Gerardo C. Glikin

Suicide gene and cytokines combined nonviral gene therapy for spontaneous canine melanoma.

Canine spontaneous melanoma is a highly aggressive tumor resistant to current therapies. We evaluated the safety, efficacy and antitumor effects of direct intratumor injections of lipoplexes encoding herpes simplex thymidine kinase coadministrated with ganciclovir, and irradiated transgenic xenogeneic cells secreting 20–30 μg day−1 of human granulocyte–macrophage colony-stimulating factor and interleukin-2. Toxicity was minimal or absent in all patients. This combined treatment (CT) induced tumor regression and a pronounced immune cell infiltration. The objective responses (47%: 21/45) averaged 80% of tumor mass loss. Local CT also induced systemic antitumor response evidenced by complete remission of one pulmonary metastasis and by the significantly higher percentage of metastasis-free patients (76: 34/45)) until the study ending compared to untreated (UC: 29%, 5/17), surgery-treated (CX: 48%, 11/23) or suicide gene-treated controls (SG: 56%, 9/16) (Fishers exact test). CT significantly improved median survival time: 160 (57–509) days compared to UC (69 (10–169)), CX (82 (43–216)) or SG (94 (46–159)). CT also increased (P<0.00001, Kaplan–Meier analysis) metastasis-free survival: >509 (57–509) days with respect to UC: 41 (10–169), CX: 133 (43–216) and SG: >159 (41–159). Therefore, CT controlled tumor growth by delaying or preventing distant metastasis, thereby significantly extending survival and recovering the quality of life.

Cytokine-enhanced vaccine and interferon-β plus suicide gene as combined therapy for spontaneous canine sarcomas

Eleven soft tissue- and five osteosarcoma canine patients were subjected to: (i) periodic subcutaneous injection of irradiated xenogeneic cells secreting hGM-CSF and hIL-2 mixed with allogeneic or autologous tumor homogenates; and (ii) injections of cIFN-β and HSVtk-carrying lipoplexes and ganciclovir, marginal (after surgery) and/or intratumoral (in the case of partial tumor resection, local relapse or small surface tumors). This treatment alone (4 patients) or as surgery adjuvant (12 patients), was safe and well tolerated. In those patients presenting local disease (6/11), the suicide gene plus cIFN-β treatment induced local antitumor activity evidenced by the objective responses (3 complete, 2 partial) and stable diseases (2). In addition, the treatment prevented or delayed local relapse, regional metastases (lymph nodes developed only in 3/16) and distant metastases (0/16), suggesting a strong systemic antitumor immunity. The most encouraging result was the long survival times of 10 patients (>1 year, with good quality of life).

Suicide gene therapy on spontaneous canine melanoma: correlations between in vivo tumors and their derived multicell spheroids in vitro

To validate the use of multicellular spheroids to predict the efficacy of herpes simplex thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene therapy in the respective in vivo tumors, we established and characterized 15 melanoma-derived cell lines from surgically excised melanoma tumors. Three HSVtk-lipofected cell lines were not sensitive to GCV in any culture configuration, other five displayed similar sensitivity as monolayers or spheroids, and only one resulted more sensitive when grown as spheroids. Other six cell lines manifested a relative multicellular resistance (MCR) phenotype growing as spheroids, compared with the same cells growing as monolayers. The reverse correlation between the MCR and the monolayers survival to HSVtk/GCV suggests that one of the main causes of MCR would be the rapid cell repopulation after suicide gene treatment. The high correlation of MCR with the spheroids radial growth and with the mitotic index of the respective originary tumors supported this re-growth involvement. A remarkable finding was the high correlation in HSVtk/GCV sensitivity between in vivo tumor and the corresponding derived cell lines growing as spheroids (R2=0.85). This strongly encourages the implementation of spheroids as highly realistic experimental model for optimizing and predicting the in vivo response of the respective tumors to therapeutic strategies.

