Methods to Evaluate Cell Growth, Viability, and Response to Treatment in a Tissue Engineered Breast Cancer Model

Abstract

The use of in vitro, engineered surrogates in the field of cancer research is of interest for studies involving mechanisms of growth and metastasis, and response to therapeutic intervention. While biomimetic surrogates better model human disease, their complex composition and dimensionality make them challenging to evaluate in a real-time manner. This feature has hindered the broad implementation of these models, particularly in drug discovery. Herein, several methods and approaches for the real-time, non-invasive analysis of cell growth and response to treatment in tissue-engineered, three-dimensional models of breast cancer are presented. The tissue-engineered surrogates used to demonstrate these methods consist of breast cancer epithelial cells and fibroblasts within a three dimensional volume of extracellular matrix and are continuously perfused with nutrients via a bioreactor system. Growth of the surrogates over time was measured using optical in vivo (IVIS) imaging. Morphologic changes in specific cell populations were evaluated by multi-photon confocal microscopy. Response of the surrogates to treatment with paclitaxel was measured by optical imaging and by analysis of lactate dehydrogenase and caspase-cleaved cytokeratin 18 in the perfused medium. Each method described can be repeatedly performed during culture, allowing for real-time, longitudinal analysis of cell populations within engineered tumor models.

Applicability of drug response metrics for cancer studies using biomaterials

“…The easiest way to distinguish multiple cell populations is by using cells that express a reporter transgene or labeling the different cells with nontoxic dyes. Measurement of total fluorescence or bioluminescence provides an estimation of the labeled cell number over the course of drug treatment [48,49]. However, because dead cells may remain within 3D cultures, total fluorescence readings may be less accurate.…”

Endothelial PAI-1 (Plasminogen Activator Inhibitor-1) Blocks the Intrinsic Pathway of Coagulation, Inducing the Clearance and Degradation of FXIa (Activated Factor XI)

“…7 Tissue specimens from diagnostic surgery can be procured and manipulated to obtain primary human cells which can be immortalized for long term use by treating with human telomerase. 8 Immortalized primary cells retain many traits from the primary tissue specimen while still undergoing multiple passages, and they can recapitulate cancer cell signaling and extracellular matrix (ECM) remodeling. 8 The ability to incorporate primary samples into in vitro models of each metastatic stage has the potential to transform these devices into more precise and impactful predictors of clinical outcomes.…”

Quantifying Fluorescently Labeled Ceramide Levels in Human Sarcoma Cell Lines in Response to a Sphingomyelin Synthase Inhibitor

“…This method is an effective means of screening the biological consequence of inhibiting enzymes in the ceramide metabolism pathway. While this method has focused exclusively on the effects of inhibiting sphingomyelin synthase, there are multiple pathways involved in ceramide metabolism (Figure 1) and similar approaches have been used to investigate inhibitors of ceramidase and sphingosine kinase [2,3,6,7,8]. One limitation of the study is that because there are multiple pathways responsible for ceramide metabolism, inhibiting one arm of the pathway does not fully prevent the metabolism of ceramide, which could result in the loss of the fluorescent signal even in the presence of a potent and specific inhibitor.…”

“…There are copious ways of measuring cell viability and events associated with apoptosis during pharmacological screening of small-molecule inhibitors [8,9], but these methods do not pinpoint a mechanism of action of how a cell arrived at that end fate. Understanding the molecular underpinnings of how a drug works will lead to more precise applications in precision medicine and circumvent future developments of resistance.…”