Lymphocytes In Tumor-bearing Mice Research Articles

The Efficacy of Fosbretabulin Disodium Combined with Radiofrequency Ablation in Lung Cancer.

Radiofrequency ablation (RFA) is a technology that uses radiofrequency thermal effect to induce coagulation necrosis of tumor tissue under the guidance of imaging. However, distant metastasis of tumor cells caused by tumor angiogenesis can lead to incomplete tumor clearing. In this study, LLC1 cell line was used for the construction of subcutaneous xenografts. Either 10 mg/kg or 20 mg/kg Fosbretabulin disodium (FBTD) was intragastrically administered every 2 days for a week. RFA was performed at the end of medication. The proportion of T cells was examined by flow cytometry. Serum IgG and IgA levels of mice were examined by ELISA. Expression of certain genes was estimated by qRT-PCR assay. In this study, we demonstrated that FBTD was able to significantly enhance RFA-induced immune function in tumor-bearing mice by upregulating RFA-induced CD8+ killer T cells. Consistently, 10 mg/kg or 20 mg/kg FBTD therapy upregulated the percentage of IFNγ+ and TNFα+ CD8+ tumor infiltrating lymphocytes in tumor-bearing mice compared to the RFA alone or FBTD alone group. Mechanistically, we reported that FBTD inhibited the RFA-induced PD-1 and PD-L1 upregulation in vivo. In conclusion, we demonstrated that FBTD promoted the antitumor effects of RFA in lung tumor-bearing mice in this study.

Synergistic photothermal cancer immunotherapy by Cas9 ribonucleoprotein-based copper sulfide nanotherapeutic platform targeting PTPN2

Photothermal therapy (PTT) is a promising strategy for the treatment of advanced malignant neoplasm. However, the anti-tumor efficacy by PTT alone is insufficient to control tumor growth and metastasis. Here, we report a multifunctional nanotherapeutic system exerting a combined PTT and immunotherapy to synergistically enhance the therapeutic effect on melanoma. In particular, we selected the semiconductor nanomaterial copper sulfide (CuS), which served not only as a near-infrared (NIR) light-triggered photothermal converter for tumor hyperthermia but as a basic carrier to modify Cas9 ribonucleoprotein targeting PTPN2 on its surface. Efficient PTPN2 depletion was observed after the treatment of CuS-RNP@PEI nanoparticles, which caused the accumulation of intratumoral infiltrating CD8 T lymphocytes in tumor-bearing mice and upregulated the expression levels of IFN-ᵧ and TNF-α in tumor tissue, thus sensitizing tumors to immunotherapy. In addition, the effect worked synergistically with tumor ablation and immunogenic cell death (ICD) induced by PTT to amplify anti-tumor efficacy. Taken together, this exogenously controlled method provides a simple and effective treatment option for advanced malignant neoplasm.

Angiotensin II contributes to intratumoral immunosuppression via induction of PD-L1 expression in non-small cell lung carcinoma

The formation of an immunosuppressive microenvironment and up-regulation of PD-L1 protein are the main causes of tumor immune escape. Previous reports suggest that Angiotensin II (Ang II) can modulate the immune status of tumor microenvironment in non-small cell lung cancer (NSCLC), but the underlying mechanism remains not fully understood. Here we demonstrated that AngII treatment causes the reduction of intratumoral infiltrating CD4 T lymphocytes in tumor-bearing mice, increases the accumulation of immunosuppressive granulocytes and TAMs in tumor tissue, and upregulates the expression levels of immunosuppressive marker genes. In addition, AngII/AGTR1 axis triggers cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability by human antigen R (HuR), an AU-rich element (ARE)-binding protein. Collectively, AngII/AGTR1 signaling promotes the tumor immunosuppressive microenvironment by upregulating PD-L1 in NSCLC, the mechanism of which is largely accounted by HuR-mediated PD-L1 mRNA stabilization.

Impact of 5-Fu/oxaliplatin on mouse dendritic cells and synergetic effect with a colon cancer vaccine.

