Novel Complex of PD-L1 Aptamer and Albumin Enhances Antitumor Efficacy In Vivo.

The PD-1/PD-L1 pathway blockade can generate a good clinical response by reducing immunosuppression and provoking durable antitumor immunity. In addition to antibodies, aptamers can also block the interaction between PD-1 and PD-L1. For the in vivo application, however, free aptamers are usually too small in size and quickly removed from blood via glomerular filtration. To avoid renal clearance of aptamer, we conjugated the PD-L1 aptamer to albumin to form a larger complex (BSA-Apt) and evaluated whether BSA-Apt would enhance the in vivo antitumor efficacy. The PD-L1 aptamer was thiol-modified and conjugated to the amino group of BSA via a SMCC linker. The average size of BSA-Apt was 11.65 nm, which was above the threshold for renal clearance. Functionally, BSA-Apt retained the capability of the PD-L1 aptamer to bind with PDL1-expressing tumor cells. Moreover, both the free aptamer and BSA-Apt augmented the PBMC-induced antitumor cytotoxicity in vitro. Furthermore, BSA-Apt generated a significantly stronger antitumor efficacy than the free PD-L1 aptamer in vivo without raising systemic toxicity. The results indicate that conjugating the PD-L1 aptamer to albumin may serve as a promising strategy to improve the in vivo functionality of the aptamer and that BSA-Apt may have application potential in cancer immunotherapy.

A mutation in PHKG1 causes high drip loss and low meat quality in Chinese Ningdu yellow chickens.

With increasing societal development and the concurrent improvement in people's quality of life, meat consumption has gradually changed from a focus on "quantity" to "quality". Broiler production is increasingly used as a means to improve meat quality by altering various characteristics, especially its genetic factors. However, until now, little has been known about the genetic variants related to meat quality traits in Chinese purebred chicken populations. To better understand these genetic underpinnings, a total of 17 traits related to meat quality and carcass were measured in 325 Chinese Ningdu yellow chickens. We performed DNA sequencing to detect nucleotide mutations, after which we conducted association studies between PHKG1 gene polymorphisms and traits related to meat quality and carcass. Results indicated a large phenotypic variation in meat quality traits. More specifically, the single nucleotide polymorphism (SNP) rs15845448 was significantly associated with drip loss at 24 h (P = 8.04 × 10-6) and 48 h (P = 5.47 × 10-6), pH (P = 2.39 × 10-3), and meat color L* (P = 9.88 × 10-3). Moreover, the SNP rs15845448 reduced 24 h and 48 h drip loss by 3.62 and 5.97%, respectively. However, no significant associations were found between rs15845448 and carcass traits (P > 0.05). Furthermore, a haplotype block containing 2 adjacent SNPs (rs15845448 and rs15845450) was identified. This block displayed 4 distinct haplotypes that had significant association with drip loss at 24 h and 48 h, pH, and meat color L*. Collectively, these results provide new insights into the genetic basis of meat quality in Chinese Ningdu yellow chickens. Moreover, the significance of SNP rs15845448 could be incorporated into the selection programs involving this breed.

Novel Complex of PD-L1 Aptamer and Holliday Junction Enhances Antitumor Efficacy in Vivo.

Blocking the PD-1/PD-L1 pathway can diminish immunosuppression and enhance anticancer immunity. PD-1/PD-L1 blockade can be realized by aptamers, which have good biocompatibility and can be synthesized in quantity economically. For in vivo applications, aptamers need to evade renal clearance and nuclease digestion. Here we investigated whether DNA nanostructures could be used to enhance the function of PD-L1 aptamers. Four PD-L1 aptamers (Apt) were built into a Holliday Junction (HJ) to form a tetravalent DNA nanostructure (Apt-HJ). The average size of Apt-HJ was 13.22 nm, which was above the threshold for renal clearance. Apt-HJ also underwent partial phosphorothioate modification and had improved nuclease resistance. Compared with the monovalent PD-L1 aptamer, the tetravalent Apt-HJ had stronger affinity to CT26 colon cancer cells. Moreover, Apt-HJ markedly boosted the antitumor efficacy in vivo vs. free PD-L1 aptamers without raising systemic toxicity. The results indicate that multiple aptamers attached to a DNA nanostructure may significantly improve the function of PD-L1 aptamers in vivo.

Targeted Treatment of Colon Cancer with Aptamer-Guided Albumin Nanoparticles Loaded with Docetaxel.

