Antitumor Ability Research Articles

Antrodia cinnamomea Enhances Chemo-Sensitivity of 5-FU and Suppresses Colon Tumorigenesis and Cancer Stemness via Up-Regulation of Tumor Suppressor miR-142-3p.

5-Fluorouracil (5-FU) regimen remains the backbone of the first-line agent to treat colon cancer, but often these patients develop resistance. Cancer stem cells (CSC’s) are considered as one of the key contributors in the development of drug resistance and tumor recurrence. We aimed to provide preclinical evidence for Antrodia cinnamomea (AC), as a potential in suppressing colon cancer CSC’s to overcome 5-FU drug-resistant. In-vitro assays including cell viability, colony formation, AC + 5-FU drug combination index and tumor sphere generation were applied to determine the inhibitory effect of AC. Mouse xenograft models also incorporated to evaluate in vivo effect of AC. AC treatment significantly inhibited the proliferation, colony formation and tumor sphere generation. AC also inhibited the expression of oncogenic markers (NF-κB, and C-myc), EMT/metastasis markers (vimentin and MMP3) and stemness associated markers (β-catenin, SOX-2 and Nanog). Sequential treatment of AC and 5-FU synergized and reduces colon cancer viability both in vivo and in vitro. Mechanistically, AC mediated anti-tumor effect was associated with an increased level of tumor suppressor microRNAs especially, miR142-3p. AC can be a potent synergistic adjuvant, down-regulates cancer stemness genes and enhances the antitumor ability of 5-FU by stimulating apoptosis-associated genes, suppressing inflammation and metastasis genes through miR142-3p in colon cancer.

Highly biocompatible thermosensitive nanocomposite gel for combined therapy of hepatocellular carcinoma via the enhancement of mitochondria related apoptosis

Primary hepatocellular carcinoma (HCC) is a common malignant tumor. Surgery is the main treatment, but HCC patients have a potential risk of tumor recurrence. Besides, many limitations arise during the application of single first-line antitumor drugs. Here, we selected Pluronic F-127 and sodium alginate (SA) to prepare a thermosensitive gel (Gel). The optimal synergistic ratio of PTX and DOX on the SMMC-7721 cells was 1: 2 (w/w), calculated by the Chou–Talalay analysis. Then, PTX and DOX coloaded liposomes (PD-LPs) with such drugs ratios presented enhanced anticancer ability in vitro. Upon local injection, the PD-LPs Gel formed a nanoparticles reservoir at tumor via sol–gel transformation, while exhibiting a long-term effective anti-tumor ability in vivo. The relative tumor volume after the PD-LPs Gel treatment was reduced over 62%. Effective mitochondria related apoptosis induction was observed. Therefore, the local delivery of PD-LPs Gel can be a promising alternative method for the HCC therapy.

Abstract 1512: Nicotine promotes brain metastasis of lung cancer by inducing M1/M2 conversion and suppressing the innate immune response of microglia

