Xenograft Model Of Human Ovarian Cancer Research Articles

Antitumor Activity of Dehydroxymethylepoxyquinomicin (DHMEQ) in Monotherapy and Combination with Cisplatin in the SKOV-3 Ovarian Cancer Model

The objective of this study was to evaluate the antitumor activity of DHMEQ as monotherapy and in combination with cisplatin in a human ovarian cancer xenograft model. Cisplatin was used as a comparator. To create the xenograft model, human ovarian cancer cells (SKOV-3 line) were subcutaneously implanted into immunodeficient mice. The study was conducted on female SCID Beige C.B-17 Cg-Prkdcscid Lystbg/Crl mice. Antitumor activity was determined by comparing tumor growth inhibition (TGI) in the treatment groups to that in the control group. Results showed that daily intraperitoneal administration of DHMEQ at a dose of 14 mg/kg following a single intraperitoneal dose of cisplatin at 4 mg/kg reduced tumor growth in the SKOV-3 cell line xenograft model.

Abstract 6441: Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer

Abstract Background. HSF1 helps cancer cells cope with multiple stresses caused by oncogene activation. Methods. See details below. Results. We discovered the bisamide NXP800 as an inhibitor of HSF1-mediated transcription through phenotypic screening and med chem optimization. Gene expression microarray analysis of human cancer cell lines and tumor xenografts treated with bisamide inhibitors indicated activation of the ATF4 axis of the ISR as a key mechanism of action. In SK-OV-3 ovarian cancer xenografts, NXP800 showed clear PK/PD relationships with increased expression of ATF4 transcriptional target genes alongside decreased expression of HSF1 transcriptional targets. NXP800 also caused tumor regressions in this model. Expanding in additional human ovarian cancer xenograft models, we observed efficacy in five and a complete lack of response in three others. The sensitive models all had homozygous deleterious mutations in the ARID1A gene, whereas the non-responding xenografts were all wild type (WT). ARID1A is a component of the SWI/SNF chromatin remodelling complex involved in repression and activation of target genes. Subsequent screening of the large Sanger human cancer cell line panel confirmed ARID1A as the most significant common disease-related alteration predicting sensitivity to NXP800 in ovarian cancer cell lines. This predictive relationship was confirmed in an ARID1A isogenic HCT-116 cell line pair; sensitivity was greater in the homozygous ARID1A mutant cells compared to WT, resulting in PARP cleavage in the mutant cells only. In vivo there was no effect in WT HCT-116 xenografts whereas growth inhibition was observed in the homozygous mutant cells, resulting in significantly smaller tumors. In addition, the induction of ATF4 target genes was stronger and more prolonged in the mutant cells. It is known that whereas short term ATF4 activation is adaptive, persistent activation can promote the induction of apoptosis. CHIP-seq analysis confirmed clear relationships between ARID1A status, ATF4 and HSF1 promoter occupancy, and the distribution of BRG1 and RNA pol II at target sites, although these were often gene-specific and complex. A relatively simple example is the regulation of the INHBE gene (expression of which is a PD biomarker). In untreated samples there is no binding of ATF4, BRG1 and RNA pol II at the INHBE promoter. NXP800 treatment results in the recruitment of BRG1, ATF4 and RNA pol II in ARID1A mutant TOV-21G cells but not in the RMGI WT cells, leading to increased expression in the mutant cells only. Conclusions. We propose that ARID1A loss alters the binding and recruitment of ATF4 and HSF1 leading to the altered and prolonged expression of ATF4 target genes and increased sensitivity to NXP800. NXP800 is currently in phase Ib for the treatment of ARID1A mutant platinum resistant ovarian cancer (NCT05226507). Citation Format: Robert H. te Poele, Marissa Powers, Swee Sharp, Emmanuel de Billy, Maria Taskinen, Loredana Pellegrino, Sharon Gowan, Asadh Miah, Angela Hayes, Matthew Cheeseman, Keith Jones, Suzanne Eccles, Florence Raynaud, Paul Clarke, Paul Workman. Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6441.

