Evidence for rRNA 2'-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes.

Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

Protocol for real-time monitoring of CD8+ T and myeloid cell behavior in human high-grade serous ovarian cancer slices.

Studying cell behavior in live human tumors is crucial to understand and improve response to immunotherapies. Here, we present a protocol to slice human ovarian tumors ex vivo, maintain their viability for 24 h, and monitor the behavior of CD8+ T and myeloid cells in real time. Furthermore, we detail procedures to semi-automatically analyze cell movements and aggregate and process behavior data. This protocol can potentially be applied for multiple tumor types and mouse cancer models. For complete details on the use and execution of this protocol, please refer to Laforets et al.1.

Human 3D Ovarian Cancer Models Reveal Malignant Cell-Intrinsic and -Extrinsic Factors That Influence CAR T-cell Activity.

In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell-intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFß-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell-cell and cell-matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.

Semi-supervised analysis of myeloid and T cell behavior in ex vivo ovarian tumor slices reveals changes in cell motility after treatments.

Studies of the high-grade serous ovarian cancer (HGSOC) tumor microenvironment, the most lethal gynecological cancer, aim to enhance the efficiency of established therapies. Cell motility is an important process of anti-tumor response. Using ex vivo human and mouse HGSOC tumor slices combined with time-lapse imaging, we assessed the motility of CD8+ T and myeloid cells. We developed a semi-supervised analysis of cell movements, identifying four cell behaviors: migrating, long migrating, static, and wobbling. Tumor slices were maintained 24h ex vivo, retaining viability and cell movements. Ex vivo treatments with lipopolysaccharide altered CD8+ T and myeloid cell behavior. In vivo chemotherapy reduced ex vivo cell movements in human and mouse tumors and differentially affected CD8+ T and myeloid cells in chemo-sensitive and chemo-resistant mouse models. Ex vivo tumor slices can extend in vivo mouse studies to human, providing a stepping stone to translate mouse cancer studies to clinical trials.

Immune Mechanisms of Resistance to Cediranib in Ovarian Cancer.

This article investigates mechanisms of resistance to the VEGF receptor inhibitor cediranib in high-grade serous ovarian cancer (HGSOC), and defines rational combination therapies. We used three different syngeneic orthotopic mouse HGSOC models that replicated the human tumor microenvironment (TME). After 4 to 5 weeks treatment of established tumors, cediranib had antitumor activity with increased tumor T-cell infiltrates and alterations in myeloid cells. However, continued cediranib treatment did not change overall survival or the immune microenvironment in two of the three models. Moreover, treated mice developed additional peritoneal metastases not seen in controls. Cediranib-resistant tumors had intrinsically high levels of IL6 and JAK/STAT signaling and treatment increased endothelial STAT3 activation. Combination of cediranib with a murine anti-IL6 antibody was superior to monotherapy, increasing mouse survival, reducing blood vessel density, and pSTAT3, with increased T-cell infiltrates in both models. In a third HGSOC model, that had lower inherent IL6 JAK/STAT3 signaling in the TME but high programmed cell death protein 1 (PD-1) signaling, long-term cediranib treatment significantly increased overall survival. When the mice eventually relapsed, pSTAT3 was still reduced in the tumors but there were high levels of immune cell PD-1 and Programmed death-ligand 1. Combining cediranib with an anti-PD-1 antibody was superior to monotherapy in this model, increasing T cells and decreasing blood vessel densities. Bioinformatics analysis of two human HGSOC transcriptional datasets revealed distinct clusters of tumors with IL6 and PD-1 pathway expression patterns that replicated the mouse tumors. Combination of anti-IL6 or anti-PD-1 in these patients may increase activity of VEGFR inhibitors and prolong disease-free survival.

The Tumor Microenvironment of Clear-Cell Ovarian Cancer.

Some patients with advanced clear-cell ovarian cancer (CCOC) respond to immunotherapy; however, little is known about the tumor microenvironment (TME) of this relatively rare disease. Here, we describe a comprehensive quantitative and topographical analysis of biopsies from 45 patients, 9 with Federation Internationale des Gynaecologistes et Obstetristes (FIGO) stage I/II (early CCOC) and 36 with FIGO stage III/IV (advanced CCOC). We investigated 14 immune cell phenotype markers, PD-1 and ligands, and collagen structure and texture. We interrogated a microarray data set from a second cohort of 29 patients and compared the TMEs of ARID1A-wildtype (ARID1Awt) versus ARID1A-mutant (ARID1Amut) disease. We found significant variations in immune cell frequency and phenotype, checkpoint expression, and collagen matrix between the malignant cell area (MCA), leading edge (LE), and stroma. The MCA had the largest population of CD138+ plasma cells, the LE had more CD20+ B cells and T cells, whereas the stroma had more mast cells and αSMA+ fibroblasts. PD-L2 was expressed predominantly on malignant cells and was the dominant PD-1 ligand. Compared with early CCOC, advanced-stage disease had significantly more fibroblasts and a more complex collagen matrix, with microarray analysis indicating "TGFß remodeling of the extracellular matrix" as the most significantly enriched pathway. Data showed significant differences in immune cell populations, collagen matrix, and cytokine expression between ARID1Awt and ARID1Amut CCOC, which may reflect different paths of tumorigenesis and the relationship to endometriosis. Increased infiltration of CD8+ T cells within the MCA and CD4+ T cells at the LE and stroma significantly associated with decreased overall survival.

Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance.

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5'-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that "man-made" fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.

Translational reprogramming of colorectal cancer cells induced by 5-fluorouracil through a miRNA-dependent mechanism.

5-Fluorouracil (5-FU) is a widely used chemotherapeutic drug in colorectal cancer. Previous studies showed that 5-FU modulates RNA metabolism and mRNA expression. In addition, it has been reported that 5-FU incorporates into the RNAs constituting the translational machinery and that 5-FU affects the amount of some mRNAs associated with ribosomes. However, the impact of 5-FU on translational regulation remains unclear. Using translatome profiling, we report that a clinically relevant dose of 5-FU induces a translational reprogramming in colorectal cancer cell lines. Comparison of mRNA distribution between polysomal and non-polysomal fractions in response to 5-FU treatment using microarray quantification identified 313 genes whose translation was selectively regulated. These regulations were mostly stimulatory (91%). Among these genes, we showed that 5-FU increases the mRNA translation of HIVEP2, which encodes a transcription factor whose translation in normal condition is known to be inhibited by mir-155. In response to 5-FU, the expression of mir-155 decreases thus stimulating the translation of HIVEP2 mRNA. Interestingly, the 5-FU-induced increase in specific mRNA translation was associated with reduction of global protein synthesis. Altogether, these findings indicate that 5-FU promotes a translational reprogramming leading to the increased translation of a subset of mRNAs that involves at least for some of them, miRNA-dependent mechanisms. This study supports a still poorly evaluated role of translational control in drug response.