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Cooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in Kras G12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with Kras G12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with Kras G12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis.

Background: To develop a vaccine-based immunotherapy for sarcoma, we evaluated a mixture of heat shock proteins (mHSPs) as a vaccine for sarcoma treatment in a mouse model. Heat shock protein/peptides (HSP/Ps) are autoimmune factors that can induce both adaptive and innate immune responses; HSP/Ps isolated from tumors can induce antitumor immune activity when used as vaccines., Methods: In this study, we evaluated the effects of mHSP/Ps on prophylactic antitumor immunity. We extracted mHSP/Ps, including HSP60, HSP70, GP96, and HSP110, from the mouse sarcoma cell lines S180 and MCA207 using chromatography. The immunity induced by mHSP/Ps was assessed using flow cytometry, ELISPOT, lactate dehydrogenase release, and enzyme-linked immunosorbent assay., Results: Of S180 sarcoma-bearing mice immunized with mHSP/Ps isolated from S180 cells, 41.2% showed tumor regression and long-term survival, with a tumor growth inhibition rate of 82.3% at 30 days. Of MCA207 sarcoma-bearing mice immunized with mHSP/Ps isolated from MCA207 cells, 50% showed tumor regression and long-term survival with a tumor growth inhibition rate of 79.3%. All control mice died within 40 days. The proportions of natural killer cells, CD8+, and interferon-γ-secreting cells and tumor-specific cytotoxic T-lymphocyte activity were increased in the immunized group., Conclusions: Vaccination with a polyvalent mHSP/P cancer vaccine can induce an immunological response and a marked antitumor response to autologous tumors. This mHSP/P vaccine exerted greater antitumor effects than did HSP70, HSP60, or tumor lysates alone.

Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor (TNF)-α is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better-tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha-V integrins, has both anti-angiogenic and direct anti-tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra-peritoneal (i.p.) injections of cilengitide or saline 2 hr before and 3 hr after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a three to sevenfold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting., (Copyright © 2012 UICC.)

The chemopreventive and therapeutic efficacy of the cyclooxygenase (COX) inhibitor ibuprofen (IB) and of sulfasalazine (SASP), a drug that targets the antioxidant xc- system, were exploited in the experimental model of 3-methylcholantrene (3-MCA)-induced mouse sarcoma. The chemopreventive treatments gave unsatisfactory results because administration of IB one day after the 3-MCA injection only slightly delayed the tumor development, whereas SASP dispensed under the same conditions resulted in accelerated tumorigenesis. Similarly, the therapeutic treatment with either drug, administrated daily from the tumor detection, decreased the proliferation rate of tumor cells and increased the survival of treated mice only at a low extent. Remarkably, the combined chemopreventive treatment with IB and therapeutic treatment with SASP displayed a better efficacy, with strong delay of sarcoma growth, reduced tumor size and increased survival of treated mice. The two drugs target not only tumor cells but also tumor-associated macrophages that were dramatically decreased in the tumor infiltrate of mice subjected to the combined treatment. The synergistic effects of the association between a broad anti-inflammatory compound, such as IB, and a redox-directed drug, such as SASP, shed new light in the role of inflammation and of the redox response in chemical tumorigenesis and point to the combined chemopreventive plus therapeutic treatment with IB and SASP as a promising novel approach for antitumor therapy.

An ideal anticancer strategy should target only the malignant cells but spare the normal ones. In this regard, we established a platform, consisting of an antigen-delivering vehicle and a protein vaccine, for developing an immunotherapeutic approach with the potential for eliminating various cancer types. Mesenchymal stem cells (MSCs) have been demonstrated capable of targeting tumors and integrating into the stroma. Moreover, we have developed a protein vaccine PE(ΔIII)-E7-KDEL3 which specifically recognized E7 antigen and elicited immunity against cervical cancer. Taking advantage of tumor-homing property of MSCs and PE(ΔIII)-E7-KDEL3, we used E6/E7-immortalized human MSCs (KP-hMSCs) as an E7 antigen-delivering vehicle to test if this protein vaccine could effectively eliminate non-E7-expressing tumor cells. Animals which received combined treatment of KP-hMSCs and PE(ΔIII)-E7-KDEL3 demonstrated a significant inhibition of tumor growth and lung-metastasis when compared to PE(ΔIII)-E7-KDEL3 only and KP-hMSCs only groups. The efficiency of tumor suppression correlated positively to the specific immune response induced by PE(ΔIII)-E7-KDEL3. In addition, this combined treatment inhibited tumor growth via inducing apoptosis. Our findings indicated that KP-hMSCs could be used as a tumor-targeting device and mediate antitumor effect of PE(ΔIII)-E7-KDEL3. We believe this strategy could serve as a platform for developing a universal vaccine for different cancer types.

Immunotherapy using dendritic cells (DCs) has the potential to activate both T cells and NK cells. We previously demonstrated the long-lasting antitumor responses by NK cells following immunization with bone marrow-derived DCs. In the current study, we demonstrate that long-term antitumor NK responses require endogenous DCs and a subset of effector memory CD4(+) T (CD4(+) T(EM)) cells. One month after DC immunization, injection of a tumor into DC-immunized mice leads to an increase in the expression of CXCL10 by endogenous DCs, thus directing NK cells into the white pulp where the endogenous DCs bridged CD4(+) T(EM) cells and NK cells. In this interaction, CD4(+) T(EM) cells express CD40L, which matures the endogenous DCs, and produce cytokines, such as IL-2, which activates NK cells. These findings suggest that DC vaccination can sustain long-term innate NK cell immunity but requires the participation of the adaptive immune system.

The interaction between cancer vaccine adjuvants and myeloid-derived suppressor cells (MDSCs) is currently poorly understood. Very small size proteoliposomes (VSSP) are a nanoparticulated adjuvant under investigation in clinical trials in patients with renal carcinoma, breast cancer, prostate cancer, and cervical intraepithelial neoplasia grade III. We found that VSSP adjuvant induced a significant splenomegaly due to accumulation of CD11b(+)Gr-1(+) cells. However, VSSP-derived MDSCs showed a reduced capacity to suppress both allogeneic and Ag-specific CTL response compared with that of tumor-induced MDSCs. Moreover, splenic MDSCs isolated from tumor-bearing mice treated with VSSP were phenotypically more similar to those isolated from VSSP-treated tumor-free mice and much less suppressive than tumor-induced MDSCs, both in vitro and in vivo. Furthermore, different from dendritic cell vaccination, inoculation of VSSP-based vaccine in EG.7-OVA tumor-bearing mice was sufficient to avoid tumor-induced tolerance and stimulate an immune response against OVA Ag, similar to that observed in tumor-free mice. This effect correlated with an accelerated differentiation of MDSCs into mature APCs that was promoted by VSSP. VSSP used as a cancer vaccine adjuvant might thus improve antitumor efficacy not only by stimulating a potent immune response against tumor Ags but also by reducing tumor-induced immunosuppression.