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting

Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumors. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies. Here, we describe CAR T cells targeting tumor-associated glycoprotein-72 (TAG72), utilizing the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. CAR T cell-mediated IFNγ production facilitated by IL-12 signaling is required for tumor cell killing, which is recapitulated by engineering an optimized membrane-bound IL-12 (mbIL12) molecule in CAR T cells. These T cells show improved antigen-dependent T cell proliferation and recursive tumor cell killing in vitro, with robust in vivo efficacy in human ovarian cancer xenograft models. Locoregional administration of mbIL12-engineered CAR T cells promotes durable anti-tumor responses against both regional and systemic disease in mice. Safety and efficacy of mbIL12-engineered CAR T cells is demonstrated using an immunocompetent mouse model, with beneficial effects on the immunosuppressive tumor microenvironment. Collectively, our study features a clinically-applicable strategy to improve the efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting regional and systemic disease.

A combined network pharmacology and molecular biology approach to investigate the potential mechanisms of G-M6 on ovarian cancer

Ginsenoside 3β,12β,21α,22β-Hydroxy-24-norolean-12-ene (G-M6), a phase I metabolite of anti-tumor medication 20(R)-25-methoxyl-dammarane-3β,12β,20-triol (AD-1), beats the parent drug in anti-ovarian cancer efficacy. The mechanism of action for ovarian cancer, however, is uncertain. Using network pharmacology, human ovarian cancer cells and nude mouse ovarian cancer xenotransplantation model, the anti-ovarian cancer mechanism of G-M6 was preliminarily explored in this study. The PPAR signal pathway is the key signal pathway of the G-M6 anti-ovarian cancer mechanism, according to data mining and network analysis. Docking tests demonstrated that the bioactive chemical G-M6 was capable of forming a stable bond with the PPARγ target protein capsule. Using human ovarian cancer cells and xenograft model of ovarian cancer to evaluate the anticancer activity of G-M6. The IC50 value of G-M6 was 5.83±0.36, lower than AD-1 and Gemcitabine. The tumor weight of the RSG 80 mg/kg group (C), G-M6 80 mg/kg group (I), and RSG 80 mg/kg + G-M6 80 mg/kg group (J) after the intervention was as follows: C < I < J. The tumor inhibition rates of groups C, I, and J were 28.6%, 88.7%, and 92.6%, respectively. When RSG and G-M6 are combined to treat ovarian cancer, q = 1.00 is calculated according to King's formula, which indicates that RSG and G-M6 have additive effects. Its molecular mechanism may involve the up-regulation of PPARγ and Bcl-2 protein expressions, and the down-regulation of Bax, Cytochrome C (Cyt. C), Caspase-3, and Caspase-9 protein expressions. These findings serve as a reference for further research into the processes behind ginsenoside G-M6′s ovarian cancer therapy.

Engineering Bifunctional Calcium Alendronate Gene‐Delivery Nanoneedle for Synergistic Chemo/Immuno‐Therapy Against HER2 Positive Ovarian Cancer

Ovarian cancer is the most lethal gynecological malignancy. Most patients are diagnosed at an advanced stage with widespread peritoneal dissemination and ascites. Bispecific T-cell engagers (BiTEs) have demonstrated impressive antitumor efficacy in hematological malignancies, but the clinical potency is limited by their short half-life, inconvenient continuous intravenous infusion, and severe toxicity at relevant therapeutic levels in solid tumors. To address these critical issues, the design and engineering of alendronate calcium (CaALN) based gene-delivery system is reported to express therapeutic level of BiTE (HER2×CD3) for efficient ovarian cancer immunotherapy. Controllable construction of CaALN nanosphere and nanoneedle is achieved by the simple and green coordination reactions that the distinct nanoneedle-like alendronate calcium (CaALN-N) with a high aspect ratio enabled efficient gene delivery to the peritoneum without system in vivo toxicity. Especially, CaALN-N induced apoptosis of SKOV3-luc cell via down-regulation of HER2 signaling pathway and synergized with HER2×CD3 to generate high antitumor response. In vivo administration of CaALN-N/minicircle DNA encoding HER2×CD3 (MC-HER2×CD3) produces sustained therapeutic levels of BiTE and suppresses tumor growth in a human ovarian cancer xenograft model. Collectively, the engineered alendronate calcium nanoneedle represents a bifunctional gene delivery platform for the efficient and synergistic treatment of ovarian cancer.

Exploiting the Receptor-Binding Domains of R-Spondin 1 to Target Leucine-Rich Repeat-Containin G-Coupled Protein Receptor 5-Expressing Stem Cells in Ovarian Cancer.