The aim of the present study was to investigate the effects of 5-fluorouracil (5-Fu) and oxaliplatin on the function and activation pathways of mouse dendritic cells (DCs), and to clarify whether 5-Fu/oxaliplatin combined with the CD1d-MC38/α-galactosylceramide (α-GC) tumor vaccine exhibits synergistic effects on the treatment of colon cancer in mice. The combination of the Toll like receptor (TLR) ligands and/or 5-Fu/oxaliplatin was added into myeloid-derived DCs in vitro culture. DC phenotypic changes were detected by flow cytometry, and the secretion of DC cytokines was detected by cytometric bead array (CBA). A MC38 mouse colon cancer model was constructed and the DCs were isolated from the spleen, tumor tissue and lymph nodes following intraperitoneal injection of 5-Fu/oxaliplatin. The cell phenotypes were detected by flow cytometry. The tumor infiltrating leukocytes, splenocytes and lymph node cells were co-cultured with the dead MC38 tumor cells, and the secretion levels of interferon-γ (IFN-γ) were detected. 5-Fu/oxaliplatin combined with our previously developed CD1d-MC38/α-GC tumor vaccine was used to inhibit the growth of MC38 colon cancer in mice, and the tumor growth rate and survival time were recorded. 5-Fu/oxaliplatin exerted no significant effect on the expression of the stimulating phenotypes of DCs in vitro, while it could reduce the expression of programmed death ligand 1/2 (PD-L1/L2) and promote interleukin-12 (IL-12) secretion by DCs. Furthermore 5-Fu/oxaliplatin was beneficial to the differentiation of T-helper 1 (Th1) cells. 5-Fu/oxaliplatin further enhanced the stimulating phenotypic expression of DCs in tumor bearing mice, decreased PD-L1/L2 expression, and specifically activated the lymphocytes. The CD1d-MC38/α-GC tumor vaccine combined with 5-Fu/oxaliplatin could exert a synergistic role that resulted in a significant delay of the tumor growth rate, and an increase in the survival time of tumor bearing mice. 5-Fu/oxaliplatin decreased the expression of the DC inhibitory phenotypes PD-L1/L2, promoted DC phenotypic maturation in tumor bearing mice, activated the lymphocytes of tumor bearing mice, and exerted synergistic effects with the CD1d-MC38/α-GC colon cancer tumor vaccine.

Inhibition of IL-17A in tumor microenvironment augments cytotoxicity of tumor-infiltrating lymphocytes in tumor-bearing mice.

It remains controversial whether IL-17A promotes or inhibits cancer progression. We hypothesized that IL-17A that is locally produced in the tumor microenvironment has an important role in angiogenesis and tumor immunity. We investigated the effect of inhibiting IL-17A at tumor sites on tumor growth and on local and systemic anti-tumor immunity. MC38 or B16 cells were inoculated subcutaneously into mice, and intratumoral injection of an adenovirus vector expressing siRNA against the mouse IL-17A gene (Ad-si-IL-17) significantly inhibited tumor growth in both tumor models compared with control mice. Inhibition of IL-17A at tumor sites significantly suppressed CD31, MMP9, and VEGF expression in tumor tissue. The cytotoxic activity of CD8+ T cells from tumor-infiltrating lymphocytes in mice treated with Ad-si-IL-17 was significantly higher than in control mice; however, CD8+ T cells from splenocytes had similar activity levels. Suppression of IL-17A at tumor sites led to a Th1-dominant environment, and moreover, eliminated myeloid-derived suppressor cells and regulatory T cells at tumor sites but not in splenocytes. In conclusion, blockade of IL-17A at tumor sites helped suppress tumor growth by inhibiting angiogenesis as well as cytotoxic T lymphocytes activation at tumor sites.

Increased Frequency of Foxp3+ Regulatory T Cells in Mice with Hepatocellular Carcinoma

The CD4+CD25+ regulatory T cell (Treg) is a special kind of T cell subset. Studies have showed that Treg cells are involved in a number of physiological processes and pathologic conditions such as autoimmune diseases, transplantation tolerance and cancer. Tregs with unique capacity for immune inhibition can impair anti-tumour immunity and help tumor cells to escape from immune surveillance. The aim of our study was to investigate whether Tregs are involved in hepatocellular carcinoma (HCC). A BABL/C mouse with HCC in situ model was established to evaluate the Treg existence in carcinoma tissues and the changes of Tregs in spleen using flow cytometry and immunohistochemistry methods. Granzyme B expression in carcinoma tissues was analyzed by immunohistochemistry to investigate the tumor local immune status. The proportion of CD4+CD25+/CD4+ spleen lymphocytes of tumor bearing mice (18.8% ± 1.26%) was found to be significantly higher than that in normal mice (9.99% ± 1.90%) (P<0.01 ). Immunohistochemistry of spleen tissue also confirmed that there was an increase in Treg in tumor-bearing mice, while in carcinomas it showed Treg cells to be present in tumor infiltrating lymphocyte areas while Granzyme B was rarely observed. Anti-tumour immunity was suppressed, and this might be associated with the increase of Tregs. Our observations suggest that the CD4+CD25+Treg/ CD4+ proportion in spleen lymphocytes can be a sensitive index to evaluate the change of Tregs in hepatocellular carcinoma mice and the Treg may be a promising therapeutic target for cancer.