Both experimental and clinical data indicate that the sympathetic nervous system may affect the development of certain tumors. To test this, in the present study we combined in vivo and in vitro approaches to study the effect of the sympathetic nervous system on proliferation of BP6-TU2 fibrosarcoma cells. First, we investigated the effect of 6-hydroxydopamine-induced sympathectomy on tumor development and survival of tumor-bearing rats. One week after chemical sympathectomy, we injected the BP6-TU2 fibrosarcoma cells intraperitoneally into male Wistar rats. The sympathectomy significantly reduced the incidence of intraperitoneal tumors and resulted in significantly improved survival of tumor-bearing rats compared to those with intact sympathetic innervation. Using immunohistochemical methods, we found neuron-specific enolase immunopositive structures within fibrosarcoma tissue, indicating innervation of tumors. Finally, an in vitro study showed elevated proliferation of BP6-TU2 fibrosarcoma cells in response to adding norepinephrine to the culture medium. Our findings indicate that sympathetic nerves directly potentiate the proliferation of BP6-TU2 fibrosarcoma cells in rats.

A large number of active principles from traditional medicinal plants have been reported to have chemopreventive properties. In the present study, therapeutic efficacy of an aqueous extract of Indigofera aspalathoides against growth of transplanted experimental fibrosarcomas in Wistar strain male albino rats was tested. Tumors which appeared about six weeks after implantation were highly localized and were maintained by serial transplantation. Rats were divided into four groups. Group I served as normal control animals. Group II were fibrosarcoma bearing animals. Group III were animals with fibrosarcoma treated with Indigofera aspalathoides aqueous extracts at a dose of 250 mg/kg. b. w. per day for 30 days. Group IV animals were treated with aqueous extract of Indigofera aspalathoides alone. Reduction in tumor weight was noted in Group III as compared to II. The levels of cytochrome C in liver and kidney, the levels of cytochrome P450 and cytochrome b5 in liver microsomes, phase I biotransformation enzymes NADPH-cytochrome P450, NADPH-cytochrome b5, and aniline hydroxylase, and the phase II enzymes glutathione-S-transferase and UDP glucuronyl transferase indicated that their modulation played a role in the therapeutic efficacy of Indigofera aspalathoides against experimental fibrosarcoma.

3-Methylcholanthrene (MCA)-induced sarcomas have been used as conventional tools for investigating immunosurveillance against tumor development. However, MCA-induced sarcoma is not always an ideal model for the study of the human cancer system because carcinomas and not sarcomas are the dominant types of human cancers. To resolve this problem, we established a novel and simple method to induce mouse squamous cell carcinomas (SCCs). As well known, the subcutaneous injection of MCA caused the formation of sarcomas at 100% incidence. However, we here first succeeded at inducing SCC at 60% of incidence within 2 months by a single intra-dermal injection of MCA. Using this primary SCC model, we demonstrated the critical role of interferon (IFN)-gamma-dependent type 1 immunity in immunosurveillance against SCC from the following results: (i) The incidence of SCC was accelerated in IFN-gamma-deficient mice compared with that in wild-type mice; (ii) In vivo injection of CpG-oligodeoxynucleotides (CpG-ODN) caused a marked reduction in the incidence of SCC in parallel with the activation of type 1-dependent antitumor immunity and (iii) The antitumor activity of CpG-ODN was significantly decreased in IFN-gamma-deficient mice. Thus, our established MCA-induced mouse SCC model could be a powerful tool for evaluating immunosurveillance mechanisms during the development of SCC and might result in a novel strategy to address immunosurveillance mechanisms of human cancer.

The objective of the present study was to isolate a lectin from fresh fruiting bodies of the mushroom Pleurotus citrinopileatus and examine it for various biological activities. The isolation procedure comprised ion exchange chromatography on DEAE-cellulose, CM-celluloses, and Q-Sepharose, and gel filtration on Superdex 75. A homodimeric 32.4 kDa lectin displaying high hemagglutinating activity was isolated with over 110 fold of purification. Its N-terminal amino acid sequence, QYSQMAQVME, has not been reported for other lectins. The lectin was unadsorbed on DEAE-cellulose in 0.001 M NH4HCO3 buffer (pH 9.4), but adsorbed on CM-cellulose in 0.001 M NH4OAc buffer (pH 4.8) and eluted by approximately 0.05 M NaCl in the same buffer. The lectin was subsequently adsorbed on Q-Sepharose and eluted by a linear gradient of 0-0.2 M NaCl in 10 mM NH4HCO3 buffer (pH 8.5). The lectin was obtained in a purified form after gel filtration by fast protein liquid chromatography on Superdex 75. The hemagglutinating activity of the lectin was inhibited by maltose, O-nitrophenyl-beta-d-galactopyranoside, O/P-nitrophenyl-beta-d-glucuronide and insulin. It was stable at temperatures up to 60 degrees C, and in NaOH and HCl solutions up to 0.1 M and 0.006 M concentration, respectively. It was sensitive to inhibition by HgCl2 and potentiation by AlCl3. The lectin exerted potent antitumor activity in mice bearing sarcoma 180, and caused approximately 80% inhibition of tumor growth when administered intraperitonealy at 5 mg/kg daily for 20 days. It elicited a mitogenic response from murine splenocytes in vitro with the maximal response at a lectin concentration of 2 microM. The lectin inhibited HIV-1 reverse transcriptase with an IC50 of 0.93 microM. It was devoid of antifungal activity.

Homeopathy is considered as one modality for cancer therapy. However, there are only very few clinical reports on the activity of the drugs, as well as in experimental animals. Presently we have evaluated the inhibitory effects of potentized homeopathic preparations against N'-nitrosodiethylamine (NDEA) induced hepatocellular carcinoma in rats as well as 3-methylcholanthrene-induced sarcomas in mice. We have used Ruta, Hydrastis, Lycopodium and Thuja, which are commonly employed in homeopathy for treating cancer. Administration of NDEA in rats resulted in tumor induction in the liver and elevated marker enzymes such as gamma-glutamyl transpeptidase, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and alkaline phosphatase in the serum and in liver. Concomitant administration of homeopathic drugs retarded the tumor growth and significantly reduced the elevated marker enzymes level as revealed by morphological, biochemical and histopathological evaluation. Out of the four drugs studied, Ruta 200c showed maximum inhibition of liver tumor development. Ruta 200c and phosphorus 1M were found to reduce the incidence of 3-methylcholanthrene-induced sarcomas and also increase the life span of mice harboring the tumours. These studies demonstrate that homeopathic drugs, at ultra low doses, may be able to decrease tumor induction by carcinogen administration. At present we do not know the mechanisms of action of these drugs useful against carcinogenesis.

The evidence has been produced that immunological mechanisms are involved in the known anticancer phenomenon of T. cruzi. Non-inbred albino mice were immunized with avirulent cultures of three strains and seven clones and then transplanted a tumor--sarcoma-180 or Ehrlich's adenocarcinoma. The used cultures induced the generation of T. cruzi antibodies whose level peaked by postimmunization days 50-60: the titers being 1:40-1:80 and the spread among the mice being 60%. Concurrently, immunization against T. cruzi provided a certain oncoprotective effect. In the immunized mice, the sizes of sarcoma-180 and adenocarcinoma were 1.5-2.0 and 2.0-2.5 times, respectively, less than those in the non-immunized ones. The antitumor protection was directly related to the murine blood T. cruzi antibody level during which implantation of a tumor occurred. At the peak of an immune response, the effect was 2 times higher than that in the early postimmunization periods. T. cruzi strains that were more immunogenic than clones ensured a more significant oncoprotection. The latter was more considerable in mice having antibody titers of 1:40-1:80 than in those with antibody titers of 1:10-1:20 and particularly in the animals showing no humoral response to the administration of the parasites at all.

