In vivo fluorescence flow cytometry reveals that the nanoparticle tumor vaccine OVA@HA-PEI effectively clears circulating tumor cells

Tumor vaccine therapy offers significant advantages over conventional treatments,including reduced toxic side effects.However,it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods.Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors,thereby limiting their clinical efficacy.In this study,we developed a nanoparticle-based tumor vaccine,OVA@HA-PEI,utilizing ovalbumin(OVA)as the presenting antigen and hyaluronic acid(HA)and polyethyleneimine(PEI)as adjuvants and carriers.This formulation significantly enhanced the proliferation of immune cells and cytokines,such as CD3,CD8,interferon-,and tumor necrosis factor-,in vivo,effectively activating an immune response against B16–F10 tumors.In vivofluorescenceflow cytometry(IVFC)has already become an effective method for monitoring circulating tumor cells(CTCs)due to its direct,noninvasive,and long-term detection capabilities.Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1(PD-1)antibody.The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone.Additionally,this combination therapy substantially reduced the number of CTCs in vivo,increased the clearance rate of CTCs by the immune system,and slowed tumor progression.Thesefindings greatly enhance the clinical application prospects of IVFC and tumor vaccines.

Enhancement of antitumor vaccine in ablated hepatocellular carcinoma by high-intensity focused ultrasound

AIM:To investigate whether tumor debris created by high-intensity focused ultrasound(HIFU)could trigger antitumor immunity in a mouse hepatocellular carcinoma model. METHODS:Twenty C57BL/6J mice bearing H22 hepatocellular carcinoma were used to generate antitumor vaccines.Ten mice underwent HIFU ablation,and the remaining 10 mice received a sham-HIFU procedure with no ultrasound irradiation.Sixty normal mice were randomly divided into HIFU vaccine,tumor vaccine and control groups.These mice were immunized with HIFU-generated vaccine,tumor-generated vaccine,and saline,respectively.In addition,20 mice bearing H22 tumors were successfully treated with HIFU ablation. The protective immunity of the vaccinated mice was investigated before and after a subsequent H22 tumor challenge.Using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay,the cytotoxicity of splenic lymphocytes co-cultured with H22 cells wasdetermined in vitro before the tumor challenge,and tumor volume and survival were measured in vivo after the challenge in each group.The mechanism was also explored by loading the vaccines with bone marrowderived dendritic cells(DCs). RESULTS:Compared to the control,HIFU therapy, tumor-generated and HIFU-generated vaccines significantly increased cytolytic activity against H22 cells in the splenocytes of the vaccinated mice(P<0.001). The tumor volume was significantly smaller in the HIFU vaccine group than in the tumor vaccine group(P <0.05)and control group(P<0.01).However,there was no tumor growth after H22 rechallenge in the HIFU therapy group.Forty-eight-day survival rate was 100%in mice in the HIFU therapy group,30%in both the HIFU vaccine and tumor vaccine groups,and 20% in the control group,indicating that the HIFU-treated mice displayed significantly longer survival than the vaccinated mice in the remaining three groups(P< 0.001).After bone marrow-derived DCs were incubated with HIFU-generated and tumor-generated vaccines, the number of mature DCs expressing MHC-Ⅱ + ,CD80 + and CD86 + molecules was significantly increased,and interleukin-12 and interferon-γlevels were significantly higher in the supernatants when compared with immature DCs incubated with mouse serum(P<0.001). However,no differences of the number of mature DCs and cytokine levels were observed between the HIFU- generated and tumor-generated vaccines(P>0.05). CONCLUSION:Tumor debris remaining after HIFU can improve tumor immunogenicity.This debris releases tumor antigens as an effective vaccine to develop host antitumor immune response after HIFU ablation.

STUDY ON SOLIDIFIED TUMOR VACCINE PREPARED FROM AUTOGENOUS CANCEROUS TISSUE

In order to evaluate the immune effects of hyperthermically solidified tumor vaccine (STV) prepared from autogenous caucerous tissue,experimental study on mice and clinical application were performed. It was found that Balb/c mice immunized with STV could resist the attack of 3×108 cells of H22 liver cancer with an survival rate of 93.3% (28/30) and the 2-year survival rate of the patients treated with STV prepared from autogenous cancerous tissue was 60%(18/30)as compared with 13.5% (5/37)of the control(P<0.01).