Leucine-rich repeat-containing G-protein-coupled receptor (LGR5) and LGR6 mark epithelial stem cells in normal tissues and tumors. They are expressed by stem cells in the ovarian surface and fallopian tube epithelia from which ovarian cancer arises. High-grade serous ovarian cancer is unique in expressing unusually high levels of LGR5 and LGR6 mRNA. R-spondins are the natural ligands for LGR5 and LGR6 to which they bind with nanomolar affinity. To target stem cells in ovarian cancer, we used the sortase reaction to site-specifically conjugate the potent cytotoxin monomethyl auristatin E (MMAE) via a protease sensitive linker to the two furin-like domains of RSPO1 (Fu1-Fu2) that mediate its binding to LGR5 and LGR6 and their co-receptors Zinc And Ring Finger 3 and Ring Finger Protein 43 via a protease-cleavable linker. An immunoglobulin Fc domain added to the N-terminal end served to dimerize the receptor-binding domains so that each molecule carries two MMAE. The resulting molecule, FcF2-MMAE, demonstrated: 1) selective LGR5-dependent low nanomolar cytotoxicity against ovarian cancer cells in vitro; 2) selectivity that was dependent on binding to both the LGR receptors and ubiquitin ligase co-receptors; 3) favorable stability and plasma pharmacokinetic properties when administered intravenously with an elimination half-life of 29.7 hours; 4) selective inhibition of LGR5-rich as opposed to isogenic LGR5-poor tumors in vivo; and, 5) therapeutic efficacy in three aggressive wild-type human ovarian cancer xenograft models. These results demonstrate the successful use of the Fu1-Fu2 domain of RSPO1 as a drug carrier and the ability of FcF2-MMAE to target cells in tumors that express stem cell markers. SIGNIFICANCE STATEMENT: FcF2-MMAE is a novel cancer therapeutic that exploits the high-affinity binding domains of RSPO1 to target monomethyl auristatin E to tumor stem cells that express LGR5. FcF2-MMAE has low nanomolar LGR5-dependent cytotoxicity in vitro, favorable pharmacokinetics, and differential efficacy in an isogenic LGR5-poor versus LGR5-rich ovarian cancer xenograft model when given on a weekly schedule.

Fluorochrome Selection for Imaging Intraoperative Ovarian Cancer Probes.

The identification and removal of all gross and microscopic tumor to render the patient disease free represents a huge challenge in ovarian cancer treatment. The presence of residual disease is an independent negative prognostic factor. Herein, we describe the synthesis and the “in vitro” evaluation of compounds as cyclooxygenase (COX)-1 inhibitors, the COX-1 isoform being an ovarian cancer biomarker, each bearing fluorochromes with different fluorescence features. Two of these compounds N-[4-(9-dimethylimino-9H-benzo[a]phenoxazin-5-ylamino) butyl]-2-(3,4-bis(4-methoxyphenyl)isoxazol-5-yl)acetamide chloride (RR11) and 3-(6-(4-(2-(3,4-bis(4-methoxyphenyl)isoxazole-5-yl)acetamido)butyl)amino-6-oxohexyl)-2-[7-(1,3-dihydro-1,1-dimethyl-3-ethyl 2H-benz[e]indolin-2-yl-idene)-1,3,5-heptatrienyl]-1,1-dimethyl-3-(6-carboxilato-hexyl)-1H-benz[e]indolium chloride, 23 (MSA14) were found to be potent and selective inhibitors of cyclooxygenase (COX)-1 “in vitro”, and thus were further investigated “in vivo”. The IC50 values were 0.032 and 0.087 µM for RR11 and 23 (MSA 14), respectively, whereas the COX-2 IC50 for RR11 is 2.4 µM while 23 (MSA14) did not inhibit COX-2 even at a 50 µM concentration. Together, this represented selectivity index = 75 and 874, respectively. Structure-based virtual screening (SBVS) performed with the Fingerprints for Ligands and Proteins (FLAP) software allowed both to differentiate highly active compounds from less active and inactive structures and to define their interactions inside the substrate-binding cavity of hCOX1. Fluorescent probes RR11 and 23 (MSA14), were used for preliminary near-infrared (NIR) fluorescent imaging (FLI) in human ovarian cancer (OVCAR-3 and SKOV-3) xenograft models. Surprisingly, a tumor-specific signal was observed for both tested fluorescent probes, even though this signal is not linked to the presence of COX-1.

