HT-29 Xenografts Research Articles

Abstract 2182: Preclincial in vitro and in vivo combination therapies for mutant IDH1R132 tumors with BAY 1436032

Abstract Background: Isocitrate dehydrogenase (IDH) mutations are drivers of different tumors. The IDH1R132 mutation is frequently found in glioma (80%), chondrosarcoma (CS, 45%), intrahepatic cholangiocarcinoma (ICC, 20%), acute myeloid leukemia (AML, 7%), and with low incidences in other solid tumors. Although clinical therapies of mutant IDH inhibition were recently approved for the treatment of AML, few patients benefit from a single therapy with a mutant IDH inhibitor and most of them relapse. Here we report preclinical in vitro and in vivo data on combination therapies of the mIDH1R132 inhibitor BAY 1436032 with standard of care chemotherapies. Methods: in vitro: The sarcoma cell line HT1080 (IDH1R132C), together with two IDH1 wildtype sarcoma cell lines, the SW1353 (IDH2R172S) and SW872 (IDHwt) cell lines, were treated with BAY 1436032 alone and in combination with different standard of care drugs. in vivo: The patient derived xenograft (PDX) ICC model LIXFC2084 (IDH1R132L), the melanoma PDX model MEXF1341 (IDH1R132C) (MEXF1341), the PDX AML model 13PB020 (IDH1R132C) and a sarcoma cell line derived xenograft model HT1080 (IDH1R132C) were included. All models were treated with BAY 1436032 alone and in combination with different standard of care (SOC) drugs. Results: The in vitro treatment with BAY 1436032 unexpectedly increased growth in HT1080 (IDH1R132C) cells with no effect to the control cell lines SW1353 and SW872. In contrast, in vivo, combination of BAY1436032 with everolimus had additive anti-tumor efficacy and was synergistic with cisplatin or the MEK inhibitor refametinib in the treatment of HT1080 xenografts. The combination of BAY 1436032 and refametinib was also additive in the treatment of a melanoma xenograft with IDH1R132C mutation. Another additive efficacy of BAY 1436032 and gemcitabine or cisplatin could be observed in an ICC xenograft model and further in the combination with everolimus in the same model. The combination of BAY 1436032 together with azacitidine in a PDX AML model was also synergistic. Conclusion: These results highlight the combinatorial potential of the mIDH1R132 inhibitor BAY 1436032 with standard of care therapy and the importance of microenvironmental factors, which has to be taken into account when dealing with mutant IDH tumors. Citation Format: Stefan Kaulfuss, Julia Zaman, Anuhar Chaturvedi, Holger Hess-Stumpp, Jennifer Höde, Ricarda Biber, Renan Borowicz, Michael Heuser, Andreas von Deimling, Stefan Pusch. Preclincial in vitro and in vivo combination therapies for mutant IDH1R132 tumors with BAY 1436032 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2182.

2,7-naphthyridinone-based MET kinase inhibitors: A promising novel scaffold for antitumor drug development

As part of our effort to develop new molecular targeted antitumor drug, a novel 2,7-naphthyridone-based MET kinase inhibitor, 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)- 3-fluorophenyl)amino)-2-(4-fluorophenyl)-2,7-naphthyridin-1(2H)-one (13f), was identified. Knowledge of the binding mode of BMS-777607 in MET led to the design of new inhibitors that utilize novel 2,7-naphthyridone scaffold to conformationally restrain the key pharmacophoric groups (block C). Detailed SAR studies resulted in the discovery of a new MET inhibitor 13f, displaying favorable in vitro potency and oral bioavailability. More importantly, 13f exhibited excellent in vivo efficacy (tumor growth inhibition/TGI of 114% and 95% in 50 mg/kg, respectively) both in the U-87 MG and HT-29 xenograft models. The favorable drug-likeness of 13f indicated that 2,7-naphthyridinone may be used a promising novel scaffold for antitumor drug development. The preclinical studies of 13f are under way.

