HT-29 Tumor Research Articles

Preclinical evaluation of [99mTc]Tc-labeled anti-EpCAM nanobody for EpCAM receptor expression imaging by immuno-SPECT/CT.

Overexpression of epithelial cell adhesion molecule (EpCAM) plays essential roles in tumorigenesis and tumor progression in almost all epithelium-derived cancer. Monitoring EpCAM expression in tumors can be used for the diagnosis, staging, and prognosis of cancer patients, as well as guiding the individualized treatment of EpCAM-targeted drugs. In this study, we described the synthesis and evaluation of a site-specifically [99mTc]Tc-labeled EpCAM-targeted nanobody for the SPECT/CT imaging of EpCAM expression. We first prepared the [99mTc]Tc-HYNIC-G4K; then, it was site-specifically connected to EpCAM-targeted nanobody NB4. The in vitro characteristics of [99mTc]Tc-NB4 were investigated in HT-29 (EpCAM positive) and HL-60 (EpCAM negative) cells, while the in vivo studies were performed using small-animal SPECT/CT in the subcutaneous tumor models and the lymph node metastasis model to verify the specific targeting capacity as well as the potential applications of [99mTc]Tc-NB4. [99mTc]Tc-NB4 displayed a high EpCAM specificity both in vitro and in vivo. SPECT/CT imaging revealed that [99mTc]Tc-NB4 was cleared rapidly from the blood and normal organs except for the kidneys, and HT-29 tumors were clearly visualized in contrast with HL-60 tumors. The uptake value of [99mTc]Tc-NB4 in HT-29 tumors was increased continuously from 3.77 ± 0.39%ID/g at 0.5h to 5.53 ± 0.82%ID/g at 12h after injection. Moreover, the [99mTc]Tc-NB4 SPECT/CT could clearly image tumor-draining lymph nodes. [99mTc]Tc-NB4 is a broad-spectrum, specific, and sensitive SPECT radiotracer for the noninvasive imaging of EpCAM expression in the epithelium-derived cancer and revealed a great potential for the clinical translation.

Celecoxib-Induced Modulation of Colon Cancer CD133 Expression Occurs through AKT Inhibition and Is Monitored by 89Zr Immuno-PET.

We developed an immuno-PET technique that monitors modulation of tumor CD133 expression, which is required for the success of CD133-targeted therapies. Methods. Anti-CD133 antibodies were subjected to sulfhydryl moiety-specific 89Zr conjugation. 89Zr-CD133 IgG was evaluated for specific activity and radiolabel stability. Colon cancer cells underwent binding assays and Western blotting. Biodistribution and PET studies were performed in mice. Results. 89Zr-CD133 IgG showed excellent target specificity with 97.2 ± 0.7% blocking of HT29 cell binding by an excess antibody. Intravenous 89Zr-CD133 IgG followed biexponential blood clearance and showed CD133-specific uptake in HT29 tumors. 89Zr-CD133 IgG PET/CT and biodistribution studies confirmed high HT29 tumor uptake with lower activities in the blood and normal organs. In HT29 cells, celecoxib dose-dependently decreased CD133 expression and 89Zr-CD133 IgG binding that reached 19.9 ± 2.1% (P < 0.005) and 50.3 ± 10.9% (P < 0.001) of baseline levels by 50 μM, respectively. Celecoxib treatment of mice significantly suppressed tumor CD133 expression to 67.5 ± 7.8% of controls (P < 0.005) and reduced tumor 89Zr-CD133 IgG uptake from 15.5 ± 1.4% at baseline to 12.3 ± 2.0%ID/g (P < 0.01). Celecoxib-induced CD133 reduction in HT29 cells and tumors was associated with substantial suppression of AKT activation. There were also reduced HIF-1α accumulation and IκBα/NFκB phosphorylation. Conclusion. 89Zr-CD133 IgG PET provides high-contrast tumor imaging and monitors celecoxib treatment-induced modulation of tumor CD133 expression, which was found to occur through AKT inhibition. This technique may thus be useful for screening drugs that can effectively suppress colon cancer stem cells.

Noninvasive Evaluation of EGFR Expression of Digestive Tumors Using 99mTc-MAG3-Cet-F(ab')2-Based SPECT/CT Imaging.

