchymal phenotypic characteristics across a range of tumor histologies using a validated, high-resolution digital microscopic immunofluorescence assay (IFA) that incorporates ß-catenin detection and cellular morphology to delineate carcinoma cells from stromal fibroblasts and that quantitates the individual and colocalized expression of the epithelial marker E-cadherin (E) and the mesenchymal marker vimentin (V) at subcellular resolution ("EMT-IFA"). We report the discovery of ß-catenin+ cancer cells that coexpress E-cadherin and vimentin in core-needle biopsies from patients with various advanced metastatic carcinomas, wherein these cells are transitioning between strongly epithelial and strongly mesenchymal-like phenotypes. Treatment of carcinoma models with anticancer drugs that differ in their mechanism of action (the tyrosine kinase inhibitor pazopanib in MKN45 gastric carcinoma xenografts and the combination of tubulin-targeting agent paclitaxel with the BCR-ABL inhibitor nilotinib in MDA-MB-468 breast cancer xenografts) caused changes in the tumor epithelial-mesenchymal character. Moreover, the appearance of partial EMT or mesenchymal-like carcinoma cells in MDA-MB-468 tumors treated with the paclitaxel-nilotinib combination resulted in upregulation of cancer stem cell (CSC) markers and susceptibility to FAK inhibitor. A metastatic prostate cancer patient treated with the PARP inhibitor talazoparib exhibited similar CSC marker upregulation. Therefore, the phenotypic plasticity conferred on carcinoma cells by EMT allows for rapid adaptation to cytotoxic or molecularly targeted therapy and could create a form of acquired drug resistance that is transient in nature. SIGNIFICANCE: Despite the role of EMT in metastasis and drug resistance, no standardized assessment of EMT phenotypic heterogeneity in human carcinomas exists; the EMT-IFA allows for clinical monitoring of tumor adaptation to therapy.

Therapeutic vulnerability of an in vivo model of alveolar soft part sarcoma (ASPS) to antiangiogenic therapy.

In vivo growth of alveolar soft part sarcoma (ASPS) was achieved using subcutaneous xenografts in sex-matched nonobese diabetic severe combined immunodeficiency mice. One tumor, currently at passage 6, has been maintained in vivo for 32 months and has maintained characteristics consistent with those of the original ASPS tumor including (1) tumor histology and staining with periodic acid Schiff/diastase, (2) the presence of the ASPL-TFE3 type 1 fusion transcript, (3) nuclear staining with antibodies to the ASPL-TFE3 type 1 fusion protein, (4) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS, (5) stable expression of signature ASPS gene transcripts and finally, the development and maintenance of a functional vascular network, a hallmark of ASPS. The ASPS xenograft tumor vasculature encompassing nests of ASPS cells is highly reactive to antibodies against the endothelial antigen CD34 and is readily accessible to intravenously administered fluorescein isothiocyanate-dextran. The therapeutic vulnerability of this tumor model to antiangiogenic therapy, targeting vascular endothelial growth factor and hypoxia-inducible factor-1 alpha, was examined using bevacizumab and topotecan alone and in combination. Together, the 2 drugs produced a 70% growth delay accompanied by a 0.7 net log cell kill that was superior to the antitumor effect produced by either drug alone. In summary, this study describes a preclinical in vivo model for ASPS which will facilitate investigation into the biology of this slow growing soft tissue sarcoma and demonstrates the feasibility of using an antiangiogenic approach in the treatment of ASPS.

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.

Novel antibody reagents for characterization of drug- and tumor microenvironment-induced changes in epithelial-mesenchymal transition and cancer stem cells.

The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation-mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous hHGF knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.

Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts.

We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1alpha protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1alpha protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1alpha inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

"Vasocrine" formation of tumor cell-lined vascular spaces: implications for rational design of antiangiogenic therapies.

