Tumor-targeted gene delivery of tumor necrosis factor-alpha induces tumor necrosis and tumor regression without systemic toxicity

We have recently developed surface-shielded transferrin-polyethylenimine (Tf-PEI)/DNA delivery systems that target reporter gene expression to distant tumors after systemic application. In the present study, we used surface-shielded Tf-PEI/DNA complexes for delivering the gene for a highly potent cytokine, tumor necrosis factor-alpha (TNFalpha). TNFalpha is known for its ability to induce hemorrhagic tumor necrosis and tumor regression. However, the therapeutic application of TNFalpha is hampered by its high systemic toxicity dictating the need to target TNFalpha activity to the tumor. Systemic application of surface-shielded Tf-PEI complexes with the TNFalpha gene resulted in preferential expression of TNFalpha in the tumor without detectable TNFalpha serum levels, in contrast to the application of nontargeted complexes. Tumor-targeted TNFalpha gene delivery induced pronounced hemorrhagic tumor necrosis and inhibition of tumor growth in three murine tumor models of different tissue origins, Neuro2a neuroblastoma, MethA fibrosarcoma, and M-3 melanoma, with complete tumor regressions observed in the MethA model. No systemic TNF-related toxicity was observed due to the localization of the TNFalpha activity to the tumor. Targeted gene therapy may be an attractive strategy applicable to highly active, yet toxic, molecules such as TNFalpha.     

Targeting CCL2 with systemic delivery of neutralizing antibodies induces prostate cancer tumor regression in vivo

The identification of novel tumor-interactive chemokines and the associated insights into the molecular and cellular basis of tumor-microenvironment interactions have continued to stimulate the development of targeted cancer therapeutics. Recently, we have identified monocyte chemoattractant protein 1 (MCP-1; CCL2) as a prominent regulator of prostate cancer growth and metastasis. Using neutralizing antibodies to human CCL2 (CNTO888) and the mouse homologue CCL2/JE (C1142), we show that treatment with anti-CCL2/JE antibody (2 mg/kg, twice weekly i.p.) attenuated PC-3Luc-mediated overall tumor burden in our in vivo model of prostate cancer metastasis by 96% at 5 weeks postintracardiac injection. Anti-CCL2 inhibition was not as effective as docetaxel (40 mg/kg, every week for 3 weeks) as a single agent, but inhibition of CCL2 in combination with docetaxel significantly reduced overall tumor burden compared with docetaxel alone, and induced tumor regression relative to initial tumor burden. These data suggest an interaction between tumor-derived chemokines and host-derived chemokines acting in cooperation to promote tumor cell survival, proliferation, and metastasis.     

Oral delivery of tumor-targeting Salmonella for cancer therapy in murine tumor models

Tumor-targeting bacteria have been investigated intensively in recent years as anticancer agents. To ensure the reliability of infection, bacteria have conventionally been injected intravenously or intraperitoneally into animals or humans. However, systemic infection of bacteria is rather inconvenient and carries the risk of obvious toxicity. Here we tested whether Salmonella typhimurium VNP20009, a tumor-targeting strain, could be administrated orally for tumor therapy. Tumor-targeting potential, antitumor effects, as well as toxicity of orally administrated VNP20009 were investigated in this study. Oral delivery of VNP20009 not only exhibited high tumor-targeting potential, but also led to a significant anticancer effect by delaying tumor growth and prolonging survival in murine tumor models. As part of combination therapy, orally administrated bacteria notably improved the antitumor effect of cyclophosphamide. In vitro and in vivo studies showed that VNP20009 significantly induced tumor cell apoptosis. No obvious toxicity was observed during the treatments with oral inoculation of VNP20009. Comparative analysis of toxicity in tumor-bearing and tumor-free mice further revealed that orally administrated Salmonella had high safety compared to conventional systemic infection of bacteria. The findings indicated that oral administration of tumor-targeting bacteria is effective and safe. This approach provides a novel avenue in the application of bacteria as a potential antitumor agent.     

Systemic delivery of liposomal short-chain ceramide limits solid tumor growth in murine models of breast adenocarcinoma.