Enhanced tumor retention of NTSR1-targeted agents by employing a hydrophilic cysteine cathepsin inhibitor

We explored the approach of using an analog of E−64, a well-known and hydrophilic cysteine cathepsin (CC) inhibitor, as a potent cysteine cathepsin-trapping agent (CCTA) to improve the tumor retention of low-molecular-weight, receptor-targeted radiopharmaceuticals. The synthesized hydrophilic CCTA-incorporated, NTSR1-targeted agents demonstrated a substantial increase in cellular retention upon uptake into the NTRS1-positive HT-29 human colon cancer cell line. Similarly, biodistribution studies using HT-29 xenograft mice revealed a significant and substantial increase in tumor retention for the CCTA-incorporated, NTSR1-targeted agent. The intracellular trapping mechanism of the CCTA-incorporated agents by macromolecular adduct formation was confirmed using multiple in vitro and in vivo techniques. Furthermore, utilization of the more hydrophilic CCTA greatly increased the hydrophilicity of the resulting NTSR1-targeted constructs leading to substantial decreases in most non-target tissues in contrast to our previously reported dipeptidyl acyloxymethyl ketone (AOMK) constructs. This work further confirms that the CCTA trapping approach can make significant improvements in the clinical potential of NTSR1-and other receptor-targeted radiopharmaceuticals.

Nicotinamide phosphoribosyltransferase is a molecular target of potent anticancer agents identified from phenotype-based drug screening

Phenotypic screening in drug discovery has been revived with the expectation of providing promising lead compounds and drug targets and improving the success rate of drug approval. However, target identification remains a major bottleneck in phenotype-based drug discovery. We identified the lead compounds K542 and K405 with a selective inhibition of cell viability against sphingosine-1-phosphate lyase 1 (SGPL1)-transduced ES-2 cells by phenotypic screening. We therefore performed an in vivo pharmacological examination and observed the antitumor activity of K542 in an HT-1080 tumor-bearing mouse xenograft model. SGPL1 was expected to be a therapeutic target in some cancers, suggesting that these lead molecules might be promising candidates; however, their mechanisms of action still remain unexplained. We therefore synthesized the affinity probe Ind-tag derived from K542 and identified the proteins binding to Ind-tag via a pull-down experiment. Proteomics and biochemical analyses revealed that the target molecule of these lead compounds was Nicotinamide phosphoribosyltransferase (NAMPT). We established K542-resistant DLD-1 and HT-1080 cells, and genetic analyses of these cells identified a missense mutation in the NAMPT-encoding gene. This enzymatic experiment clearly showed that K393 exerts enzymatic inhibition against NAMPT. These proteomics, genetics and biochemical analyses clarified that compounds K542 and K405 were NAMPT inhibitors.

Aqueous extract of clove inhibits tumor growth by inducing autophagy through AMPK/ULK pathway.

Cloves (Syzygium aromaticum), a traditional Chinese medicinal herb, displays broad biological activity. In the present study, the aqueous extract of clove (AEC) was prepared, and its anticancer affects were studied. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-zolium (MTS) analysis revealed that AEC was able to inhibit cancer cell growth in vitro on several cancer cell lines; the IC50 is around 150μg/ml for human pancreatic ASPC-1 and human colon HT-29 cancer cells. Treatment of the cancer cells with AEC also diminished the colony formation significantly in both human pancreatic ASPC-1 cancer cells and human colon HT-29 cancer cells. In vivo study revealed that AEC inhibited the tumor growth significantly in HT-29 xenograft mice model. Transmission electron microscope, flow cytometry assay, and fluorescence microscope analysis confirmed that AEC is capable of inducing cell autophagy. Further study showed that AMPK/ULK pathway plays an important role in AEC-induced autophagy in cancer cells. Analysis of AEC components was performed by liquid chromatograph mass spectrometer approach, and more than nine constitutes were identified in AEC fraction. The study provides evidence that AEC has potential to be developed as a novel anticancer agent or as an adjuvant in cancer chemotherapy.

