Micromilling is an underutilized technique for fabricating microfluidic platforms that is well-suited for the diverse needs of the biologic community. This technique, however, produces culture surfaces that are considerably rougher than in commercially available culture platforms and the hydrophilicity of these surfaces can vary considerably depending on the choice of material. In this study, we evaluated the impact of surface topography and hydrophilicity in milled microfluidic devices on the cellular phenotype and function of primary human macrophages. We found that the rough culture surface within micromilled systems affected the phenotype of macrophages cultured in these devices. However, the presence, type, and magnitude of this effect was dependent on the surface hydrophilicity as well as exposure to chemical polarization signals. These findings confirm that while milled microfluidic systems are an effective platform for culture and analysis of primary macrophages, the topography and hydrophilicity of the culture surface within these systems should be considered in the planning and analysis of any macrophage experiments in which phenotype is relevant.

Deregulation of apoptotic pathways plays a central role in cancer pathogenesis. X-linked inhibitor of apoptosis protein (XIAP), is an antiapoptotic molecule, whose elevated expression has been observed in tumor specimens from patients with prostate carcinoma. Studies in human cancer cell culture models and xenograft tumor models have demonstrated that loss of XIAP sensitizes cancer cells to apoptotic stimuli and abrogates tumor growth. In view of these findings, XIAP represents an attractive antiapoptotic therapeutic target for prostate cancer. To examine the role of XIAP in an immunocompetent mouse cancer model, we have generated transgenic adenocarcinoma of the mouse prostate (TRAMP) mice that lack XIAP. We did not observe a protective effect of Xiap deficiency in TRAMP mice as measured by tumor onset and overall survival. In fact, there was an unexpected trend toward more aggressive disease in the Xiap-deficient mice. These findings suggest that alternative mechanisms of apoptosis resistance are playing a significant oncogenic role in the setting of Xiap deficiency. Our study has implications for XIAP-targeting therapies currently in development. Greater understanding of these mechanisms will aid in combating resistance to XIAP-targeting treatment, in addition to optimizing selection of patients who are most likely to respond to such treatment. Cell Death and Differentiation (2008) Apoptosis is a process of cell death that is tightly regulated by a cadre of both pro-and antiapoptotic proteins. In contrast to healthy cells, a hallmark of cancerous cells is the acquired capacity to evade this process of programmed cell death. 1,2 The acquisition of genetic lesions leading to oncogene activation normally triggers a program of apoptosis or senescence. Additionally, the tumor microenvironment often exposes malignant cells to apoptotic stimuli, such as hypoxia or activation of death receptors. Thus, suppression of the pathway leading to cell death has been suggested as a necessarily early event in the development of neoplasia.Execution of the apoptotic cell death process is carried out by caspases, a family of cysteine aspartate proteases. 3,4 During apoptosis, loss of mitochondrial integrity or engagement of death receptors leads to the activation of initiator caspase-9 or -8, respectively. In either case, the initiator caspases cleave and activate effector caspases, including caspase-3 or -7. The cascade of caspase cleavage is regulated by X-linked inhibitor of apoptosis protein (XIAP). XIAP belongs to the IAP family, characterized by containing at least one zinc-binding baculovirus IAP repeat. 5 The only member of the IAP family that potently inhibits caspase activity, XIAP has been demonstrated to directly inhibit caspases-3, -7 and -9, blocking both intrinsic and extrinsic apoptotic signals. 6 Given its role in apoptosis, there has been much interest in understanding the role of XIAP in cancer and evaluating XIAP as a therapeutic target. [7][8][9] XIAP overexpression has been reported in a variety of human ca...

Background Neuroendocrine prostate cancer (NEPC) is an aggressive subtype, the presence of which changes the prognosis and management of metastatic prostate cancer. Methods We performed analytical validation of a Circulating Tumor Cell (CTC) multiplex RNA qPCR assay to identify the limit of quantification (LOQ) in cell lines, synthetic cDNA, and patient samples. We next profiled 116 longitudinal samples from a prospectively collected institutional cohort of 17 patients with metastatic prostate cancer (7 NEPC, 10 adenocarcinoma) as well as 265 samples from 139 patients enrolled in 3 adenocarcinoma phase II trials of androgen receptor signaling inhibitors (ARSIs). We assessed a NEPC liquid biomarker via the presence of neuroendocrine markers and the absence of androgen receptor (AR) target genes. Results Using the analytical validation LOQ, liquid biomarker NEPC detection in the longitudinal cohort had a per-sample sensitivity of 51.35% and a specificity of 91.14%. However, when we incorporated the serial information from multiple liquid biopsies per patient, a unique aspect of this study, the per-patient predictions were 100% accurate, with a receiver-operating-curve (ROC) AUC of 1. In the adenocarcinoma ARSI trials, the presence of neuroendocrine markers, even while AR target gene expression was retained, was a strong negative prognostic factor. Conclusion Our analytically validated CTC biomarker can detect NEPC with high diagnostic accuracy when leveraging serial samples that are only feasible using liquid biopsies. Patients with expression of NE genes while retaining AR-target gene expression may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype. Funding NIH (DP2 OD030734, 1UH2CA260389, R01CA247479, and P30 CA014520), Department of Defense (PC190039 and PC200334), and Prostate Cancer Foundation (Movember Foundation — PCF Challenge Award).

Immune tolerance to self-antigens can limit robust anti-tumor immune responses in the use of tumor vaccines. Expression of novel tumor associated antigens can improve immune recognition and lysis of tumor cells. The cancer-testis antigen (CTA) family of proteins has been hypothesized to be an ideal class of antigens due to tumor-restricted expression, a subset of which have been found to induce antibody responses in patients with prostate disease. We demonstrate that CTA expression is highly inducible in five different Prostate Cancer (PC) cell lines using a hypomethylating agent 5-Aza-2′-deoxycytidine (5AZA) and/or a histone deacetylase inhibitor LBH589. These CTAs include NY-ESO1, multiple members of the MAGE and SSX families and NY-SAR35. A subset of CTAs is synergistically induced by the combination of 5AZA and LBH589. We developed an ex vivo organ culture using human PC biopsies for ex vivo drug treatments to evaluate these agents in clinical samples. These assays found significant induction of SSX2 in 9/9 distinct patient samples and NY-SAR35 in 7/9 samples. Further, we identify expression of SSX2 in circulating tumor cells (CTC) from patients with advanced PC. These results indicate that epigenetic modifying agents can induce expression of a broad range of neoantigens in human PC and may serve as a useful adjunctive therapy with novel tumor vaccines and checkpoint inhibitors.