1α, 25 Dihydroxyvitamin D (1,25(OH)2D) inhibits the T cell suppressive function of myeloid derived suppressor cells (MDSC).

Myeloid derived suppressor cells (MDSC) suppress the ability of cytotoxic T cells to attack and clear tumor cells from the body. The active form of vitamin D, 1,25 dihydroxyvitamin D (1,25(OH)2D), regulates myeloid cell biology and previous research showed that in mouse models 1,25(OH)2D reduced the tumor level of CD34+ cells, an MDSC precursor, and reduced metastasis. We tested whether MDSC are vitamin D target cells by examining granulocytic- (G-MDSC) and monocytic (M-MDSC) MDSC from tumors, spleen, and bone marrow. Vitamin D receptor (VDR) mRNA levels are low in MDSC from bone marrow and spleen but are 20-fold higher in tumor MDSC. At all sites, M-MDSC have 4-fold higher VDR mRNA expression than G-MDSC. Bone marrow MDSC were induced to differentiate in vitro into tumor MDSC-like cells by treating with IFN-γ, IL-13, and GM-CSF for 48 h. This treatment significantly elevated Arg1 and Nos2 levels, activated the T cell-suppressive function of MDSC, increased VDR expression 50-fold, and made the MDSC responsive to 1,25(OH)2D treatment. Importantly, 1,25(OH)2D treatment reduced the T cell suppressive capacity of cytokine-induced total MDSC and M-MDSC by ≥70 % and tumor-derived M-MDSC by 30-50 %. Consistent with this finding, VDR deletion (KO) increased T cell suppressive function of in vitro M-MDSC by 30 % and of tumor-derived M-MDSC by 50 % and G-MDSC by 400 %. VDR KO did not alter Nos2 mRNA levels but significantly increased Arg1 mRNA levels in tumor M-MDSC by 100 %. In contrast, 1,25(OH)2D treatment reduced nitric oxide production in both in vitro derived M- and G- MDSC. The major finding of this study is that 1,25(OH)2D signaling through the VDR decreases the immunosuppressive capability of MDSC. Collectively, our data suggest that activation of vitamin D signaling could be used to suppress MDSC function and release a constraint on T-cell mediated clearance of tumor cells.

Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth.

Protein arginine methyltransferase 5 (PRMT5) is an emerging epigenetic enzyme that mainly represses transcription of target genes via symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3. Accumulating evidence suggests that PRMT5 may function as an oncogene to drive cancer cell growth by epigenetic inactivation of several tumor suppressors. Here, we provide evidence that PRMT5 promotes prostate cancer cell growth by epigenetically activating transcription of the androgen receptor (AR) in prostate cancer cells. Knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor reduces the expression of AR and suppresses the growth of multiple AR-positive, but not AR-negative, prostate cancer cells. Significantly, knockdown of PRMT5 in AR-positive LNCaP cells completely suppresses the growth of xenograft tumors in mice. Molecular analysis reveals that PRMT5 binds to the proximal promoter region of the AR gene and contributes mainly to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter by its interaction with Sp1, the major transcription factor responsible for AR transcription, and forms a complex with Brg1, an ATP-dependent chromatin remodeler, on the proximal promoter region of the AR gene. Furthermore, PRMT5 expression in prostate cancer tissues is significantly higher than that in benign prostatic hyperplasia tissues, and PRMT5 expression correlates positively with AR expression at both the protein and mRNA levels. Taken together, our results identify PRMT5 as a novel epigenetic activator of AR in prostate cancer. Given that inhibiting AR transcriptional activity or androgen synthesis remains the major mechanism of action for most existing anti-androgen agents, our findings also raise an interesting possibility that targeting PRMT5 may represent a novel approach for prostate cancer treatment by eliminating AR expression.

Abrogating cholesterol esterification suppresses growth and metastasis of pancreatic cancer.

Cancer cells are known to execute reprogramed metabolism of glucose, amino acids and lipids. Here, we report a significant role of cholesterol metabolism in cancer metastasis. By using label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, mediated by acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme. Expression of ACAT-1 showed a correlation with poor patient survival. Abrogation of cholesterol esterification, either by an ACAT-1 inhibitor or by shRNA knockdown, significantly suppressed tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Mechanically, ACAT-1 inhibition increased intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum stress and caused apoptosis. Collectively, our results demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification.

