Non-viral-mediated gene transfer of OX40 ligand for tumor immunotherapy.

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.

Molecules promoting circulating clusters of cancer cells suggest novel therapeutic targets for treatment of metastatic cancers.

Treatment of metastatic disease remains among the most challenging tasks in oncology. One of the early events that predicts a poor prognosis and precedes the development of metastasis is the occurrence of clusters of cancer cells in the blood flow. Moreover, the presence of heterogeneous clusters of cancerous and noncancerous cells in the circulation is even more dangerous. Review of pathological mechanisms and biological molecules directly involved in the formation and pathogenesis of the heterotypic circulating tumor cell (CTC) clusters revealed their common properties, which include increased adhesiveness, combined epithelial-mesenchymal phenotype, CTC-white blood cell interaction, and polyploidy. Several molecules involved in the heterotypic CTC interactions and their metastatic properties, including IL6R, CXCR4 and EPCAM, are targets of approved or experimental anticancer drugs. Accordingly, analysis of patient survival data from the published literature and public datasets revealed that the expression of several molecules affecting the formation of CTC clusters predicts patient survival in multiple cancer types. Thus, targeting of molecules involved in CTC heterotypic interactions might be a valuable strategy for the treatment of metastatic cancers.

Cell-specific expression of the FAP gene is regulated by enhancer elements.

Fibroblast activation protein (FAP) is an integral membrane serine protease that acts as both dipeptidyl peptidase and collagenase. In recent years, FAP has attracted considerable attention due to its specific upregulation in multiple types of tumor cell populations, including cancer cells in various cancer types, making FAP a potential target for therapy. However, relatively few papers pay attention to the mechanisms driving the cell-specific expression of the FAP gene. We found no correlation between the activities of the two FAP promoter variants (short and long) and the endogenous FAP mRNA expression level in several cell lines with different FAP expression levels. This suggested that other mechanisms may be responsible for specific transcriptional regulation of the FAP gene. We analyzed the distribution of known epigenetic and structural chromatin marks in FAP-positive and FAP-negative cell lines and identified two potential enhancer-like elements (E1 and E2) in the FAP gene locus. We confirmed the specific enrichment of H3K27ac in the putative enhancer regions in FAP-expressing cells. Both the elements exhibited enhancer activity independently of each other in the functional test by increasing the activity of the FAP promoter variants to a greater extent in FAP-expressing cell lines than in FAP-negative cell lines. The transcription factors AP-1, CEBPB, and STAT3 may be involved in FAP activation in the tumors. We hypothesized the existence of a positive feedback loop between FAP and STAT3, which may have implications for developing new approaches in cancer therapy.

Efficient Selection of Enhancers and Promoters from MIA PaCa-2 Pancreatic Cancer Cells by ChIP-lentiMPRA.

A library of active genome regulatory elements (putative promoters and enhancers) from MIA PaCa-2 pancreatic adenocarcinoma cells was constructed using a specially designed lentiviral vector and a massive parallel reporter assay (ChIP-lentiMPRA). Chromatin immunoprecipitation of the cell genomic DNA by H3K27ac antibodies was used for primary enrichment of the library for regulatory elements. Totally, 11,264 unique genome regions, many of which are capable of enhancing the expression of the CopGFP reporter gene from the minimal CMV promoter, were identified. The regions tend to be located near promoters. Based on the proximity assay, we found an enrichment of highly expressed genes among those associated with three or more mapped distal regions (2 kb distant from the 5'-ends of genes). It was shown significant enrichment of genes related to carcinogenesis or Mia PaCa-2 cell identity genes in this group. In contrast, genes associated with 1-2 distal regions or only with proximal regions (within 2 kbp of the 5'-ends of genes) are more often related to housekeeping functions. Thus, ChIP-lentiMPRA is a useful strategy for creating libraries of regulatory elements for the study of tumor-specific gene transcription.

Pancreatic Lineage Specifier PDX1 Increases Adhesion and Decreases Motility of Cancer Cells.

Intercellular interactions involving adhesion factors are key operators in cancer progression. In particular, these factors are responsible for facilitating cell migration and metastasis. Strengthening of adhesion between tumor cells and surrounding cells or extracellular matrix (ECM), may provide a way to inhibit tumor cell migration. Recently, we demonstrated that PDX1 ectopic expression results in the reduction of pancreatic cancer line PANC-1 cell motility in vitro and in vivo, and we now provide experimental data confirming the hypothesis that suppression of migration may be related to the effect of PDX1 on cell adhesion. Cell migration analyses demonstrated decreased motility of pancreatic Colo357 and PANC-1 cell lines expressing PDX1. We observed decreased expression levels of genes associated with promoting cell migration and increased expression of genes negatively affecting cell motility. Expression of the EMT regulator genes was only mildly induced in cells expressing PDX1 during the simulation of the epithelial-mesenchymal transition (EMT) by the addition of TGFß1 to the medium. PDX1-expressing cancer cell lines showed increased cell adhesion to collagen type I, fibronectin, and poly-lysine. We conclude that ectopic expression of PDX1 reduces the migration potential of cancer cells, by increasing the adhesive properties of cells and reducing the sensitivity to TGFß1-induced EMT.