Cytokine-enhanced vaccine and suicide gene therapy as adjuvant treatments of metastatic melanoma in a horse

EQUINE melanomas occur most commonly in grey horses five years of age or older ([Smith and others 2002][1]). Affected horses often have encapsulated nodules, where malignant and benign melanomas can be distinguished by microscopy. The features of the disease in grey horses include spontaneous

A laboratory scale device for microencapsulation of genetically engineered cells into alginate beads

The microencapsulation of recombinant cells, widely used for in vitro high-density cell culture, is a novel and potentially cost-effective method of in vivo heterologous protein delivery, where the protein producing cells are immunologically protected from tissue rejection. We report here a simple, reliable and inexpensive laboratory method to generate calcium alginate microcapsules containing genetically engineered, interleukin-2 expressing, Chinese hamster ovary (CHO) cells.

Interferon-β lipofection II. Mechanisms involved in cell death and bystander effect induced by cationic lipid-mediated interferon-β gene transfer to human tumor cells

We evaluated the cytotoxic effects (apoptosis, necrosis and early senescence) of human interferon-β (hIFNβ) gene lipofection. The cytotoxicity of hIFNβ gene lipofection resulted equivalent to that of the corresponding addition of the recombinant protein (rhIFNβ) on human tumor cell lines derived from Ewings sarcoma (EW7 and COH) and colon (HT-29) carcinomas. However, it was stronger than rhIFNβ on melanoma (M8) and breast adenocarcinoma (MCF7). To reveal the mechanisms involved in these differences, we compared the effects of hIFNβ gene and rhIFNβ protein on EW7 and M8 (sensitive and resistant to rhIFNβ protein, respectively). Lipofection with hIFNβ gene caused a mitochondrial potential decrease simultaneous with an increase of oxidative stress in both cell lines. However, rhIFNβ protein displayed the same pattern of response only in EW7-sensitive cell line. The great bystander effect of the hIFNβ gene lipofection, involving the production of reactive oxygen species, would be among the main causes of its success. In EW7, this effect killed >60% of EW7 cell population, even though only 1% of cells were expressing the transgene. As hIFNβ gene was effective even in the rhIFNβ protein-resistant M8 cell line and in a way not limited by low lipofection efficiency, these results strongly support the clinical potential of this approach.

Interferon-β gene transfer induces a strong cytotoxic bystander effect on melanoma cells

A local gene therapy scheme for the delivery of type I interferons could be an alternative for the treatment of melanoma. We evaluated the cytotoxic effects of interferon-β (IFNβ) gene lipofection on tumor cell lines derived from three human cutaneous and four canine mucosal melanomas. The cytotoxicity of human IFNβ gene lipofection resulted higher or equivalent to that of the corresponding addition of the recombinant protein (rhIFNβ) to human cells. IFNβ gene lipofection was not cytotoxic for only one canine melanoma cell line. When cultured as monolayers, three human and three canine IFNβ-lipofected melanoma cell lines displayed a remarkable bystander effect. As spheroids, the same six cell lines were sensitive to IFNβ gene transfer, two displaying a significant multicell resistance phenotype. The effects of conditioned IFNβ-lipofected canine melanoma cell culture media suggested the release of at least one soluble thermolabile cytotoxic factor that could not be detected in human melanoma cells. By using a secretion signal-free truncated human IFNβ, we showed that its intracellular expression was enough to induce cytotoxicity in two human melanoma cell lines. The lower cytoplasmatic levels of reactive oxygen species detected after intracellular IFNβ expression could be related to the resistance displayed by one human melanoma cell line. As IFNβ gene transfer was effective against most of the assayed melanomas in a way not limited by relatively low lipofection efficiencies, the clinical potential of this approach is strongly supported.

Interferon-β lipofection I. Increased efficacy of chemotherapeutic drugs on human tumor cells derived monolayers and spheroids.

