Nano-volume well array chip for large-scale propagation and high-resolution analysis of individual cancer stem cells

Copyright: © 2014 Clausell-Tormos J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Cellular heterogeneity represents an increasingly appreciated aspect for research in life science. To address this issue, we have developed a nano-volume well array chip that allows larger-scale isolation and propagation of single cells. Notably, the chip enables single-cell analysis of freshly isolated primary cells at a high-resolution. With an average height of 130 ± 10 μm and an average diameter of 80 ± 10 μm, each nano-volume well can hold up to 0.4 nL of volume, and is compatible with both adherent as well as 3D suspension cultures. Simultaneous time-lapse imaging of thousands of nano-volume wells allows to monitor cell division, as well as tracking of cell fate, and/or alterations in the microscopic cellular morphology and/or markers expression. To demonstrate its application, we employed the system for propagating and tracking of Cancer Stem Cells (CSCs). CSCs could be monitored over three consecutive days by time-lapse high-resolution imaging at the single-cell level. We could demonstrate that non-CSCs do not dedifferentiate into CSCs, while CSCs were able to give rise to both CSCs and non-CSCs by undergoing symmetric and asymmetric division, respectively. Altogether, we have developed a novel nano-volume well array chip that significantly ameliorates clonal propagation and high-resolution image analysis of rare cells

Statistical Modeling of Single Target Cell Encapsulation

High throughput drop-on-demand systems for separation and encapsulation of individual target cells from heterogeneous mixtures of multiple cell types is an emerging method in biotechnology that has broad applications in tissue engineering and regenerative medicine, genomics, and cryobiology. However, cell encapsulation in droplets is a random process that is hard to control. Statistical models can provide an understanding of the underlying processes and estimation of the relevant parameters, and enable reliable and repeatable control over the encapsulation of cells in droplets during the isolation process with high confidence level. We have modeled and experimentally verified a microdroplet-based cell encapsulation process for various combinations of cell loading and target cell concentrations. Here, we explain theoretically and validate experimentally a model to isolate and pattern single target cells from heterogeneous mixtures without using complex peripheral systems.Wallace H. Coulter Foundation (Young Investigator in Bioengineering Award)National Institutes of Health (U.S.) (Grant R01AI081534)National Institutes of Health (U.S.) (Grant R21AI087107

Efficacy of Sym004 in Patients With Metastatic Colorectal Cancer With Acquired Resistance to Anti-EGFR Therapy and Molecularly Selected by Circulating Tumor DNA Analyses: A Phase 2 Randomized Clinical Trial.

IMPORTANCE: Acquired resistance to anti-EGFR therapy (epidermal growth factor receptor) is frequently due to RAS and EGFR extracellular domain (ECD) mutations in metastatic colorectal cancer (mCRC). Some anti-EGFR-refractory patients retain tumor EGFR dependency potentially targetable by agents such as Sym004, which is a mixture of 2 nonoverlapping monoclonal antibodies targeting EGFR. OBJECTIVE: To determine if continuous blockade of EGFR by Sym004 has survival benefit. DESIGN, SETTING, AND PARTICIPANTS: Multicenter, phase 2, randomized, clinical trial comparing 2 regimens of Sym004 with investigator's choice from March 6, 2014, through October 15, 2015. Circulating tumor DNA (ctDNA) was analyzed for biomarker and tracking clonal dynamics during treatment. Participants had wild-type KRAS exon 2 mCRC refractory to standard chemotherapy and acquired resistance to anti-EGFR monoclonal antibodies. INTERVENTIONS: Participants were randomly assigned in a 1:1:1 ratio to Sym004, 12 mg/kg/wk (arm A), Sym004, 9 mg/kg loading dose followed by 6 mg/kg/wk (arm B), or investigator's choice of treatment (arm C). MAIN OUTCOMES AND MEASURES: Overall survival (OS). Secondary end points included preplanned exploratory biomarker analysis in ctDNA. RESULTS: A total of 254 patients were randomized (intent-to-treat [ITT] population) (median age, 63 [range, 34-91] years; 63% male; n\u2009=\u2009160). Median OS in the ITT population was 7.9 months (95% CI, 6.5-9.9 months), 10.3 months (95% CI, 9.0-12.9 months), and 9.6 months (95% CI, 8.3-12.2 months) for arms A, B, and C, respectively (hazard ratio [HR], 1.31; 95% CI, 0.92-1.87 for A vs C; and HR, 0.97; 95% CI, 0.68-1.40 for B vs C). The ctDNA revealed high intrapatient genomic heterogeneity following anti-EGFR therapy. Sym004 effectively targeted EGFR ECD-mutated cancer cells, and a decrease in EGFR ECD ctDNA occurred in Sym004-treated patients. However, this did not translate into clinical benefit in patients with EGFR ECD mutations, likely owing to co-occurring resistance mechanisms. A subgroup of patients was defined by ctDNA (RAS/BRAF/EGFR ECD-mutation negative) associated with improved OS in Sym004-treated patients in arm B compared with arm C (median OS, 12.8 and 7.3 months, respectively). CONCLUSIONS AND RELEVANCE: Sym004 did not improve OS in an unselected population of patients with mCRC and acquired anti-EGFR resistance. A prospective clinical validation of Sym004 efficacy in a ctDNA molecularly defined subgroup of patients with refractory mCRC is warranted. TRIAL REGISTRATION: clinicaltrialsregister.eu Identifier: 2013-003829-29

Microdroplet-Enabled Highly Parallel Co-Cultivation of Microbial Communities

Microbial interactions in natural microbiota are, in many cases, crucial for the sustenance of the communities, but the precise nature of these interactions remain largely unknown because of the inherent complexity and difficulties in laboratory cultivation. Conventional pure culture-oriented cultivation does not account for these interactions mediated by small molecules, which severely limits its utility in cultivating and studying “unculturable” microorganisms from synergistic communities. In this study, we developed a simple microfluidic device for highly parallel co-cultivation of symbiotic microbial communities and demonstrated its effectiveness in discovering synergistic interactions among microbes. Using aqueous micro-droplets dispersed in a continuous oil phase, the device could readily encapsulate and co-cultivate subsets of a community. A large number of droplets, up to ∼1,400 in a 10 mm×5 mm chamber, were generated with a frequency of 500 droplets/sec. A synthetic model system consisting of cross-feeding E. coli mutants was used to mimic compositions of symbionts and other microbes in natural microbial communities. Our device was able to detect a pair-wise symbiotic relationship when one partner accounted for as low as 1% of the total population or each symbiont was about 3% of the artificial community