Dual PI3K-BRD4 Inhibitor SF1126 Inhibits Colorectal Cancer Cell Growth in Vitro and in Vivo

Bromodomain-containing protein 4 (BRD4) and phosphatidylinositol 3-kinase (PI3K) are key oncogenic cascades in colorectal cancer (CRC). SF1126 is a novel and potent PI3K-BRD4 dual inhibitor. CRC cells and human colon epithelial cells were treated with SF1126. Cell survival was tested by MTT and soft agar colony formation assays. Cell proliferation was tested by BrdU ELISA method. Cell apoptosis was tested by a TUNEL staining method and Histone DNA ELISA. Western blotting was utilized to test the signaling proteins. A HT-29 xenograft mice model was established to study the anti-tumor activity of SF1126 in vivo. SF1126 potently inhibited the survival, proliferation, and progression of the cell cycle in an established CRC cell line (HT-29) and primary human colon cancer cells. Significant activation of apoptosis was detected in SF1126-treated CRC cells. In CRC cells, SF1126 blocked Akt-mammalian target of rapamycin (mTOR) complex1/2 signaling and downregulated BRD4 target proteins (Myc and cyclin D1). Further studies showed that SF1126 activated p38 signaling in CRC cells. In contrast, the p38 inhibitors or p38 short hairpin RNA inhibited SF1126-induced cytotoxicity and apoptosis in CRC cells. In vivo, subcutaneous administration of SF1126 significantly inhibited HT-29 xenograft tumor growth in nude mice. SF1126 inhibits CRC cell growth possibly by targeting PI3K-Akt-mTOR, BRD4, and p38 signaling.

Synthesis, Characterization, and Evaluation of Near-IR Boron Dipyrromethene Bioconjugates for Labeling of Adenocarcinomas by Selectively Targeting the Epidermal Growth Factor Receptor.

A series of five boron dipyrromethene (BODIPY) bioconjugates containing an epidermal growth factor receptor (EGFR)-targeted pegylated LARLLT peptide and/or a glucose or biotin ethylene diamine group were synthesized, and the binding capability of the new conjugates to the extracellular domain of EGFR was investigated using molecular modeling, surface plasmon resonance, fluorescence microscopy, competitive binding assays, and animal studies. The BODIPY conjugates with a LARLLT peptide were found to bind specifically to EGFR, whereas those lacking the peptide bound weakly and nonspecifically. All BODIPY conjugates showed low cytotoxicity (IC50 > 94 μM) in HT-29 cells, both in the dark and upon light activation (1.5 J/cm2). Studies of nude mice bearing subcutaneous human HT-29 xenografts revealed that only BODIPY conjugates bearing the LARLLT peptide showed tumor localization 24 h after intravenous administration. The results of our studies demonstrate that BODIPY bioconjugates bearing the EGFR-targeting peptide 3PEG-LARLLT show promise as near-IR fluorescent imaging agents for colon cancers overexpressing EGFR.

Development of a recombinant human IL-15·sIL-15Rα/Fc superagonist with improved half-life and its antitumor activity alone or in combination with PD-1 blockade in mouse model

