Intensified NK cell therapy in combination with low-dose chemoradiotherapy against human colorectal cancer.

The therapeutic potential of adoptive natural Killer (NK) cells immunotherapy in combination with chemoradiotherapy, the main treatment modality for colorectal cancer (CRC), has not yet been explored. Here, we aimed to investigate the efficacy of NK cells to potentiate primary tumor control and improve survival outcomes, especially in combination with low-dose chemoradiotherapy. Ex vivo activated NK cells (> 90% purity) from healthy donors were obtained. NK cells were administered intravenously to the CRC-bearing mice and intensified in vivo in combination with low-dose 5-fluorouracil (0.5 mg/kg or 1 mg/Kg) and irradiated tumors with low doses (2 Gy or 4 Gy). Real-time NK cell cytotoxicity demonstrated a synergistic killing effect of a combination of low-dose chemoradiotherapy, mainly through NKp30 and NKG2D, showing a decrease in NK cell degranulation after blocking NKG2D and NKp30. In vivo tumor characteristics after combination treatment showed decreased CD112, CD155, MICA, and MICB expression. Under the combination strategy, 70% of the mice had free lung metastasis and 90% without secondary gross tumors, indicating suppressed distant metastasis to lung and axillary regions. This combination therapy resulted in significantly synergistic antitumor activity against primary solid tumors compared to chemoradiotherapy only. Furthermore, the intensified NK cell administration showed significantly better primary tumor control and survival outcomes than the non-intensified NK cell administration in a human colorectal HT-29 model treated with low-dose chemoradiotherapy. Optimized NK cell therapy combined with low-dose chemoradiotherapy can provide effective therapeutic potential for intractable cold human colorectal cancer.

Hint from an Enzymatic Reaction: Superoxide Dismutase Models Efficiently Suppress Colorectal Cancer Cell Proliferation.

Superoxide dismutases (SODs) are essential antioxidant enzymes that prevent massive superoxide radical production and thus protect cells from damage induced by free radicals. However, this concept has rarely been applied to directly impede the function of driver oncogenes, thus far. Here, leveraging efforts from SOD model complexes, we report the novel finding of biomimetic copper complexes that efficiently scavenge intracellularly generated free radicals and, thereby, directly access the core consequence of colorectal cancer suppression. We conceived four structurally different SOD-mimicking copper complexes that showed distinct disproportionation reaction rates of intracellular superoxide radical anions. By replenishing SOD models, we observed a dramatic reduction of intracellular reactive oxygen species (ROS) and adenine 5'-triphosphate (ATP) concentrations that led to cell cycle arrest at the G2/M stage and induced apoptosis in vitro and in vivo. Our results showcase how nature-mimicking models can be designed and fine-tuned to serve as a viable chemotherapeutic strategy for cancer treatment.

A sugar modified amphiphilic cationic nano-adjuvant ceased tumor immune suppression and rejuvenated peptide vaccine induced antitumor immunity in cervical cancer.

Human papilloma virus (HPV), one of the most common cancer-causing viruses, accounts for more than 90% of human anal and cervical cancers. Clinical studies have focused on adjuvant therapy with vaccines to improve therapeutic outcomes in patients with late-stage HPV-related cancers. In the present study, a mannose receptor (CD206) targeting a lithocholic acid-modified polyethylenimine (PEI) nano-adjuvant delivering the toll-like receptor 7/8 agonist, resiquimod (R848) (mLAPMi-R848), in a HPV E6- and E7-expressing TC-1 tumor murine model was developed. Peritumoral administration of mLAPMi resulted in enhanced accumulation in tumor/tumor-draining lymph nodes and significantly targeted antigen presenting cells like macrophage and dendritic cells. PEI-based nanocarriers can exploit the adjuvant potency of R848 and improve the antitumor immunity. Hence, co-administration of mLAPMi-R848 along with an E6E7 peptide in TC-1 tumor mice eradicated tumor burden and elicited splenocyte-induced cytotoxicity in TC-1 cancer cells. In a bilateral TC-1 tumor model, administration of mLAPMi-R848 and E6E7 peptide significantly suppressed both primary and secondary tumor burdens and improved the overall survival rate. Immune cell profiling revealed elevated levels of mature DCs and CD8+ T cells but reduced levels of tumor-associated immunosuppressive cells (TAICs) like myeloid derived suppressor cells (MDSCs) and regulatory T (Treg) cells in distal tumors. Overall, this study demonstrated that mLAPMi-R848 has improved the antitumor immunity of the peptide antigen against HPV-induced cancers by targeted immunodulation of antigen presenting cells (APCs) and reducing TAICs. Furthermore, this nano-adjuvant has the potential to offer a new treatment option for patients with cervical cancer and can be applied for the treatment of other HPV induced cancers.