Expression of EMT-Related Genes in Hybrid E/M Colorectal Cancer Cells Determines Fibroblast Activation and Collagen Remodeling.

Collagen, the main non-cellular component of the extracellular matrix (ECM), is profoundly reorganized during tumorigenesis and has a strong impact on tumor behavior. The main source of collagen in tumors is cancer-associated fibroblasts. Cancer cells can also participate in the synthesis of ECM; however, the contribution of both types of cells to collagen rearrangements during the tumor progression is far from being clear. Here, we investigated the processes of collagen biosynthesis and remodeling in parallel with the transcriptome changes during cancer cells and fibroblasts interactions. Combining immunofluorescence, RNA sequencing, and second harmonic generation microscopy, we have explored the relationships between the ratio of epithelial (E) and mesenchymal (M) components of hybrid E/M cancer cells, their ability to activate fibroblasts, and the contributions of both cell types to collagen remodeling. To this end, we studied (i) co-cultures of colorectal cancer cells and normal fibroblasts in a collagen matrix, (ii) patient-derived cancer-associated fibroblasts, and (iii) mouse xenograft models. We found that the activation of normal fibroblasts that form dense collagen networks consisting of large, highly oriented fibers depends on the difference in E/M ratio in the cancer cells. The more-epithelial cells activate the fibroblasts more strongly, which correlates with a dense and highly ordered collagen structure in tumors in vivo. The more-mesenchymal cells activate the fibroblasts to a lesser degree; on the other hand, this cell line has a higher innate collagen remodeling capacity. Normal fibroblasts activated by cancer cells contribute to the organization of the extracellular matrix in a way that is favorable for migratory potency. At the same time, in co-culture with epithelial cancer cells, the contribution of fibroblasts to the reorganization of ECM is more pronounced. Therefore, one can expect that targeting the ability of epithelial cancer cells to activate normal fibroblasts may provide a new anticancer therapeutic strategy.

Novel Histone-Based DNA Carrier Targeting Cancer-Associated Fibroblasts.

Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFRß). In this study, we fused histone H2A with PDGFRß-binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFRß-positive and PDGFRß-negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFRß-positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFRß-positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFRß-positive tumor stromal cells.

Construction of a combinatorial library of chimeric tumor-specific promoters.

Gene therapy is a fast-developing field of molecular medicine. New, effective, and cancer-specific promoters are in high demand by researchers seeking to treat cancer through expression of therapeutic genes. Here, we created a combinatorial library of tumor-specific chimeric promoter modules for identifying new promoters with desired functions. The library was constructed by randomly combining promoter fragments from eight human genes involved in cell proliferation control. The pool of chimeric promoters was inserted into a lentiviral expression vector upstream of the CopGFP reporter gene, transduced into A431 cells, and enriched for active promoters by cell sorting. The enriched library contained a remarkably high proportion of active and tumor-specific promoters. This approach to generating combinatorial libraries of chimeric promoters may serve as a useful tool for selecting highly specific and effective promoters for cancer research and gene therapy.

DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity.

BACKGROUND: Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome. RESULTS: Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV-tk) gene in a vector expressing also the neoR gene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations. CONCLUSIONS: We demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.

A new technique for selective identification and mapping of enhancers within long genomic sequences.

We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust and high-throughput approach to the mapping of enhancers. The selection procedure is based on the ability of such sequences to activate a minimal promoter that drives expression of a selective gene. To this end a mixture of short DNA fragments derived from the segment of interest was cloned in a retroviral vector containing the neomycin phosphotransferase II gene under control of a cytomegalovirus (CMV) minimal promoter. The pool of retroviruses obtained was used to infect HeLa cells and then to select neomycin-resistant colonies containing constructs with enhancer-like sequences. The pool of the genomic fragments was rescued by PCR and cloned, forming a library of the potential enhancers. Fifteen enhancer-like fragments were selected from 1-Mb human genome locus, and enhancer activity of 13 of them was verified in a transient transfection reporter gene assay. The sequences selected were found to be predominantly located near 5' regions of genes or within gene introns.