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Purpose: Epacadostat, an indole 2,3 dioxygenase 1 (IDO1) inhibitor, proposed to shift the tumor microenvironment toward an immune-stimulated state, showed early promise in melanoma but has not been studied in sarcoma. This study combined epacadostat with pembrolizumab, which has modest activity in select sarcoma subtypes., Patients and Methods: This phase II study enrolled patients with advanced sarcoma into five cohorts including (i) undifferentiated pleomorphic sarcoma (UPS)/myxofibrosarcoma, (ii) liposarcoma (LPS), (iii) leiomyosarcoma (LMS), (iv) vascular sarcoma, including angiosarcoma and epithelioid hemangioendothelioma (EHE), and (v) other subtypes. Patients received epacadostat 100 mg twice daily plus pembrolizumab at 200 mg/dose every 3 weeks. The primary endpoint was best objective response rate (ORR), defined as complete response (CR) and partial response (PR), at 24 weeks by RECIST v.1.1., Results: Thirty patients were enrolled [60% male; median age 54 years (range, 24-78)]. The best ORR at 24 weeks was 3.3% [PR, n = 1 (leiomyosarcoma); two-sided 95% CI, 0.1%-17.2%]. The median PFS was 7.6 weeks (two-sided 95% CI, 6.9-26.7). Treatment was well tolerated. Grade 3 treatment-related adverse events occurred in 23% (n = 7) of patients. In paired pre- and post-treatment tumor samples, no association was found between treatment and PD-L1 or IDO1 tumor expression or IDO-pathway-related gene expression by RNA sequencing. No significant changes in serum tryptophan or kynurenine levels were observed after baseline., Conclusions: Combination epacadostat and pembrolizumab was well tolerated and showed limited antitumor activity in sarcoma. Correlative analyses suggested that inadequate IDO1 inhibition was achieved., (©2023 American Association for Cancer Research.)

Intestinal-type gastric cancer often results from Helicobacter pylori infection through intestinal metaplasia, a transdifferentiated premalignant phenotype. Because H. pylori virulence factor CagA has been associated with aberrant expression of the transcription factor CDX1, which regulates intestinal differentiation, we explored its relationship with H. pylori infection and function during gastric carcinogenesis in normal gastric epithelial cells and gastric cancer cell lines. Infection of HFE 145 cells with CagA + H. pylori increased expression of CDX1, as well as the epithelial-to-mesenchymal transition (EMT) markers Snail and Slug, increased invasion and migration, but those effects were not found in HFE 145 cells infected with CagA-deficient H. pylori . CDX1 overexpression increased expression of the intestinal markers Villin, sucrose isomaltase (SI), and MUC2, induced spheroid formation, and enhanced expression of the stem cell markers CD44, SOX2, Oct4, and Nanog, while CDX1 knockdown inhibited proliferation and intestinal stemness. Treatment of CDX1-expressing cells with metformin, an antidiabetic drug known to decrease the risk of gastric cancer, decreased expression of EMT and stemness markers, and reduced spheroid formation. In a murine xenograft model, combining metformin or shCDX1 with cisplatin reduced tumor growth, increased caspase-3 cleavage, and reduced expression of CD44 and MMP-9 to a greater degree than cisplatin alone. Patients with more advanced intestinal metaplasia staging exhibited higher CDX1 expression than those with earlier intestinal metaplasia staging ( P = 0.039), and those with H. pylori tended to have more CDX1 expression than noninfected patients ( P = 0.061). Finally, human tissue samples with higher CDX1 levels showed prominent CD44/SOX2 expression. Our findings indicate CagA + H. pylori -induced CDX1 expression may enhance gastric cancer tumorigenesis and progression, and support therapeutic targeting of CDX1 in gastric cancer. IMPLICATIONS: This study shows that CDX1 contributes to the tumorigenesis and progression of gastric cancer and suggests the potential of targeting CDX1 to treat this malignancy., (©2019 American Association for Cancer Research.)