Bioinspired nonheme iron complex that triggers mitochondrial apoptotic signalling pathway specifically for colorectal cancer cells.

The activation of dioxygen is the keystone of all forms of aerobic life. Many biological functions rely on the redox versatility of metal ions to perform reductive activation-mediated processes entailing dioxygen and its partially reduced species including superoxide, hydrogen peroxide, and hydroxyl radicals, also known as reactive oxygen species (ROS). In biomimetic chemistry, a number of synthetic approaches have sought to design, synthesize and characterize reactive intermediates such as the metal-superoxo, -peroxo, and -oxo species, which are commonly found as key intermediates in the enzymatic catalytic cycle. However, the use of these designed complexes and their corresponding intermediates as potential candidates for cancer therapeutics has scarcely been endeavored. In this context, a series of biomimetic first-row transition metal complexes bearing a picolylamine-based water-soluble ligand, [M(HN3O2)]2+ (M = Mn2+, Fe2+, Co2+, Cu2+; HN3O2 = 2-(2-(bis(pyridin-2-ylmethyl)amino)ethoxy)ethanol) were synthesized and characterized by various spectroscopic methods including X-ray crystallography and their dioxygen and ROS activation reactivity were evaluated in situ and in vitro. It turned out that among these metal complexes, the iron complex, [Fe(HN3O2)(H2O)]2+, was capable of activating dioxygen and hydrogen peroxide and produced the ROS species (e.g., hydroxyl radical). Upon the incubation of these complexes with different cancer cells, such as cervical, breast, and colorectal cancer cells (MDA-MB-231, AU565, SK-BR-3, HeLa S3, HT-29, and HCT116 cells), only the iron complex triggered cellular apoptosis specifically for colorectal cancer cells; the other metal complexes show negligible anti-proliferative activity. More importantly, the biomimetic complexes were harmless to normal cells and produced less ROS therein. The use of immunocytochemistry combined with western blot analysis strongly supported that apoptosis occurred via the intrinsic mitochondrial pathway; in the intracellular network, [Fe(HN3O2)(H2O)]2+ resulted in (i) the activation and/or production of ROS species, (ii) the induction of intracellular impaired redox balance, and (iii) the promotion of the mitochondrial apoptotic signaling pathway in colorectal cancer cells. The results have implications for developing novel biomimetic complexes in cancer treatments and for designing potent candidates with cancer-specific antitumor activity.

Heat-Confined Tumor-Docking Reversible Thermogel Potentiates Systemic Antitumor Immune Response During Near-Infrared Photothermal Ablation in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) features immunologically "cold" tumor microenvironments with limited cytotoxic T lymphocyte (CTL) infiltration. Although ablation therapies have demonstrated modulation of "cold" TNBC tumors to inflamed "hot" tumors, recruitment of myeloid derived suppressor cells (MDSCs) at the tumors post ablation therapies prevents the infiltration of CTLs and challenge the antitumor potentials of T-cell therapies. Here, a thermal ablation immunotherapy strategy is developed to prevent the immune suppressive effects of MDSCs during photothermal ablation and induce a durable systemic antitumor immunity to eradicate TNBC tumors. An injectable pluronic F127/hyaluronic acid (HA)-based hydrogel embedded with manganese dioxide (BM) nanoparticles and TLR7 agonist resiquimod (R848) (BAGEL-R848), is synthesized to induce in situ laser-assisted gelation of the hydrogel and achieve desired ablation temperatures at a low laser-exposure time. Upon 808-nm laser irradiation, a significant reduction in the tumor burden is observed in BAGEL-R848-injected 4T1 tumor-bearing mice. The ablation induced immunogenic cell death and sustained release of R848 from BAGEL-R848 promotes dendritic cell maturation and reduced MDSCs localization in tumors. In addition, inflammatory M1 macrophages and CD8+IFN+ CTL are enriched in distant tumors in bilateral 4T1 tumor model, preventing metastatic tumor growth and signifying the potential of BAGEL-R848 to treat TNBC.