Oral administration of fluoxetine alters the proliferation/apoptosis balance of lymphoma cells and up-regulates T cell immunity in tumor-bearing mice

Antidepressants have a controversial role with regard to their influence on cancer and immunity. Recently, we showed that fluoxetine administration induces an enhancement of the T-cell mediated immunity in naïve mice, resulting in the inhibition of tumor growth. Here we studied the effects of fluoxetine on lymphoma proliferation/apoptosis and immunity in tumor bearing-mice. We found an increase of apoptotic cells (active Caspase-3 +) and a decrease of proliferative cells (PCNA +) in tumors growing in fluoxetine-treated animals. In addition, differential gene expressions of cell cycle and death markers were observed. Cyclins D3, E and B were reduced in tumors from animals treated with fluoxetine, whereas the tumor suppressor p53 and the cell cycle inhibitors p15/INK4B, p16/INK4A and p27/Kip1 were increased. Besides, the expression of the antiapoptotic factor Bcl-2 and the proapoptotic factor Bad were lower and higher respectively in these animals. These changes were accompanied by increased IFN-γ and TNF-α levels as well as augmented circulating CD8 + T lymphocytes in tumor-bearing mice treated with the antidepressant. Therefore, we propose that the up-regulation of T-cell mediated antitumor immunity may be contributing to the alterations of tumor cell proliferation and apoptosis thus resulting in the inhibition of tumor progression.

Metronomic Chemotherapy Enhances Antitumor Effects of Cancer Vaccine by Depleting Regulatory T Lymphocytes and Inhibiting Tumor Angiogenesis

Although cancer vaccines are emerging as innovative methods for cancer treatment, these alone have limited potential for treating measurable tumor burden. Thus, the importance of identifying anticancer strategies with greater potency is necessary. The chimeric DNA vaccine CTGF/E7 (connective tissue growth factor linked to the tumor antigen human papillomavirus 16 E7) generates potent E7-specific immunity and antitumor effects. We tested immune-modulating doses of chemotherapy in combination with the CTGF/E7 DNA vaccine to treat existing tumors in mice. Metronomic low doses of paclitaxel, not the maximal tolerable dose, are synergistic with the antigen-specific DNA vaccine. Paclitaxel, given in metronomic sequence with the CTGF/E7 DNA vaccine enhanced the vaccine's potential to delay tumor growth and decreased metastatic tumors in vivo better than the CTGF/E7 DNA vaccine alone. The two possible mechanisms of metronomic paclitaxel chemotherapy are the depletion of regulatory T cells and the inhibition of tumor angiogenesis rather than direct cancer cell cytolytic effects. Results indicate that combination treatment of metronomic chemotherapy and antigen-specific DNA vaccine can induce more potent antigen-specific immune responses and antitumor effects. This provides an immunologic basis for further testing in cancer patients.

GM-CSF up-regulates the expression of CCL2 by T lymphocytes in mammary tumor-bearing mice

MCP-1/CCL2 (monocyte chemoattractant protein-1/CC chemokine ligand 2) is a beta or CC chemokine that is expressed by a variety of cell types, including fibroblasts, endothelial, smooth muscle, and glial cells. In addition, cells involved in immunity, such as monocytes/macrophages, neutrophils, and eosinophils have also been shown to express this chemoattractant. Using a murine model of the D1-DMBA-3 mammary adenocarcinoma, we demonstrated the unique production of CCL2 by splenic T lymphocytes from tumor-bearing animals. Because this tumor produces GM-CSF, and this factor is also up-regulated in the B lymphocytes of tumor-bearing mice, we looked at the ability of GM-CSF to induce CCL2 production by T cells. Treatment of normal and tumor bearers' T cells with GM-CSF resulted in an increased secretion of this chemokine. This up-regulation was seen with or without stimulation by Concanavalin A, although these treatments were additive in their effects. The induction of CCL2 was studied at the molecular level by analyzing the effect(s) of a variety of physiological and pharmacological agents on cultured T cells. These results suggest that the tumor-derived factor GM-CSF activates various signaling pathways within splenic T cells to up-regulate CCL2 expression.