PURPOSE: Chemotherapy of colon cancer needs improvement to mitigate the severe adverse effects (AEs) associated with the cytotoxic drugs. The aim of this study is to develop a novel targeted drug delivery system (TDDS) with practical application potential for colon cancer treatment. METHODS: The TDDS was built by loading docetaxel (DTX) in albumin nanoparticles (NPs) that were functionalized with nucleolin-targeted aptamers (AS1411). RESULTS: The TDDS (Apt-NPs-DTX) had an average size of 62 nm and was negatively charged with a zeta potential of -31.2 mV. DTX was released from the albumin NP with a typical sustained release profile. Aptamer-guided NPs were preferentially ingested by nucleolin-expressing CT26 colon cancer cells vs the control cells. In vitro cytotoxicity study showed that Apt-NPs-DTX significantly enhanced the killing of CT26 colon cancer cells. Importantly, compared with non-targeted drug delivery, Apt-NPs-DTX treatment significantly improved antitumor efficacy and prolonged the survival of CT26-bearing mice, without raising systemic toxicity. CONCLUSION: The results suggest that Apt-NPs-DTX has potential in the targeted treatment of colon cancer.

Targeted Therapy of Colon Cancer by Aptamer-Guided Holliday Junctions Loaded with Doxorubicin.

PURPOSE: Chemotherapy is the primary treatment for advanced colon cancer, but its efficacy is often limited by severe toxicities. Targeted therapy in the form of selectively drug delivery system (SDDS) is an important strategy to reduce adverse effects. Here, we aim to design a novel SDDS with potential for practical application using biocompatible components and scalable production process, for targeted delivery of doxorubicin (Dox) to colon cancer cells. METHODS: The SDDS was made of a self-assembled DNA nano-cross (Holliday junction, or HJ) functionalized by four AS1411 aptamers (Apt-HJ) and loaded with Dox. RESULTS: Apt-HJ had an average size of 12.45 nm and a zeta potential of -11.6 mV. Compared with the monovalent AS1411 aptamer, the quadrivalent Apt-HJ showed stronger binding to target cancer cells (CT26). A complex of Apt-HJ and doxorubicin (Apt-HJ-Dox) was formed by intercalating Dox into the DNA structure of Apt-HJ, with each complex carrying approximately 17 Dox molecules. Confocal microscopy revealed that Apt-HJ-Dox selectively delivered Dox into CT26 colon cancer cells but not the control cells. Moreover, Apt-HJ-Dox achieved targeted killing of CT26 cancer cells in vitro and reduced the damage to control cells. Importantly, compared with free Dox, Apt-HJ-Dox significantly enhanced the antitumor efficacy in vivo without boosting the adverse effects. CONCLUSION: These results suggest that Apt-HJ-Dox has application potential in targeted treatment of colon cancer.

Selection of a novel DNA aptamer against OFA/iLRP for targeted delivery of doxorubicin to AML cells.

The standard treatment for most acute myeloid leukemia (AML) is chemotherapy, which is often associated with severe adverse effects. One strategy to reduce the adverse effects is targeted therapy that can selectively deliver anticancer drugs to tumor cells. Immature laminin receptor protein (OFA/iLRP) is a potential target for AML treatment, because it is over-expressed on the surface of AML cells but under-expressed in normal tissue. In this study, we developed the first aptamer for OFA/iLRP and explored its potential as a targeting ligand for delivery of doxorubicin (Dox) to AML cells in vitro. The selected aptamer (AB3) was a 59-base DNA oligonucleotides. It bound to OFA/iLRP structure with a Kd of 101 nM and had minimal cross-reactivity to albumin, trypsin, or ovalbumin. Moreover, AB3 could bind to OFA/iLRP-positive AML cells but not the OFA/iLRP-negative control cells. An aptamer-doxorubicin (Apt-Dox) complex was formed by intercalating doxorubicin into the DNA structure of AB3. Apt-Dox selectively delivered Dox to OFA/iLRP-positive AML cells but notably decreased the drug intake by OFA/iLRP-negative control cells. In addition, cytotoxicity study revealed that Apt-Dox efficaciously destroyed the OFA/iLRP-positive AML cells, but significantly reduced the damage to control cells. The results indicate that the OFA/iLRP aptamer AB3 may have application potential in targeted therapy against AML.

