Antitumor activity of interleukin 12 against interleukin 2-transduced mouse glioma cells

We subcutaneously inoculated parental and glioma cells genetically engineered to express interleukin-2 (SR/IL-2) into syngeneic mice. The tumor growth of the transfectants was slower than that of the parental cells. We then stereotactically inoculated transfectants into the brains of mice. The survival of the mice injected with parental cells was shorter than that of the mice inoculated with transfectants. SR/IL-2 cells were inoculated subcutaneously into the flank of mice, after which rmIL-12 was administered intraperitoneally (i.p.). The resultant transient tumor growth was followed by regression. rmIL-12 or saline were then injected i.p. into mice that had been inoculated in the brain with SR/IL-2 cells. There was no significant difference in survival time between the treated and control groups.     

Macrophages as effector cells in interleukin 12-induced T cell-dependent tumor rejection

Interleukin (IL)-12 activates a T-cell-dependent antitumor immune response that is able to eradicate established large tumors in a number of immunogenic tumor models. The effector mechanisms in these dramatic antitumor responses have not yet been identified. In this report, we show that the effector mechanism of IL-12-induced rejection of established MCA207 tumors is unique in that it is not dependent on perforin, Fas/Fas ligand, and nitric oxide. Study of cyclophosphamide plus IL-12 (Cy + IL-12)-induced rejection of ascites Sa1 tumor demonstrates that macrophages are the predominant immune cell infiltration in the ascites. These macrophages possess nonspecific tumoricidal activity in vivo as immune distinct MCA207 tumor cells inoculated i.p., but not s.c., in mice bearing regressing Sa1 ascites tumors after Cy + IL-12 therapy are rejected. Furthermore, Cy + IL-12-treated Sa1 ascites cells or macrophages, but not spleen macrophages from the same mouse or inflammatory macrophages induced by thioglycollate, are able to suppress the development of immune-irrelevant s.c. tumors in a Winn assay. These macrophages kill various tumor cells in a contact-dependent manner in vitro, and the cytotoxicity is preserved after fixation with paraformaldehyde. These results demonstrate that activated macrophages function as effector cells in an IL-12-induced, T-cell-dependent eradication of established tumors through a novel contact-dependent, paraformaldehyde fixation-resistant, apoptosis-inducing mechanism.     

Immunotherapy of a murine colon cancer with syngeneic spleen cells, immune RNA and tumor antigen

In combination with xenogeneic immune RNA (l-RNA) and tumor antigen (TA), syngeneic spleen cells inhibited the growth of a N-methyl-N-nitrosourethane-induced colon carcinoma (CT-26) in BALB/c mice. The sequential administration of 1 X 10(7) spleen cells, l mg of anti-CT-26 l-RNA and 0.4 mg of CT-26 TA resulted in a 20% survival rate of mice bearing established CT-26 tumors. On the other hand, administration of the spleen cells only, l-RNA only or TA only was not effective in inhibiting tumor growth. Similarly, when any one of the three agents (l-RNA, TA and spleen cells) was omitted, no anti-tumor effect was obtained. A higher dose (1 X 10(8) and a lower dose(1 X 10(6) of spleen cells decreased the anti-tumor effect of this combination therapy. A higher dose (2 mg) and a lower dose (0.5 mg) of l-RNA, as well as a higher dose of TA (0.8 mg) had no influence on the anti-tumor effect. However, a lower dose of TA (0.2 mg) in combination with spleen cells and l-RNA decreased the anti-tumor effect. When all three agents were administered at higher or lower doses, no anti-tumor effect was obtained. When mice bearing CT-26 tumors were treated with spleen cells, plus l-RNA directed against a syngeneic but antigenically different tumor (BP/B/5) and BP/B/5/TA, with spleen cells plus CT 26 I-RNA and BP/B/5TA, or with spleen cells plus BP/B/5 l-RNA and CT-26 TA, no anti-tumor effect was observed. These results indicate that the anti-tumor responses observed were due to tumor-specific immune responses. In conclusion, in order to obtain growth retardation or regression of established CT-26 tumor transplants, the sequential administration of all three agents (spleen cells, l-RNA and TA) was required. The optimal doses of each agent were found to be 1 X 10(7) spleen cells, 0.5 mg or 1 mg of l-RNA and 0.4 mg of tumor antigen. Higher or lower doses decreased the anti-tumor effect. Tumor-specific immune reactions appeared to be involved.     

