A method to culture human alveolar rhabdomyosarcoma cell lines as rhabdospheres demonstrates an enrichment in stemness and notch signaling

Slemmons, Katherine K.; Deel, Michael D.; Lin, Yi-Tzu; Oristian, Kristianne M.; Kuprasertkul, Nina; Genadry, Katia C.; Chen, Po‐Han; Chi, Jen‐Tsan; Linardic, Corinne M.

doi:10.1242/bio.050211

Cited by 4 publications

(5 citation statements)

References 39 publications

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“…Therefore, we hypothesized that FPRMS, contrary to FNRMS but similarly to acute lymphoblastic leukemia and melanoma [48,51], possess a high abundance of cells with tumor initiating potential. Corroborating this hypothesis, expression of the pluripotency genes NANOG, OCT4, and SOX2 was homogeneous in all analyzed monolayer cultures of FPRMS cell lines, as also confirmed by Slemmons et al [49].…”

Section: Discussionsupporting

confidence: 84%

“…Although we could maintain three out of seven FPRMS lines for five weeks under the new established sphere conditions, the observed increase in sphere numbers as well as the upregulation of the core stem cell genes compared to the monolayer cell culture was very moderate and not consistent for all the analyzed cell lines. Recently, Slemmons et al established other sphere culture conditions able to maintain two FPRMS cell lines in suspension [ 49 ]. Although the spheres were not passaged at clonal level, they showed upregulation of NANOG, OCT4, and SOX2 after serial passaging when compared to the monolayer culture, but not with xenograft derived cells.…”

Section: Discussionmentioning

confidence: 99%

“…The sphere medium used by Slemmons et al contained eight times more bFGF and four times more EGF than the medium used in our study. Instead of PDGF they added insulin to the neurobasal media supplemented with B27 [ 49 ]. We consciously used the minimal concentration of growth factors allowing passaging of the spheres in order to exclude a media-dependent induced cell plasticity.…”

Section: Discussionmentioning

confidence: 99%

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High Frequency of Tumor Propagating Cells in Fusion-Positive Rhabdomyosarcoma

Generali

Satheesha

Bode

et al. 2021

Genes

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Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Fusion-positive RMS (FPRMS), expressing the PAX3/7-FOXO1, has a worse prognosis compared to the more common fusion-negative RMS (FNRMS). Although several studies reported hierarchical organization for FNRMS with the identification of cancer stem cells, the cellular organization of FPRMS is not yet clear. In this study we investigated the expression of key stem cell markers, developed a sphere assay, and investigated the seven most common FPRMS cell lines for subpopulations of tumor propagating cancer stem-like cells, also called cancer stem cells (CSCs). Moreover, loss- and gain-of-functions of the stem cell genes SOX2, OCT4, and NANOG were investigated in the same cells. Single-cell clonal analysis was performed in vitro as well as in vivo. We found that no stable CSC subpopulation could be enriched in FPRMS. Unlike depletion of PAX3-FOXO1, neither overexpression nor siRNA-mediated downregulation of SOX2, OCT4, and NANOG affected physiology of RMS cells. Every single subclone-derived cell clone initiated tumor growth in mice, despite displaying considerable heterogeneity in gene expression. FPRMS appears to contain a high frequency of tumor propagating stem-like cells, which could explain their higher propensity for metastasis and relapse. Their dependency on PAX3-FOXO1 activity reinforces the importance of the fusion protein as the key therapeutic target.

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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High Frequency of Tumor Propagating Cells in Fusion-Positive Rhabdomyosarcoma

Generali

Satheesha

Bode

et al. 2021

Genes

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“…Fourth, inhibition of ALDH1A1 expression or activity had effects similar to those of EHMT1 depletion. These observations are consistent with the finding that ARMS tumorspheres (rhabdospheres) display stem cell characteristics [29].…”

Section: Discussionsupporting

confidence: 92%

“…Tumorsphere formation was accomplished by slightly modifying previously described methods [28,29]. In brief, RH41 (25 000 cells per well) or RH30 (20 000 cells per well) cells were grown in neurobasal medium (Gibco, Waltham, MA, USA) enriched with 2X or 1X B27 (Invitrogen), 40 ng/ml bFGF (Invitrogen), 80 ng/ ml EGF (Sigma), and 50 μg/ml insulin (Sigma).…”