Novel Bispecific Aptamer Enhances Immune Cytotoxicity Against MUC1-Positive Tumor Cells by MUC1-CD16 Dual Targeting.

A promising strategy in cancer immunotherapy is the employment of a bispecific agent that can bind with both tumor markers and immunocytes for recruitment of lymphocytes to tumor sites and enhancement of anticancer immune reactions. Mucin1 (MUC1) is a tumor marker overexpressed in almost all adenocarcinomas, making it a potentially important therapeutic target. CD16 is expressed in several types of immunocytes, including NK cells, γδ-T cells, monocytes, and macrophages. In this study, we constructed the first bispecific aptamer (BBiApt) targeting both MUC1 and CD16. This aptamer consisted of two MUC1 aptamers and two CD16 aptamers linked together by three 60 nt DNA spacers. Compared with monovalent MUC1 or CD16 aptamers, BBiApt showed more potent avidity to both MUC1-positive tumor cells and CD16-positive immunocytes. Competition experiments indicated that BBiApt and monovalent aptamers bound to the same sites on the target cells. Moreover, BBiApt recruited more CD16-positive immunocytes around MUC1-positive tumor cells and enhanced the immune cytotoxicity against the tumor cells in vitro. The results suggest that, apart from bispecific antibodies, bispecific aptamers may also potentially serve as a novel strategy for targeted enhancement of antitumor immune reactions against MUC1-expressing malignancies.

Selection of a novel CD19 aptamer for targeted delivery of doxorubicin to lymphoma cells.

CD19 is overexpressed in most human B cell malignancies and considered an important tumor marker for diagnosis and treatment. Aptamers are oligonucleotides that may potentially serve as tumor-homing ligand for targeted cancer therapy with excellent affinity and specificity. In this study, we selected a novel CD19 aptamer (LC1) that was a 59-nucleotide single strand DNA. The aptamer could bind to recombinant CD19 protein with a Kd of 85.4 nM, and had minimal cross reactivity to bovine serum albumin (BSA) or ovalbumin (OVA). Moreover, the aptamer was found capable of binding with the CD19-positive lymphoma cells (Ramos and Raji), but not the CD19-negative cell lines (Jurkat and NB4). An aptamer-doxorubicin complex (Apt-Dox) was also formulated, and selectively delivered doxorubicin to CD19-positive lymphoma cells in vitro. The results indicate that aptamer LC1 can recognize CD19-positive tumor cells and may potentially function as a CD19-targeting ligand.

Aptamer-guided DNA tetrahedron as a novel targeted drug delivery system for MUC1-expressing breast cancer cells in vitro.

Mucin 1 (MUC1) is an important molecular target for cancer treatment because it is overexpressed in most adenocarcinomas. In this study, a new MUC1-targeted drug delivery system was assembled using an aptamer (Apt) that could recognize MUC1 and a DNA tetrahedron (Td) that could carry doxorubicin (Dox) within its DNA structure. The complex thus formed (Apt-Td) had an average size of 12.38 nm and was negatively charged. Similar to the MUC1 aptamer, the Apt-Td could preferentially bind with MUC1-positive MCF-7 breast cancer cells. A drug loading experiment revealed that each Apt-Td complex could carry approximately 25 Dox molecules. Moreover, Apt-Td selectively delivered Dox into the MUC1-positive breast cancer cells but reduced Dox uptake by the MUC1-negative control cells. Dox-loaded Apt-Td also induced a significantly higher cytotoxicity to the MUC1-positive cancer cells versus the MUC1-negative control cells in vitro (p<0.01). These results suggest that Apt-Td may potentially serve as a drug carrier in the targeted treatment of MUC1-expressing breast cancers.

In vitro enhancement of dendritic cell-mediated anti-glioma immune response by graphene oxide.

Malignant glioma has extremely poor prognosis despite combination treatments with surgery, radiation, and chemotherapy. Dendritic cell (DC)-based immunotherapy may potentially serve as an adjuvant treatment of glioma, but its efficacy generally needs further improvement. Here we explored whether graphene oxide (GO) nanosheets could modulate the DC-mediated anti-glioma immune response in vitro, using the T98G human glioma cell line as the study model. Pulsing DCs with a glioma peptide antigen (Ag) generated a limited anti-glioma response compared to un-pulsed DCs. Pulsing DCs with GO alone failed to produce obvious immune modulation effects. However, stimulating DCs with a mixture of GO and Ag (GO-Ag) significantly enhanced the anti-glioma immune reaction (p < 0.05). The secretion of interferon gamma (IFN-γ) by the lymphocytes was also markedly boosted by GO-Ag. Additionally, the anti-glioma immune response induced by GO-Ag appeared to be target-specific. Furthermore, at the concentration used in this study, GO exhibited a negligible effect on the viability of the DCs. These results suggested that GO might have potential utility for boosting a DC-mediated anti-glioma immune response.