Expression of secretory phospholipase A2 in colon tumor cells potentiates tumor growth

Secretory phospholipase A2 (sPLA2-IIA) has been shown to attenuate intestinal tumorigenesis in Apc(Min) mice, demonstrating that it is a tumor modifier. To further explore the actions of sPLA2-IIA in tumorigenesis, sPLA2-IIA was overexpressed in two cell lines where it is normally absent, the murine colon tumor cell line AJ02nm0, and human colon carcinoma cell line HCT-116. Two allelic variants of sPLA2-IIA were tested in this study; sPLA2-IIA(AKR) and sPLA2-IIA(SWR), which are derived from AKR/J and SWR/J mice, respectively, and differ by a single amino acid at position 63 in the calcium- and receptor-binding domain. There was no change in cell-doubling time for either allele when compared to vector controls. Furthermore, sodium butyrate and arachidonic acid (AA)-induced cell death were unchanged in control and transfected cells. Addition of the sPLA2 substrate, palmitoyl-arachidonoyl-phosphatidic acid (PAPA), to AJ02nm0 cells resulted in a modest (12%-24%), but significant (P < 0.01), inhibition of growth that was dependent on sPLA2-IIA expression. However, when AJ02nm0 and HCT-116 cells were injected subcutaneously (sc) into nude mice, Pla2g2a expression resulted in a 2.5-fold increase in tumor size. In addition, sPLA2-IIA expressing HCT-116 tumors were found to be more infiltrative than controls. We conclude that the ability of sPLA2-IIA to slow tumor cell growth is dependent upon the availability of substrate, and that in some instances sPLA2-IIA may actually enhance tumor growth. Mechanisms that may account for differences between the tumor explant model versus the Apc(Min) model of intestinal cancer are discussed.     

Dendritic cell vaccination with xenogenic polypeptide hormone induces tumor rejection in neuroendocrine cancer

PURPOSE: No relevant breakthrough has yet been achieved in the identification of tumor antigens in many neuroendocrine cancer types that exist, such as malignant gastrinoma, insulinoma, or medullary thyroid carcinoma. The aim of this study was to proof the concept of dendritic cell immunization with a tumor cell-specific polypeptide hormone as a target molecule in a transgenic mouse model for medullary thyroid carcinoma (Ret/Cal mice). EXPERIMENTAL DESIGN: Ret/Cal mice were repeatedly immunized for up to 6 months with amino acid-modified (xenogenic) calcitonin-pulsed dendritic cells. Xenogenic calcitonin was chosen for immunization due to its higher immunogenicity as compared with murine calcitonin. RESULTS: Lymph nodes from control protein-immunized mice did not show any macroscopic abnormalities, whereas tumor peptide-treated mice revealed in general profoundly enlarged lymph nodes. In tetramer analysis of paratumorous lymph nodes, 1.9% to 3.1% of all infiltrating CD8(+) T cells were specific for one of three tumor epitopes tested. Analysis of the activated IFN-gamma-secreting component in splenic cells revealed an average of 2.8% tumor epitope-specific CD8(+) cells. Immunohistochemistry revealed strong CD8(+) tumor infiltration in calcitonin-vaccinated mice. In addition, these cells also showed strong in vitro lysis capacity at up to 63.3%. Most importantly, calcitonin-immunized mice revealed largely diminished tumor outgrowth (-74.3%) compared with control mice (P < 0.0001). Likewise, serum calcitonin levels in calcitonin-vaccinated Ret/Cal mice were lower than in the control group. CONCLUSION: These results have a major effect, as they are the first to establish a role for xenogenic polypeptide hormones as target molecules for immunotherapy in endocrine malignancies.     

Cell interactions in adoptive immune rejection of a syngeneic tumor

Studies of adoptive tumor neutralization (the Winn test) in BALB/c mice rendered immune to the Simian Virus 40-induced tumor mKSA indicate that: a) the reaction is extremely specific; b) there is a very close correlation between immunity detected by the Winn test and the ability of the intact animal to reject tumor; and c) immunity reflected by the ⁵¹chromium lymphocytotoxicity assay (CRA) and microcytotoxicity test (MCI) correlate poorly with in vivo resistance to tumor challenge and the Winn test. Investigation of the contribution of the immune donor lymphocytes to the reaction demonstrated that immune thymus-dependent lymphocytes (T cells) were essential, but other donor cell populations were not. Proliferation of immune donor cells was not required. Recipient T cells do not participate in the reaction. We propose that donor T cells do not attack the tumor directly, but must interact with recipient cells to generate an effector response. Some of the parameters of the postulated cooperative interaction are discussed.     

