The Hippo Pathway Effectors YAP/TAZ-TEAD Oncoproteins as Emerging Therapeutic Targets in the Tumor Microenvironment.

Various cancer cell-associated intrinsic and extrinsic inputs act on YAP/TAZ proteins to mediate the hyperactivation of the TEAD transcription factor-based transcriptome. This YAP/TAZ-TEAD activity can override the growth-limiting Hippo tumor-suppressor pathway that maintains normal tissue homeostasis. Herein, we provide an integrated summary of the contrasting roles of YAP/TAZ during normal tissue homeostasis versus tumor initiation and progression. In addition to upstream factors that regulate YAP/TAZ in the TME, critical insights on the emerging functions of YAP/TAZ in immune suppression and abnormal vasculature development during tumorigenesis are illustrated. Lastly, we discuss the current methods that intervene with the YAP/TAZ-TEAD oncogenic signaling pathway and the emerging applications of combination therapies, gut microbiota, and epigenetic plasticity that could potentiate the efficacy of chemo/immunotherapy as improved cancer therapeutic strategies.

Targeting hypoxia-induced tumor stemness by activating pathogen-induced stem cell niche defense.

Tumor hypoxia and oxidative stress reprograms cancer stem cells (CSCs) to a highly aggressive and inflammatory phenotypic state of tumor stemness. Previously, we characterized tumor stemness phenotype in the ATP Binding Cassette Subfamily G Member 2 (ABCG2)-positive migratory side population (SPm) fraction of CSCs exposed to extreme hypoxia followed by reoxygenation. Here, we report that post-hypoxia/reoxygenation SPm+/ABCG2+ CSCs exerts defense against pathogen invasion that involves bystander apoptosis of non-infected CSCs. In an in vitro assay of cancer cell infection by Bacillus Calmette Guerin (BCG) or mutant Mycobacterium tuberculosis (Mtb) strain 18b (Mtb-m18b), the pathogens preferentially replicated intracellular to SPm+/ABCG2+ CSCs of seven cell lines of diverse cancer types including SCC-25 oral squamous cancer cell line. The conditioned media (CM) of infected CSCs exhibited direct anti-microbial activity against Mtb and BCG, suggesting niche defense against pathogen. Importantly, the CM of infected CSCs exhibited marked in vitro bystander apoptosis toward non-infected CSCs. Moreover, the CM-treated xenograft bearing mice showed 10- to 15-fold reduction (p < 0.001; n = 7) in the number of CSCs residing in the hypoxic niches. Our in vitro studies indicated that BCG-infected SPm+/ABCG2+ equivalent EPCAM+/ABCG2+ CSCs of SCC-25 cells underwent pyroptosis and released a high mobility group box protein 1 (HMGB1)/p53 death signal into the tumor microenvironment (TME). The death signal can induce a Toll-like receptor 2/4-mediated bystander apoptosis in non-infected CSCs by activating p53/MDM2 oscillation and subsequent activation of capase-3-dependent intrinsic apoptosis. Notably, SPm+/ABCG2+ but not SP cells undergoing bystander apoptosis amplified the death signal by further release of HMGB1/p53 complex into the TME. These results suggest that post-hypoxia SPm+/ABCG2+ CSCs serve a functional role as a tumor stemness defense (TSD) phenotype to protect TME against bacterial invasion. Importantly, the CM of TSD phenotype undergoing bystander apoptosis may have therapeutic uses against CSCs residing in the hypoxic niche.

Correction: Next-generation multimodality of nutrigenomic cancer therapy: sulforaphane in combination with acetazolamide actively target bronchial carcinoid cancer in disabling the PI3K/Akt/mTOR survival pathway and inducing apoptosis.

[This corrects the article DOI: 10.18632/oncotarget.28011.].

Repositioning of Old Drugs for Novel Cancer Therapies: Continuous Therapeutic Perfusion of Aspirin and Oseltamivir Phosphate with Gemcitabine Treatment Disables Tumor Progression, Chemoresistance, and Metastases.