Selection of a novel DNA aptamer for assay of intracellular interferon-gamma.

Interferon-gamma (IFN-γ) is a glycoprotein generated by lymphocytes that possesses anti-tumor, antiviral and immunomodulatory functions. IFN-γ assays are broadly employed in immunological research and clinical diagnostic tests. Intracellular IFN-γ staining, in particular, is an important immune assay that allows simultaneous determination of cellular phenotype and antigen-specific T cell response. Aptamers have great potential for molecule detection and can bind to target molecules with high affinity and specificity. In this study, a novel 59-mer DNA aptamer (B1-4) was developed for assay of intracellular IFN-γ. The selected aptamer bound to IFN-γ with a Kd of 74.5 nM, with minimal cross-reactivity to albumin. The aptamer was also found capable of binding with paraformaldehyde-fixed IFN-γ. Moreover, B1-4 could enter permeated and paraformaldehyde-fixed lymphocytes, and bound to intracellular IFN-γ produced by these cells. When FITC-labeled B1-4 was used to stain a group of lymphocytes, the average fluorescence of the cells was positively correlated with the number of PMA-stimulated lymphocytes within the group. A standard curve could thus be established for assessing the fraction of IFN-γ-producing cells in a cluster of lymphocytes. The selected aptamer hence provides a novel approach for assaying intracellular IFN-γ generated by a group of lymphocytes, and may have application potential in both scientific research and clinical laboratory test.

Genistein attenuates vascular endothelial impairment in ovariectomized hyperhomocysteinemic rats.

Hyperhomocysteinemia (HHcy) is a well-known independent risk factor for vascular diseases in the general population. This study was to explore the effect of genistein (GST), a natural bioactive compound derived from legumes, on HHcy-induced vascular endothelial impairment in ovariectomized rats in vivo. Thirty-two adult female Wistar rats were assigned randomly into four groups (n = 8): (a) Con: control; (b) Met: 2.5% methionine diet; (c) OVX + Met: ovariectomy + 2.5% methionine diet; (d) OVX + Met + GST: ovariectomy + 2.5% methionine diet + supplementation with genistein. After 12 wk of different treatment, the rats' blood, toracic aortas and liver samples were collected for analysis. Results showed that high-methionine diet induced both elevation of plasma Hcy and endothelial dysfunction, and ovariectomy deteriorated these injuries. Significant improvement of both functional and morphological changes of vascular endothelium was observed in OVX + Met + GST group; meanwhile the plasma Hcy levels decreased remarkably. There were significant elevations of plasma ET-1 and liver MDA levels in ovariectomized HHcy rats, and supplementation with genistein could attenuate these changes. These results implied that genistein could lower the elevated Hcy levels, and prevent the development of endothelial impairment in ovariectomized HHcy rats. This finding may shed a novel light on the anti-atherogenic activities of genistein in HHcy patients.

Novel HER2 aptamer selectively delivers cytotoxic drug to HER2-positive breast cancer cells in vitro.

BACKGROUND: Aptamer-based tumor targeted drug delivery system is a promising approach that may increase the efficacy of chemotherapy and reduce the related toxicity. HER2 protein is an attractive target for tumor-specific drug delivery because of its overexpression in multiple malignancies, including breast, gastric, ovarian, and lung cancers. METHODS: In this paper, we developed a new HER2 aptamer (HB5) by using systematic evolution of ligands by exponential enrichment technology (SELEX) and exploited its role as a targeting ligand for delivering doxorubicin (Dox) to breast cancer cells in vitro. RESULTS: The selected aptamer was an 86-nucleotide DNA molecule that bound to an epitope peptide of HER2 with a Kd of 18.9 nM. The aptamer also bound to the extracellular domain (ECD) of HER2 protein with a Kdof 316 nM, and had minimal cross reactivity to albumin or trypsin. In addition, the aptamer was found to preferentially bind to HER2-positive but not HER2-negative breast cancer cells. An aptamer-doxorubicin complex (Apt-Dox) was formulated by intercalating Dox into the DNA structure of HB5. The Apt-Dox complex could selectively deliver Dox to HER2-positive breast cancer cells while reducing the drug intake by HER2-negative cells in vitro. Moreover, Apt-Dox retained the cytotoxicity of Dox against HER2-positive breast cancer cells, but reduced the cytotoxicity to HER2-negative cells. CONCLUSIONS: The results suggest that the selected HER2 aptamer may have application potentials in targeted therapy against HER2-positive breast cancer cells.