Purpose This study is aimed at investigating the feasibility of cetuximab (Cet) F(ab′)2 fragment- (Cet-F(ab′)2-) based single photon emission tomography/computed tomography (SPECT/CT) for assessing the epidermal growth factor receptor (EGFR) expression in digestive tumor mouse models. Methods Cet-F(ab′)2 was synthesized using immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) protease and purified with protein A beads. The product and its in vitro stability in normal saline and 1% bovine serum albumin were analyzed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The EGFR expression in the human colon tumor cell line HT29 and the human stomach tumor cell line MGC803 were verified using western blotting and immunocytochemistry. Cet-F(ab′)2 was conjugated with 5(6)-carboxytetramethylrhodamine succinimidyl ester to demonstrate its binding ability to the MGC803 and HT29 cells. Cet-F(ab′)2 was conjugated with NHS-MAG3 for 99mTc radiolabeling. The best imaging time was determined using a biodistribution assay at 1, 4, 16, and 24 h after injection of the 99mTc-MAG3-Cet-F(ab′)2 tracer. Furthermore, 99mTc-MAG3-Cet-F(ab′)2 SPECT/CT was performed on MGC803 and HT29 tumor-bearing nude mice. Results HT29 cells had low EGFR expression while MGC803 cell exhibited the high EGFR expression. Cet-F(ab′)2 and intact cetuximab showed similar high binding ability to MGC803 cells but not to HT29 cells. Cet-F(ab′)2 and 99mTc-MAG3-Cet-F(ab′)2 showed excellent in vitro stability. The biodistribution assay showed that the target to nontarget ratio was the highest at 16 h (17.29 ± 5.72, n = 4) after tracer injection. The 99mTc-MAG3-Cet-F(ab′)2-based SPECT/CT imaging revealed rapid and sustained tracer uptake in MGC803 tumors rather than in HT29 tumors with high image contrast, which was consistent with the results in vitro. Conclusion SPECT/CT imaging using 99mTc-MAG3-Cet-F(ab′)2 enables the evaluation of the EGFR expression in murine EGFR-positive tumors, indicating the potential utility for noninvasive evaluation of the EGFR expression in tumors.

Immunomodulatory and antitumor activities of the exopolysaccharide produced by potential probiotic Lactobacillus plantarum YW11 in a HT-29 tumor-burdened nude mouse model

In this study, the exopolysaccharides (EPSs)-producing L. plantarum YW11 was initially evaluated for its tolerance to simulated artificial gastric juice and bile salts, and cell surface hydrophobicity. Subsequent study using a HT-29 tumor-burdened nude mouse model showed that the EPS had potent immunomodulatory and antitumor activities. Administration of the EPS had no influence on body weight of the mice, and it could significantly prevent HT-29 tumor cells induced acute liver and kidney damages. The purified EPS at a dose of 5 mg/mL was able to enhance effectively the killing activities of splenic natural killer cells and cytotoxic T lymphocyte, and to stimulate the secretion of serum cytokines IL-2 and TNF-α. As evidenced by a cell culture assay, the EPS exhibited obvious inhibition on growth of colon cancer HT-29 cells treated with 600 μg/mL of the EPS for 72 h with an inhibition rate of 58.02%. Administration of the EPS obviously inhibited growth of HT-29 xenograft tumor to an extent near that of the positive control (5-Fu, 25 mg/kg bw). Therefore, the results of this study indicated the important roles of the EPS in the potential probiotic functions of the strain.

Chemical Compounds, Antitumor and Antimicrobial Activities of Dry Ethanol Extracts from Koelreuteria paniculata Laxm.

