When breast cancer patients start to exhibit resistance to hormonal therapy or chemotherapy, the mTOR inhibitor everolimus can be considered as an alternative therapeutic agent. Everolimus can deregulate the PI3K/AKT/mTOR pathway and affect a range of cellular functions. In some patients, the agent does not exhibit the desired efficacy and, even worse, not without the associated side effects. This study assessed the use of immunofluorescence (IF) as a modality to fill this unmet need of predicting the efficacy of everolimus prior to administration. Cell viability and MTT assays based on IF intensities of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 on breast cancer cells (Hs578T, MCF7, BT474, MDA-MB-231) and patient-derived cell culture from metastatic sites (ABC-82T and ABC-16TX1) were interrogated. Results show that independent pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 IF expressions can classify data into different groups: everolimus sensitive and resistant. The combined IF baseline intensity of these proteins is predictive of the efficacy of everolimus, and their intensities change dynamically when cells are resistant to everolimus. Furthermore, mTOR resistance is not only consequence of the AKT/mTOR pathway but also through the LKB1 or MAPK/ERK pathway. The LKB1 and pho-GSK3β may also be potential predictive markers for everolimus.

Rare cells in the blood often have rich clinical significance. Although their isolation is highly desirable, this goal remains elusive due to their rarity. This paper presents a systemic approach to isolate and characterize trophoblasts from the maternal circulation. A microfluidic rare cell disc assay (RaCDA) was designed to process an extremely large volume of up to 15 mL of blood in 30 min, depleting red blood cells (RBCs) and RBC-bound white blood cells (WBC) while isolating trophoblasts in the collection chip. To minimize cell loss, on-disc labeling of cells with fluorescent immuno-staining identified the trophoblasts. Retrieval of trophoblasts utilized an optimized strategy in which multiple single cells were retrieved within the same micropipette column, with each cell encapsulated in a fluid volume (50 nL) separated by an air pocket (10 nL). Further, whole-genome amplification (WGA) amplified contents from a few retrieved cells, followed by quality control (QC) on the success of WGA via housekeeping genes. For definitive confirmation of trophoblasts, short-tandem repeat (STR) of the WGAamplified content was compared against STR from maternal WBC and amniocytes from amniocentesis. Results showed a mean recovery rate (capture efficiency) of 91.0% for spiked cells with a WBC depletion rate of 99.91%. The retrieval efficiency of single target cells of 100% was achieved for up to four single cells retrieved per micropipette column. Comparison of STR signatures revealed that the RaCDA can retrieve trophoblasts from the maternal circulation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.