Recombinant human interleukin-15 (IL-15) is a potent cancer immunotherapeutic candidate due to its excellent immune stimulating effects. Previous work demonstrated that IL-15 appeared with short half-life in circulation system, while the complex with its receptor can prolong the half-life as well as benefit its activities in vivo. Therefore, IL-15 complex was more favorably considered for clinical development. Herein we developed IL-15·sIL-15Rα/Fc, a complex comprising of IL-15 and the extracellular region of its receptor alpha subunit which fused to Immunoglobulin G (IgG1) Fc to further prolong the half-life in plasma. Through transient gene expression in HEK293 cells, we expressed the superagonist by co-transfection of plasmids encoding IL-15 and sIL-15Rα/Fc respectively, yielding 36 mg/L of product after purification. Pharmacokinetic study demonstrated that the combination profoundly prolonged the half-life of IL-15 to 13.1 h in mice, about 18 folds longer than that of IL-15 monomer which is around 0.7 h. The bioactivity of the superagonist was characterized by CTLL-2 cells proliferation assay in vitro, showing its capability of stimulating the expansion of memory CD8+ T cells (cluster of differentiation) in mouse spleen. Using a HT-29 xenograft NOD-SCID mouse model, we observed tumor growth inhibition in all groups that received the superagonist, indicating its anti-tumor efficacy via stimulating infused human immune cells. In addition, combo cancer treatment by IL-15·sIL-15Rα/Fc and programmed death-1 (PD-1) antibody have shown stronger inhibitory effects as compared with treatment with either single molecule. Therefore, we developed IL-15·sIL-15Rα/Fc to be a long half-life potential cancer immunotherapy candidate that can be applied alone or in synergy with PD-1/PD-L1 blockade.

Bicyclic Peptides as a New Modality for Imaging and Targeting of Proteins Overexpressed by Tumors.

Molecular imaging of cancers using probes specific for tumor-associated target proteins offers a powerful solution for providing information regarding selection of targeted therapy, patient stratification, and response to therapy. Here we demonstrate the power of bicyclic peptides as targeting probes, exemplified with the tumor-overexpressed matrix metalloproteinase MT1-MMP as a target. A bicyclic peptide with subnanomolar affinity towards MT1-MMP was identified, and its radioconjugate showed selective tumor uptake in an HT1080 xenograft mouse model. Proteolytic stabilization of the peptide by chemical modification significantly enhanced the in vivo tumor signal [from 2.5%ID/g to 12%ID/g at 1 hour post injection (p.i.)]. Studies using mouse xenograft models with different cell lines show a robust correlation between tumor signals and in vivo MT1-MMP expression levels. Fatty acid modification of the bicyclic peptide extended its circulating half-life, resulting in increased tumor signals (36%ID/g at 6 hours p.i.). Comparative work with an equipotent radiolabeled MT1-MMP targeting antibody demonstrated starkly differential biodistribution and tumor accumulation properties, with the tumor signal slowly increasing to 6.2%ID/g within 48 hours. The rapid tumor penetration characteristics of bicyclic peptides, coupled with high potency and chemical versatility, thus offer high-contrast imaging probes for clinical diagnostics with compelling additional potential in targeted therapy.Significance: This work demonstrates the potential of bicyclic peptides as a platform for the development of high-contrast imaging probes for potential use in clinical cancer diagnostics and molecularly targeted therapeutics.

Induction of G0/G1 Phase Cell Cycle Arrest and Apoptosis by <i>Aegiceras corniculatum</i> in Human Colorectal Cancer via Activation of FoxO Signalling

Background: Aegiceras corniculatum (Linn.) Blanco, commonly known as black mangrove, river mangrove, kacang-kacang, and Narikandam, is a mangrove plant, found from India through Southeast Asia to southern China and Australia. Aegiceras corniculatum is known to exhibit anticancer effects against different types of cancer; however, the anticancer activity and underlying mechanisms of action of A. corniculatum leaf extract on colorectal cancer (CRC) have not been elucidated. Methods: In the present study, a colony-forming assay, western blot, flow cytometry and a xenografts model were applied to investigate the effects of n-butanol extract of A. corniculatum leaves (NACL) on CRC in vitro and in vivo. Findings: The results showed that NACL inhibits the viability and proliferation of CRC cells in a dose-dependent manner. Besides, NACL also induces cell apoptosis and cell cycle arrest by activating Forkhead box proteins and controlling the cell cycle checkpoint pathways, which are associated with the caspase-dependent mitochondrial apoptotic cascades and Bcl-2 family proteins. More importantly, the tumour sizes in HT-29 xenograft nude mice decreased after treatment with NACL in vivo. Significance: These findings demonstrate that A. corniculatum leaf extracts have potent anti-cancer activities across different colorectal and other solid tumor cell lines, via regulating cell cycle and apoptosis pathway, thus it has the potential to be developed as an anticancer therapy to enhance other standard of cares in the clinic for patients with CRC. Funding Statement: The work was supported by Guangxi scientific research and technology development plan project (No. GKH15104001-11), Guangxi Science and Technology Plan Projects (No. GKAD17195025), and Guangxi Key Laboratory Construction Project (No.17-259-20). Declaration of Interests: All authors state there are no conflicts of interest to disclose. Ethics Approval Statement: Experimental conditions and procedures involving animals were approved by the Institutional Animal Ethics Committee (IAEC) of Guangxi Medical University (Nanning, Guangxi, China). The experiments were carried out in accordance with the Laboratory Animal Use Guidelines of IAEC. Animal handling followed the National Animal Welfare Law of China.

