Splenocytes Of Tumor-bearing Mice Research Articles

Royal Jelly Nanoparticle Alleviates Experimental Model of Breast Cancer Through Suppressing Regulatory T Cells and Upregulating TH1 Cells.

Royal jelly, a natural product from bees' hypopharyngeal glands, is commonly used in biomedicine due to its antioxidant and anti-tumor activities. The aim of this study was to compare royal jelly in free form and loaded in layered double hydroxide (LDH) nanoparticle for the treatment of breast cancer with a focus on Th1 and T regulatory parameters in an animal model. Nanoparticles were produced using the coprecipitation method and characterized using DLS, FTIR, and SEM techniques. Forty female BALB/c mice were inoculated with 7.5 x 105 4T1 cells and treated with royal jelly in free and nanoparticle form. Clinical signs and tumor volume were assessed weekly. The effect of royal jelly products on the serum level of IFN-γ and TGF-β was measured by ELISA. In addition, the mRNA expression of these cytokines and Th1 and regulatory T cells' transcription factors (T-bet and FoxP3) was assessed by real-time PCR in the splenocytes of tumor-bearing mice. The physicochemical analysis of nanoparticles confirmed the synthesis of LDH nanoparticles and loading of royal jelly into the LDH structures (RJ-LDH). Animal studies showed that royal jelly and RJ-LDH significantly reduced the size of tumor in BALB/c mice. Additionally, treatment with RJ-LDH significantly inhibited TGF-β and increased IFN-γ production. The data also revealed that RJ-LDH inhibited the differentiation of regulatory T cells, while promoting Th1 cell differentiation via regulating their master transcription factors. These results indicated that royal jelly and RJ-LDH could inhibit breast cancer progression by in-hibiting regulatory T cells and expansion of Th1 cell. Furthermore, the current study demonstrated the therapeutic efficacy of royal jelly is enhanced by LDH nanoparticles; hence, RJ-LDH is significantly more efficient than Free-RJ in the treatment of breast cancer.

Abstract A22: Cervical cancer immunomodulation through stat3 and p65 NFκb: Effects beyond the tumor microenvironment

Abstract Chronic signaling by molecules generated in the tumor microenvironment has local and systemic effects on the immune system. We have previously shown that cervical cancer cells express cytokines as IL-6 and G-CSF, among other factors. These cytokines have been associated with cancer progression in several types of cancer, either by promoting chronic inflammation and/or tolerance. Trying to better understand how cells from the immune system and tumor integrate signals generated by the tumor microenvironment, we have investigated the activation status of signaling pathways involved in cell proliferation and survival, inflammation and immune responses. We observed a cohort of cervical cancer patients, all of whom had cervical biopsies positive for high oncogenic risk human papillomavirus (HPV). In the cervical cancer biopsies, we found constitutive expression of phosphorylated STAT3, Akt, p65 NFκB and CREB, in both the tumor and leukocyte compartments. In the peripheral blood, however, we found constitutive expression of phosphorylated STAT3 and Akt, but significantly lower expression of phosphorylated p65 NFκB than in controls. Data previously published by our laboratory using a HPV16 experimental model showed high constitutive expression of phospho-STAT3 and low negative expression of phospho-p65 NFκB in splenocytes of tumor-bearing mice compared to controls. Given the data from our patients, we hypothesized that the high STAT3 expression and low NFκB expression could have a correlation, with a biologic effect on tumor-bearing subjects. Again, using an HPV16 experimental model, we neutralized IL-6 and G-CSF expression or pharmacologically inhibited STAT3 with the NSC74859 compound. In both cases, we observed significant reduction in tumor growth, decrease in phospho-STAT3 expression (when using the neutralizing antibodies), and importantly, significant increase in phospho-p65 NFκB expression. We also observed a significant increase in T-cell infiltration in tumors from treated animals and increase in antigen-specific T-cell proliferation. Our data indicate that cytokines that signal chronically through STAT3 can inhibit antitumor immune responses through NFκB inhibition. We also suggest that inhibition of STAT3 may be used as therapeutic tool against cervical cancer and other HPV-associated cancers. Financial support: FAPESP 2014/19326-6; 2008/57889-1; CNPq 573799/2008-3. Citation Format: Renata Ariza Marques Rossetti, Gretel Rodriguez Rodriguez, Ildelfonso Alves Da Silva, Jr. Jesus De Paula Carvalho, Maria Beatriz Sartor De Faria Rosa, Luisa Lina Villa, Ana Paula Lepique. Cervical cancer immunomodulation through stat3 and p65 NFκb: Effects beyond the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A22.