The changes of CD4+CD25+/CD4+ proportion in spleen of tumor-bearing BALB/c mice

CD4+CD25+ regulatory T lymphocytes (TR) constitute 5–10% of peripheral CD4+ T cells in naive mice and humans, and play an important role in controlling immune responses. Accumulating evidences show that TR cells are involved in some physiological processes and pathologic conditions such as autoimmune diseases, transplantation tolerance and cancer, and might be a promising therapeutic target for these diseases.To evaluate the change of CD4+CD25+ TR cells in mouse tumor models, CD4+CD25+ subset in peripheral blood and spleen lymphocytes from normal or C26 colon-carcinoma-bearing BABL/c mice were analyzed by flow cytometry using double staining with CD4 and CD25 antibodies.The proportion of CD4+CD25+/CD4+ in spleen lymphocytes was found to be higher than that in peripheral blood lymphocytes in normal mice. No difference was observed in the proportion in peripheral blood lymphocytes between tumor bearing mice and normal mice, while there was a significant increase in the proportion in spleen lymphocytes in tumor bearing mice as compared with normal mice. Moreover, the proportion increased in accordance with the increase in the tumor sizes. The increase in the proportion was due to the decrease in CD4+ in lymphocytes, which is resulted from decreased CD4+CD25- subset in lymphocytes. Our observation suggests the CD4+CD25+/CD4+ proportion in spleen lymphocytes might be a sensitive index to evaluate the TR in tumor mouse models, and our results provide some information on strategies of antitumor immunotherapy targeting CD4+CD25+ regulatory T lymphocytes.

Efficient activation of antitumor immunity by IL-6 gene-modified leukemia vaccine in combination with low dose cyclophosphamide and low dose IL-2

Objective: To investigate the antitumor effect of the IL-6 gene-modified erythroleukemia cells combined with low dose cyclophosphamide (Cy) and low dose IL-2. Methods: Mice inoculated with FBL-3-IL-6 in combination with low dose IL-2 and low dose cyclophosphamide (Cy). Results: Mice received combined therapy of FBL-3-IL-6, IL-2 and Cy developed tumors most slowly and survived much longer when compared with mice in control groups, with 5 out of 8 leukemia-bearing mice being tumor free 100 days after the combined treatment. To further explain the mechanism of the antitumor effects by the combined therapy. It was found that combined therapy with low dose Cy, low dose IL-2 and FBL-3-IL-6 achieved maximal cytotoxic effects of nature killer cells and specific cytotoxic T lymphocytes, increased production IL-2, TNF and GM-CSF from spleen lymphocytes in tumor-bearing mice. Vaccination with the FBL3-IL-6 also enhanced the cytotoxic activity of the peritoneal macrophages. The results demonstrated that administration of low dose Cy and low dose IL-2 in combination with IL-6 genemodified leukemia vaccine could elicit potent antileukemia effects, and the mechanisms involved in the antitumor process may include the induction of specific and nonspecific antitumor immunity, reversal of T suppressor cells that mediated local immuno-suppression in tumor bearing mice. Conclusion: The combined therapy with cytokine gene-modified tumor vaccine, low dose of Cy and IL-2 might be a promising approach for the treatment of leukemia.

Suppression of cytotoxic T lymphocyte activity by gamma/delta T cells in tumor-bearing mice.

Spleen cells derived from tumor-bearing mice prove useful for the elucidation of the mechanism determining how tumor cells evade cytotoxic T lymphocytes (CTL) in tumor-bearing hosts. Our data indicate that inactive CTL or precursor CTL specific for tumor antigens are present among lymphocytes of tumor-bearing mice. However, their activity is inhibited by a soluble factor produced by other cells present in the same source. Inhibition of the cytolytic reaction was also detected in the culture supernatant of spleen cells obtained from normal mice, precultured in the presence of tumor cell culture supernatant and interleukin-2 (IL-2). Cell-depletion and cell-purification studies let us conclude that cells that produced the CTL-inhibitory factor (CTL-IF) were gamma/delta T cells. The gamma/delta T cells that were activated in vivo in tumor bearers were able to produce CTL-IF after isolation and in vitro culture. Maximum activation of gamma/delta T cells was achieved by antigenic stimulation and by suppression of cells that interfered with the activation of gamma/delta T cells. CTL-IF, which was assayed by use of CTL clones, did not show antigen specificity. Inhibition depended on a relatively heat- and acid-stable, but alkali-labile molecule with a molecular mass of less than 10 kDa. The latter characteristics imply that CTL-IF does not resemble any of the known lymphokines produced by gamma/delta T cells. These observations emphasize the crucial role of the gamma/delta T cells in the escape of tumor cells from the attack of tumor-specific CTL.