Abstract Approximately 50% of patients with metastatic lung cancer develop brain metastasis which is almost always fatal, and the median survival time of these patients is only a few months. Smoking is the major risk factor for lung cancer, and tobacco smoke contains a toxic mix of more than 7,000 chemical. Among those chemicals, nicotine has been shown to promote cancer progression and metastasis. However, the exact mechanism by which nicotine derives this process remain poorly understood. The communications between neoplastic cells and microglia, especially M2 type pro-tumor microglia, profoundly affect the brain metastasis progression. Therefore, we hypothesize that nicotine skews microglial polarization to M2 phenotype which promotes brain metastasis of lung cancer. We found that the brain metastasis-free survival rate was significantly decreased in current smokers compared to non-smokers. Abundant CD206+ M2 microglia were observed in brain metastatic lesions in such patients. Consistent with these results, when we transplanted the H2030 brain metastasis of lung cancer cell line (H2030BrM) into nude mice by intra-cardiac injection followed by with or without nicotine treatment, the brain metastasis-free survival was decreased in nicotine treatment group. The number of CD206+cells were also increased in the mouse brain after nicotine treatment. To test the effect of nicotine-driven microglia on tumor progression, H2030BrM cells were treated with conditioned medium (CM) generated from human microglia that were treated with or without nicotine. We found that the CM significantly increased the population of ESA+/CD44+ cancer stem cells and promoted their sphere forming abilities. The expression of SOX2 and Nanog were up-regulated when the H2030BrM cells were cultured with the CM of nicotine-treated microglia. Blocking nicotine-driven M2 microglial polarization by a STAT3 inhibitor suppressed the M2 polarization, resulting in inhibition of tumor growth. To examine the mechanism by which nicotine suppresses anti-tumor function of microglia, we measured the expression of “do not eat me” signal, SIRP on microglia and CD47 on tumor cells. H2030BrM and human microglial cells were treated with nicotine. We found that the mRNA and protein expression of SIRP were up-regulated on human microglia, while the expression of CD47 on H2030BrM were elevated by nicotine treatment. We used FACS to detect the phagocytic ability of microglial by co-culturing H2030BrM with microglial cells. We found that the anti-tumor phagocytic activities of microglia cells were significantly decreased upon nicotine treatment. In conclusion, our results suggest that nicotine promotes lung cancer brain metastasis by enhancing both tumor progression and stemness by skewing microglial polarization and inhibiting its anti-tumor phagocytic ability. Citation Format: Shih-Ying Wu, Fei Xing, Sambad Sharma, Abhishek Tyagi, Kerui Wu, Yin Liu, Dan Zhao, Ravindra Deshpande, Yusuke Shiozawa, Kounosuke Watabe. Nicotine promotes brain metastasis of lung cancer by inducing M1/M2 conversion and suppressing the innate immune response of microglia [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 1512.

Anti-Colorectal Cancer Effect via Application of Polyethylene Glycol Modified Liposomal Apatinib.

Metastatic colorectal cancer is a stumbling block in colorectal cancer management due to limited treatment approaches and unfavorable prognosis. Angiogenesis is essential in tumor development and metastasis. Vascular endothelial growth factor receptor (VEGFR) targeted anti-angiogenesis drugs have gain inspiring achievements in cancer treatment. Apatinib is a novel small molecular VEGFR-2 tyrosine kinase targeted drug with poor water-solubility, showing anti-tumor ability in some solid tumors. In this study, PEG modified liposomal Apatinib (Apatinib-Lipo/PEG) was synthesized and applied for colorectal cancer treatment. Results showed that Apatinib-Lipo/PEG could attenuate proliferation and induce apoptosis of CT26 cells. Moreover, Apatinib-Lipo/PEG significantly reduced tumor growth in colorectal peritoneal model as well as subcutaneous model without obvious toxicity. The anti-tumor mechanisms included inhibiting tumor proliferation, promoting tumor apoptosis and suppressing tumor angiogenesis. Thereafter, the application of Apatinib-Lipo/PEG would be very promising in colorectal cancer treatment.

Osteogenesis effects of magnetic nanoparticles modified-porous scaffolds for the reconstruction of bone defect after bone tumor resection.

The treatment of bone defect after bone tumor resection is a great challenge for orthopedic surgeons. It should consider that not only to inhibit tumor growth and recurrence, but also to repair the defect and preserve the limb function. Hence, it is necessary to find an ideal functional biomaterial that can repair bone defects and inactivate tumor. Magnetic nanoparticles (MNPs) have its unique advantages to achieve targeted hyperthermia to avoid damage to surrounding normal tissues and promote osteoblastic activity and bone formation. Based on the previous stage, we successfully prepared hydroxyapatite (HAP) composite poly(lactic-co-glycolic acid) (PLGA) scaffolds and verified its good osteogenic properties, in this study, we produced an HAP composite PLGA scaffolds modified with MNPs. The composite scaffold showed appropriate porosity and mechanical characteristics, while MNPs possessed excellent magnetic and thermal properties. The cytological assay indicated that the MNPs have antitumor ability and the composite scaffold possessed good biocompatibility. In vivo bone defect repair experiment revealed that the composite scaffold had good osteogenic capacity. Hence, we could demonstrate that the composite scaffolds have a good effect in bone repair, which could provide a potential approach for repairing bone defect after bone tumor excision.