Antitumoral vaccination with granulocyte-macrophage colony-stimulating factor or interleukin-12-expressing DHD/K12 colon adenocarcinoma cells

Immunomodulating gene therapy for the treatment of malignant diseases is under extensive investigation. In this study, we induced an antitumoral immune response with murine interleukin-12 (mIL-12) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cells in a model of peritoneal carcinomatosis. Intraperitoneal injection of DHD/K12 tumoral cells engineered to produce IL-12 or GM-CSF did not generate any tumors, whereas untransduced DHD/K12 cells gave rise to peritoneal carcinomatosis. IL-12-expressing DHD/K12 cells also protected against tumors derived from coinjected parental cells. To test whether cytokine-producing cells could elicit a memory antitumoral immune response, animals received a challenge with parental DHD/K12 cells 35 days after the injection of proliferating or irradiated DHD/K12 engineered cells. Under our experimental conditions, irradiated tumor cells did not generate any antitumoral immunity. In contrast, tumor development was delayed and survival increased in the animals vaccinated with cytokine-secreting proliferating cells. A specific cytotoxic T-lymphocyte response against DHD/K12 parental cells was observed after vaccination with GM-CSF-expressing cells. Our results demonstrated that intraperitoneal vaccination with IL-12- or GM-CSF-expressing adenocarcinoma cells induced a systemic immune antitumoral response that may be useful as an adjuvant therapy after surgical resection of colorectal cancer.     

Circulating tumor cells and circulating tumor DNA in colon cancer

It is known that tumor cells have the ability to penetrate into the bloodstream. The identification of such circulating tumor cells (CTC) determines the prognosis in several tumors, including colon cancer. Tumor DNA (ctDNA), which is only a part of the total circulating DNA obtained from the blood of cancer patients, is also further separated from plasma. This separation of the neoplastic derivatives of the primary tumor and metastases (CTC, ctDNA, RNA, proteome) in plasma is called “liquid biopsy.” CTC increasingly represents the pool of tumor cells that can initiate the growth of metastatic lesions, while the ctDNA provides the information about the whole tumor mass. Traditional tissue biopsy gives information based only on one small section of the primary tumor or metastasis, often retrieved before the start of treatment; however, liquid biopsy provides real-time information about the molecular disorders for the whole tumor mass and allows us to estimate the dynamics of the evolutionary tumor changes, the heterogeneity of the disease, and the effect of chemotherapy. With the possibility of obtaining multiple blood samples for analysis during the therapy, in contrast to traditional biopsy, it also allows us to evaluate the mechanisms of resistance to treatment, which in the future will perhaps lead to modification of the treatment in accordance with the detected molecular defects in tumors. Thus, this would facilitate implementing the principles of personalized therapy. In this literature review, we concentrate on liquid biopsy in patients with colon cancer.     

Lymphokine-activated killer cells: lysis of fresh syngeneic natural killer-resistant murine tumor cells by lymphocytes cultured in interleukin 2

Normal splenocytes that are cultured in the lymphokine, interleukin 2 (IL-2), for as short as 2 days develop lytic activity for fresh syngeneic natural killer-resistant tumor cells as well as natural killer-sensitive YAC cells in a 4-hr 51Cr release assay. Lymphokine-activated killer (LAK) cells do not lyse syngeneic fresh lymphocytes but do lyse syngeneic concanavalin A-induced lymphocyte blasts. Lysis is not due to the presence of lectin or xenogeneic serum and appears to be an intrinsic property of lymphocytes activated in IL-2. The activation appears universal in that lymphocytes from all strains of mice activated in this manner exhibited similar patterns of lysis for fresh tumor target cells. To characterize the cells responsible for this lysis, we analyzed the phenotypic expression of surface markers on these cells with depletion techniques using monoclonal antibody and complement. These studies indicate that the precursor of the LAK cell is Thy-1+ and nonadherent to plastic or nylon wool. Lysis of syngeneic tumor was inhibited when LAK cells were treated with an anti-Thy-1.2, or anti-Lyt-2.2 monoclonal antibody and complement but not with anti-Lyt-1.2 monoclonal antibody and complement, indicating that the observed lytic activity was due to a Thy-1+ Lyt-1-2+ cell. Furthermore, LAK cell-mediated lysis could be inhibited by the addition of anti-Lyt-2 or LFA-1 monoclonal antibody to cytotoxicity assays. Cold target inhibition analysis revealed that the syngeneic tumor cells were lysed by recognition of a determinant not present on normal lymphocytes or lymphocyte blasts. This lysis of fresh solid tumor cells by lymphoid cells grown in IL-2 may be of value in the study of tumor-host immunological interactions. The biological significance of tumor lysis by IL-2-activated cells requires further study.     

