Melanoma Spheroids Grown Under Neural Crest Cell Conditions Are Highly Plastic Migratory/Invasive Tumor Cells Endowed with Immunomodulator Function

International audienceBACKGROUND: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. CONCLUSION/SIGNIFICANCE: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability

Yeast Gis2 and its human ortholog CNBP are novel components of stress-induced RNP granules.

Although a CCTG expansion in the gene encoding the zinc knuckle protein CNBP causes a common form of muscular dystrophy, the function of both human CNBP and its putative budding yeast ortholog Gis2 remain poorly understood. Here we report the protein interactions of Gis2 and the subcellular locations of both Gis2 and CNBP. We found that Gis2 exhibits RNA-dependent interactions with two proteins involved in mRNA recognition, the poly(A) binding protein and the translation initiation factor eIF4G. We show that Gis2 is a component of two large RNA-protein granules, processing bodies and stress granules, which contain translationally repressed mRNAs. Consistent with a functional ortholog, CNBP also associates with the poly(A) binding protein and accumulates in stress granules during arsenite treatment of human cells. These results implicate both Gis2 and CNBP in mRNA handling during stress

Gis2 accumulates in P-bodies and stress granules during stationary phase.

<p>(A and B) Yeast strains expressing chromosomal Gis2-mCh and the indicated (A) P-body or (B) stress granule markers were grown for 4 days in glucose-containing media and examined using confocal microscopy. Scale bar, 5 µm.</p

Gis2 associates with proteins involved in translation initiation.

<p>(A) After tandem affinity purification, eluates from an untagged strain and a strain expressing Gis2-TAP were fractionated in a SDS-polyacrylamide gel and proteins visualized by silver staining. Lane 1, molecular size markers. Sizes are in kDa. (B) Lysates of an untagged strain and a strain expressing Gis2-GFP were subjected to immunoprecipitation with anti-GFP antibodies. Prior to immunoprecipitation, Gis2-GFP lysates were incubated with the indicated amounts of RNase A. Proteins in immunoprecipitates were detected by Western blotting with antibodies against Pab1, eIF4G1, and eIF4G2. The efficiency of immunoprecipitation was determined by re-probing with anti-GFP. As a negative control, the blot was reprobed to detect Pgk1. (C) Lysates of untagged and <i>Gis2-(FLAG</i>)<i>₃</i> strains expressing Pab1-GFP, eIF4G1-GFP or eIF4G2-GFP were subjected to immunoprecipitation with anti-GFP antibodies. After Western blotting, Gis2-(FLAG)₃ was detected with anti-FLAG antibodies. To examine immunoprecipitation efficiency, Pab1-GFP, eIF4G1-GFP and eIF4G2-GFP were detected with anti-GFP antibodies. Pgk1 was detected as a negative control.</p

Cycloheximide enhances the rate of disappearance of CNBP-positive foci.

<p>(A and B) To induce P-bodies and stress granules, HeLa cells expressing RFP-DCP1a and RFP-RCK and untransfected cells were incubated with 500 µM arsenite for 30 minutes. Following arsenite treatment, cells were allowed to recover in fresh medium for 45 minutes in the (A) absence or (B) presence of cycloheximide. After fixation, transfected cells were subjected to immunofluorescence to detect CNBP (top and middle panels) or CNBP and TIAR (bottom panels) and examined using confocal microscopy.</p

A small fraction of Gis2 sediments with polyribosomes.

<p>(A and B) <i>GIS2-GFP</i> cell lysates were prepared in the presence (A) or absence (B) of cycloheximide and fractionated in 15–50% sucrose gradients. Fractions were collected while monitoring OD₂₅₄. Proteins were subjected to Western blotting to detect Gis2-GFP, Pab1, eIF4G1, eIF4G2 and ribosomal proteins L1A and L1B. (C and D) <i>GIS2-GFP</i> cell lysates prepared in the presence of cycloheximide were either untreated (C) or incubated with 5 U/µl micrococcal nuclease (D) prior to sedimentation. Fractions from each gradient were analyzed in two gels as indicated by the lines.</p

The Immigrant Partnership and Advocacy Curricular Kit: A Comprehensive Train-the-Trainer Curriculum in Immigrant and Refugee Health.

The number of immigrants and refugees in the United States is growing, yet many trainees and clinicians feel unprepared to manage the diverse needs of this population. This perspective piece describes the development of the Immigrant Partnership and Advocacy Curricular Kit (I-PACK) by the Midwest Consortium of Global Child Health Educators. I-PACK is an adjunct to the Consortiums sugarprep.org global health curricular materials. Using Kerns six-step approach to curriculum development, they developed eight modules in immigrant and refugee health that incorporate interactive learning activities. The I-PACK was launched as an open-access resource in September 2020. As of September 2021, the curriculum has been freely available at sugarprep.org/i-pack and downloaded from educators in 15 countries. The I-PACK curriculum can address a growing need in medical education to empower learners and clinicians to provide competent and compassionate care for immigrants and refugees

Gis2 accumulates in P-bodies and stress granules during glucose depletion.

<p>(A and B) Yeast strains expressing chromosomal Gis2-mCh and (A) the P-body markers Dcp2-GFP and Edc3-GFP or (B) the stress granule markers Pab1-GFP, eIF4G1-GFP, eIF4G2-GFP and Pub1-GFP were grown in glucose-containing media, then resuspended in fresh media that either lacked or contained glucose. After 10 minutes, cells were observed using confocal microscopy. In glucose media (right column), no Gis2-mCh foci were observed; thus only the merged panels are shown. Bars, 5 µm.</p

Polysome profiles following glucose deprivation of yeast cells.

<p>(A–F) Wild-type and the indicated mutant strains were grown in glucose-containing media until early logarithmic phase, pelleted, and resuspended in glucose-containing media (left panels) or in media lacking glucose (right panels) and grown for an additional 10 minutes. Lysates were fractionated in 15–50% sucrose gradients and the positions of ribosomal subunits, monoribosomes and polyribosomes detected by monitoring OD₂₅₄ during collection. (A) wild-type, (B) <i>gis2</i>Δ , (C) <i>pat1</i>Δ, (D) <i>gis2</i>Δ<i>pat1</i>Δ, (E) <i>dhh1</i>Δ, (F) <i>gis2</i>Δ<i>dhh1</i>Δ cells. To ensure reproducibility, each mutant was analyzed at least twice. (G) The P/M ratio was determined for wild-type, <i>gis2</i>Δ, <i>dhh1</i>Δ and <i>gis2</i>Δ <i>dhh1</i>Δ strains as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052824#pone.0052824-Clarkson1" target="_blank">[63]</a> following 10 minutes of glucose depletion. P/M ratios for wild-type and <i>gis2</i>Δ strains were determined from three biological replicates, while the P/M ratios for <i>dhh1</i>Δ and <i>dhh1</i>Δ <i>gis2</i>Δ strains were determined from four replicates. Asterisk, p<.05, two-tailed paired t-test.</p

Natural killer cell crosstalk with allogeneic human cardiac-derived stem/progenitor cells controls persistence

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