Immunization with serological identification of Ags by recombinant expression cloning (SEREX)-defined self-Ags leads to generation/activation of CD4+ CD25+ regulatory T cells with suppressive activities and enhanced expression of Foxp3. This is associated with increased susceptibility to pulmonary metastasis following challenge with syngeneic tumor cells and enhanced development of 3-methylcholanthrene-induced primary tumors. In contrast, coimmunization with the same SEREX-defined self-Ags mixed with a CTL epitope results in augmented CTL activity and heightened resistance to pulmonary metastasis, both of which depend on CD4+ Th cells. These active regulatory T cells and Th cells were derived from two distinct CD4+ T cell subsets, CD4+ CD25+ T cells and CD4+ CD25- T cells, respectively. In the present study, IFN-gamma was found to abrogate the generation/activation of CD4+ CD25+ regulatory T cells by immunization with SEREX-defined self-Ag. CD4+ CD25+ T cells from these IFN-gamma-treated mice failed to exhibit immunosuppressive activity as measured by 1) increased number of pulmonary metastasis, 2) enhanced development of 3-methylcholanthrene-induced primary tumors, 3) suppression of peptide-specific T cell proliferation, and 4) enhanced expression of Foxp3. The important role of IFN-gamma produced by CD8+ T cells was shown in experiments demonstrating that CD4+ CD25+ T cells cotransferred with CD8+ T cells from IFN-gamma(-/-) mice, but not from wild-type BALB/c mice, became immunosuppressive and enhanced pulmonary metastasis when recipient animals were subsequently immunized with a SEREX-defined self-Ag and a CTL epitope. These findings support the idea that IFN-gamma regulates the generation/activation of CD4+ CD25+ regulatory T cells.

The C5a anaphylatoxin protein plays a central role in inflammation associated with complement activation. This protein is commonly regarded as one of the most potent inducers of the inflammatory response and a C5a peptide agonist was used as a molecular adjuvant. However, the full length C5a protein has not been tested as a potential tumor therapy. In this report, we describe the creation of a mini-gene construct that directs C5a expression to any cell of interest. Functional expression could be demonstrated in the murine mammary sarcoma, EMT6. When C5a expressing cells were injected into syngeneic mice, most C5a-expressing clones had significantly reduced tumor growth. Further characterization of a clone expressing low levels of C5a demonstrated that one-third of mice injected with this line had complete tumor regression. The mice whose tumors regressed were immune to subsequent challenge with unmodified EMT6 cells, suggesting that a component of the innate immune response can be used to augment adaptive immunity. Cellular analyses demonstrated that a significant difference in actual tumor cell number could be detected as early as day 10. A block in cell cycle progression was evident at all time points and high levels of apoptosis were observed early in the regression event. These data demonstrate that the complement protein C5a can play a significant protective role in tumor immunity.

The CD8(+) T cell response to Moloney-murine leukemia virus (M-MuLV)-induced Ags is almost entirely dominated by the exclusive expansion of lymphocytes that use preferential TCRVbeta chain rearrangements. In mice lacking T cells expressing these TCRVbeta, we demonstrate that alternative TCRVbeta can substitute for the lack of the dominant TCRVbeta in the H-2-restricted M-MuLV Ag recognition. We show that, at least for the H-2(b)-restricted response, the shift of TCR usage is not related to a variation of the immunodominant M-MuLV epitope recognition. After virus immunization, all the potentially M-MuLV-reactive lymphocytes are primed, but only the deletion of dominant Vbeta rescues the alternative Vbeta response. The mechanism of clonal T cell "immunodomination" that guides the preferential Vbeta expansion is likely the result of a proliferative advantage of T cells expressing dominant Vbeta, due to differences in TCR affinity and/or cosignal requirements. In this regard, a CD8 involvement is strictly required for the virus-specific cytotoxic activity of CTL expressing alternative, but not dominant, Vbeta gene rearrangements. The ability of T cells expressing alternative TCRVbeta rearrangements to mediate tumor protection was evaluated by a challenge with M-MuLV tumor cells. Although T cells expressing alternative Vbeta chains were activated and expanded, they were not able to control tumor growth in a long-lasting manner due to their incapacity of conversion and accumulation in the T central memory pool.

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that plays an important role in leukocytes homing to lymphoid tissues. The ability of SLC to co-localize both T cells and dendritic cells formed the rationale to evaluate its utility in cancer immunotherapy. The in vivo antitumor effect of murine SLC (mSLC) has been well documented, but little is known about that of human SLC (hSLC). To investigate the antitumor efficiency in vivo of hSLC, the hSLC gene was artificially synthesized and induced to express as a soluble form in Escherichia coli. After purification, the purity of the recombinant human SLC (rhSLC) protein was above 95% by SDS-PAGE analysis. The K(d) of rhSLC binding to peripheral blood lymphocytes (PBLs) was 0.2186 +/- 0.02675 microM as assessed by FACS, and the maximal chemotactic index of rhSLC was 9.49 at 100 nM as assessed by in vitro chemotaxis assay. Then genomic sequences of hSLC and mSLC, and of human CCR7 (hCCR7) and murine CCR7 (mCCR7), the receptor for SLC, were aligned. It was found that hSLC and mSLC share 70.72% identity and hCCR7 and mCCR7share 86.77% identity. Furthermore, we found that rhSLC could chemoattract murine peripheral blood mononuclear cells (PBMCs) in vitro. On the basis of these facts, immune competent mice inoculated with S180 sarcoma cells were chosen as an in vivo model. Intratumoral injections of rhSLC inhibited tumor growth and increased survival. These findings suggest that, despite its incapability to bind to either human or murine CXCR3, which is related to angiostasis, rhSLC can induce an antitumor response in vivo by another route. This report proves that rhSLC has a potent tumor-inhibition ability that makes it a promising candidate agent in cancer immunotherapy.

Adoptive transfer of tumor-specific effector T cells induces regression of advanced tumors and induces a long term memory response; however, the origin of this response has not been clearly defined. In this study Thy1.2+ mice bearing advanced MCA-205 tumors were treated with sublethal total body irradiation, followed by adoptive transfer of congenic Thy1.1+ T cells that had been sensitized to tumor in vivo and then activated ex vivo with anti-CD3, IL-2, and IL-7. Splenocytes were recovered >140 days after the initial therapy, and the L-selectinlow memory cell subset was separated into host Thy1.2+ and transferred Thy1.1+ cells and restimulated ex vivo. Both adoptively transferred Thy1.1+ cells as well as reconstituted host Thy1.2+ cells could specifically eliminate MCA-205 pulmonary metastases. Interestingly, hosts with partial responses followed by tumor recurrence nevertheless harbored memory cells that could be isolated and numerically amplified ex vivo to regenerate potent effector function. Memory cells were recovered after adoptive transfer into lymphodepleted nontumor-bearing hosts, indicating that they were not dependent on continued Ag exposure. These experiments establish that rapid ex vivo expansion of tumor Ag-primed T cells does not abrogate their capacity to become long-lived memory cells. Moreover, immune-mediated tumor regression coincident with lymphoid reconstitution produces another wave of host memory cells. These data suggest an approach to rescuing antitumor immune function even in hosts with long-standing progressive tumor through restorative ex vivo activation.

