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.

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.

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.

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.

Inhibition of bioenergetic metabolism by the combination of metformin and 2-deoxyglucose highly decreases viability of feline mammary carcinoma cells.

Feline mammary carcinoma (FMC) is a highly aggressive pathology that has been proposed as an interesting model of breast cancer disease, especially for the hormone refractory subgroup. Recently, cancer cell metabolism has been described as a hallmark of cancer cells. Here, we investigate the effects and mechanism of metabolic modulation by metformin (MET, anti-diabetic drug), 2-deoxyglucose (2DG, hexokinase inhibitor) or a combination of both drugs, MET/2DG on two established FMC cells lines: AlRB (HER2 (3+) and Ki67<5%) and AlRATN (HER2 (-) and Ki67>15%). We found that treatments significantly decreased both FMC cells viability by up to 80%. AlRB resulted more sensitive to 2DG than AlRATN (IC50: 3.15 vs 6.32mM, respectively). The combination of MET/2DG potentiated the effects of the individually added drugs on FMC cells. In addition, MET/2DG caused an increased in intracellular oxidants, autophagic vesicles and completely inhibited colony formation. Conversely, only MET significantly altered plasma membrane integrity, presented late apoptotic/necrotic cells and increased both glucose consumption and lactate concentration. Our results support further studies to investigate the potential use of this metabolic modulation approach in a clinical veterinary setting.

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.

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.