In vitro tumor cell culture models have illuminated the potential therapeutic utility of elevating the intracellular concentration of the antimitogenic and proapoptotic sphingolipid, ceramide. However, although cell-permeable, short-chain ceramide is an effective apoptotic agent in vitro, its use as an in vivo, systemically delivered therapeutic is limited by its inherent lipid hydrophobicity and physicochemical properties. Here, we report that the systemic i.v. delivery of C6-ceramide (C6) in a pegylated liposomal formulation significantly limited the growth of solid tumors in a syngeneic BALB/c mouse tumor model of breast adenocarcinoma. Over a 3-week treatment period, a well-tolerated dose of 36 mg/kg liposomal-C6 elicited a >6-fold reduction in tumor size compared with empty ghost liposomes. Histologic analyses of solid tumors from liposomal-C6-treated mice showed a marked increase in the presence of apoptotic cells, with a coincident decrease in cellular proliferation and in the development of a microvessel network. Liposomal-C6 accumulated within caveolae and mitochondria, suggesting putative mechanisms by which ceramide induces selective cancer cell cytotoxicity. A pharmacokinetic analysis of systemic liposomal-C6 delivery showed that the pegylated liposomal formulation follows first-order kinetics in the blood and achieves a steady-state concentration in tumor tissue. Confirming the therapeutic utility of i.v. liposomal-C6 administration, we also shown diminution of solid tumor growth in a human xenograft model of breast cancer. Together, these results indicate that bioactive ceramide analogues can be incorporated into pegylated liposomal vehicles for improved solubility, drug delivery, and antineoplastic efficacy.     

Development of a flexible and specific gene delivery system for production of murine tumor models.

To develop models of human cancer we have expressed the avian retroviral receptor, TVA, under a variety of mammalian promoters in transgenic mice, thus rendering mice susceptible to infection with avian leukosis virus-derived gene vectors. TVA-based retroviral gene transfer offers advantages over current murine models of human cancer. A single transgenic mouse line can be used to evaluate multiple genetic lesions, individually and in combination. Furthermore, mutant genes are introduced somatically into animals, as occurs in the majority of naturally occurring tumors. Because the avian viral vectors replicate only in avian cells, the viral receptor in infected transgenic mouse cells remains available for multiple rounds of infection with different ASLV vectors. We discuss the theoretical and practical aspects of using recombinant avian retroviruses with TVA transgenic mice to generate cancer models.     

Mitomycin C combination therapy against murine tumor systems. Effectiveness with cyclophosphamide and methotrexate

Mitomycin C (MMC) has been evaluated in combination with several antitumor agents. Full dose response curves were established for all drugs and drug combinations. Synergy was shown with MMC plus either cyclophosphamide (CYC) or methotrexate (MTX). In testing MMC and CYC against P388 leukemia, the combined treatment yielded a 75% rate of long-term survivors at the optimal level, compared to no survivors at the optimal level of the best single agent, CYC, alone. There was no increased toxicity among the combination-treated animals. Large increases in lifespan were obtained against L1210 and B16. Maximally tolerated doses of the single agents could be combined without increased toxicity. The combination of MMC and MTX was synergistic against ip L1210 and P388 leukemias. The responses of mice bearing L1210 to treatment on days 1, 5, and 9 respectively, were 42% ILS for 3.0 mg/kg MMC; 96% ILS for 15 mg/kg MTX; 172% ILS with four out of ten survivors for 3.0 mg/kg MMC plus 15 mg/kg MTX. MMC and adriamycin (ADR) were found to be synergistic against B16 melanoma at one schedule but not against another schedule, or against colon carcinoma 26. No improvements over optimal nontoxic single agent therapy were seen for chlorambucil, 5-fluorouracil, dibromodulcitol, cis-diaminedichloroplatinum or 4-'(9-acridinylamino) methansulfon-M-anisidide. On the basis of these data, recommendations were made for clinical trials for MMC plus either CYC or MTX against lung, breast, and colon tumors.     

环磷酰胺与顺铂联合治疗肿瘤患者剂量优化试验研究

目的 探讨环磷酰胺与顺铂联合化疗最佳给药剂量.方法 对120例卵巢癌和恶性淋巴瘤患者按完全随机分组法分为6组,分别按照环磷酰胺（CTX）500 mg/m2＋顺铂（DDP） 40 mg/m2、CTX 500 mg/m2＋ DDP 50 mg/ m2、CTX 500 mg/m2＋ DDP 60 mg/m2、CTX 600 mg/m2＋ DDP 40 mg/m2、CTX 600 mg/ m2＋ DDP 50 mg/m2、CTX 600 mg/m2＋ DDP 60 mg/m2静脉给药,每周1次,连用2周,比较各组疗效、不良反应.结果 各组间疗效差异无统计学意义（P＞0.05）.高剂量化疗组（CTX 500 mg/m2＋DDP 60 mg/m2、CTX 600 mg/m2＋ DDP 60 mg/m2）白细胞抑制率较高,分别为72.6％、79.3％,淋巴细胞损伤最大,血肌酐、血尿素氮、丙氨酸氨基转移酶、天冬氨酸氨基转移酶水平显著下降,而最低剂量化疗组（CTX 500 mg/m2＋DDP 40 mg/ m2）血象在正常范围,肾功能、肝功能损伤发生率低.结论 CTX与DDP联合化疗最佳给药剂量分别为500、40 mg/m2.     

