Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment

Roberge, Cassandra L.; Barroso, Margarida; Corr, David T.

doi:10.3791/64030-v

Cited by 3 publications

(4 citation statements)

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“…We expected, based on previous studies by ourselves and others, ,, and anticipated that 3D tumor cell aggregates in a spheroid morphology would lead to higher IC 50 values. Hence, dose–response biological activity assays were extended for BT-474 (Figure A) and MDA-MB-231 (Figure B) cell lines to 3D tumor aggregates, prepared using liquid-overlay with Matrigel following previous work by our group. ,, Briefly, MDA-MB-231 or BT-474 cell lines, selected for their stark differences in morphology and cell receptor presence, were seeded in a 96-well plate in the presence of Matrigel, centrifuged to form 3D tumor spheroids, and allowed to incubate for 4 days. Following 24 h treatment of MDA-MB-231 3D spheroids, the IC 50 value for 1 was 140 μg/mL, representing a 1.7-fold increase over that of 2D monolayer cultures (Table ).…”

Section: Resultsmentioning

confidence: 99%

“…Matrigel (2.5% v/v) was added to each well, and, immediately after, the plate was centrifuged for 10 min at 1000 rpm to ensure collection of a cell pellet at the bottom of each well. Plates were incubated, and cell aggregates were cultured over a 4 day maturation period prior to compound testing. ,, Compounds were individually solubilized in 100% DMSO, followed by dilutions to concentrations from 10,000 to 80 μg/mL in 1:2 serial dilutions; 10 μL of each corresponding drug concentration was added to each well in triplicatethus, each concentration has been diluted 10-fold due to the 100 μL of media present in the cell from cell seeding. Care was taken such that the final concentration of DMSO was no more than 1% v/v per well.…”

Section: Materials and Experimental Methodsmentioning

confidence: 99%

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Molecularly Engineered Surfactin Analogues Induce Nonapoptotic-Like Cell Death and Increased Selectivity in Multiple Breast Cancer Cell Types

et al. 2023

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Surfactin, a negatively charged amphiphilic lipopeptide biosurfactant, is synthesized by the bacterium Bacillus subtilis. It consists of a cyclic heptapeptide and an 11−15C β-hydroxy fatty acid. To probe how the modification of the molecular skeleton of surfactin influences its selectivity and activity against breast cancer, six synthetic surfactins were generated. Modifications were accomplished by conjugating amine-functionalized molecules to the Glu and Asp carboxyl moieties of the heptapeptide. The resulting synthetic surfactins provided a diverse series of molecules with differences in charge, size, and hydrophilicity. After purification and structural analysis, insights into biological activity and specificity were generated for each compound. Dosedependent growth inhibition was determined for four tumorigenic breast cancer cell lines in monolayer and spheroid morphologies, as well as nontumorigenic fibroblasts and sheep erythrocytes, which were utilized to determine selectivity indices. Results indicated that two compounds, which have amplified anionic charge, had increased activity on breast cancer, with reduced activity on nontumorigenic fibroblasts and erythrocytes. Cationic derivative surf-ethylenediamine has increased activity on all cell lines tested. Novel correlations between dose−response activities and physicochemical properties of all compounds determined that there is a significant correlation between the critical micelle concentration and activity against multiple cell lines.

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Section: Resultsmentioning

confidence: 99%

Section: Materials and Experimental Methodsmentioning

confidence: 99%

Molecularly Engineered Surfactin Analogues Induce Nonapoptotic-Like Cell Death and Increased Selectivity in Multiple Breast Cancer Cell Types

et al. 2023

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“…While the assays performed herein did not allow for inferences into the location of dead/ dying cells, future work should seek to expand these results by incorporating information about cell viability with respect to location. A recently published technique from our lab uses the same OCT imaging modality with more in-depth Imaris image analysis to track regional cell death in drugged aggregate models, 60 which is expected to help provide a clearer picture as to where a drug is acting most strongly.…”

