Dual-mRNA Delivery Using Tumor Cell Lysate-Based Multifunctional Nanoparticles as an Efficient Colon Cancer Immunogene Therapy.

Background: Messenger RNA (mRNA)-based immunogene therapy holds significant promise as an emerging tumor therapy approach. However, the delivery efficiency of existing mRNA methods and their effectiveness in stimulating anti-tumor immune responses require further enhancement. Tumor cell lysates containing tumor-specific antigens and biomarkers can trigger a stronger immune response to tumors. In addition, strategies involving multiple gene therapies offer potential optimization paths for tumor gene treatments. Methods: Based on the previously developed ideal mRNA delivery system called DOTAP-mPEG-PCL (DMP), which was formed through the self-assembly of 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and methoxypoly (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL), we introduced a fused cell-penetrating peptide (fCPP) into the framework and encapsulated tumor cell lysates to form a novel nanovector, termed CLSV system (CLS: CT26 tumor cell lysate, V: nanovector). This system served a dual purpose of facilitating the delivery of two mRNAs and enhancing tumor immunogene therapy through tumor cell lysates. Results: The synthesized CLSV system had an average size of 241.17 nm and a potential of 39.53 mV. The CLSV system could not only encapsulate tumor cell lysates, but also deliver two mRNAs to tumor cells simultaneously, with a transfection efficiency of up to 60%. The CLSV system effectively activated the immune system such as dendritic cells to mature and activate, leading to an anti-tumor immune response. By loading Bim-encoded mRNA and IL-23A-encoded mRNA, CLSV/Bim and CLSV/IL-23A complexes were formed, respectively, to further induce apoptosis and anti-tumor immunity. The prepared CLSV/dual-mRNA complex showed significant anti-cancer effects in multiple CT26 mouse models. Conclusion: Our results suggest that the prepared CLSV system is an ideal delivery system for dual-mRNA immunogene therapy.

[Tumor cell lysate with low content of HMGB1 enhances immune response of dendritic cells against lung cancer in mice].

OBJECTIVE: To assess the effect of tumor cell lysate (TCL) with low high-mobility group B1 (HMGB1) content for enhancing immune responses of dendritic cells (DCs) against lung cancer. METHODS: TCLs with low HMGB1 content (LH-TCL) and normal HMGB1 content (NH-TCL) were prepared using Lewis lung cancer (LLC) cells in which HMGB1 was inhibited with 30 nmol/L glycyrrhizic acid (GA) and using LLC cells without GA treatment, respectively. Cultured mouse DCs were exposed to different doses of NH-TCL and LH-TCL, using PBS as the control. Flow cytometry was used to detect the expressions of CD11b, CD11c and CD86 and apoptosis of the stimulated DCs, and IL-12 levels in the cell cultures were detected by ELISA. Mouse spleen cells were co-cultured with the stimulated DCs, and the activation of the spleen cells was assessed by detecting CD69 expression using flow cytometry; TNF-ß production in the spleen cells was detected with ELISA. The spleen cells were then co-cultured with LLC cells at the effector: target ratios of 5:1, 10:1 and 20:1 to observe the tumor cell killing. In the animal experiment, C57/BL6 mouse models bearing subcutaneous LLC xenograft received multiple injections with the stimulated DCs, and the tumor growth was observed. RESULTS: The content of HMGB1 in the TCL prepared using GA-treated LLC cells was significantly reduced (P < 0.01). Compared with NH-TCL, LH-TCL showed a stronger ability to reduce apoptosis (P < 0.001) and promote activation and IL- 12 production in the DCs. Compared with those with NH-TCL stimulation, the DCs stimulated with LH-TCL more effectively induced activation of splenic lymphocytes and enhanced their anti-tumor immunity (P < 0.05). In the cell co-cultures, the spleen lymphocytes activated by LH-TCL-stimulated DCs showed significantly enhanced LLC cell killing activity (P < 0.01). In the tumor-bearing mice, injections of LH-TCL-stimulated DCs effectively activated host anti-tumor immunity and inhibited the growth of the tumor xenografts (P < 0.05). CONCLUSION: Stimulation of the DCs with LH-TCL enhances the anti-tumor immune activity of the DCs and improve the efficacy of DCbased immunotherapy for LLC in mice.