Metastatic pancreatic cancer has an invariably fatal outcome, with an estimated median progression-free survival of approximately six months employing our best combination chemotherapeutic regimens. Once drug resistance develops, manifested by increased primary tumor size and new and growing metastases, patients often die rapidly from their disease. Emerging evidence indicates that chemotherapy may contribute to the development of drug resistance through the upregulation of epithelial-mesenchymal transition (EMT) pathways and subsequent cancer stem cell (CSC) enrichment. Neuraminidase-1 (Neu-1) regulates the activation of several receptor tyrosine kinases implicated in EMT induction, angiogenesis, and cellular proliferation. Here, continuous therapeutic targeting of Neu-1 using parenteral perfusion of oseltamivir phosphate (OP) and aspirin (ASA) with gemcitabine (GEM) treatment significantly disrupts tumor progression, critical compensatory signaling mechanisms, EMT program, CSC, and metastases in a preclinical mouse model of human pancreatic cancer. ASA- and OP-treated xenotumors significantly inhibited the metastatic potential when transferred into animals.

Next Generation of Cancer Drug Repurposing: Therapeutic Combination of Aspirin and Oseltamivir Phosphate Potentiates Gemcitabine to Disable Key Survival Pathways Critical for Pancreatic Cancer Progression.

Resistance to chemotherapeutics and high metastatic rates contribute to the abysmal survival rate in patients with pancreatic cancer. An alternate approach for treating human pancreatic cancer involves repurposing the anti-inflammatory drug, aspirin (ASA), with oseltamivir phosphate (OP) in combination with the standard chemotherapeutic agent, gemcitabine (GEM). The question is whether treatment with ASA and OP can sensitize cancer cells to the cytotoxicity induced by GEM and limit the development of chemoresistance. To assess the key survival pathways critical for pancreatic cancer progression, we used the AlamarBlue cytotoxicity assay to determine the cell viability and combination index for the drug combinations, flow cytometric analysis of annexin V apoptosis assay to detect apoptotic and necrotic cells, fluorometric QCM™ chemotaxis migration assay to assess cellular migration, fluorometric extracellular matrix (ECM) cell adhesion array kit to assess the expression of the ECM proteins, scratch wound assay using the 96-well WoundMaker™, and the methylcellulose clonogenic assay to assess clonogenic potential. The combination of ASA and OP with GEM significantly upended MiaPaCa-2 and PANC-1 pancreatic cancer cell viability, clonogenic potential, expression of critical extracellular matrix proteins, migration, and promoted apoptosis. ASA in combination with OP significantly improves the effectiveness of GEM in the treatment of pancreatic cancer and disables key survival pathways critical to disease progression.

The Next-Generation of Combination Cancer Immunotherapy: Epigenetic Immunomodulators Transmogrify Immune Training to Enhance Immunotherapy.

Cancer immunotherapy harnesses the immune system by targeting tumor cells that express antigens recognized by immune system cells, thus leading to tumor rejection. These tumor-associated antigens include tumor-specific shared antigens, differentiation antigens, protein products of mutated genes and rearrangements unique to tumor cells, overexpressed tissue-specific antigens, and exogenous viral proteins. However, the development of effective therapeutic approaches has proven difficult, mainly because these tumor antigens are shielded, and cells primarily express self-derived antigens. Despite innovative and notable advances in immunotherapy, challenges associated with variable patient response rates and efficacy on select tumors minimize the overall effectiveness of immunotherapy. Variations observed in response rates to immunotherapy are due to multiple factors, including adaptative resistance, competency, and a diversity of individual immune systems, including cancer stem cells in the tumor microenvironment, composition of the gut microbiota, and broad limitations of current immunotherapeutic approaches. New approaches are positioned to improve the immune response and increase the efficacy of immunotherapies, highlighting the challenges that the current global COVID-19 pandemic places on the present state of immunotherapy.

Next-generation multimodality of nutrigenomic cancer therapy: sulforaphane in combination with acetazolamide actively target bronchial carcinoid cancer in disabling the PI3K/Akt/mTOR survival pathway and inducing apoptosis.

