Huaiyu Ma

Targeted Therapy with a Salmonella Typhimurium Leucine-Arginine Auxotroph Cures Orthotopic Human Breast Tumors in Nude Mice

We report here a modified auxotrophic strain of Salmonella typhimurium that can target and cure breast tumors in orthotopic mouse models. We have previously reported development of a genetically modified strain of S. typhimurium, selected for prostate tumor targeting and therapy in vivo. The strain, termed S. typhimurium A1, selectively grew in prostate tumors in xenograft models causing tumor regression. In contrast, normal tissue was cleared of these bacteria even in immunodeficient athymic mice with no apparent side effects. A1 is auxotrophic (leucine-arginine dependent) but apparently receives sufficient nutritional support only from tumor tissue. The ability to grow in viable tumor tissue may account, in part, for the unique antitumor efficacy of the strain. In the present report, to increase tumor-targeting capability of A1, the strain was reisolated after infection of a human colon tumor growing in nude mice. The tumor-isolated strain, termed A1-R, had increased targeting for tumor cells in vivo as well as in vitro compared with A1. Treatment with A1-R resulted in highly effective tumor targeting, including viable tumor tissue and significant tumor shrinkage in mice with s.c. or orthotopic human breast cancer xerographs. Survival of the treated animals was significantly prolonged. Forty percent of treated mice were cured completely and survived as long as non-tumor-bearing mice. These results suggest that amino acid auxotrophic virulent bacteria, which selectively infect and attack viable tumor tissue, are a promising approach to cancer therapy.

Monotherapy with a tumor-targeting mutant of Salmonella typhimurium cures orthotopic metastatic mouse models of human prostate cancer

Bacterial infection occasionally has a marked therapeutic effect on malignancies, as noted as early as the 19th century. Recently, there have been attempts to develop cancer treatment by using tumor-targeting bacteria. These treatments were developed to deliver therapeutic molecules specifically to tumors. Researchers used anaerobic microorganisms that preferentially grew in necrotic tumor areas. However, the resulting tumor killing was, at best, limited. We have developed a far more effective bacterial cancer therapy by targeting viable tumor tissue by using Salmonella typhimurium leu-arg auxotrophs. Although these bacteria grow in viable as well as necrotic areas of tumors, the nutritional auxo trophy severely restricts growth in normal tissue. In the current study, we measured the antitumor efficacy of the S. typhimurium A1-R mutant, which is auxotrophic for leu-arg and has increased antitumor virulence selected by tumor passage. A1-R was used to treat metastatic PC-3 human prostate tumors that had been orthotopically implanted in nude mice. GFP was used to image tumor and metastatic growth. Of the 10 mice with the PC-3 tumors that were injected weekly with S. typhimurium A1-R, 7 were alive and well at the time the last untreated mouse died. Four A1-R-treated mice remain alive and well 6 months after implantation. Ten additional nontumor-bearing mice were injected weekly to determine the toxicity of S. typhimurium A1-R. No toxic effects were observed. The approach described here, where bacterial monotherapy effectively treats metastatic prostate tumors, is a significant improvement over previous bacterial tumor-therapy strategies that require combination with toxic chemotherapy.

Potent and highly selective hypoxia-activated achiral phosphoramidate mustards as anticancer drugs.

A series of achiral hypoxia-activated prodrugs were synthesized on the basis of the DNA cross-linking toxin of the prodrug, ifosfamide. The hypoxia-selective cytotoxicity of several of the compounds was improved over previously reported racemic mixtures of chiral bioreductive phosphoramidate prodrugs. Prodrugs activated by 2-nitroimidazole reduction demonstrated up to 400-fold enhanced cytotoxicity toward H460 cells in culture under hypoxia versus their potency under aerobic conditions. Compounds were further assessed for their stability to cytochrome P450 metabolism using a liver microsome assay. The 2-nitroimidazole containing lead compound 3b (TH-302) was selectively potent under hypoxia and stable to liver microsomes. It was active in an in vivo MIA PaCa-2 pancreatic cancer orthotopic xenograft model as a monotherapy and demonstrated dramatic efficacy when used in combination with gemcitabine, extending survival with one of eight animals tumor free at day-44. Compound 3b has emerged as a promising antitumor agent that shows excellent in vivo efficacy and is currently being evaluated in the clinic.

