Particulate mediators of the bystander effect linked to suicide and interferon-ß transgene expression in melanoma cells.

In the context of comparative oncology, melanoma cells derived from companion animal tumors are good models for optimizing and predicting their in vivo response to therapeutic strategies. Here, we report that human, canine, and feline melanoma cells driven to death by bleomycin, interferon-ß gene, or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) treatment significantly increased their internal granularity. This fact correlated with the release of a heterogeneous collection of nano- and micro-sized granules as revealed by transmission electron microscopy. While killing lipofected cells, the expressed transgenes and their derived products were incorporated into these granules that were isolated by differential centrifugation. These particulate factors (PFs) were able to transfer, in a dose- and time-dependent manner, appreciable levels of therapeutic genes, related proteins, and drugs. Thus, when recipient cells of SG-carrying PFs were exposed to ganciclovir, this prodrug was efficiently activated, eliminating them. These PFs kept the functionality of their cargo, even after being subjected to adverse conditions, such as the presence of DNase, freezing, or heating. Since our in vitro system did not include any of the immune mechanisms that could provide additional antitumor activity, the chemo-gene treatments amplified by these delivery bags of therapeutic agents offer a great clinical potential.

Combination of cytokine-enhanced vaccine and chemo-gene therapy as surgery adjuvant treatments for spontaneous canine melanoma.

After 6 years of follow-up treating 364 canine melanoma patients, we present here results about the proof-of-concept, safety, and efficacy of a new surgery adjuvant combined gene therapy. The adjuvant treatment (AT) group was divided in three arms as follows: (i) complete surgery plus vaccine (CS-V), (ii) complete surgery plus combined treatment (CS-CT), and (iii) partial surgery plus combined treatment (PS-CT). Besides the genetic vaccines composed by tumor extracts and lipoplexes carrying human interleukin-2 and granulocyte-macrophage colony-stimulating factor genes, the patients were subjected to combined treatment received in the post-surgical bed injections of lipid-complexed thymidine kinase suicide gene plus ganciclovir and canine interferon-ß gene plus bleomycin. As compared with surgery-only treated controls (So), CS-CT and CS-V treatments significantly increased the fraction of local disease-free (from 20 to 89 and 74%) and distant metastases-free patients (M0: from 45 to 87 and 84%). Although less effective than CS arms, PS-CT arm demonstrated a significantly improved control of metastatic disease (M0: 80%) compared with So (M0: 44%). In addition, AT produced a significant 9.3- (CS-CT), 6.5- (CS-V), and 5.4-fold (PS-CT) increase of overall survival as compared with their respective So controls. In general terms, the AT changed a lethal disease into a chronic disease where 70% of CS-CT, 51% of CS-V, and 14% of PS-CT patients died of melanoma unrelated causes. These surgery adjuvant treatments delayed or prevented post-surgical recurrence and distant metastasis, and improved disease-free and overall survival while maintaining quality of life. These successful outcomes encourage assaying a similar scheme for human melanoma.

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

We evaluated the effects of expression of interferon-ß (IFNß) 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 IFNß 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 IFNß gene-lipofected Ol cells, and with recombinant human IFNß on unlipofected SB2 cells. Furthermore, IFNß 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 IFNß expression, suggesting the involvement of ROS as mediators of IFNß 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 IFNß for cancer therapy.

Therapeutic potential of the cytosine deaminase::uracil phosphoribosyl transferase/5-fluorocytosine suicide system for canine melanoma.

We tested the efficacy of a yeast cytosine deaminase::uracil phosphoribosyl transferase/5-fluorocytosine (CDU/5-FC) non-viral suicide system on eight established canine melanoma cell lines. Albeit with different degree of sensitivity 5 days after lipofection, this system was significantly efficient killing melanoma cells, being four cell lines highly, two fairly and two not very sensitive to CDU/5-FC (their respective IC50 ranging from 0.20 to 800 µM 5-FC). Considering the relatively low lipofection efficiencies, a very strong bystander effect was verified in the eight cell lines: depending on the cell line, this effect accounted for most of the induced cell death (from 70% to 95%). In our assay conditions, we did not find useful interactions either with the herpes simplex thymidine kinase/ganciclovir suicide system (in sequential or simultaneous modality) or with cisplatin and bleomycin chemotherapeutic drugs. Furthermore, only two cell lines displayed limited useful interactions of the CDU/5-FC either with interferon-ß gene transfer or the proteasome inhibitor bortezomib respectively. These results would preclude a wide use of these combinations. However, the fact that all the tested cells were significantly sensitive to the CDU/5-FC system encourages further research as a gene therapy tool for local control of canine melanoma.