Effects of exercise training and supplementation with selenium nanoparticle on T-helper 1 and 2 and cytokine levels in tumor tissue of mice bearing the 4 T1 mammary carcinoma.

Physical exercise decreases the incidence of breast cancer and also improves survival in breast cancer patients. However, the mechanistic basis of these protective effects of exercise is not well known. Changes in tumor cytokines, such as oncostatin-M (OSM), have been associated with modulation of antitumor immune responses in breast cancer. Exercise and antioxidants such as selenium affect both antitumor immune responses as well as tumor cytokine expression. Thus, the aim of this study was to determine the effects of aerobic exercise training (AET) and selenium nanoparticle (SeNP) administration on T-helper 1 and 2 and tumor tissue cytokines in mice bearing the 4 T1 mammary carcinoma. We examined the effects of 6 wk of AET and SeNP administration (100 μg three times/wk) on tumor size, concentration of tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-γ, IL-4 and OSM in tumor tissue and INF-γ and IL-4 in splenocytes of 64 mice bearing the 4 T1 mammary carcinoma. AET increased OSM levels in tumor tissue. Moreover, AET increased levels of TNF-α in tumor tissue, whereas SeNP supplementation decreased IL-4 levels tumor tissue. Also, the combination of AET and SeNP administration decreased tumor volume and increased T-helper 1 cytokines in the splenocytes of tumor-bearing mice. These findings suggest that the combination of AET and SeNP supplementation effects antitumor immune responses in splenocytes, whereas AET induced antitumor cytokines, such as OSM and TNF-α in tumor tissue.

Homoharringtonine induced immune alteration for an Efficient Anti-tumor Response in Mouse Models of Non-small Cell Lung Adenocarcinoma Expressing Kras Mutation

Homoharringtonine (HHT), an inhibitor of protein synthesis, has been used to treat leukemia. Its therapeutic effects on non-small cell lung adenocarcinoma carrying KRAS mutation and their immune system are less understood. The present study examined the therapeutic efficacy and the immune effects of HHT in two murine lung tumor models, xenograft and transgenic, carrying the Kras mutation G12D and G12C respectively. HHT exhibited efficient anticancer activity, significantly suppressing lung tumor growth in vitro and in vivo. The levels of 22 cytokines and chemokines in splenocytes of tumor-bearing mice were examined. Interleukin-12 expression was lower in splenocytes of HHT-treated mice when compared to the controls as demonstrated by a cytokine array and an enzyme-linked immunosorbent assay. The expression levels of CD80, CD86, and CD69 in B220+ B cells from splenocytes of HHT-treated mice were higher than that of control mice in two mouse tumor models. Furthermore, antitumor effect of HHT was attenuated with depletion of B cells. Increased numbers of CD80+ and CD86+ B cells were observed in the mice treated with narciclasine, another translation inhibitor. In conclusion, HHT changed the features of immune cells, and exhibited efficient anti-tumor activity against lung tumor carrying mutant Kras expression.

STAT3 Knockdown in B16 Melanoma by siRNA Lipopolyplexes Induces Bystander Immune Response In Vitro and In Vivo

