Systematic Analysis of Different Cell Spheroids with a Microfluidic

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Systematic Analysis of Different Cell Spheroids with a Microfluidic Device Using Scanning Electrochemical Microscopy and Gene Expression Profiling Liang Zhao, Mi Shi, Yang Liu, Xiaonan Zheng, Jidong Xiu, Yingying Liu, Lu Tian, Hongjuan Wang, Meiqin Zhang, and Xueji Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00376 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019

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Analytical Chemistry

Systematic Analysis of Different Cell Spheroids with a Microfluidic Device Using Scanning Electrochemical Microscopy and Gene Expression Profiling

Liang Zhao†*, Mi Shi†, Yang Liu, Xiaonan Zheng, Jidong Xiu, Yingying Liu, Lu Tian, Hongjuan Wang, Meiqin Zhang*, Xueji Zhang*,

Institute of Precision Medicine and Health, Research Center for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, Beijing Key Laboratory for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing 100083, China.

Author information: †: Contributed equally to this work and are considered as co-first authors *: Corresponding Authors: E-mail: [email protected] E-mail: [email protected] E-mail: [email protected]

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: The 3D cell spheroid is an emerging tool that allows better recapitulating in vivo scenario with multiple factors, such as tissue-like morphology, and membrane protein expression that intimately coordinates with enzyme activity, thus providing a psychological environment for tumorigenesis study. For analyzing different spheroids, conventional optical imaging may be hampered by the need of fluorescent labeling, which could cause toxicity side effects. As an alternative approach, scanning electrochemical microscopy (SECM) enables the label-free imaging. However, SECM for cell spheroid imaging is currently suffering from incapability of systematically analyzing the cell aggregates from spheroids generation, electrochemical signal gaining, and the gene expression on different individual cell spheroids. Herein, we developed a top-removable microfluidic device for cell aggregates yielding and SECM imaging methodology to analyze heterotypic 3D cell spheroids on a single device. This technique allows not only on-chip culturing cell aggregates but also SECM imaging of the spheroids after opening the chip and subsequent qPCR assay of corresponding clusters. Employing the micro-pits arrays (85x4) with a top withdrawable microfluidic layer, uniformly sized breast tumor cell and fibroblasts spheroids can be simultaneously produced on a single device. By leveraging voltage-switching mode SECM (VSM-SECM) at different potentials of dual mediators, we evaluated alkaline phosphatase (ALP) without disturbance of substrate morphology for distinguishing the tumor aggregates from stroma. Moreover, this method also enables


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gene expression profiling on individual tumor or stromal spheroids. Therefore, this new strategy can seamlessly bridge SECM measurements and molecular biological analysis.

Main text Conventionally, the monolayer cancer cells are well studied for understanding tumor development.1 This two dimensional (2D) cell niche based method, however, suffered from inherent limitations for lacking physiological three dimensional (3D) scenario that is naturally related to tumor development.2, 3 Thus, for more accurately recapitulating tumor architecture, the implementation of 3D in vitro tumor models may serve as a new strategy that enables researchers to explore cancer drug response,4 tumorigenesis,5 and other tumor biology.6 As a 3D tumor model, the non-scaffold-based tumor cultures are self-assembled from tumor cells, commonly known as spheroids, which produces several common features that are similar with the solid tumor in vivo such as cellular heterogeneity, cell-cell signaling, hypoxia, membrane protein distribution, and gene expression patterns.7-10 Many new technologies to generate cell spheroids have been developed.11-13 Among these methods, micro-fabrication based spheroids generation is considered as a promising platform due to the large-scale production of well-controlled aggregates with homogeneous morphology under highly reproducible conditions.14,

15

However, due to the thickness of the cell spheroid, turbid solution and debris, and the need to label non-fluorescent molecules such as membrane proteins, which could disturb the regulation of gene expression, the optical observation is hard to detour the bottleneck for tagging cell aggregates.16 Moreover, such characterization is required for gaining



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quantitative biological data such as corresponding gene expression. Therefore, there is an urgent demand for developing alternative ways to explore cellular spheroids. In contrast with fluorescence labeling for quantification of cellular protein, scanning electrochemical microscopy (SECM) allows for in-situ observation of single cell, embryonic stem cell spheroid,20 human tissue