Isorhapontigenin shows strong anti-melanoma cell activity, and induces anti-tumor immunity

Abstract Background Melanoma is one of the deadliest forms of skin cancer, and its incidence has continued to rise during the past decade. Isorhapontigenin (ISO) is a new derivative of stilbene compound, isolated from Chinese herb Gnetum Cleistostachyum with capability of antitumor. Purpose The potential anti-melanoma activity, impact on genes’ expression, as well as the influence of immune response against melanoma of ISO were detected in our studies in order to clarify its anti-melanoma effect and the corresponding mechanism. Methods Cytotoxicity of ISO on melanoma cell line B16F10 was studied by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cell cycle analysis by flow cytometry, annexin V staining, RT-qPCR and Western blot were used to elucidate the mechanism of action of ISO at the cellular level. The in vivo anti-tumor ability of ISO was measured in C57BL/6 mouse syngraft models. Results ISO induced dose-dependent inhibitory effect on the proliferation of B16F10 cell by arresting cells at G0/G1 phase promoting the apoptosis of the cell, which was associated with an increase in the Bax:Bcl-2 ratio and decrease in the proliferative-related genes CYCLIN D1 and SURVIVIN in vitro. In addition, ISO could inhibit the tumor immune escape by promoting the expression of MHC molecules on the membrane of tumor cells, and decreased the infiltration of Tregs and MDSCs which involved in suppressing the anti-tumor effects of immune system. On the other hand, ISO increased the infiltration of T cells, NK cells and NKT cells, which was able to enhance the anti-tumor immune response. Conclusion Taken together, these findings indicate that ISO may be useful as a treatment for Melanoma and thus warrants further investigation.

Silencing long non-coding RNA HOTAIR exerts anti-oncogenic effect on human acute myeloid leukemia via demethylation of HOXA5 by inhibiting Dnmt3b

BackgroundAs an aggressive hematological malignancy, acute myeloid leukemia (AML) remains a dismal disease with poor prognosis. Long non-coding RNAs (lncRNAs) have been widely reported to be involved in tumorigenesis of AML. Here, we define an important role of lncRNA HOTAIR in AML in relation to HOXA5 methylation.MethodsFirstly, the expression of HOTAIR was examined in AML samples and cells collected. Next, gain- or loss-of function experiments were conducted in AML cells to explore the effect of HOTAIR on AML. Then, relationship among HOXA5 promoter methylation, HOTAIR and Dnmt3b was measured. Expression of HOXA5 and cell proliferation/apoptosis-related genes was also detected. A last, in vivo assay was performed to assess the tumor formation in nude mice in order to explore the roles of HOTAIR and HOXA5 in cell apoptosis and proliferation.ResultsLncRNA HOTAIR was found to be upregulated in AML cells and tissues. With silencing of HOTAIR and overexpression of HOXA5, AML cell proliferation was decreased while the apoptosis was induced. Furthermore, HOTAIR was observed to recruit Dnmt3b and to increase HOXA5 promoter methylation. Moreover, silencing HOTAIR and upregulating HOXA5 were found to induce apoptosis and reduce proliferation of AML cells in vivo.ConclusionOur findings highlight the anti-tumor ability of HOTAIR silencing in AML, suggesting that silencing HOTAIR was able to inhibit AML progression through HOXA5 promoter demethylation by decreasing Dnmt3b.

Hexahistidine-metal assemblies: A promising drug delivery system

It is of considerable interest to construct an ideal drug delivery system (i.e., high drug payload, minimal cytotoxicity, rapid endocytosis, and lysosomal escape) under mild conditions for disease treatment, tissue engineering, bioimaging, etc. Inspired by the coordinative interactions between histidine and metal ions, we present the facile synthesis of hexahistidine (His6)-metal assembly (HmA) particles under mild conditions for the first time. The HmA particles presented a high loading capacity, a wide variety of loadable drugs, minimal cytotoxicity, quick internalization, the ability to bypass the lysosomes, and rapid intracellular drug release. In addition, HmA encapsulation largely improved the antitumor ability of camptothecin (CPT) relative to free CPT. By capitalizing on these promising features in drug delivery, HmA will have great potential in various biomedical fields. Statement of SignificanceIt is of considerable interest to construct an ideal drug delivery system (i.e., high drug payload, minimal cytotoxicity, rapid endocytosis, and lysosomal escape) under mild conditions. Inspired by the coordinative interactions between histidine and metal ions, we present for the first time the facile synthesis of Hexahistidine (His6)-metal assembly (HmA) particles under mild conditions. The HmA particles exhibited a high loading capacity, a wide variety of loadable drugs, minimal cytotoxicity, quick internalization, the ability to bypass the lysosomes, and rapid intracellular drug release. By capitalizing on these promising features in drug delivery, HmA will have great potential in various biomedical fields.