Section: Tumorsphere Assaysmentioning

confidence: 99%

EHMT1 promotes tumor progression and maintains stemness by regulating ALDH1A1 expression in alveolar rhabdomyosarcoma

Nachiyappan

Soon

Lim

et al. 2022

The Journal of Pathology

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Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. Cancer stem cells (CSCs) are seeds for tumor relapse and metastasis. However, pathways that maintain stemness genes are not fully understood. Here, we report that the enzyme euchromatic histone lysine methyltransferase 1 (EHMT1) is expressed in primary and relapse ARMS tumors. EHMT1 suppression impaired motility and induced differentiation in ARMS cell lines and reduced tumor progression in a mouse xenograft model in vivo. RNA sequencing of EHMT1-depleted cells revealed downregulation of ALDH1A1 that is associated with CSCs. Consistent with this, inhibition of ALDH1A1 expression and activity mimicked EHMT1 depletion phenotypes and reduced tumorsphere formation. Mechanistically, we demonstrate that EHMT1 does not bind to the ALDH1A1 promoter but activates it by stabilizing C/EBPβ, a known regulator of ALDH1A1 expression. Our findings identify a role for EHMT1 in maintenance of stemness by regulating ALDH1A1 expression and suggest that targeting ALDH + cells is a promising strategy in ARMS.

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HDAC3 genetic and pharmacologic inhibition radiosensitizes fusion positive rhabdomyosarcoma by promoting DNA double-strand breaks

Cassandri,

Porrazzo,

Pomella

et al. 2024

Cell Death Discov.

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Radiotherapy (RT) plays a critical role in the management of rhabdomyosarcoma (RMS), the prevalent soft tissue sarcoma in childhood. The high risk PAX3-FOXO1 fusion-positive subtype (FP-RMS) is often resistant to RT. We have recently demonstrated that inhibition of class-I histone deacetylases (HDACs) radiosensitizes FP-RMS both in vitro and in vivo. However, HDAC inhibitors exhibited limited success on solid tumors in human clinical trials, at least in part due to the presence of off-target effects. Hence, identifying specific HDAC isoforms that can be targeted to radiosensitize FP-RMS is imperative. We, here, found that only HDAC3 silencing, among all class-I HDACs screened by siRNA, radiosensitizes FP-RMS cells by inhibiting colony formation. Thus, we dissected the effects of HDAC3 depletion using CRISPR/Cas9-dependent HDAC3 knock-out (KO) in FP-RMS cells, which resulted in Endoplasmatic Reticulum Stress activation, ERK inactivation, PARP1- and caspase-dependent apoptosis and reduced stemness when combined with irradiation compared to single treatments. HDAC3 loss-of-function increased DNA damage in irradiated cells augmenting H2AX phosphorylation and DNA double-strand breaks (DSBs) and counteracting irradiation-dependent activation of ATM and DNA-Pkcs as well as Rad51 protein induction. Moreover, HDAC3 depletion hampers FP-RMS tumor growth in vivo and maximally inhibits the growth of irradiated tumors compared to single approaches. We, then, developed a new HDAC3 inhibitor, MC4448, which showed specific cell anti-tumor effects and mirrors the radiosensitizing effects of HDAC3 depletion in vitro synergizing with ERKs inhibition. Overall, our findings dissect the pro-survival role of HDAC3 in FP-RMS and suggest HDAC3 genetic or pharmacologic inhibition as a new promising strategy to overcome radioresistance in this tumor.

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A method to culture human alveolar rhabdomyosarcoma cell lines as rhabdospheres demonstrates an enrichment in stemness and notch signaling

Cited by 4 publications

References 39 publications

High Frequency of Tumor Propagating Cells in Fusion-Positive Rhabdomyosarcoma

High Frequency of Tumor Propagating Cells in Fusion-Positive Rhabdomyosarcoma

EHMT1 promotes tumor progression and maintains stemness by regulating ALDH1A1 expression in alveolar rhabdomyosarcoma

HDAC3 genetic and pharmacologic inhibition radiosensitizes fusion positive rhabdomyosarcoma by promoting DNA double-strand breaks

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