Koelreuteria paniculata Laxm. is used in traditional medicine and has various established biological activities, however, the species is considered to be a potentially invasive alien tree species for Bulgarian flora. However, there is still much to be studied about the phytochemical and biological characteristics of the species. The present study aimed to determine the chemical composition of the ethanol extracts of aerial plant parts, by GC-MS analysis, and to thereby evaluate their in vitro antitumor and antibacterial properties. All three extracts were tested against the HT-29 and PC3 tumor cell lines using the MTT assay. Fifty-six components were identified from leaf, flower, and stem bark extracts, and over 10% were the following constituents: pyrogallol, α-terpinyl acetate, neryl acetate, and α-terpinyl isobutanoate. The oxygenated monoterpenes predominated in the extracts, followed by the oxygenated aliphatics and phenylpropanoids. Significant antiproliferative activity on the HT-29 cell line (IC50–21.44 µg/mL and 23.63 µg/mL, respectively) was found for the flower and leaf extracts. Antibacterial activity was established for the following bacteria strains: Bacillus subtilis ATCC 6633, Bacillus cereus NCTC 10320, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 6027, and Proteus vulgaris ATCC 6380. The stem bark and flower extracts showed better antimicrobial potential. K. paniculata could be considered as a potential source of biologically active substances with antitumor and antibacterial properties.

Imidazole Analogs of Vascular-Disrupting Combretastatin A-4 with Pleiotropic Efficacy against Resistant Colorectal Cancer Models.

Specific targeting of the tumoral vasculature by vascular-disrupting agents (VDA), of which combretastatin A-4 (CA-4) is a main representative, has been considered a new therapeutic strategy against multidrug-resistant tumors. In addition, CA-4 and analogs are tubulin-targeting agents and can exert direct antitumor effects by different mechanisms. Herein, we analyzed a series of synthetic CA-4 analogs featuring N-methylimidazole-bridged Z-alkenes with different halo- or amino-substituted aryl rings in vitro and in vivo, focusing on models of colorectal cancer. Combined in vitro/in vivo structure–activity relationship studies using cell lines and xenograft tumors susceptible to VDA-induced vascular damage demonstrated a clear association of cytotoxic and vascular-disrupting activity with the ability to inhibit tubulin polymerization, which was determined by specific substitution constellations. The most active compounds were tested in an extended panel of colorectal cancer (CRC) cell lines and showed activity in CA-4-resistant and chemotherapy-resistant cell lines. The bromo derivative brimamin was then compared with the known fosbretabulin (CA-4P) by activity tests on DLD-1- (multidrug-resistant) and HT29- (CA-4-resistant) derived xenograft tumors. Treatment did not induce pronounced vascular-disrupting effects in these tumors. Histological analyses revealed distinct tumor substructures and vessel compositions of DLD-1/HT29 tumors, which clearly differed from the tumor models susceptible to VDA treatment. Even so, brimamin effectively retarded the growth of DLD-1 tumors, overcoming their resistance to standard treatment, and it inhibited the outgrowth of disseminated HT29 tumor cells in an experimental metastasis model. In conclusion, combretastatin analogous N-methylimidazoles proved capable of inducing vascular-disrupting effects, comparable to those of CA-4P. In addition, they showed antitumor activities in models of drug-resistant colorectal cancer, independent of vascular-disrupting effects.

Pharmacologic Ascorbate Enhances the Therapeutic Index of ATM-Inhibitor Based Chemoradiation for Colorectal Cancer

Ataxia telangiectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury.Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57bl/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors.Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH- is H202 mediated as it is completely attenuated by inducible catalase expression. P-AscH- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH-significantly decreased nuclear localization of activated pATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT.P-AscH- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH- enhances DNA DSBs by stimulating an H202 flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

DNA aptamer S11e recognizes fibrosarcoma and acts as a tumor suppressor

Fibrosarcoma is a serious malignant mesenchymal tumor with strong invasiveness, high recurrence, and poor prognosis. Currently, surgical resection is the main treatment for fibrosarcoma. However, due to the lack of specific biomarkers, the inability to accurately diagnose fibrosarcoma can lead to sub-optimal surgical outcomes and decreased survival. Here, we seek to address this translational barrier and we show that DNA aptamer S11e was able to recognize fibrosarcoma cells (HT1080) but not human embryonic lung fibroblast cells with Kd values in the nanomolar range. In addition, we found that S11e discerned tumors in HT1080 xenograft mouse models and tumor tissues from fibrosarcoma patients. Furthermore, we demonstrated that S11e internalized into HT1080 cells independent of the lysosome pathway and located in mitochondria. Moreover, we revealed that S11e promoted the apoptosis of HT1080 cells and inhibited HT1080 cell migration. Finally, we investigated the biologically functional cellular target of S11e using a mass spectrometry approach, and identified that Diablo/SMAC protein is a cellular binding protein of S11e, by interacting to which S11e inhibited HT1080 cell migration and invasion. Taken together, these results provide the evidence that S11e may be useful for early diagnosis, targeted therapy, and prognostication of fibrosarcoma.