Downregulation of survivin by adenovirus-mediated shRNA promotes apoptosis in skin cancer cells.

BackgroundSurvivin, a member of the inhibitor of apoptosis protein family, is highly expressed in many cancers and has important roles in inhibiting apoptosis by blocking caspase activation. However, its antitumor effects remain largely unknown. Here we explore the function of survivin in skin cancer.MethodsWe used qPCR and Western blot to examine survivin expression in skin cancer patients and cell line. We generated several survivin shRNA constructs and tested the effects of survivin shRNA on cancer cell viability using MTT assay, flow cytometry, and TUNEL assay.ResultsWe found that survivin was upregulated in both skin cancer patients and skin cancer cell line A431. Knockdown survivin via shRNA inhibited cancer cell proliferation and promoted apoptosis in both A431 cell and in vivo xenograft tumor mouse model. The antitumor effect is comparable to resveratrol, a drug known to inhibit cancer progression. Moreover, we showed that inhibition of survivin was able to increase the expression of cleaved caspase 7/caspase 9 and activate the ataxia-telangiectasia mutated-NF-κB pathway in A431 cells.ConclusionSurvivin-shRNA possesses antitumor abilities in vitro and in vivo by inhibiting the proliferation and promoting apoptosis of A431 cells. It may serve as a potential anticancer target for skin cancer therapy in the future.

Delanzomib, a novel proteasome inhibitor, sensitizes breast cancer cells to doxorubicin-induced apoptosis.

BackgroundDelanzomib, a novel proteasome inhibitor, has demonstrated promising efficacy and antitumor ability in human multiple myeloma cell lines and patient‐derived cells. However, the potential therapeutic effects of delanzomib on breast cancer remain unknown. In this study, we show that delanzomib has antitumor effects and synergizes with doxorubicin (Dox) in human breast cancer cell lines.MethodsCell proliferation assay and flow cytometry were used to evaluate cell viability and apoptosis in eight human breast cancer cell lines after treatment with delanzomib or Dox. Essential molecules of the p53, MAPK, and apoptosis signaling pathways were analyzed by Western blotting.ResultsDelanzomib induced cell death and demonstrated synergism with Dox in all tested breast cancer cell lines. In addition, delanzomib enhanced the Dox‐induced phosphorylation of p38/JNK and the expression of transcriptional target proteins of p53, such as p21, p27, NOXA, and PUMA.ConclusionThe combined regimen of the proteasome inhibitor delanzomib with Dox chemotherapy may become an effective strategy for breast cancer therapy.

Anchorage of curcumin onto PVP enhances anti-tumor effect of curcumin

Curcumin, a natural polyphenol compound, exhibits wide range of pharmacological activities. However, its poor aqueous solubility, weak stability, and rapid metabolism limit its potential development. In this study, solid dispersion of curcumin with PVP was developed. This study aimed to evaluate the loading capacity, stability, solubility, and anti-tumor activity of PVP-Cur solid dispersion. Scanning electron microscope (SEM), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), and fourier transform infrared (FTIR) spectroscopy were used to investigate physical characterization of PVP-Cur. Loading capacity, stability, and solubility of PVP-Cur were evaluated by spectrophotometer. The biological effects of PVP-Cur on tumor cell proliferation and apoptosis were analyzed using CCK-8 assay and 7-AAD/AnnexinV-APC assay, respectively. PVP-cur-induced activation of caspase-3 and caspase-9 were evaluated by in vitro caspase activity assay and western blotting. It was interesting to note that PVP: curcumin in the ratios of 6:1 or 8:1 exhibited the highest loading capacity. Anchorage of curcumin onto PVP increased its solubility and stability. Compared with curcumin alone, PVP-Cur displayed an increased ability in abrogating tumor cell proliferation and inducing tumor cell apoptosis via mitochondria signaling, which was attributed by the enhanced cellular uptake of PVP-Cur. Anchorage of curcumin onto PVP was an effective way to increase the solubility and stability of curcumin. PVP-Cur increased anti-tumor ability and cellular uptake relative to curcumin, indicating that PVP-Cur is a promising candidate for anti-cancer drug development.

