Svetlana Grabauskiene

CD8+ Th17 Mediate Costimulation Blockade-Resistant Allograft Rejection in T-bet-Deficient Mice

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.

A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

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, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs 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 is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h(-1) in contrast to a flow rate of 1 mL h(-1) standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs.

IL-6 Promotes Cardiac Graft Rejection Mediated by CD4+ Cells

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. Furthermore, in CD4+ cell-dominated rejection, IL-6 neutralization was effective when anti–IL-6 administration was delayed by as many as 6 d posttransplant. 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. Collectively, 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.

CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells

OBJECTIVE Overexpression of checkpoint kinase 1 (CHK1) is associated with poorer patient outcome and therapeutic resistance in multiple tumor models. Inhibition of CHK1 has been proposed as a strategy to increase the effectiveness of chemotherapeutic agents, especially in p53-deficient tumors. In this study, we evaluated the effects of a novel CHK1 inhibitor, MK-8776, in combination with pemetrexed (PMX) on cell proliferation and survival in a panel of p53 mutant non-small cell lung cancer (NSCLC) cell lines. METHODS We examined CHK1 expression in 442 resected lung adenocarcinoma specimens using Affymetrix U133A gene expression arrays. We correlated CHK1 mRNA expression with patient survival, tumor differentiation and genomic complexity. We evaluated CHK1 levels in NSCLC cell lines and identified four p53 mutant cell lines with variable CHK1 expression (H1993, H23, H1437 and H1299) based on publicly available gene expression data. We confirmed differential CHK1 mRNA and CHK1 protein levels by qRT-PCR, ELISA, Western Blot analysis (WB) and immunohistochemistry. We examined cell line sensitization to PMX in response to CHK1 inhibition with MK-8776 using WST-1 and clonogenic survival assays. RESULTS We found that elevated CHK1 expression in primary lung adenocarcinomas correlates with poor tumor differentiation and significantly worse patient survival. Tumors with elevated CHK1 mRNA levels have a higher number of gene mutations and DNA copy number gain or amplifications. CHK1 inhibition by MK-8776 enhances sensitivity of NSCLC cell lines to PMX. CHK1 mRNA and protein expression are variable among NSCLC cell lines, and cells expressing higher levels of CHK1 protein are more sensitive to the CHK1 inhibition by MK-8776 as compared to low CHK1 expressing cells. CONCLUSIONS These findings suggest that CHK1 levels may not only serve as a biomarker of poor prognosis in surgically-resected lung adenocarcinomas, but could also be a predictive marker for CHK1 inhibitor sensitivity, pending in vivo and clinical confirmation.

Pulmonary venous blood sampling significantly increases the yield of circulating tumor cells in early-stage lung cancer.

OBJECTIVE To identify circulating tumor cells (CTCs) in the blood of patients with early-stage lung cancer and to show that sampling pulmonary vein (PV) blood using microfluidic chip technology will yield significantly more CTCs. Improving early detection of lung cancer is critical to improving lung cancer survival. Reproducible detection of CTCs is limited currently in early stage tumors. METHODS Patients undergoing pulmonary resection had PV blood drawn before resection. Peripheral blood was sampled at preoperative, intraoperative, and postoperative times. Samples were analyzed on microfluidic chips using antibody-based capture. RESULTS A total of 32 patients with primary lung cancer were evaluated. Twenty patients had 1 or more CTCs detected in at least 1 sample (62.5%). The mean number of CTCs from peripheral vein sources at the preoperative, intraoperative, and postoperative time points was 1.3, 1.9, and 0.6 respectively. The average number of CTCs in the PV was 340.0 (range, 0.0-5422.50; P > .01). When PV CTCs were present, the number of CTCs was correlated with pathological tumor size (P = .0236). The number of PV CTCs was not correlated with any other clinical feature (eg, smoking status, preoperative or postoperative stage). Furthermore, the number of PV CTCs was significantly higher when preoperative bronchoscopic biopsy was performed, compared with computed tomography-guided biopsy (P = .0311). Seven patients had evidence of CTC clusters, or microemboli. CONCLUSIONS 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.

Checkpoint kinase 1 protein expression indicates sensitization to therapy by checkpoint kinase 1 inhibition in non-small cell lung cancer.

BACKGROUND When presenting with advanced stage disease, lung cancer patients have <5% 5-y survival. The overexpression of checkpoint kinase 1 (CHK1) is associated with poorer outcomes and may contribute to therapy resistance. Targeting CHK1 with small-molecule inhibitors in p53 mutant tumors might improve the effectiveness of chemotherapy and radiotherapy in non-small cell lung cancer (NSCLC). METHODS We evaluated CHK1 messenger RNA and protein levels in multiple NSCLC cell lines. We assessed cell line sensitization to gemcitabine, pemetrexed, and radiotherapy by CHK1 inhibition with the small molecule AZD7762 using proliferation and clonogenic cell survival assays. We analyzed CHK1 signaling by Western blotting to confirm that AZD7762 inhibits CHK1. RESULTS We selected two p53 mutant NSCLC cell lines with either high (H1299) or low (H1993) CHK1 levels for further analysis. We found that AZD7762 sensitized both cell lines to gemcitabine, pemetrexed, and radiotherapy. Chemosensitization levels were greater, however, for the higher CHK1 protein expressing cell line, H1299, when compared with H1993. Furthermore, analysis of the CHK1 signaling pathway showed that H1299 cells have an increased dependence on the CHK1 pathway in response to chemotherapy. There was no increased sensitization to radiation in H1299 versus H1993. CONCLUSIONS CHK1 inhibition by AZD7762 preferentially sensitizes high CHK1 expressing cells, H1299, to anti-metabolite chemotherapy as compared with low CHK1 expressing H1993 cells. Thus, CHK1 inhibitors may improve the efficacy of standard lung cancer therapies, especially for those subgroups of tumors harboring higher expression levels of CHK1 protein.