Tumor-associated carbohydrate antigens are typically perceived as inadequate targets for generating tumor-specific cellular responses. Lectin profile reactivity and crystallographic studies demonstrate that MHC class I molecules can present to the immune system posttranslationally modified cytosolic peptides carrying O-beta-linked N-acetylglucosamine (GlcNAc). Here we report that a peptide surrogate of GlcNAc can facilitate an in vivo tumor-specific cellular response to established Meth A tumors that display native O-GlcNAc glycoproteins on the tumor cell surface. Peptide immunization of tumor-bearing mice had a moderate effect on tumor regression. Inclusion of interleukin 12 in the immunization regimen stimulated complete elimination of tumor cells in all of the mice tested, whereas interleukin 12 administration alone afforded no tumor growth inhibition. Adoptive transfer of immune T cells into tumor-bearing nude mice indicates a role for CD8+ T cells in tumor regression. This work postulates that peptide mimetics of glycosylated tumor rejection antigens might be further developed for immune therapy of cancer.

The application of pulsed electric fields to cells is extended to include nonthermal pulses with shorter durations (10-300 ns), higher electric fields (< or =350 kV/cm), higher power (gigawatts), and distinct effects (nsPEF) compared to classical electroporation. Here we define effects and explore potential application for nsPEF in biology and medicine. As the pulse duration is decreased below the plasma membrane charging time constant, plasma membrane effects decrease and intracellular effects predominate. NsPEFs induced apoptosis and caspase activation that was calcium-dependent (Jurkat cells) and calcium-independent (HL-60 and Jurkat cells). In mouse B10-2 fibrosarcoma tumors, nsPEFs induced caspase activation and DNA fragmentation ex vivo, and reduced tumor size in vivo. With conditions below thresholds for classical electroporation and apoptosis, nsPEF induced calcium release from intracellular stores and subsequent calcium influx through store-operated channels in the plasma membrane that mimicked purinergic receptor-mediated calcium mobilization. When nsPEF were applied after classical electroporation pulses, GFP reporter gene expression was enhanced above that observed for classical electroporation. These findings indicate that nsPEF extend classical electroporation to include events that primarily affect intracellular structures and functions. Potential applications for nsPEF include inducing apoptosis in cells and tumors, probing signal transduction mechanisms that determine cell fate, and enhancing gene expression.

Peptide elution and expression cloning methods have been used to identify T cell-recognized antigens for which no molecular information is available. We identified a unique tumor antigen peptide pRL1a, IPGLPLSL that is recognized by CTL on BALB/c RL male 1 leukemia by peptide elution. The sequence of the peptide corresponded to the normally untranslated 5' region of akt. Cytotoxicity was generated in BALB/c spleen cells by in vivo and in vitro sensitization with pRL1a peptide in the form of multiple antigen peptide (MAP), but not the original form. pRL1a MAP immunization had a significant growth-inhibitory effect. pRL1a MAP was mostly internalized into the endosomal compartment of antigen-presenting cells, leaked to the cytosol, and degraded, and the pRL1a peptide produced was presented through the MHC class I pathway. In vivo depletion of CD4 T cells from tumor-inoculated BALB/c mice caused RL male 1 regression. Overexpression of the RLakt molecule seemed to induce CD4 immunoregulatory cells, which resulted in progressive RL male 1 growth in BALB/c mice. In vivo administration of anti-CD25 mAb (PC61) caused regression of RL male 1, suggesting that CD4(+) CD25(+) immunoregulatory cells were involved in the tumor growth. Recently, we improved the sensitivity and the efficacy of T cell antigen cloning from cDNA expression libraries by using large- and small-scale ELISPOT assays. Using the IFN-gamma ELISPOT method, we obtained a cDNA clone S35 of 937 bp recognized by AT-1 CTL on BALB/c Meth A sarcoma. S35 was a part of the retinoic acid-regulated nuclear matrix-associated protein (ramp). AT-1 CTL recognized the peptide LGAEAIFRL, which was derived from a newly created open reading frame due to the exon 14 extension.

p53 is overexpressed by half of all cancers, and is an attractive target for a vaccine approach to immunotherapy. p53 overexpression is frequently the result of point mutations, which leaves the majority of the protein in its wild-type form. Therefore, the majority of p53 sequence is wild type, making it a self-protein for which tolerance plays a role in limiting immune responses. To overcome tolerance to p53, we have expressed wild-type murine p53 in the nonpathogenic attenuated poxvirus, modified vaccinia virus Ankara (recombinant modified vaccinia virus Ankara expressing wild-type murine p53 (rMVAp53)). Mice immunized with rMVAp53 vaccine developed vigorous p53-specific CTL responses. rMVAp53 vaccine was evaluated for its ability to inhibit the outgrowth of the syngeneic murine sarcoma Meth A, which overexpresses mutant p53. Mice were inoculated with a lethal dose (5 x 10(5) cells injected s.c.) of Meth A tumor cells and vaccinated by i.p. injection 3 days later with 5 x 10(7) PFU of rMVAp53. The majority of mice remained tumor free and resistant to rechallenge with Meth A tumor cells. We wished to determine whether rMVAp53 immunization could effect the rejection of an established, palpable Meth A tumor. In subsequent experiments, mice were injected with 10(6) Meth A tumor cells, and treated 6 days later with anti-CTLA-4 Ab (9H10) and rMVAp53. The majority of treated mice had complete tumor regression along with lasting tumor immunity. In vivo Ab depletion confirmed that the antitumor effect was primarily CD8 and to a lesser extent CD4 dependent. These experiments demonstrate the potential of a novel cell-free vaccine targeting p53 in malignancy.

Unlabelled: The effect of parenterally-administered histamine dihydrochloride (histamine), the role of the histamine H2-receptor and the importance of histamine administration routes on the in vivo growth of a rat Leydig cell sarcoma (LTW) were explored., Materials and Methods: Wistar/Furth rats with LTW tumours transplanted into subcutaneous and liver tissue received treatment by daily subcutaneous injections or by an osmotic pump for 10 days., Results: Subcutaneous injections of histamine (0.5 mg/kg) reduced the liver tumour weight by 46+/-8% (p=0.0002) and subcutaneous tumour weight by 41+/-12% (p=0.026) versus animals receiving subcutaneous saline injections. Histamine continuously administered by osmotic pumps at doses of 0.5, 2 and 20 mg/kg/24 hour, did not reduce tumour growth. Ranitidine (50 mg/kg s.c.), inhibited the anti-tumour effect observed by subcutaneous histamine injections. In conclusion, H2-receptor-mediated tumour growth inhibition was accomplished by bolus injections of histamine.

Amifostine was investigated for its ability to inhibit spontaneous metastases formation using the well-characterized murine sarcoma, Sa-NH. Amifostine was administered intraperitoneally at a dose of 50 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8-8.5 mm in diameter. Amifostine was again administered immediately after surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later. Twenty-one days later, animals were evaluated for the presence of spontaneously developed pulmonary metastases. Nontumor-bearing control animals were sham treated using the same dosing and surgery schedules. Treatment with amifostine appeared to slightly delay tumor growth, that is, 13 vs. 12 days for tumors to reach an average diameter of 8 mm. Amifostine reduced both the incidence of pulmonary metastases formed in experimental animals from 77% to 57% (p < 0.05), and their average number per animal from 12.8 +/- 5.4 (SEM) to 2.9 +/- 1.1 (SEM). The effect of amifostine exposure on serum levels of the angiogenesis inhibitor angiostatin was also determined using Western blot analysis. Consistent with the antimetastatic effect, exposure of animals to 50 mg/kg of amifostine resulted in a 4-fold enhanced serum level of angiostatin above control levels. This phenomenon occurred in tumor-bearing and nontumor-bearing animals. The effects of amifostine on matrix metalloproteinase (MMP) enzymatic activity was also determined using gelatin zymography. Conditioned growth medium collected from Sa-NH cells grown to confluency was exposed to various concentrations of SH, i.e., 2-[(aminopropyl)amino]ethane-thiol (WR-1065), the active thiol form of amifostine, for either 30 min or 18 hr. WR-1065, as a function of increasing dose and time, inhibited the enzymatic activities of MMP-2 and MMP-9. At a concentration and time of exposure likely to be achieved in vivo, that is, 40 microM and 30 min, MMP-2 and MMP-9 activities were reduced to between 30% and 40% of control values. Consistent with these affects, WR-1065 was also found to be effective in inhibiting the ability of Sa-NH cells to migrate through Matrigel membranes. After an 18-hr exposure under in vitro conditions, WR-1065 at concentrations of 4, 40 and 400 microM, and 4 mM, inhibited Sa-NH migration to 11%, 44%, 81% and 97% of control values, respectively. The abilities of amifostine and its active thiol WR-1065 to stimulate angiostatin production in mice, and to inhibit the MMP enzymatic activities and invasion ability of Sa-NH cells under in vitro conditions, are consistent with the observed antimetastatic effects exhibited against Sa-NH tumors growing in vivo., (Copyright 2002 Wiley-Liss, Inc.)