Transcriptome analysis upon potassium usnate exposure reveals ATF3-induced apoptosis in human gastric and colon cancer cells.

BACKGROUND: Potassium usnate (KU), a water-soluble form of usnic acid, shows anticancer activity. However, the underlying mechanisms have not been fully elucidated. PURPOSE: We aimed to identify the pathways involved in anticancer effects of KU in human gastric cancer (GC) and colorectal cancer (CRC) cells using RNA-sequencing (RNA-seq) based transcriptome analysis. STUDY DESIGN: We analyzed the cytotoxic effects of KU to identify the common molecular events in GC and CRC cells upon KU exposure using unbiased approaches. METHODS: Cell viability assays and western blot experiments were used to examine apoptotic changes, cell cycle arrest, and endoplasmic reticulum (ER) stress-induced cellular responses in KU-treated cells. Total RNA from KU-treated human GC and CRC cells was prepared for RNA-seq analysis. Gene ontology term and gene set enrichment analyses were used to identify the key mediators of the cytotoxic effects of KU. The expression of ER stress-induced apoptotic markers was evaluated using quantitative reverse-transcription PCR and western blot analysis. Chromatin immunoprecipitation assays for ATF3 and H3K27ac, and ATF3 knockdown were employed to verify the underlying molecular mechanisms. The inhibitory effect of KU on tumor growth in vivo was validated with metastatic tumor nodule formations in a mouse liver model. RESULTS: KU exerted cytotoxicity in human GC and CRC cells through the activation of the ER stress-induced apoptotic pathway. KU stimulated ATF3 expression, an important mediator of molecular events of apoptosis. ATF3 binds to the promoter region of ATF3, CHOP, GADD34, GADD45A, DR5, and PUMA genes and subsequently promoted apoptotic events. Knockdown of ATF3 significantly reduced the expression of ATF3 target genes and the cytotoxic effects of KU. The intraperitoneal injection of KU induced ATF3 and the apoptosis of implanted colon cancer cells, resulting in reduced metastatic tumor growth in the mouse livers. CONCLUSION: KU exerts cytotoxic effects in human GC and CRC cells by triggering ER stress-induced apoptosis via an ATF3 dependent pathway.

Potassium usnate, a water-soluble usnic acid salt, shows enhanced bioavailability and inhibits invasion and metastasis in colorectal cancer.

Usnic acid (UA), a lichen secondary substance, has considerable anticancer activity in vitro, whereas its effect in vivo is limited. Here, potassium usnate (KU) was prepared by the salinization of UA to enhance its water solubility. KU showed increased bioavailability compared with UA in the tumor, liver, and plasma of a CT26 syngeneic mouse tumor xenograft model after oral administration, as determined by LC-MS/MS analysis. KU exhibited potent anticancer effects on colorectal cancer cells and inhibited liver metastasis in an orthotopic murine colorectal cancer model. KU treatment downregulated the epithelial-mesenchymal markers Twist, Snail, and Slug and the metastasis-related genes CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL in cells and tumor tissues. The present results suggest the potential application of the water-soluble form of UA, KU, in anticancer therapy.

Acetonic extracts of the endolichenic fungus EL002332 isolated from Endocarpon pusillum exhibits anticancer activity in human gastric cancer cells.

