Biology subjects

Pelletier, R.

Publications and source records attributed to Pelletier, R..

6 recordsLinked to original sources

bioRxiv2025

Mechanisms of tumor persistence in metastatic melanoma following successful immunotherapy

Tumor dormancy is thought to enable cancer recurrence, but the settings and mechanisms of dormancy in patients are poorly characterized. Both immunogenic tumor mass dormancy, involving continued tumor cell proliferation and balanced immune-mediated cell death, and cell-level quiescence have been observed in preclinical models. Here, we demonstrate the existence of mass dormancy rather than quiescence in long-term stable residual lesions in melanoma patients treated with immune checkpoint inhibitors (ICI). In a large stable lesion subjected to detailed spatial profiling, the proportion of proliferating tumor cells is similar to that in site-matched tumors from patients progressing after ICI. Residual stable lesions from other patients, judged to contain only scar tissue upon clinical pathology review, also contained nests of dividing tumor cells surrounded by active immune cells. These findings demonstrate the presence of viable tumor cells and mass dormancy in persistent stable lesions, a finding with implications for disease monitoring and management.

cancer biology↗

bioRxiv2025

Multiomic profiling of a unique in-transit melanoma cohort identifies melanoma differentiation as predictor of tumor progression and therapy response

Melanoma patients with in-transit metastasis (ITM), a stage of disease where melanoma has metastasized to sites in between the primary lesion and draining lymph node, vary significantly in their clinical outcomes, but the biology driving differential outcomes in ITM is poorly understood. To elucidate the mechanisms of differential outcomes, we utilized multimodal molecular profiling (WES, RNA-seq, highly multiplexed immunofluorescence, spatial transcriptomics) in 1) evolutionary analysis of longitudinal tumor samples and 2) identifying prognostic tumor intrinsic and microenvironmental features in a unique cohort of patients with unresectable ITM. Among other findings, we observed a persistent dedifferentiated AXL/NGFR clonal lineage pre-existing and following immune checkpoint blockade in in-transit and distant metastases. Concordantly, we found that low pigmentation and high T cell exhaustion signatures were independently associated with distant progression. Our findings highlight tumor cell state and immune dysfunction as key predictors and potential biomarkers of metastatic risk in ITM. STATEMENT OF SIGNIFICANCEWhat drives distant progression in melanoma is unclear. Analyzing tumor and immune features in a rare in-transit melanoma patient cohort, we identify biological signals highlighting how immune and tumor states observable in pre-distant metastasis melanomas shape long-term outcomes, and nominate potential prognostic biomarkers.

cancer biology↗

bioRxiv2025

Spatial determinants of tumor cell dedifferentiation and plasticity in primary cutaneous melanoma

Localized cutaneous melanoma can be cured by excision but success critically depends on early detection and risk assessment of primary lesions. However, their initiation, progression, and immunology remain poorly understood, partly due to high intra- and inter-tumor heterogeneity. We studied this heterogeneity using spatial profiling in over 300 histological domains, each representing a single progression stage, and found that 200-600 cell neighborhoods from a single melanoma can be as different in RNA and protein expression as neighborhoods from different tumors. These differences are not stochastic, however, and disease progression can be mapped at the neighborhood level onto a cell state landscape defined by the activity of the melanocyte master regulator MITF and genes associated with a dedifferentiated neural crest phenotype. Position in this landscape is influenced by proximity to immune cells, perivascular environments, and other tissue features, but no single association is absolute, giving rise to complex spatial patterns.