Chimeric antigen receptor T cells engineered to secrete CD40 agonist antibodies enhance antitumor efficacy

BackgroundAlthough chimeric antigen receptor (CAR)-T cell therapy has been remarkably successful for haematological malignancies, its efficacy against solid tumors is limited. The combination of CAR-T cell therapy with immune checkpoint inhibitors (CPIs), such as PD-1, PD-L1, and CTLA-4 antibodies, is a promising strategy for enhancing the antitumor efficacy of CAR-T cells. However, because most patients acquire resistance to CPIs, investigating other strategies is necessary to further improve the antitumor efficacy of CAR-T cell therapy for solid tumors. Recently, CD40 agonist antibodies showed potential antitumor efficacy by activating the CD40 pathway.ResultsBased on the piggyBac transposon system, rather than the widely used viral vectors, we constructed a meso3-CD40 CAR-T targeting region III of mesothelin (MSLN) that possessed the ability to secrete anti-CD40 antibodies. Compared with meso3 CAR-T cells, which did not secrete the anti-CD40 antibody, meso3-CD40 CAR-T cells secreted more cytokines and had a relatively higher proportion of central memory T (TCM) cells after stimulation by the target antigen. In addition, compared with meso3 CAR-T cells, meso3-CD40 CAR-T cells had a more powerful cytotoxic effect on target cells at a relatively low effector-to-target ratio. More importantly, we demonstrated that the antitumor activity of meso3-CD40 CAR-T cells was enhanced in a human ovarian cancer xenograft model in vivo.ConclusionsIn conclusion, these results highlight anti-CD40-secreting CAR-T cells generated by nonviral vectors as a potential clinical strategy for improving the efficacy of CAR-T cell therapies.

Abstract 122: Targeting forward and reverse EphB4/EFNB2 signaling by a peptide with dual functions

Although chloroquine administration in vivo following haemorrhage in mice decreases tumour necrosis factor-alpha (TNF-alpha) release by macrophage (M phi), the mechanism remains unknown. To study this, peritoneal M phi (pM phi) from unmanipulated, sham-operated and post-haemorrhage mice were isolated, treated with 0.13 mg/ml chloroquine for 2 hr, and then stimulated with lipopolysaccharide (LPS) for 48 hr. Pretreatment of pM phi from various groups of mice with chloroquine resulted in 75-90% inhibition of TNF-alpha release, determined by bioassay. Total RNA was isolated from pM phi and murine M phi-derived cell lines (P388D1 and RAW 264.7), stimulated with LPS for 0.5 or 1 hr, respectively, and Northern blot analysis for TNF-alpha mRNA performed. Chloroquine inhibited TNF-alpha mRNA expression without interfering with mRNA stability, suggesting that this agent reduces M phi TNF-alpha release by disrupting TNF-alpha gene transcription.

Verticillin A Causes Apoptosis and Reduces Tumor Burden in High-Grade Serous Ovarian Cancer by Inducing DNA Damage

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy in women worldwide and the fifth most common cause of cancer-related deaths among U.S. women. New therapies are needed to treat HGSOC, particularly because most patients develop resistance to current first-line therapies. Many natural product and fungal metabolites exhibit anticancer activity and represent an untapped reservoir of potential new agents with unique mechanism(s) of action. Verticillin A, an epipolythiodioxopiperazine alkaloid, is one such compound, and our recent advances in fermentation and isolation are now enabling evaluation of its anticancer activity. Verticillin A demonstrated cytotoxicity in HGSOC cell lines in a dose-dependent manner with a low nmol/L IC50 Furthermore, treatment with verticillin A induced DNA damage and caused apoptosis in HGSOC cell lines OVCAR4 and OVCAR8. RNA-Seq analysis of verticillin A-treated OVCAR8 cells revealed an enrichment of transcripts in the apoptosis signaling and the oxidative stress response pathways. Mass spectrometry histone profiling confirmed reports that verticillin A caused epigenetic modifications with global changes in histone methylation and acetylation marks. To facilitate in vivo delivery of verticillin A and to monitor its ability to reduce HGSOC tumor burden, verticillin A was encapsulated into an expansile nanoparticle (verticillin A-eNP) delivery system. In an in vivo human ovarian cancer xenograft model, verticillin A-eNPs decreased tumor growth and exhibited reduced liver toxicity compared with verticillin A administered alone. This study confirmed that verticillin A has therapeutic potential for treatment of HGSOC and that encapsulation into expansile nanoparticles reduced liver toxicity.