Monotherapy with a Tumor-Targeting Mutant of S. typhimurium Inhibits Liver Metastasis in a Mouse Model of Pancreatic Cancer

Cancer of the exocrine pancreas is the fourth leading cause of cancer deaths in the United States. Currently, surgical resection is the only hope for cure. The majority of patients present with locally-advanced or metastatic disease. The most common site for distant metastasis is the liver. We report here a modified auxotrophic strain of S. typhimurium that can target and inhibit the growth of liver metastasis in a mouse model of pancreatic cancer. This strain of S. typhimurium is auxotrophic (leucine-arginine dependent) but apparently receives sufficient nutritional support from tumor tissue. To increase tumor targeting ability and tumor killing efficacy, this strain was further modified by re-isolation from a tumor growing in a nude mouse and termed A1-R. In the present study, we demonstrate the efficacy of locally- as well as systemically-administered A1-R on liver metastasis of pancreatic cancer. Mice treated with A1-R given locally via intrasplenic injection or systemically via tail vein injection had a much lower hepatic and splenic tumor burden compared with control mice. Systemic treatment with intravenous A1-R also increased survival time. All results were statistically significant. This study suggests the clinical potential of bacterial treatment of a critical metastatic target of pancreatic cancer.

In vitro/in vivo biorecognition of lectin-immobilized fluorescent nanospheres for human colorectal cancer cells.

Peanut agglutinin (PNA)-immobilized polystyrene nanospheres with surface poly(N-vinylacetamide) (PNVA) chains encapsulating coumarin 6 were designed as a novel colonoscopic imaging agent. PNA was a targeting moiety that binds to beta-D-galactosyl-(1-3)-N-acetyl-D-galactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. PNVA was immobilized with the aim of reducing nonspecific interactions between imaging agents and normal tissues. Coumarin 6 was encapsulated into nanosphere cores to provide endoscopically detectable fluorescence intensity. After incubation of imaging agents with human cells, the fluorescence intensity of imaging agent-bound cells was estimated quantitatively. The average fluorescence intensity of any type of colorectal cancer cell used in this study was higher than that of small intestinal epithelial cells that had not exposed the carbohydrate. The in vivo performance of imaging agents was subsequently evaluated using a human colorectal cancer orthotopic animal model. Imaging agent-derived strong fluorescence was observed at several sites of the large intestinal mucosa in the tumor-implanted nude mice after the luminal side of the colonic loop was contacted with imaging agents. In contrast, when mice that did not undergo tumor implantation were used, the fluorescence intensity on the mucosal surface was extremely low. Data indicated that imaging agents bound to colorectal cancer cells and the cancer cell-derived tumors with high affinity and specificity.

Efficacy against lung metastasis with a tumor-targeting mutant of Salmonella typhimurium in immunocompetent mice

Salmonella typhimurium double leu-arg auxotrophs have been shown to be highly effective as antitumor agents in nude mouse models of human metastatic cancer. In order to proceed to clinical development of the S. typhimurium double auxotroph, termed A1-R, it is necessary to evaluate antitumor efficacy in immunocompetent mice. In the present study, we have observed the efficacy of A1-R on the Lewis lung (LLC) carcinoma in vitro as well as in C57BL/6 (C57) immunocompetent mice. In vitro, A1-R treatment of LLC began to induce cell death within one hour. Various doses and schedules of A1-R were administered to C57 mice implanted with LLC, including bolus single intravenous injection; medium dose with weekly intravenous administration and metronomic treatment with small intravenous doses twice a week. Bolus treatment was toxic to the immunocompetent host, in contrast to nude mice. Lower-dose weekly doses and metronomic doses were well tolerated by the immunocompetent host. Weekly intravenous injection with 2 x 107 bacteria and twice a week intravenous injection with 107 bacteria significantly inhibited metastasis formation, while bolus injection was toxic. Intra-thoracic administration was carried out with 108 bacteria A1-R injected into Lewis lung-bearing C57 mice weekly for three weeks. Lung metastasis was significantly inhibited by intrathoracic bacterial administration, without toxicity. The results in this report, demonstrating the anti-metastatic efficacy of S. typhimurium A1-R in immunocompetent mice, indicate the clinical potential of bacterial therapy of cancer.

Detection of early colorectal cancer imaged with peanut agglutinin-immobilized fluorescent nanospheres having surface poly(N-vinylacetamide) chains.

Peanut agglutinin (PNA)-immobilized fluorescent nanospheres were designed as a novel imaging agent for colonoscopy. PNA is a targeting moiety that binds to beta-D-galactosyl-(1-3)-N-acetyl-D-galactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. The in vivo performance of the imaging agent was evaluated using a human colorectal cancer orthotopic animal model. Human colorectal adenocarcinoma cell lines, HT-29, HCT-116, and LS174T, were implanted on the cecal serosa of immune-deficient mice. A loop of the tumor-bearing cecum was made, and the luminal side was treated with the imaging agent. Strong fluorescence was observed at several sites of the cecal mucosa, irrespective of cancer cell type. Microscopic histological evaluation of the cecal mucosa revealed that bright areas with fluorescence derived from the imaging agent and dark areas without the fluorescence well denoted the presence and absence, respectively, of the invasion of implanted cancer cells on the mucosal side. This good correlation showed that PNA-immobilized fluorescent nanospheres recognized millimeter-sized tumors on the cecal mucosa with high affinity and specificity.