Our previous work showed that in a mouse model of gastric adenocarcinoma with loss of p53 and Cdh1 that adding oncogenic Kras (a.k.a. Tcon mice) accelerates tumorigenesis and metastasis. Here, we sought to examine KRAS activation in epithelial-to-mesenchymal transition (EMT) and generation of cancer stem-like cells (CSC). Transduction of nontransformed HFE-145 gastric epithelial cells with oncogenic KRAS G12V significantly decreased expression of the epithelial marker E-cadherin, increased expression of the mesenchymal marker vimentin and the EMT transcription factor Slug, and increased migration and invasion by 15- to 17-fold. KRAS G12V also increased expression of self-renewal proteins such as Sox2 and increased spheroid formation by 2.6-fold. In tumor-derived organoids from Tcon mice, Kras knockdown decreased spheroid formation, expression of EMT-related proteins, migration, and invasion; similar effects, as well as reversal of chemoresistance, were observed following KRAS knockdown or MEK inhibition in patient tumor-derived gastric adenocarcinoma cell lines (AGS and KATOIII). KRAS inhibition in gastric adenocarcinoma spheroid cells led to reduced AGS flank xenograft growth, loss of the infiltrative tumor border, fewer lung metastases, and increased survival. In a tissue microarray of human gastric adenocarcinomas from 115 patients, high tumor levels of CD44 (a marker of CSCs) and KRAS activation were independent predictors of worse overall survival. In conclusion, KRAS activation in gastric adenocarcinoma cells stimulates EMT and transition to CSCs, thus promoting metastasis. IMPLICATIONS: This study provides rationale for examining inhibitors of KRAS to block metastasis and reverse chemotherapy resistance in gastric adenocarcinoma patients., (©2019 American Association for Cancer Research.)

Purpose: Lauren diffuse-type gastric adenocarcinomas (DGAs) are generally genomically stable. We identified lysine (K)-specific methyltransferase 2C ( KMT2C ) as a frequently mutated gene and examined its role in DGA progression., Experimental Design: We performed whole exome sequencing on tumor samples of 27 patients with DGA who underwent gastrectomy. Lysine (K)-specific methyltransferase 2C ( KMT2C ) was analyzed in DGA cell lines and in patient tumors., Results: KMT2C was the most frequently mutated gene (11 of 27 tumors [41%]). KMT2C expression by immunohistochemistry in tumors from 135 patients with DGA undergoing gastrectomy inversely correlated with more advanced tumor stage ( P = 0.023) and worse overall survival ( P = 0.017). KMT2C shRNA knockdown in non-transformed HFE-145 gastric epithelial cells promoted epithelial-to-mesenchymal transition (EMT) as demonstrated by increased expression of EMT-related proteins N-cadherin and Slug. Migration and invasion in gastric epithelial cells following KMT2C knockdown increased by 47- to 88-fold. In the DGA cell lines MKN-45 and SNU-668, which have lost KMT2C expression, KMT2C re-expression decreased expression of EMT-related proteins, reduced cell migration by 52% to 60%, and reduced cell invasion by 50% to 74%. Flank xenografts derived from KMT2C-expressing DGA organoids, compared with wild-type organoids, grew more slowly and lost their infiltrative leading edge. EMT can lead to the acquisition of cancer stem cell (CSC) phenotypes. KMT2C re-expression in DGA cell lines reduced spheroid formation by 77% to 78% and reversed CSC resistance to chemotherapy via promotion of DNA damage and apoptosis., Conclusions: KMT2C is frequently mutated in certain populations with DGA. KMT2C loss in DGA promotes EMT and is associated with worse overall survival., (©2018 American Association for Cancer Research.)