Synergistic Effect of a Mesothelin-Targeted 227Th Conjugate in Combination with DNA Damage Response Inhibitors in Ovarian Cancer Xenograft Models.

Targeted 227Th conjugates (TTCs) represent a new class of therapeutic radiopharmaceuticals for targeted α-therapy. They comprise the α-emitter 227Th complexed to a 3,2-hydroxypyridinone chelator conjugated to a tumor-targeting monoclonal antibody. The high energy and short range of the α-particles induce antitumor activity, driven by the induction of complex DNA double-strand breaks. We hypothesized that blocking the DNA damage response (DDR) pathway should further sensitize cancer cells by inhibiting DNA repair, thereby increasing the response to TTCs. Methods: This article reports the evaluation of the mesothelin (MSLN)-TTC conjugate (BAY 2287411) in combination with several DDR inhibitors, each of them blocking different DDR pathway enzymes. MSLN is a validated cancer target known to be overexpressed in mesothelioma, ovarian, lung, breast, and pancreatic cancer, with low expression in normal tissue. In vitro cytotoxicity experiments were performed on cancer cell lines by combining the MSLN-TTC with inhibitors of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related (ATR), DNA-dependent protein kinase, and poly[adenosine diphosphate ribose] polymerase (PARP) 1/2. Further, we evaluated the antitumor efficacy of the MSLN-TTC in combination with DDR inhibitors in human ovarian cancer xenograft models. Results: Synergistic activity was observed in vitro for all tested inhibitors (inhibitors are denoted herein by the suffix “i”) when combined with MSLN-TTC. ATRi and PARPi appeared to induce the strongest increase in potency. Further, in vivo antitumor efficacy of the MSLN-TTC in combination with ATRi or PARPi was investigated in the OVCAR-3 and OVCAR-8 xenograft models in nude mice, demonstrating synergistic antitumor activity for the ATRi combination at doses demonstrated to be nonefficacious when administered as monotherapy. Conclusion: The presented data support the mechanism-based rationale for combining the MSLN-TTC with DDR inhibitors as new treatment strategies in MSLN-positive ovarian cancer.

Targeted Drug Delivery and Image-Guided Therapy of Heterogeneous Ovarian Cancer Using HER2-Targeted Theranostic Nanoparticles.

Cancer heterogeneity and drug resistance limit the efficacy of cancer therapy. To address this issue, we have developed an integrated treatment protocol for effective treatment of heterogeneous ovarian cancer.Methods: An amphiphilic polymer coated magnetic iron oxide nanoparticle was conjugated with near infrared dye labeled HER2 affibody and chemotherapy drug cisplatin. The effects of the theranostic nanoparticle on targeted drug delivery, therapeutic efficacy, non-invasive magnetic resonance image (MRI)-guided therapy, and optical imaging detection of therapy resistant tumors were examined in an orthotopic human ovarian cancer xenograft model with highly heterogeneous levels of HER2 expression.Results: We found that systemic delivery of HER2-targeted magnetic iron oxide nanoparticles carrying cisplatin significantly inhibited the growth of primary tumor and peritoneal and lung metastases in the ovarian cancer xenograft model in nude mice. Differential delivery of theranostic nanoparticles into individual tumors with heterogeneous levels of HER2 expression and various responses to therapy were detectable by MRI. We further found a stronger therapeutic response in metastatic tumors compared to primary tumors, likely due to a higher level of HER2 expression and a larger number of proliferating cells in metastatic tumor cells. Relatively long-time retention of iron oxide nanoparticles in tumor tissues allowed interrogating the relationship between nanoparticle drug delivery and the presence of resistant residual tumors by in vivo molecular imaging and histological analysis of the tumor tissues. Following therapy, most of the remaining tumors were small, primary tumors that had low levels of HER2 expression and nanoparticle drug accumulation, thereby explaining their lack of therapeutic response. However, a few residual tumors had HER2-expressing tumor cells and detectable nanoparticle drug delivery but failed to respond, suggesting additional intrinsic resistant mechanisms. Nanoparticle retention in the small residual tumors, nevertheless, produced optical signals for detection by spectroscopic imaging.Conclusion: The inability to completely excise peritoneal metastatic tumors by debulking surgery as well as resistance to chemotherapy are the major clinical challenges for ovarian cancer treatment. This targeted cancer therapy has the potential for the development of effective treatment for metastatic ovarian cancer.