Doxorubicin-loaded red blood cells reduced cardiac toxicity and preserved anticancer activity

Doxorubicin (DOX) is one of the most widely used anticancer agents. DOX is known for inducing cardiotoxicity, resulting in the long-term development of heart failure. Intravascular delivery of DOX may benefit from the carriage by red blood cells (RBCs), as they can limit the systemic toxicity while delivering the DOX to the tumor. This study proposes a methodology for the synthesis of electrophoretically DOX-loaded red blood cells (RBC-DOX), as well as the assessment of its antitumorigenic effects in human colon cancer cells (HT-29), and in colon cancer xenograft models. In addition, healthy mice without tumors were dosed with RBC-DOX to assess cardiotoxicity via assessment of indexes of cardiac function after multiple doses of RBC-DOX. The HT-29 IC50 was found to be lower for RBC-DOX compared to free DOX. Tumor volume for the RBC-DOX group was smaller than the free DOX groups in HT-29 xenografts models. Statistically higher concentrations of DOX were found in the liver, spleen, and lungs for the RBC-DOX group compared to the free DOX group. However, the heart and the skin had statistically lower DOX concentrations for the RBC-DOX group compared to the free DOX group, with no significant differences in tumor biodistribution. All hemodynamic and cardiac function parameters were closer to control parameters for the RBC-DOX treated compared to for the free DOX-treated mice. These results suggest that RBC-DOX can be an alternative to prolong treatments with DOX, with superior antitumorigenic effects, decreased myelosuppression, and limited cardiac toxicity compared to equivalent doses of free DOX.

Noninvasive evaluation of 18F-FDG/18F-FMISO-based Micro PET in monitoring hepatic metastasis of colorectal cancer

This study aimed to explore the application of two radiotracers (18F-fluorodeoxyglucose (FDG) and 18F-fluoromisonidazole (FMISO)) in monitoring hepatic metastases of human colorectal cancer (CRC). Mouse models of CRC hepatic metastases were established by implantation of the human CRC cell lines LoVo and HT29 by intrasplenic injection. Wound healing and Transwell assays were performed to examine cell migration and invasion abilities. Radiotracer-based cellular uptake in vitro and micro-positron emission tomography imaging of liver metastases in vivo were performed. The incidence of liver metastases in LoVo-xenografted mice was significantly higher than that in HT29-xenografted ones. The SUVmax/mean values of 18F-FMISO, but not 18F-FDG, in LoVo xenografts were significantly greater than in HT29 xenografts. In vitro, LoVo cells exhibited stronger metastatic potential and higher radiotracer uptake than HT29 cells. Mechanistically, the expression of HIF-1α and GLUT-1 in LoVo cells and LoVo tumor tissues was remarkably higher than in HT29 cells and tissues. Linear regression analysis demonstrated correlations between cellular 18F-FDG/18F-FMISO uptake and HIF-1α/GLUT-1 expression in vitro, as well as between 18F-FMISO SUVmax and GLUT-1 expression in vivo. 18F-FMISO uptake may serve as a potential biomarker for the detection of liver metastases in CRC, whereas its clinical use warrants validation.

Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo.

Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.

Novel N-1 substituted fluoroquinolones inhibit human topoisomerase I activity and exhibit anti-proliferative activity.

Fluoroquinolone-class agents selectively target the bacterial type IIA topoisomerases DNA gyrase and topoisomerase IV, with a few exceptions that target eukaryotic type IIA topoisomerases. Fluoroquinolones bind and stabilize type IIA topoisomerase-DNA covalent complexes that contain a double-strand break. This unique mode of action is referred to as 'topoisomerase poisoning'. We discovered that two novel fluoroquinolones having aryl functionality at the N-1 position, UITT-3-217 (217) and UITT-3-227 (227), could inhibit the catalytic activity of human topoisomerase II without stabilizing topoisomerase-DNA complexes, i.e., without poisoning it. Surprisingly, these compounds are more effective in inhibiting the catalytic activities of human and bacterial topoisomerase I. The National Cancer Institute's 60 human tumor cell lines screen revealed significant anti-proliferative activities with 217 and 227 against the majority of 60 cancer cell lines. A proof of concept in vivo efficacy study using an HT-29 xenograft model of human colorectal cancer showed that 217 could inhibit the proliferation of human colorectal cancer cells to a degree comparable to fluorouracil in mice. Although 227 also exhibited anti-proliferative activity, it was not as effective as 217 in this xenograft model. These novel fluoroquinolones may serve as promising lead compounds for the development of new anticancer drugs.

Umbilical Cord Blood-Derived Natural Killer Cells Combined with Bevacizumab for Colorectal Cancer Treatment.

Colorectal cancer (CRC) is among the cancers with the highest incidence globally, and it currently ranks as the fourth leading cause of cancer-related deaths worldwide. Novel strategies for the treatment of advanced CRC are urgently needed, and adoptive transfer of allogeneic natural killer (NK) cells represents an attractive option. In this study, we successfully expanded NK cells from umbilical cord blood (UCB) with membrane-bound interleukin (IL)-21, termed eUCB-NK cells. eUCB-NK cells efficiently lysed CRC cell lines in vitro and secreted significantly higher levels of interferon-γ, tumor necrosis factor-α, granulocyte-macrophage colony stimulating factor, and chemokine ligand 3 compared with IL-2-stimulated NK cells. Adoptive transfer of these NK cells significantly inhibited the growth of HT29 xenografts, whereas LoVo tumors were not effectively controlled with eUCB-NK cells. Higher numbers of NK cells inside HT29 tumors, not seen in LoVo tumors, might contribute to the differences in response to eUCB-NK cells. Bevacizumab increased extravasation of adoptively transferred NK cells into LoVo tumors and improved the therapeutic activity of eUCB-NK cells. These results justify clinical translation of UCB-derived NK cell-based therapeutics, used alone or in combination with bevacizumab, as a novel treatment option for patients with CRC.

Abstract 5811: Evaluation of antitumor efficacy of CA102N, H-Nim and hyaluronic acid (HA) conjugate, alone or in combination with 5-fluorouracil (5-FU) in in vivo human colorectal cancer models

