2D culture as a model for drug testing often turns to be clinically futile. Therefore, 3D cultures (3Ds) show potential to better model responses to drugs observed in vivo. In preliminary studies, using melanoma (B16F10) and renal (RenCa) cancer, we confirmed that 3Ds better mimics the tumor microenvironment. Here, we evaluated how the proposed 3D mode of culture affects tumor cell susceptibility to anti-cancer drugs, which have distinct mechanisms of action (everolimus, doxorubicin, cisplatin). Melanoma spheroids showed higher resistance to all used drugs, as compared to 2D. In an RCC model, such modulation was only observed for doxorubicin treatment. As drug distribution was not affected by the 3D shape, we assessed the expression of MDR1 and mTor. Upregulation of MDR1 in RCC spheroids was observed, in contrast to melanoma. In both models, mTor expression was not affected by the 3D cultures. By NGS, 10 genes related with metabolism of xenobiotics by cytochrome p450 were deregulated in renal cancer spheroids; 9 of them were later confirmed in the melanoma model. The differences between 3D models and classical 2D cultures point to the potential to uncover new non-canonical mechanisms to explain drug resistance set by the tumor in its microenvironment.

Compared to other mammalian species, porcine oocytes and embryos are characterized by large amounts of lipids stored mainly in the form of droplets in the cytoplasm. The amount and the morphology of lipid droplets (LD) change throughout the preimplantation development, however, relatively little is known about expression of genes involved in lipid metabolism of early embryos. We compared porcine and bovine blastocyst stage embryos as well as dissected inner cell mass (ICM) and trophoblast (TE) cell populations with regard to lipid droplet storage and expression of genes functionally annotated to selected lipid gene ontology terms using RNA-seq. Comparing the number and the volume occupied by LD between bovine and porcine blastocysts, we have found significant differences both at the level of single embryo and a single blastomere. Aside from different lipid content, we found that embryos regulate the lipid metabolism differentially at the gene expression level. Out of 125 genes, we found 73 to be differentially expressed between entire porcine and bovine blastocyst, and 36 and 51 to be divergent between ICM and TE cell lines. We noticed significant involvement of cholesterol and ganglioside metabolism in preimplantation embryos, as well as a possible shift towards glucose, rather than pyruvate dependence in bovine embryos. A number of genes like DGAT1, CD36 or NR1H3 may serve as lipid associated markers indicating distinct regulatory mechanisms, while upregulated PLIN2, APOA1, SOAT1 indicate significant function during blastocyst formation and cell differentiation in both models.

Renal cell carcinoma (RCC) is one of the most common cancers worldwide with a nearly non-symptomatic course until the advanced stages of the disease. RCC can be distinguished into three subtypes: papillary (pRCC), chromophobe (chRCC) and clear cell renal cell carcinoma (ccRCC) representing up to 75% of all RCC cases. Detection and RCC monitoring tools are limited to standard imaging techniques, in combination with non-RCC specific morphological and biochemical read-outs. RCC subtype identification relays mainly on results of pathological examination of tumor slides. Molecular, clinically applicable and ideally non-invasive tools aiding RCC management are still non-existent, although molecular characterization of RCC is relatively advanced. Hence, many research efforts concentrate on the identification of molecular markers that will assist with RCC sub-classification and monitoring. Due to stability and tissue-specificity miRNAs are promising candidates for such biomarkers. Here, we performed a meta-analysis study, utilized seven NGS and seven microarray RCC studies in order to identify subtype-specific expression of miRNAs. We concentrated on potentially oncocytoma-specific miRNAs (miRNA-424-5p, miRNA-146b-5p, miRNA-183-5p, miRNA-218-5p), pRCC-specific (miRNA-127-3p, miRNA-139-5p) and ccRCC-specific miRNAs (miRNA-200c-3p, miRNA-362-5p, miRNA-363-3p and miRNA-204-5p, 21-5p, miRNA-224-5p, miRNA-155-5p, miRNA-210-3p) and validated their expression in an independent sample set. Additionally, we found ccRCC-specific miRNAs to be differentially expressed in ccRCC tumor according to Fuhrman grades and identified alterations in their isoform composition in tumor tissue. Our results revealed that changes in the expression of selected miRNA might be potentially utilized as a tool aiding ccRCC subclass discrimination and we propose a miRNA panel aiding RCC subtype distinction.