Preclinical Evaluation of Chimeric Antigen Receptor–Modified T Cells Specific to Epithelial Cell Adhesion Molecule for Treating Colorectal Cancer

Chimeric antigen receptor-modified T cells (CAR-T cells) have emerged as a promising cancer immunotherapy for solid tumors. Epithelial cell adhesion molecule (EpCAM) is overexpressed in a variety of tumors and is recognized as a biomarker for circulating tumor cells and cancer stem cells, representing an attractive target for adoptive T-cell immunotherapy. This study generated third-generation CAR-T cells with redirected specificity to EpCAM (EpCAM CAR-T) by lentiviral vector. The study demonstrated that EpCAM CAR-T cells can elicit lytic cytotoxicity to target cells in an EpCAM-dependent manner and secrete cytotoxic cytokines, including interferon gamma and tumor necrosis factor alpha. Furthermore, adoptive transfer of EpCAM CAR-T cells significantly delayed tumor growth and formation in xenograft models. In addition, the safety evaluation showed that CAR-T cells have no systemic toxicity in mice. The data confirmed the antitumor ability and safety of CAR-T cells targeting EpCAM and may provide a new target for CAR-T cell therapies in treating solid tumors.

Highly Effective Treatment of CD38 Positive Experimental Lymphoma with 225Actinium-Daratumumab

Daratumumab is a human cytolytic antibody specific for CD38 that is used clinically for treatment of patients with multiple myeloma (MM). Current therapeutic regimens require multiple injections over months of treatment, therefor increasing the potency of daratumumab to shorten the length of treatment would be very advantageous. 225Ac is an α-particle emitting radionuclide that has potent cytotoxic activities over relatively short distances, allowing for precise targeting of a lethal dose of radiation. Previously we have established that labeling daratumumab with 225Ac increased more than 10-fold its ability to kill MM cell lines in vitro. In this study we evaluated the therapeutic potential of 225Ac-daratumumab by treating mice with established CD38-positive tumors. Mice deficient in T- and B-cells were injected subcutaneously with human tumor (Daudi) cells and once tumors reached an average volume of ∼200 mm3, mice were treated with 225Ac-daratumumab. To determine the localization of daratumumab within the tumor-bearing mice it was labeled with 111In, which can be easily imaged and is used as a surrogate to estimate the localization of 225Ac-daratumumab. The distribution of the 111In-daratumumab was then followed for 10 days using a microSPECT/CT scanner. To evaluate the antitumor ability of the 225Ac-daratumumab, tumor-bearing mice were injected with 225Ac- daratumumab at a dose of 400 nCi/0.3 μg of antibody, and 200 nCi/0.3 μg of antibody. As a control, mice were injected with either saline or an equivalent amount of unlabeled daratumumab. In addition, a group of mice was also treated with 30 times greater dose of unlabeled daratumumab (10 μg) - a dose which was previously shown to be effective against established tumors. 111Indaratumumab begun to accumulate in the tumor 24 hours after intraperitoneal injection and by 7 days was exclusively present in the tumor. The growth of the tumors in mice treated with 400 nCi/0.3 μg was significantly retarded compared to mice treated with equal concentration of unlabeled daratumumab or saline. Tumor growth was similar between mice treated with 400 nCi/0.3 μg of 225Ac-daratumumab and mice treated with 10 μg of unlabeled daratumumab. In conclusion, this study shows that labeling daratumumab with 225Ac increases its antitumor activity at least 30-fold. This study suggests that 225Ac labeling daratumumab increases its potency and could greatly reduce the amount of daratumumab needed for treatment in the clinic. This study also highlights the potential of targeting α-emitters to tumors as a viable therapeutic approach.