OBJECTIVE: Aberrations in the PI3K/AKT/mTOR survival pathway in many cancers are the most common genomic abnormalities. The phytochemical and bioactive agent sulforaphane (SFN) has nutrigenomic potential in activating the expression of several cellular protective genes via the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 is primarily related to mechanisms of endogenous cellular defense and survival. The efficacy of SFN in combination with acetazolamide (AZ) was investigated in reducing typical H727 and atypical H720 BC survival, migration potential, and apoptosis in vitro and in vivo preclinical xenograft tissues. MATERIALS AND METHODS: Microscopic imaging, immunocytochemistry, wound healing assay, caspase-cleaved cytokeratin 18 (M30, CCK18) CytoDeath ELISA assay, immunofluorescence labeling assays for apoptosis, hypoxia, Western Blotting, Tunnel assay, measurement of 5-HT secretion by carbon fiber amperometry assay, quantitative methylation-specific PCR (qMSP), morphologic changes, cell viability, apoptosis activity and the expression levels of phospho-Akt1, Akt1, HIF-1α, PI3K, p21, CAIX, 5-HT, phospho-mTOR, and mTOR in xenografts derived from typical H727 and atypical H720 BC cell lines. RESULTS: Combining AZ+SFN reduced tumor cell survival compared to each agent alone, both in vitro and in vivo xenograft tissues. AZ+SFN targeted multiple pathways involved in cell cycle, serotonin secretion, survival, and growth pathways, highlighting its therapeutic approach. Both H727 and H720 cells were associated with induction of apoptosis, upregulation of the p21 cell cycle inhibitor, and downregulation of the PI3K/Akt/mTOR pathway, suggesting that the PI3K/Akt/mTOR pathway is a primary target of the AZ+SFN combination therapy. CONCLUSIONS: Combining SFN+AZ significantly inhibits the PI3K/Akt/mTOR pathway and significantly reducing 5-HT secretion in carcinoid syndrome.

3D Multicellular Stem-Like Human Breast Tumor Spheroids Enhance Tumorigenicity of Orthotopic Xenografts in Athymic Nude Rat Model.

Therapeutic targeting of stem cells needs to be strategically developed to control tumor growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes present, including cancer stem cells (CSCs). The development of 3D stem-like properties of human breast tumor spheroids in stem cell factor conditioned media was investigated in orthotopic xenografts for enhanced tumorgenicity in the athymic nude rat model. MCF-7, ZR-75-1, and MDA-MB-231 breast cancer cell lines were cultured in serum-free, stem cell factor-supplemented medium under non-adherent conditions and passaged to generate 3rd generation spheroids. The spheroids were co-cultured with fetal lung fibroblast (FLF) cells before orthotopic heterotransplantation into the mammary fat pads of athymic nude rats. Excised xenografts were assessed histologically by H&E staining and immunohistochemistry for breast cancer marker (ERB1), proliferation marker (Ki67), mitotic marker (pHH3), hypoxia marker (HIF-2α), CSC markers (CD47, CD44, CD24, and CD133), and vascularization markers (CD31, CD34). Breast cancer cells cultured in stem cell factor supplemented medium generated 3D spheroids exhibited increased stem-like characteristics. The 3D stem-like spheroids co-cultured with FLF as supporting stroma reproducibly and efficiently established orthotopic breast cancer xenografts in the athymic nude rat.

Hypoxia-Targeting Drug Evofosfamide (TH-302) Enhances Sunitinib Activity in Neuroblastoma Xenograft Models.