Therapeutic tumor vaccines-a rising star to benefit cancer patients

Malignant tumors are still a worldwide threat to human health.Tumor treatment strategies are constantly evolving,and the advent of tumor immunotherapy has brought up hope to many types of tumors,especially for those that are refractory to conventional therapies including surgery,radiotherapy,and chemotherapy.Tumor vaccines can initiate or amplify an anti-tumor immune response in tumor patients through active immunization,and therefore occupy an important position in tumor immunotherapy.The main types of tumor vaccines include tumor cell vaccines,dendritic cell vaccines,polypeptide vaccines and nucleic acid vaccines.Due to factors such as poor antigen selection and suppressive tumor microenvironment,earliest tumor vaccines on clinical trials failed to achieve satisfactory clinical effects.However,with the development of second-generation genome sequencing technologies and bioinformatics tools,it is possible to predict neoantigens generated by tumor-specific mutations and therefore prepare personalized vaccines.This article summarizes the global efforts in developing tumor vaccines and highlights several representative tumor vaccines in each category.

Advanced nano-based strategies for mRNA tumor vaccine

Tumor vaccine is a promising strategy for cancer immunotherapy by introducing tumor antigens into the body to activate specific anti-tumor immune responses.Along with the technological breakthroughs in genetic engineering and delivery systems,messenger ribonucleic acid(mRNA)technology has achieved unprecedented development and application over the last few years,especially the emergency use authorizations of two mRNA vaccines during the COVID-19 pandemic,which has saved countless lives and makes the world witness the powerful efficacy of mRNA technology in vaccines.However,unlike infectious disease vaccines,which mainly induce humoral immunity,tumor vaccines also need to activate potent cellular immunity to control tumor growth,which creates a higher demand for mRNA delivery to the lymphatic organs and antigen-presenting cells(APCs).Here we review the existing bottlenecks of mRNA tumor vaccines and advanced nano-based strategies to overcome those challenges,as well as future considerations of mRNA tumor vaccines and their delivery systems.

Converting bacteria into autologous tumor vaccine via surface biomineralization of calcium carbonate for enhanced immunotherapy

Autologous cancer vaccine that stimulates tumor-specific immune responses for personalized immunotherapy holds great potential for tumor therapy.However,its efficacy is still suboptimal due to the immunosuppressive tumor microenvironment(ITM).Here,we report a new type of bacteria-based autologous cancer vaccine by employing calcium carbonate(CaCO_(3))biomineralized Salmonella(Sal)as an in-situ cancer vaccine producer and systematical ITM regulator.CaCO_(3) can be facilely coated on the Sal surface with calcium ionophore A23187 co-loading,and such biomineralization did not affect the bioactivities of the bacteria.Upon intratumoral accumulation,the CaCO_(3) shell was decomposed at an acidic microenvironment to atenuate tumor acidity,accompanied by the release of Sal and Ca^(2+)/A23187.Specifically,Sal served as a cancer vaccine producer by inducing cancer cells'immunogenic cell death(ICD)and promoting the gap junction formation between tumor cells and dendritic cells(DCs)to promote antigen presentation.Ca^(2+),on the other hand,was intermalized into various types of immune cells with the aid of A23187 and synergized with Sal to systematically regulate the immune system,including DCs maturation,macrophages polarization,and T cells activation.As a result,such bio-vaccine achieved remarkable effcacy against both primary and metastatic tumors by eliciting potent anti-tumor immunity with full biocompatibility.This work demonstrated the potential of bioengineered bacteria as bio-active vaccines for enhanced tumor immunotherapy.