Abstract CA102N is a conjugate of HA and H-Nim (N-(4-Amino-2-phenoxyphenyl, methanesulfonamide), a derivative of cyclooxygenase-2 inhibitor Nimesulide, and a CD44 receptor targeted system currently in preclinical development for metastatic colorectal cancer. The in vivo antitumor activities of CA102N was evaluated singly or combined with 5-FU in CR5038 (Patient Derived Xenograft (PDX) and HT29 Xenograft models. Inhibitory activity was assessed according to tumor-volume changes. CA102N (i.v. 200mg/kg, BIW) monotherapy did not show significant tumor growth inhibition at either the end of the treatment (21 days) or prolonged observational (45 days) period, in CR5038 PDX-SCID metastatic model in comparison to vehicle control or 5-FU (i.v. 50mg/kg, QW). Notably, the combination therapy displayed a marked reduction in tumor growth (>70%) compared to 5-FU alone (p=0.0003) or vehicle control (p<0.0001) in addition to prolonged median survival (p<0.05). Alternatively, HT29 xenografts nude mice were found to have significantly reduced tumor burden treated with either single agents CA102N (400 mg/kg, QW) or 5-FU (50 mg/kg, QW), (p<0.012) compared to control vehicle. The drug combination further displayed a synergistic antitumoral effect and greater median survival (p <0.05) in HT29 model. CA102N was well tolerated alone or in the combination therapy. Based on the results of this study, combined treatment of CA102N and 5-FU resulted in a complete response at the regimen given. The combination of CA102N and 5-FU represents a promising potential therapeutic strategy and a new approach to reverse drug resistant in the treatment of colorectal cancer. Studies presently are ongoing to further evaluate its efficacy in CT26 murine colorectal models. Citation Format: Eskouhie Tchaparian, Louis Lin. Evaluation of antitumor efficacy of CA102N, H-Nim and hyaluronic acid (HA) conjugate, alone or in combination with 5-fluorouracil (5-FU) in in vivo human colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5811.

99mTc labeled D(LPR): A novel retro-inverso peptide for VEGF receptor-1 targeted tumor imaging

IntroductionThe aim of this study was to evaluate the ability of D(LPR), a novel retro-inverso peptidomimetic derivative for imaging colon cancer. MethodsTwo different D(LPR) analogs were designed and compared based on conjugation of HYNIC at peptide's C or N terminal and then labeled with technetium-99m using tricine/EDDA as an exchange coligands. The radiolabeled conjugates were assessed for in vitro stability in saline and serum. The VEGFR-1 and NRP-1 receptors affinity, in vitro internalization and also dissociation Constance was evaluated. SPCET imaging and biodistribution studies were performed in nude mice bearing HT-29 xenograft tumors. ResultsBoth peptides labeled with technetium-99m in high radiochemical yield (˃97%). Peptide stability studies indicated a high metabolic stability of the radiopeptides in solution and serum. In vitro blocking studies demonstrated specific binding and internalization of [99mTc]Tc-HYNIC-peptides in cultured HUVEC cells. The Kd value for 99mTc-peptide 1 and 99mTc-peptide 2 were found to be 56.8 ± 12.9 nM and 71.6 ± 17.9 nM respectively. The tumor to muscle ratio was significant at 0.5 and 1 h after injection (4.5 and 4 for 99mTc-peptide 1 and 4.9 and 4.4 for 99mTc-peptide 2 at 0.5 and 1 h p.i. respectively). SPECT imaging studies revealed that both radioconjugates had prominent activity accumulation in VEGFR-1 and NRP-1 expressing HT-29 tumors. ConclusionThis study is the first instance of using a radiolabeled retro-inverso peptide for tumor imaging which is a promising tool to improve the performance of fragile peptide probes in vivo as imaging agents and warrant further investigations in other peptide-target systems.

Low dose-rate irradiation with [3H]-labelled valine to selectively target hypoxic cells in a human colorectal cancer xenograft model

Background Earlier in vitro studies show that irradiation with an ultra-low dose-rate of 15 mGy/h delivered with [3H]-valine leads to loss of clonogenicity in hypoxic T-47D cells. Here, the aim was to determine if [3H]-valine could be used to deliver low dose-rate irradiation in a colorectal cancer model. Methods Clonogenicity was measured in cultured cancer cell line HT29 irradiated with 15 mGy/h combined with intermittent hypoxia. Mice with HT29 xenografts were irradiated by repeated injections of [3H]-valine intravenously. The activity in the tumor tissue was measured by scintillation counting and tumor growth, hypoxic fraction and tritium distribution within tumors were assessed by pimonidazole staining and autoradiography. Results Ultra-low dose-rate irradiation decreased clonogenicity in hypoxic colorectal cancer cells. In vivo, the tumor growth, hypoxic fraction and weight of the mice were similar between the treated and untreated group. Autoradiography showed no [3H]-valine uptake in hypoxic tumor regions in contrast to aerobic tissue. Conclusion Continuous low-dose-rate irradiation was well tolerated by aerobic tissue. This indicates a potential use of low dose-rate irradiation to target hypoxic tumor cells in combination with high dose-rate irradiation to eradicate the well oxygenated tumor regions. However, [3H]-valine is not the appropriate method to deliver ultra-low dose-rate irradiation in vivo.