Biodegradation of graphene-based nanomaterials in blood plasma affects their biocompatibility, drug delivery, targeted organs and antitumor ability

The extensive use of graphene-family nanomaterials (GFNs) in biomedicine and other fields has intentionally or unintentionally resulted in their introduction into the blood circulation system, but the effects of the biotransformation of GFNs in blood plasma on their biocompatibility, organ targeting, drug delivery and antitumor ability remain unclear. The present work discovered that GFN sheets were degraded in human blood plasma to holey sheets and aromatic hydrocarbons. The carbon atoms connected with oxygen-containing groups in the planes of GFNs were the initial attack sites for active substances (e.g., OH and O2-) in blood plasma. Subsequently, CC/CC bonds were broken. The reaction rate depended strongly on the extent of oxidization of GFNs. The pristine GFNs caused secondary structure damage to proteins and disturbances of cellular metabolic pathways. In contrast, the biotransformed nanomaterials presented high biocompatibility and were located in and targeted different tissues from their pristine forms, which influenced specific organ targeting therapy. The biotransformed nanomaterials also exhibited higher efficiencies of drug delivery (drug release and location) and killing tumor cells in vitro and in vivo. These findings provide insights into the application of nanomaterials in human healthcare using biotransformed nanomaterials.

Ratiometric co-encapsulation and co-delivery of doxorubicin and paclitaxel by tumor-targeted lipodisks for combination therapy of breast cancer

Combination therapy is a promising treatment for certain advanced drug-resistant cancers. Although effective inhibition of various tumor cells was reported in vitro, combination treatment requires improvement in vivo due to uncontrolled ratiometric delivery. In this study, a tumor-targeting lipodisk nanoparticle formulation was developed for ratiometric loading and the transportation of two hydrophobic model drugs, doxorubicin (DOX) and paclitaxel (PTX), in one single platform. Furthermore, a slightly acidic pH-sensitive peptide (SAPSP) incorporated into lipodisks effectively enhanced the tumor-targeting and cell internalization. The obtained co-loaded lipodisks were approximately 30 nm with a pH-sensitive property. The ratiometric co-delivery of two drugs via lipodisks was confirmed in both the drug-resistant MCF-7/ADR cell line and its parental MCF-7 cell line in vitro, as well as in a tumor-bearing mouse model in vivo compared with a cocktail solution of free drugs. Co-loaded lipodisks exerted improved cytotoxicity to tumor cells in culture, particularly to drug-resistant tumor cells at synergistic drug ratios. In an in vivo xenograft mouse model, the anti-tumor ability of co-loaded lipodisks was evidenced by the remarkable inhibitory effect on tumor growth of either MCF-7 or MCF-7/ADR tumors, which may be attributed to the increased and ratiometric accumulation of both drugs in the tumor tissues. Therefore, tumor-specific lipodisks were crucial for the combination treatment of DOX and PTX to completely exert a synergistic anti-cancer effect. It is concluded that for co-loaded lipodisks, cytotoxicity data in vitro could be used to predict their inhibitory activity in vivo, potentially enhancing the clinical outcome of synergistic therapy.

PREDIKSI INTERAKSI MOLEKUER CAPE DENGAN IL-2, CD25, IL-10, CTLA-4, IDO, TGFβ, and CCL2 DENGAN SOFTWARE DOCKING MOLEKULER

The use of herbal medicine as a nutritious ingredient to treat various diseases is still mostly done traditionally based on hereditary information. The development of technology supports the exploration of natural materials as a nutritious material with various scientific evidences. The development of computational and bioinfrmatic technology simulates interactions between various active compounds and target molecules virtually commonly known as molecular docking, a virtual screening based on structure used to predict the interaction of three-dimensional structures of small molecules (compounds) with the target molecular structure. Propolis is a substance produced by bees from resins collected from plants and combined with wax and secretions from bee salivary glands which are rich in various enzymes. In the world of medicine, propolis has been used since time immemorial and until now it has been known that propolis can be a natural medicine with anti-bacterial, anti-tumor, antioxidant and immunomodulatory abilities. Molecular docking between one of the active compounds in propolis, namely caffeic acid phenethyl ester (CAPE) with various target molecules such as IL-2, CD25, IL10, TGFβ, CTLA-4, CCL21, and IDO using the DockingServer software provides information that CAPE has inhibitory activity against IL-2, CD25, IL-10, CTLA-4, IDO, TGFβ, and CCL2 so that it has the potential to be further developed as a candidate for immunomodulatory therapy.