BACKGROUND: Endolichenic fungi are microbes that inhabit the thalli of lichens and produce various unique chemicals that can be used for pharmaceutical purposes. PURPOSE: This study screened a library of endolichenic fungal extracts to identify novel anticancer agents capable of suppressing the tumorigenicity of human cancer cells. METHODS: Active compounds were isolated from extracts of endolichenic fungi by column chromatography and reverse-phase HPLC. The anticancer effects of the extracts on cell viability was assessed with the use of MTT assay, Western blotting, fluorescence labeling of apoptotic cell, and flow cytometric analysis; and cell motility with the use of migration, invasion and soft agar colony-formation assay in vitro; and on skin and intraperitoneal mouse xenograft tumors in vivo were investigated. The therapeutic effects of the extract alone or in combination with the conventional chemoreagent docetaxel were analyzed by sulforhodamine B assay. RESULTS: Acetone extracts of EL002332, isolated from Endocarpon pusillum collected in the China desert in 2010, showed selective cytotoxicity against AGS human gastric cancer cells and CT26 mouse colon cancer cells. An active pure compound named myC was isolated from mycelium acetone extracts in a liquid culture system and showed more potent cytotoxicity than crude extracts in the AGS cell line. Especially, myC greatly increased the apoptotic cell population at the IC50 concentration and activated apoptotic signaling by regulating Bcl2 family protein expression and caspase pathway activity. EL002332 crude extracts and myC decreased AGS cell motility at sub-lethal concentrations. In vivo skin and intraperitoneal xenograft tumor experiments showed that the size of tumors and the tumor score were significantly smaller in EL002332 crude extract-treated groups than in control groups. EL002332 crude extracts showed synergistic effects with docetaxel on the AGS and TMK1 cell lines. CONCLUSION: The endolichenic fungus EL002332 has potential anticancer activity in gastric cancer and peritoneal carcinomatosis.

A Hyaluronic Acid-Conjugated Gadolinium Hepatocyte-Specific T1 Contrast Agent for Liver Magnetic Resonance Imaging.

PURPOSE: In this study, we synthesized hyaluronic acid-conjugated gadolinium (HA-diethylene triamine pentaacetic acid (DTPA)-Gd) and evaluated as hepatocyte-specific magnetic resonance imaging (MRI) contrast agent for the diagnosis of hepatic metastasis. PROCEDURES: We conducted Fourier transform (FT)-IR analysis to determine the conjugation of HA and DTPA and performed cell viability assays using NIH3T3 and FL83B cell lines. We also conducted T1-weighted MRI of HA-DTPA-Gd and gadoxetic acid to compare the paramagnetic properties of both. RESULTS: HA-DTPA-Gd had a higher efficiency in liver MRI compared with the commercially available liver-specific contrast agent (p < 0.001). HA-DTPA-Gd, which possessed a higher T1 relaxivity, showed excellent capability for the diagnosis of hepatic metastasis through an in vivo MRI study in comparison with gadoxetic acid (p < 0.001). CONCLUSION: Based on this study, we believe that HA-DTPA-Gd has promising potential for use as a contrast agent for liver MRI of hepatic metastases.

Dendritic cell-based immunotherapy for colon cancer using an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope from tumor-associated antigen 90K.

Tumor-associated antigen 90K is implicated in cell-cell and cell-extracellular matrix adhesion through its interaction with galectin-3 and integrin-ß1 and is highly expressed in malignant tissues, making it a novel target for the development of new immunotherapies. We investigated a potential immunotherapy treatment for colon cancer using 90K-specific cytotoxic T lymphocytes induced by autologous dendritic cells and pulsed with 90K peptides. We selected three peptides (90K351, 90K5 and 90K523) that bind to HLA-A*0201 molecules on the basis of their binding affinity, as determined by a peptide-T2 binding assay. Dendritic cells pulsed with 90K peptides resulted in the efficient generation of mature dendritic cells and exhibited enhanced T-cell stimulation and polarization of naive T cells toward Th1. Dendritic cells pulsed with 90K peptides generated potent cytotoxic T-lymphocytes that lysed T2 cells loaded with each 90K peptide, and 90K(+)/HLA-A2(+) colon cancer cell lines, including HCT116 and SW480, in a dose-dependent and HLA-A*0201-restricted manner. No killing was observed in 90K(+)/HLA-A2(-) DLD1 or 90K(-)/HLA-A2(-) K562 cells. Therefore, we believe that cytotoxic T-lymphocytes stimulated by 90K peptide-pulsed dendritic cells naturally recognize the 90K peptide presented by colon cancer cells in the context of HLA-A2, and kill 90K-positive tumor cells. Dendritic cells pulsed with 90K peptides led to the induction of granzyme B and perforin positive CD8(+) T cells against HCT116 and SW480 cells, but not DLD1 cells. In conclusion, 90K-specific cytotoxic T lymphocytes, generated by stimulating T cells with 90K peptide-pulsed dendritic cells, could be useful effector cells for the immunotherapy treatment of colon cancer.