Blockading a new NSCLC immunosuppressive target by pluripotent autologous tumor vaccines magnifies sequential immunotherapy

The presence of multiple immunosuppressive targets and insufficient activation and infiltration of cytotoxic T lymphocytes(CTLs)allow tumor cells to escape immune surveillance and disable anti-PD-1/PD-L1 immunotherapy.Nanobiotechnology-engineered autologous tumor vaccines(ATVs)that were camouflaged by tumor cell membrane(TCM)were designed to activate and facilitate CTLs infiltration for killing the unprotected lung tumor cells,consequently realizing the sequential immunotherapy.PDE5 was firstly screened out as a new immunosuppressive target of lung cancer in clinical practice.Immediately afterwards,phosphodiesterase-5(PDE5)and programmed cell death 1 ligand 1(PD-L1)dual-target co-inhibition was proposed to unfreeze the immunosuppressive microenvironment of NSCLC.Systematic studies validated that this ATVs-unlocked sequential immunotherapy after co-encapsulating PDE5 inhibitor and NO donor(i.e.,L-arginine)exerted robust anti-tumor effects through increasing inducible nitric oxide synthase(iNOS)expression,blockading PDE5 pathway and activating systematic immune responses,which synergistically eradicated local and abscopal lung cancers in either orthotopic or subcutaneous models.The pluripotent ATVs that enable PDE5 inhibition and sequential immunotherapy provide a new avenue to mitigate immunosuppressive microenvironment and magnify anti-PD-1/PD-L1 immunotherapy.

Tumor neoantigens: Novel strategies for application of cancer immunotherapy

Neoantigen-targeted immunotherapy is a rapidly advancing field that holds great promise for treating cancer.The recognition of antigens by immune cells is a crucial step in tumor-specific killing,and neoantigens generated by mutations in cancer cells possess high immunogenicity and are selectively expressed in tumor cells,making them an attractive therapeutic target.Currently,neoantigens find utility in various domains,primarily in the realm of neoantigen vaccines such as DC vaccines,nucleic acid vaccines,and synthetic long peptide vaccines.Additionally,they hold promise in adoptive cell therapy,encompassing tumor-infiltrating cells,T cell receptors,and chimeric antigen receptors which are expressed by genetically modified T cells.In this review,we summarized recent progress in the clinical use of tumor vaccines and adoptive cell therapy targeting neoantigens,discussed the potential of neoantigen burden as an immune checkpoint in clinical settings.With the aid of state-of-the-art sequencing and bioinformatics technologies,together with significant advancements in artificial intelligence,we anticipated that neoantigens will be fully exploited for personalized tumor immunotherapy,from screening to clinical application.

CELLULAR IMMUNITY EFFECT OF LEUKEMIA VACCINE ON TUMOR BURDEN RAT

Objective To evaluate the effect of the active specific immunotherapy with leukemia vaccine in the malignant hematopoietic diseases. Methods We established the animal models by inoculating C 57 BL/6 rats with FBL 3 erythroleukemia cells and prepared three types of tumor vaccine, which were administered on the rats respectively. The MTT colorimetric assay was adopted 2 and 4 weeks later to test the cytotoxicity of macrophage( M Φ ) and that of cytotoxic T lymphocyte(CTL) derived from the rats injected with tumor vaccines, and compared the results with the control group. Results With the growth of erythroleulemia cells in the rats, the cellular immunity was seriously depressed, and the inhibition of specific cellular immunity was later than that of non specific cellular immunity. The tumor vaccine made from inactivated tumor cells, IFA and cytokines (rGM CSF, rIL 2 and rIL 6), promote the cellular immunity of tumor burden rats, especially the specific cellular immunity more efficiently than that of tumor vaccine made from inactivated tumor cells and IFA, but the third vaccine made from inactivated tumor cells alone has no effect. Conclusion The tumor vaccine made from inactivated tumor cells with addition of IFA and cytokines (rGM CSF, rIL 2 and rIL 6) provides a promising future in the active specific immunotherapy against hematopoietic tumor.

Developing effective tumor vaccines: basis, challenges and perspectives

A remarkable advance in tumor immunology during the last decade is the elucidation of the antigenic basis of tumor recognition and destruction.A variety of tumor antigens have been identified using several strategies including conventional experiments and newly developed bioinformatics.Among these antigens,cancer/testis antigen(CT antigen)is considered to be the most promising target for immunotherapy by vaccination.Successful immunotherapy of tumors requires understanding of the natural relationship between the immune system and tumor in the status of differentiation,invasion and maturation.Continued progress in development of effective cancer vaccines depends on the identification of appropriate target antigens,the establishment of optimal immunization strategies without harmful autoimmune responses and the ability of manipulating tumor microenvironment to circumvent immune suppression and to augment the anti-tumor immune response.