Abstract IA09: Harnessing natural and engineered properties of NKT cells for cancer immunotherapy

Abstract Due to their natural anti-tumor properties and ability to preferentially localize to the neuroblastoma (NB) tumor site, Vα24-invariant natural killer T cells (NKTs) are promising candidate immune effectors for chimeric antigen receptor (CAR)-based immunotherapies targeting NB and other solid tumors. We have previously demonstrated that human NKTs expressing a CAR specific for ganglioside GD2 (CAR.GD2) mediated potent anti-tumor activity in a xenogeneic NB model in NOD/SCID/IL-2Rγnull mice. In comparison with CAR.GD2 T cells, CAR.GD2 NKTs localized more effectively to the tumor tissues and did not induce graft-versus-host disease (GvHD). Clinical development of NKT cell-based therapeutics requires overcoming two fundamental challenges: 1) the low frequency of NKTs in human peripheral blood, and 2) the limited ability of adoptively transfered NKTs/CAR-NKTs to persist in tumor-bearing animals. To address the first limitation, we have developed a cGMP protocol to isolate NKT cells from leukapheresis products using NKT-specific magnetic beads with the CliniMAX® system (Miltenyi). Isolated NKTs then undergo stimulation with CD1d-expressing antigen-presenting cells pulsed with α-galactosylceramide, retroviral transduction with a CAR-expressing vector, and rapid numeric expansion in cytokine-supplemented culture. This protocol routinely produces more than 109 CAR-NKTs within 17 days with average NKT cell purity and CAR expression of 96% and 54%, respectively. To overcome the second limitation, we incorporated the primary NKT homeostatic cytokine, IL-15, into the CAR.GD2 construct and evaluated its ability to enhance NKT cell in vivo persistence and therapeutic efficacy. Following adoptive transfer into mice bearing human NB xenografts, NKTs expressing CAR.GD2 were undetectable by three weeks whereas CAR.GD2/IL-15 NKTs underwent progressive expansion at sites of NB metastasis, reaching 32% of bone marrow cells two months after a single injection. Treatment with CAR.GD2/IL-15 NKTs resulted in a median survival of 70 days versus 48 days for mice treated with CAR.GD2 NKTs (P < 0.001). Importantly, the robust in vivo expansion and potent anti-tumor activity of CAR.GD2/IL-15 NKTs did not produce off-target toxicity or induce GvHD based on detailed pathological analysis of mouse tissues. These results have enabled the initiation of a first-in-human CAR-NKT clinical trial evaluating the safety of autologous NKTs expressing the GD2-specific CAR with IL-15 in children with relapsed or refractory neuroblastoma (NCT03294954). Citation Format: Andras Heczey, Amy N. Courtney, Ho Ngai, Gengwen Tian, Simon N. Robinson, Gianpietro Dotti, Leonid S. Metelitsa. Harnessing natural and engineered properties of NKT cells for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA09.

Chemotherapy of Breast Cancer Cells Using Novel pH-Responsive Cellulose-Based Nanocomposites.

Purpose: The objective of the current study was to compare the anticancer efficacy of doxorubicin-loaded cellulose based magnetic (Fe3O4), zinc oxide (ZnO) nanoparticles on and free doxorubicin (DOX) on MCF-7 breast cancer cells. Methods: Novel pH-sensitive cellulose-graft poly acrylic acid based Fe3O4 (Cellulose-g-PAAg- PAcMNPs) and ZnO (Cellulose-g-PAA-g-PAcZnO) nanocomposites were synthesized via polymerization of acrylic acid and modified 3-(trimethoxysilyl) propyl methacrylate onto the cellulosic backbone via reversible addition-fragmentation chain transfer (RAFT) method. Results: Cellulose-g-PAA-g-PAcMNPs and Cellulose-g-PAA-g-PAcZnO nanocarriers with mean diameter of 15 and 38 nm were prepared successfully. DOX was loaded effectively to the ZnO and Fe3O4 nanocarriers via complexing and electrostatic force with great encapsulation efficiency of 99.07% and 98.92%, respectively. DOX-loaded nanocarriers showed obvious pHdependent tumor specific drug release pattern. MTT assay results indicated that IC50 of the DOX loaded Cellulose-g-PAA-g-PAcZnO, DOX loaded Cellulose-g-PAA-g-PAcMNPs and free DOX after 48 hours treatment with MCF7 cell lines were about 24.03, 49.27 and 99.76 μg mL−1, respectively. Therefore both DOX nanoformulations significantly increase antitumor ability compared to free DOX (P < 0.05). The results of MTT assay and DAPI staining revealed that DOX-loaded Cellulose-g-PAA-g-PAcZnO NPs show higher chemotherapy efficiency in MCF7 breast cancer cell line compare to the DOX-loaded Cellulose-g-PAA-g-PAcMNPs due to high interaction of ZnO with DOX. Conclusion: The formation of the complexes between the DOX and ZnO nanoparticles at the chelating sites of the quinone and the phenolic oxygen molecules of DOX, lead to more sustained drug release and enhanced chemotherapy effectiveness by increasing the intracellular concentration of DOX.