Inhibition of adherence of Mycobacterium avium complex and Mycobacterium tuberculosis to fibronectin on the respiratory mucosa.

Mycobacterium species adhere to the respiratory mucosa via mucus and fibronectin of extracellular matrix exposed by damaged epithelium. We have investigated whether inhibiting adherence to fibronectin influences subsequent infection of human respiratory tissue by Mycobacterium avium complex and Mycobacterium tuberculosis. Human respiratory tissue was pretreated with mycobacterial fibronectin attachment proteins prior to infection with M. avium complex and M. tuberculosis and the number of recoverable bacteria over time was compared to untreated controls. Inhibition significantly reduced recovery of M. avium complex at 15min (P= 0.02), 7days (P = 0.04), and 14 days (P= 0.03); whereas recovery of M. tuberculosis was only reduced at 15 min (P = 0.01) and not at later timepoints. We conclude that M. avium complex and M. tuberculosis infection of the mucosa proceeds by different mechanisms, since M. tuberculosis infection is independent of fibronectin adherence.

Interleukin 10 induced augmentation of delayed-type hypersensitivity (DTH) enhances Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediated antitumour activity.

Intravesical BCG therapy is effective in the treatment of superficial bladder cancer. Both clinical and experimental results suggest a role for cytokines and delayed-type hypersensitivity (DTH) in BCG-induced antitumour immunity. We characterized the modulatory effects of BCG on bladder cytokine expression and determined the relationship between DTH and BCG antitumour activity. The bladders of mice were instilled with BCG through a catheter. Bladder tissue RNA and urine were collected for evaluation of cytokine expression using reverse transcriptase-polymerase chain reaction (RT-PCR) and/or ELISA. IFN-gamma and TNF-alpha, the two major cytokines associated with DTH, were efficiently induced by BCG. IL10, an important down-regulator of DTH, was also induced by BCG. Constitutive levels of IL4 and IL5 were observed, but neither IL4 nor IL5 were modulated by BCG. Similar results were observed in the kinetic analysis of urinary cytokines in patients after intravesical BCG therapy. Production of Th1 (T helper type 1) cytokines (IFN-gamma, IL2 and IL12) preceded that of the Th2 (T helper type 2) cytokine IL10. A tendency toward higher ratios of IFN-gamma versus IL10 for BCG responders also was observed. In animal studies the absence of IL10 abrogated either by antibody inhibition or the use of genetically modified, IL10 deficient (IL10-/-) mice resulted in enhanced DTH responses. Under conditions of enhanced DTH, a significant enhancement in antitumour activity was observed. These data demonstrate that DTH and its associated mononuclear infiltration and cytokine production are important to the antitumour activity of intravesical BCG therapy, and suggest that effects to diminish IL10 production may have therapeutic value.

Interaction of Mycobacterium tuberculosis with human respiratory mucosa.

OBJECTIVE: Endobronchial infection is associated with pulmonary tuberculosis in the majority of cases. We have investigated the adherence of Mycobacterium tuberculosis to the human respiratory mucosa. DESIGN: Organ cultures constructed with human tissue were infected with M. tuberculosis in the presence or absence of mycobacterial fibronectin attachment cell surface proteins and examined by scanning electron microscopy. RESULTS: M. tuberculosis adhered mainly to extracellular matrix (ECM) in areas of mucosal damage, but not to ciliated mucosa, intact extruded cells, basement membrane or collagen fibres. Bacteria also adhered to fibrous but not globular mucus and occasionally to healthy unciliated mucosa, open tight junctions and to extruded cells that had degenerated, exposing their contents. There was a significant reduction (p<0.05) in the number of bacteria adhering to ECM after pre-incubation of bacteria with fibronectin and after pre-incubation of the tissue with M. avium fibronectin attachment protein (FAP) and M. bovis antigen 85B protein, in a concentration dependent manner. The combined effect of FAP and antigen 85B protein was significantly greater than either protein alone. Bacterial adherence to fibrous mucus was not influenced by fibronectin. CONCLUSION: We conclude that M. tuberculosis adheres to ECM in areas of mucosal damage at least in part via FAP and antigen 85B protein.

