Acoustic and Magnetic Techniques for the Isolation and Analysis of Cells in Microfluidic Platforms

Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management.

The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis.

The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments.

Development of a Novel c-MET-Based CTC Detection Platform.

UNLABELLED: Amplification of the MET oncogene is associated with poor prognosis, metastatic dissemination, and drug resistance in many malignancies. We developed a method to capture and characterize circulating tumor cells (CTC) expressing c-MET using a ferromagnetic antibody. Immunofluorescence was used to characterize cells for c-MET, DAPI, and pan-CK, excluding CD45(+) leukocytes. The assay was validated using appropriate cell line controls spiked into peripheral blood collected from healthy volunteers (HV). In addition, peripheral blood was analyzed from patients with metastatic gastric, pancreatic, colorectal, bladder, renal, or prostate cancers. CTCs captured by c-MET were enumerated, and DNA FISH for MET amplification was performed. The approach was highly sensitive (80%) for MET-amplified cells, sensitive (40%-80%) for c-MET-overexpressed cells, and specific (100%) for both c-MET-negative cells and in 20 HVs. Of 52 patients with metastatic carcinomas tested, c-MET CTCs were captured in replicate samples from 3 patients [gastric, colorectal, and renal cell carcinoma (RCC)] with 6% prevalence. CTC FISH demonstrated that MET amplification in both gastric and colorectal cancer patients and trisomy 7 with gain of MET gene copies in the RCC patient. The c-MET CTC assay is a rapid, noninvasive, sensitive, and specific method for detecting MET-amplified tumor cells. CTCs with MET amplification can be detected in patients with gastric, colorectal, and renal cancers. IMPLICATIONS: This study developed a novel c-MET CTC assay for detecting c-MET CTCs in patients with MET amplification and warrants further investigation to determine its clinical applicability. Mol Cancer Res; 14(6); 539-47. ©2016 AACR.

Competing Actions of Type 1 Angiotensin II Receptors Expressed on T Lymphocytes and Kidney Epithelium during Cisplatin-Induced AKI.

Inappropriate activation of the renin-angiotensin system (RAS) contributes to many CKDs. However, the role of the RAS in modulating AKI requires elucidation, particularly because stimulating type 1 angiotensin II (AT1) receptors in the kidney or circulating inflammatory cells can have opposing effects on the generation of inflammatory mediators that underpin the pathogenesis of AKI. For example, TNF-α is a fundamental driver of cisplatin nephrotoxicity, and generation of TNF-α is suppressed or enhanced by AT1 receptor signaling in T lymphocytes or the distal nephron, respectively. In this study, cell tracking experiments with CD4-Cre mT/mG reporter mice revealed robust infiltration of T lymphocytes into the kidney after cisplatin injection. Notably, knockout of AT1 receptors on T lymphocytes exacerbated the severity of cisplatin-induced AKI and enhanced the cisplatin-induced increase in TNF-α levels locally within the kidney and in the systemic circulation. In contrast, knockout of AT1 receptors on kidney epithelial cells ameliorated the severity of AKI and suppressed local and systemic TNF-α production induced by cisplatin. Finally, disrupting TNF-α production specifically within the renal tubular epithelium attenuated the AKI and the increase in circulating TNF-α levels induced by cisplatin. These results illustrate discrepant tissue-specific effects of RAS stimulation on cisplatin nephrotoxicity and raise the concern that inflammatory mediators produced by renal parenchymal cells may influence the function of remote organs by altering systemic cytokine levels. Our findings suggest selective inhibition of AT1 receptors within the nephron as a promising intervention for protecting patients from cisplatin-induced nephrotoxicity.

Single-cell functional analysis of parathyroid adenomas reveals distinct classes of calcium sensing behaviour in primary hyperparathyroidism.

Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder caused by a failure of calcium sensing secondary to tumour development in one or more of the parathyroid glands. Parathyroid adenomas are comprised of distinct cellular subpopulations of variable clonal status that exhibit differing degrees of calcium responsiveness. To gain a clearer understanding of the relationship among cellular identity, tumour composition and clinical biochemistry in PHPT, we developed a novel single cell platform for quantitative evaluation of calcium sensing behaviour in freshly resected human parathyroid tumour cells. Live-cell intracellular calcium flux was visualized through Fluo-4-AM epifluorescence, followed by in situ immunofluorescence detection of the calcium sensing receptor (CASR), a central component in the extracellular calcium signalling pathway. The reactivity of individual parathyroid tumour cells to extracellular calcium stimulus was highly variable, with discrete kinetic response patterns observed both between and among parathyroid tumour samples. CASR abundance was not an obligate determinant of calcium responsiveness. Calcium EC50 values from a series of parathyroid adenomas revealed that the tumours segregated into two distinct categories. One group manifested a mean EC50 of 2.40 mM (95% CI: 2.37-2.41), closely aligned to the established normal range. The second group was less responsive to calcium stimulus, with a mean EC50 of 3.61 mM (95% CI: 3.45-3.95). This binary distribution indicates the existence of a previously unappreciated biochemical sub-classification of PHPT tumours, possibly reflecting distinct etiological mechanisms. Recognition of quantitative differences in calcium sensing could have important implications for the clinical management of PHPT.