Different vaccine constructs based on DNA vaccines and viral recombinant vaccines expressing mouse p53 were compared for induction of protective immune responses to challenge with a sarcoma cell line that expresses high levels of mutated p53 protein. Viral recombinant vaccines based on E1-deleted adenovirus or vaccinia virus recombinants expressing p53 with wild-type sequences in the mutational hotspot domain and a single mutation in the tetramerization domain (p53(mu338)) failed to induce protection against progression of this tumor cell line. A DNA vaccine expressing a form of p53 carrying the same point mutations as the tumor cell line showed low efficacy that was comparable to that of a DNA vaccine expressing p53(mu338). Efficacy of the DNA vaccine was augmented upon expressing p53(mu338) as a fusion protein linked to a viral leader sequence. Other modifications such as fusion to the signal sequence of the lysosome-associated membrane protein (LAMP) or ubiquitin failed to improve the efficacy of the vaccine to p53. Protection mediated by CD4(+) and CD8(+) T cells was specific for p53.

Although CD8(+) T cells play a central role as immune effectors, CD4(+) T cells act to control the activation and persistence of the CD8(+) T cell response in autoimmune disease, antiviral immunity, and experimental systems with immunogenic model tumor Ag. However, little information is available on the effects of CD4(+) T cells on the function of endogenous CD8(+) T lymphocytes recognizing authentic tumor rejection Ag with limited immunogenicity. We report here that the prophylactic or postchallenge administration of T helper Th1-type and Th2-type CD4(+) clones specific for an unmutated rejection Ag (murine P815AB, resembling tumor-specific shared Ag in humans) leads to the induction of P815AB-specific reactivity in vivo and concomitant tumor destruction, with quantitative rather than qualitative differences characterizing the antitumor activity of Th1 vs Th2 cells. Because the transferred CD4(+) cells lacked direct antitumor activity in vitro and required the de novo generation of P815AB-specific CD8(+) T cells in vivo, these findings suggest that CD4(+) lymphocytes can enhance the ability of host APC to initiate an endogenous CD8(+) T cell response to authentic, poorly immunogenic tumor rejection Ag.

Mice made transgenic (Tg) for a rat anti-mouse CD4 Ab (GK mice) represent a novel CD4-deficient model. They not only lack canonical CD4 cells in the periphery, but also lack the residual aberrant Th cells that are found in CD4-/- mice and MHC class II-/- mice. To analyze the role of CD4 help and costimulation for CTL induction against alloantigens, we have assessed the surface and functional phenotype of CD8 cells in vivo (e.g., clearance of allogeneic P815 cells) and in vitro. In our CD4-deficient GK mice, CTL responses to allogeneic P815 cells were induced, albeit delayed, and were sufficient to eliminate P815 cells. Induction of CTL and elimination of allogeneic P815 cells were inhibited both in the presence and absence of CD4 cells by temporary CD40 ligand blockade. This indicated that direct interaction of CD40/CD40L between APCs and CD8 cells may be an accessory signal in CTL induction (as well as the indirect pathway via APC/CD4 interaction). Furthermore, whereas in CTLA4Ig single Tg mice P815 cells were rejected promptly, in the double Tg GK/CTLA4Ig mice CTL were not induced and allogeneic P815 cells were not rejected. These findings suggest that CD40/CD40L is involved in both CD4-dependent and CD4-independent pathways, and that B7/CD28 is pivotal in the CD4-independent pathway of CTL induction against allogeneic P815 cells.

Effective antitumor immune responses against cerebral malignancies have been demonstrated in several models, but precise cellular function of specific effector cells is poorly understood. We have explored this topic by analyzing the MHC class I-restricted T cell response elicited after implantation of HLA-CW3-transfected P815 mastocytoma cells (P815-CW3) in syngeneic mice. In this model, tumor-specific CTLs use a distinctive repertoire of TCRs that allows ex vivo assessment of the response by immunophenotyping and TCR spectratyping. Thus, for the first time in a brain tumor model, we are able to directly visualize ex vivo CTLs specific for a tumor-expressed Ag. Tumor-specific CTLs are detected in the CNS after intracerebral implantation of P815-CW3, together with other inflammatory cells. Moreover, despite observations in other models suggesting that CTLs infiltrating the brain may be functionally compromised and highly dependent upon CD4 T cells, in this syngeneic P815-CW3 model, intracerebral tumors were efficiently rejected, whether or not CD4 T cells were present. This observation correlated with potent ex vivo cytotoxicity of brain-infiltrating CTLs, specific for the immunodominant epitope CW3170-179 expressed on P815-CW3 tumor cells.

We have previously reported that CTL were demonstrable early after inoculation of CMS5 fibrosarcoma cells, but that they disappeared within 3 wk. These mice were unable to reject a challenge with CMS5 tumor cells. Other studies demonstrated cell surface phenotype and signaling abnormalities of cells within the spleen. Since we assumed that such an environment would make it more difficult to elicit antitumor immune responses via immunotherapy, we asked whether resection of the tumor could reverse these abnormalities. Although early after tumor cell inoculation tumor resection leads to the development of immunity, the effect at late time points has not been studied critically. To test this, mice were inoculated s.c. with CMS5 cells and after 28 days the tumors were resected. We observed a gradual normalization of the cellular phenotype of the spleen. In particular, there was a decrease in the number of Mac1+/Gr1(high) cells and an increase in the number of CD3+ cells in the spleen within 24-48 h of tumor resection. By day 10, these values were normal. Levels of p56lck increased as well. The functional implications of these changes were illustrated by the reduced growth rate or the complete rejection of a challenge of tumor cells in the resected mice. Both CD4+ and CD8+ cells were involved in the restoration of tumor immunity. Our results suggested that tumor resection not only led to the reversal of immune suppression, but also unmasked a population of primed T cells able to mediate protective immunity.