Immunization with type 5 adenovirus recombinant for a tumor antigen in combination with recombinant canarypox virus (ALVAC) cytokine gene delivery induces destruction of established prostate tumors.

Prostate-specific antigen (PSA) is expressed by prostate epithelial cells and has a highly restricted tissue distribution. Prostatic malignancies in 95% of patients continue to express PSA, making this antigen a good candidate for targeted immunotherapy. The goals of our studies are to generate a recombinant PSA adenovirus type 5 (Ad5-PSA) that is safe and effectively activates a PSA-specific T-cell response capable of eliminating prostate cancer cells, and to characterize the immunologic basis for this rejection. Here we show that immunization of mice with Ad5-PSA induced PSA-specific cellular and humoral immunity that was protective against a subcutaneous challenge with RM11 prostate cancer cells expressing PSA (RM11psa), but not mock-transfected RM11 tumor cells (RM11neo). Mice immunized with recombinant adenovirus type 5 encoding beta-galactosidase (Ad5-lacZ) did not generate protective immunity. Antitumor activity was predominantly mediated by CD8(+) T lymphocytes. Although Ad5-PSA immunization prior to RM11psa challenge was protective, Ad5-PSA immunization alone was not able to control the growth of existing RM11psa tumors. In contrast, established RM11psa tumors ranging in size from 500 to 1,000 mm(3) were efficiently eliminated if Ad5-PSA priming was followed 7 days later by intratumoral injection of recombinant canarypox viruses (ALVAC) encoding interleukin-12 (IL-12), IL-2, and tumor necrosis factor-alpha. In this case, antitumor immunity was still dominated by CD8(+) T lymphocytes, but natural killer cells became necessary for a maximal response. These data provide information on the effector cell populations in a protective immune response to prostate cancer and demonstrate the utility of an Ad5-PSA vaccine combined with cytokine gene delivery to eliminate large established tumors that are refractory to other interventional methods.

Vaccines in urologic malignancies.

The prospect of activating the immune system to combat neoplastic disease has stimulated the interest of clinicians and scientists for over 100 years. Despite a few notable exceptions (especially with urologic malignancies), immunotherapy has not fully reached its considerable therapeutic potential for the treatment of cancer. Tumors undoubtedly express antigens that may act as targets for antitumor immunity, and advances in molecular biology and tumor immunology have recently revived the possibility of a cancer vaccine. This improved understanding has resulted in numerous successes with active immunotherapy in animal models and has facilitated the clinical testing of cancer vaccines. Ongoing advances in the identification of unique, tumor-specific antigens and their presentation to stimulate T cells will be necessary for the emergence of these novel vaccine therapies for cancer patients. Herein we review the current concepts of tumor immunology, including observations on cell types probably involved with the immune surveillance of tumors, the presentation and recognition of "foreign" antigens, and possible mechanisms of tumor escape from the immune response, all of which are critical to the understanding of new initiatives for cancer vaccine therapy. Finally, we review some of the successes and limitations of vaccine therapy for urologic malignancies.

Inhibition of murine prostate tumor growth and activation of immunoregulatory cells with recombinant canarypox viruses.