Quercetin suppresses DNA double-strand break repair and enhances the radiosensitivity of human ovarian cancer cells via p53-dependent endoplasmic reticulum stress pathway.

Quercetin is proven to have anticancer effects for many cancers. However, the role of tumor suppressor p53 on quercetin’s radiosensitization and regulation of endoplasmic reticulum (ER) stress response in this process remains obscure. Here, quercetin exposure resulted in ER stress, prolonged DNA repair, and the expression of p53 protein; phosphorylation on serine 15 and 20 increased in combination with X-irradiation. Quercetin pretreatment could potentiate radiation-induced cell death. The combination of irradiation and quercetin treatment aggravated DNA damages and caused typical apoptotic cell death; as well the expression of Bax and p21 elevated and the expression of Bcl-2 decreased. Knocking down of p53 could reverse all the above effects under quercetin in combination with radiation. In addition, quercetin-induced radiosensitization was through stimulation of ATM phosphorylation. In human ovarian cancer xenograft model, combined treatment of quercetin and radiation significantly restrained the growth of tumors, accompanied with the activation of p53, CCAAT/enhancer-binding protein homologous protein, and γ-H2AX. Overall, these results indicated that quercetin acted as a promising radiosensitizer through p53-dependent ER stress signals.

Combined Gene Therapy Using AdsVEGFR2 and AdsTie2 With Chemotherapy Reduces the Growth of Human Ovarian Cancer and Formation of Ascites in Mice.

Ovarian cancer is highly dependent on tumor microvessels and angiogenesis regulated by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) and angiopoietins (Ang) and their Tie receptors. We studied the efficacy of adenoviral (Ad) gene therapy with soluble VEGFR2 and Tie2 combined with paclitaxel and carboplatin for the treatment of ovarian cancer. An intraperitoneal human ovarian cancer xenograft model in nude mice (n = 44) was used in this study. Gene therapy was given intravenously when the presence of sizable tumors was confirmed in magnetic resonance imaging. The study groups were as follows: AdCMV as a control (group I), AdCMV with chemotherapy (group II), AdsVEGFR2 and AdsTie2 (group III), and AdsVEGFR2 and AdsTie2 with chemotherapy (group IV). Antitumor effectiveness was assessed by overall tumor growth, ascites, immunohistochemistry, microvessel density, and sequential magnetic resonance imaging analyses. AdsVEGFR2 and AdsTie2 gene therapy (group III) significantly reduced tumor weights as compared with group II (P = 0.007). Accumulation of ascites was significantly reduced when the mice were treated with AdsVEGFR2 and AdsTie2 gene therapy or with combined gene therapy and chemotherapy as compared with controls (P = 0.029 and P = 0.010, respectively). Vascular endothelial growth factor and Ang2 levels in ascites fluid were elevated after the gene therapy. Combined inhibition of VEGF/VEGFR2 and Ang/Tie2 pathways provided efficient therapy for ovarian cancer in mice. In addition, antiangiogenic gene therapy has potential as a treatment for the accumulation of ascites.

Loss of the novel mitochondrial protein FAM210B promotes metastasis via PDK4-dependent metabolic reprogramming

Recent advances in tumor metabolism have revealed that metabolic reprogramming could dramatically promote caner metastasis. However, the relation and mechanism between metastasis and metabolic reprogramming are not thoroughly explored. Cell proliferation, colony formation, and invasion analysis were performed to evaluate the role of FAM210B in human cancer cells. Human ovarian cancer xenograft model was used to determine the effects of inhibiting FAM210B by shRNA on tumor metastasis. Microarray analysis was used to determine the target genes of FAM210B. FAM210B cellular localization was performed by mitochondria isolation and mitochondria protein extraction. To detect FAM210B-mediated metabolic reprogramming, oxygen consumption rate and extracellular acidification rate were measured. Our previous study screened a novel cancer progression-suppressor gene, FAM210B, which encodes an outer mitochondrial membrane protein, by the suppression of mortality by antisense rescue technique (SMART). Here we demonstrated that FAM210B loss was significantly associated with cancer metastasis and decreased survival in a clinical setting. Additionally, it was found that low expression of FAM210B was significantly correlated with decreased survival and enhanced metastasis in vivo and in vitro, and the loss of FAM210B led to an increased mitochondrial respiratory capacity and reduced glycolysis through the downregulation of pyruvate dehydrogenase kinase 4 (PDK4), which activated the EMT program and enhanced migratory and invasive properties. Collectively, our data unveil a potential metabolic target and mechanism of cancer metastasis.