Gastric adenocarcinoma is the third leading cause of cancer-related death worldwide, but no models exist to readily investigate distant metastases that are mainly responsible for mortality in this disease. Here we report the development of a genetically engineered mouse model of gastric adenocarcinoma tumorigenesis based on Kras G12D expression plus inactivation of E-cadherin ( Cdh1 ) and p53 in the gastric parietal cell lineage. Intestinal and diffuse gastric tumors arise rapidly in this model that displays a median survival of 76 days. Tumors occur throughout the stomach, with metastases documented in lymph nodes, lung, and liver. Mice otherwise identical but retaining one wild-type Cdh1 allele exhibited longer survival with only 20% penetrance of invasive tumors and no apparent lung or liver metastases. Notably, increased RAS activity and downstream MAPK signaling was observed in stomachs only when E-cadherin was absent. This model offers a valuable tool to investigate gastric adenocarcinoma subtypes where RAS/MAPK pathway activation and E-cadherin attenuation are common. Cancer Res; 77(19); 5349-59. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Rac1, a Rho GTPase family member, is dysregulated in a variety of tumor types including gastric adenocarcinoma, but little is known about its role in cancer stem-like cells (CSCs). Therefore, Rac1 activity and inhibition were examined in gastric adenocarcinoma cells and mouse xenograft models for epithelial-to-mesenchymal transition (EMT) and CSC phenotypes. Rac1 activity was significantly higher in spheroid-forming or CD44 + gastric adenocarcinoma CSCs compared with unselected cells. Rac1 inhibition using Rac1 shRNA or a Rac1 inhibitor (NSC23766) decreased expression of the self-renewal transcription factor, Sox-2, decreased spheroid formation by 78%-81%, and prevented tumor initiation in immunodeficient mice. Gastric adenocarcinoma CSCs had increased expression of the EMT transcription factor Slug, 4.4- to 8.3-fold greater migration, and 4.2- to 12.6-fold greater invasion than unselected cells, and these increases could be blocked completely with Rac1 inhibition. Gastric adenocarcinoma spheroid cells were resistant to 5-fluorouracil and cisplatin chemotherapy, and this chemotherapy resistance could be reversed with Rac1 shRNA or NSC23766. The PI3K/Akt pathway may be upstream of Rac1, and JNK may be downstream of Rac1. In the MKN-45 xenograft model, cisplatin inhibited tumor growth by 50%, Rac1 inhibition by 35%, and the combination by 77%. Higher Rac1 activity, in clinical specimens from gastric adenocarcinoma patients who underwent potentially curative surgery, correlated with significantly worse survival ( P = 0.017). In conclusion, Rac1 promotes the EMT program in gastric adenocarcinoma and the acquisition of a CSC state. Rac1 inhibition in gastric adenocarcinoma cells blocks EMT and CSC phenotypes, and thus may prevent metastasis and augment chemotherapy. Implications: In gastric adenocarcinoma, therapeutic targeting of the Rac1 pathway may prevent or reverse EMT and CSC phenotypes that drive tumor progression, metastasis, and chemotherapy resistance. Mol Cancer Res; 15(8); 1106-16. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: The Lauren diffuse type of gastric adenocarcinoma (DGA), as opposed to the intestinal type (IGA), often harbors mutations in RHOA, but little is known about the role of RhoA in DGA., Experimental Design: We examined RhoA activity and RhoA pathway inhibition in DGA cell lines and in two mouse xenograft models. RhoA activity was also assessed in patient tumor samples., Results: RhoA activity was higher in DGA compared with IGA cell lines and was further increased when grown as spheroids to enrich for cancer stem-like cells (CSCs) or when sorted using the gastric CSC marker CD44. RhoA shRNA or the RhoA inhibitor Rhosin decreased expression of the stem cell transcription factor, Sox2, and decreased spheroid formation by 78% to 81%. DGA spheroid cells had 3- to 5-fold greater migration and invasion than monolayer cells, and this activity was Rho-dependent. Diffuse GA spheroid cells were resistant in a cytotoxicity assay to 5-fluorouracil and cisplatin chemotherapy, and this resistance could be reversed with RhoA pathway inhibition. In two xenograft models, cisplatin inhibited tumor growth by 40% to 50%, RhoA inhibition by 32% to 60%, and the combination by 77% to 83%. In 288 patient tumors, increased RhoA activity correlated with worse overall survival in DGA patients (P = 0.017) but not in IGA patients (P = 0.612)., Conclusions: RhoA signaling promotes CSC phenotypes in DGA cells. Increased RhoA activity is correlated with worse overall survival in DGA patients, and RhoA inhibition can reverse chemotherapy resistance in DGA CSC and in tumor xenografts. Thus, the RhoA pathway is a promising new target in DGA patients., (©2015 American Association for Cancer Research.)