Proliferation index and pseudoprogression as predictors of the therapeutic efficacy of suicide gene therapy for canine melanoma.

In our veterinary clinical trials, the combination of systemic immunotherapy with local herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) treatment induced tumor pseudoprogression as part of a strong local antitumor response. This phenomenon could be owing to tumor inflammation, increased vascular permeability and to different tumor growth rates before, during and after SG therapy. The proliferation index (PI: the fraction of viable cells in S, G2/M, and hyperdiploid phases) would reflect the in-vivo and in-vitro proportion of proliferating melanoma cells in the absence of treatment (PIB) or in response to SG (PISG). The extent of in-vivo and in-vitro melanoma cells responses to SG exhibited a reverse correlation with PIB and a direct correlation with PISG. Then, the final SG outcome depended on the balance between PIB-dependent 'regrowth resistance' versus 'regrowth sensitivity' to SG treatment. In all the cell lines derived from canine tumors presenting partial responses to SG treatment, PISG prevailed over PIB. Conversely, as more aggressive was the tumor (greater PIB of the cell line), the more the balance displacement towards 'regrowth resistance' over SG 'regrowth sensitivity'. All these parameters could have a prognostic value for SG treatment response and provide a glimpse at the clinical benefit of this therapy.

Mechanisms Enhancing the Cytotoxic Effects of Bleomycin plus Suicide or Interferon-ß Gene Lipofection in Metastatic Human Melanoma Cells.

BACKGROUND: Three metastatic human melanoma cell lines generated from patient removed lymph nodes and spleen metastasis were established in our laboratory. OBJECTIVE: To investigate the mechanisms enhancing the cytotoxic effects of Bleomycin (BLM), herpes simplex virus thymidine kinase/ganciclovir Suicide Gene (SG) and human interferon-ß gene (hIFNß) lipofection in early passages of these melanoma cell lines. METHODS: In these cell lines, we determined: cytotoxicity, bystander effect, lipofection efficiencies, apoptosis, necrosis, senescence, colony forming capacity and mitochondrial membrane depolarization after treatments. RESULTS: The three assayed cell lines displayed sensitivity to single and combined BLM/gene treatments. BLM improved the antitumor and anti-clonogenic effects of SG and hIFNß genes. Considering the low lipofection efficiencies (<10%), one of the main causes of the SG and hIFNß gene effectiveness was their bystander effect. In one of these cell lines, this effect eradicated up to 60% of the cells although <1% expressed the transgene. In the three cell lines, BLM alone or combined with SG or hIFNß gene significantly increased the percentage of cells exhibiting membrane compromise, DNA damage, and senescence. Interestingly, the strong BLM/hIFNß gene combination was able to generate from 73% to 98% of non-viable cells. The high proportion of senescent cells induced by BLM alone or combined with genes strongly decreased the clonogenic capacity of surviving cells. CONCLUSION: The presented results indicate that BLM improves the antitumor effects of SG and hIFNß transgene expression. Altogether, these findings strongly support the clinical potential of these combined approaches.

Combination of Suicide and Cytokine Gene Therapies as Surgery Adjuvant for Canine Mammary Carcinoma.

The incidence of canine mammary carcinoma varies with age, breed, and spay status, being among the main tumors appearing in intact female dogs. Thirty-six canine mammary carcinoma patients received injections of canine interferon-ß (cIFN-ß) and HSV-thymidine kinase/ganciclovir (HSV-tk/GCV) carrying lipoplexes, into the tumor bed, immediately after surgery. Next, they started periodic subcutaneous injections of lipoplexes carrying a human granulocyte-macrophage colony stimulating factor and interleukin-2 mixed with allogeneic mammary carcinoma extracts. This combined strategy was safe and well tolerated. In addition, only two out of 26 patients treated with complete surgery developed a local relapse, and 0 out of 29 stage II and III patients displayed distant metastases, suggesting both local and systemic antitumor activities. The most encouraging result was the long survival times: 22 > 1 year (where 13 > 2 and 4 > 3 years), while maintaining a good quality of life. The preliminary results in five patients presenting with local disease, an additional HSV-tk/GCV plus cIFN-ß gene treatment induced local antitumor activity, evidenced by four objective responses (one complete, three partial) and one stable disease. This successful outcome supports further studies to validate this approach not only for canine veterinary patients, but also for translation to human patients.