Establishment of an In Vivo Xenograft Mouse Model of a Subcutaneous Submillimeter HT-29 Tumor Formed from a Single Spheroid Transplanted Using Radiation-Crosslinked Gelatin Hydrogel Microwell

Colorectal cancer is a frequent cause of death worldwide. The detection and treatment of small nodules are crucial for improving survival of colorectal cancer patients. Submillimeter tumors are useful tools for developing novel methods to approach this issue. However, there are no suitable in vivo models that allow easy monitoring of the growth of these tumors. This study established a xenograft mouse model of subcutaneous submillimeter tumors with human colorectal cancer HT-29 cells. We transplanted a single spheroid formed by HT-29 cells expressing red fluorescent protein (RFP) (HT-29-RFP). Additionally, we adopted our newly developed radiation-crosslinked gelatin hydrogel microwells (rGHMs), which can be used as a culture base to form spheroids and as a transplantation scaffold with biocompatibility and biodegradability. Spheroids approximately 700 μm in size were uniformly created in seven days in the respective rGHMs. Every single spheroid was extracted either with or without rGHM and transplanted into the subcutis of severe combined immunodeficiency (SCID) mice (n = 4). After 21 days, the spheroids inoculated together with rGHM successfully formed uniform subcutaneous submillimeter tumor xenografts that were observable in vivo in a stereoscopic fluorescence microscope in all transplanted mice. In contrast, spheroids transplanted without rGHM also developed small tumors in all mice but showed higher variability in size than those transplanted with rGHM. During transplantation, the rGHM ensured easy handling and stabilization of the position of a single spheroid. Inoculation of spheroids with rGHM in the nude mice was similarly examined (n = 4), showing that only one out of four mice formed tumors. In conclusion, rGHM effectively formed spheroids and created uniformed xenografted submillimeter tumors of HT-29-RFP in SCID mice. Our model could provide a useful platform to develop medicines and methods for detection and treatment of small nodules of colorectal cancer.

Glucosinolate-Enriched Fractions from Maca (Lepidium meyenii) Exert Myrosinase-Dependent Cytotoxic Effects against HepG2/C3A and HT29 Tumor Cell Lines

The consumption of glucosinolate (GL)-rich foods, including Brassica vegetables, such as mustard, broccoli, and maca, is associated with decreased risk of developing cancer. The GL content in maca, which is recognized as a “superfood”, is approximately 100-times higher than that in other brassicas. Although maca is a potential dietary source of GLs, limited studies have examined the bioactivity of maca GLs using the combination of chemical characterization and bioassays. In this study, the fractions (Lm-II and Lm-III) rich in intact GLs (glucotropaeolin and glucolimnanthin) were isolated and characterized from maca ethanolic extracts using chromatography and mass spectrometry. Additionally, the growth-inhibitory effects of Lm-II and Lm-II fractions against hepatocellular carcinoma (HepG2/C3A) and colon adenocarcinoma (HT29) cell lines were examined in the absence or presence of myrosinase (MYR). Fractions lacking low molecular weight sugars dose-dependently exerted cytotoxic effects in the presence of MYR. The half-maximal inhibitory concentration values of Lm-II and Lm-III against HepG2/C3A were 118.8 and 69.9 µg/mL, respectively, while those against HT29 were 102.6 and 71.5 µg/mL, respectively. These results suggest that the anticancer properties of maca can be attributed to GLs and corroborate the categorization of maca as a “superfood.” Supplemental data for this article is available online at https://doi.org/10.1080/01635581.2021.1952444

Abstract 270: Enhanced anti-tumor effect and tolerance of novel irinotecan (sn-38) nanoparticle with double core-shell micelle technology