This study was aimed at creating a more effective tumor cell vaccine by suppressing Ii protein in the presence of MHC class II molecules within a cancer cell. Absence of the Ii protein, which normally blocks the antigenic-peptide-binding site of MHC class II molecules at synthesis in the endoplasmic reticulum, presumably increases the range of cancer-related epitopes presented to CD4+ helper T cells. Effective suppression of Ii protein was achieved with an antisense, phosphorothioate oligonucleotide, which was selected on the basis of (1) the RNase H activation assay, (2) an assay for Ii protein suppression, and (3) a test for potency with respect to the extent of base sequence ("sequence walking"). The SaI murine sarcoma, which is MHC-class-I+ and MHC-class-II-, Ii-protein-, upon transfection with genes for either interferon gamma or the MHC class II transactivator, came to express MHC class II molecules and Ii protein. In each line of transfected tumor cells, the antisense oligonucleotide profoundly suppressed Ii protein in 35%-55% cells, without affecting expression of MHC class II molecules. Inoculation of mice with such Ii-protein-suppressed tumor vaccine cells, after either formaldehyde fixation or X-irradiation, led to much greater protection against challenge with the parental SaI sarcoma than did inoculation with untreated cells. This approach to cancer cell vaccination can be applied in a wide range of human tumors.

To assess the role of the p53 tumor suppressor gene in skin carcinogenesis by UV radiation, mice constitutively lacking one or both copies of the functional p53 gene were compared to wild-type mice for their susceptibility to UV carcinogenesis. Heterozygous mice showed greatly increased susceptibility to skin cancer induction, and homozygous p53 knockout mice were even more susceptible. Accelerated tumor development in the heterozygotes was not associated with loss of the remaining wild-type allele of p53, as reported for tumors induced by other carcinogens, but in many cases was associated with UV-induced mutations in p53. Tumors arose on the ears and dorsal skin of mice of all three genotypes, and homozygous knockout mice also developed ocular tumors, mainly melanomas. Skin tumors in the p53 knockout mice were predominately squamous cell carcinomas and were associated with premalignant lesions resembling actinic keratoses, whereas those in the heterozygous and wild-type mice were mainly sarcomas. These results demonstrate the importance of p53 in protecting against UV-induced cancers, particularly in the eye and epidermis.

The purpose was to observe the effect on sarcomagenesis of 3 substances reported to inhibit neoplastic growth--interferon alpha-2/alpha-1 hybrid (IFN), 5-iodo-2-deoxyuridine (IUdR) and methylglyoxal bis(guanylhydrazone) (MGBG). Inhibitory effect might help diminish the sarcoma risk of human implants. The substances were applied respectively to groups of 25mm cellulose filters which were implanted subcutaneously 1 per animal in randomly assigned respective groups of 50 female BALB/c mice. The implant sites were palpated weekly. On detection of a tumour the animal was sacrificed. The number of tumours arising and the accumulated weeks of exposure to the implants were recorded per group and compared to those of controls with untreated filters. Tumour incidence in the 2 IFN groups was 33/45 and 35/48 mice--160 per cent that of the controls, 22/48 (chi-square p < 0.05). In the IUdR group tumour incidence was 24/44 mice--194 per cent that of controls (p < 0.05), and in the MGBG group 15/43--122 per cent that of controls (p < 0.75). Although the substances inhibit tumour growth in man, they did not inhibit but increased film sarcomagenesis, not significant for MGBG. Observation of the effects of such substances with dual neoplastic activity may furnish clues to the control processes of neoplasia.

We studied the effect of protein-bound polysaccharide PSK on metastatic colonization of BALB/c mice after intravenous injections of different syngeneic murine H-2 positive and H-2 negative tumor clones. The tumor lines used were different clones from chemically induced fibrosarcomas (GR9.B9, an H-2 negative clone from GR9 tumor, and B7.1.B4, an H-2 positive clone from B7.1 tumor). These clones were selected because of their different sensitivity to NK cytotoxicity, which was related to MHC class I expression. Pretreatment of mice with PSK inhibited metastatic colonization derived from B9 H-2 negative tumor cells. In contrast, lung colonization of PSK treated mice injected with B7.1.B4 H-2 positive tumor cells was higher, and differences in the number of colonies between untreated and PSK treated mice were small. In several experiments the effect of PSK was attenuated to a greater degree when high numbers of cells were injected. Abrogation of NK cells with anti-asialo GM1 serum significantly increased (in all tumors and at different cell doses) the number of metastatic colonies in comparison with untreated mice injected with tumors, regardless of the cell dose used. These results clearly suggest that NK cell activation in vivo by the protein bound polysaccharide PSK abrogates metastasis formation in mice. Abrogation was dependent on the H-2 phenotype even when pretreatment consisted of a single dose of PSK. This effect, related to the NK sensitivity of the tumor target, can be used to predict the effect of PSK in vivo.

We have recently reported that interleukin 18 (IL-18) pretreatment induces immunologically mediated antitumor effects in BALB/c mice injected i.p. with syngeneic Meth A sarcoma. In this study, mice were pretreated with IL-18 before Meth A transplantation, and immunocompetency in pretreated or untreated tumor-bearing mice (TBM) 3, 9, and 15 days after transplantation was compared with that of normal mice. On day 3, pretreated TBM mitogen-stimulated spleen cells produced significantly decreased levels of IL-2 and IFN-gamma during 24-h culture. In contrast, IL-10 and granulocyte macrophage colony-stimulating factor productions were significantly enhanced in pretreated TBM cultures, and natural killer (NK) cell activity was also significantly augmented. Splenomegaly was also observed in the pretreated TBM on day 3, and the proliferating cells were identified as asialo GM1+ cells by flow cytometry. Cytotoxic activity of pretreated TBM spleen cells after a 5-day mixed lymphocyte-tumor cell culture did not differ from that of untreated TBM and normal mice on day 3 but was significantly enhanced on days 9 and 15 compared with that observed in normal mice and untreated TBM. Concurrently, the production of IL-2 and of IL-10 recovered and decreased, respectively, and NK activity dropped to normal levels. The effects of IL-18 on cytokine production and NK activity observed on day 3 treated TBM were also reproduced in normal mice. In conclusion, IL-18 seems to enhance the generation of NK activity early after tumor transplantation and simultaneously induces an increase and a decrease in the production of IL-10 and IL-2, respectively. As NK activity subsides to normal levels and IL-10 synthesis decreases, IL-2 synthesis is restored, and cytolytic cell activity is significantly enhanced. These results provide new insight into the immunologically mediated antitumor effects of IL-18.

Inbred adult female rats were immunized against syngeneic ST-FeSV induced sarcoma cells. ST-FeSV was injected subcutaneously into 57 neonates (vaccinated) born from these immunized females and into 60 non-vaccinated syngeneic neonates. Serum thymic factor (FTS) was injected subcutaneously into 10 of vaccinated and 30 of non-vaccinated rats. Sarcomas developed in 40.4% (19/47) of vaccinated (A), 20.0% (2/10) of vaccinated FTS injected (B), 63.3% (19/30) of non-vaccinated FTS injected (C), and 76.7% (23/30) of non-treated (D) rats. By AB immunostaining using antibody to v-fes product (P85), sarcomas developed in 10 of 13 rats of group C tested, and 3 of 6 rats of group D tested were positive, but those in 7 rats of group A and 2 rats of group B tested were all negative. Lung metastasis was observed in rats of all groups except those of B group. All sera of animals that developed sarcomas were positive to P85 in Western blot analysis. These results showed that FTS augmented suppressive effects on sarcoma development in hosts immune to the viral oncogene product.

Iscador, an extract from the semi-parasitic plant Viscum album, was found to inhibit 20-methylcholanthrene-induced carcinogenesis in mice. Intraperitoneal administration of Iscador (1 mg/dose) twice weekly for 15 weeks could completely inhibit 20-methylcholanthrene-induced sarcoma in mice and protect these animals from tumour-induced death. Iscador was found to be effective even at lowered doses. After administration of 0.166, 0.0166 and 0.00166 mg/dose 67, 50 and 17% of animals respectively did not develop sarcoma.