Persistent activation of STAT3 plays a major role in cancer progression and immune escape. Therefore, targeting STAT3 in tumors is essential to enhance/reactivate antitumor immune response. In our previous studies, we demonstrated the efficacy of stearic acid-modified polyethylenimine (PEI-StA) in promoting small interfering RNA (siRNA) silencing of STAT3 in B16.F10 melanoma in vitro and in vivo. In the current study, we examine the immunologic impact of this intervention. Toward this goal, the infiltration and activation of lymphocytes and dendritic cells (DCs) in the tumor mass were assessed using flow cytometry. Moreover, the levels of IFN-γ, IL-12, and TNF-α in homogenized tumor supernatants were determined. Moreover, mixed lymphocytes reaction using splenocytes of tumor-bearing mice was used to assess DC functionality on siRNA/lipopolyplexes intervention. Our results demonstrated up to an approximately fivefold induction in the infiltration of CD3+ cells in tumor mass on STAT3 knockdown with high levels of CD4+, CD8+, and NKT cells. Consistently, DC infiltration in tumor milieu increased up to approximately fourfold. Those DCs were activated, in an otherwise suppressive microenvironment, as evidenced by a high expression of costimulatory molecules CD86 and CD40. ELISA analysis revealed a significant increase in IFN-γ, IL-12, and TNF-α. Moreover, mixed lymphocytes reaction demonstrated alloreactivity of these DCs as assessed by high T-cell proliferation and IL-2 production. Our results suggest a bystander immune response after local STAT3 silencing by siRNA. This strategy could be beneficial as an adjuvant therapy along with current cancer vaccine formulations.

Abstract 2705: Glycoprotein 90K's antitumor effects through activation of the dendritic cells

Abstract The secreted glycoprotein, 90K is also known as Mac-2 binding protein belonging to the scavenger receptor cysteine-rich domain superfamily that is implicated in immune defense and immune regulation. We examined the relation between over-expressed 90K in tumor cells and immune activation. Here, we investigated whether over-expressed 90K enable the activation of signal transduction pathways and subsequent modulation of DC-maturation status. In vitro cell functional assay, transduction of 90K to dendritic cells was achieved by adenoviral system. Also, we tried to make a mouse model with cancer overexpressing 90K and cancer knockdowned 90K. Then, we analyzed the infiltrated immune cell into tumor region and splenocytes of tumor bearing mice. Dendritic cells transfected with 90K showed IL12p70 and IFN-gamma incensement in DC-cytokine profile and induction of Th1 responses. Tumor infiltrating cells (TIL) into splenocytes were increased in tumor bearing mice knockdowned 90K. Our data indicate that 90K can induce robust maturation of dendritic cells and tumor regression through recruiting TIL. We suggest that 90K may play role in a new target for the treatment of cancer by immune modulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2705. doi:10.1158/1538-7445.AM2011-2705

Vascular disrupting agent DMXAA enhances the antitumor effects generated by therapeutic HPV DNA vaccines.

Antigen-specific immunotherapy using DNA vaccines has emerged as an attractive approach for the control of tumors. Another novel cancer therapy involves the employment of the vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In the current study, we aimed to test the combination of DMXAA treatment with human papillomavirus type 16 (HPV-16) E7 DNA vaccination to enhance the antitumor effects and E7-specific CD8+ T cell immune responses in treated mice. We determined that treatment with DMXAA generates significant therapeutic effects against TC-1 tumors but does not enhance the antigen-specific immune responses in tumor bearing mice. We then found that combination of DMXAA treatment with E7 DNA vaccination generates potent antitumor effects and E7-specific CD8+ T cell immune responses in the splenocytes of tumor bearing mice. Furthermore, the DMXAA-mediated enhancement or suppression of E7-specific CD8+ T cell immune responses generated by CRT/E7 DNA vaccination was found to be dependent on the time of administration of DMXAA and was also applicable to other antigen-specific vaccines. In addition, we determined that inducible nitric oxide synthase (iNOS) plays a role in the immune suppression caused by DMXAA administration before DNA vaccination. Our study has significant implications for future clinical translation.

The effect of vaccination with the lysate of heat‐shocked tumor cells on nitric oxide production in BALB/c mice with fibrosarcoma tumor