Real-Time GFP Intravital Imaging of the Differences in Cellular and Angiogenic Behavior of Subcutaneous and Orthotopic Nude-Mouse Models of Human PC-3 Prostate Cancer

There are two major types of mouse xenograft models of cancer: subcutaneous implantation and orthotopic implantation. Subcutaneous transplant models are widely used with both cancer cell lines and human‐tumor specimens. Recently, subcutaneous models of patient tumors, termed patient‐derived xenographs (PDX) have become highly popular and have acquired such names as “Avatar” and “Xenopatients.” However, such s.c. models rarely metastasize and are therefore not patient‐like. In contrast, orthotopic models have the capability to metastasize. If intact fragments of tumor tissue are implanted by surgical orthotopic implantation (SOI), the metastatic potential can match that of the donor patient. The present study images in real time, using green fluorescent protein (GFP) expression, the very different tumor behavior at the orthotopic and subcutaneous sites of human prostate cancer PC‐3 in athymic nude mice. By day‐2 after tumor implantation, the orthotopic tumor is already highly vascularized and the cancer cells have begun to migrate out of the tumor. In contrast, the subcutaneous tumor only begins to be vascularized by day‐3 and cells do not migrate from the tumor. Angiogenesis is much more extensive in the orthotopic tumor throughout the 2‐week observation period. The orthotopic PC‐3‐GFP tumor progresses very rapidly and distinct metastasis have appeared in lymph nodes by day‐3 which rapidly appear in many areas of the abdominal cavity including portal lymph nodes by day‐7. At day‐14, no invasion or metastasis was observed with the s.c. tumor even when the animal was extensively explored. These results explain why orthotopic tumors mimimc clinical metastatic tumors in nude mice and why subcutaneous tumors do not. J. Cell. Biochem. 117: 2546–2551, 2016.

High antimetastatic efficacy of MEN4901/T-0128, a novel camptothecin carboxymethyldextran conjugate.

The antimetastatic activity of a novel camptothecan conjugate, MEN4901/T-0128, in which 7-ethyl-10-aminopropyloxy-camptothecin (T-2513) is bound to a biodegradable carboxymethyldextran via a Gly-Gly-Gly linker, was observed in this study. High antimetastatic activity of MEN4901/T-0128 was demonstrated in a clinically-relevant orthotopic mouse model of human colon cancer. MEN4901/T-0128 and irinotecan were compared for anti-metastatic activity as well as efficacy against the primary tumor. An imageable, metastatic model was made by surgical orthotopic implantation (SOI) of the green fluorescent protein (GFP)-expressing HT-29 tumor in nude mice. MEN4901/T-0128 and irinotecan were administered intravenously at various doses and schedules. MEN4901/T-0128, with treatment beginning on d 49 after SOI, was highly effective on lymph node metastasis as well as against the primary tumor. Both GFP imaging and histology demonstrated a markedly lower metastatic incidence of lymph nodes in all MEN4901/T-0128 treated mice compared with irinotecan-treated and untreated mice. At the most efficacious dose of MEN4901/T-0128, only 1 of 12 animals had lymph node metastasis compared with 19 of 20 in the control group. The present study demonstrates the principle that when a camptothecan is conjugated to an appropriate polymer, the drug can become extremely effective with important clinical potential for antimetastatic therapy, a most urgent need.

Toxicology and efficacy of tumor-targeting Salmonella typhimurium A1-R compared to VNP 20009 in a syngeneic mouse tumor model in immunocompetent mice

Salmonella typhimurium A1-R (S. typhimurium A1-R) attenuated by leu and arg auxotrophy has been shown to target multiple types of cancer in mouse models. In the present study, toxicologic and biodistribution studies of tumor-targeting S. typhimurium A1-R and S. typhimurium VNP20009 (VNP 20009) were performed in a syngeneic tumor model growing in immunocompetent BALB/c mice. Single or multiple doses of S. typhimurium A1-R of 2.5 × 105 and 5 × 105 were tolerated. A single dose of 1 × 106 resulted in mouse death. S. typhimurium A1-R (5 × 105 CFU) was eliminated from the circulation, liver and spleen approximately 3-5 days after bacterial administration via the tail vein, but remained in the tumor in high amounts. S. typhimurium A1-R was cleared from other organs much more rapidly. S. typhimurium A1-R and VNP 20009 toxicity to the spleen and liver was minimal. S. typhimurium A1-R showed higher selective targeting to the necrotic areas of the tumors than VNP20009. S. typhimurium A1-R inhibited the growth of CT26 colon carcinoma to a greater extent at the same dose of VNP20009. In conclusion, we have determined a safe dose and schedule of S. typhimurium A1-R administration in BALB/c mice, which is also efficacious against tumor growth. The results of the present report indicate similar toxicity of S. typhimurium A1-R and VNP20009, but greater antitumor efficacy of S. typhimurium A1-R in an immunocompetent animal. Since VNP2009 has already proven safe in a Phase I clinical trial, the present results indicate the high clinical potential of S. typhimurium A1-R.