Abstract SN-38 is a potent active metabolite of irinotecan with wide range of anti-tumor effect acknowledged in pancreatic, ovarian, lung, breast cancer models as well as colorectal cancer. Studies suggest irinotecan has extremely low solubility in biological fluid, and since irinotecan is a prodrug with the low conversion rate to the pharmaceutically active form, SN-38, the indication is limited to colorectal cancer. SN Bioscience has developed stable and safe SN-38 nano-particle injection (SNB-101) with approved biopolymers with double core-shell micelle technology and conducted preclinical studies in various tumor models for the assessment of efficacy. Outcomes suggest reduction in the tumor weight and inhibition of the growth rate in the every human tumor cell lines, revealing that 20 mg/kg (as SN-38) of SNB-101 is more effective in reducing tumor size than its comparative substance (Irinotecan HCl, 50 mg/kg). The results indicate the MTD of SNB-101 could be increased by least 2.67 folds through new drug delivery system by nano-particle injection. To further investigate the antitumor activity of SNB-101, we have applied the therapy in combination with different kinds of medications in the additional models. First, we have examined synergistic antitumor activity of SNB-101 in combined therapy with Docetaxel in various mouse xenograft models of gastric cancer (Hs746T), breast cancer (MDA-MB-231), and lung cancer (A549). As results, we have established the combination use of SNB-101 plus Docetaxel had significantly greater antitumor activity against all the monotherapy control groups. Second, we have continued to investigate the advantages of combination treatment of SNB-101 plus Radiotherapy (5Gy) in mice xenograft derived with SCLC (NCI-H69) and colorectal cancer (HCT116). From these studies, promising outcome of antitumor action was also noted, recapitulating excellent therapeutic effect of SNB-101 alone and in combination with Radiotherapy against lung cancer and colorectal cancer cells. Furthermore, an additional study with combination of SNB-101 plus an anti-vascular endothelial growth factor humanized monoclonal antibody (Bevacizumab) showed significantly stronger anti-tumor activities against the HT-29 xenograft model than did the monotherapy with either agent. As the addition of Bevacizumab to SNB-101 monotherapy is associated with profound inhibition of HT-29 tumor growth progression, the consistent reduction in tumor weights was also observed. Although tolerability of the substance in clinical studies is well known, there are some limitations in correlating the results of pre-clinical models to in-depth comprehension of human model; thus, we hope to further investigate the pharmacological action of SNB-101 in new regimens and indications in the next step - First in Human trial. Citation Format: Jong Oh Kim, Eun Kyung Choi, Seong Yun Jeong, Jung Jin Hwang, Eun Sung Jun, Jae-Min Kim, Hong-Mei Zheng, Younghwan Park, Siyoung Jung. Enhanced anti-tumor effect and tolerance of novel irinotecan (sn-38) nanoparticle with double core-shell micelle technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 270.

Abstract 1847: Anti-GARP antibody DS-1055a augments antitumor immunity by depleting highly suppressive GARP+ regulatory T cells

Abstract Immune checkpoint blockers (ICBs) have drastically changed the clinical care of cancer; however, the population of patients who can benefit is relatively small because of intrinsic or acquired resistance to immune therapy. To evade immune destruction, tumors exploit several distinct strategies including immunosuppressive cells such as regulatory T (Treg) cells. Treg cells, an essential component for maintaining self-tolerance, inhibit antitumor immunity, consequently hindering protective cancer immunosurveillance and hampering effective antitumor immune responses in tumor-bearing hosts. It is often reported that a high ratio of Treg cells to effector CD8+ T cells is associated with poor prognosis in patients with cancer, and Treg cells represent one of the most important factors associated with resistance to ICBs, suggesting that Treg cells represent a new promising target for anti-cancer therapy. In this study, we performed a comprehensive screening to identify a tumor-infiltrating Treg cell-specific molecule among immune cell subsets in humans. Glycoprotein A repetitions predominant (GARP) was identified as a suitable target for selective Treg cell depletion in the tumor microenvironment (TME) of multiple caner types. In the periphery, GARP was selectively induced in Treg cells, but not in effector T cells, by polyclonal stimulation in humans. DS-1055a, a novel afucosylated anti-human GARP antibody, effectively depleted GARP+ Treg cells, leading to the activation of effector T cells. Moreover, DS-1055a decreased FOXP3+CD4+ T cell counts in the TME and exhibited remarkable antitumor activity in a human peripheral blood mononuclear cell-transplanted humanized mouse model bearing HT-29 tumors. From these results, we propose that DS-1055a can be a new Treg cell-targeted regent that augments antitumor T cell immunity by depleting GARP+ Treg cells. Currently, a Phase I study of DS-1055a is on-going (NCT04419532). Citation Format: Kazuki Satoh, Yoichi Kobayashi, Kaori Fujimaki, Masato Hata, Shinko Hayashi, Saori Ishida, Daisuke Sugiyama, Takahiko Sato, Lim Kyungtaek, Megumi Miyamoto, Shiho Kozuma, Michinori Kadokura, Kenichi Wakita, Kazuki Hirahara, Masato Amano, Ichiro Watanabe, Atsushi Okamoto, Andrea Tuettenberg, Helmut Jonuleit, Atsushi Tanemura, Shoichi Maruyama, Toshinori Agatsuma, Teiji Wada, Hiroyoshi Nishikawa. Anti-GARP antibody DS-1055a augments antitumor immunity by depleting highly suppressive GARP+ regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1847.

