scholarly journals Multicellular tumor spheroids- an effective in vitro model for understanding drug resistance in head and neck cancer

2019 ◽  
Author(s):  
Mohammad Azharuddin ◽  
Karin Roberg ◽  
Ashis Kumar Dhara ◽  
Mayur Vilas Jain ◽  
Padraig D’arcy ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Head And Neck ◽  
Cancer Cells ◽  
Present Report ◽  
Treatment Success ◽  
Integrated Approach ◽  
Drug Uptake ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids

AbstractA hallmark of cancer is the ability to develop resistance against therapeutic agents. Therefore, developing effective in vitro strategies to identify drug resistance remains of paramount importance for treatment success. A way cancer cells achieve drug resistance is through the expression of efflux pumps that actively pump drugs out of the cells. To date, several studies have investigated the potential of using 3D multicellular tumor spheroids (MCSs) to assess drug resistance; however, a unified system that uses MCSs to differentiate between multi drug resistant (MDR) and non-MDR cells does not exist. In the present report, we have used MCSs obtained from post-diagnosed, pre-treated (PDPT) patient derived head and neck squamous cancer cells that often become treatment resistant, to develop an integrated approach combining clinical drug response and cytotoxicity screening, real-time drug uptake monitoring, and drug transporter activity assessment using flow cytometry in the presence and absence of their respective specific inhibitors. The present report shows a comparative response to MDR, drug efflux capability, and reactive oxygen species (ROS) activity to assess the resistance profile of PDPT patient-derived MCSs and two-dimensional cultures of the same set of cells. We show that MCSs serve as robust and reliable models for the clinical evaluation of drug resistance. Our proposed strategy can thus have potential clinical applicability for profiling drug resistance in cancers with unknown resistance profiles, which consequentially can indicate benefit from downstream therapy.

scholarly journals Dissecting multi drug resistance in head and neck cancer cells using multicellular tumor spheroids

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mohammad Azharuddin ◽  
Karin Roberg ◽  
Ashis Kumar Dhara ◽  
Mayur Vilas Jain ◽  
Padraig Darcy ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Head And Neck ◽  
Cancer Cells ◽  
Cell Lines ◽  
Treated Patient ◽  
Drug Uptake ◽  
Multi Drug Resistance ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids

AbstractOne of the hallmarks of cancers is their ability to develop resistance against therapeutic agents. Therefore, developing effective in vitro strategies to identify drug resistance remains of paramount importance for successful treatment. One of the ways cancer cells achieve drug resistance is through the expression of efflux pumps that actively pump drugs out of the cells. To date, several studies have investigated the potential of using 3-dimensional (3D) multicellular tumor spheroids (MCSs) to assess drug resistance; however, a unified system that uses MCSs to differentiate between multi drug resistance (MDR) and non-MDR cells does not yet exist. In the present report we describe MCSs obtained from post-diagnosed, pre-treated patient-derived (PTPD) cell lines from head and neck squamous cancer cells (HNSCC) that often develop resistance to therapy. We employed an integrated approach combining response to clinical drugs and screening cytotoxicity, monitoring real-time drug uptake, and assessing transporter activity using flow cytometry in the presence and absence of their respective specific inhibitors. The report shows a comparative response to MDR, drug efflux capability and reactive oxygen species (ROS) activity to assess the resistance profile of PTPD MCSs and two-dimensional (2D) monolayer cultures of the same set of cell lines. We show that MCSs provide a robust and reliable in vitro model to evaluate clinical relevance. Our proposed strategy can also be clinically applicable for profiling drug resistance in cancers with unknown resistance profiles, which consequently can indicate benefit from downstream therapy.

scholarly journals A Simple Three-Dimensional In Vitro Culture Mimicking the In Vivo-Like Cell Behavior of Bladder Patient-Derived Xenograft Models

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1304
Author(s):  
Robson Amaral ◽  
Maike Zimmermann ◽  
Ai-Hong Ma ◽  
Hongyong Zhang ◽  
Kamilla Swiech ◽  
...  
Keyword(s):  
Drug Resistance ◽  
In Vitro Culture ◽  
Cancer Cells ◽  
Low Cost ◽  
Maintenance Cost ◽  
Tumor Spheroids ◽  
Patient Derived Xenograft ◽  
Pdx Models