BACKGROUND: Immunization with modified tumor cells carrying recombinant immunomodulatory genes is being explored as cancer immunotherapy. In this study, we examine whether canarypox ALVAC viruses carrying immunostimulatory cytokine genes (granulocyte-macrophage colony-stimulating factor, interleukin 2, interleukin 12, and tumor necrosis factor-alpha) can induce antitumor immunity (to rechallenge) in the RM-1 model of a highly aggressive, weakly immunogenic murine prostate cancer. METHODS: For antitumor activity studies, RM-1 murine prostate cancer cells were infected with the parental ALVAC virus or one or two recombinant ALVAC-cytokine viruses and then injected into male C57BL/6 mice. For rechallenge studies, other mice were first given an injection subcutaneously with irradiated (nonproliferating) recombinant ALVAC-infected RM-1 cells and then (10 days later) with untreated RM-1 cells. For the determination of which immune cells were required for antitumor activity, mice were immunodepleted of CD4, CD8, or natural killer (NK) NK1.1 cells with the corresponding monoclonal antibodies and were then given an injection of ALVAC-cytokine-infected RM-1 cells. For all experiments, tumor outgrowth and animal survival were monitored. RESULTS: After subcutaneous injection into mice, RM-1 cells infected with one (except ALVAC-interleukin 2) or two ALVAC-cytokine recombinants had statistically significantly greater antitumor activity than RM-1 cells infected with parental ALVAC (P<.001 for all; two-sided test). The antitumor activity of RM-1 cells infected with any two ALVAC-cytokine recombinants was greater than, but not statistically significantly different from, that of RM-1 cells infected with any one ALVAC-cytokine recombinant. NK1.1 cells were necessary for antitumor activity, but tumor-specific CD4(+) regulatory T cells were also induced that inhibited CD8(+) RM-1-specific cytotoxic T cells, resulting in the lack of immunity to a rechallenge by RM-1 cells. DISCUSSION: Canarypox viruses can transfer immunostimulatory cytokine genes into RM-1 prostate cancer cells. When such cells were injected into mice, the cytokines induced an antitumor response against this highly aggressive, weakly immunogenic tumor. This response, however, did not protect the mouse against a rechallenge with RM-1 cells because suppressor CD4(+) T cells were induced that inhibited tumor-specific CD8(+) cytotoxic T cells.

NK cells are essential for effective BCG immunotherapy.

Adjuvant intravesical bacillus Calmette-Guérin (BCG) therapy is a well-established and successful adjuvant immunotherapy in the treatment of superficial bladder cancer. Although the function of natural killer (NK) cells in other immunotherapeutic regimens (e.g., lymphokine-activated killer [LAK] cell or interleukin-2 [IL-2] therapy) has been established, the contribution of NK cells to effective BCG immunotherapy is not clear. We used a human in vitro system to analyze the role of NK cells in BCG-induced cellular cytotoxicity. After stimulation of mononuclear cells with BCG for 7 days, these BCG-activated killer (BAK) cells displayed substantial cytotoxicity against bladder tumor cells. Magnetic depletion experiments and fluorescence activated cell sorting revealed that NK cells were the major effector cell population. To address NK cell function in vivo, we studied a syngeneic orthotopic murine bladder cancer model and compared BCG immunotherapy in C57BL/6 wild-type mice, NK-deficient beige mice and mice treated with anti-NK1.1 monoclonal antibody. Four weekly instillations of viable BCG significantly prolonged survival in wild-type mice compared with control mice treated with solvent alone. In contrast, BCG therapy was completely ineffective in NK-deficient beige mice and in mice treated with anti-NK1.1 monoclonal antibody. These findings suggest a key role for NK cells during BCG immunotherapy.

Evaluation of gene transfer efficiency by viral vectors to murine bladder epithelium.

PURPOSE: In pre-clinical gene therapy studies of bladder cancer there is tremendous variation in the ability of viral vectors to deliver genetic material to bladder epithelium. Possible explanations for this variability may involve the physical parameters of delivering vectors in these experimental models. We examined the effects of intravesical volume and pressure during instillation as well as chemical modification of the bladder epithelium on subsequent gene expression in the bladder in mice. MATERIALS AND METHODS: Female C57B1/6 mice underwent intravesical instillation of the replication restricted canarypox virus (ALVAC) recombinant for the reporter genes luciferase or beta-galactosidase. Similar viral titers were instilled at different volumes and a pressure transducer measured intravesical pressure when the vector was instilled. Also, various agents, including 0.6 N hydrochloric acid, 0.4% oxychlorosene, poly-L-lysine and 0.25 M. ammonium chloride, were used to modify the bladder surface before vector instillation and then assayed for transgene expression. RESULTS: As expected, maximum intravesical pressure measured during instillation was significantly greater in mice instilled with a higher volume (33.1 versus 9.8 mm. Hg). Significantly more gene expression was detected in bladders instilled with a higher volume of viral vectors (p <0.05). Likewise, higher instillation pressures resulted in higher transgene expression in distant organs. Modification of the bladder epithelium with agents such as oxychlorosene and poly-L-lysine resulted in elevated gene expression with only minimal increases in systemic activity. CONCLUSIONS: Significant differences in gene expression are achieved by varying physical parameters during intravesical instillation. Increased gene expression associated with larger volume instillation may be responsible for some reported variability of gene transfer to the bladder. Alternate manipulations, such as modifying the bladder surface, may be done to enhance gene transfer to the urothelium without increasing systemic distribution.

