Intralesional delivery of dendritic cells engineered to express T-bet promotes protective type 1 immunity and the normalization of the tumor microenvironment.

T-bet (Tbx21), a T-box transcription factor, has been previously identified as a master regulator of type 1 T cell polarization. We have also recently shown that the genetic engineering of human dendritic cells (DCs) to express human T-bet cDNA yields type 1-polarizing APCs in vitro (1). In the present study, murine CD11c(+) DCs were transduced with a recombinant adenovirus encoding full-length murine T-bets (DC.mTbets) and analyzed for their immunomodulatory functions in vitro and in vivo. Within the range of markers analyzed, DC.mTbets exhibited a control DC phenotype and were indistinguishable from control DCs in their ability to promote allogenic T cell proliferation in MLR in vitro. However, DC.mTbets were superior to control DCs in promoting Th1 and Tc1 responses in vitro via a mechanism requiring DC-T cell interaction or the close proximity of these two cell types and that can only partially be explained by the action of DC-elaborated IL-12p70. When injected into day 7 s.c. CMS4 sarcoma lesions growing in syngenic BALB/c mice, DC.mTbets dramatically slowed tumor progression (versus control DCs) and extended overall survival via a mechanism dependent on both CD4(+) and CD8(+) T cells and, to a lesser extent, asialoGM1(+) NK cells. DC.mTbet-based therapy also promoted superior tumor-specific Tc1 responses in the spleens and tumor-draining lymph nodes of treated animals, and within the tumor microenvironment it inhibited the accumulation of CD11b(+)Gr1(+) myeloid-derived suppressor cells and normalized CD31(+) vascular structures. These findings support the potential translational utility of DC.Tbets as a therapeutic modality in the cancer setting.

Vaccination against a hit-and-run viral cancer.

Cancers with viral aetiologies can potentially be prevented by antiviral vaccines. Therefore, it is important to understand how viral infections and cancers might be linked. Some cancers frequently carry gammaherpesvirus genomes. However, they generally express the same viral genes as non-transformed cells, and differ mainly in also carrying oncogenic host mutations. Infection, therefore, seems to play a triggering or accessory role in disease. The hit-and-run hypothesis proposes that cumulative host mutations can allow viral genomes to be lost entirely, such that cancers remaining virus-positive represent only a fraction of those to which infection contributes. This would have considerable implications for disease control. However, the hit-and-run hypothesis has so far lacked experimental support. Here, we tested it by using Cre-lox recombination to trigger transforming mutations in virus-infected cells. Thus, 'floxed' oncogene mice were infected with Cre recombinase-positive murid herpesvirus-4 (MuHV-4). The emerging cancers showed the expected genetic changes but, by the time of presentation, almost all lacked viral genomes. Vaccination with a non-persistent MuHV-4 mutant nonetheless conferred complete protection. Equivalent human gammaherpesvirus vaccines could therefore potentially prevent not only viral genome-positive cancers, but possibly also some cancers less suspected of a viral origin because of viral genome loss.

Peripheral blood and lymphatic organs in BALB/c mice during the progressive and regressive phases of Moloney sarcoma development.

Blood cell counts, differential blood cell counts and weights of the spleen and peripheral lymph nodes draining the area of lesions induced by Moloney sarcoma virus inoculation into the quadriceps shank muscles of inbred BALB/c mice were examined at various stages of tumor development and regression. The blood cell count remained constant through the observation period up to 27 days post tumor development and regression. Differential counts revealed some changes in the cellular composition of the peripheral blood. The most pronounced was an increase of neutrophiles at the stage of tumor development, and their decline with tumour regression. The enlargement of the spleen and of the popliteal lymph nodes draining the tumour site, at the peak of tumor development on day 13 post MSV inoculation, involved at least a doubling of splenic weight, and a much greater weight increase for lymph nodes. This was a long-lasting, although declining event, extending beyond tumor regression.

Establishment of productively infected walleye dermal sarcoma explant cells.