Novel MUC1 aptamer selectively delivers cytotoxic agent to cancer cells in vitro.

Chemotherapy is a primary treatment for cancer, but its efficacy is often limited by the adverse effects of cytotoxic agents. Targeted drug delivery may reduce the non-specific toxicity of chemotherapy by selectively directing anticancer drugs to tumor cells. MUC1 protein is an attractive target for tumor-specific drug delivery owning to its overexpression in most adenocarcinomas. In this study, a novel MUC1 aptamer is exploited as the targeting ligand for carrying doxorubicin (Dox) to cancer cells. We developed an 86-base DNA aptamer (MA3) that bound to a peptide epitope of MUC1 with a K(d) of 38.3 nM and minimal cross reactivity to albumin. Using A549 lung cancer and MCF-7 breast cancer cells as MUC1-expressing models, MA3 was found to preferentially bind to MUC1-positive but not MUC1-negative cells. An aptamer-doxorubicin complex (Apt-Dox) was formulated by intercalating doxorubicin into the DNA structure of MA3. Apt-Dox was found capable of carrying doxorubicin into MUC1-positive tumor cells, while significantly reducing the drug intake by MUC1-negative cells. Moreover, Apt-Dox retained the efficacy of doxorubicin against MUC1-positive tumor cells, but lowered the toxicity to MUC1-negative cells (P<0.01). The results suggest that the MUC1 aptamer may have potential utility as a targeting ligand for selective delivery of cytotoxic agent to MUC1-expressing tumors.

Novel aptamer-nanoparticle bioconjugates enhances delivery of anticancer drug to MUC1-positive cancer cells in vitro.

MUC1 protein is an attractive target for anticancer drug delivery owing to its overexpression in most adenocarcinomas. In this study, a reported MUC1 protein aptamer is exploited as the targeting agent of a nanoparticle-based drug delivery system. Paclitaxel (PTX) loaded poly (lactic-co-glycolic-acid) (PLGA) nanoparticles were formulated by an emulsion/evaporation method, and MUC1 aptamers (Apt) were conjugated to the particle surface through a DNA spacer. The aptamer conjugated nanoparticles (Apt-NPs) are about 225.3 nm in size with a stable in vitro drug release profile. Using MCF-7 breast cancer cell as a MUC1-overexpressing model, the MUC1 aptamer increased the uptake of nanoparticles into the target cells as measured by flow cytometry. Moreover, the PTX loaded Apt-NPs enhanced in vitro drug delivery and cytotoxicity to MUC1(+) cancer cells, as compared with non-targeted nanoparticles that lack the MUC1 aptamer (P<0.01). The behavior of this novel aptamer-nanoparticle bioconjugates suggests that MUC1 aptamers may have application potential in targeted drug delivery towards MUC1-overexpressing tumors.

Carbon nanotubes enhance cytotoxicity mediated by human lymphocytes in vitro.

With the expansion of the potential applications of carbon nanotubes (CNT) in biomedical fields, the toxicity and biocompatibility of CNT have become issues of growing concern. Since the immune system often mediates tissue damage during pathogenesis, it is important to explore whether CNT can trigger cytotoxicity through affecting the immune functions. In the current study, we evaluated the influence of CNT on the cytotoxicity mediated by human lymphocytes in vitro. The results showed that while CNT at low concentrations (0.001 to 0.1 µg/ml) did not cause obvious cell death or apoptosis directly, it enhanced lymphocyte-mediated cytotoxicity against multiple human cell lines. In addition, CNT increased the secretion of IFN-γ and TNF-α by the lymphocytes. CNT also upregulated the NF-κB expression in lymphocytes, and the blockage of the NF-κB pathway reduced the lymphocyte-mediated cytotoxicity triggered by CNT. These results suggest that CNT at lower concentrations may prospectively initiate an indirect cytotoxicity through affecting the function of lymphocytes.

Phytoestrogen α-zearalanol improves vascular function in ovariectomized hyperhomocysteinemic rats.