Revolutionizing gastric cancer treatment:The potential of immunotherapy

Gastric cancer,a prevalent malignancy worldwide,ranks sixth in terms of frequency and third in fatality,causing over a million new cases and 769000 annual deaths.Predominant in Eastern Europe and Eastern Asia,risk factors include family medical history,dietary habits,tobacco use,Helicobacter pylori,and Epstein-Barr virus infections.Unfortunately,gastric cancer is often diagnosed at an advanced stage,leading to a grim prognosis,with a 5-year overall survival rate below 5%.Surgical intervention,particularly with D2 Lymphadenectomy,is the mainstay for early-stage cases but offers limited success.For advanced cases,the National Comprehensive Cancer Network recommends chemotherapy,radiation,and targeted therapy.Emerging immunotherapy presents promise,especially for unresectable or metastatic cases,with strategies like immune checkpoint inhibitors,tumor vaccines,adoptive immunotherapy,and nonspecific immunomodulators.In this Editorial,with regards to the article“Advances and key focus areas in gastric cancer immunotherapy:A comprehensive scientometric and clinical trial review”,we address the advances in the field of immunotherapy in gastric cancer and its future prospects.

Immunotherapeutic effects of dendiritic cells vaccine pulsed with tumor cell lysate in mice with pancreatic carcinoma

Objective To observe the immunotherapeutic effects of dendritic cells vaccine pulsed with tumor cell lystate on mice with pancreatic carcinoma. Methods Dendritic cells (MTSC4) were pulsed with tumor cells lysate. The immune preventative and immnotherapeutic effects of DC vaccines on mice with pancreatic carcinoma were assessed. Results After vaccination of the DC vaccines,mice remained tumor-free for at least 25 days in DCs vaccines group,but in other groups the subcutaneous implantation tumorigenesis were found beginning 3 to 9 days. CTL stimulated by DC vaccines effected cytolytic activity against pancreatic carcinoma cells. The survival period was obviously prolonged in DCs vaccines group (56 ±9)d than in other groups P【0.01) and tumors (1.4 ±0.8)g in DCs vaccines group were significantly smaller than that in other groups (P 【 0. 05). Conclusion Tumor cell lysate-pulsed dendrtic cells vaccines can induce a specific and effective immune response against pancreatic carcinoma cell implanted in mice.

Enhancing Antitumor by Immunization with Fusion of Dendritic Cells and Engineered Tumor Cells

A novel approach for a dentritic cells (DCs) based tumor vaccine was developed for the formation of hybrid engineered J558 after fusion with DCs. To make the hybrid tumor vaccine generate more efficient specific CTL cytotoxicity against wild type tumor cells, we genetically engineered tumor cells with mIL 12 gene prior to the cell fusion. mIL 12 was detected at 870±60 pg/(10 5 cells/ml) in the culture supernatants and the fusion ratio was about 30 % by the co focal microscopic analysis. Vaccination of mice with DCs fused with engineered J558 induced more efficient tumor specific CTL cytotoxicity against wild type tumor cells in vitro and with efficient antitumor immunity in vivo . These results suggest that this approach of using DCs fused with engineered tumor cells could be applied in clinical settings of DCs based cancer vaccines.

Anti-Tumor Effect of Heat Shock Protein 70-Peptide Complexes on A-549 Cells

Objective： To investigate the anti-tumor immunity （HSP70-PC） from human lung cancer tissue. Methods in vitro of heat shock protein 70-peptide complexes HSP70-PC was purified from lung tumor tissues and corresponding non-tumor lung samples with the methods of ADP-affinity chromatography, DEAE ion-exchange chromatography and Western-blot. The activation and proliferation of PBMC induced by different HSP70-PC and tumor cytotoxic reactivity to A549 cells in vitro were measured by the MTT cell proliferation assay. Results： The purified HSP70-PC had a very high purity found by SDS-PAGE and Western-blot. Human lymphocytes were sensitized efficiently by HSP70 preparation purified from lung cancer tissues and a definite cytotoxicity to A-549 cells was observed. There was significant difference with HSP70-PC purified from lung cancer, compared with the control group （P〈0.001）. Conclusion： High purity of HSP70-PC could be achieved from tumor tissues in this study. HSP70-PC purified from human tumor tissues can induce anti-tumor immunity in vitro mainly implemented by eliciting CTL immunity.