Cutting edge: restoration of the ability to generate CTL in mice immune to adenovirus by delivery of virus in a collagen-based matrix.

Viruses are commonly used for the delivery of genes coding for tumor-associated Ags to elicit tumor-specific immune responses. The success of viral vectors has been limited in preclinical and clinical trials in part because of antiviral immunity. We investigated the ability of a collagen-based matrix (Gelfoam; Pharmacia and Upjohn, Kalamazoo, MI) to improve CTL activation by recombinant adenovirus. The data show that coinjection of Gelfoam with type 5 adenovirus recombinant for prostate-specific Ag (Ad5-PSA) enhanced CTL activation. Ad5-PSA priming in Gelfoam also abrogated the inhibitory effects of adenoviral immunity on CTL activation in mice naive to PSA but immune to adenovirus. Finally, Gelfoam enhanced immunization in a self-Ag model using type 5 adenovirus recombinant for membrane-bound OVA (Ad5-mOVA) in rat insulin promoter (RIP)-mOVA-transgenic mice. Thus, Gelfoam enhances CTL activation by recombinant viral vectors in a setting where preformed Ab to the virus is present and also in a tolerant self-Ag model.

Biomarker distribution after injection into the canine prostate: implications for gene therapy.

OBJECTIVE: To evaluate the distribution of biomarkers after transrectal injection into the canine prostate and to report a method for enhancing the distribution of gene expression. MATERIALS AND METHODS: Carbon black was first used to evaluate the histopathological distribution in canine prostate of single or multiple injections via the transurethral, transperineal and transrectal routes. The distribution of canarypox virus (ALVAC) vector-delivered gene expression was then compared using both fluid-phase injection techniques and delivery in a solid carrier composed of a gelatine sponge matrix. RESULTS: After transurethral administration, carbon black was detected as scattered particles in ducts and acini, mostly in the periphery of the gland. Direct transrectal injection of carbon black resulted in a localized collection at the site of injection, with only a minimal peri-acinar distribution. Transrectal injection of the fluid-phase (virus suspended in diluent) ALVAC vector encoding the beta-galactosidase gene resulted in a similar distribution, with limited gene expression at the site of injection and in the needle track. Delivery of the same number of virus particles in the gelatine sponge matrix resulted in qualitatively greater gene expression. CONCLUSIONS: Direct injection of the canine prostate with biomarkers, including viral vectors, in the fluid-phase results in very localized gene expression, while the distribution was more widespread after delivery in a gelatine sponge matrix.

APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells.

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.

Role of animal models in understanding intravesical therapy with bacille Calmette-Guérin.

Animal models provide a vehicle for understanding basic biological questions. Through the use of animal models, adequate control of experimental design is possible so that rigorous experiments can be performed to test a hypothesis. In the case of testing therapeutic mechanisms, it is important to select a model that is most analogous to the clinical setting so that observations can be readily transferred to clinical studies for validation. In mechanistic studies of Mycobacterium bovis strain (bacille Calmette-Guérin) therapy for bladder cancer, the orthotopic animal model most closely mimics the clinical treatment of bladder cancer.

Stress and symptomatology in patients with interstitial cystitis: a laboratory stress model.