Docetaxel-loaded thermoresponsive conjugated linoleic acid-incorporated poloxamer hydrogel for the suppression of peritoneal metastasis of gastric cancer.

We evaluated the potential of a thermoresponsive hydrogel consisting of conjugated linoleic acid-coupled Pluronic F-127 (Plu-CLA) as a controlled release, intraperitoneal delivery system for docetaxel with the aim of treating peritoneal dissemination of gastric cancer. Previously, we established a peritoneal metastasis model that involves the injection of BALB/c mice with TMK1 human gastric cancer cells. One week after the TMK1 cells were injected, the mice were injected intraperitoneally with docetaxel alone or docetaxel-loaded Plu-CLA. Tumor progression and response to therapy were monitored by micro-positron emission tomography. The total number of peritoneal tumors and the ascites volume were also measured. Compared with docetaxel alone, the combination of docetaxel and Plu-CLA (docetaxel-Plu-CLA) significantly and synergistically reduced tumor cell survival. Docetaxel-Plu-CLA showed excellent anti-tumor activity, inducing apoptosis more potently than docetaxel alone. Docetaxel-Plu-CLA also significantly reduced the number of peritoneal metastatic nodules and increased survival in the peritoneal gastric cancer xenograft model. Our results show that intraperitoneal administration of docetaxel-Plu-CLA synergistically inhibits peritoneal metastasis and prolongs survival in a peritoneal gastric cancer model. Therefore, Plu-CLA is a potential intraperitoneal-route carrier for hydrophobic docetaxel for the effective treatment of peritoneal metastatic gastric cancer.

Enhanced anti-cancer effect of 5-fluorouracil loaded into thermo-responsive conjugated linoleic acid-incorporated poloxamer hydrogel on metastatic colon cancer models.

Conjugated linoleic acid-coupled Pluronic F127 (Plu-CLA) is an effective drug delivery system with numerous advantages and anti-cancer activity. 5-FU administered in Plu-CLA hydrogel (P-FU) led to the significant enhancement of tumor growth suppression and cellular apoptosis. Moreover, growth of hepatic and intraperitoneal metastases in vivo was significantly reduced in mice treated with P-FU. Therefore, Plu-CLA could be a potential intraperitoneal carrier for hydrophilic 5-FU for the effective treatment of metastatic colon cancer.

Induction of multiple myeloma-specific cytotoxic T lymphocyte stimulation by dendritic cell pulsing with purified and optimized myeloma cell lysates.

We investigated the possibility of immunotherapy for multiple myeloma (MM) using myeloma-specific cytotoxic T lymphocytes (CTLs) that were stimulated in vitro by dendritic cells (DCs) pulsing with purified and optimized myeloma lysates. CD14(+) cells were cultured in the presence of GM-CSF and IL-4. On day 6, the immature DCs were pulsed with the purified myeloma cell lysates, and then maturation of the DCs was induced by the addition of a cytokine cocktail. There were no differences in the phenotypic expressions of mature DCs that were generated by pulsing with CD138(+) cell lysates or total cell lysates. In optimization of the concentration of myeloma lysates, DCs pulsed with 10 microg/mL of myeloma lysate had greater allogeneic T-cell stimulatory capacities than those pulsed with higher concentrations of myeloma lysates. The CTL lines generated by DCs pulsed with myeloma lysates demonstrated potent cytotoxic activities against autologous target cells, but not against HLA-A2(-) cell lines or K562 cell lines. The DCs pulsed with myeloma lysates demonstrated a higher stimulatory capacity for autologous CTL compared with mature nonpulsed DCs. These results suggested that the DCs pulsed with purified and optimized myeloma cell lysates could generate potent myeloma-specific CTLs for approaches in MM.