Novel anti-GARP antibody DS-1055a augments anti-tumor immunity by depleting highly suppressive GARP+ regulatory T cells.

Regulatory T (Treg) cells, which are essential for maintaining self-tolerance, inhibit anti-tumor immunity, consequently hindering protective cancer immunosurveillance, and hampering effective anti-tumor immune responses in tumor-bearing hosts. Here, we show that depletion of Treg cells via targeting glycoprotein A repetitions predominant (GARP) induces effective anti-tumor immune responses. GARP was specifically expressed by highly suppressive Treg cells in the tumor microenvironment (TME) of multiple cancer types in humans. In the periphery, GARP was selectively induced in Treg cells, but not in effector T cells, by polyclonal stimulation. DS-1055a, a novel afucosylated anti-human GARP monoclonal antibody, efficiently depleted GARP+ Treg cells, leading to the activation of effector T cells. Moreover, DS-1055a decreased FoxP3+CD4+ T cells in the TME and exhibited remarkable anti-tumor activity in humanized mice bearing HT-29 tumors. We propose that DS-1055a is a new Treg-cell-targeted cancer immunotherapy agent with augmentation of anti-tumor immunity.

The combination of lactoferrin and linolenic acid inhibits colorectal tumor growth through activating AMPK/JNK-related apoptosis pathway.

Colorectal cancer is a common cause of death with few available therapeutic strategies, and the preventative complexes in adjunctive therapy are urgently needed. Increasing evidences have shown that natural ingredients, including lactoferrin, oleic acid, docosahexaenoic acid (DHA) and linolenic acid, possess anti-inflammatory and anti-tumor activities. However, investigations and comparisons of their combinations in colorectal tumor model have not been reported, and the mechanism is still unrevealed. In the study, we examined the viability, migration, invasion and apoptosis of HT29 cells to choose the proper doses of these components and to select the effective combination in vitro. BALB/c nude mice bearing colorectal tumor were used to explore the role of selected combination in inhibiting tumor development in vivo. Additionally, metabonomic detection was performed to screen out the specific changed metabolitesand related pathway. The results demonstrated that lactoferrin at 6.25 μM, oleic acid at 0.18 mM, DHA at 0.18 mM, and linolenic acid at 0.15 mM significantly inhibited the viabilities of HT29 cells (p < 0.05). The combination of lactoferrin (6.25 μM) + linolenic acid (0.15 mM) exhibited the strongest activity in inhibiting the migration and invasion of HT29 cells in vivo and suppressing tumor development in vitro (p < 0.05). Furthermore, the lactoferrin + linolenic acid combination activated p-AMPK and p-JNK, thereby inducing apoptosis of HT29 cells (p < 0.05). The present study was the first to show that lactoferrin + linolenic acid combination inhibited HT29 tumor formation by activating AMPK/JNK related pathway.

Pharmacologic ascorbate enhances the therapeutic index of ATM-inhibitor based chemoradiation for colorectal cancer.

3609 Background: Ataxia telangectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury. Methods: Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57blj/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors. Results: Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH- is H202 mediated as it is completely attenuated by inducible catalase expression. P-AscH- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH-significantly decreased nuclear localization of activated P-ATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT. Conclusions: P-AscH- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH- enhances DNA DSBs by stimulating an H202 flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

RRx-001 Increases Erythrocyte Preferential Adhesion to the Tumor Vasculature.