Antiangiogenic therapy has shown promising results in preclinical and clinical trials. However, tumor cells acquire resistance to this therapy by gaining ability to survive and proliferate under hypoxia induced by antiangiogenic therapy. Combining antiangiogenic therapy with hypoxia-activated prodrugs can overcome this limitation. Here, we have tested the combination of antiangiogenic drug sunitinib in combination with hypoxia-activated prodrug evofosfamide in neuroblastoma. In vitro, neuroblastoma cell line SK-N-BE(2) was 40-folds sensitive to evofosfamide under hypoxia compared to normoxia. In IV metastatic model, evofosfamide significantly increased mice survival compared to the vehicle (P=.02). In SK-N-BE(2) subcutaneous xenograft model, we tested two different treatment regimens using 30 mg/kg sunitinib and 50 mg/kg evofosfamide. Here, sunitinib therapy when started along with evofosfamide treatment showed higher efficacy compared to single agents in subcutaneous SK-N-BE(2) xenograft model, whereas sunitinib when started 7 days after evofosfamide treatment did not have any advantage compared to treatment with either single agent. Immunofluorescence of tumor sections revealed higher number of apoptotic cells and hypoxic areas compared to either single agent when both treatments were started together. Treatment with 80 mg/kg sunitinib with 50 mg/kg evofosfamide was significantly superior to single agents in both xenograft and metastatic models. This study confirms the preclinical efficacy of sunitinib and evofosfamide in murine models of aggressive neuroblastoma. Sunitinib enhances the efficacy of evofosfamide by increasing hypoxic areas, and evofosfamide targets hypoxic tumor cells. Consequently, each drug enhances the activity of the other.

Manganese-enhanced MRI of minimally gadolinium-enhancing breast tumors.

PURPOSE: To investigate the potential of manganese (Mn)-enhanced MRI for sensitive detection and delineation of tumors that demonstrate little enhancement on Gd-DTPA. MATERIALS AND METHODS: Eighteen nude rats bearing 1 to 2 cm in diameter orthotopic breast tumors (ZR75 and LM2) were imaged on a 3 Tesla (T) clinical scanner. Gd-DTPA was administered intravenously and MnCl2 subcutaneously, both at 0.05 mmol/kg. T1 -weighted imaging and T1 measurements were performed precontrast, 10 min post-Gd-DTPA, and 24 h post-MnCl2 . Tumors were excised and histologically assessed using H&E (composition and necrosis) and CD34 (vascularity). RESULTS: Most tumors (78%) demonstrated little enhancement (< 20% change in R1 ) on Gd-DTPA. MnCl2 administration achieved greater and more uniform enhancement throughout the tumor mass (i.e., not restricted to the tumor periphery), with R1 changing over 20% in 72% of tumors. MnCl2 -induced R1 changes compared with Gd-induced changes were significantly greater in both ZR75 (P < 0.01) and LM2 tumors (P < 0.05). Histology confirmed very low vascularity in both tumor models, and necrotic areas were well delineated only on Mn-enhanced MRI. CONCLUSION: Mn-enhanced MRI is a promising approach for detection of low-Gd-enhancing tumors.

Carbonic anhydrase II mediates malignant behavior of pulmonary neuroendocrine tumors.

In normal lung, the predominant cytoplasmic carbonic anhydrase (CA) isozyme (CAII) is highly expressed in amine- and peptide-producing pulmonary neuroendocrine cells where its role involves CO2 sensing. Here, we report robust cytoplasmic expression of CAII by immunohistochemistry in the tumor cells of different native neuroendocrine tumor (NET) types, including typical and atypical carcinoids and small-cell lung carcinomas, and in NET and non-NET tumor cell lines. Because, in both pulmonary neuroendocrine cell and related NETs, the hypercapnia-induced secretion of bioactive serotonin (5-hydroxytryptamine) is mediated by CAII, we investigated the role of CAII in the biological behavior of carcinoid cell line H727 and the type II cell-derived A549 using both in vitro clonogenicity and in vivo xenograft model. We show that short hairpin RNA-mediated down-regulation of CAII resulted in significant reduction in clonogenicity of H727 and A549 cells in vitro, and marked suppression of tumor growth in vivo. CAII-short hairpin RNA cell-derived xenografts showed significantly reduced mitosis (phosphohistone H3 marker) and proliferation associated antigen Ki-67 (Ki67 marker), and significantly increased apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Using an apoptosis gene array, we found no association with caspases 3 and 8, but with a novel association of CAII-mediated apoptosis with specific mitochondrial apoptosis-associated proteins. Furthermore, these xenografts showed a significantly reduced vascularization (CD31 marker). Thus, CAII may play a critical role in NET lung tumor growth, angiogenesis, and survival, possibly via 5-hydroxytryptamine, known to drive autocrine tumor growth. As such, CAII is a potential therapeutic target for the difficult-to-treat lung NETs.