Adeno-associated viral vector-mediated expression of endostatin inhibits tumor growth and metastasis in an orthotropic pancreatic cancer model in hamsters

We examined the feasibility of using adeno-associated virus (AAV)-mediated systemic delivery of endostatin in gene therapy to treat metastasis of pancreatic cancer. We established an animal model of orthotopic metastatic pancreatic cancer in which the pancreatic cancer cell line PGHAM-1 was inoculated into the pancreas of Syrian golden hamsters. Transplanted cells proliferated rapidly and metastasized to the liver. An AAV vector expressing endostatin (5 x 10(10) particles) was injected intramuscularly into the left quadriceps or intravenously into the portal vein. These routes of vector administration were evaluated by comparing various parameters of tumor development. Intramuscular injection of the vector modestly increased the serum endostatin level. The numbers of metastases and the incidence of hemorrhagic ascites were decreased in the treated animals. In contrast, the serum concentration of endostatin was significantly increased after intraportal injection of the vector. The antitumor effects on all parameters (including the size and microvessel density of primary pancreatic tumors, the sizes and number of liver metastases, and the incidence of hemorrhagic ascites) were significant. These results suggest that systemic delivery of endostatin represents a potentially effective treatment for pancreatic cancer and liver metastases. The route of vector administration influences the efficacy of AAV-mediated endostatin expression. Intraportal injection of the AAV vector appears to be more effective as an antiangiogenic gene therapy for pancreatic cancer.     

Dynamics of tumor cell clonogen repopulation in a murine sarcoma treated with cyclophosphamide

Experiments were performed to establish the extent and kinetics of tumor cell repopulation in a murine sarcoma, designated SA-NH, treated with cyclophosphamide (CY). Mice bearing 8-mm leg tumors were treated with 200 mg/kg CY which caused a transient tumor regression. Changes in the absolute clonogen content of tumors was determined by the change in TCD₅₀ values (50% tumor control) obtained under hypoxic conditions of local tumor irradiation at different times after CY treatment until tumors regrew to the pretreatment size. For comparison, hypoxic TCD₅₀ values were determined during the growth of tumors not treated with CY. CY greatly depleted tumors of clonogenic cells as manifested by the reduction in the control TCD₅₀ value of 64.5 Gy to 32.8 Gy 1 day after CY treatment. The reduced TCD₅₀ value remained unchanged for 2 weeks after treatment with CY, at which time the TCD₅₀ began to rapidly increase, continuing until the end of the observation period of 21 days when tumors reached the pretreatment size. In contrast, there was a constant but slower increase in TCD₅₀ values during the growth of tumors not treated with CY. The daily increase in TCD₅₀ was more than twice as high in CY-treated than in CY-untreated tumors: 4.5 Gy/day versus 2.1 Gy/day. This implies that the rate of clonogen production in CY-treated tumors was twice as high as that of unperturbed tumors. The possibility that tumors regrowing after CY treatment might consist of cells cross-resistant to irradiation was rejected by experiments showing that tumors grown from cells taken from CY-recurrent tumors were not significantly more resistant than control tumors of the same size. Further studies revealed that systemic effects produced by CY are conducive to tumor cell proliferation but the contribution of this mechanism to the observed increased rate of tumor cell repopulation in the recurrent tumors is uncertain.     

Eradication of human non-Hodgkin's lymphoma in SCID mice by BCL-2 antisense oligonucleotides combined with low-dose cyclophosphamide.