Preferential homing of tumor-infiltrating lymphocytes in tumor-bearing mice.

In view of the current interest in the use of lymphoid cells in adoptive immunotherapy of patients with advanced cancer, we have studied the homing patterns of various lymphoid effector cells in mammary-tumor-bearing mice. Single-cell suspensions of total splenocytes, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells were prepared from the spleens of C3H/OuJ mice. Tumor-infiltrating lymphocytes (TIL) were isolated from mammary adenocarcinomas excised from retired breeder females of the same substrain. Effector cells were labeled with indium-111 and injected via a tail vein into female C3H/OuJ mice bearing one or more mammary tumors. Twenty-four hours after administration, total splenocytes, NK cells, and LAK cells distributed themselves evenly between normal mammary tissue and mammary adenocarcinomas. Only TIL had a higher concentration in tumors than in corresponding normal mammary tissue. The ability of the different lymphocyte preparations to lyse YAC-1 cells was determined by means of a 4-h 51Cr-release cytotoxicity assay. Cells harvested from LAK cell cultures and further enriched by centrifugation through a discontinuous Percoll gradient and interleukin-2 (IL-2)-stimulated TIL demonstrated the highest levels of cytotoxicity, while total splenocytes and fresh TIL were characterized by the lowest levels. Since IL-2-stimulated TIL were highly cytotoxic and exhibited better tumor localization than both NK cells and LAK cells in this system, they may be the lymphoid effectors of choice for adoptive immunotherapy of advanced cancer.

Establishment of experimental malignant glioma-specific cytotoxic T lymphocyte clone by T cell growth factor.

In an attempt to facilitate the long-term proliferative growth and subsequent cloning of cytotoxic T lymphocytes (CTL's) against syngeneic murine 203-glioma (20-methylcholanthrene-induced ependymoblastoma of C57BL/6 mouse origin), sensitized T lymphocytes from tumor-bearing mice were cultured in the presence of T cell growth factor (TCGF). Of five clones established by a limiting dilution technique, two clones (G-CTLL 1 and 2) exhibited tumor-specific cytotoxicity. G-CTLL 1 cells, which possessed much higher cytotoxic activity than G-CTLL 2 cells, were further analyzed. G-CTLL 1 cells were maintained in a TCGF-dependent exponential proliferative culture for over 18 months and continued to mediate an extremely high cytotoxic activity with the target specificity (50- to 100-fold increases over the peak cytotoxic activity of sensitized T lymphocytes in tumor-bearing mice). Their phenotypes of surface antigens were Thy-1+ (weak positive), Lyt-1.-2.+3+, and asialo-GM1-, and their cytotoxicity was blocked by adding only anti-Lyt-2 monoclonal antibodies. These results indicated that the cloned cells originated from CTL's. The cloned cells were characterized by the production of immune interferon with the glioma antigen-stimulation, suggesting that the immune interferon could enhance the cytotoxic activity of the CTL clone at the site of a clone-target cell recognition event.

Fluorescence polarization assay of mitogenic response of lymphocytes

Fluorescence polarization measurement was applied to detect the impaired Con A-induced mitogenic responses in tumor-bearing mice. Spleen lymphocytes from normal C3H/HeN mice and from those inoculated 1×106 Ehrlich ascites tumor cells were treated with Con A and examined for their fluorescence polarization changes (P-value) using fluorescein diacetate and a polarization spectrofluorometer. Murine spleen cells when stimulated by 5μg/ml of Con A at 37°C for 45 min showed a high polarization change with a reduction rate of p-value, 0.79±0.03 (n=33). This Con A-induced polarization change of spleen cells was almost completely inhibited by α-methylmannoside, a competitive inhibitor of Con A-binding to the cell surface. Spleen lymphocytes from tumor-bearing mice showed much less polarization changes with a reduction rate of P-value, 0.91±0.04 (n=33). This low fluorescence polarization change appeared as early as 3 days after tumor inoculation. Impairement of mitogenic responses as revealed by 3H-thymidine uptake in tumor-bearing mice also appeared 3 days after tumor inoculation. These results indicate that the fluorescence polarization measurement may be a valuable tools for the detection of impaired mitogenic responses of lymphocytes in tumor-bearing mice.