Recent clinical trials of cancer immunogene therapy in companion animals.

This mini-review presents the results of veterinary clinical trials on immunogene therapy published from 2014 to 2016. A variety of tumors, among them melanoma (canine and equine), mastocytoma (canine), mammary adenocarcinoma (canine) and fibrosarcoma (feline) were treated by using diverse strategies. Non-viral vectors were usually employed to transfer genes of cytokines, suicide enzymes and/or tumor associated antigens. In general terms, minor or no adverse collateral effects were related to these procedures, and treated patients frequently improved their conditions (better quality of life, delayed or suppressed recurrence or metastatic spread, increased survival). Some of these new methodologies have a promising future if applied as adjuvant treatments of standard approaches. The auspicious results, derived from immunogene therapy studies carried out in companion animals, warrant their imperative usage in veterinary clinical oncology. Besides, they provide a strong preclinical basis (safety assays and proofs of concept) for analogous human clinical trials.

Bortezomib Enhances the Antitumor Effects of Interferon-ß Gene Transfer on Melanoma Cells.

BACKGROUND: Malignant melanoma is a fast growing form of skin cancer with increasing global incidence. Clinically, canine malignant melanoma and human melanoma share comparable treatment-resistances, metastatic phenotypes and site selectivity. OBJECTIVE: Both interferon-ß (IFNß) and bortezomib (BTZ) display inhibitory activities on melanoma cells. Here, we evaluated the cytotoxic effects of the combination of BTZ and IFNß gene lipofection on cultured melanoma cell lines. METHOD: Cell viability determined by the acid phosphatase method, cell migration mesasured by the wound healing assay, DNA fragmentation and cell cycle by flow cytometry after propidium iodide staining and reactive oxygen species (ROS) production by H2DCF-DA fluorescence. RESULTS: Four canine mucosal (Ak, Br, Bk and Ol) and two human dermal (A375 and SB2) melanoma cell lines were assayed. BTZ sub-pharmacological concentrations (5 nM) enhanced the cytotoxic effects of IFNß transgene expression on melanoma cells monolayers and spheroids. The combination was also more effective than the single treatments when assayed for clonogenic survival and cell migration. The combined treatment produced a significant raise of apoptosis evidenced by DNA fragmentation as compared to either BTZ or IFNß gene lipofection single treatments. Furthermore, BTZ significantly increased the intracellular ROS generation induced by IFNß gene transfer in melanoma cells, an effect that was reversed by the addition of the ROS inhibitor N-acetyl-L-cystein. CONCLUSION: The present work encourages further studies about the potential of the combination of interferon gene transfer with proteasome inhibitors as a new combined therapy for malignant melanoma, both in veterinary and/or human clinical settings.

Therapeutic potential of bleomycin plus suicide or interferon-ß gene transfer combination for spontaneous feline and canine melanoma.

We originated and characterized melanoma cell lines derived from tumors of two feline and two canine veterinary patients. These lines reestablished the morphology, physiology and cell heterogeneity of their respective parental tumors. We evaluated the cytotoxicity of bleomycin (BLM) alone, or combined with interferon-ß (IFN-ß) or HSVtk/GCV suicide gene (SG) lipofection on these cells. Although the four animals presented stage III disease (WHO system), SG treated feline tumors displayed stable disease in vivo, while the canine ones exhibited partial response. Their derived cell lines reflected this behavior. Feline were significantly more sensitive than canine cells to IFN-ß gene transfer. BLM improved the antitumor effects of both genes. The higher levels of reactive oxygen species (ROS) significantly correlated with membrane and DNA damages, emphasizing ROS intervention in apoptotic and necrotic cell death. After 3 days of BLM alone or combined with gene treatments, the colony forming capacity of two canine and one feline treatments survivor cells almost disappeared. Taken together, these results suggest that the treatments eradicated tumor initiating cells and support the clinical potential of the tested combinations.

The combination of bleomycin with suicide or interferon-ß gene transfer is able to efficiently eliminate human melanoma tumor initiating cells.