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas in walleye fish. Virus expression is tightly regulated and limited to accessory gene transcripts throughout tumour development. During tumour regression, this regulation is lost and the replication of virus is greatly enhanced. Cultured walleye fibroblasts infected in vitro do not produce significant quantities of infectious virus. Tissue culture cells established by explantation of tumour cells were found to harbour WDSV provirus and to express accessory and structural proteins. The sequence of the provirus showed little variation from a previous WDSV isolate. Retroviral particles were isolated from supernatants from these cells and were able to transfer infection to uninfected walleye fibroblasts. In addition to the virus present in supernatants, much of the virus was cell associated and liberated only by sonication. This virus was found at internal cellular membranes, including mitochondria, and was infectious.

Adenovirus E1A, not human papillomavirus E7, sensitizes tumor cells to lysis by macrophages through nitric oxide- and TNF-alpha-dependent mechanisms despite up-regulation of 70-kDa heat shock protein.

Expression of adenovirus (Ad) serotype 2 or 5 (Ad2/5) E1A or human papillomavirus (HPV)16 E7 reportedly sensitizes cells to lysis by macrophages. Macrophages possess several mechanisms to kill tumor cells including TNF-alpha, NO, reactive oxygen intermediates (ROI), and Fas ligand (FasL). E1A sensitizes cells to apoptosis by TNF-alpha, and macrophages kill E1A-expressing cells, in part through the elaboration of TNF-alpha. However, E1A also up-regulates the expression of 70-kDa heat shock protein, a protein that inhibits killing by TNF-alpha and NO, thereby protecting cells from lysis by macrophages. Unlike E1A, E7 does not sensitize cells to killing by TNF-alpha, and the effector mechanism(s) used by macrophages to kill E7-expressing cells remain undefined. The purpose of this study was to further define the capacity of and the effector mechanisms used by macrophages to kill tumor cells that express Ad5 E1A or HPV16 E7. We found that Ad5 E1A, but not HPV16 E7, sensitized tumor cells to lysis by macrophages. Using macrophages derived from mice unable to make TNF-alpha, NO, ROI, or FasL, we determined that macrophages used NO, and to a lesser extent TNF-alpha, but not FasL or ROI, to kill E1A-expressing cells. Through the use of S-nitroso-N-acetylpenicillamine, which releases NO upon exposure to an aqueous environment, E1A was shown to directly sensitize tumor cells to NO-induced death. E1A sensitized tumor cells to lysis by macrophages despite up-regulating the expression of 70-kDa heat shock protein. In summary, E1A, but not E7, sensitized tumor cells to lysis by macrophages. Macrophages killed E1A-expressing cells through NO- and TNF-alpha-dependent mechanisms.

Activity of the vascular targeting agent combretastatin A-4 disodium phosphate in a xenograft model of AIDS-associated Kaposi's sarcoma.

Combretastatin A4 disodium phosphate (CA4DP) was evaluated in a xenograft model of AIDS-KS. KS xenografts were highly vascular, showing brisk mitotic activity, focal areas of necrosis, and intervening fibrovascular septae. Neoplastic cells were large or spindle-shaped, with vesicular nuclei and modest pleomorphism. Multiple junctions, microvillous-like projections, abortive lumina and rare Weibel Palade bodies were revealed by electron microscopy. Treatment with CA4DP (100 mg/kg) resulted in rapid onset of vascular effects that within 4 h resulted in an almost complete vascular shutdown in these tumors. Histological evaluation showed morphological damage within a few hours after treatment, followed by extensive necrosis which increased to approximately 90% by 24 h. At this time, viable tumor cells were evident only at the periphery of the tumor. These findings demonstrate not only the marked susceptibility of the KS model to CA4DP but also its potential application in studies related to the pathogenesis and therapy of AIDS-KS.

CpG island protects Rous sarcoma virus-derived vectors integrated into nonpermissive cells from DNA methylation and transcriptional suppression.

CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation.

Adenovirus-mediated p53 gene therapy inhibits human sarcoma tumorigenicity.