Tumor cell lysate with low content of HMGB1 enhances immune response of dendritic cells against lung cancer in mice / 南方医科大学学报

OBJECTIVE@#To assess the effect of tumor cell lysate (TCL) with low high-mobility group B1 (HMGB1) content for enhancing immune responses of dendritic cells (DCs) against lung cancer.@*METHODS@#TCLs with low HMGB1 content (LH-TCL) and normal HMGB1 content (NH-TCL) were prepared using Lewis lung cancer (LLC) cells in which HMGB1 was inhibited with 30 nmol/L glycyrrhizic acid (GA) and using LLC cells without GA treatment, respectively. Cultured mouse DCs were exposed to different doses of NH-TCL and LH-TCL, using PBS as the control. Flow cytometry was used to detect the expressions of CD11b, CD11c and CD86 and apoptosis of the stimulated DCs, and IL-12 levels in the cell cultures were detected by ELISA. Mouse spleen cells were co-cultured with the stimulated DCs, and the activation of the spleen cells was assessed by detecting CD69 expression using flow cytometry; TNF-β production in the spleen cells was detected with ELISA. The spleen cells were then co-cultured with LLC cells at the effector: target ratios of 5:1, 10:1 and 20:1 to observe the tumor cell killing. In the animal experiment, C57/BL6 mouse models bearing subcutaneous LLC xenograft received multiple injections with the stimulated DCs, and the tumor growth was observed.@*RESULTS@#The content of HMGB1 in the TCL prepared using GA-treated LLC cells was significantly reduced (P < 0.01). Compared with NH-TCL, LH-TCL showed a stronger ability to reduce apoptosis (P < 0.001) and promote activation and IL- 12 production in the DCs. Compared with those with NH-TCL stimulation, the DCs stimulated with LH-TCL more effectively induced activation of splenic lymphocytes and enhanced their anti-tumor immunity (P < 0.05). In the cell co-cultures, the spleen lymphocytes activated by LH-TCL-stimulated DCs showed significantly enhanced LLC cell killing activity (P < 0.01). In the tumor-bearing mice, injections of LH-TCL-stimulated DCs effectively activated host anti-tumor immunity and inhibited the growth of the tumor xenografts (P < 0.05).@*CONCLUSION@#Stimulation of the DCs with LH-TCL enhances the anti-tumor immune activity of the DCs and improve the efficacy of DCbased immunotherapy for LLC in mice.

Myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated tumor cell lysates enhance antitumor activity in murine lung cancer.

The present study aimed to investigate the efficacy of a myeloid dendritic cell (mDCs) and plasmacytoid (p)DC combined vaccine loaded with heat-treated cancer cell lysates against lung cancer cells. The mDCs and pDCs were selected using magnetic bead sorting. Antigen loading was performed by adding heat-treated Lewis lung cancer cell lysates to mDC, pDC or mDC+pDC (1:1). Surface expression of CD80, CD86, CD40 and major histocompatibility complex (MHC)-II molecules were determined using flow cytometry, and the secretion of cytokines IL-12, IL-6 and TNF-α were assessed using ELISA assays. The effect of the mDC and pDC vaccine on cytotoxic T lymphocytes (CTLs) against tumor cells was investigated. Tumor-bearing nude mice were intravenously injected with the mDC and pDC combined vaccine. Tumor tissues were collected for hematoxylin and eosin and TUNEL staining. Loading with tumor cell lysate significantly upregulated the surface expression of costimulatory molecules MHC-II on DCs and enhanced secretions of IL-6, IL-12 and TNF-α by DCs. In addition, the tumor cell lysate-loaded mDC and pDC combined vaccine significantly promoted lymphocyte proliferation and enhanced CTL-mediated cytotoxicity against Lewis lung cancer cells compared with mDC or pDC treatment alone. Furthermore, intravenous injection of the mDC and pDC combined vaccine into tumor-bearing nude mice significantly inhibited subcutaneous tumor growth and induced necrosis and apoptosis within the tumor tissue. Overall, the pDC and mDC combination vaccine loaded with heat-treated Lewis lung cancer cell lysate had a synergistic effect on the induction of T lymphocyte proliferation and antitumor efficacy, which may be associated with the upregulation of co-stimulatory molecules and cytokine secretions.

