RESUMEN
Background: Immune checkpoint blockade (ICB) is rapidly becoming a standard of care in the treatment of many cancer types. However, the subset of patients who respond to this type of therapy is limited. Another way to promote antitumoral immunity is the use of immunostimulatory molecules, such as cytokines or T cell co-stimulators. The systemic administration of immunotherapeutics leads to significant immune-related adverse events (irAEs), therefore, the localized antitumoral action is needed. One way to achieve this is intratumoral non-viral gene-immune therapy, which allows for prolonged and localized gene expression, and multiple drug administration. In this study, we combined the previously described non-viral gene delivery system, PEG-PEI-TAT copolymer, PPT, with murine OX40L-encoding plasmid DNA. Methods: The resulting OX40L/PPT nanoparticles were characterized via gel mobility assay, dynamic light scattering analysis and in vitro transfection efficiency evaluation. The antitumoral efficacy of intratumorally (i.t.) administered nanoparticles was estimated using subcutaneously (s.c.) implanted CT26 (colon cancer), B16F0 (melanoma) and 4T1 (breast cancer) tumor models. The dynamics of stromal immune cell populations was analyzed using flow cytometry. Weight loss and cachexia were used as irAE indicators. The effect of combination of i.t. OX40L/PPT with intraperitoneal PD-1 ICB was estimated in s.c. CT26 tumor model. Results: The obtained OX40L/PPT nanoparticles had properties applicable for cell transfection and provided OX40L protein expression in vitro in all three investigated cancer models. We observed that OX40L/PPT treatment successfully inhibited tumor growth in B16F0 and CT26 tumor models and showed a tendency to inhibit 4T1 tumor growth. In B16F0 tumor model, OX40L/PPT treatment led to the increase in antitumoral effector NK and T killer cells and to the decrease in pro-tumoral myeloid cells populations within tumor stroma. No irAE signs were observed in all 3 tumor models, which indicates good treatment tolerability in mice. Combining OX40L/PPT with PD-1 ICB significantly improved treatment efficacy in the CT26 subcutaneous colon cancer model, providing protective immunity against CT26 colon cancer cells. Conclusion: Overall, the anti-tumor efficacy observed with OX40L non-viral gene therapy, whether administered alone or in combination with ICB, highlights its potential to revolutionize cancer gene therapy, thus paving the way for unprecedented advancements in the cancer therapy field.
Asunto(s)
Inmunoterapia , Ligando OX40 , Animales , Ligando OX40/genética , Ratones , Inmunoterapia/métodos , Línea Celular Tumoral , Femenino , Terapia Genética/métodos , Nanopartículas , Técnicas de Transferencia de Gen , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microambiente Tumoral/inmunología , Polietileneimina/química , Humanos , Melanoma Experimental/terapia , Melanoma Experimental/inmunología , Polietilenglicoles/químicaRESUMEN
Chemokines are involved in the humoral regulation of body homeostasis. Changes in the blood level of chemokines were found in cancer, atherosclerosis, diabetes, and other systemic diseases. It is essential to distinguish the effects of co-morbid pathologies and cancer on the level of chemokines in the blood. We aimed to analyze, by multiplex cytometry, the levels of chemokines in the blood of healthy young volunteers as well as of intact mice and mice with CT26 colon and Pan02 pancreatic tumors. Two types of chemokines were identified both in human and murine plasmas: homeostatic ones, which were found in high concentrations (>100 pg/mL), and inducible ones, which can be undetectable or determined at very low levels (0−100 pg/mL). There was a high variability in the chemokine levels, both in healthy humans and mice. To analyze chemokine levels during tumor growth, C57BL/6 and BALB/c were inoculated with Pan02 or CT26 tumor cells, accordingly. The tumors significantly differed in the growth and the mortality of mice. However, the blood chemokine levels did not change in tumor-bearing mice until the very late stages. Taken collectively, blood chemokine level is highly variable and reflects in situ homeostasis. Care should be taken when considering chemokines as prognostic parameters or therapeutic targets in cancer.
RESUMEN
BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system. METHODS: We studied the antitumor efficacy of a PEI (polyethylenimine)-PEG (polyethylene glycol) copolymer carrying the HSVtk gene combined in one vector with granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA. The system HSVtk-GM-CSF/PEI-PEG was tested in vitro in various mouse and human cell lines, ex vivo and in vivo using mouse models. RESULTS: We showed that the HSVtk-GM-CSF/PEI-PEG system effectively inhibited the growth of transplanted human and mouse tumors, suppressed metastasis and increased animal lifespan. CONCLUSIONS: We demonstrated that appreciable tumor shrinkage and metastasis inhibition could be achieved with a simple and low toxic chemical carrier - a PEI-PEG copolymer. Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Neoplasias/terapia , Polímeros/química , Timidina Quinasa/genética , Timidina Quinasa/uso terapéutico , Animales , Cationes , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ganciclovir/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Humanos , Sitios Internos de Entrada al Ribosoma/genética , Lípidos , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neoplasias/patología , Polietilenglicoles/química , Polietileneimina/química , Simplexvirus/enzimologíaRESUMEN
For a 140-kb human genome locus, an analysis of the distribution of Dam methylase accessible sites, DNase I sensitive and resistant regions, unmethylated CpG sites and acetylated histone H3 molecules was performed and compared with transcriptional activity of the genes within the locus. A direct correlation was found between the extent of Dam methylation and C5 cytosine (CpG) methylation. It was also demonstrated that promoter regions of all highly and moderately transcribed genes are highly accessible to methylation by Dam methylase. In contrast, promoters of non-transcribed genes showed a very low extent of Dam methylation. Promoter regions of non-transcribed genes were also highly CpG methylated, and the promoter and more distant 5'-regions of the housekeeping gene COX6B1 were substantially CpG-demethylated. Some highly Dam methylase accessible regions are present in the intergenic regions of the locus suggesting that the latter contain either unidentified non-coding transcripts or extended regulatory elements like locus control regions.