TGF-ß1 induces EMT reprogramming of porcine bladder urothelial cells into collagen producing fibroblasts-like cells in a Smad2/Smad3-dependent manner.

Activation of fibroblasts and their differentiation into myofibroblasts, excessive collagen production and fibrosis occurs in a number of bladder diseases. Similarly, conversion of epithelial cells into mesenchymal cells (EMT) has been shown to increase fibroblasts like cells. TGF-ß1 can induce the EMT and the role of TGF-ß1-induced EMT during bladder injury leading to fibrosis and possible organ failure is gaining increasing interest. Here we show that EMT and fibrosis in porcine bladder urothelial (UC) cells are Smad dependent. Fresh normal porcine bladder urothelial cells were grown in culture with or without TGF-ß1 and EMT markers were assessed. TGF-ß1 treatment induced changes in cellular morphology as depicted by a significant decrease in the expression of E-cadherin and corresponding increase in N-cadherin and α-SMA. We knocked down Smad2 and Smad3 by Smad specific siRNA. Downregulation of E-cadherin expression by TGF-ß1 was Smad3-dependent, whereas N-cadherin and α-SMA were dependent on both Smad2 and Smad3. Connective tissue growth factor (CTGF/CCN2), matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) has been shown to play important roles in the pathogenesis of fibrosis. Induction of these genes by TGF-ß1 was found to be time dependent. Upregulation of CTGF/CCN2 by TGF-ß1 was Smad3 dependent; whereas MMP-2 was Smad2 dependent. Smad2 and Smad3 both participated in MMP-9 expression. TGF-ß1 reprogrammed mesenchymal fibroblast like cells robustly expressed collagen I and III and these was inhibited by SB-431542, a TGF-ß receptor inhibitor. Our results indicate that EMT of porcine bladder UC cells is TGF-ß1 dependent and is mediated through Smad2 and Smad3. TGF-ß1 may be an important factor in the development of bladder fibrosis via an EMT mechanism. This identifies a potential amenable therapeutic target.

Combination of carbonic anhydrase inhibitor, acetazolamide, and sulforaphane, reduces the viability and growth of bronchial carcinoid cell lines.

BACKGROUND: Bronchial carcinoids are pulmonary neuroendocrine cell-derived tumors comprising typical (TC) and atypical (AC) malignant phenotypes. The 5-year survival rate in metastatic carcinoid, despite multiple current therapies, is 14-25%. Hence, we are testing novel therapies that can affect the proliferation and survival of bronchial carcinoids. METHODS: In vitro studies were used for the dose-response (AlamarBlue) effects of acetazolamide (AZ) and sulforaphane (SFN) on clonogenicity, serotonin-induced growth effect and serotonin content (LC-MS) on H-727 (TC) and H-720 (AC) bronchial carcinoid cell lines and their derived NOD/SCID mice subcutaneous xenografts. Tumor ultra structure was studied by electron microscopy. Invasive fraction of the tumors was determined by matrigel invasion assay. Immunohistochemistry was conducted to study the effect of treatment(s) on proliferation (Ki67, phospho histone-H3) and neuroendocrine phenotype (chromogranin-A, tryptophan hydroxylase). RESULTS: Both compounds significantly reduced cell viability and colony formation in a dose-dependent manner (0-80 µM, 48 hours and 7 days) in H-727 and H-720 cell lines. Treatment of H-727 and H-720 subcutaneous xenografts in NOD/SCID mice with the combination of AZ + SFN for two weeks demonstrated highly significant growth inhibition and reduction of 5-HT content and reduced the invasive capacity of H-727 tumor cells. In terms of the tumor ultra structure, a marked reduction in secretory vesicles correlated with the decrease in 5-HT content. CONCLUSIONS: The combination of AZ and SFN was more effective than either single agent. Since the effective doses are well within clinical range and bioavailability, our results suggest a potential new therapeutic strategy for the treatment of bronchial carcinoids.

Tumor dynamics in response to antiangiogenic therapy with oral metronomic topotecan and pazopanib in neuroblastoma xenografts.