Mutations of the p53 tumor-suppressor gene are the most frequent genetic abnormality in soft tissue sarcomas. Because these rare tumors also respond poorly to standard chemotherapy and bear a 50% 5-year mortality rate, we investigated the possible therapeutic benefits of p53 gene restoration in sarcomas. We constructed Ad5p53, which is an E1A-deleted, replication-deficient adenovirus expressing a cytomegalovirus promoter-driven wild-type p53 cDNA with a Flag sequence tag. SKLMS-1 human leiomyosarcoma cells containing a mis-sense p53 point mutation were effectively transduced with Ad5p53. Increasing levels of Flag-p53 protein, as well as dose-dependent p21Cip1 induction, were observed through a dose range of 10-500 plaque-forming units (PFU)/cell. In vitro administration of Ad5p53 as a single 100 PFU/cell dose caused 40-60% growth inhibition of SKLMS-1 cells at posttreatment days 4, 6, and 8 compared with untreated or viral control treated-cells (P < .05, Student's t test). Relative to these same controls, in vivo treatment of SKLMS-1-bearing severe combined immunodeficient mice with 6 x 10(9) PFU of Ad5p53 by intratumoral injection resulted in a 35-day tumor growth delay and complete tumor regression in 40% of mice (P < .05, Student's t test). The expression of virally derived p53 mRNA in Ad5p53-treated tumor tissues was detected in treated tumor specimens by reverse transcriptase polymerase chain reaction. Reduced intratumoral cellularity and the presence of p53 staining in adjacent normal tissue, consistent with delivery of exogenous p53 to the tumor target, were evident only in Ad5p53-treated tumors after immunohistochemical staining for p53. These results indicate that wild-type p53 gene restoration in sarcomas retards tumor growth and may come to be usefully applied to the clinical treatment of this disease as a single regimen or in combination with conventional therapies.

Investigations on the aminoacid content of tumor associated antigens of rat sarcoma cells induced by virus.

In our previous work (Alexandrov et al., 1996) was reported that the rat sarcoma cells induced by SR-RSV express two tumor associated antigens (TAA). The one TAA has a molecular weight of 52 kD and is detected by the help of a monoclonal antibody 2C2 only on the outer side of the plasma membrane of the sarcoma cells. The other antigen, with molecular weight of 28 kD, is expressed on the outher and inner side of the membrane. The antigens were isolated as a pure fraction by polyacrylamide electrophoresis and prepared for aminoacid analysis after that. The consisting 16 bound aminoacids were in different amounts. Both antigens are rich in glycine and poor in aromatic and sulphur-containing aminoacids. The presence of glucosamine and galactosamine in the antigens proves their glycoprotein nature. The received data show that the both TAA-s differ not only in molecular weights, place of expression and functional activity, but also in the amount of the bound aminoacids which constitute their proteins.

Studies on the pathology, especially brain lesions, induced by R7, a spontaneous mutant of Moloney murine sarcoma virus 124.

We have recently isolated R7, a spontaneous Moloney murine sarcoma virus (MoMuSV) 124 variant. Molecular cloning and sequence analysis showed that, relative to MoMuSV 124, R7 has an extra repeat in each enhancer and a truncated mos gene in frame with the truncated gag coding sequence. This report presents a detailed study on the pathology induced by R7. R7 induced not only sarcomas with well developed angiomatous components but also brain lesions. Brain lesions were observed in all less-than-48-hour-old BALB/c mice inoculated with greater than 2 x 10(5) R7 focus-forming units (FFUs). R7 was detected in all brains examined by day 9 after inoculation, and brain lesions were observed in two of four mice examined by day 14 after inoculation. Light microscopy of brains revealed that approximately 15% of the lesions were unenclosed blood pools of varying sizes containing red blood cells and inflammatory cells spreading into surrounding brain tissues. The remainder of the brain lesions had tumor cells. These lesions ranged from a few enlarged vascular endothelial cells intermixed with blood cells to large circumscribed lesions consisting of well developed tangled masses of vessels surrounded by blood pools. Activated astrocytes surrounded and infiltrated the tumors. In addition, the thymus of R7-infected mice regressed significantly and precipitously due to apoptosis (especially of cortical thymocytes) at the end stage of the disease.

