Biology subjects

Moyano-Galceran, L.

Publications and source records attributed to Moyano-Galceran, L..

4 recordsLinked to original sources

bioRxiv2025

Generation of an in vitro 3D multicellular culture model of ovarian high-grade serous carcinoma

The development of translational ovarian cancer models to investigate and overcome treatment resistance accounting for the impact of the tumor microenvironment is critical. Here, we present a protocol to establish a multicellular culture model that retains both genetic complexity and the microenvironment of patient tumors, is amenable for molecular and phenotypic analyses, and high throughput drug testing. We describe steps for culturing and characterizing stromal cells derived from cryopreserved and fresh samples and detail procedures for combining them with organoids. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/686515v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@792c12org.highwire.dtl.DTLVardef@13c6680org.highwire.dtl.DTLVardef@6d73adorg.highwire.dtl.DTLVardef@1c56834_HPS_FORMAT_FIGEXP M_FIG C_FIG Before you beginThis protocol describes the in vitro generation of a complex 3D multicellular model (MC), mimicking relevant cellular and extracellular matrix (ECM) interactions in metastatic ovarian high-grade serous carcinoma (HGSC). First, cultures of stromal cells (cancer-associated fibroblasts (CAF), mesothelial cells and adipocytes) are generated. CAF and mesothelial cell cultures are established from fresh tumor tissues and/or from cryopreserved tissue digest and ascites fluid. Next, the identity of the stromal cells is evaluated using relevant markers, and the validated cultures are expanded and cryopreserved. Adipocytes are isolated from fresh tumor tissues and cultured in suspension for a short period before 3D embedding. Finally, previously established patient-derived cancer organoids1 are combined with relevant components of the tumor microenvironment (TME)2, including Type I collagen (main ECM protein in omental metastases) and stromal cells (Figure 1). The resulting MC model, which is viable for at least 14 days, can be used in various downstream applications. Here, we provide detailed protocols for two of them: high throughput drug sensitivity testing and single-cell RNA sequencing (scRNA-seq). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/686515v1_fig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@13c3a1dorg.highwire.dtl.DTLVardef@10b56f6org.highwire.dtl.DTLVardef@17a6e8forg.highwire.dtl.DTLVardef@f09b27_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Overview of the samples and culture conditions used to establish stromal cell cultures, and their integration with patient-derived organoids to generate the MC model. C_FIG

cancer biology↗

bioRxiv2022

Efficacy and safety of glycosphingolipid SSEA-4 targeting CAR-T cells in solid tumors

Chimeric antigen receptor (CAR) T-cell immunotherapies for solid tumors face critical challenges such as heterogeneous antigen expression. We characterized SSEA-4 cell-surface glycolipid as a target for CAR-T cell therapy. SSEA-4 is mainly expressed during embryogenesis but is also found in several cancer types making it an attractive tumor-associated antigen. Anti-SSEA-4 CAR-T cells were generated and assessed pre-clinically in vitro and in vivo for anti-tumor response and safety. SSEA-4 CAR-T cells effectively eliminated SSEA-4 positive cells in all tested cancer cell lines whereas SSEA-4 negative cells lines were not targeted. In vivo efficacy and safety studies using NSG mice and the high-grade serous ovarian cancer cell line OVCAR4 demonstrated a remarkable and specific anti-tumor response at all CAR-T cell doses used. At high T cell doses, CAR-T cell-treated mice showed signs of health deterioration after a follow-up period. However, severity of toxicity was reduced with delayed onset when lower CAR-T cell doses were used. Our data demonstrate the efficacy of anti-SSEA-4 CAR-T therapy; however, safety strategies, such as dose-limiting and/or equipping CAR-T cells with combinatorial antigen recognition should be implemented for its potential clinical translation.

cancer biology↗

bioRxiv2022

Mechanical confinement and DDR1 signalling synergise to regulate collagen-induced apoptosis in rhabdomyosarcoma cells

Fibrillar collagens promote cell proliferation, migration, and survival in various epithelial cancers and are generally associated with tumour aggressiveness. However, the impact of fibrillar collagens on soft tissue sarcoma behaviour remains poorly understood. Unexpectedly, we find here that fibrillar collagen-related gene expression is associated with favourable patient prognosis in rhabdomyosarcoma. By developing and using collagen matrices with distinct stiffness and in vivo-like microarchitectures, we uncover that the activation of DDR1 has pro-apoptotic and integrin {beta}1 pro-survival function, specifically in 3D rhabdomyosarcoma cell cultures. We demonstrate that rhabdomyosarcoma cell-intrinsic or extrinsic matrix remodelling promotes cell survival. Mechanistically, we find that the 3D-specific collagen-induced apoptosis results from a dual DDR1-independent and a synergistic DDR1-dependent TRPV4-mediated response to mechanical confinement. Altogether, our results indicate that dense microfibrillar collagen-rich microenvironments are detrimental to rhabdomyosarcoma cells through an apoptotic response orchestrated by the induction of DDR1 signalling and mechanical confinement. This mechanism helps to explain the preference of rhabdomyosarcoma cells to grow in and metastasise to low fibrillar collagen microenvironments such as the lung.

cancer biology↗

bioRxiv2021

Regulatory FOXP3+ T cells in uterine sarcomas are associated with favorable prognosis, low extracellular matrix expression and reduced YAP activation

PurposeUterine sarcomas are rare but deadly malignancies without effective treatment. The goal of this study was to characterize and identify potential mechanisms underlying observed variations in the immune microenvironment of different sarcoma subtypes, using integrated clinicopathological and molecular methods. Experimental designFifty-eight cases of uterine sarcoma with full clinicopathological annotation were analyzed for their immune landscape in the tumor microenvironment, gene, and protein expression. Cases included leiomyosarcoma (LMS; n=13), low-grade endometrial stromal sarcoma (ESS; n=16), undifferentiated uterine sarcoma (UUS; n=26), and YWHAE-FAM22 translocation-bearing ESS (YFAM; n=3). Image analysis was used to quantify immune cells and immune regulatory proteins. Gene ontology and network enrichment analysis of matching transcriptomic data was used to relate over- and under expressed genes to pathways and further to the immune phenotype and clinicopathological findings. ResultsImmune cell characterization revealed overall prevalence of regulatory T cells and the pro-tumor M2-like macrophages. Cytotoxic T cells were only found in ESS and UUS tumors. Expression of immune regulatory proteins was heterogeneous, with PD-L1 being undetectable. Hierarchical clustering of patients showed four immune signatures independent of tumor type, where infiltration of non-exhausted FOXP3+ cells and M1-like macrophages were associated with greater overall survival. High CD8+/FOXP3+ ratio in UUS and ESS was associated with poor survival and upregulation of extracellular matrix (ECM)-related genes and proteins and YAP nuclear localization. ConclusionsUterine sarcomas present distinct immune signatures with prognostic value, independent of tumor type. This study suggests that the ECM is a potential regulator of the immune microenvironment in uterine sarcomas.

cancer biology↗