Long-term recording from the chorda tympani nerve in rats.

Cuff electrodes with headcap connectors were implanted around the rat chorda tympani nerve. Whole nerve recordings under anesthesia were made from these nerves every week to chemical, thermal and tactile stimuli applied to the anterior tongue. The signal/noise ratio of these recordings was similar to acute recordings from the chorda tympani nerve, and the nerves were spontaneously active. Responses to chemical as well as thermal and mechanical stimulation of the tongue were recorded as early as 2 and 3 weeks after implantation and recordings from the same nerve were made for more than 3 months. These results have demonstrated the feasibility of making long-term chronic recordings of chemosensory activity in the chorda tympani nerve. The cuff electrode has great potential to provide correlative information between neurophysiological and behavioral data.

Poor Prognosis Indicated by Venous Circulating Tumor Cell Clusters in Early-Stage Lung Cancers

Early detection of metastasis can be aided by circulating tumor cells (CTC), which also show potential to predict early relapse. Because of 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 with preoperative Pe (P < 0.0001) and intraoperative 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 preoperative 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. Cancer Res; 77(18); 5194-206. ©2017 AACR.

Abstract 1581: A study of pulmonary and peripheral vein blood as sources of circulating tumor cells in early lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Lung cancer is one of the most prevalent cancers world-wide. It is usually diagnosed at an advanced stage with low survival rates. Early detection and intervention can have a profound impact in improving survival. Circulating tumor cells (CTCs), or cells shed by the tumor into the blood circulation, offer a less invasive method of tumor biopsy. Surgical lobectomy for early stage lung cancers provides access to the pulmonary vein (PV) draining the affected lobe, and is presumably an enriched source of CTCs shed by the primary lung tumor. Analyzing peripheral (Pe) and pulmonary vein (PV) blood combined could potentially shed light onto optimal markers and disease progression. We hypothesize that blood from the pulmonary vein has a higher abundance of CTCs than peripheral blood, thereby providing a larger yield of CTCs from early lung cancer patients for downstream analysis. A cohort of greater than 30 lung cancer patients was enrolled in a pilot study. PV and Pe blood specimens from these patients were obtained at different time points in the perioperative time period, and were processed through a high-throughput microfluidic device, the OncoBean Chip that isolates CTCs by immuno-affinity based capture. A higher number of PV CTCs were detected than from Pe. In addition, rare CTC clusters were observed in the PV blood in some cases, which were further characterized for protein expression. Gene expression analysis was performed for comparison of CTCs obtained from these two different sources. Evaluating different CTC sources along the venous drainage of a tumor could potentially offer an insight into CTC biology and spread of the disease. Citation Format: Vasudha Murlidhar, Rishindra M. Reddy, Ebrahim Azizi, Lili Zhao, Svetlana Grabauskiene, Zhuo Zhang, Nithya Ramnath, Jules Lin, Andrew C. Chang, Philip W. Carrott, William R. Lynch, Mark B. Orringer, Max S. Wicha, Nallasivam Palanisamy, David G. Beer, Sunitha Nagrath. A study of pulmonary and peripheral vein blood as sources of circulating tumor cells in early lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1581. doi:10.1158/1538-7445.AM2015-1581

Abstract 3069: Radial flow microfluidic device for high-throughput affinity-based isolation of circulating tumor cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process accounting for the majority of cancer-related deaths. They offer a non-invasive biopsy technique to study tumors and are shown to be useful prognostic indicators. Because of their rarity in the bloodstream, microfluidic isolation techniques are complex and time-consuming, and provide yields of CTCs insufficient or non-viable for studies other than enumeration. Additionally, due to the low processing speeds, the volumes of blood processed remain limited and can be a hindrance to obtaining higher yields of CTCs and their potential use as biomarkers of early diagnosis. Here we report a novel high throughput microfluidic technology, the OncoBean Chip, employing radial flow that introduces a varying shear profile across the device and enables efficient cell capture by affinity at high flow rates. The cell recovery from whole blood was validated with cancer cell lines H1650 and MCF7, and the OncoBean Chip achieved an efficiency >80% at a throughput of 10 mL/hr, a flow rate yet achieved only in physical size based separation techniques. A cell viability of 92.91% shows that the cells recovered at high throughput could still be used for downstream analysis. Clinical competence was demonstrated in blood specimens from breast, pancreatic and lung cancer patients. The OncoBean Chip thus holds potential applications in the diagnosis of early stage cancers, where the low numbers of CTCs could be enriched using this novel device. Citation Format: Vasudha Murlidhar, Rishindra M. Reddy, Mina Zeinali, Svetlana Grabauskiene, Mostafa Ghannad-Rezaie, Max S. Wicha, Diane M. Simeone, Nithya Ramnath, Sunitha Nagrath. Radial flow microfluidic device for high-throughput affinity-based isolation of circulating tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2014-3069