Haploid male germ cells show no susceptibility to transformation by simian virus 40 large tumour antigen in transgenic mice.

Cell-type specific tumorigenesis can be induced in transgenic mice by the directed expression of simian virus 40 (SV 40) large tumour antigen (TAg). In an attempt to determine the susceptibility of haploid male germ cells to neoplastic transformation by this oncogene, transgenic mice were generated that harboured a chimeric gene composed of the SV40 T antigen genes fused to the 2.3-kb 5' flanking sequences of the rat proacrosin gene. It was previously shown that this regulatory sequence is able specifically to direct the expression of CAT reporter gene in male germ cells with the onset of translation in early haploid male germ cells. The transgene showed regulated expression in male germ cells. Although T antigen immunostaining was detected specifically in spermatids, no testicular pathology was observed. This indicates that spermatids show no susceptibility to transformation by oncogene TAg. However, in about 10% of animals of two independent transgenic lines, we could find non-testicular tumours in abdomen with a sarcoma-like structure in advanced age which showed SV40 TAg expression.

Transcriptional analysis of walleye dermal sarcoma virus (WDSV).

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas of walleye that develop and regress on a seasonal basis. WDSV contains, in addition to gag, pol, and env, three open reading frames (ORFs) designated ORF A, ORF B, and ORF C. The polymerase chain reaction technique was used to amplify and clone cDNAs representing subgenomic viral mRNAs isolated from developing (fall) and regressing (spring) tumors. Nine different singly or multiply spliced viral transcripts were identified and all were found to utilize a common 5' leader sequence. This leader sequence is spliced to the pol/env junction or downstream of env to generate singly spliced transcripts. Multiply spliced transcripts contain the 5' leader, the pol/env junction, and sequences derived from the 3' end of the genome. One multiply spliced transcript was isolated with the potential to encode the full-length ORF A protein. In addition, WDSV produced mRNAs that utilize alternative splice acceptor sites which would allow synthesis of five variant forms of the ORF A protein. In contrast, the ORF B protein is postulated to arise from a singly spliced transcript with the potential to encode the entire open reading frame. Spliced subgenomic transcripts representing ORF C mRNAs were not identified, suggesting that ORF C may be encoded from the full-length viral genomic transcript. We estimate that at least a 100-fold lower amount of the accessory/regulatory subgenomic transcripts exists in developing vs regressing tumors. These results demonstrate that WDSV undergoes an elaborate pattern of mRNA splicing similar to that of other complex retroviruses.

Role of p53 mutation in polyomavirus-induced tumorigenesis.

Small DNA tumour viruses, such as simian virus 40 (SV40), papilloma viruses and adenoviruses, encode proteins that form complexes with and inactivate the p53 and retinoblastoma (RB) proteins. This convergent evolution reflects the common need of these viruses to inactivate these two important regulators of cell cycle progression and cell survival. Polyomavirus, a close relative of SV40, is different. Its large T protein complexes only with RB, not with p53. We have examined whether this is compensated by the frequent appearance of p53 mutations in polyomavirus-induced tumours. We tested the p53 status of 15 polyomavirus-induced sarcomas. Two sarcomas were p53-negative while six carried mutant p53. Another six sarcomas expressed low levels of wild-type p53. One tumour expressed high levels of wild-type p53 protein as shown by DNA sequencing and immunofluorescence staining. MDM2 amplification was not detected in any of the tumours, but Northern blotting showed that MDM2 was overexpressed in at least two tumours that expressed wild-type p53 and in one tumour that expressed both wild-type and mutant p53. Treatment with the DNA-damaging agent mitomycin C caused p53 protein accumulation followed by induction of MDM2 and WAF1/p21 mRNA in four of the tumours expressing wild-type p53, indicating that p53-mediated transcriptional activation was unaltered in these tumours. However, p53-mediated transactivation of WAF1/p21 was impaired in the wild-type p53-expressing tumours that expressed elevated levels of MDM2. These results demonstrate that p53 mutation and inactivation are frequently but not invariably involved in polyomavirus-induced tumorigenesis.