Experiments were conducted to evaluate the effects of alprazolam (1 mg/kg i.p.) on the development of autochthonous tumors induced by the Moloney sarcoma virus (MSV) in BALB/c female mice subjected to stress. Enhancement of MSV-induced tumor incidence and growth was observed, together with a delay in the usual prompt regression of the tumors, when programmed white sound anxiety-stress was administered immediately following MSV i.m. inoculation. However, a depressant effect on tumor size and incidence was observed when stress was administered before virus inoculation. Treatment with alprazolam was found to reverse partially the adverse effects of postinoculation stress, and also to inhibit the beneficial effects of the preinoculation administration of stress on tumor development. Pretreatment with Ro 15-1788 (10 mg/kg s.c.), a central benzodiazepine antagonist, resulted in a suppression on both effects of alprazolam in stressed mice.

The effects of injection of 6 day-old homogenate of chicken embryo tissue on sarcoma of Pliss have been investigated. It was found to drastically inhibit said malignancy in rats and to increase their survival. Histological study revealed extensive necrosis of tumor tissue as well as formation of fibro-vascular structures showing characteristic leukocyte-macrophageal infiltration in the treated animals unlike untreated controls and those injected with cyclophosphamide. A link between activation of cellular immunity and antitumor effects of embryonal tissue homogenate is suggested.

In sarcomagenesis in rats infected neonatally with feline sarcoma virus (ST-FeSV), v-fes product (P85) was previously shown by us to be a predictive and preventive determinant. In order to explore the part played by P85 in tumor suppression, DNA was extracted from precancerous granulomas and from slow or rapid growing sarcomas induced by neonatal injection of the virus. The v-fes signal from extracted DNA was analyzed by PCR-SSCP. The prototype v-fes gene signal was detected in most lesions and found to be generally amplified in rapid growing sarcomas and in some granulomas. Several v-fes homologs showing varying mobilities in gel were seen in most sarcomas and some granulomas with or without the prototype v-fes signal. In slow growing sarcomas and granulomas induced in hosts that were immunized with ST-FeSV induced syngeneic sarcoma and proved to carry IgG antibody to P85, the prototype v-fes gene was found to be down-regulated and v-fes homologs were found to be reduced in number or eliminated. These results suggest that the development of v-fes mutations is associated with the growth potential of cells carrying the v-fes gene, and that host immunity to v-onc product influences the development of virogene rearrangements and results in slow and suppressed growth of tumors caused by neonatal infection with retrovirus.

We investigated the effect of combined treatment with anti-LFA-1 and anti-ICAM-1 monoclonal antibodies (MAbs) in the immune reaction to Moloney-murine-sarcoma-virus(M-MSV)-induced tumors, which spontaneously regress due to the generation of a strong virus-specific cytotoxic-T-lymphocyte(CTL) response. Repeated systemic administration of both MAbs to M-MSV-injected mice enhanced tumor growth and delayed regression, while treatment with a single MAb had a similar, though less pronounced, effect. The immune depression achieved could not be attributed to lymphocyte depletion, because no reduction in the total number of leukocytes was detected in the peripheral blood or spleen of these mice. However, anti-LFA-I MAb, alone or in combination with anti-ICAM-I MAb, prevented lymphocyte homing in tumor-draining lymph nodes. Cytofluorimetric analysis disclosed a profound down-modulation of LFA-I and ICAM-I molecule expression on T cells following in vivo MAb treatment. Moreover, in anti-LFA-I MAb-treated mice, the receptor was coated to saturation, while anti-ICAM-I MAb treatment brought about ICAM-I-molecule-coating levels below saturation. Evaluation of M-MSV-specific CTL precursor (p) frequency in lymphoid organs of mice receiving combined MAb treatment showed that CTL generation was greatly reduced 10 days after M-MSV injection, and returned to control levels by day 15. Our findings indicate that systemic administration of MAbs to LFA-I and ICAM-I molecules brings about a strong immune suppressive effect which is mainly due to a block in T-lymphocyte re-circulation, and activation by tumor cells. However, this immune-depressive effect is only temporary, and strictly dependent on continuous MAb administration. Thus, our data suggest that treatment with anti-LFA-I and anti-ICAM-I MAbs combined is unable to induce T-cell tolerance in a highly immunogenic system.

Zinostatin stimalamer (ZSS) is a new anticancer agent derived from neocarzinostatin (NCS), which is synthesized by conjugation of one molecule of NCS and two molecules of poly(styrene-co-maleic acid). ZSS exhibited potent in vitro and in vivo antitumor activity in preclinical experiments, and a clinical trial of the intra-arterial administration of ZSS with iodized oil on hepatocellular carcinoma showed potent antitumor activity. We investigated the effect of ZSS and NCS on antitumor resistance and found that pretreatment with either drug suppressed the growth of MethA tumors in Balb/c mice and induced tumor eradication when given separately by single administration at therapeutic doses between 1 day and 4 weeks before tumor transplantation. The findings that the cytocidal activity of these drugs was not detected in vivo at the time of tumor transplantation and that tumor regression was preceded by a period of transient growth suggested that tumor regression was due to host-mediated antitumor activity induced by these drugs. Pretreatment with ZSS or NCS also suppressed the growth of Colon 26 carcinoma and Sarcoma 180. The finding that NCS showed the same effect as ZSS suggests that poly(styrene-comaleic acid) is not essential for the induction of host-mediated antitumor activity. Furthermore, apo-ZSS, which lacks cytocidal activity, did not induce antitumor activity. From this, it is suggested that the cytocidal effect of ZSS involves the induction of host-mediated antitumor resistance. In athymic Balb/c nu/nu mice, pretreatment with ZSS or NCS did not induce tumor eradication, suggesting that mature T lymphocytes play an important role in tumor eradication. Challenging MethA was rejected without transient growth in mice that had been cured of MethA, but challenging Colon 26 was not, showing that anti-MethA resistance was augmented selectively in the MethA-eradicated mice. Splenocytes from MethA-bearing mice pretreated with the drug showed tumor-neutralizing activity beginning 14 days after tumor transplantation. Tumor-neutralizing activity was only induced after MethA transplantation. The effector cells of this tumor-neutralizing activity were Thy1.2+ T lymphocytes that had been passed through a nylon-wool column, but no significant augmentation of cell-mediated cytotoxic activity of splenocytes from MethA-eradicated mice was observed in vitro.

Many tumors express peptides that are potentially immunogenic; however, the host's immune system is often not sufficiently stimulated to mediate tumor rejection. The inability to mount a potent antitumor immune response has often been attributed to the lack of generation of sufficient tumor-specific T cell help. Efforts in this laboratory to improve tumor-specific immunity have therefore focused on improving the generation of tumor-reactive T helper cells. Previous studies have suggested that immunity to the murine SaI sarcoma can be significantly improved if the tumor is engineered to express syngeneic MHC class II molecules, and thereby directly present tumor peptides to Th lymphocytes. In the present study we demonstrate that vaccination with class II+ SaI transfectants results in immunity that is extremely effective against high-dose challenges of wild-type SaI tumor. The immunity induced by immunization with these transfectants is also exceptionally long-lived (greater than 6 months) and radiation resistant, suggesting that tumor-specific memory T cells are generated. The resulting immunity is specific for the immunizing tumor and protects autologous mice against challenges of both ascites and solid SaI variants. Depletion and adoptive transfer studies confirm the role of CD4+ T cells in the induced immunity, supporting the hypothesis that improving the generation of Th cells enhances the antitumor immune response. Inasmuch as irradiated or paraformaldehyde-fixed transfectants are as effective as live transfectants in stimulating tumor rejection, these genetically engineered tumor cells may serve as useful vaccines against wild-type neoplasms.