Metronomic chemotherapy, combined with targeted antiangiogenic drugs, has demonstrated significant anticancer efficacy in various studies. Though, tumors do acquire resistance. Here, we have investigated the effect of prolonged therapy with oral metronomic topotecan and pazopanib on tumor behavior in a neuroblastoma mouse xenograft model. SK-N-BE(2) xenograft-bearing mice were treated with either of the following regimens (daily, orally): vehicle (control), 150 mg/kg pazopanib, 1.0 mg/kg topotecan, and combination of topotecan and pazopanib. Planned durations of treatment for each regimen were 28, 56, and 80 days or until the end point, after which animals were sacrificed. We found that only combination-treated animals survived until 80 days. Combination halted tumor growth for up to 50 days, after which gradual growth was observed. Unlike single agents, all three durations of combination significantly lowered microvessel densities compared to the control. However, the tumors treated with the combination for 56 and 80 days had higher pericyte coverage compared to control and those treated for 28 days. The proliferative and mitotic indices of combination-treated tumors were higher after 28 days of treatment and comparable after 56 days and 80 days of treatment compared to control. Immunohistochemistry, Western blot, and real-time polymerase chain reaction revealed that combination treatment increased the hypoxia and angiogenic expression. Immunohistochemistry for Glut-1 and hexokinase II expression revealed a metabolic switch toward elevated glycolysis in the combination-treated tumors. We conclude that prolonged combination therapy with metronomic topotecan and pazopanib demonstrates sustained antiangiogenic activity but also incurs resistance potentially mediated by elevated glycolysis.

Spatio-temporal distribution of Smads and role of Smads/TGF-ß/BMP-4 in the regulation of mouse bladder organogenesis.

Although Shh, TGF-ß and BMP-4 regulate radial patterning of the bladder mesenchyme and smooth muscle differentiation, it is not known what transcription factors, local environmental cues or signaling cascades mediate bladder smooth muscle differentiation. We investigated the expression patterns of signaling mediated by Smad2 and Smad3 in the mouse embryonic bladder from E12.5 to E16.5 by using qRT-PCR, in situ hybridization and antibodies specifically recognizing individual Smad proteins. The role of Smad2 and Smad3 during smooth muscle formation was examined by disrupting the Smad2/3 signaling pathway using TßR1 inhibitor SB-431542 in organ culture system. qRT-PCR results showed that R-Smads, Co-Smad and I-Smads were all expressed during bladder development. RNA ISH for BMP-4 and immunostaining of TGF-ß1 showed that BMP-4 and TGF-ß1 were expressed in the transitional epithelium, lamina propia and muscularis mucosa. Smad1, Smad5 and Smad8 were first expressed in the bladder epithelium and continued to be expressed in the transitional epithelium, muscularis mesenchyme and lamina propia as the bladder developed. Smad2, Smad3 and Smad4 were first detected in the bladder epithelium and subsequently were expressed in the muscularis mesenchyme and lamina propia. Smad6 and Smad7 showed overlapping expression with R-Smads, which are critical for bladder development. In bladder explants (E12.5 to E16.5) culture, Smad2 and Smad3 were found localized within the nuclei, suggesting critical transcriptional regulatory effects during bladder development. E12.5 to E16.5 bladders were cultured with and without TßR1 inhibitor SB-431542 and assessed by qRT-PCR and immunofluorescence. After three days in culture in SB-431542, α-SMA, Smad2 and Smad3 expressions were significantly decreased compared with controls, however, with no significant changes in the expression of smooth muscle myosin heavy chain (SM-Myh. Based on the Smad expression patterns, we suggest that individual or combinations of Smads may be necessary during mouse bladder organogenesis and may be critical mediators for bladder smooth muscle differentiation.

Preclinical models for pediatric solid tumor drug discovery: current trends, challenges and the scopes for improvement.