SRR-SB3, a disulfide-containing macrolide that inhibits a late stage of the replicative cycle of human immunodeficiency virus.

From a series of macrocyclic diamides possessing the disulfide linkage, only SRR-SB3, a compound that complexes with zinc, was found to inhibit human immunodeficiency virus type 1 (HIV-1; strain IIIB) replication at a concentration of 1.8 to 6.5 micrograms/ml in MT-4, CEM, and peripheral blood mononuclear cells. SRR-SB3 was toxic to MT-4 cells at a concentration of 15.9 micrograms/ml, resulting in a selectivity index of 9 in these cells. This macrolide was also effective against various other HIV-1 strains, including clinical isolates and HIV-1 strains resistant to protease inhibitors and nucleoside and nonnucleoside reverse transcriptase inhibitors. It was also active against various HIV-2 strains, simian immunodeficiency virus (strain MAC251), and Moloney murine sarcoma virus, but not against viruses other than retroviruses. In addition, the compound was found to inhibit chronic HIV-1 infections in vitro. The compound in combination with other antiviral agents, such as zidovudine, zalcitabine, and stavudine, showed an effect that was between additive and synergistic. Time-of-addition experiments indicated that SRR-SB3 acts at a late stage of the HIV-1 replicative cycle.

Neutral metoclopramide induces tumor cytotoxicity and sensitizes ionizing radiation of a human lung adenocarcinoma and virus induced sarcoma in mice.

Radiation induced cytotoxicity was potentiated by neutralized metoclopramide (nMCA; Neu-Sensamide, Oxigene Inc) when a human lung adenocarcinoma (H2981) transplanted into scid mice and an adeno-type 12 virus induced mouse sarcoma (A12B3) inoculated into CBA mice were exposed in vivo to low dose radiation at single doses of 1 and 2 Gy respectively. However, when the radiation dose was increased to 6, 10 or 18 Gy (single dose) and combined with a single dose nMCA (2 mg/kg), tumor cytotoxicity was not sensitized by the combination treatment. A fractionated dose of ionizing radiation (3 x 1 Gy) in combination with nMCA at a repeated dose of 3 x 10 mg/kg body weight (1 dose/day, i.m.) significantly increased cytotoxicity in H2981 compared with radiation given alone. nMCA alone also had a statistically significant dose dependent cytotoxic effect on H2981 growth when it was administered as repeated doses (8 doses) at 2 mg/kg or 10 mg/kg (1 dose every second day), and a similar result was achieved at 20 mg/kg but not at 2 and 10 mg/kg in the A12B3 tumor. In addition, the tumor volume at the start of treatment was important for the anti-tumor effect of nMCA (i.e. the larger initial tumor volume gave less effect on tumor growth). Taken together, our data propose that the mode of action of nMCA is different from radiation, and hence the two mechanisms are at least additive when in combination with lower radiation doses. The data further suggest that the cytotoxic mechanism is consistent with potentiating apoptosis because low and repeated doses of radiation (1-2 Gy), which are known to increase cytotoxicity by apoptosis, are sensitized by nMCA but not high doses and nMCA has more potent anti-tumor effects against H2981 tumors which have a higher constitutive apoptotic fraction of cells than A12B3.

The susceptibility to cytotoxic T lymphocyte mediated lysis of chemically induced sarcomas from immunodeficient and normal mice.