Red blood cells (RBCs) serve a variety of functions beyond mere oxygen transport both in health and pathology. Notably, RRx-001, a minimally toxic pleiotropic anticancer agent with macrophage activating and vascular normalization properties currently in Phase III trials, induces modification to RBCs which could promote vascular adhesion similar to sickle cells. This study assessed whether RBCs exposed to RRx-001 adhere to the tumor microvasculature and whether this adhesion alters tumor viability. We next investigated the biomechanics of RBC adhesion in the context of local inflammatory cytokines after treatment with RRx-001 as a potential mechanism for preferential tumor aggregation. Human HEP-G2 and HT-29 tumor cells were subcutaneously implanted into nu/nu mice and were infused with RRx-001-treated and Technetium-99m (99mTc)-labeled blood. RBC adhesion was quantified in an in vitro human umbilical vein endothelial cell (HUVEC) assay under both normoxic and hypoxic conditions with administration of either lipopolysaccharide (LPS) or Tumor necrosis alpha (TNF) to mimic the known inflammation in the tumor microenvironment. One hour following administration of 99mTc labeled RBCs treated with 10 mg/kg RRx-001, we observed an approximate 2.0-fold and 1.5-fold increase in 99mTc-labeled RBCs compared to vehicle control in HEPG2 and HT-29 tumor models, respectively. Furthermore, we observed an approximate 40% and 36% decrease in HEP-G2 and HT-29 tumor weight, respectively, following treatment with RRx-001. To quantify RBC adhesive potential, we determined , or the shear stress required for 50% disassociation of RBCs from HUVECs. After administration of TNF-α under normoxia, was determined to be 4.5 dynes/cm2 (95% CI: 4.3–4.7 dynes/cm2) for RBCs treated with 10 M RRx-001, which was significantly different (p < 0.05) from in the absence of treatment. Under hypoxic conditions, the difference of with (4.8 dynes/cm2; 95% CI: 4.6–5.1 dynes/cm2) and without (2.6 dynes/cm2; 95% CI: 2.4–2.8 dynes/cm2) 10 M RRx-001 treatment was exacerbated (p = 0.05). In conclusion, we demonstrated that RBCs treated with RRx-001 preferentially aggregate in HEP-G2 and HT-29 tumors, likely due to interactions between RRx-001 and cysteine residues within RBCs. Furthermore, RRx-001 treated RBCs demonstrated increased adhesive potential to endothelial cells upon introduction of TNF-α and hypoxia suggesting that RRx-001 may induce preferential adhesion in the tumor but not in other tissues with endothelial dysfunction due to conditions prevalent in older cancer patients such as heart disease or diabetic vasculopathy.

The Chemokine CX3CL1 Improves Trastuzumab Efficacy in HER2 Low-Expressing Cancer In Vitro and In Vivo.

A crucial mode of action of trastuzumab is the labeling of HER2-positive (HER2+) tumor cells for the eradication by natural killer (NK) cells, a process called antibody-dependent cellular cytotoxicity (ADCC). However, despite widespread HER2 expression among cancer entities, only a fraction, with robust HER2 overexpression, benefits from trastuzumab therapy. ADCC requires both sufficient lymphocytic infiltration and close binding of the immune cells to the antibody-tagged tumor cells. We hypothesized that the chemokine CX3CL1 could improve both processes, as it is synthesized as a membrane-bound, adhesive form that is eventually cleaved into a soluble, chemotactic protein. Here, we show that CX3CL1 overexpression is a positive prognostic marker in breast cancer. CX3CL1 overexpression attracted tumor-suppressive lymphocytes, including NK cells, and inhibited tumor growth and lung metastasis in the syngeneic 4T1 breast cancer mouse model. In HER2+ SKBR3, MDA-MB-453, and HT-29 tumor cells, CX3CL1 overexpression increased NK cell-mediated cytotoxicity in vitro and acted synergistically with trastuzumab. Even though CX3CL1 did not further improve trastuzumab efficacy in vivo in the trastuzumab-sensitive MDA-MB-453 model, it compensated for NK-cell depletion and prolonged survival. In the HER2 low-expressing HT-29 model, however, CX3CL1 overexpression not only prolonged survival time but also overcame trastuzumab resistance in a partly NK cell-dependent manner. Taken together, these findings identify CX3CL1 as a feasible pharmacologic target to enable trastuzumab therapy in HER2 low-expressing cancers and render it a potential predictive biomarker to determine therapy responders.