INTRODUCTION: The enhancement in pediatric cancer survival achieved in the past few decades has been confined to low- and moderate-risk cancers, whereas no notable improvement in survival was observed in high-risk and advanced-stage childhood cancers. High attrition rate of candidate drugs in clinical trials is a major hurdle in the development of effective therapies for pediatric solid tumors. In order to reduce the failure rate of candidate drugs in clinical trials, more effective strategies are needed to enhance the predictability of preclinical testing. AREAS COVERED: The authors have described the current trends in preclinical drug development for treating pediatric solid tumors. Furthermore, the authors review their limitations and the available remedies, with regards to choice of models, pharmacokinetic considerations and the criteria for assessing the long-term efficacy of a candidate drug. EXPERT OPINION: In many solid tumors, common differences between pediatric and adult cancers have been observed, and therefore, clinical trials for pediatric solid tumors must be conducted on the basis of preclinical observations in pediatric solid tumor models. There is a need to invest in extensive preclinical testing on pediatric solid tumor models. None of the preclinical models can fully recapitulate the human cancers. Therefore, these limitations must be considered while conducting a preclinical trial. The dose and schedule of drugs used for preclinical testing must be clinically relevant. While testing the efficacy of drugs, the markers of apoptosis, drug resistance, hypoxia and tumor-initiating cells can inform us about the long-term therapeutic response of a cancer.

Circulating endothelial cells and circulating endothelial precursor cells in patients with osteosarcoma.

BACKGROUND: Circulating endothelial cells (CECs) have been detected at increased numbers in patients with solid cancers. CECs have not been systematically evaluated in patients with osteosarcoma. PROCEDURE: Patients 12 months to 30 years of age with newly diagnosed high-grade osteosarcoma were eligible for this prospective cohort study. Patients provided a single blood sample at study entry for CEC quantification by flow cytometry at a single reference laboratory. CECs were defined as CD146+, CD31+, CD45-, and CD133-. CEC progenitor cells (CEPs) were defined as CD146+, CD31+, CD45-, and CD133+. RESULTS: Eighteen patients enrolled (11 males; median age 16 years; range 5-21 years). CEC counts did not differ between patients with osteosarcoma compared to seven pediatric healthy controls (median 645 cells/ml, range 60-5,320 cells/ml vs. 1,670 cells/ml, range 330-4,700 cells/ml, respectively; P = 0.12). CEP counts did not differ between patients compared to controls (median 126 cells/ml, range 0-5,320 cells/ml vs. median 260 cells/ml, range 0-10,670 cells/ml, respectively; P = 0.69). CEC and CEP counts did not correlate with metastatic status, tumor size, or histologic response to neoadjuvant chemotherapy. CONCLUSIONS: CEC and CEP levels are not increased in patients with osteosarcoma compared to healthy controls. CECs and CEPs do not correlate with clinical features of osteosarcoma. Alternative novel markers of disease burden and response are needed in this disease.

Metronomic oral topotecan with pazopanib is an active antiangiogenic regimen in mouse models of aggressive pediatric solid tumor.

PURPOSE: Low dose metronomic (LDM) chemotherapy, combined with VEGF signaling pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth in many adult preclinical cancer models. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, a VEGF receptor inhibitor, in three pediatric extracranial solid tumor mouse models. EXPERIMENTAL DESIGN: In vitro dose-response study of topotecan and pazopanib was conducted on several neuroblastoma, osteosarcoma, and rhabdomyosarcoma cell lines. In vivo antitumor efficacies of the LDM topotecan and pazopanib as single agents and in combination were tested on 4 subcutaneous xenograft models and on 2 neuroblastoma metastatic models. Circulating angiogenic factors such as circulating endothelial cells (CEC), circulating endothelial pro genitor cells (CEP), and microvessel densities were used as surrogate biomarker markers of antiangiogenic activity. RESULTS: In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, combination of topotecan + pazopanib (TP + PZ) showed significant antitumor activity and significant enhancement in survival compared with the respective single agents in all models. Reductions in viable CEP and/or CEC levels and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. Pharmacokinetic studies of both drugs did not reveal any drug-drug interaction. CONCLUSION: Metronomic administration of TP + PZ showed a statistically significant antitumor activity compared with respective single agents in pediatric tumor mouse models and represent a valid option as a maintenance therapy in aggressive pediatric solid tumors.