Tumor microenvironments shape aggressiveness and are largely maintained by the conditions of angiogenesis formation. Thus, endothelial cells’ (ECs) biological reactions are crucial to understand and control the design of efficient therapies. In this work, we used models of ECs to represent a breast cancer tumor site as well as the same, healthy tissue. Cells characterization was performed at the transcriptome and protein expression levels, and the cells functional biological responses (angiogenesis and permeability) were assessed. We showed that the expression of proteins specific to ECs (ACE+, VWF+), their differentiation (CD31+, CD 133+, CD105+, CD34-), their adhesion properties (ICAM-1+, VCAM-1+, CD62-L+), and their barrier formation (ZO-1+) were all downregulated in tumor-derived ECs. NGS-based differential transcriptome analysis confirmed CD31-lowered expression and pointed to the increase of Ephrin-B2 and SNCAIP, indicative of dedifferentiation. Functional assays confirmed these differences; angiogenesis was impaired while permeability increased in tumor-derived ECs, as further validated by the distinctly enhanced VEGF production in response to hypoxia, reflecting the tumor conditions. This work showed that endothelial cells differed highly significantly, both phenotypically and functionally, in the tumor site as compared to the normal corresponding tissue, thus influencing the tumor microenvironment.

LINE-1 (L1) retrotransposons are mobile genetic elements that create new genomic insertions by a copy-paste mechanism involving L1 RNA/RNP intermediates. L1 encodes two ORFs, of which L1-ORF2p nicks genomic DNA and reverse transcribes L1 mRNA using the nicked DNA as a primer which base-pairs with poly(A) tail of L1 mRNA. To better understand the importance of non-templated L1 3′ ends’ dynamics and the interplay between L1 3′ and 5′ ends, we investigated the effects of genomic knock-outs and temporal knock-downs of XRN1, DCP2, and other factors. We hypothesized that in the absence of XRN1, the major 5′→3′ exoribonuclease, there would be more L1 mRNA and retrotransposition. Conversely, we observed that loss of XRN1 decreased L1 retrotransposition. This occurred despite slight stabilization of L1 mRNA, but with decreased L1 RNP formation. Similarly, loss of DCP2, the catalytic subunit of the decapping complex, lowered retrotransposition despite increased steady-state levels of L1 proteins. In both XRN1 and DCP2 depletions we observed shortening of L1 3′ poly(A) tails and their increased uridylation by TUT4/7. We explain the observed reduction of L1 retrotransposition by the changed qualities of non-templated L1 mRNA 3′ ends demonstrating the important role of L1 3′ end dynamics in L1 biology.

MicroRNAs are post-transcriptional regulators of gene expression, and disturbances of their expression are the basis of many pathological states, including cancers. The miRNA pattern in the context of tumor microenvironment explains mechanisms related to cancer progression and provides a potential target of modern therapies. Here we show the miRNA pattern in renal cancer focusing on hypoxia as a characteristic feature of the tumor microenvironment and dysregulation of PTEN, being a major tumor suppressor. Methods comprised the CRSPR/Cas9 mediated PTEN knockout in the Renca kidney cancer cell line and global miRNA expression analysis in both in vivo and in vitro (in normoxic and hypoxic conditions). The results were validated on human cancer models with distinct PTEN status. The increase in miR-210-3p in hypoxia was universal; however, the hypoxia-induced decrease in PTEN was associated with an increase in miR-221-3p, the loss of PTEN affected the response to hypoxia differently by decreasing miR-10b-5p and increasing miR-206-3p. In turn, the complete loss of PTEN induces miR-155-5p, miR-100-5p. Upregulation of miR-342-3p in knockout PTEN occurred in the context of the whole tumor microenvironment. Thus, effective identification of miRNA patterns in cancers must consider the specificity of the tumor microenvironment together with the mutations of key suppressors.