Establishment of a Tumor-bearing Mouse Model Stably Expressing EGFP Labeled Human MUC1 VNTRs

Two eukaryotic vectors expressing 9 tandem repeats of human MUCI（VNTR）, VR1012-VNTR, and pEGFP-VNTR, were constructed by cloning VNTR gene into VR1012 and pEGFP, respectively. VNTR stably expressing murine Lewis lung carcinoma（LLC） cell line（VNTR^＋ LLC） was established by Lipofectamine-mediated transfection of pEGFP-VNTR into LLC cells. The EGFP expression was observed under a fluorescent microscope and VNTR expression in VNTR^＋ LLC cells was confirmed by means of Western blotting. A syngenic graft tumor model was generated by subcutaneous injection of VNTR^＋ LLC cells into C57/BL6 mice and tumor size increased rapidly with time and in a cell qumber dependent manner. VNTR mRNA expression in the tumor formed was confirmed by RT-PCR. After the third immunization mice were challenged subcutaneously with 5×10^5 VNTR^＋ LLC cells, a significant reduction of subcutaneous tumor growth was observed in the groups immunized with VNTR plasmid DNA compared with that in the groups immunized with the vector DNA alone. Thus, the suppression of subcutaneous tumor was antigen-specific. This model is useful for the development of tumor vaccines targeting MUCI VNTRs.

GM-CSF GENE OR B7-1 GENE MODIFIED MURINE EL-4 CELLS VACCINE

Objective: To study the vaccine potency of gene-modified tumor cells. Methods: The EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene or B7-1 gene. The effect of gene transduction on antitumor immunity was investigated. Results: Flow cytometry analysis showed that expression of their surface marker between wild-type EL-4 cells and gene transduced tumor cells was the same except for CD80 positive in B7-1 gene transduced cells. GM-CSF gene or B7-1 gene transduced EL-4 cells resulted in remarkable loss of tumorigenicity in syngenetic mice. The systemic protective immunity was induced against the challenge with EL-4/wt cells. Therapeutic vaccine with EL-4/GM-CSF or EL/7-1 cells could retard the growth of established early-stage EL-4/wt tumor significantly, but not retard the growth of late-stage EL-4/wt tumor. Irradiated GM-CSF gene transduced EL-4 cells showed strong vaccine effect against EL-4 cell challenge, but irradiated B7-1 gene transduced EL-4 cells showed weak vaccine effect. Remarkable cooperative antitumor effect against EL-4 cell challenge was observed when both irradiated EL-4/GM-CSF and EL-4/B7-1 were inoculated together. Conclusion: GM-CSF gene or B7-1 gene transduced BL-4 cells can be used as a good tumor vaccine. The combination of the two kinds of vaccine may have potential application value in human cancer treatment.

Establishment of a Tumor-bearing Mouse Model Stably Expressing Human Tumor Antigens Survivin and MUC1 VNTRs

The eukaryotic vectors VR1012 expressing survivin or 33 tandem repeats of human mucin 1（MUC1）（VNTRs）,namely,VR1012-S and VR1012-VNTR（VNTR=variable number of tandem repeat）,were constructed by cloning survivin and VNTR genes into VR1012,respectively.The eukaryotic vector pEGFP expressing survivin and MUC1 VNTRs fusion gene pEGFP-MS was also constructed.Mouse melanoma cell line（B16） stably expressing survivin and MUC1 VNTRs（MS ＋ B16） was established by Lipofectamine-mediated transfection of pEGFP-MS into B16 cells.EGFP expression in MS ＋ B16 cells was observed using a fluorescent microscope and survivin and MUC1 VNTRs（MS） expression was confirmed by means of Western blot analysis.A syngenic graft tumor model was generated by subcutaneous injection of MS ＋ B16 cells into C57/BL6 mice and tumor size increased rapidly with time in a cell number dependent manner.After the third immunization,mice were challenged subcutaneously with 5×l0 5 MS ＋ B16 cells.Compared with that of the negative control immunized with phosphate-buffered saline（PBS）,a significant reduction of tumor growth was observed in groups immunized with survivin plasmid DNA and MUC1 VNTRs plasmid DNA.Thus,the suppression of subcutaneous tumor was antigen-specific.This model is useful for the development of tumor vaccines targeting survivin and MUCI VNTRs.

