Microfluidic Biopsy Trapping Device for the Real-Time Monitoring of Tumor Microenvironment

<div><p>The tumor microenvironment is composed of cellular and stromal components such as tumor cells, mesenchymal cells, immune cells, cancer associated fibroblasts and the supporting extracellular matrix. The tumor microenvironment provides crucial support for growth and progression of tumor cells and affects tumor response to therapeutic interventions. To better understand tumor biology and to develop effective cancer therapeutic agents it is important to develop preclinical platforms that can faithfully recapitulate the tumor microenvironment and the complex interaction between the tumor and its surrounding stromal elements. Drug studies performed in vitro with conventional two-dimensional cancer cell line models do not optimally represent clinical drug response as they lack true tumor heterogeneity and are often performed in static culture conditions lacking stromal tumor components that significantly influence the metabolic activity and proliferation of cells. Recent microfluidic approaches aim to overcome such obstacles with the use of cell lines derived in artificial three-dimensional supportive gels or micro-chambers. However, absence of a true tumor microenvironment and full interstitial flow, leads to less than optimal evaluation of tumor response to drug treatment. Here we report a continuous perfusion microfluidic device coupled with microscopy and image analysis for the assessment of drug effects on intact fresh tumor tissue. We have demonstrated that fine needle aspirate biopsies obtained from patient-derived xenograft models of adenocarcinoma of the lung can successfully be analyzed for their response to ex vivo drug treatment within this biopsy trapping microfluidic device, wherein a protein kinase C inhibitor, staurosporine, was used to assess tumor cell death as a proof of principle. This approach has the potential to study tumor tissue within its intact microenvironment to better understand tumor response to drug treatments and eventually to choose the most effective drug and drug combination for individual patients in a cost effective and timely manner.</p></div

Microfluidic device design.

<p>(A) Exploded view of the device showing the main body made from PDMS containing the 1 mm diameter inlet and outlets punched for each of the 10 channels. The main body is then sealed to a 25 x 75 mm glass slide by plasma gas treatment. Tubing is inserted into each of the inlet and outlets of the device. (B) Fully assembled PDMS prototype. (C) Solid Works rendering of a channel in the device showing the central post arrangement used to trap each FNAB tissue sample. The channel is 10 mm long, 600 μm wide and 125 μm in height. Each post is 150 μm long, 75 μm wide and 125 μm in height.</p

Assessment of small molecule drug perfusion in a tumor FNA fragment using Doxorubicin HCL.

<p>(A) Drug perfusion after an 8-hour period showing distribution of drug indicated by green fluorescent intensity spatially similar throughout the FNAB tissue sample. (B) Drug perfusion after a 24-hour period showing intensity peaks spatially similar throughout the FNAB tissue sample and the formation of apoptotic bodies.</p

Calcein AM dye validation and FNAB sample viability assessment.

<p>Calcein AM dye validation and FNAB sample viability assessment.</p

Comparison of the calculated viability index of the tumor FNAB samples during 5 days of treatment.

<p>(A) Comparison of the mean averaged VI of negative control group and staurosporine treated group from the baseline over the 5 day exposure period with n = 18 within each group. The difference in means between each treatment group was statistically significant, except for the baseline. (B.1, B.3) A 3-dimensional view of a representative FNAB sample from the negative control group at day 1 and day 5 respectively. (B.2, B.4) Histogram of the relative intensity values across the negative control FNAB samples for day 1 and day 5, respectively. (C.1, C.3) A 3-dimensional view of a representative FNAB sample from the 50 μM staurosporine treated group at day 1 and day 5 respectively. (C.2, C.4) Histogram of the relative intensity values across the 50 μM staurosporine treated FNAB samples for day 1 and day 5 respectively.</p

Evaluation of antibody perfusion through the tumor FNAB on device.

<p>(A) 10x Phase contrast image of FNAB sample in trap of device and 10x fluorescent z-axis images (z5, z7, z9, z11) 24 hours post the staining procedure using isotype control antibodies for both EpCAM (red-Cy5) and CD44 (green-FITC). (B) 10x Phase contrast image of FNAB sample in trap of device and 10x fluorescent z-axis images (z5, z7, z9, z11) 24 hours post the staining procedure using EpCAM (red-Cy5) and CD44 (green-FITC).</p

HDACi treatment specifically inhibits CHK1 expression and upregulates its downstream signaling proteins CDC25A, CDC25C, and CDC2, involved in G₂ cell cycle checkpoint control.

<p><i>A</i>, A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of CDC2 (pCDC2 ^Y15), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. <i>B</i>, PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 ^Y15), serine-216 phosphorylation of CDC25C (pCDC25c ^S216), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. <i>C</i>, drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. <i>D</i>, PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. <i>E</i>, A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 ^Y15, and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.</p

Purvalanol A (Pur A) pretreatment diminishes the cytotoxic effect of LBH589 in A549 cells.

<p>Cells treated with LBH589 40 nM with or without Pur A (10 µM) pretreatment were analyzed by flow cytometry for cell cycle distribution or by Western blot to determine drug-mediated changes in cPARP. β-actin was used as a loading control.</p

LBH589 treatment leads to mitotic abnormalities and cytokinesis failures.

<p>A549 cells were treated with vehicle (control) or 40 nM LBH589 for 24 hours. <i>A</i>, arrows show bi- or multinucleated cells with impaired cytokinesis in LBH589-treated NSCLC cells. <i>B</i>, cells were fixed and stained with DAPI or cleaved poly (ADP-ribose) polymerase (cPARP) antibody (×100 or ×400 magnification). <i>C</i>, Western blot analysis demonstrating that HDAC inhibition by LBH589 causes histone H3 phosphorylation (H3-P10), histone H4 acetylation (Acety-H4), and PARP cleavage (cPARP) in A549 cells treated with LBH589 for 24 hours. β-actin was used as loading control.</p

LBH589 and Chk1 inhibitor treatment shows a synergistic effect in NSCLC cells.

<p><i>A</i>, A549 cells were treated with LBH589 40 nM and/or a UCN-01 (250 nM) for 24 hours, cell extracts were prepared, and Western blot analysis was performed with PARP (t: total, c: cleaved). Experiments were repeated at least 3 times, and a representative experiment is shown. <i>B</i>, A549 cells were treated with LBH589 and UCN-01 either alone or in combination at a constant ratio (1∶40) for 72 hours. Drug concentrations are indicated on the horizontal axis and plotted against cell viability of control wells, which was arbitrarily set at 100% viability for each experiment. Error bars represent ± SD of 4 replicate wells. <i>C</i>, combined effects of LBH589 and Chk1 inhibitor UCN-01 were quantified with the Chou and Talalay combination index (CI) method (40). The CI used for drug combination analyses was determined by the isobologram equation (see text). Ranking symbols (+/−) indicate average calculated Chou and Talalay combination index (CI) range (+++, strong synergism).</p