Honokiol-enhanced cytotoxic T lymphocyte activity against cholangiocarcinoma cells mediated by dendritic cells pulsed with damage-associated molecular patterns.

BACKGROUND: Cholangiocarcinoma or biliary tract cancer has a high mortality rate resulting from late presentation and ineffective treatment strategy. Since immunotherapy by dendritic cells (DC) may be beneficial for cholangiocarcinoma treatment but their efficacy against cholangiocarcinoma was low. We suggest how such anti-tumor activity can be increased using cell lysates derived from an honokiol-treated cholangiocarcinoma cell line (KKU-213L5). AIM: To increase antitumour activity of DCs pulsed with cell lysates derived from honokiol-treated cholangiocarcinoma cell line (KKU-213L5). METHODS: The effect of honokiol, a phenolic compound isolated from Magnolia officinalis, on choangiocarcinoma cells was investigated in terms of the cytotoxicity and the expression of damage-associated molecular patterns (DAMPs). DCs were loaded with tumour cell lysates derived from honokiol-treated cholangiocarcinoma cells their efficacy including induction of T lymphocyte proliferation, proinflammatory cytokine production and cytotoxicity effect on target cholangiocarcinoma cells were evaluated. RESULTS: Honokiol can effectively activate cholangiocarcinoma apoptosis and increase the release of damage-associated molecular patterns. DCs loaded with cell lysates derived from honokiol-treated tumour cells enhanced priming and stimulated T lymphocyte proliferation and type I cytokine production. T lymphocytes stimulated with DCs pulsed with cell lysates of honokiol-treated tumour cells significantly increased specific killing of human cholangiocarcinoma cells compared to those associated with DCs pulsed with cell lysates of untreated cholangiocarcinoma cells. CONCLUSION: The present findings suggested that honokiol was able to enhance the immunogenicity of cholangiocarcinoma cells associated with increased effectiveness of DC-based vaccine formulation. Treatment of tumour cells with honokiol offers a promising approach as an ex vivo DC-based anticancer vaccine.

[Anti-lung cancer effect of myeloid and plasmacytoid dendritic cell combined vaccines loaded with tumor cell lysates in vitro].

Objective: To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated Lewis lung cancer cell lysates for treatment of lung cancer in mice. Methods: Bone marrow cells were induced by the recombinant mouse fms-like tyrosine kinase receptor 3 ligand (rmFlt3-L) in vitro, myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC∶pDC=1∶1 were stimulated with heat-treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ (MHC-Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin-12 (IL-12), interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). Results: The lymphocyte proliferation in mice stimulated with mDC+ pDC group loaded with heat-treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively (P<0.05). When the ratio of effector cells: target cells (E∶T) was 10∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P<0.05). When the ratio of E∶T was 20∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 54.77%±3.28%, significantly higher than 35.25%±1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P<0.05). When the ratio of E∶T was 40∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 73.01%±0.91%, significantly higher than 51.36%±0.58% of mDC group and 22.65%±1.28% of pDC group, respectively (P<0.05). With the rate of E∶T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC-Ⅱ of mDC: pDC=1 group pulsed with heat-treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL-6 cytokine concentrations of mDC+ pDC group, mDC group and pDC group loaded with heat-treated Lewis lung cancer cell lysates were (586.67±52.52) pg/ml, (323.33±67.14) pg/ml and (166.67±16.07) pg/ml, respectively. The concentrations of IL-12 in each group were (2 568.75±119.24) pg/ml, (2 156.25±120.55) pg/ml and (672.92±31.46) pg/ml, respectively. The concentrations of TNF-α in each group were (789.33±48.08) pg/ml, (584.89±116.49) pg/ml and (291.56±40.73) pg/ml, respectively. The concentrations of IL-6, IL-12 and TNF-α secreted by mDC+ pDC group were much higher than those of mDC group and pDC group under the same culture conditions (P<0.05). Conclusions: The mDCs and pDCs combined vaccines pulsed with heat-treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti-tumor effect is related with up-regulation of co-stimulatory molecules and increased secretion of cytokines.