Cancers overexpressing Bcl-2 protein, which prevents programmed cell death (apoptosis), are less sensitive to stresses that produce cellular damage, including chemotherapy. If the level of Bcl-2 protein can be reduced sufficiently using antisense oligonucleotides (ASOs) targeting the gene message, then cytotoxic agents may be rendered more effective in eliminating disease and increasing cure rate. Preclinical studies in SCID mice bearing Bcl-2 overexpressing systemic human B-cell lymphoma (DoHH2) were undertaken to support development of a clinical trial. These data confirm that a combination of an ASO (5 mg/kg) targeting bcl-2 and a low dose of cyclophosphamide (35 mg/kg) was an effective strategy, leading to the eradication of the DoHH2 cells in vivo and cure of the animals. When mice deficient in natural killer cell activity were treated with an ASO, similar results were observed, suggesting that ASO stimulation of the host immune system was not a significant factor in elimination of lymphoma cells. These studies indicate that therapeutic strategies involving the use of an ASO targeting bcl-2 in combination with a cytotoxic agent may improve clinical outcomes.     

拓扑替康联合环磷酰胺治疗儿童神经母细胞瘤

目的观察评价拓扑替康联合环磷酰胺治疗神经母细胞瘤的疗效和毒性.方法14例神经母细胞瘤患儿,应用拓扑替康联合环磷酰胺联合静脉用药.大剂量应用环磷酰胺[70mg/(kg·d)×2 d]和拓扑替康[2mg/(m2·d)×3 d].其中12例治疗2～13个周期,进行疗效和不良反应评价.结果12例患者中,进展3例,稳定及缓解9例,总有效率为75%.不良反应主要为骨髓抑制和消化道反应,但对症治疗后,无相关性死亡.结论拓扑替康联合环磷酰胺治疗儿童神经母细胞瘤安全有效.     

Antitumoral efficacy by systemic delivery of heparin conjugated polyethylenimine-plasmid interleukin-15 complexes in murine models of lung metastasis

Gene therapy shows promising application in cancer therapy, but the lack of an ideal gene delivery system is still a tough challenge for cancer gene therapy. Previously, we prepared a novel cationic nanogel, heparin-polyethylenimine (HPEI), which had potential application in gene delivery. In the present study, we constructed a plasmid with high expression efficiency of interleukin-15 (IL15) and investigated the effects HPEI-plasmid IL15 (HPEI-pIL15) complexes on the distribution level of the lung. We then evaluated the anticancer effect of HPEI-pIL15 complexes on lung metastases of B16-F10 melanoma and CT26 colon carcinoma. These results demonstrated that intravenous injection of the HPEI-pIL15 complex exhibited the highest plasmid distribution level in the lung compared with that of PEI2K-pIL15 and PEI25K-pIL15, and mice treated with HPEI-pIL15 had a lower tumor metastasis index compared with other treatment groups. Moreover, the number of natural killer cells, which were intermingled among the tumor cells, and the level of tumor necrosis factor-α and interferon-γ in the serum also increased in the pIL15-treated mice. Furthermore, the cytotoxic activity of spleen cells also increased significantly in the HPEI-pIL15 group. In addition, induction of apoptosis and inhibition of cell proliferation in lung tumor foci in the HPEI-pIL15 group was observed. Taken together, treating lung metastasis cancer with the HPEI nanogels delivered by plasmid IL15 might be a new and interesting cancer gene therapy protocol.     

MHC independent anti-tumor immune responses induced by Hsp70-enriched exosomes generate tumor regression in murine models

The ideal cancer vaccine should work regardless of MHC types but currently the barrier generated by MHC specificity hampers the development of human cancer vaccines, requesting to identify strong immunogenic molecules that can induce anti-cancer immune responses without being affected by MHC polymorphism. Tumor-derived exosomes are small membrane vesicles containing tumor antigens as well as other immunologically important molecules such as MHC molecules and heat shock proteins (HSPs). Because of their potential immunogenicity, the plausible utility of tumor-derived exosomes as an MHC independent cancer vaccine was proposed. Here, we investigated whether Hsp70-enriched tumor exosomes can induce stronger immunogenicity as compared to normal tumor-derived exosomes in autologous as well as allogeneic murine models in vitro and in vivo. Western blotting showed that the exosomes of heat-treated tumor cells (HS Exo) contained higher amounts of Hsp70 than the exosomes of untreated cells (CNTL Exo). In both MHC type-identical and -irrelevant antigen-presenting cell models in vitro, HS Exo triggered the increased expressions of MHC class II molecules. Crucially, HS Exo performed greater therapeutic capability in regressing pre-established MHC type-identical and -irrelevant tumors than CNTL Exo in vivo. The analyses of anti-tumor function in allogeneic mouse model demonstrated that HS Exo elicited Th1-polarized immune responses defined by the increased productions of IgG2a and IFN-γ. In summary, the Hsp70-enriched exosomes extracted from heat-treated tumors induced strong Th1 immune responses, resulting in eliminating cancer cells in allogeneic hosts in vivo. These results indicate that HS Exo is a potent MHC independent cell-free cancer therapeutic agent that can be developed for clinical trials.     