Purpose: Gastric cancers may harbor a subset of cells with cancer stem cell (CSC) properties, including chemotherapy resistance, and CD44 is a gastric CSC marker. The Hedgehog (HH) pathway is a key developmental pathway that can be subverted by CSCs during tumorigenesis. Here, we examine the role of HH signaling in CD44(+) gastric cancer cells., Experimental Design: Gastric cancer cell lines, tumor xenografts, and patient tumors were examined., Results: Gastric cancer cell lines AGS, MKN-45, and NCI-N87 grown as spheroids or sorted for CD44(+) were found to have upregulation of HH pathway proteins. HH inhibition using Smoothened (Smo) shRNA or vismodegib (VIS) decreased spheroid formation and colony formation. CD44(+) cells, compared with unselected cells, were also resistant to 5-fluorouracil and cisplatin chemotherapy, and this resistance was reversed in vitro and in xenografts with Smo shRNA or VIS. CD44(+) cells also had significantly more migration, invasion, and anchorage-independent growth, and these properties could all be blocked with HH inhibition. Clinical tumor samples from a phase II trial of chemotherapy with or without VIS for advanced gastric cancer were analyzed for CD44 expression. In the chemotherapy alone group, high CD44 expression was associated with decreased survival, whereas in the chemotherapy plus VIS group, high CD44 expression was associated with improved survival., Conclusions: HH signaling maintains CSC phenotypes and malignant transformation phenotypes in CD44(+) gastric cancer cells, and HH inhibition can reverse chemotherapy resistance in CD44(+) cells. Gastric cancer is a heterogeneous disease, and the strategy of combining chemotherapy with HH inhibition may only be effective in tumors with high CD44 levels., (©2014 American Association for Cancer Research.)

Unlabelled: Intratumoral hypoxia and expression of hypoxia-inducible factor-1α (HIF-1α) correlate with metastasis and poor survival in patients with sarcoma. We show here that hypoxia controls sarcoma metastasis through a novel mechanism wherein HIF-1α enhances expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). We show that loss of HIF-1α or PLOD2 expression disrupts collagen modification, cell migration, and pulmonary metastasis (but not primary tumor growth) in allograft and autochthonous LSL-Kras(G12D/+); Trp53(fl/fl) murine sarcoma models. Furthermore, ectopic PLOD2 expression restores migration and metastatic potential in HIF-1α-deficient tumors, and analysis of human sarcomas reveals elevated HIF1A and PLOD2 expression in metastatic primary lesions. Pharmacologic inhibition of PLOD enzymatic activity suppresses metastases. Collectively, these data indicate that HIF-1α controls sarcoma metastasis through PLOD2-dependent collagen modification and organization in primary tumors. We conclude that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell dissemination., Significance: Undifferentiated pleomorphic sarcoma (UPS) is a commonly diagnosed and particularly aggressive sarcoma subtype in adults, which frequently and fatally metastasizes to the lung. Here, we show the potential use of a novel therapeutic target for the treatment of metastatic UPS, specifi cally the collagen-modifying enzyme PLOD2.