Therapeutic potential of an anti-HER2 single chain antibody\u2013DM1 conjugates for the treatment of HER2-positive cancer

Antibody–drug conjugates (ADCs) take the advantage of monoclonal antibodies to selectively deliver highly potent cytotoxic drugs to tumor cells, which have become a powerful measure for cancer treatment in recent years. To develop a more effective therapy for human epidermal growth factor receptor 2 (HER2)-positive cancer, we explored a novel ADCs composed of anti-HER2 scFv–HSA fusion antibodies conjugates with a potent cytotoxic drug DM1. The resulting ADCs, T-SA1–DM1 and T-SA2–DM1 (drug-to-antibody ratio in the range of 3.2–3.5) displayed efficient inhibition in the growth of HER2-positive tumor cell lines and the half-maximal inhibitory concentration on SKBR-3 and SKOV3 cells were both at the nanomolar levels in vitro. In HER2-positive human ovarian cancer xenograft models, T-SA1–DM1 and T-SA2–DM1 also showed remarkable antitumor activity. Importantly, three out of six mice exhibited complete remission without regrowth in the high-dose group of T-SA1–DM1. On the basis of the analysis of luminescence imaging, anti-HER2 scFv–HSA fusion antibodies, especially T-SA1, showed strong and rapid tumor tissue penetrability and distribution compared with trastuzumab. Collectively, the novel type of ADCs is effective and selective targeting to HER2-positive cancer, and may be a promising antitumor drug candidate for further studies.

Melittin-MIL-2 fusion protein as a candidate for cancer immunotherapy.

BackgroundCytokine fusion protein that modulates the immune response holds great potential for cancer immunotherapy. IL-2 is an effective treatment against advanced cancers. However, the therapeutic efficacy of IL-2 is limited by severe systemic toxicity. Several mutants recombinant IL-2 can increase antitumor activity and minimize systemic toxicity. Melittin is an attractive anticancer candidate because of its wide-spectrum lytic properties. We previously generated a bifunctional fusion protein melittin-MIL-2, composed of melittin and a mutant IL-2. The melittin-MIL-2 inhibited the growth of human ovarian cancer SKOV3 cells in vitro and in vivo tumor growth. However, whether this antitumor effect could also be used in cancer immunotherapy was unknown. To assess its cancer immunotherapy potential, we further investigated its more effective antitumor immune response and antitumor effect against cancers of different tissue origins in vitro and in vivo.MethodsThe specific IL-2 activity of the melittin-MIL-2 fusion protein was tested on the cytokine growth dependent cell line CTLL-2. The cytolytic activity was detected by standard 4-h 51Cr-release assays. PBMC stimulation in response to the melittin-MIL-2 was determined by IFN-γ release assay. We observed the cancer cell proliferation of different tissue origins by MTT assay. The ability of melittin-MIL-2 to inhibit tumor growth in vivo was evaluated by using human liver (SMMC-7721 cancer cells), lung (A549 cancer cells) and ovarian (SKOV3 cancer cells) cancer xenograft models. To assess the immunity within the tumor microenvironment, the level of some cytokines including IFN-γ, TNF-α, IL-12 and IL-4 was analyzed by ELISA. We injected the MDA-MB-231 cells and the melittin-MIL-2 into mice, and the anti-metastatic effect was examined by counting nodules in the lung.ResultsThe melittin-MIL-2 was more effective in inducing T cell and NK-cell cytotoxicity. The fusion protein significantly increased IFN-γ production in PBMCs. In vitro, the melittin-MIL-2 mediated immune cells killing or directly killed the cancer cell lines of different tissue origins. In vivo, the fusion protein exhibited stronger inhibition on the growth of transplanted human tumors compared to rIL-2. The melittin-MIL-2 treatment promoted the IFN-γ secretion in tumor tissues and decreased the immunosuppressive cells in vivo. Furthermore, the fusion protein reduced lung metastasis of breast cancer.ConclusionsThis study provides the evidence that the melittin-MIL-2 can produce stronger immune stimulation and antitumor effects, and the fusion protein is a potent candidate for cancer immunotherapy.

