Jose Ignacio Varillas

Circulating tumor cells (CTCs) are an important biomarker for cancer prognosis and treatment monitoring. However, the heterogeneity of the physical and biological properties of CTCs limits the efficiency of various approaches used to isolate small numbers of CTCs from billions of normal blood cells. To address this challenge, we developed a lateral filter array microfluidic (LFAM) device to integrate size‐based separation with immunoaffinity‐based CTC isolation. The LFAM device consists of a serpentine main channel, through which most of a sample passes, and an array of lateral filters for CTC isolation. The unique device design produces a two‐dimensional flow, which reduces nonspecific, geometric capture of normal cells as typically observed in vertical filters. The LFAM device was further functionalized by immobilizing antibodies that are specific to the target cells. The resulting devices captured pancreatic cancer cells spiked in blood samples with (98.7±1.2) % efficiency and were used to isolate CTCs from patients with metastatic colorectal cancer.

Circulating tumor cells (CTCs) are an important biomarker for cancer prognosis and treatment monitoring. However, the heterogeneity of the physical and biological properties of CTCs limits the efficiency of various approaches used to isolate small numbers of CTCs from billions of normal blood cells. To address this challenge, we developed a lateral filter array microfluidic (LFAM) device to integrate size‐based separation with immunoaffinity‐based CTC isolation. The LFAM device consists of a serpentine main channel, through which most of a sample passes, and an array of lateral filters for CTC isolation. The unique device design produces a two‐dimensional flow, which reduces nonspecific, geometric capture of normal cells as typically observed in vertical filters. The LFAM device was further functionalized by immobilizing antibodies that are specific to the target cells. The resulting devices captured pancreatic cancer cells spiked in blood samples with (98.7±1.2) % efficiency and were used to isolate CTCs from patients with metastatic colorectal cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) requires multimodal therapeutic approaches and disease monitoring for effective treatment. Liquid biopsy biomarkers, including circulating tumor cells (CTCs) and cancer stem-like cells (CSCs), hold promise for evaluating treatment response promptly and guiding therapeutic modifications.

The characterization of single cells within heterogeneous populations has great impact on both biomedical sciences and cancer research. By investigating cellular compositions on a broad scale, pertinent outliers may be lost in the sample set. Alternatively, an investigation focused on the behavior of specific cells, such as circulating tumor cells (CTCs), will reveal genetic biomarkers or phenotypic characteristics associated with cancer and metastasis. On average, CTC concentration in peripheral blood is extremely low, as few as one to two per billion of healthy blood cells. Consequently, the critical element lacking in many methods of CTC detection is accurate cell capture efficiency at low concentrations. To simulate CTC isolation, researchers usually spike small amounts of tumor cells to healthy blood for separation. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. We report our study on an innovative apparatus and method designed for low-cost, precise, and replicable single-cell spiking (SCS). Our SCS method operates solely from capillary aspiration without the reliance on external laboratory equipment. To ensure that our method does not affect the viability of each cell, we investigated the effects of surface membrane tensions induced by aspiration. Finally, we performed affinity-based CTC isolation using human acute lymphoblastic leukemia cells (CCRF-CEM) spiked into healthy whole blood with the SCS technique. The results of the isolation experiments demonstrate the reliability of our method in generating low-concentration cell samples.