Tumors induce new blood vessel growth primarily from host organ microvascular endothelial cells (EC), and microvasculature differs significantly between the lung and liver. Vascular endothelial growth factor (VEGF or VEGF-A) promotion of tumor angiogenesis is thought to be mediated primarily by VEGF receptor-2 (VEGFR-2). In this study, VEGFR-2 antibody (DC101) inhibited growth of RenCa renal cell carcinoma lung metastases by 26%, whereas VEGFR-1 antibody (MF-1) had no effect. However, VEGFR-2 neutralization had no effect on RenCa liver metastases, whereas VEGFR-1 neutralization decreased RenCa liver metastases by 31%. For CT26 colon carcinoma liver metastases, inhibition of both VEGFR-1 and VEGFR-2 was required to induce growth delay. VEGFR-1 or VEGFR-2 inhibition decreased tumor burden not by preventing the establishment of micrometastases but rather by preventing vascularization and growth of micrometastases by 55% and 43%, respectively. VEGF induced greater phosphorylation of VEGFR-2 in lung ECs and of VEGFR-1 in liver ECs. EC proliferation, migration, and capillary tube formation in vitro were suppressed more by VEGFR-2 inhibition for lung EC and more by VEGFR-1 inhibition for liver EC. Collectively, our results indicate that liver metastases are more reliant on VEGFR-1 than lung metastases to mediate angiogenesis due to differential activity of VEGFRs on liver EC versus lung EC. Thus, therapies inhibiting specific VEGFRs should consider the targeted sites of metastatic disease., (©2010 AACR.)

Tumors implanted near the scapulae have been shown to grow four times faster than the same tumors implanted at the iliac crest. Although there were marked differences in the vascularization of tumors from these two different sites, the mechanism controlling regional angiogenesis was not identified. Here, we show site-specific growth of intraperitoneal tumor implants in the mouse abdomen. Our data indicate that the angiogenic response of the host differs significantly between the upper and lower sites in the mouse abdomen and reveal that the expansion of tumor mass is restricted to sites with low angiogenic responses, such as the bowel mesentery in the lower abdomen. We show that, in this model, this suppression of angiogenesis is due to an expression gradient of thrombospondin-1 (TSP-1), a potent endogenous angiogenesis inhibitor. Mice with a targeted deletion of TSP-1 no longer show regional restriction of tumor growth. The physiologic relevance of these findings may be seen in patients with peritoneal carcinomatosis, whereby tumors spread within the peritoneal cavity and show differential growth in the upper and lower abdomen. We hypothesize that the difference in tumor growth in these patients may be due to a gradient of TSP-1 expression in stroma. Finally, our studies suggest that upregulation of TSP-1 in tumor cells is one method to suppress the growth of tumors in the upper abdomen., (© 2010 AACR.)

Metastasis relies on angiogenesis for tumor expansion. Tumor angiogenesis is restrained by a variety of endogenous inhibitors, including thrombospondin 1 (TSP1). The principal antiangiogenic activity of TSP1 resides in a domain containing three TSP1 repeats (3TSR), and TSP1 cleavage is regulated, in part, by the metalloproteinase ADAMTS1. In this study, we examined the role of TSP1 and ADAMTS1 in controlling metastatic disease in the liver and lung. TSP1 overexpression inhibited metastatic growth of colon or renal carcinoma cells in liver but not lung. Metastatic melanoma in liver grew more rapidly in Tsp1-null mice compared with controls, whereas in lung grew similarly in Tsp1-null mice or controls. Recombinant TSP1 was cleaved more efficiently in lysates from liver than lung. ADAMTS1 inhibition by neutralizing antibody, small interfering RNA, or genetic deletion abrogated cleavage activity. To confirm that lack of cleavage of TSP1 ablated its antiangiogenic function in the lung, we generated colon cancer cells stably secreting only the 3TSR domain and found that they inhibited formation of both liver and lung metastases. Collectively, our results indicate that the antiangiogenic activity of TSP1 is differentially regulated by ADAMTS1 in the liver and lung, emphasizing the concept that regulation of angiogenesis is varied in different tissue environments.