We evaluated the effect of hIFNβ gene transfer alone or in combination with different antineoplastic drugs commonly used in cancer treatment. Five human tumor-derived cell lines were cultured as monolayers and spheroids. Four cell lines (Ewing sarcomas EW7 and COH, melanoma M8 and mammary carcinoma MCF-7) were sensitive to hIFNβ gene lipofection. Although this effect appeared in both culture configurations, spheroids showed a relative multicellular resistance (insensitive colon carcinoma HT-29 excluded). EW7 and M8 hIFNβ-expressing cells were exposed to different concentrations of bleomycin, bortezomib, carboplatin, doxorubicin, etoposide, methotrexate, paclitaxel and vincristine in both configuration models. In chemotherapy-sensitive EW7 monolayers, the combination of hIFNβ gene and antineoplastic drugs displayed only additive or counteractive (methotrexate) effects, suggesting that cytotoxic mechanisms triggered by hIFNβ gene lipofection could be saturating the signaling pathways. Conversely, in chemotherapy-resistant EW7 spheroids or M8 cells, the combination of hIFNβ with drugs that mainly operate at the genotoxic level (doxorubicin, methotrexate and paclitaxel) presented only additive effects. However, drugs that also increase pro-oxidant species can complement the antitumor efficacy of the hIFNβ gene and clearly caused potentiated effects (bleomycin, bortezomib, carboplatin, etoposide and vincristine). The great bystander effect induced by hIFNβ gene lipofection could be among the main causes of its effectiveness, because only 1 or 2% of EW7 or M8 hIFNβ-expressing cells killed more than 60 or 80% of cell population, respectively.

Interferon-β gene transfer inhibits melanoma cells adhesion and migration.

HighlightsInterferon‐&bgr; expression inhibits melanoma cells adhesion to ECM proteins and collagen.Interferon‐&bgr; expression inhibits melanoma cells migration in monolayers and spheroids.These effects are mediated by the induced raise of ROS following interferon‐&bgr; gene lipofection and expression. ABSTRACT We evaluated the effects of expression of interferon‐&bgr; (IFN&bgr;) after lipofection on melanoma cells adhesion and migration. Three canine mucosal (Ak, Br and Ol) and one human dermal (SB2) melanomas were assayed. By means of the wound healing assay, we found a significant inhibitory effect of canine IFN&bgr; gene expression on cells migration in Br and Ol monolayers. This effect could be reproduced on unlipofected Ol cells with conditioned culture media obtained from canine IFN&bgr; gene‐lipofected Ol cells, and with recombinant human IFN&bgr; on unlipofected SB2 cells. Furthermore, IFN&bgr; gene expression of the four tested tumor cells significantly inhibited their adhesion to extracellular matrix (ECM) proteins and their spreading from multicellular spheroids onto gelatin coating. The addition of catalase reverted the increase of reactive oxygen species (ROS) in Ol cells and the inhibition of cell migration in monolayers (Ol) and spheroids (Ol an SB2) produced by canine and human IFN&bgr; expression, suggesting the involvement of ROS as mediators of IFN&bgr; action on the cells interactions with ECM. Together with its known immune, antiangiogenic and cytotoxic effects, the present data strongly support more studies exploring the clinical potential of IFN&bgr; for cancer therapy.

Cytotoxic effects induced by interferon-ω gene lipofection through ROS generation and mitochondrial membrane potential disruption in feline mammary carcinoma cells

Progress in comparative oncology promises advances in clinical cancer treatments for both companion animals and humans. In this context, feline mammary carcinoma (FMC) cells have been proposed as a suitable model to study human breast cancer. Based on our previous data about the advantages of using type I interferon gene therapy over the respective recombinant DNA derived protein, the present work explored the effects of feline interferon-ω gene (fIFNω) transfer on FMC cells. Three different cell variants derived from a single spontaneous highly aggressive FMC tumor were successfully established and characterized. Lipofection of the fIFNω gene displayed a significant cytotoxic effect on the three cell variants. The extent of the response was proportional to ROS generation, mitochondrial membrane potential disruption and calcium uptake. Moreover, a lower sensitivity to the treatment correlated with a higher malignant phenotype. Our results suggest that fIFNω lipofection could offer an alternative approach in veterinary oncology with equal or superior outcome and with less adverse effects than recombinant fIFNω therapy.