Peter J. Attayek

Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma.

Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90±8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm(2)) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 µL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm(2)) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization.

In Vitro Polarization of Colonoids to Create an Intestinal Stem Cell Compartment

The polarity of proliferative and differentiated cellular compartments of colonic crypts is believed to be specified by gradients of key mitogens and morphogens. Indirect evidence demonstrates a tight correlation between Wnt- pathway activity and the basal-luminal patterning; however, to date there has been no direct experimental manipulation demonstrating that a chemical gradient of signaling factors can produce similar patterning under controlled conditions. In the current work, colonic organoids (colonoids) derived from cultured, multicellular organoid fragments or single stem cells were exposed in culture to steep linear gradients of two Wnt-signaling ligands, Wnt-3a and R-spondin1. The use of a genetically engineered Sox9-Sox9EGFP:CAGDsRED reporter gene mouse model and EdU-based labeling enabled crypt patterning to be quantified in the developing colonoids. Colonoids derived from multicellular fragments cultured for 5 days under a Wnt-3a or a combined Wnt-3a and R-spondin1 gradient were highly polarized with proliferative cells localizing to the region of the higher morphogen concentration. In a Wnt-3a gradient, Sox9EGFP polarization was 7.3 times greater than that of colonoids cultured in the absence of a gradient; and the extent of EdU polarization was 2.2 times greater than that in the absence of a gradient. Under a Wnt-3a/R-spondin1 gradient, Sox9EGFP polarization was 8.2 times greater than that of colonoids cultured in the absence of a gradient while the extent of EdU polarization was 10 times greater than that in the absence of a gradient. Colonoids derived from single stem cells cultured in Wnt-3a/R-spondin1 gradients were most highly polarized demonstrated by a Sox9EGFP polarization 20 times that of colonoids grown in the absence of a gradient. This data provides direct evidence that a linear gradient of Wnt signaling factors applied to colonic stem cells is sufficient to direct patterning of the colonoid unit in culture.

Isolation and in vitro culture of rare cancer stem cells from patient-derived xenografts of pancreatic ductal adenocarcinoma.

Described is the construction of a large array of releasable microstructures (micropallets) along with screening and isolation protocols for sorting rare, approximately 1 in 10,000, cancer stem cells (CSCs) from a heterogeneous cell population. A 10.1 × 7.1 cm array of micropallets (50 × 50 × 75 μm structures and 25 μm micropallet gap) was fabricated on a large glass substrate, providing an array of approximately 1.3 million releasable microstructures. Image analysis algorithms were developed to permit array screening for identification of fluorescently labeled cells in less than 15 min using an epifluorescent wide-field microscope with a computer controlled translational stage. Device operation was tested by culturing HeLa cells transfected with green fluorescent protein (GFP) admixed with wild-type HeLa cells at ratios of 1:10(4) to 1:10(6) on the array followed by screening to identify flourescent cells. Micropallets containing cells of interest were then selectively released by a focused laser pulse and collected on a numbered poly(dimethylsiloxane) (PDMS) substrate with high viability. A direct comparison of this technology with fluorescence-activated cell sorting (FACS) demonstrated that micropallet arrays offered enhanced post sorting purity (100%), yield (100%), and viability (94-100%) for rare cell isolation. As a demonstration of the technologys value, pancreatic tumor cells from Panc-1 cell lines and patient-derived xenografts were screened for the presence of CD24, CD44, and CD326: surface markers of pancreatic CSCs. Following cell isolation and culture, 63 ± 23% of the isolated Panc-1 cells and 35% of sorted human xenograft cells formed tumor spheroids retaining high expression levels of CD24, CD44, and CD326. The ability to isolate rare cells from relatively small sample sizes will facilitate our understanding of cell biology and the development of new therapeutic strategies.

Array-Based Platform To Select, Release, and Capture Epstein–Barr Virus-Infected Cells Based on Intercellular Adhesion

Microraft arrays were developed to select and separate cells based on a complex phenotype, weak intercellular adhesion, without knowledge of cell-surface markers or intracellular proteins. Since the cells were also not competent to bind to a culture surface, a method to encapsulate nonadherent cells within a gelatin plug on the concave microraft surface was developed, enabling release and collection of the cells without the need for cell attachment to the microraft surface. After microraft collection, the gelatin was liquified to release the cell(s) for culture or analysis. A semiautomated release and collection device for the microrafts demonstrated 100 ± 0% collection efficiency of the microraft while increasing throughput 5-fold relative to that of manual release and collection. Using the microraft array platform along with the gelatin encapsulation method, single cells that were not surface-attached were isolated with a 100 ± 0% efficiency and a 96 ± 4% postsort single-cell cloning efficiency. As a demonstration, Epstein-Barr virus-infected lymphoblastoid cell lines (EBV-LCL) were isolated based on their intercellular adhesive properties. The identified cell colonies were collected with a 100 ± 0% sorting efficiency and a postsort viability of 87 ± 3%. When gene expression analysis of the EBV latency-associated gene, EBNA-2, was performed, there was no difference in expression between blasting or weakly adhesive cells and nonblasting or nonadhesive cells. Microraft arrays are a versatile method enabling separation of cells based on complicated and as yet poorly understood cell phenotypes.

Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9

Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation.

In Vitro Generation of Mouse Colon Crypts

Organoid culture has had a significant impact on in vitro studies of the intestinal epithelium; however, the exquisite architecture, luminal accessibility, and lineage compartmentalization found in vivo has not been recapitulated in the organoid systems. We have used a microengineered platform with suitable extracellular matrix contacts and stiffness to generate a self-renewing mouse colonic epithelium that replicates key architectural and physiological functions found in vivo, including a surface lined with polarized crypts. Chemical gradients applied to the basal-luminal axis compartmentalized the stem/progenitor cells and promoted appropriate lineage differentiation along the in vitro crypt axis so that the tissue possessed a crypt stem cell niche as well as a layer of differentiated cells covering the luminal surface. This new approach combining microengineered scaffolds, native chemical gradients, and biophysical cues to control primary epithelium ex vivo can serve as a highly functional and physiologically relevant in vitro tissue model.

Identification and isolation of antigen-specific cytotoxic T lymphocytes with an automated microraft sorting system

The simultaneous measurement of T cell function with recovery of individual T cells would greatly facilitate characterizing antigen-specific responses both in vivo and in model systems. We have developed a microraft array methodology that automatically measures the ability of individual T cells to kill a population of target cells and viably sorts specific cells into a 96-well plate for expansion. A human T cell culture was generated against the influenza M1p antigen. Individual microrafts on a 70 × 70 array were loaded with on average 1 CD8+ cell from the culture and a population of M1p presenting target cells. Target cell killing, measured by fluorescence microscopy, was quantified in each microraft. The rates of target cell death among the individual CD8+ T cells varied greatly; however, individual T cells maintained their rates of cytotoxicity throughout the time course of the experiment enabling rapid identification of highly cytotoxic CD8+ T cells. Microrafts with highly active CD8+ T cells were individually transferred to wells of a 96-well plate, using a needle-release device coupled to the microscope. Three sorted T cells clonally expanded. All of these expressed high-avidity T cell receptors for M1p/HLA*02:01 tetramers, and 2 of the 3 receptors were sequenced. While this study investigated single T cell cytotoxicity rates against simple targets with subsequent cell sorting, future studies will involve measuring T cell mediated cytotoxicity in more complex cellular environments, enlarging the arrays to identify very rare antigen specific T cells, and measuring single cell CD4+ and CD8+ T cell proliferation.

A dosimetric model for the heterogeneous delivery of radioactive nanoparticles In vivo: A feasibility study

AbstractᅟAccurate and quantitative dosimetry for internal radiation therapy can be especially challenging, given the heterogeneity of patient anatomy, tumor anatomy, and source deposition. Internal radiotherapy sources such as nanoparticles and monoclonal antibodies require high resolution imaging to accurately model the heterogeneous distribution of these sources in the tumor. The resolution of nuclear imaging modalities is not high enough to measure the heterogeneity of intratumoral nanoparticle deposition or intratumoral regions, and mathematical models do not represent the actual heterogeneous dose or dose response. To help answer questions at the interface of tumor dosimetry and tumor biology, we have modeled the actual 3-dimensional dose distribution of heterogeneously delivered radioactive nanoparticles in a tumor after systemic injection.Methods24 h after systemic injection of dually fluorescent and radioactive nanoparticles into a tumor-bearing mouse, the tumor was cut into 342 adjacent sections and imaged to quantify the source distribution in each section. The images were stacked to generate a 3D model of source distribution, and a novel MATLAB code was employed to calculate the dose to cells on a middle section in the tumor using a low step size dose kernel.ResultsThe average dose calculated by this novel 3D model compared closely with standard ways of calculating average dose, and showed a positive correlation with experimentally determined cytotoxicity in vivo. The high resolution images allowed us to determine that the dose required to initiate radiation-induced H2AX phosphorylation was approximately one Gray. The nanoparticle distribution was further used to model the dose distribution of two other radionuclides.ConclusionsThe ability of this model to quantify the absorbed dose and dose response in different intratumoral regions allows one to investigate how source deposition in different tumor areas can affect dose and cytotoxicity, as well as how characteristics of the tumor microenvironment, such as hypoxia or high stromal areas, may affect the potency of a given dose.