PURPOSE: Although patients with interstitial cystitis frequently report symptom exacerbation due to stress, to our knowledge this association has not been empirically examined. We evaluated the effects of a laboratory mental stress challenge on symptoms of urgency and pain in patients with interstitial cystitis and healthy controls. MATERIALS AND METHODS: A total of 14 females with interstitial cystitis and 14 age matched controls participated in a laboratory session, including a 60-minute baseline measurement, 25 minutes of mental stress tasks and 75 minutes of recovery. Acute symptoms of pain and urgency were assessed at voiding 15 minutes before the stressor, and 25, 70 and 100 minutes after stressor onset. Chronic symptoms were evaluated by questions from the Interstitial Cystitis Data Base survey. RESULTS: Patients reported significantly greater pain and urgency than controls at all 4 voidings (p <0.005). Pain increased in patients from the prestressor point to 25 minutes after stressor onset (p <0.005), remained elevated at 70, and decreased between 70 and 100. At 100 minutes patient pain remained significantly elevated above baseline (p = 0.018). Patient urgency was significantly elevated over baseline by 70 minutes after stressor onset (p <0.001) and significantly decreased between 70 and 100 minutes (p <0.002). Controls had no symptom changes with stress. CONCLUSIONS: These findings indicate that an acute stressor evokes increased symptoms of pain and urgency in patients with interstitial cystitis but not in controls. Findings are consistent with sympathetic effects on inflammatory processes in interstitial cystitis. However, further evaluation of the mechanisms underlying stress related interstitial cystitis symptom exacerbation is needed to provide a more comprehensive understanding of these phenomena.

Adenoviral-mediated transfer of the TNF-related apoptosis-inducing ligand/Apo-2 ligand gene induces tumor cell apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines that induces apoptosis in a variety of cancer cells. The results presented in this study demonstrate that introduction of the human TRAIL gene into TRAIL-sensitive tumor cells using an adenoviral vector leads to the rapid production and expression of TRAIL protein, and subsequent death of the tumor cells. Tumor cell death was mediated by an apoptotic mechanism, as evidenced by the activation of caspase-8, cleavage of poly(ADP-ribose) polymerase, binding of annexin V, and inhibition by caspase inhibitor zVAD-fmk. These results define a novel method of using TRAIL as an antitumor therapeutic, and suggest the potential use for an adenovirus-encoding TRAIL as a method of gene therapy for numerous cancer types in vivo.

Viral vector delivery in solid-state vehicles: gene expression in a murine prostate cancer model.

BACKGROUND: Although there are increasingly more clinical trials involving gene therapy, efficient gene transfer remains a major hurdle to success. To enhance the efficiency of delivery of viral vectors in gene therapy protocols, we evaluated the effect of various matrices to act as a vehicle for recombinant virus during intratumoral injection. METHODS: The ability of several vehicles (catgut spacer, polyglycolic acid, chromic catgut, and gelatin sponge matrix) to deliver the canarypox virus ALVAC to the cells of the murine prostate cancer cell line RM-1 was studied in vitro and in vivo. ALVAC recombinants encoding the murine cytokines interleukin 2 (IL-2), interleukin 12 (IL-12), and tumor necrosis factor-alpha (TNF-alpha) were used to assess enhancement of antitumor activity after intratumoral inoculation. Confirmatory experiments were conducted by use of another mouse prostate cancer cell line, RM-11, and a mouse bladder cancer cell line, MB-49. All statistical tests were two-sided. RESULTS: The gelatin sponge matrix proved to be the most effective solid-state vehicle for delivering viral vectors to cells in culture. In addition, this matrix statistically significantly enhanced expression of ALVAC-delivered reporter genes in tumor models when compared with fluid-phase delivery of virus (P =.037 for the RM-1 model and P =.03 for the MB-49 model). Statistically significant growth inhibition of established tumors was observed when a combination of the three recombinant ALVAC viruses expressing IL-2, IL-12, and TNF-alpha was delivered with the matrix in comparison with 1) fluid-phase intratumoral injection of the ALVAC recombinants, 2) no treatment, or 3) treatment with parental ALVAC (all P<.05). CONCLUSIONS: Viral vector delivery in a solid-state vehicle resulted in improved recombinant gene expression in vivo and translated to greater inhibition of tumor growth in an immunotherapy protocol for heterotopic tumor nodules. The efficient delivery of reporter genes described herein may prove useful in many solid tumor gene therapy protocols.