The generation of leukemic dendritic cells from acute myeloid leukemia cells is potentiated by the addition of CD40L at the terminal maturation stage.

Leukemic dendritic cells (DCs) that are derived from acute myeloid leukemia (AML) cells display low-level expression of several key molecules. We investigated the optimal combination of cytokines needed to generate potent leukemic DCs from AML cells in vitro. AML cells were cultured in the presence of the following combinations of cytokines: Group A, granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-4 (IL-4) + tumor necrosis factor-alpha (TNF-alpha); Group B, GM-CSF + IL-4 + CD40L; and Group C, CD40L addition at the terminal maturation point of cells that were grown as for Group A. The AML cells showed clear upregulation of CD80, CD83, CD86, CD40, and HLA-DR expression under all culture conditions, without significant differences between these groups. However, the addition of CD40L (as in Group C) showed a slight upregulation in the expression of CD83 and CD86 on leukemic DCs. The leukemic DCs in Groups A and B had higher allogeneic T-cell stimulatory capacities than untreated AML cells, and the addition of CD40L (Group C) enhanced this effect. The function of the cytotoxicity-stimulating autologous T cells was also augmented by the addition of CD40L (Group C). These results suggest that AML cells may be used to generate leukemic DCs using various cytokine combinations, and that the most potent, mature leukemic DCs are generated by the addition of CD40L to terminal-stage AML cultures that are grown in the presence of conventional cytokine combinations.

Immunotherapy using autologous monocyte-derived dendritic cells pulsed with leukemic cell lysates for acute myeloid leukemia relapse after autologous peripheral blood stem cell transplantation.

Although a second stem cell transplantation (SCT) can be used as salvage therapy in patients with relapsing leukemia after SCT, most of these patients have a poor outcome. We tried clinical vaccination using monocyte-derived dendritic cells (DCs) pulsed with leukemic lysates to treat relapsing acute myeloid leukemia (AML) after autologous SCT. To generate DCs, CD14+ cells isolated from peripheral blood stem cell products were cultured in AIM-V in the presence of GM-CSF and IL-4. Adding TNF-alpha on day 6 induced maturation of the DCs, which were harvested on day 8 or 9. The DCs were incubated with tumor lysate and KLH for 2 hr at 37 degrees C. After certifying the absence of microorganisms and endotoxins, the patients received four DC vaccinations at two- to three-week intervals. Two patients received four DC vaccinations with means of 7.8 x 10(6) and 9 x 10(6) DCs at two- to three-week intervals. The DC vaccinations were well tolerated with no apparent side effects. After the vaccinations, the patients showed immunological responses with positive delayed-type hypersensitivity skin reaction and increasing autologous T cells stimulatory capacity to the DCs; however, the BM blast percentage of the patients did not improve. The results suggest that DCs are a feasible cellular therapy for relapsing AML after autologous SCT.

Generation of cytotoxic donor CD8+ T cells against relapsing leukemic cells following allogeneic transplantation by stimulation with leukemic cell- or leukemic lysate pulsed donor cell-derived dendritic cells.

To treat leukemia relapse after allogeneic hematopoietic stem cell transplantation (HSCT), we investigated the possibility of immunotherapy using donor CD8+ T cells that were generated by stimulating leukemic cell-derived dendritic cells (leukemic-DCs) or leukemic cell lysate pulsed donor cell-derived DCs (donor-DCs). Leukemic- and donor-DCs were generated from mononuclear cells of patients and CD14+ cells of HLA-matched donors, respectively. The expression of CD80, CD83, CD86, CD1a, and CD40 on leukemic-DCs was significantly lower than that on donor-DCs. Donor-DCs exhibited a higher capacity to stimulate allogeneic T cells compared with leukemic-DCs. Donor CD8+ T cells stimulated by leukemic- or donor-DCs were more cytotoxic than unprimed CD8+ T cells, and slightly higher cytotoxicity was observed with donor-DCs compared to leukemic-DCs. This study indicates that leukemic- or donor-DCs pulsed with leukemic cell lysates can effectively prime donor cytotoxic T cells in vitro, and that they may be used as a potential alternative tool for treating leukemic patients who relapse after allogeneic HSCT.