using-optical-tweezers-for-the-generation-of-hybrid-spheroids

Using Optical Tweezers for the Generation of Hybrid Spheroids

Two-dimensional (2D) cell culture is still among the most commonly used models in preclinical cancer research. However, the three-dimensional (3D) in ...

Wrocław Medical University

Photodynamic therapy (PDT) is a low-invasive method of treatment of various diseases, mainly neoplastic conditions. PDT has been experimentally combined with multiple treatment methods. In this study, we tested a combination of 5-aminolevulinic acid (5-ALA) mediated PDT with thalidomide (TMD), which is a drug presently used in the treatment of plasma cell myeloma. TMD and PDT share similar modes of action in neoplastic conditions. Using 4T1 murine breast carcinoma and 2H11 murine endothelial cells lines as an experimental tumor model, we tested 5-ALA-PDT and TMD combination in terms of cytotoxicity, apoptosis, Vascular Endothelial Growth Factor (VEGF) expression, and, in 2H11 cells, migration capabilities by wound healing assay. We have found an enhancement of cytotoxicity in 4T1 cells, whereas, in normal 2H11 cells, this effect was not statistically significant. The addition of TMD decreased the production of VEGF after PDT in 2H11 cell line. Our results reveal enhanced effectiveness of 5-ALA-PDT with TMD treatment compared to 5-ALA-PDT or TMD treatment alone. The addition of TMD may be a promising proceeding of the anti-tumor effect of PDT by decreasing the VEGF concentration in the culture medium. Further studies, including testing on different cell lines, are needed to confirm this assumption.

The Assessment of the Combined Treatment of 5-ALA Mediated Photodynamic Therapy and Thalidomide on 4T1 Breast Carcinoma and 2H11 Endothelial Cell Line.

Hypoxia is a common feature in most tumors, including hematological malignancies. There is a lack of studies on hypoxia- and physioxia-induced global proteome changes in lymphoma. Here, we sought to explore how the proteome of diffuse large B-cell lymphoma (DLBCL) changes when cells are exposed to acute hypoxic stress (1% of O) and physioxia (5% of O) for a long-time. A total of 8239 proteins were identified by LC-MS/MS, of which 718, 513, and 486 had significant changes, in abundance, in the Ri-1, U2904, and U2932 cell lines, respectively. We observed that changes in B-NHL proteome profiles induced by hypoxia and physioxia were quantitatively similar in each cell line; however, differentially abundant proteins (DAPs) were specific to a certain cell line. A significant downregulation of several ribosome proteins indicated a translational inhibition of new ribosome protein synthesis in hypoxia, what was confirmed in a pathway enrichment analysis. In addition, downregulated proteins highlighted the altered cell cycle, metabolism, and interferon signaling. As expected, the enrichment of upregulated proteins revealed terms related to metabolism, HIF1 signaling, and response to oxidative stress. In accordance to our results, physioxia induced weaker changes in the protein abundance when compared to those induced by hypoxia. Our data provide new evidence for understanding mechanisms by which DLBCL cells respond to a variable oxygen level. Furthermore, this study reveals multiple hypoxia-responsive proteins showing an altered abundance in hypoxic and physioxic DLBCL. It remains to be investigated whether changes in the proteomes of DLBCL under normoxia and physioxia have functional consequences on lymphoma development and progression.

Proteomic-Based Analysis of Hypoxia- and Physioxia-Responsive Proteins and Pathways in Diffuse Large B-Cell Lymphoma.

B-cell non-Hodgkin lymphomas (B-NHLs) are the most common lymphoproliferative malignancy. Despite targeted therapies, the bone marrow involvement remains a challenge in treating aggressive B-NHLs, partly due to the protective interactions of lymphoma cells with mesenchymal stromal cells (MSCs). However, data elucidating the relationship between MSCs and B-NHLs are limited and inconclusive due to the lack of reproducible in vitro three-dimensional (3D) models. Here, we developed and described a size-controlled and stable 3D hybrid spheroids of Ri-1 (diffuse large B-cell lymphoma, DLBCL) and RAJI (Burkitt lymphoma, BL) cells with HS-5 fibroblasts to facilitate research on the crosstalk between B-NHL cells and MSCs.