Amphiphilic dendrimer engineered nanocarrier systems for co-delivery of siRNA and paclitaxel to matrix metalloproteinase-rich tumors for synergistic therapy

Combinations of chemotherapeutics with small interfering RNA (siRNA) can incorporate the advantages of their different mechanisms to exert a synergetic effect. A safe and effective vehicle for simultaneous delivery of the components to tumor cells is a prerequisite for obtaining the optimum effect. We developed an amphiphilic dendrimer engineered nanocarrier system (ADENS) for co-delivering paclitaxel and siRNA for cancer treatment. This nanocarrier possesses a unique hollow core/shell structure in which siRNA is incorporated in the hydrophilic cavity and large quantities of paclitaxel are stored in the hydrophobic interlayer, while the outer PEG layer serves to prolong the circulation time. Further modification by tumor microenvironment-sensitive polypeptides (TMSP) significantly enhanced the cellular uptake, tumor penetration and tumor accumulation of the ADENS by a tumor microenvironment-triggered mechanism. TMSP-ADENS had prominent therapeutic effects at a relatively low drug dose both in vitro and in vivo. In A375 xenograft mice, TMSP-ADENS/siRNA/PTX showed the highest VEGF mRNA inhibition rate of 73% and suppressed tumor growth and relapse, while Taxol did not show an effect on tumor relapse. The anti-tumor and anti-angiogenic effects were further confirmed in an HT-1080 xenograft tumor model. Our findings, combined with the known biodegradability and tunable physicochemical properties of these polymers, suggest that this TMSP-ADENS can be a robust co-delivery system for cancer combination therapy in the future.

Abrus agglutinin stimulates BMP-2-dependent differentiation through autophagic degradation of β-catenin in colon cancer stem cells.

Eradicating cancer stem cells (CSCs) in colorectal cancer (CRC) through differentiation therapy is a promising approach for cancer treatment. Our retrospective tumor-specimen analysis elucidated alteration in the expression of bone morphogenetic protein 2 (BMP-2) and β-catenin during the colon cancer progression, indicating that their possible intervention through "forced differentiation" in colon cancer remission. We reveal that Abrus agglutinin (AGG) induces the colon CSCs differentiation, and enhances sensitivity to the anticancer therapeutics. The low dose AGG (max. dose = 100 ng/mL) decreased the expression of stemness-associated molecules such as CD44 and β-catenin in the HT-29 cell derived colonospheres. Further, AGG augmented colonosphere differentiation, as demonstrated by the enhanced CK20/CK7 expression ratio and induced alkaline phosphatase activity. Interestingly, the AGG-induced expression of BMP-2 and the AGG-induced differentiation were demonstrated to be critically dependent on BMP-2 in the colonospheres. Similarly, autophagy-induction by AGG was associated with colonosphere differentiation and the gene silencing of BMP-2 led to the reduced accumulation of LC3-II, suggesting that AGG-induced autophagy is dependent on BMP-2. Furthermore, hVps34 binds strongly to BMP-2, indicating a possible association of BMP-2 with the process of autophagy. Moreover, the reduction in the self-renewal capacity of the colonospheres was associated with AGG-augmented autophagic degradation of β-catenin through an interaction with the autophagy adaptor protein p62. In the subcutaneous HT-29 xenograft model, AGG profoundly inhibited the growth of tumors through an increase in BMP-2 expression and LC3-II puncta, and a decrease in β-catenin expression, confirming the antitumor potential of AGG through induction of differentiation in colorectal cancer.