304 Background: Circulating tumor cells (CTCs) represent real time dissemination of solid tumors. Historically, CTC enumeration has been challenging to perform successfully in pancreatic cancer (PCa). Using a previously published microfluidic device designed to increase cell capture, we analyzed CTC characteristics in a cohort of newly diagnosed patients with metastatic PCa. Methods: After provision of informed consent, blood samples (7-10mL) were collected within 1 month of treatment initiation. Specimens were processed within 6 hours of collection. A UF developed microfluidic device was used to capture and identify EpCam+ CTCs. Isolated cells were verified as CTCs consistent with FDA-approved definitions (cytokeratin+, DAPI+, CD45-). Statistical analysis was performed using Scheffe’s method along with analysis of variance to test interactions between the clinical demographics and groups. The study was approved by the UF IRB. Results: A cohort of 27 patients had CTC determination at treatment onset with multi-agent chemotherapy for metastatic PCa. Cohort characteristics included: median age 65 (range 29-76), 63% male, 92% white, 85% with intact primary pancreatic tumor, pancreatic tumor location: head (n = 12), body (n = 5), tail (n = 6), number of sites of metastasis: 1 (n = 18), 2 (n = 7), 3 (n = 2), and median albumin 3.9 g/dl. Median CTC for the entire cohort was 7/mL of blood (range 2-12). CTCs were detected and quantified in all patients analyzed. In those with intact primary tumors, there were no significant differences in CTCs based on age (≥65), sex, albumin (≥3.5 g/dl), or number of metastatic organ sites (1, 2, or 3). However, primary tumor location in the head vs. tail was different (mean 7.7 vs. 4.3/mL; p = 0.02; CI -5.78 – [-] 0.41). Conclusions: This analysis demonstrates the feasibility of measuring CTCs in patients with metastatic PCa. CTC amount does not appear impacted by systemic tumor burden or demographic differences. However, anatomic differences in primary tumor location may be important in CTC studies. Further analysis of a larger and more diverse patient data set is planned.

318 Background: The collection and enumeration of circulating tumor cells (CTCs) are feasible in metastatic breast, prostate and colorectal cancers. CTCs have diagnostic, prognostic and treatment monitoring implications, but have been challenging to detect in pancreatic cancer (PCa). We have proven feasibility of CTCs in PCa. This study is a pilot to assess use of CTCs as a surrogate for imaging response. Methods: Blood samples (7-10mL) from newly diagnosed metastatic PCa were collected within 1 month of treatment initiation (T₀) and at repeat scan after 2mo of treatment (T₁). Specimens were processed within 6 hours of collection. A UF developed microfluidic device was used to capture and identify EpCam+ CTCs. Isolated cells were verified as CTCs consistent with FDA-approved definitions (cytokeratin+, DAPI+, CD45-). CTC enumeration and changes were correlated with scan RECIST results at T₀ and T₁. The study was approved by the UF IRB. Results: A total of 16 patients had matched pair CTCs collected and analyzed (Table 1). For the entire cohort, the median CTC count at T₀ was 7/mL of blood (range 2-11.5/mL) and decreased to 4.5/mL (range 2-8.5/mL) at T₁ (36% reduction). CTCs were reduced to some degree in all patients with therapy. Disease control at T₁ was confirmed in 13/16 (81%) with stable disease (n=11), partial (n=1) or complete (n=1) response. Progressive disease was noted in 3 patients. CTCs were reduced by 35% in radiographic stable or responders vs 24% in those with clear progressive disease. Conclusions: This study demonstrated the feasibility of microfluidic technology to reliably and consistently identify CTCs in advanced PCa. While all patients had reduced CTCs with therapy, a trend towards higher reductions were in those with radiographic stable or improved disease. Continued assessment of CTCs in PCa for treatment monitoring are warranted and are ongoing. [Table: see text]