Section: 5d Aggregate Resultsmentioning

confidence: 99%

Sophorolipid Candidates Demonstrate Cytotoxic Efficacy against 2D and 3D Breast Cancer Models

et al. 2023

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Sophorolipids are biosurfactants derived from the nonpathogenic yeasts such as Starmerella bombicola with potential efficacy in anticancer applications. Simple and cost-effective synthesis of these drugs makes them a promising alternative to traditional chemotherapeutics, pending their success in preliminary drug-screening. Drug-screening typically utilizes 2D cell monolayers due to their simplicity and ease of high-throughput assessment. However, 2D assays fail to capture the complexity and 3D context of the tumor microenvironment and have consequently been implicated in the high percentage of drugs investigated in vitro that later fail in clinical trials. Herein, we screened two sophorolipid candidates and a clinically-used chemotherapeutic, doxorubicin, on in vitro breast cancer models ranging from 2D monolayers to 3D spheroids, employing optical coherence tomography to confirm these morphologies. We calculated corresponding IC50 values for these drugs and found one of the sophorolipids to have comparable toxicities to the chemotherapeutic control. Our findings show increased drug resistance associated with model dimensionality, such that all drugs tested showed that 3D spheroids exhibited higher IC50 values than their 2D counterparts. These findings demonstrate promising preliminary data to support the use of sophorolipids as a more affordable alternative to traditional clinical interventions and demonstrate the importance of 3D tumor models in assessing drug response.

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“…At approximately 70% confluence, SKOV3 cells expressing pmiRFP680 or pemiRFP670 were detached from their flasks via trypsin (ATCC) for the preparation of tumor spheroids using liquid overlay technique [11,12] Cells were resuspended at 2.5 e5 cells/mL in McCoy's media containing 5% soluble Matrigel matrix (Corning, NY, USA). Matrigel is a common media additive designed to assist with aggregate formation.…”

Section: D Tumor Spheroid Modelsmentioning

confidence: 99%

Multiplexed fluorescence lifetime imaging of near-infrared fluorescent proteins in 3D tumor spheroids

Sherry,

Chavez,

Kanai

et al. 2024

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XV

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The development of fluorescent proteins emitting in the near infrared (NIR) range (i.e., 650 nm-950 nm) has improved our capabilities for lifetime multiplexing and fluorescence imaging in vivo. Wavelengths in the NIR window experience reduced scattering and increased penetration depth through living tissue. Additionally, autofluorescence of cells and tissues is less prevalent in the NIR range, further improving signal to noise ratio. We performed fluorescence lifetime imaging (FLI) on breast cancer (AU565) and ovarian cancer (SKOV3) cell lines expressing the NIR fluorescent proteins (FPs), miRFP680 and emiRFP670. Confocal microscopy with time-correlated single-photon counting (TCSPC) reveals unique fluorescence decays for these NIR FPs, allowing for lifetime-based multiplexing on a single channel. Despite similar emission spectra, we were able to unmix fluorescence signals from a co-culture of SKOV3 expressing emiRFP670 and AU565 expressing miRFP680 based upon their unique fluorescence decays. We then generated 3D liquid overlay tumor spheroids using SKOV3 expressing emiRFP670 or miRFP680 for lifetime imaging via mesoscopic fluorescence molecular tomography (MFMT). 2D lifetime values and images acquired from MFMT corroborated our findings. Future investigation includes 3D light sheet mesoscopic imaging of tumor spheroids, as well as imaging of in vivo tumor xenografts expressing NIR-FPs. The long wavelengths and unique fluorescence lifetimes of emiRFP670 and miRFP680 make them ideal for multiplexed imaging, as well as for defining tumor volumes in vivo, while also leveraging the benefits of NIR imaging.

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Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment

Cited by 3 publications

References 0 publications

Molecularly Engineered Surfactin Analogues Induce Nonapoptotic-Like Cell Death and Increased Selectivity in Multiple Breast Cancer Cell Types

Molecularly Engineered Surfactin Analogues Induce Nonapoptotic-Like Cell Death and Increased Selectivity in Multiple Breast Cancer Cell Types

Sophorolipid Candidates Demonstrate Cytotoxic Efficacy against 2D and 3D Breast Cancer Models

Multiplexed fluorescence lifetime imaging of near-infrared fluorescent proteins in 3D tumor spheroids

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