Induction of potent antitumor immunity by intratumoral injection of interleukin 23-transduced dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in priming immune responses to tumor. Interleukin (IL)-23 can act directly on DC to promote immunogenic presentation of tumor peptide in vitro. Here, we evaluated the combination of bone marrow-derived DC and IL-23 on the induction of antitumor immunity in a mouse intracranial glioma model. DCs can be transduced by an adenoviral vector coding single-chain mouse IL-23 to express high levels of bioactive IL-23. Intratumoral implantation of IL-23-expressing DCs produced a protective effect on intracranial tumor-bearing mice. The mice consequently gained systemic immunity against the same tumor rechallenge. The protective effect of IL-23-expressing DCs was comparable with or even better than that of IL-12-expressing DCs. IL-23-transduced DC (DC-IL-23) treatment resulted in robust intratumoral CD8(+) and CD4(+) T-cell infiltration and induced a specific TH1-type response to the tumor in regional lymph nodes and spleen at levels greater than those of nontransduced DCs. Moreover, splenocytes from animals treated with DC-IL-23 showed heightened levels of specific CTL activity. In vivo lymphocyte depletion experiments showed that the antitumor immunity induced by DC-IL-23 was mainly dependent on CD8(+) T cells and that CD4(+) T cells and natural killer cells were also involved. In summary, i.t. injection of DC-IL-23 resulted in significant and effective systemic antitumor immunity in intracranial tumor-bearing mice. These findings suggest a new approach to induce potent tumor-specific immunity to intracranial tumors. This approach may have therapeutic potential for treating human glioma.     

Partial purification of C-C36 tumor rejection antigen molecules commonly expressed on syngeneic murine colon tumors, C-C36 and C-C26

The tumor-specific transplantation antigens (TSTAs) of BALB/c mouse colon tumor C-C36 were characterized by gel filtration (TKS-gel FPLC), ion exchange chromatography (Mono Q FPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The cell surface antigens were solubilized noncytolytically with 2.5% n-butanol. It was demonstrated that the C-C36 antigens responsible for tumor rejection activity against C-C36 cells had a molecular weight of less than 200,000, and were eluted at 0.4-0.5M NaCl. On the other hand, the cross TSTAs expressed on C-C26 cells were eluted 0.2M NaCl. SDS-PAGE analysis of the partially purified antigens showed that the C-C36 TSTAs had four bands corresponding to molecular weights of 75,000, 68,000, 60,000 and 40,000. In contrast, the cross TSTAs expressed on C-C26 cells gave several major bands corresponding to 110,000, 94,000, 60,000 and 38,000, in addition to faint bands of 75,000 and 68,000. These results indicate that, although the partially purified C-C36 and C-C26 colon tumor antigens inducing rejection of C-C36 tumor challenge in syngeneic hosts showed differences in the ionic strength at which they were eluted, there are three common molecules with molecular weights of 75,000, 68,000 and 60,000, suggesting that these common molecules might be the tumor antigens responsible for the C-C36 colon tumor rejection.     

Overexpression of p65/RelA potentiates curcumin-induced apoptosis in HCT116 human colon cancer cells