Development and Characterization of 3D Hybrid Spheroids for the Investigation of the Crosstalk Between B-Cell Non-Hodgkin Lymphomas and Mesenchymal Stromal Cells.

Interactions between stromal and lymphoma cells in the bone marrow are closely related to drug resistance and therapy failure. Physiologically relevant pre-clinical three-dimensional (3D) models recapitulating lymphoma microenvironmental complexity do not currently exist. In this study, we proposed a scheme for optically controlled hybrid lymphoma spheroid formation with the use of optical tweezers (OT). Following the preparation of stromal spheroids using agarose hydrogel, two aggressive non-Hodgkin lymphoma B-cell lines, Ri-1 (DLBCL) and Raji (Burkitt lymphoma), were used to conduct multi-cellular spheroid formation driven by in-house-developed fluorescence optical tweezers. Importantly, the newly formed hybrid spheroid preserved the 3D architecture for the next 24 h. Our model was successfully used for the evaluation of the influence of the anticancer agents doxorubicin (DOX), ibrutinib (IBR), and AMD3100 (plerixafor) on the adhesive properties of lymphoma cells. Importantly, our study revealed that a co-treatment of DOX and IBR with AMD3100 affects the adhesion of B-NHL lymphoma cells.

Formation of Lymphoma Hybrid Spheroids and Drug Testing in Real Time with the Use of Fluorescence Optical Tweezers.

 Acute myeloid leukemia (AML) is a heterogeneous disease highly resistant to chemotherapeutic agents. Leukemia stem cells (LSCs) can enter a dormant state and avoid apoptosis in the protective niche of the bone marrow (BM) microenvironment. Moreover, bone marrow stromal cells protect leukemia cells by promoting pro-survival signaling pathways and drug resistance. Therefore, attenuating interactions between leukemia cells and BM cells may have a positive therapeutic effect.  In this work, we hypothesized that sondages may inhibit the adhesion of leukemia cells to the bone marrow by inhibiting the Hedgehog (Hh) signaling pathway. The Hedgehog pathway is a key therapeutic target in AML due to its role in leukemic cell growth and survival.  We investigated the effects of sonidegib on the adhesion of individual OCI-AML3 cells to a bone marrow stromal spheroid derived from the HS-5 cell line. For this purpose, we precisely determined the minimum cell-to-cell adhesion time using optical tweezers under normoxic (21% of O) and hypoxic (1% of O) conditions.  Our results demonstrated that sonidegib significantly increased the minimum cell-to-cell adhesion time necessary for leukemic cells to establish adhesive bonds with bone marrow stromal cells, thereby indicating a reduction in their adhesive properties. Additionally, we showed that sonidegib is particularly effective at hypoxic oxygen concentrations.  The results obtained in this study suggest that sonidegib, through its modulation of the Hedgehog signaling pathway, holds promise as a potential therapeutic approach to target leukemic cell adhesion within the bone marrow microenvironment.

Article	Year
The Assessment of the Combined Treatment of 5-ALA Mediated Photodynamic Therapy and Thalidomide on 4T1 Breast Carcinoma and 2H11 Endothelial Cell Line. Molecules (Basel, Switzerland)\| PubMed ID: 33171718	2020
Proteomic-Based Analysis of Hypoxia- and Physioxia-Responsive Proteins and Pathways in Diffuse Large B-Cell Lymphoma. Cells\| PubMed ID: 34440794	2021
Development and Characterization of 3D Hybrid Spheroids for the Investigation of the Crosstalk Between B-Cell Non-Hodgkin Lymphomas and Mesenchymal Stromal Cells. OncoTargets and therapy\| PubMed ID: 35747403	2022
Formation of Lymphoma Hybrid Spheroids and Drug Testing in Real Time with the Use of Fluorescence Optical Tweezers. Cells\| PubMed ID: 35805197	2022
Sonidegib Inhibits the Adhesion of Acute Myeloid Leukemia to the Bone Marrow in Hypoxia: An Optical Tweezer Study. Biomedicines\| PubMed ID: 40149555	2025

Katarzyna Gdesz-Birula

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