Patient-derived xenograft (PDX) models allow for personalized drug selection and the identification of drug resistance mechanisms in cancer cells. However, PDX models present technical disadvantages, such as long engraftment time, low success rate, and high maintenance cost. On the other hand, tumor spheroids are emerging as an in vitro alternative model that can maintain the phenotype of cancer cells long enough to perform all assays and predict a patient’s outcome. The present work aimed to describe a simple, reproducible, and low-cost 3D in vitro culture method to generate bladder tumor spheroids using human cells from PDX mice. Cancer cells from PDX BL0293 and BL0808 models, previously established from advanced bladder cancer, were cultured in 96-well round-bottom ultra-low attachment (ULA) plates with 5% Matrigel and generated regular and round-shaped spheroids (roundness > 0.8) with a diameter larger than 400 μm and a hypoxic core (a feature related to drug resistance in solid tumors). The responses of the tumor spheroids to the antineoplastic drugs cisplatin, gemcitabine, and their combination were similar to tumor responses in in vivo studies with PDX BL0293 and BL0808 mice. Therefore, the in vitro 3D model using PDX tumor spheroids appears as a valuable tool that may predict the outcome of in vivo drug-screening assays and represents a low-cost strategy for such purpose.

scholarly journals Confocal Microscopy in Conjunction With Haemocytometry to Evaluate the Imperative Physical Characteristics of Multicellular Tumor Spheroids

2021 ◽  
Author(s):  
Aziz UR RAHMAN
Keyword(s):  
Confocal Microscopy ◽  
Drug Uptake ◽  
Live Cells ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Oriented Growth ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Method

Abstract Background: Tumor tissues resist penetration of therapeutic molecules. Multicellular tumor spheroids (MCTSs) were used as an in vitro tumor model. The aim of this study was to determine the growth of MCTSs with the age of spheroids, which could be applied and compared with in vivo drug uptake and penetration. Method: Spheroids were generated by liquid overlay techniques, and their diameter was measured by confocal microscopy for up to two weeks. The trypan blue exclusion method was used to count dead and live cells separately via a hemocytometer. Results: The pentaphysical characteristics of spheroids, including diameter, cell number, volume per cell, viability status, and estimated shell of viable and core of dead cells, were determined. The growth of spheroids was linear over the first week but declined in the 2nd week, which may be due to an overconcentration of dead cells and degraded products inside the spheroids, hence lowering the ratio of live cells in spheroids. Compaction of spheroids occurs from day 3 to day 7, with the mature spheroids having a low amount of extracellular space compared to intracellular volume. Conclusion: Age-oriented growth of MCTSs provides a rationale to predict less rapid penetration as spheroids get older and could be correlated with in vivo tumors to predict pharmaceutical and therapeutic intervention.

scholarly journals Single cell organization and cell cycle characterization of DNA stained multicellular tumor spheroids

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karl Olofsson ◽  
Valentina Carannante ◽  
Madoka Takai ◽  
Björn Önfelt ◽  
Martin Wiklund
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Drug Distribution ◽  
In Vitro Models ◽  
Biological Information ◽  
Dimensional Structure ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Nuclear Segmentation

AbstractMulticellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.

scholarly journals Ionizing radiation affects the composition of the proteome of extracellular vesicles released by head-and-neck cancer cells in vitro

2019 ◽  
Vol 60 (3) ◽  
pp. 289-297 ◽  
Author(s):  
Agata Abramowicz ◽  
Anna Wojakowska ◽  
Lukasz Marczak ◽  
Malgorzata Lysek-Gladysinska ◽  
Mateusz Smolarz ◽  
...  
Keyword(s):  
Head And Neck Cancer ◽  
Ionizing Radiation ◽  
Head And Neck ◽  
Cancer Cells ◽  
Extracellular Vesicles ◽  
Neck Cancer

scholarly journals The Effect of Tau and Taxol on Polymerization of MCF7 Microtubules In Vitro

2022 ◽  
Vol 23 (2) ◽  
pp. 677
Author(s):  
Mitra Shojania Feizabadi ◽  
Venise Jan Castillon
Keyword(s):  
Breast Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Tau Protein ◽  
Molecular Motors ◽  
Resistance Mechanism ◽  
Underlying Mechanism ◽  
Beta Tubulin ◽  
Associated Proteins