We explored the potential of a chemogene therapy combination to eradicate melanoma tumor initiating cells, key producers of recurrence and metastatic spread. Three new human melanoma cell lines, two obtained from lymph nodes and one from spleen metastasis were established and characterized. They were cultured as monolayers and spheroids and, in both spatial configurations they displayed sensitivity to single treatments with bleomycin (BLM) or human interferon-ß (hIFNß) gene or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) lipofection. However, the combination of bleomycin with SG or hIFNß gene transfer displayed greater antitumor efficacy. The three cell lines exhibited a proliferative behavior consistent with melan A and gp100 melanoma antigens expression, and BRAF V600E mutation. BLM and both genetic treatments increased the fraction of more differentiated and treatment-sensitive cells. Simultaneously, they significantly decreased the sub-population of tumor initiating cells. There was a significant correlation between the cytotoxicity of treatments with BLM and gene transfer and the fraction of cells exhibiting (i) high proliferation index, and (ii) high intracellular levels of reactive oxygen species. Conversely, the fraction of cells surviving to our treatments closely paralleled their (i) colony and (ii) melanosphere forming capacity. A very significant finding was that the combination of BLM with SG or hIFNß gene almost abrogated the clonogenic capacity of the surviving cells. Altogether, the results presented here suggest that the combined chemo-gene treatments are able to eradicate tumor initiating cells, encouraging further studies aimed to apply this strategy in the clinic.

Interferon-ß 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-ß (IFNß) gene lipofection on tumor cell lines derived from three human cutaneous and four canine mucosal melanomas. The cytotoxicity of human IFNß gene lipofection resulted higher or equivalent to that of the corresponding addition of the recombinant protein (rhIFNß) to human cells. IFNß gene lipofection was not cytotoxic for only one canine melanoma cell line. When cultured as monolayers, three human and three canine IFNß-lipofected melanoma cell lines displayed a remarkable bystander effect. As spheroids, the same six cell lines were sensitive to IFNß gene transfer, two displaying a significant multicell resistance phenotype. The effects of conditioned IFNß-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 IFNß, 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 IFNß expression could be related to the resistance displayed by one human melanoma cell line. As IFNß 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.

Cytokine-Enhanced Vaccine and Interferon-ß plus Suicide Gene Therapy as Surgery Adjuvant Treatments for Spontaneous Canine Melanoma.

We present here a nonviral immunogene therapy trial for canine malignant melanoma, an aggressive disease displaying significant clinical and histopathological overlapping with human melanoma. As a surgery adjuvant approach, it comprised the co-injection of lipoplexes bearing herpes simplex virus thymidine kinase and canine interferon-ß genes at the time of surgery, combined with the periodic administration of a subcutaneous genetic vaccine composed of tumor extracts and lipoplexes carrying the genes of human interleukin-2 and human granulocyte-macrophage colony-stimulating factor. Following complete surgery (CS), the combined treatment (CT) significantly raised the portion of local disease-free canine patients from 11% to 83% and distant metastases-free (M0) from 44% to 89%, as compared with surgery-only-treated controls (ST). Even after partial surgery (PS), CT better controlled the systemic disease (M0: 82%) than ST (M0: 48%). Moreover, compared with ST, CT caused a significant 7-fold (CS) and 4-fold (PS) rise of overall survival, and >17-fold (CS) and >13-fold (PS) rise of metastasis-free survival. The dramatic increase of PS metastasis-free survival (>1321 days) and CS recurrence- and metastasis-free survival (both >2251 days) demonstrated that CT was shifting a rapidly lethal disease into a chronic one. In conclusion, this surgery adjuvant CT was able of significantly delaying or preventing postsurgical recurrence and distant metastasis, increasing disease-free and overall survival, and maintaining the quality of life. The high number of canine patients involved in CT (301) and the extensive follow-up (>6 years) with minimal or absent toxicity warrant the long-term safety and efficacy of this treatment. This successful clinical outcome justifies attempting a similar scheme for human melanoma.

Herpes simplex virus thymidine kinase/ganciclovir system in multicellular tumor spheroids.