Purpose: Thrombospondin-1 (Tsp1), endostatin, and tumstatin are extracellular matrix-associated proteins that inhibit angiogenesis. We examined the mechanisms by which tumor cells may bypass the antiangiogenic effects of these endogenous regulators., Experimental Design: CT26 colon and RenCa renal carcinoma cells were stably transfected with Tsp1, endostatin, or tumstatin cDNA. Subcutaneous and metastatic tumor growth in syngeneic mice was analyzed. Expression of proangiogenic factors in resulting tumors was measured by quantitative real-time PCR. The combination of Tsp1 and vascular endothelial growth factor (VEGF) receptor-2 inhibition was also examined., Results: There was significant suppression of angiogenesis in flank tumors and liver metastases formed from cells overexpressing Tsp1, endostatin, or tumstatin. However, all tumors ultimately escaped angiogenesis inhibition. The combination of all three angiogenesis inhibitors had no additive effect beyond overexpression of a single inhibitor. Using quantitative real-time PCR, we found that VEGF and platelet-derived growth factor (PDGF)-A levels were routinely up-regulated at least 5-fold in all CT26 tumors overexpressing any antiangiogenic protein, and there were variable increases in angiopoietin 2 (Ang2), basic fibroblast growth factor, and PDGF-B. In contrast, RenCa tumors, which have high baseline levels of VEGF and PDGF-B, relied on basic fibroblast growth factor, Ang1, and PDGF-A up-regulation to counteract Tsp1 overexpression. Growth of CT26 cells with Tsp1 overexpression was suppressed when anti-VEGFR-2 treatment was added., Conclusions: Cancer cells with overexpression of three different endogenous angiogenesis inhibitor eventually escape angiogenesis inhibition by up-regulation of various proangiogenic factors. Tsp1, endostatin, and tumstatin may be functionally redundant in this system. These endogenous angiogenesis inhibitors are likely best used in combination with the blockade of proangiogenic pathways or with traditional chemotherapy or radiation therapy.

Vascular endothelial cell growth factor A (VEGF-A) and hypoxia play important roles in tumor angiogenesis. VEGF-A gene expression is up-regulated in tumors under hypoxic conditions, yet it is unclear how such up-regulation will affect the efficacy of RNA interference strategies targeting VEGF-A. Four potential short interfering RNA (siRNA) sequences for the VEGF-A gene were cloned into expression plasmids and transfected into HT1080 human fibrosarcoma cells. Stable transfection of these plasmids decreased VEGF-A mRNA levels and protein secretion by up to 99%. Our analysis of >100 hypoxia-related genes using oligonucleotide microarrays of 38 human sarcoma samples and 14 normal tissues identified distinctly different patterns of expression between sarcomas and normal tissues as assessed by hierarchical clustering analysis. Numerous hypoxia-related genes were significantly up-regulated in sarcomas including hypoxia-inducible factor 1alpha (HIF-1alpha). Exposure of wild-type HT1080 cells to 1% hypoxia resulted in HIF-1alpha up-regulation and a 74% increase in VEGF-A secretion as compared with secretion under normoxic conditions. Surprisingly, stable cell lines expressing VEGF-A siRNAs silenced VEGF-A expression equally well in hypoxia and normoxia. S.c. injection of cells with VEGF-A siRNAs into athymic nude mice led to slower-growing tumors, decreased blood vessel density, and greater apoptosis when compared with controls. Immunofluorescence analysis of tumor sections revealed areas of HIF-1alpha nuclear expression, suggesting areas of hypoxia, in both control tumors and VEGF-suppressed tumors. We conclude that hypoxia plays an important role in human sarcomas but hypoxic up-regulation of VEGF-A expression does not attenuate the efficacy of VEGF-A RNA interference.