Overexpression of Tau protein in breast cancer cells is identified as an indicator for potential resistance to taxane-based therapy. As reported findings have been obtained mostly from clinical studies, the undetermined underlying mechanism of such drug resistance needs to be thoroughly explored through comprehensive in vitro evaluations. Tau and Taxol bind to the beta tubulin site in microtubules’ structure. This is of particular interest in breast cancer, as microtubules of these cancer cells are structurally distinct from some other microtubules, such as neuronal microtubules, due to their unique beta tubulin isotype distribution. The observed changes in the in vitro polymerization of breast cancer microtubules, and the different function of some molecular motors along them, leave open the possibility that the drug resistance mechanism can potentially be associated with different responses of these microtubules to Tau and Taxol. We carried out a series of parallel experiments to allow comparison of the in vitro dual effect of Tau and Taxol on the polymerization of MCF7 microtubules. We observed a concentration-dependent demotion-like alteration in the self-polymerization kinetics of Tau-induced MCF7 microtubules. In contrast, microtubules polymerized under the simultaneous effects of Tau and Taxol showed promoted assembly as compared with those observed in Tau-induced microtubules. The analysis of our data obtained from the length of MCF7 microtubules polymerized under the interaction with Tau and Taxol in vitro suggests that the phenomenon known as drug resistance in microtubule-targeted drugs such as Taxol may not be directly linked to the different responses of microtubules to the drug. The effect of the drug may be mitigated due to the simultaneous interactions with other microtubule-associated proteins such as Tau protein. The observed regulatory effect of Tau and Taxol on the polymerization of breast cancer microtubules in vitro points to additional evidence for the possible role of tubulin isotypes in microtubules’ functions.

scholarly journals Targeting UCP2 Suppresses the FAK Signaling and Progression of Human Head and Neck Cancer Cells

2020 ◽  
pp. 1-8
Author(s):  
Yunfeng Zhao ◽  
Cherie Ann Nathan ◽  
Chunjing Zhang ◽  
Hongyan Du ◽  
Manikandan Panchatcharam ◽  
...  
Keyword(s):  
Head And Neck ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Uncoupling Protein ◽  
Human Head ◽  
Atp Production ◽  
Normal Tissues ◽  
Tumor Tissues ◽  
Lactate Formation

Background: New adjuvant therapies for human head and neck (H&N) cancer to improve the quality of life of the patients are in great demand. Our early studies have demonstrated that uncoupling protein 2 (UCP2) is upregulated in the tumor tissues of H&N cancer compared to the adjacent normal tissues; however, the role of UCP2 in H&N cancer has not been studied. Objective: In this manuscript, we aim to examine whether UCP2 contributes to H&N cancer progression in vitro. Methods: We generated UCP2 stable knockdown H&N cancer cells and detected the effects of UCP2 inhibition on cell proliferation, migration, invasion, 3D spheroid formation, and the sensitivity to a chemodrug treatment. Results: Knockdown of UCP2 suppressed the progression of H&N cancer in vitro, which might be mediated via the following mechanism: 1) increased the G1 phase whereas decreased the S phase of the cell cycle, which could be mediated by suppression of the G1/S regulators including CDK4/6 and cyclin D1. 2) Decreased mitochondrial oxygen consumption, ATP production, and lactate formation, which is consistent with the downregulation of c-Myc. 3) FAK may serve as the upstream signaling molecule, and its action was mediated by Akt and ERK. Conclusions: Our studies first demonstrate that targeting UCP2 may suppress H&N cancer progression in vitro.