Effects of disodium etidronate in murine tumor models

This study was designed to elucidate the effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) in experimental rodent tumors. EHDP had no antitumor activity against the L1210 leukemia implanted i.p. and against sarcoma 180, Lewis lung carcinoma (3LL) and Walker 256/B carcinoma injected i.p., s.c. or i.m. respectively. EHDP did not interfere with the antitumor activity of commonly used conventional chemotherapeutic agents (adriamycin, cyclophosphamide, 5-fluorouracil, bis-chloroethylnitrosourea) in the L1210 and 3LL models. EHDP reduced proportionally to the dose the hypercalcemia and hypercalciuria due to the Walker 256/B carcinoma growth. In an effort to evaluate whether EHDP-treated osseous tissues were more refractory to tumor growth, cells from sarcoma 180 and 3LL carcinoma were implanted intratibially (i.t.). Growth of 3LL cells was not consistently affected by EHDP, whereas a modest, but significant, growth inhibition was consistently observed with sarcoma 180 injected i.t. Growth of sarcoma 180 implanted i.p. or s.c. was not reduced by this drug, thus suggesting that inhibition of i.t. sarcoma 180 was in fact related to alterations of osseous tissues by EHDP. Inoculation of Walker 256/B carcinoma intra-aortically resulted in osteolytic bone lesions in the hind limbs. EHDP inhibited the formation of bone metastasis under these conditions.     

口服小剂量环磷酰胺联合卡培他滨维持治疗晚期三阴性乳腺癌的疗效

目的:观察小剂量环磷酰胺（CTX）联合卡培他滨（CAP）维持治疗晚期三阴性乳腺癌的疗效及安全性。方法:回顾性分析我院收治的经含卡培他滨方案两药联合化疗后达临床稳定的晚期三阴性乳腺癌患者68例,实验组34例给予口服环磷酰胺 50 mg/d,d8~21,每3周重复,联合卡培他滨1 000 mg/m2,bid,d1~14,休息7天。对照组34例患者仅给予口服卡培他滨单药维持治疗。观察2组患者的ORR、DCR、TTP、安全性及对生活质量的影响。结果:经维持治疗后实验组ORR 为29.4%,DCR为85.3%;对照组ORR 为14.7%,DCR为64.7%。实验组中位TTP为12个月,明显高于对照组中位TTP 6.9个月(P＜0.05)。两组的主要不良反应为手足综合征,实验组的胃肠道反应及血液学毒性稍高于对照组,均为Ⅰ-Ⅱ度。两组患者的生活质量评分均较治疗前明显提高,治疗后组间差异无统计学意义(P＞0.05)。结论:口服小剂量环磷酰胺联合卡培他滨维持治疗晚期三阴性乳腺癌有较好的疗效,不良反应可以耐受,可作为复发转移较快的晚期三阴性乳腺癌患者维持治疗的一种选择,具有一定的临床推广应用价值。     

Local low-dose interleukin-2 induces systemic immunity when combined with radiotherapy of cancer. A pre-clinical study

Tumor recurrence and outgrowth of metastases limit the therapeutical effect of radiotherapy. We have tested whether these problems can be overcome by supplementing radiotherapy with locoregional interleukin-2 (IL-2) treatment. The SL2 lymphoma and the M8013 mammary carcinoma were used. Mice bearing a 10-day-old s.c. tumor were locally irradiated and were treated daily with IL-2 peritumorally for 5 or 10 days. Low-dose IL-2 therapy improved local response (LR) and increased disease-free survival (DFS) in both tumor models following either single-dose irradiation or fractionated irradiation. For example, 93% of SL2-bearing mice treated with single-dose irradiation and 10 days of IL-2 experienced long-term DFS, compared with 17% for irradiation alone (p < 0.0001). Additionally, treatment of one tumor with irradiation +IL-2 led to anti-tumor effects in a second, untreated tumor in 80% of SL2-bearing mice. LR was increased to 100% and DFS to 70% when the second, non-irradiated tumor was also treated with peritumoral IL-2. We conclude that supplementing local radiotherapy with low doses of IL-2 results in increased local tumor control and regression of distant, non-irradiated tumors. This type of radioimmunotherapy is a promising new approach for the clinic. Int. J. Cancer 72:1003–1007, 1997. © 1997 Wiley-Liss, Inc.