The aim of this study was to investigate the effect of heat shock protein-70 (HSP-70) on splenocyte proliferation and nitric oxide (NO) production in the BALB/c mice fibrosarcoma tumor model. To do so, HSP-70 was induced in the lysate of heat-shocked tumor cells and WEHI-164 cells (mouse fibrosarcoma cell line) were injected subcutaneously into the right flank of inbred BALB/c mice to establish a tumor model. Three animal bearing tumor groups were applied: the test group; vaccinated with HSP-70 enriched tumor lysate; control group I, vaccinated with tumor lysate only; and control group II, which received PBS. Using immunoblot analysis, an increase of HSP-70 expression was detected in the lysate of heat-shocked cells in comparison with non-heat-shocked cells. The effect of the test lysate on NO production was measured both in vitro and in vivo in the peritoneal macrophages and splenocytes of tumor bearing mice, respectively. The result showed a significant increase in NO production both in vitro by peritoneal macrophages and in vivo after immunization with HSP-70 enriched tumor lysate. In addition, tumor growth was significantly postponed and the proliferation of splenocytes was increased in the test group. Our results indicate that the lysate of heat-shocked tumor cells was more potent than that of non-heat-shocked tumor cells in inducing anti-tumor immunity. Since production of NO by HSP-activated antigen presenting cells (APCs) is likely to affect innate immunity and tumor growth, the probable mechanism of postponing tumor growth would be NO production by innate immune cells. These findings provide a useful therapeutic model for developing novel approaches to cancer treatments.

Inhibition of indoleamine 2,3-dioxygenase suppresses NK cell activity and accelerates tumor growth.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme, is induced under various pathological conditions, including viral and bacterial infection, allograft rejection, cerebral ischemia, and tumor growth. We have previously reported that the expression of IDO mRNA was increased in some clinical cases of hepatocellular carcinoma in which the recurrence-free survival rate in these IDO-positive patients was significantly higher than that in patients without IDO mRNA induction in tumors. Additionally, IDO expressed in tumors was localized not to the tumor cells but instead to tumor-infiltrating cells by immunohistochemistry. In this study, in order to elucidate the mechanisms underlying anti-tumor effect of IDO, we investigated whether IDO inhibitor (1-methyl-dl-tryptophan, 1MT) affects the growth of subcutaneous B16 tumors in mice. Subsequently, the activity of natural killer (NK) cells was investigated under the conditions of inhibited IDO activity in vivo and in vitro. IDO mRNA expression of B16 cells, B16 subcutaneous tumor, sprenocytes of mice, and human NK cells were studied by reverse transcription-polymerase chain reaction. B16 subcutaneous tumor growth with or without IDO inhibition was observed and cytotoxic activity of NK cells were investigated under the conditions of inhibited IDO activity in vivo and in vitro. IDO mRNA was expressed in B16 subcutaneous tumor, splenocytes of tumor bearing mice, co-cultured splenocytes with B16, and human NK cells. On day 14, after injection of B16 melanoma cells, the sizes of tumors in IDO-inhibited mice were significantly larger than those in control mice. The cytotoxic activity of mice NK cells was reduced by IDO inhibition in vivo. In vitro inhibition of IDO, NK activity was reduced in dose-dependent manner of 1MT. In conclusion, these results indicated that IDO plays an important role in anti-tumor immunity by regulating cytotoxic activity of NK cells.

Prostaglandin E<sub>2</sub> from Macrophages of Murine Splenocyte Cultures Inhibits the Generation of Lymphokine-Activated Killer Cell Activity

The present work investigated the association between prostaglandin E2 (PGE2) from macrophages and its inhibition of murine lymphokine-activated killer (LAK) cell generation. The coculture of indomethacin with interleukin-2 (IL-2) augmented LAK cell activity in an indomethacin dose-response manner, and diminished PGE2 content in the corresponding culture supernatant in a reverse dose-response manner. The correlation between the increase in LAK cell activity and the decrease in PGE2 content was highly significant. Identical results were obtained with diclofenac. A profound inhibition of LAK cell activity by exogenous PGE2 in a dose-response manner was detected. Polyclonal anti-PGE2 antiserum augmented in a dose-dependent manner the LAK cell activity, by neutralizing PGE2 in the medium. A reduction of PGE2 content in the culture supernatant was also detected when the macrophage subpopulations were cultured and was indomethacin dose-dependent. In comparison with that of normal mouse splenocytes, the incubation of whole splenocytes of tumor-bearing mice, which contained a greater subpopulation of macrophages (24% vs. 12%), produced a greater PGE2 content and a correspondingly depressed LAK cell activity. Additionally, PGE2 reduced protein kinase C (PKC) activity along with LAK cell activity generated from macrophage-depleted T cells and natural-killer-like cells. These results overall indicate that PGE2 from macrophages in murine splenocyte cultures inhibits the LAK cell generation, and PKC may be involved in the inhibition mechanism.