Previous studies have showed that phytoestrogen α-zearalanol (α-ZAL) could antagonize homocysteine (Hcy) induced endothelin-1 (ET-1) expression, oxidative stress and apoptosis in human umbilical vein endothelial cells in vitro, however, its effect on vascular function in vivo remains to be determined. This study was designed to investigate the effects of α-ZAL on vascular function in ovariectomized (OVX) hyperhomocysteinemia (HHcy) rats and explore the mechanisms involved primarily. HHcy rat model was induced by diets containing 2.5% methionine (Met) for 12 weeks. Forty adult female Wistar rats were assigned randomly into five groups: (1) Con; (2) Met; (3) OVX+Met; (4) OVX+Met+α-ZAL; (5) OVX+Met+17ß-E(2) (17ß-estradiol). Blood was collected to analyze plasma estradiol, Hcy and ET-1. Thoracic aortas were isolated to detect its response to phenylephrine (PE) and acetylcholine (ACh) or sodium nitroprusside (SNP). Aortas eNOS expression was determined by Western blot. Thoracic aortas histological characterization was analyzed by optical microscope and scanning electron microscope (SEM). Rat plasma Hcy was significantly elevated after fed with 2.5% methionine diets, and ovariectomy aggravated this elevation. Phytoestrogen α-ZAL or 17ß-E(2) could attenuate this elevation. Plasma ET-1 levels increased significantly in ovariectomized HHcy rats, and supplement with α-ZAL or 17ß-E(2) could reverse these changes. In rats of OVX+Met group, PE elicited significantly greater contraction in a dose-dependent manner in endothelium-intact thoracic aortas rings; ACh elicited significantly less percentage relaxation. These effects were significantly attenuated by supplement with α-ZAL or 17ß-E(2). There was no significant difference between groups in relaxation induced by SNP whether endothelium intact or not. Thoracic aortas morphology study also showed severe endothelium injury in ovariectomized HHcy rats, both α-ZAL and 17ß-E(2) could attenuate this change. Aortas eNOS expression was decreased in ovariectomized HHcy rats, and supplement with α-ZAL or 17ß-E(2) could reverse these changes. These findings demonstrated that α-ZAL could effectively alleviate the impairment of endothelial cells and improve vascular function in ovariectomized HHcy rats by decreasing plasma Hcy and antagonizing decreasing of aortas eNOS expression. This protective effect is somewhat similar with 17ß-E(2).

Biocompatibility of bacterial magnetosomes: acute toxicity, immunotoxicity and cytotoxicity.

In this study, we examined the acute toxicity, immunotoxicity, and cytotoxicity of bacterial magnetosomes (BMs). LD(50) of BMs injected into the sublingual vein of SD rats was 62.7 mg/kg. Further studies with injection of 40 mg/kg BMs showed no significant difference between BM-treated and control rats in terms of routine blood exam results, liver and kidney function tests, organ coefficients of major organs, or Stimulation Index (SI) of lymph cells with ConA and/or LPS antigens. Histological examination of major organs from 40 mg/kg BM-treated rats showed no obvious pathological changes except for increased number of vacuoles in livers, and somewhat thicker interlobular septa in lungs. BMs showed little cytotoxic effect on H22, HL60, or EMT-6 cells. Growth of all three cells was neither inhibited nor stimulated by incubation with 9 microg/ml BMs, which also had no effect on DNA content, cell size, or cell membrane integrity.

Aluminum nanoparticles enhance anticancer immune response induced by tumor cell vaccine.

The application of nanomaterial in cancer treatment is promising and intriguing. Anti-tumor immunotherapy has the potential to significantly improve the prognosis of cancer treatment, though the efficacy of immunotherapy generally needs further improvement. One way to improve the efficacy is using immune adjuvants, but the adjuvants for anticancer immunotherapy have to be more potent than for prophylactic vaccines. Here, we report that compared to conventional alum adjuvant, aluminum oxide nanoparticles (nano-alum) may further enhance the anticancer effects of an immunotherapy that employs tumor cell vaccine (TCV). The average tumor size tends to be lower in animals that receive the combinational treatment of nano-alum and TCV. The anticancer cytotoxicity by the lymphocytes was also significantly higher in the treatment group that received both TCV and nano-alum. These results suggest that nano-alum may potentially serve as a potent immune adjuvant and have prospective applications in anticancer immunotherapy.