Curcumin, the yellow pigment in the spice turmeric, has potent chemopreventive activities that involve diverse molecular pathways. It is widely believed that curcumin pro-apoptotic properties are mediated by downregulation of NF kappa B (NFkappaB). The p65/RelA subunit of NFkappaB may influence cell death, in part by activation of NFkappaB anti-apoptotic target genes including X-linked inhibitor of apoptosis (XIAP), A20, bcl-xL and inhibition of sustained activation of c-Jun N-terminal kinase (JNK). We have shown previously that curcumin inhibits NFkappaB, activates JNK and promotes apoptosis in HCT116 colorectal cancer cells. Here, we show that forced overexpression of p65 does not affect curcumin-induced JNK activation. Indeed, overexpression of p65 enhanced curcumin-mediated apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and poly(ADP-ribose) polymerase (PARP) cleavage. This potentiating effect of p65 upon curcumin-mediated apoptosis was reversed by transfection of cells with an IkappaB super-repressor (DeltaNIkappaB). Curcumin treatment inhibited expression of NFkappaB anti-apoptotic target genes in mock-transfected and in p65-overexpressing HCT116 cells, although expression levels remained higher in the latter. Taken together, these results show that curcumin-mediated activation of JNK or induction of apoptosis does not require inhibition of p65. Furthermore, curcumin/p65 synergy in promotion of apoptosis cannot be attributed to active repression of NFkappaB anti-apoptotic genes.     

Histoimmunological characterization of the cellular reaction to liver metastases induced by colon cancer cells in syngeneic rats

Artificial liver metastases were produced by injecting in vitro cultivated colonic cancer cells into the mesenteric vein of syngeneic rats. An immunohistologic study of the inflammatory host cells infiltrating the tumors was carried out from 1 to 46 days after tumor cell injection, using a set of monoclonal antibodies labelling tumor cells, macrophages or lymphoid cells subpopulations. In small metastases, 3-7 days after tumor cell injection, tumor cell foci were surrounded by a dense corona of inflammatory cells, predominantly T lymphocytes and NK cells. This inflammatory corona regressed when metastases grew larger and completely disappeared around the large metastases found more than 28 days after cell injection. Inside the metastases, the tumor cells were mixed with a small number of cells with the characteristic labelling of macrophages (KiM2R+, W3/25+); however, the inside macrophages are OX8+ in contrast to macrophages found in normal liver and in the peritumoral corona. The progressive disappearance of the coronal inflammatory cells suggests that cancer cells are able to produce or to induce other cells to produce anti-inflammatory agents inhibiting the accumulation of macrophages and lymphoid cells around the tumors.     

Effect of syngeneic tumor cells bound to concanavalin A on tumor growth

Transplanted MOPC 315 solid myelomas in BALB/cCR mice were treated with single injections of 1.6 × 10⁶ syngeneic MOPC 315 cells combined either with human gamma globulin or with Concanavalin A (Con A). The group of mice which received injections of MOPC 315 cells coupled with human gamma globulin failed to show any significant response. The second group which received injections of MOPC 315 cells coated with Con A responded in variable fashion. Response was measured in terms of delay of tumor growth, differences in growth pattern, and prolongation of survival time. Slight response was observed in 8 of the 20 mice; moderate response was seen in 6 mice; 6 mice did not respond. The data obtained point to a possibly effective tumor vaccine which deserves further study.     

Characterization of tumor rejection antigen molecules of chemically induced murine colon tumor C-C26

The molecular nature of a tumor-specific transplantation antigen (TSTA) of a chemically induced BALB/c mouse colon tumor C-C26 was investigated. The antigen was noncytolytically extracted by 2.5% n-butanol treatment of the cells. Crude butanol extract from C-C26, but not from colon tumor C-C51 and fibrosarcoma Meth-A of BALB/c mice could provide protection against the challenged C-C26 tumor in the transplantation experiment. Crude butanol extract from another syngeneic colon tumor C-C36 also induced a cross-protection against the challenged C-C26 tumor. C-C26 crude butanol extract was characterized by biochemical procedures including the Sephadex G200 column, lens culinaris affinity column, and anion-exchange Mono Q fast protein liquid chromatography column, and by the enzyme digestion study of the antigens and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data indicated that C-C26 TSTA was eluted into fractions containing molecules of approximately Mr 200,000 on Sephadex G200 column chromatography. This antigen was also found in unbound fractions on a lens culinaris affinity column. The antigen was further separated into the fraction that was eluted with 0.4 M NaCl in an ionic strength on Mono Q fast protein liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction showed the molecule with a molecular weight of 30,000. The enzyme digestion study indicated that the immunogenicity of the antigen was inactivated by papain but probably not by neuraminidase treatment. These data suggest that the immunogenic moiety of C-C26 TSTA molecules is located in the peptide portions rather than in sialic acid residues or carbohydrate portions. Furthermore, there are several similarities of the molecular characteristics between C-C26 TSTA and previously reported C-C36 TSTA, such as the amenability to n-butanol extraction. Lens culinaris lectin inaffinity, and ionic strength.