Immune-mediated anti-neoplastic effect of intratumoral RSV envelope glycoprotein expression is related to apoptotic death of tumor cells but not to the size of syncytia

AIM： To promote the development of improved tumor vaccination strategies relying on the intratumoral expression of viral fusogenic membrane proteins, we elucidated whether the size of syncytia or the way tumor cells die has an effect on the therapeutic outcome. METHODS： In two syngeneic subcutaneous murine colon cancer models we assessed the anti-neoplastic effect on vector-treated and contralateral untreated tumors. RESULTS： Intratumoral injection of a replication-defective adenovirus encoding respiratory syncytial virus fusion protein （RSV-F） alone （Ad.RSV-F） or together with the attachment glycoprotein RSV-G （Ad.RSV-F/G） led to a significant growth reduction of the vector-treated and contralateral untreated tumors. The treatment response was associated with a strong tumor-specific CTL response and significantly improved survival with medians of 46 d and 44 d, respectively. Intratumoral injection of Ad,RSV-G or a soluble RSV-F encoding adenovirus （Ad.RSV-Fsol） had no significant anti-neoplastic effect. The median survival of these treabnent groups and of Ad.Null-treated control animals was about 30 d. CONCLUSION： Although in vitro transduction of colon cancer cell lines with Ad.RSV-F/G resulted in about 8-fold larger syncytia than with Ad.RSV-F, the in vivo outcome was not sig nificantly different, Transduction of murinecolon cancer cell lines with Ad.RSVoF or Ad.RSV-F/G caused apoptotic cell death, in contrast to transduction with Ad.RSV-G or Ad.RSV-Fsol, suggesting an importance of the mode of cell death. Overall, these findings provide insight into improved tumor vaccination strategies relying on the intratumoral expression of viral fusogenic membrane proteins.

Immunotherapy for advanced or recurrent hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is associated with high morbidity and mortality,and is prone to intra-and extrahepatic metastasis due to the anatomical and functional characteristics of the liver.Due to the complexity and high relapse rate associated with radical surgery or radiofrequency ablation,immune checkpoint inhibitors(ICIs)are increasingly being used to treat HCC.Several immunotherapeutic agents,along with their combinations,have been clinically approved to treat advanced or recurrent HCC.This review discusses the leading ICIs in practice and those currently undergoing randomized phase 1-3 trials as monotherapy or combination therapy.Furthermore,we summarize the rapidly developing alternative strategies such as chimeric antigen receptor-engineered T cell therapy and tumor vaccines.Combination therapy is a promising potential treatment option.These immunotherapies are also summarized in this review,which provides insights into the advantages,limitations,and novel angles for future research in establishing viable and alternative therapies against HCC.

Cocktail nano-adjuvant enhanced cancer immunotherapy based on NIR-Ⅱ-triggered in-situ tumor vaccination

Second near-infrared(NIR-Ⅱ)light triggered in-situ tumor vaccination(ISTV)represents one of the most promising strategies in boosting the whole-body antitumor immunity.While most of previously developed nano-adjuvants for NIR-Ⅱ-triggered ISTV are“all-in-one”formulations,which may indiscriminately damage both the tumor cells and the immune cells,limiting the overall effect of immune response.To overcome this obstacle,we designed a“cocktail”nano-adjuvant by physically mixing hyaluronidases(HAase)-decorated gold nanostars(HA)for NIR-Ⅱlight triggered in situ production of tumor-associated antigens and CpG functionalized gold nanospheres(CA)for immune cells activation.Compared to“all-in-one”formulation,the“cocktail”nano-adjuvants displayed a significantly stronger immune response on NIR-Ⅱlight induced dendritic cells(DCs)mutation and T cells differentiation,greater effect on tumor-growth inhibition,and higher efficacy in inhibition of pulmonary metastases.What is more,increasing the molar ratio of HA to CA led to an enhanced anticancer immune responses.This study highlight the nano-adjuvant formulation effects on the treatment of tumors with multiple targets.