Abstract POSTER-THER-1436: Image-guided and targeted therapy of advanced ovarian cancer using theranostic nanoparticles

Abstract About 80% of ovarian cancer patients are diagnosed at the advanced stage due to lack of a specific symptom for early detection. The major clinical challenges in the treatment of advanced ovarian cancer are inability of complete removal of peritoneal tumor metastases by debulking surgery, and drug resistant recurrent tumors developed in the majority of the patients. To address those clinical challenges, we have developed HER2/neu targeted magnetic iron oxide nanoparticles (IONPs) that efficiently deliver therapeutic and imaging agents into HER2 expressing ovarian cancer cells. Amphiphilic polymer coated 10 nm core size IONPs, without or with PEG modification, were conjugated with near infrared dye (NIR-830) labeled HER2 affibody or HER2 affitoxin, which is a fusing protein containing a HER2 affibody for targeting and a modified PE38 toxin as a therapeutic agent. A chemotherapy drug cisplatin, commonly used for ovarian cancer therapy, was further conjugated to carboxyl groups on the polymer coating of IONP via a coordinate bond. First, we demonstrated targeted drug delivery, and specificity and sensitivity of non-invasive tumor imaging by optical and MR imaging following intravenous (i.v.) or intraperitoneal (i.p.) delivery of the targeted theranostic IONPs in an orthotopic SKOV3 human ovarian cancer xenograft model with a high level of HER2/neu expression. Significant inhibition of the growth of primary ovarian tumor, and peritoneal and lung metastases was found after i.v. or i.p. delivery of cisplatin using 0.5 to 5 mg/Kg of cisplatin equivalent doses of HER2 affibody- or HER2 affitoxin-IONP in the tumor bearing mice once every 5 days for five injections. Furthermore, i.p. delivery of HER2 targeted theranostic IONP-cisplatin showed significantly better responses compared with i.v. delivery of the same nanoparticles. HER2 affitoxin-IONP-cisplatin treated mouse group showed enhanced anti-tumor effect compared to HER affibody-IONP-cisplatin treated mouse group. For both delivery approaches, we detected stronger tumor growth inhibitory effect on peritoneal metastatic tumors compared to the response in the primary ovarian tumors. Interestingly, we observed marked differences in the response to HER2 targeted therapy in different tumor bearing mice in the same group, and in different tumors in the same mouse. We also found that non-invasive optical imaging and MRI can be used to monitor IONP-drug delivery into tumors and to detect good responder or poor responder tumors during the treatment. Therefore, the HER2 targeted and image-guided treatment approach using theranostic IONPs has the potential for further development of a new biomarker targeted therapy for significantly improving survival of the ovarian cancer patients Citation Format: Lily Yang, Minati Satpathy, Liya Wang, Rafal Zielinski, Weiping Qian, Malgorzata Lipowska, Jacek Capala, Andrew Wang, Hui Mao. Image-guided and targeted therapy of advanced ovarian cancer using theranostic nanoparticles [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1436.

46 Broad-spectrum preclinical combination activity of eribulin combined with various anticancer agents in human breast cancer, lung cancer, ovarian cancer, and melanoma xenograft models

and Q-PCR. The status of TP53 in ALL cells was determined by Sanger sequencing. Results: Nine ALL cell lines exhibited different susceptibilities to Aurora kinase inhibitors (AKIs). Cells sensitive to AKIs underwent apoptosis at an IC50 of approximately 10 to 30 nM and displayed a phenotype of Aurora-A inhibition, whereas cells resistant to Aurora kinase inhibitors (with an IC50 more than 10 mM) accumulated polyploidy, which may have resulted from Aurora-B inhibition. Drug susceptibility was not correlated with the expression level or activation status of Aurora kinases. RS4;11 and MV4;11 cells, which contain the MLL-AF4 gene, were both sensitive to Aurora-A inhibitors. CDKN1A might govern the drug responsiveness of ALL cell lines in a TP53-independent manner. Primary ALL cells with MLL-AF4 and CDKN1A expression were sensitive to AKIs. Conclusions: Our study suggests that Aurora-A kinase inhibitors may have clinical utility in MLL-AF4-positive ALL. CDKN1A can be used as a biomarker to determine drug responsiveness in MLL-AF4-positive ALL.