Anti-lung cancer effect of myeloid and plasmacytoid dendritic cell combined vaccines loaded with tumor cell lysates in vitro / 中华肿瘤杂志

Objective@#To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated Lewis lung cancer cell lysates for treatment of lung cancer in mice.@*Methods@#Bone marrow cells were induced by the recombinant mouse fms-like tyrosine kinase receptor 3 ligand (rmFlt3-L) in vitro, myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC∶pDC=1∶1 were stimulated with heat-treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ (MHC-Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin-12 (IL-12), interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA).@*Results@#The lymphocyte proliferation in mice stimulated with mDC+ pDC group loaded with heat-treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively (P<0.05). When the ratio of effector cells: target cells (E∶T) was 10∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P<0.05). When the ratio of E∶T was 20∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 54.77%±3.28%, significantly higher than 35.25%±1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P<0.05). When the ratio of E∶T was 40∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 73.01%±0.91%, significantly higher than 51.36%±0.58% of mDC group and 22.65%±1.28% of pDC group, respectively (P<0.05). With the rate of E∶T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC-Ⅱ of mDC: pDC=1 group pulsed with heat-treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL-6 cytokine concentrations of mDC+ pDC group, mDC group and pDC group loaded with heat-treated Lewis lung cancer cell lysates were (586.67±52.52) pg/ml, (323.33±67.14) pg/ml and (166.67±16.07) pg/ml, respectively. The concentrations of IL-12 in each group were (2 568.75±119.24) pg/ml, (2 156.25±120.55) pg/ml and (672.92±31.46) pg/ml, respectively. The concentrations of TNF-α in each group were (789.33±48.08) pg/ml, (584.89±116.49) pg/ml and (291.56±40.73) pg/ml, respectively. The concentrations of IL-6, IL-12 and TNF-α secreted by mDC+ pDC group were much higher than those of mDC group and pDC group under the same culture conditions (P<0.05).@*Conclusions@#The mDCs and pDCs combined vaccines pulsed with heat-treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti-tumor effect is related with up-regulation of co-stimulatory molecules and increased secretion of cytokines.

Thymoquinone Potently Enhances the Activities of Classically Activated Macrophages Pulsed with Necrotic Jurkat Cell Lysates and the Production of Antitumor Th1-/M1-Related Cytokines.

Antitumor activity of classically activated macrophage (Mϕ) may be impaired within the tumors, spleen, and bone marrow. Thus, it is possible to boost its antitumor activity after its pulsing with necrotic tumor cell lysates combined with an adjuvant. We set out to determine the potential adjuvant effects of thymoquinone (TQ; 2-isopropyl-5-methyl-1,4-benzoquinone, C10H12O2) on both functional activities of classically activated Mϕs, pulsed or not with necrotic Jurkat T cell line lysates (NecrJCL), and the balance of antitumor cytokines (ATCs) versus immunosuppressive cytokines (ISCs) during crosstalk with autologous human CD4+ T cells. We found that TQ treatment resulted in a significant upregulation of phagocytic activity, respiratory burst, the production of interleukin-2 (IL-2), IL-6, and IL-17 in NecrJCL-pulsed Mϕ co-culture system, and, conversely, in downregulation of the production of IL-6, IL-17, nitric oxide (NO), and arginase activity in nonpulsed TQ-treated Mϕs co-culture system. In addition, TQ has also shown low upregulation effect on the production of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-1ß, pathogen killing capacity and H2O2 in NecrJCL-pulsed Mϕs co-cultures. Moreover, TQ significantly downregulated arginase activity, and significantly upregulated inducible NO synthase (iNOS) activity-to-arginase activity ratio in NecrJCL-pulsed Mϕ co-cultures. Furthermore, TQ downregulated IL-10-to-IL-17 ratio and total cellular cholesterol content (ttcCHOL), but upregulated the ratios of IL-1ß-to-IL-4, IL-1ß-to-IL-10, IFN-γ-to-IL-4, IFN-γ-to-IL-10, TNF-α-to-IL-4, TNF-α-to-IL-10, and combined proinflammatory cytokines (PICs)-to-anti-inflammatory cytokines (AICs) in NecrJCL-pulsed Mϕs co-culture system, whereas significant differences were highlighted only for IL-10-to-IL-17, IFN-γ-to-IL-10, and PICs-to-AICs ratios. Our outcomes demonstrated that TQ can act as potent adjuvant for enhancing both the functional activities of NecrJCL-pulsed Mϕ and the production of ATCs during their interplay with CD4+ T cells.