Inhibition of mouse RM-1 prostate cancer and B16F10 melanoma by the fusion protein of HSP65 & STEAP1 186-193

The research of tumor vaccine plays a crucial role in tumor immunotherapy. This study has constructed and prepared a fusion protein vaccine of heat shock protein 65 (HSP65) and the octapeptide epitope 186-193 of the six transmembrane epithelial antigen of the prostate 1 (STEAP1 186-193), and investigated the inhibitory effect of the fusion protein on mouse RM-1 prostate cancer and B16F10 melanoma xenografts. The fusion protein His-HSP65-STEAP1 186-193 (HHST1), His-HSP65-2×STEAP1 186-193 (HHST2) and His-HSP65-6×STEAP1 186-193 (HHST6) were obtained by setting different copy number of STEAP1 186-193 and adding His purification tag before HSP65. Firstly the inhibitory effect of fusion protein on mouse RM-1 prostate cancer xenografts has been studied, which could be the basis of the study the inhibitory effect of the best fusion protein on mouse B16F10 melanoma xenografts. All studies compared with the fusion protein His-HSP65 (HHSP65), the fusion proteins HHST1, HHST2 and HHST6 all could significantly inhibit the growth of mouse RM-1 prostate cancer xenografts. In addition, the fusion protein HHST2 was proved to be the best compared with the fusion proteins HHST1 and HHST6 (P<0.05). Apart from this, compared with the fusion protein HHSP65, the fusion protein HHST2 also significantly inhibited the growth of mouse beared B16F10 melanoma. The results above indicate that HSP65 and STEAP1 186-193 can significantly inhibit the growth of mouse RM-1 prostate cancer and B16F10 melanoma xenografts, and the appropriate increase of copy number can effectively improve that the fusion protein has an excellent anti-tumor ability.

Antitumor activity and ability to prevent acrylamide formation in fried foods of asparaginase from soybean root nodules.

A novel asparaginase (designated srnASNase) has been purified from soybean root nodules and identified by MALDI-TOF/TOF-MS. And the enzymatic properties, antitumor activity and the ability to prevent acrylamide formation in fried foods of srnASNase were evaluated. SrnASNase had high specific activity (531.37 U/mg) toward L-asparagine under optimum conditions (pH 8.0 and 40°C), no activity toward L-glutamine and D-glutamine, but trace activity toward D-asparagine. It was stable in the pH range of 7.0-9.0 and up to 40°C. The Km and Vmax of srnASNase were 0.36mM and 51.64mM/min, respectively. Further, in vitro anti-proliferative activity on human cancer cells assay showed that srnASNase was superior to commercial asparaginase in solution by controlling the tumor cell growth with time. In addition, srnASNase showed more effective acrylamide mitigation than commercial asparaginase in fried foods. These results indicate that srnASNase is a potential candidate for applications in the food processing and pharmaceutical industry. PRACTICAL APPLICATIONS: L-asparaginase (L-asparagine amidohydrolase; EC 3.5.1.1) is an enzyme that catalyzes the hydrolysis of the amide group of the side-chain of L-asparagine into aspartic acid and ammonia. It has long been used as a primary component in the treatment of acute lymphoblastic leukemia (All) and other related blood cancers. Apart from its clinical usage, L-asparaginase has attracted more attention in the food processing industries as a promising acrylamide-mitigating agent in recent years. This research revealed that soybean root nodules might be good sources of novel asparaginase.