Early detection of metastasis can be aided by circulating tumor cells (CTCs), which also show potential to predict early relapse. Due to the limited CTC numbers in peripheral blood in early stages, we investigated CTCs in pulmonary vein blood accessed during surgical resection of tumors. Pulmonary vein (PV) and peripheral vein (Pe) blood specimens from patients with lung cancer were drawn during the perioperative period and assessed for CTC burden using a microfluidic device. From 108 blood samples analyzed from 36 patients, PV had significantly higher number of CTCs compared to pre-operative Pe (p<0.0001) and intra-operative Pe (p<0.001) blood. CTC clusters with large number of CTCs were observed in 50% of patients, with PV often revealing larger clusters. Long term surveillance indicated that presence of clusters in pre-operative Pe blood predicted a trend toward poor prognosis. Gene expression analysis by RT-qPCR revealed enrichment of p53 signaling and extracellular matrix involvement in PV and Pe samples. Ki67 expression was detected in 62.5% of PV samples and 59.2% of Pe samples, with the majority (72.7%) of patients positive for Ki67 expression in PV having single CTCs as opposed to clusters. Gene ontology analysis revealed enrichment of cell migration and immune-related pathways in CTC clusters, suggesting survival advantage of clusters in circulation. Clusters display characteristics of therapeutic resistance, indicating the aggressive nature of these cells. Thus, CTCs isolated from early stages of lung cancer are predictive of poor prognosis and can be interrogated to determine biomarkers predictive of recurrence.

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer-related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time-consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here we report a high throughput microfluidic technology, the OncoBean Chip, employing radial flow that introduces a varying shear profile across the device enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood was validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL hr−1 in contrast to a flow rate of 1 mL hr−1 standardly reported with other microfluidic devices. Cells were recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application was demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic and lung cancer patients. Comparable CTC numbers were recovered in all the samples at the two flow rates demonstrating the ability of the technology to perform at high-throughputs.

While studying Th responses induced by cardiac transplantation, we observed that mice deficient in the Th1 transcription factor T-bet (T-bet−/−) mount both Th1 and Th17 responses, whereas wild-type recipients mount only Th1 responses. Cells producing both IFN-γ and IL-17 were readily detectable within the rejecting graft of T-bet−/− recipients, but were absent from the spleen, indicating that the in vivo microenvironment influences Th function. In addition, disrupting CD40-CD40L costimulatory interactions was highly effective at prolonging allograft survival in WT mice, but ineffective in T-bet−/− recipients. In this study, we report that CD8+ Th17 mediate costimulation blockade-resistant rejection in T-bet−/− allograft recipients. Depleting CD8+ cells or neutralizing IL-17 or the Th17-inducing cytokine IL-6 ablated the Th17 response and reversed costimulation blockade-resistant graft rejection. Neutralizing IL-4 in IFN-γ−/− allograft recipients did not induce Th17, suggesting that T-bet, rather than IL-4 and IFN-γ (known inhibitors of Th17), plays a critical role in negatively regulating Th17 in the transplant setting.

With a single vein draining the entire tumor basin, lung cancers are unique, allowing the high-yield isolation of CTCs from the PV. This method may facilitate future studies to improve the detection and analysis of early-stage lung CTCs.

IL-6 mediates numerous immunologic effects relevant to transplant rejection; however its specific contributions to these processes are not fully understood. To this end, we neutralized IL-6 in settings of acute cardiac allograft rejection associated with either CD8+ or CD4+ cell dominant responses. In a setting of CD8+ cell dominant graft rejection, IL-6 neutralization delayed the onset of acute rejection while decreasing graft infiltrate and inverting anti-graft Th1/Th2 priming dominance in recipients. IL-6 neutralization markedly prolonged graft survival in the setting of CD4+ cell mediated acute rejection and was associated with decreased graft infiltrate, altered Th1 responses, and reduced serum alloantibody. Further, in CD4+ cell dominated rejection, IL-6 neutralization was effective when anti-IL-6 administration was delayed by as many as six days post-transplant. Finally, IL-6 deficient graft recipients were protected from CD4+ cell dominant responses suggesting that IL-6 production by graft recipients, rather than grafts, is necessary for this type of rejection. Cumulatively, these observations define IL-6 as a critical promoter of graft infiltration and a shaper of T cell lineage development in cardiac graft rejection. In light of these findings, the utility of therapeutics targeting IL-6 should be considered for preventing cardiac allograft rejection.