(158i) Anticancer Drug Evaluation in MCF7 Spheroids Cultured in a 3D-Printed Miniaturized Continuous Stirred Tank Reactor (mCSTR)

Cancer continues to be one of the deadliest diseases worldwide, with 600,000 cancer-related deaths reported in 2019 in the USA alone. Despite enormous efforts to find new therapeutic cancer targets and cancer therapies, the current platforms for evaluating drug candidate safety and efficacy have serious limitations (i.e., cost, reproducibility, availability, and ethical concerns). Furthermore, these platforms often provide unreliable results. Improved cancer research models are therefore needed to achieve better predictive results for the efficacy of new therapeutic compounds in humans.

Here, we describe a simple and robust system for anti-cancer drug screening. The system is based on the use of a 3D-printed miniaturized (3 mL volume) continuous stirred tank mini-reactor (mCSTR) that enables the extended culture (20–30 days) of breast cancer spheroids for evaluation of anti-cancer drugs. The off-center agitation system of this CSTmR enables homogeneous chaotic mixing at low speeds (100 rpm) as confirmed by computational fluid dynamics (CFD) simulation and tracer studies under batch and continuous operation.

We have cultured populations of small MCF7 cancer spheroids, fabricated using conventional methods, under continuous flow conditions at different flow rates (to attain different hydraulic residence times HRT) and with various inlet substrate concentrations. Here, the residence time (HRT) and the inlet concentration are used to modulate the growth dynamics and glucose consumption of the spheroids. For instance, we monitored the progression of size, shape, genetic expression of relevant cancer biomarkers, and glucose utilization for 30 days in continuous cultures perfused with culture media (glucose concentration of 1.02 mg µL^-1 mg µL^-1) at 2.3 µL min^-1 (HRT=22h). Under these conditions, a pseudo steady state was reached, as suggested by the nearly constant glucose concentration within the tank (and the indicators of size, shape, and expression).

We also independently evaluated the effect of three different anti-cancer drugs (docetaxel, the taxol paclitaxel and the anthracycline doxorubicin) in continuously cultured spheroid populations. We continuously fed each drug (at two different doses) from day five, while monitoring the glucose concentration within the tank as an overall indicator of the progression of cell viability with time. The endpoint of each experiment was determined when glucose consumption ceased. Endpoint evaluation of spheroids exposed to different drugs and doses included morphological observations by scanning electron microscopy (SEM), viability determined by Live/Dead assays, and drug cytotoxicity as assessed by lactate dehydrogenase (LDH) activity and changes in the expression of specific genes related to drug resistance and apoptosis (BCL2, BAX, ABCB1, ABCC1 and ABCG2).

The MCF7 spheroids showed distinctive dose-dependent responses to each drug. Our results suggest that this mCSTR system is a suitable platform for the continuous perfusion and evaluation of anti-cancer drugs. Moreover, the system is highly customizable, as a wide spectrum of experiments can be implemented under continuous flow conditions simply by varying residence times, inlet concentrations, and spheroid densities. Likewise, this system could represent an option for the culture of cancer biopsies for personalized medicine applications.