Circulating tumor cells (CTCs) have shown potential for cancer diagnosis and prognosis. Affinity-based CTC isolation methods have been proved to be efficient for CTC detection in clinical blood samples. One of the popular choices for affinity-based CTC isolation is to immobilize capture agents onto an array of microposts in microchannels, providing high CTC capture efficiency due to enhanced interactions between tumor cells and capture agents on the microposts. However, how the cells interact with microposts under different flow conditions and what kind of capture pattern results from the interactions have not been fully investigated; a full understanding of these interactions will help to design devices and choose experimental conditions for higher CTC capture effeciency. We report our study on their interaction and cell distribution patterns around microposts under different flow conditions. Human acute lymphoblastic leukemia cells (CCRF-CEM) were used as target cancer cells in this study, while the Sgc8 aptamer that has specific binding with CCRF-CEM cells was employed as a capture agent. We investigated the effects of flow rates and micropost shapes on the cell capture efficiency and capture patterns on microposts. While a higher flow rate decreased cell capture efficiency, we found that the capture pattern around microposts also changed, with much more cells captured in the front half of a micropost than at the back half. We also found the ratio of cells captured on microposts to the cells captured by both microposts and channel walls increased as a function of the flow rate. We compared circular microposts with an elliptical shape and found that the geometry affected the capture distribution around microposts. In addition, we have developed a theoretical model to simulate the interactions between tumor cells and micropost surfaces, and the simulation results are in agreement with our experimental observation.

OBJECTIVES/SPECIFIC AIMS: The goal of this research is to use circulating tumor cells (CTC) enumeration and characterization to monitor anticancer treatment response. Emerging evidence strongly suggests the implications that epithelial-to-mesenchymal transition may have in cancer metastasis. Consequently, we hope to elucidate the significance of mesenchymal and stem-like CTCs in the peripheral blood of metastatic pancreatic cancer patients by analyzing the prevalence and frequency trends of CD133+ CTCs, as they relate to clinical events. We also hope to determine if there is a correlation between EpCAM+ CTCs and CD133+ CTCs numbers with tumor size, disease stage, and patient clinical outcome. METHODS/STUDY POPULATION: Blood samples of patients with metastatic pancreatic cancer (stage IV) were obtained from the University of Florida Health Cancer Center after informed consent through an IRB-approved protocol. CTC capture, characterization, and enumeration was performed on the blood of these cancer patients during their anticancer treatment. Patients had blood drawn for this purpose at time points aligned with clinical phlebotomy (every 2 weeks). CTC capture was performed by introducing treated patient blood samples into antibody-functionalized microdevices. The PDMS devices were functionalized by immobilizing either anti-EpCAM or anti-CD133, through an avidin-biotin complex. After capture, cells were fixated and permeabilized with 4% paraformaldehyde and 0.2% Triton X-100, respectively. Three-color immunocytochemistry (anti-cytokeratin-FITC, anti-CD45-PE, and DAPI) was performed to identify CTCs from nonspecifically captured blood cells. To be counted as a CTC, based on the FDA-approved technical definition, a cell with the appropriate cell size and morphology must be nucleated (DAPI+), express cytokeratin (CK+), and lack the leukocytic CD45 marker (CD45−). RESULTS/ANTICIPATED RESULTS: We tested the clinical utility of the device for monitoring the response of patients with advanced pancreatic cancer to a chemotherapy treatment consisting of anticancer drugs including 5-fluorouracil, leucovorin, oxaliplatin, and dasatinib. We have detected EpCAM+ CTCs in 47/47 (100%) and CD133+ CTCs in 41/47 (87.2%) of blood samples, coming from a cohort of 16 patients. We studied the correlation between the CTC numbers and the clinical result of patients in the study. We found that the size and changes in the size of the primary tumor (confirmed by CT scans) correlated with the frequency and increase/decrease trends in the number of CTCs detected. We expect to find some relationship between the number of detected CD133+ CTCs, or rather stem-like CTCs, and the clinical outcome of patients (eg, disease progression leading to withdrawal from study). DISCUSSION/SIGNIFICANCE OF IMPACT: Enumeration of patient CTCs and stem-like CTCs at different stages of a patient’s treatment may correlate with disease stage and prognosis, and prove useful in monitoring early recurrence, patient-specific treatment response, and newly acquired resistances; all of which would aid in providing guidance for the next step in clinical intervention. This type of liquid biopsy technology has great potential to make an impact in the future of personalized medicine and point-of-care diagnostics, as well as become a sturdy tool for translational research.