Here we report that B16F10 murine melanoma cells mimic endothelial cell behavior and the angiogenic process in vitro and in vivo. Cord formation in vitro by tumor cells is stimulated by hypoxia and vascular endothelial growth factor (VEGF) and inhibited by antibodies against VEGF and the VEGF KDR receptor (VEGF receptor 2). We define regulation of tumor cell-derived vascular space formation by these vasoactive compounds as "vasocrine" stimulation. ICRF 159 (Razoxane; NSC 129943) prevents tumor cell but not endothelial cell cord formation in vitro, and the antiangiogenic drug TNP-470 (NSC 642492) inhibits endothelial but not tumor cell cord formation in vitro. Both drugs inhibit formation of blood-filled vascular spaces in vivo. These results bear on the anticipated action of ICRF 159 in human clinical trials and novel strategies for targeting tumor blood supplies.

Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays.

PURPOSE: Rational development of targeted MET inhibitors for cancer treatment requires a quantitative understanding of target pharmacodynamics, including molecular target engagement, mechanism of action, and duration of effect. EXPERIMENTAL DESIGN: Sandwich immunoassays and specimen handling procedures were developed and validated for quantifying full-length MET and its key phosphospecies (pMET) in core tumor biopsies. MET was captured using an antibody to the extracellular domain and then probed using antibodies to its C-terminus (full-length) and epitopes containing pY1234/1235, pY1235, and pY1356. Using pMET:MET ratios as assay endpoints, MET inhibitor pharmacodynamics were characterized in MET-amplified and -compensated (VEGFR blockade) models. RESULTS: By limiting cold ischemia time to less than two minutes, the pharmacodynamic effects of the MET inhibitors PHA665752 and PF02341066 (crizotinib) were quantifiable using core needle biopsies of human gastric carcinoma xenografts (GTL-16 and SNU5). One dose decreased pY1234/1235 MET:MET, pY1235-MET:MET, and pY1356-MET:MET ratios by 60% to 80% within 4 hours, but this effect was not fully sustained despite continued daily dosing. VEGFR blockade by pazopanib increased pY1235-MET:MET and pY1356-MET:MET ratios, which was reversed by tivantinib. Full-length MET was quantifiable in 5 of 5 core needle samples obtained from a resected hereditary papillary renal carcinoma, but the levels of pMET species were near the assay lower limit of quantitation. CONCLUSIONS: These validated immunoassays for pharmacodynamic biomarkers of MET signaling are suitable for studying MET responses in amplified cancers as well as compensatory responses to VEGFR blockade. Incorporating pharmacodynamic biomarker studies into clinical trials of MET inhibitors could provide critical proof of mechanism and proof of concept for the field. Clin Cancer Res; 22(14); 3683-94. ©2016 AACR.

In vivo antitumor efficacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), a water-soluble geldanamycin derivative.

PURPOSE: To describe the preclinical basis for further development of 17-dimethyl aminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG, NSC 707545). METHODS: In vitro proliferation assays, and in vivo model studies in metastatic pancreatic carcinoma and subcutaneous xenograft melanoma and small-cell lung carcinoma models. RESULTS: 17-DMAG emerged from screening studies as a potent geldanamycin analog, with the average concentration inhibiting the growth of the NCI anticancer cell line drug screen by 50% being 0.053 microM. "Head to head" comparison with 17-allylamino-17-demethoxygeldanamycin (17-AAG, NSC 330507) revealed 17-DMAG to possess potent activity against certain cell types, e.g., MDA-MB-231 breast carcinoma and HL60-TB leukemia which were relatively insensitive to 17-AAG. Evidence of oral bioavailability of 17-DMAG in a saline-based formulation prompted more detailed examination of its antitumor efficacy in vivo. 17-DMAG inhibited the growth of the AsPC-1 pancreatic carcinoma xenografts growing as intrahepatic metastases at doses of 6.7-10 mg/kg twice daily for 5 days administered orally under conditions where 17-AAG was without activity. 17-DMAG in an aqueous vehicle at 7.5-15 mg/kg per day for 3 days on days 1-3, 8-10 and 13-17, or 1-5 and 8-12 showed evidence of antitumor activity by the parenteral and oral routes in the MEXF 276 and MEXF 989 melanomas and by the parenteral route in the LXFA 629 and LXFS 650 adenocarcinoma and small-cell carcinoma models. The latter activity was comparable to the historical activity of 17-AAG. CONCLUSIONS: Taken together, the in vivo activity of 17-DMAG supports the further development of this water-soluble and potentially orally administrable geldanamycin congener.