Half-Sandwich Ir(III) Complex of N1-Pyridyl-7-azaindole Exceeds Cytotoxicity of Cisplatin at Various Human Cancer Cells and 3D Multicellular Tumor Spheroids

2018 ◽  
Vol 37 (16) ◽  
pp. 2749-2759 ◽  
Author(s):  
Pavel Štarha ◽  
Zdeněk Trávníček ◽  
Hana Crlíková ◽  
Ján Vančo ◽  
Jana Kašpárková ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Human Cancer ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Human Cancer Cells ◽  
Half Sandwich

PEAK1 promotes invasion and metastasis and confers drug resistance in breast cancer

2021 ◽  
Author(s):  
xingang wang ◽  
YAN ZHENG ◽  
YU WANG
Keyword(s):  
Breast Cancer ◽  
Drug Resistance ◽  
Cell Growth ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Multi Drug Resistance ◽  
Cancer Tissues

Abstract Background and AimsPseudopodium-enriched atypical kinase 1 (PEAK1) has reported to be upregulated in human malignancies and related with poor prognosis. Enhanced PEAK1 expression facilitates tumor cell survival, invasion, metastasis and chemoresistance. However, the role of PEAK1 in breast cancer is not clear. Here, we investigated the PEAK1 expression in breast cancer and analyzed its relation with clinicopathological status and chemotherapy resistance to the neoadjuvant chemotherapy (NAC). We also investigated the role of PEAK1 on breast cancer cells in vitro and in vivo. MethodsImmunohistochemistry (IHC) was performed in 112 surgical resected breast cancer tissues. The associations between clinicopathological status, multi-drug resistance and PEAK1 expression were determined. Effect of PEAK1 overexpression or down-expression on proliferation, colony formation, invasion, migration, metastasis and Doxorubicin sensitivity in the MCF-7 cells in vitro and in vivo was detected. ResultsPEAK1 was overexpressed in breast cancer tissues and NAC -resistant breast cancer tissues. High PEAK1 expression was related with tumor size, high tumor grade, T stage, LN metastasis, recurrence, Ki-67 expression, Her-2 expression and multi-drug resistance. Targeting PEAK1 inhibited cell growth, invasion, metastasis and reversed chemoresistance to Doxorubicin in breast cancer cells in vitro and in vivo. ConclusionHigh PEAK1 expression was associated with invasion, metastasis and chemoresistance of breast cancers. Furthermore, targeting PEAK1 could inhibit cell growth and metastasis, and reverse chemoresistance in breast cancer cells, which provides an effective treatment strategies for breast cancer.

scholarly journals CAFs affect the proliferation and treatment response of head and neck cancer spheroids during co-culturing in a unique in vitro model

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Mustafa Magan ◽  
Emilia Wiechec ◽  
Karin Roberg
Keyword(s):  
Cell Proliferation ◽  
Tumor Cell ◽  
Head And Neck ◽  
Treatment Response ◽  
Tumor Cells ◽  
In Vitro Model ◽  
Tumor Spheroids ◽  
Vitro Model ◽  
The Impact

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors for which the overall survival rate worldwide is around 60%. The tumor microenvironment, including cancer-associated fibroblasts (CAFs), is believed to affect the treatment response and migration of HNSCC. The aim of this study was to create a biologically relevant HNSCC in vitro model consisting of both tumor cells and CAFs cultured in 3D to establish predictive biomarkers for treatment response, as well as to investigate the impact of CAFs on phenotype, proliferation and treatment response in HNSCC cells. Methods Three different HNSCC patient-derived tumor cell lines were cultured with and without CAFs in a 3D model. Immunohistochemistry of the proliferation marker Ki67, epidermal growth factor receptor (EGFR) and fibronectin and a TUNEL-assay were performed to analyze the effect of CAFs on both tumor cell proliferation and response to cisplatin and cetuximab treatment in tumor spheroids (3D). mRNA expression of epithelial-mesenchymal transition (EMT) and cancer stem cells markers were analyzed using qRT-PCR. Results The results demonstrated increased cell proliferation within the tumor spheroids in the presence of CAFs, correlating with increased expression of EGFR. In spheroids with increased expression of EGFR, a potentiated response to cetuximab treatment was observed. Surprisingly, an increase in Ki67 expressing tumor cells were observed in spheroids treated with cisplatin for 3 days, correlating with increased expression of EGFR. Furthermore, tumor cells co-cultured with CAFs presented an increased EMT phenotype compared to tumor cells cultured alone in 3D. Conclusion Taken together, our results reveal increased cell proliferation and elevated expression of EGFR in HNSCC tumor spheroids in the presence of CAFs. These results, together with the altered EMT phenotype, may influence the response to cetuximab or cisplatin treatment.

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