Infection of tumor cells by herpes simplex virus 1 (HSV-1) results in cell destruction and production of progeny virion in a process referred to as viral oncolysis. In this study, an HSV-1 mutant (HSV1yCD) was engineered such that the viral ribonucleotide reductase gene is disrupted by sequences encoding yeast cytosine deaminase, which efficiently metabolizes the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). HSV1yCD-infected cells convert 5-FC to 5-FU, which enhances cytotoxicity without significantly reducing viral replication and oncolysis. Oncolysis by a replicating HSV-1 mutant combined with therapeutic transgene delivery represents a new paradigm; HSV1yCD-infected cells are destroyed by viral replication, and uninfected cells are subjected to bystander killing from both progeny virion and extracellular diffusion of 5-FU. In contrast, HSV1yCD-mediated bioactivation of another prodrug, ganciclovir, impairs viral replication. HSV1yCD administered into the portal venous system replicates preferentially in liver metastases rather than normal liver. The anti-neoplastic activity of HSV1yCD combined with systemic 5-FC administration is greater than that achieved with HSV-1 replication alone. Combination oncolysis and prodrug bioactivation leads to significant prolongation of survival in mice with diffuse liver metastases.

Herpes simplex virus type 1 (HSV-1) replication within tumors can mediate tumor regression (oncolysis). The genetically engineered, HSV-1 mutant rRp450 does not express viral ribonucleotide reductase and is therefore replication conditional. During the course of infection, rRp450 expresses the cytochrome P450 transgene and HSV-1 thymidine kinase gene, thereby enabling it to bioactivate the prodrugs cyclophosphamide and ganciclovir, respectively. rRp450 replication in hepatocellular carcinoma (HCC) cells is cytotoxic and liberates progeny virion that infect adjacent tumor cells. rRp450-mediated oncolysis is enhanced in the presence of cyclophosphamide, whereas it is inhibited in the presence of ganciclovir. As a consequence of defective viral ribonucleotide reductase expression, the yield of rRp450 progeny virions from infection of HCC cells is 3 to 4 log orders greater than that from infection of normal hepatocytes. This is associated with dramatic tumor reduction of diffuse HCC after a single intravascular administration of rRp450. rRp450 holds the promise of the dual therapeutic benefit of selective oncolysis and P450 transgene delivery.

Angiogenesis is required for tumor formation. Several studies have demonstrated that tumor angiogenesis is regulated by a balance between proangiogenesis and antiangiogenesis factors and that this balance varies in different organ environments. To investigate whether expression of an angiogenesis inhibitor by cancer cells could alter this balance and prevent tumor formation in different organ environments, we engineered stable transfectants from RenCa mouse renal carcinoma cells and SW620 human colon carcinoma cells to constitutively secrete a mouse endostatin protein with c-myc and polyhistidine (His) tags. Production and secretion of the endostatin-c-myc-His fusion protein by endostatin-transfected cells were confirmed by immunofluorescence staining and Western blot analysis. The endostatin transfectants and control transfectants, stably transfected with a control plasmid, had similar in vitro growth rates compared with their parental cell lines. Conditioned medium from endostatin-transfected cells inhibited human umbilical vein endothelial cell proliferation by 36-51% compared with conditioned medium from control cells. After inoculation into mice, flank tumors from endostatin-transfected cells were 73-91% smaller than flank tumors from control cells after 3 weeks. Inoculation of a cell mixture containing 25% endostatin-transfected cells and 75% control cells resulted in inhibition of flank tumor formation as effective as after inoculation of 100% endostatin-transfected cells. Formation of lung metastases by RenCa endostatin-transfected cells and formation of liver metastases by SW620 endostatin-transfected cells were dramatically inhibited compared with formation of metastases by control cells. These findings demonstrate that endostatin can inhibit tumor formation in different organ environments and that gene delivery of endostatin into even a minority of tumor cells may be an effective strategy to prevent progression of micrometastases to macroscopic disease.