Feline sarcoma virus of Snyder-Theilen strain (ST-FeSV) induces sarcomas in Wistar/Ma rats following neonatal virus injection. Induced tumors express the viral oncogene product (P85) and elicit in hosts the specific serum anti-P85 antibody detectable by Western blot analysis. Syngeneic adult female rats were immunized with an ST-FeSV induced sarcoma that was 100% transplantable to syngeneic adult rats. Newborns from immunized rats (vaccinated rats) were found to carry anti-P85 in their sera at birth. Following neonatal injection of the virus to vaccinated and non-vaccinated control rats, tumor incidence was found to be lower and survival time significantly longer in vaccinated rats than in controls (p < 0.01). A nonapeptide known to be thymic hormone (FTS) showed suppressive effects on tumor development. These results indicate that tumors caused by perinatal retrovirus infection may be suppressed by efficient elicitation of cell-mediated immune response against the product of oncogene of the causative virus.

9-(2-Phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) is a broad-spectrum antiviral agent with potent activity against DNA viruses and retroviruses. We now demonstrate that PMEDAP is highly efficacious when given orally to mice infected with either Moloney murine sarcoma virus (MSV), Friend leukemia virus (FLV), or murine cytomegalovirus (MCMV). PMEDAP markedly delayed MSV-induced tumor initiation when administered orally at 50, 100, or 250 mg/kg/day during 5 subsequent days. At the highest dose (250 mg/kg/day), PMEDAP completely prevented tumor formation in the MSV-infected animals. PMEDAP also caused 84-96% inhibition of FLV-induced splenomegaly when given orally to FLV-infected mice at 50-250 mg/kg/day. These PMEDAP treatment regimens were also markedly effective in increasing the survival rate of MCMV-infected mice. Intraperitoneal PMEDAP achieved a comparable antiviral activity at 2- to 5-fold lower doses than oral PMEDAP. However, the therapeutic index (ratio of the toxic dose to the antivirally effective dose) of oral PMEDAP was substantially higher than that of intraperitoneal PMEDAP. Oral PMEDAP at doses of 100, 250, or 500 mg/kg resulted in plasma PMEDAP levels of 0.5-2.5 micrograms/ml, which were sustained for 3 or 6 hours after administration and may account for the high antiviral efficacy achieved.

Adhesive interactions between tumor cells and host tissue occur at several stages of metastasis. Such interactions might be inhibited by microbial metabolites resembling the binding regions of matrix molecules. Certain metabolite sequences including Gly, Asp, Arg, and Ser (GAAS) proved to be critical for cell interactions, e.g. with fibronectin. In vitro, the rosette formation of murine pulmonary cells and sarcoma L-1 cells decreased significantly in the presence of Propionibacterium acnes-metabolites rich in GAAS. In vivo, coinjection of Propionibacterium acnes-metabolites and sarcoma L-1 cells significantly inhibited the formation of lung colonies in BALB/c mice. The inhibition of lung colonization by these metabolites appeared to be noncytotoxic and obviously did not result from impairment of cellular tumorigenicity.

Manganese superoxide dismutase (MnSOD) scavenges toxic superoxide radicals produced in the mitochondria. Transfection of the human MnSOD gene into mouse C3H 10T1/2 cells resulted in production of active MnSOD, which was properly transported into mitochondria. Overexpression of MnSOD protected cells from radiation-, but not chemically-induced neoplastic transformation. This finding demonstrates that oxidative stress that occurs in the mitochondria plays an important role in the development of neoplastic transformation.

Topical application of 100 mg/kg body weight of Ixora javanica flower extract inhibited the growth and delayed the onset of papilloma formation in mice initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted using croton oil. The extract at the same dose, when administered orally inhibited the growth of subcutaneously injected 20-methylcholanthrene (MCA)-induced soft tissue fibrosarcomas significantly. Oral administration of 200 mg/kg of the extract inhibited the growth of intraperitoneally transplanted sarcoma-180 and Ehrlich ascites carcinoma tumours besides showing an increase in the life span of the treated mice. Toxicity studies showed that the blood urea nitrogen levels were elevated post treatment. The active compounds responsible for the above inhibitory effects on tumour growth were identified as ferulic acid (4-hydroxy-3-methoxy cinnamic acid) and its regionmer 3-hydroxy-4-methoxy cinnamic acid.

MCA-102, a murine sarcoma previously reported to be non-immunogenic in C57/BL6 murine tumor models was used in a tumor vaccine preparation which included liposome encapsulated IL-2 as an adjuvant. C57/BL6 mice were immunized in the right hind footpad with irradiated MCA-102 murine sarcoma cells on days 0, 7, and 21 with or without IL-2 liposome adjuvant at 25,000 IL-2 units/injection. Mice were challenged with live tumor in the right flank on day 35. Survival of mice given IL-2 liposomes with irradiated MCA-102 cells was significantly prolonged over mice given tumor antigen with saline, and non-immunized mice. In addition, mice which received the IL-2 liposome adjuvant also had prolonged survival over those mice immunized with the additional control adjuvants of free IL-2 or dimyristoyl phosphatidyl choline (DMPC) lipid in the form of empty liposomes. IL-2 liposome plus tumor antigen also yielded a significant local protective response against live MCA-102 tumor challenge. When live tumor was injected into the site of previous immunizations on day 21 after two immunizations, the IL-2 liposome adjuvant group showed significantly delayed local growth of tumor compared to animals immunized without adjuvant, or with the adjuvants of empty liposomes or free IL-2. Finally, immunized mice were challenged with irradiated tumor cells and saline intradermally in the ears and delayed type hypersensitivity (DTH), an indicator of helper T cell response, was measured.(ABSTRACT TRUNCATED AT 250 WORDS)

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) and 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) are selectively inhibitory to human immunodeficiency virus and other retroviruses. We have now investigated the effects of different PMEA and PMEDAP treatment schedules in newborn mice infected with Moloney murine sarcoma virus (MSV). Administration of a single dose of PMEA or PMEDAP on the day of MSV inoculation conferred a greater protective effect against MSV-induced tumor formation than when this dose was divided over two, four or seven injections per week. Also, the therapeutic index of PMEA and PMEDAP was increased if administered as a single dose. Furthermore, PMEA and PMEDAP afforded a marked antiviral protection if administered within one day before MSV infection. Thus, single doses of PMEA or PMEDAP, when administered shortly before or after MSV infection, appear to be effective in preventing the manifestations of the retroviral disease.

Administration of estradiol-dipropionate (EP) after the cessation of 1,2-dimethylhydrazine (DMH) treatment increased the incidence of uterine sarcomas in CBA mice from 32.5 (DMH alone) to 62.5%. Ascorbic acid (AA) (0.3% in drinking water) given simultaneously with EP decreased the tumour incidence to 35%. Sodium ascorbate did not exert an inhibiting effect. AA inhibited the increase of uterine weight produced in mice by EP and did not influence the growth of mouse transplantable uterine sarcomas. The mechanisms of the antiestrogenic effects of AA are discussed.