cancer biology↗

bioRxiv2025

Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

Primary cultures of Human Hepatocytes (PHH) are the gold standard to investigate drug-hepatotoxicity in vitro, however, large-scale studies using these primary liver cells are not possible because of the shortage in liver biopsies. HepaRG model is often considered as the closest surrogate to PHH for toxicity studies in vitro. However, differentiated HepaRG cells express very low levels of the cytochrome P450 2D6 (CYP2D6) protein, which is essential for the biotransformation of nearly 25% of drugs on the market. To overcome this limitation, infection of progenitor HepaRG cells were performed using lentiviral particles containing a transgene encoding a single mRNA translated into a polypeptide undergoing proteolytic cleavage via the T2A peptide to produce both CYP2D6 and GFP. Differentiated HepaRG cells transduced with lentivirus stably expressed GFP and catalytically active human CYP2D6 enzyme at levels close to those found in high PHH metabolizers. As expected, CYP2D6 protein was found mostly located in the endoplasmic reticulum. Using the CYP2D6 transgenic HepaRG cells, we showed that tramadol was metabolized in both, N- and O-desmethyl tramadol as observed in human serum in contrast with the production of N-desmethyl tramadol only in parental HepaRG cells via the CYP3A4 catalytic activity. Similarly, after perhexiline (PHX) treatments, higher IC50 were found in CYP2D6 expressing HepaRG cells associated to lower mitochondrial damages compared to those found in parental cells for the same PHX concentrations. Gene profiling between parental and transgenic cells demonstrated that the CYP2D6 expressing HepaRG cells had kept their ability to proliferate and differentiate with low impact on the expression of the hepatocyte specific functions. However, we identified a limited set of genes such as NXF3 and TRIM63, which were up-regulated by the mRNA encoded by the lentiviral transgene. Together, these data confirmed that the CYP2D6 transgenic HepaRG cells represent a suitable optimized transgenic model of HepaRG cells to evaluate biotransformation and toxicity of specific compounds metabolized by CYP2D6.

cell biology↗

bioRxiv2024

Lipid-directed covalent fluorescent labeling of plasma membranes for long-term imaging, barcoding and manipulation of cells

Fluorescent probes for cell plasma membrane (PM) are generally based on amphiphilic anchors that incorporate non-covalently into biomembranes. Therefore, they are not compatible with fixation and permeabilization, presence of serum, or cell co-culture because of their exchange with the medium. Here, we report a concept of lipid-directed covalent labeling of PM, which exploits transient binding to lipid membrane surface generating high local dye concentration, thus favoring covalent ligation to random proximal membrane proteins. This concept yielded a class of fluorescent probes for PM (MemGraft), where a cyanine dye (Cy3 and Cy5) bears at its two ends low-affinity membrane anchor and reactive group: an activated ester or a maleimide. We found that MemGraft probes with these reactive groups provide efficient PM labelling, in contrast to a series of control compounds, including commercial Cy3-based labels of amino and thiol groups, revealing the crucial role of the membrane anchor combined with high reactivity of activated ester and a maleimide groups. In contrast to conventional PM probes, based on non-covalent interactions, MemGraft labelling approach is compatible with cell fixation, permeabilization, trypsinization and presence of serum. The latter allows long-term cell tracking and video imaging of cell PM dynamics without signs of phototoxicity. The covalent strategy also enables staining and long-term tracking of co-cultured cells labelled in different colors without probes exchange. Moreover, combination of different ratios of MemGraft-Cy3 and MemGraft-Cy5 probes enabled long-term cell barcoding in at least 5 color codes, important for tracking and visualizing multiple cells populations. Ultimately, we found that MemGraft strategy enables efficient biotinylation of cell surface, opening the path to cell surface engineering and cell manipulation.

biochemistry↗

bioRxiv2024

PRAME expression in melanoma is negatively regulated by TET2-mediated DNA hydroxymethylation

Preferentially Expressed Antigen in Melanoma (PRAME) and Ten-Eleven Translocation (TET) dioxygenase-mediated 5-hydroxymethylcytosine (5hmC) are emerging melanoma biomarkers. We observed an inverse correlation between PRAME expression and 5hmC levels in benign nevi, melanoma in situ, primary invasive melanoma, and metastatic melanomas via immunohistochemistry and multiplex immunofluorescence: nevi exhibited high 5hmC and low PRAME, whereas melanomas showed the opposite pattern. Single-cell multiplex imaging of melanoma precursors revealed that diminished 5hmC coincides with PRAME upregulation in premalignant cells. Analysis of TCGA and GTEx databases confirmed a negative relationship between TET2 and PRAME mRNA expression in melanoma. Additionally, 5hmC levels were reduced at the PRAME 5 promoter in melanoma compared to nevi, suggesting a role for 5hmC in PRAME transcription. Restoring 5hmC levels via TET2 overexpression notably reduced PRAME expression in melanoma cell lines. These findings establish a function of TET2-mediated DNA hydroxymethylation in regulating PRAME expression and demonstrate epigenetic reprogramming as pivotal in melanoma tumorigenesis. TeaserMelanoma biomarker PRAME expression is negatively regulated epigenetically by TET2-mediated DNA hydroxymethylation

cancer biology↗