Oxyphenisatin acetate (NSC 59687) triggers a cell starvation response leading to autophagy, mitochondrial dysfunction, and autocrine TNFα-mediated apoptosis.

Oxyphenisatin (3,3-bis(4-hydroxyphenyl)-1H-indol-2-one) and several structurally related molecules have been shown to have in vitro and in vivo antiproliferative activity. This study aims to confirm and extend mechanistic studies by focusing on oxyphenisatin acetate (OXY, NSC 59687), the pro-drug of oxyphenisatin. Results confirm that OXY inhibits the growth of the breast cancer cell lines MCF7, T47D, HS578T, and MDA-MB-468. This effect is associated with selective inhibition of translation accompanied by rapid phosphorylation of the nutrient sensing eukaryotic translation initiation factor 2α (eIF2α) kinases, GCN2 and PERK. This effect was paralleled by activation of AMP-activated protein kinase (AMPK) combined with reduced phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6K and 4E-BP1. Microarray analysis highlighted activation of pathways involved in apoptosis induction, autophagy, RNA/protein metabolism, starvation responses, and solute transport. Pathway inhibitor combination studies suggested a role for AMPK/mTOR signaling, de novo transcription and translation, reactive oxygen species (ROS)/glutathione metabolism, calcium homeostasis and plasma membrane Na(+) /K(+) /Ca(2+) transport in activity. Further examination confirmed that OXY treatment was associated with autophagy, mitochondrial dysfunction, and ROS generation. Additionally, treatment was associated with activation of both intrinsic and extrinsic apoptotic pathways. In the estrogen receptor (ER) positive MCF7 and T47D cells, OXY induced TNFα expression and TNFR1 degradation, indicating autocrine receptor-mediated apoptosis in these lines. Lastly, in an MCF7 xenograft model, OXY delivered intraperitoneally inhibited tumor growth, accompanied by phosphorylation of eIF2α and degradation of TNFR1. These data suggest that OXY induces a multifaceted cell starvation response, which ultimately induces programmed cell death.

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.

Increased antitumor activity of bevacizumab in combination with hypoxia inducible factor-1 inhibition.

Inhibition of hypoxia inducible factor-1 (HIF-1) is an attractive therapeutic strategy to target the tumor microenvironment. However, HIF-1 inhibitors may have limited activity as single agents and combination therapies may be required. We tested the hypothesis that HIF-1 inhibition in a hypoxic-stressed tumor microenvironment, which could be generated by administration of antiangiogenic agents, may result in a more pronounced therapeutic effect. The activity of bevacizumab, either alone or in combination with the HIF-1alpha inhibitor topotecan, was evaluated in U251-HRE xenografts. Tumor tissue was collected at the end of treatment and changes in tumor oxygenation, angiogenesis, proliferation, apoptosis, HIF-1alpha levels, HIF-1 target genes, and DNA damage were evaluated. Bevacizumab decreased microvessel-density and increased intratumor-hypoxia, but did not induce apoptosis. Moreover, bevacizumab alone caused a significant increase of HIF-1-dependent gene expression in tumor tissue. Addition of a low dose of daily topotecan to bevacizumab significantly inhibited tumor growth, relative to mice treated with topotecan or bevacizumab alone (P < 0.01). The addition of topotecan to bevacizumab was also associated with profound inhibition of HIF-1 transcriptional activity, significant inhibition of proliferation, and induction of apoptosis. Importantly, DNA damage induced by topotecan alone was not augmented by addition of bevacizumab, suggesting that increased cytotoxic activity did not account for the increased antitumor effects observed. These results strongly suggest that combination of anti-vascular endothelial growth factor antibodies with HIF-1 inhibitors is an attractive therapeutic strategy targeting in the hypoxic tumor microenvironment.