A panel of sarcomas induced with 3-methylcholanthrene in normal and immunodeficient mice was studied for their capacity to present antigen by the endogenous, MHC class I restricted pathway. Lymphocytic choriomeningitis virus was used to infect cultured tumour cells, and the infected cells were tested for susceptibility to cytolysis by virus specific cytotoxic T cells. Tumour cells originating from tumours induced in immunocompetent C.B.-17 mice presented virus antigen more efficiently than tumour cells from immunodeficient SCID mice. No significant difference in virus antigen presentation was found between tumours from nude and nu/+ BALB/c mice. The sensitivity of target cells from the individual tumours to cytotoxic T lymphocyte (CTL) mediated lysis correlated negatively with their sensitivity to natural killer (NK) cell mediated lysis. There was a positive correlation between the sensitivity to CTL mediated lysis and surface expression of the MHC class I molecule Ld of the tumour cells. Tumour cells incapable of in vitro presentation of viral antigen to specific cytotoxic T cells originated from tumours known from previous experiments to be readily accepted after transplantation to immunocompetent, histocompatible recipients.

BK virus-induced osteosarcoma (Os515) as a model of human osteosarcoma.

BK virus was isolated by Gardner et al in 1971 from the urine of an immunosuppressed patient with a kidney allograft. Antibodies to this virus are ubiquitous in the general populations worldwide, but the oncogenic capacity of BKV in humans had not been reported. The virus transformed in vitro permissive human and non-permissive animal cells, and the transformed cells had the T antigen. Intracerebral and intravenous inoculation of BKV in newborn hamsters induced malignant tumours (mainly ependymomas, malignant insulinomas, and osteosarcomas). Subcutaneous and intraperitoneal routes were also effective. The virus was only rescued from a few tumours by fusion with human embryonic cells or Vero cells. Brain tumours appeared earlier and osteosarcomas developed in animals which survived for more than 6 months. Many of the osteosarcomas were bony and grew slowly with frequent lung metastases, and a few osteosarcomas were soft and grew rapidly without lung metastases. Experimental targeting chemotherapy with doxorubicin (DX)-containing immunoliposomes was performed against Os515 osteosarcoma. In in vitro experiments, DX-Lip-MoAb29 showed a more significant inhibitory effect on cultured Os515 cells than free Dx and DX-Lip. DX-Lip DNP had less effect. In in vivo experiments, DX-Lip-MoAb29 suppressed the growth of Os515 tumour isografts in hamsters and prolonged the survival of recipients more significantly than free DX.

Anti-L-selectin monoclonal antibody treatment in mice enhances tumor growth by preventing CTL sensitization in peripheral lymph nodes draining the tumor area.

To examine the in vivo contribution of L-selectin in the sensitization of tumor-specific CTL, we investigated the effects of treatment with the anti-L-selectin monoclonal antibody (MAb) MEL-14 on the immune response to Moloney-murine sarcoma virus (M-MSV)-induced tumors, which exhibit spontaneous regression following generation of a strong virus-specific CTL response. Daily systemic administration of MEL-14 for 10 days to M-MSV-injected mice gave rise to larger sarcomas that persisted for a longer time, compared with those arising in control mice injected with virus only. The enhanced tumor growth could not be attributed to cytotoxic activity on leukocytes by MEL-14 since no reduction in the total cell number was detected in peripheral blood and spleen of MAb-treated mice. Evaluation of the immunological response in MAb-treated animals revealed a strong reduction in the generation of virus-specific CTL precursors (CTLp) in tumor-draining peripheral lymph nodes (PLN) 10 and 15 days after M-MSV injection, while in spleen, where lymphocyte localization is independent of L-selectin expression, CTLp generation was only delayed. By day 20, when tumors had begun to regress, the CTLp number showed a marked increase in both spleen and local PLN, where naive recirculating CTL could now enter because L-selectin was no longer down-regulated or blocked by the injected MAb. Our findings indicate that functional inactivation of L-selectin by MEL-14 treatment prevented migration of naive L-selectin+CTL through high endothelial venules (HEV) and their accumulation in PLN draining the tumor area, thereby precluding the initiation of a tumor-specific CTL response that takes place primarily at this site.