Enhanced antitumor immunity by targeting dendritic cells with tumor cell lysate-loaded chitosan nanoparticles vaccine.

Whole tumor cell lysates (TCL) have been implemented as tumor antigens for cancer vaccine development, although clinical outcomes of TCL-based antitumor immunotherapy remain unsatisfactory. In order to improve the efficacy of TCL-based vaccines, biomaterials have been employed to enhance antigen delivery and presentation. Here, we have developed chitosan nanoparticles (CTS NPs) with surface mannose (Man) moieties for specific dendritic cells (DCs) targeting (Man-CTS NPs). The Man-CTS NPs were then loaded with TCL generated from B16 melanoma cells (Man-CTS-TCL NPs) for in vitro and in vivo assessment. Potency of the Man-CTS-TCL NPs as cancer vaccine was also assessed in vivo by immunization of mice with Man-CTS-TCL NPs followed by re-challenge with B16 melanoma cell inoculation. We have shown here that Man-CTS-TCL NPs promote bone marrow-derived dendritic cells (BMDCs) maturation and antigen presentation in vitro. In vivo evaluation further demonstrated that the Man-CTS-TCL NPs were readily taken up by endogenous DCs within the draining lymph node (DLN) following subcutaneous administration accompanied by increasing in serum IFN-γ and IL-4 levels. Tumor growth was also significantly delayed in mice primed with Man-CTS-TCL NPs vaccine, attributable at least in part to cytotoxic T lymphocytes response. Moreover, Man-CTS-TCL NPs vaccine also exhibited therapeutic effects in mice with melanoma. Thus, we report here the Man-CTS-TCL NPs as effective anti-tumor vaccine for cancer immunotherapy.

Antitumor Effect of Dendritic Cell Pulsed with Tumor Cell Lysate Against Hepatic Metastases in Murine Model

PURPOSE: Activated bone marrow-derived dendritic cells (DCs) express high level of MHC class I and II molecules as well as intercellular adhesion molecule and B7, required for T cell activation. This study was designed to examine whether DCs pulsed with tumor lysates were capable of inducing tumor specific CTLs. METHODS: To generate mature DCs, bone marrow cells of female BALB/c mice were cultured in the presence of GM-CSF and IL-4. Mature DCs were idenfied by surface expression of MHC class II molecules and costimulatory molecules. By FACS analysis, it was found that most DCs highly expressed B7-1, B-7-2 and CD40 as well as MHC class II molecules. BAlB/c were immunized subcutaneously. Cytolytic activity was determined by chromium release assay using splenocytes harvested from immunized mice 7 days after the immunization, Cytolytic activity was measured against CT-26 and RAG tumor cells. In vivo protection experiment was performed. Mice were immunized subcutaneously wity DCs pulsed with CT-26 lysates (1x10(6) per mouse) and were challenged intrahepatically with wild type CT-26 (5x10(4) per mouse) two weeks following immunization. Three weeks after the challenge, animals were euthanized for identification of hepatic tumors. RESULTS: Lysis of CT-26 cells were significantly greater with the splenocytes from the immunized mice. Incidence and mean volume of hepatic cancer in the immunized group were 50% (5/10) and 78+/-22 mm3. These results were significantly different from those from control groups:100% (10/10) and 1014.5+/-667.8 mm3 in media treated group, 90% (9/10) and 855.5+/-270.6 mm3 in mice treated with irradiated CT-26, 100% (10/10) and 994 255 mm3 in the animals treated with DC alone. CONCLUSIONS: DCs pulsed with CT-26 lysates could successfully induce antitumor immunity in the BLAB/c against syngeneic CT-26 carcinoma cells. Pulsing method was so simple that neither genetic engineerings nor cellular fusion were not necessary. Even though the present study did not conduct survival experiments, it was thought that clinical application of DC-based immunotherapy could be expedited by pulsing of tumor lysate into the DCs.