We have developed multicellular spheroids (MCS) established from LM05e and LM3 spontaneous Balb/c-murine mammary adenocarcinoma and B16 C57-murine melanoma derived cell lines as an in vitro model to study the efficacy of the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide system. We demonstrated for the first time that HSVtk-expressing cells assembled as MCS manifested a GCV resistance phenotype compared to the same cells grown as sparse monolayers. HSVtk-expressing LM05e, LM3 and B16 spheroids were 16-, three- and nine-fold less sensitive to GCV than their respective monolayers, even though they could express transgenes 10-, eight- and five-fold more efficiently. Mixed populations of HSVtk- and their respective beta gal-expressing cells displayed a cell-type specific bystander effect that was higher in monolayers than in MCS. However, HSVtk-expressing cells in two- or three-dimensional cultures were always significantly more sensitive to GCV than the beta gal-expressing counterparts, supporting the feasibility of this suicide approach in vivo. We present evidence showing that HSVtk-expressing tumor cells, when transferred from monolayers to MCS, displayed: (i) lower GCV cytotoxic activity and bystander effect; (ii) higher and efficient expression of genes transferred as lipoplexes; (iii) lower cell proliferation rates; and (iv) changes in intracellular Bax/Bcl-xL rheostat of mitochondria-mediated apoptosis.

Suicide plus immune gene therapy prevents post-surgical local relapse and increases overall survival in an aggressive mouse melanoma setting.

In an aggressive B16-F10 murine melanoma model, we evaluated the effectiveness and antitumor mechanisms triggered by a surgery adjuvant treatment that combined a local suicide gene therapy (SG) with a subcutaneous genetic vaccine (Vx) composed of B16-F10 cell extracts and lipoplexes carrying the genes of human interleukin-2 and murine granulocyte and macrophage colony stimulating factor. Pre-surgical SG treatment, neither alone nor combined with Vx was able to slow down the fast evolution of this tumor. After surgery, both SG and SG + Vx treatments, significantly prevented (in 50% of mice) or delayed (in the remaining 50%) post-surgical recurrence, as well as significantly prolonged recurrence-free (SG and SG + Vx) and overall median survival (SG + Vx). The treatment induced the generation of a pseudocapsule wrapping and separating the tumor from surrounding host tissue. Both, SG and the subcutaneous Vx, induced this envelope that was absent in the control group. On the other hand, PET scan imaging of the SG + Vx group suggested the development of an effective systemic immunostimulation that enhanced (18)FDG accrual in the thymus, spleen and vertebral column. When combined with surgery, direct intralesional injection of suicide gene plus distal subcutaneous genetic vaccine displayed efficacy and systemic antitumor immune response without host toxicity. This suggests the potential value of the assayed approach for clinical purposes.

Cationic lipid:DNA complexes allow bleomycin uptake by melanoma cells.

Bleomycin is a chemotherapeutic agent barely diffusible through the plasmatic membrane. We evaluated DNA/cationic lipids complexes (lipoplexes) as mediators of its uptake in four spontaneous canine melanoma derived cell lines (Ak, Bk, Br and Rkb). Cell survival after lipofection plus or minus bleomycin was determined by the acid phosphatase method and the cellular uptake of lipoplexes, carrying the E. coli ß-galactosidase gene, was evidenced by SYBR Green I staining. The four cell lines resulted sensitive to the bleomycin/lipoplexes system in both spatial configurations. Survival rates values were lower than 20% in monolayers of the four tested lines and lower than 30% in three lines (Ak, Bk and Rkb) when grown as spheroids. The sensitization to bleomycin depended on lipoplexes in Ak and Rkb while Bk (in both spatial configurations) and Br (as monolayers) were sensitive to bleomycin alone. Although some degree of sensitivity to bleomycin was induced by cationic lipids alone in Ak and Rkb monolayers, the maximal bleomycin effects appeared in the presence of lipoplexes. The sensitization was independent of transcriptional activity. The co-administration of lipoplexes diminished bleomycin IC50: 10-fold in Ak and Rkb monolayers; and sensitized the Ak and Rkb resistant spheroids. The bleomycin cytotoxic effects depended on lipoplexes concentration and diminished when cells were incubated at 8°C. Our results suggest that lipoplexes sensitize cells to bleomycin, increasing its uptake by an active transport mechanism, such as endocytosis. The bleomycin/lipoplexes system appears as a promising combination of chemotherapy and non-viral cancer gene therapy.

Cytokine-enhanced vaccine and interferon-ß 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-ß 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-ß 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).