MgB2 powders and bioevaluation of their interaction with planktonic microbes, biofilms, and tumor cells

Commercial nanopowders of MgB2 were characterized from the viewpoint of granulometric distribution, structure, microstructure, and pH behavior in water. The powders are very different: a higher amount of the MgB2 phase with a lower tendency for agglomeration determines a higher rate of pH-increase. A higher rate of pH-increase usually produces a stronger antimicrobial activity against Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Candida parapsilosis reference strains. The variation of the pH-increase rate suggests the possibility of temporo-spatial control of MgB2 bioactivity, although the contribution of other factors should not be neglected. Remarkably, the efficiency of the MgB2 powders is higher against biofilms than on microbes in the planktonic state. Further, our experiments confirm the antimicrobial efficiency of MgB2 in the in vitro tests against 29 methicillin resistant clinical S. aureus isolates and 33 vancomycin resistant E. faecium/faecalis strains, but in this case the biofilms are more resistant than planktonic cells. The MgB2 treatment of infected mice led to a significant decrease of E. coli colonization in liver, spleen and peritoneal liquid and it also caused changes in the intestinal microbiota. The activity of powders on HeLa and HT-29 tumor cell lines was assessed by inverted microscopy, flow cytometry, and evaluation of the cellular cycle. MgB2 inhibits tumor cell growth influencing DNA synthesis (S-phase). The obtained results indicate that the tested powders could provide promising solutions for the development of large-spectrum multifunctional antimicrobial and anti-biofilm agents, and/or for anti-cancer therapies.

Chitosan-coated poly(є-caprolactone) nanocapsules for mucoadhesive applications of perillyl alcohol

ABSTRACT Perillyl alcohol (POH) is an essential oil found in several plants and presents significant therapeutic and chemo-preventive activity against some tumors. However, it exhibits low aqueous solubility and thereby reduced oral absorption and bioavailability. Nanoencapsulation of POH can be an important tool to overcome such limitations. In this work, chitosan was used for coating poly (ε-caprolactone) nanocapsules for mucoadhesive applications of POH. The nanocapsules were obtained by the nanoprecipitation method and presented a mean diameter of about 330 nm, spherical morphology, polydispersity index of 0.14, zeta potential of +32 mV, and POH encapsulation efficiency of 56%. POH was prolonged released from nanocapsules. In mucoadhesion studies, nanocapsules showed higher adsorption to mucin compared to free POH, possibly due to electrostatic interactions between chitosan and mucin. In the cytotoxicity assay, POH-loaded nanocapsules presented higher IC50 than free POH; however, they were more selective for HT-29 tumor cells than for normal Vero cells. The results demonstrate the potential application of chitosan-coated nanocapsules for the oral administration of POH intended for cancer therapy.

“One-stitch” bioorthogonal prodrug activation based on cross-linked lipoic acid nanocapsules

Bioorthogonal prodrug activation is fascinating but suffers from staggered administration of prodrug and trigger, which would not only reduce the therapeutic effect but bring great inconvenience for clinical application. Herein, we report a new cross-linked lipoic acid nanocapsules (cLANCs) based two-component bioorthogonal nanosystem for “one-stitch” prodrug activation. Due to the reversible stability of cLANCs, the loaded prodrug and trigger cannot release in advance while can react upon arrival in the tumor tissue. Moreover, the cLANCs would be degraded into dihydrolipoic acid in tumor cells to potentiate the anticancer effect of the drug synthesized in situ. The data showed that the new bioorthogonal system held a killing effect 1.63 times higher than that of parent drug 3 against human colorectal tumor cells (HT29) and a tumor inhibitory rate 34.2% higher than that of 3 against HT29 tumor xenograft model with negligible side effects. The biodistribution study showed that the “one-stitch” prodrug activation exhibited a selective accumulation of 3 in the tumor tissue compared with free 3 group (34.2 μg vs 3.56 μg of 3/g of tissue). This two-component bioorthogonal nanosystem based on cross-linked lipoic acid nanocapsules constitutes the first example of “one-stitch” bioorthogonal prodrug activation.