Drug resistant pancreatic cancer cells exhibit altered biophysical interactions with stromal fibroblasts in imaging studies of 3D co-culture models.

Interactions between tumor and stromal cells are well known to play a prominent roles in progression of pancreatic ductal adenocarcinoma (PDAC). As knowledge of stromal crosstalk in PDAC has evolved, it has become clear that cancer associated fibroblasts can play both tumor promoting and tumor suppressive roles through a combination of paracrine crosstalk and juxtacrine interactions involving direct physical contact. Another major contributor to dismal survival statistics for PDAC is development of resistance to chemotherapy drugs. Though less is known about how the acquisition of chemoresistance impacts upon tumor-stromal crosstalk. Here, we use 3D co-culture geometries to recapitulate juxtacrine interactions between epithelial and stromal cells. In particular, extracellular matrix (ECM) overlay cultures in which stromal cells (pancreatic stellate cells, or normal human fibroblasts) are placed adjacent to PDAC cells (PANC1), result in direct heterotypic cell adhesions accompanied by dramatic fibroblast contractility which leads to highly condensed macroscopic multicellular aggregates as detected using particle image velocimetry (PIV) analysis to quantify cell velocities over the course of time lapse movie sequences. To investigate how drug resistance impacts these juxtacrine interactions we contrast cultures in which PANC1 are substituted with a drug resistant subline (PANC1-OR) previously established in our lab. We find that heterotypic cell-cell interactions are highly suppressed in drug-resistant cells relative to the parental PANC1 cells. To investigate further we conduct RNA-seq and bioinformatics analysis to identify differential gene expression in PANC1 and PANC1-OR, which shows that negative regulation of cell adhesion molecules, consistent with increased epithelial mesenchymal transition (EMT), is also consistent with loss of hetrotypic cell-cell contact necessary for the contractile behavior observed in drug naïve cultures. Overall these findings elucidate the role of drug-resistance in inhibiting an avenue of stromal crosstalk which is associated with tumor suppression and also help to establish cell culture conditions useful for further mechanistic investigation.

An assessment of the limits of detection, sensitivity and specificity of three devices for public health-based drug checking of fentanyl in street-acquired samples.

BACKGROUND: Fentanyl has caused rapid increases in US and Canadian overdose deaths, yet its presence in illicit drugs is often unknown to consumers. This study examined the validity in identifying the presence of fentanyl of three portable devices that could be used in providing drug checking services and drug supply surveillance: fentanyl test strips, a hand-held Raman Spectrometer, and a desktop Fourier-Transform Infrared Spectrometer. METHODS: In Fall 2017, we first undertook an assessment of the limits of detection for fentanyl, then tested the three devices' sensitivity and specificity in distinguishing fentanyl in street-acquired drug samples. Utilizing test replicates of standard fentanyl reference material over a range of increasingly lower concentrations, we determined the lowest concentration reliably detected. To establish the sensitivity and specificity for fentanyl, 210 samples (106 fentanyl-positive, 104 fentanyl-negative) previously submitted by law enforcement entities to forensic laboratories in Baltimore, Maryland, and Providence, Rhode Island, were tested using the devices. All sample testing followed parallel and standardized protocols in the two labs. RESULTS: The lowest limit of detection (0.100 mcg/mL), false negative (3.7%), and false positive rate (9.6%) was found for fentanyl test strips, which also correctly detected two fentanyl analogs (acetyl fentanyl and furanyl fentanyl) alone or in the presence of another drug, in both powder and pill forms. While less sensitive and specific for fentanyl, the other devices conveyed additional relevant information including the percentage of fentanyl and presence of cutting agents and other drugs. CONCLUSION: Devices for fentanyl drug checking are available and valid. Drug checking services and drug supply surveillance should be considered and researched as part of public health responses to the opioid overdose crisis.