21, 22

17-19

without interfering with cell’s

environment or metabolism.23-25 Regarding tumor study, researchers have employed SECM to study 2D cultured tumor in many aspects, including multidrug resistance,26, 27 membrane protein expression,28, 29 internalization,30 and the glucose uptaken.31 Recently, SECM has also been employed for detecting cellular senescence,32 and label-free dead/live assay on 3D tumor model.33 However, although most of these methods are readily applicable to investigate homo-typical tumor cells or cell aggregates, in-depth characterization of the tumor/non-tumor cell spheroids from label-free SECM imaging to a seamless analysis of the gene expression of specific markers remains challenge.34 To address above problems, by leveraging microfluidic chip, we describe a new systematic analysis workflow for studying heterotypic 3D cell spheroids from aggregates generation,

independent

cultivation,

label-free

voltage-switching

imaging35,

and

downstream comparative RNA analysis of individual cell clumps. Our strategy is illustrated in figure 1, where we used a dismantlable microfluidic device for generating cell spheroids (figure 1a). As a proof of concept, the membrane-bound protein, alkaline phosphatase (ALP), was chosen as the pluripotent marker detected label-freely by SECM interrogation. In this system (figure 1b), PAPP can firstly be catalyzed by ALP,


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located on tumor cell membrane, yielding the PAP, which was then oxidized into p-quinone imine (PQI) at Pt microelectrode. It should be noted that the electrochemical current at the constant-height mode, which may result in artifacts, is affected by both the underlying surface topography and electrochemically active species PAP. To accomplish a more precise electrochemical measurement, it is imperative to address this topographical convolution problem. Therefore, we employed the dual-mediator36 based scanning during SECM measurement. As a highly charged cation, Ruhex ([Ru(NH3)6]3+) is a cell-impermeable redox mediator that allows for recording a pure negative feedback, which is analogous to surface topography as shown in figure 1 (c). We thus implemented Ruhex for solely measuring the signal from the micro-pit topography, for analyzing the net ALP activity.27,

37

Those corresponding cell clusters can be consequently retrieved for

qPCR assay as shown in figure 1 (d) to (e). Our microfluidic chip consists of 4 channels (W x H = 600 x 190 μm), with the interval distance of two channels is 450 μm (figure 2a, b), where up to four types of cell spheroids can be cultured. There are four weir-arrays (85 micro-wells each) on bottom with 100 μm in diameter and height of each pit (Fig. S-1, S-2). The MCF-7 micro-tumor (~90 μm in diameter, red) and fibroblast cell spheroids (~75 μm in diameter, green) was clearly positioned in assigned culture lanes, indicating that our design can successfully fulfill the heterogeneous spheroids generation by using the microfluidic partition (shown in figure 2c). In addition, we found that, after peeling off the top microchannels, only few cell aggregates got lost during this process (figure 2d, S-2), which facilitated subsequent



analysis without losing analytical specimen. Consequently, to assess the ALP activity, we performed the SECM scanning on the corresponded region (figure 2f), at applied potential +0.3 V with the scan rate of 400 μm/s (with a diameter of 25 μm at +0.3 V vs. Ag QRE, the increment distance was 8 μm, with an interval time of 0.02 s, in a HEPES based solution, with 25 mM HEPES, 4.7 mM PAPP and 1mM Hexaammineruthenium (III) chloride) for achieving a 2800 μm x 400 μm image. The current response for oxidation of PAP observed over the tumor cell spheroids is significantly larger than that over the empty micro-pit or stromal fibroblast spheroids (figure 2f, i) in adjacent lanes (edge to edge distance of 450 μm). In addition, M-shape peak of current can only be observed for the microwells with tumor cell spheroids (figure 2i), which is probably due to the coffee ring effect driven edge-accumulation of PAP, which was continuously generated inside the microwell.38 Thus, we propose that the M-shape current peak can be used to indicate the ALP activity of cell spheroids in a micro-weir. We then confirmed that the signal of empty microwell was merely aroused from the ALP hydrolyzed PAP diffusion by scanning native cell-free micro pit structures (figure S-3, details were shown in supporting information). Also, we simulated the local PAP flux using COMSOL Multiphysics 5.3 (see details in supporting information). The simulation result (shown in figure S4) showed that after 15 minutes diffusion of PAP, its concentration in empty neighbor microwell (edge to edge distance of 100 μm) was about 60-70% of that with spheroids in, which agreed with the experimental current response (Fig. S-3). However, it should be noted that the faradaic microelectrode current monitored


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during SECM imaging is concomitantly affected by the morphological changing of substrate and the electrochemical activity of the underlying surface. After the ALP measurement, we rescanned the same region by switching the work potential from +0.3 V to -0.35 V vs. Ag QRE (at which [Ru(NH3)6]3+ reduction was a diffusion-limited reaction) to gain the topographical image of the substrate35 as shown in figure 2(g, j). In figure 2j, it clearly showed that four cell aggregates and five empty microwells can be evidently identified from the current. In other words, pits without cells are “deeper pits”. The current response over those deeper pits (vacant microwells) is larger (~30% higher) than the weir that has been occupied (figure 2j) because it progressively decreases with increasing cell spheroid height as a result of the hindered diffusion of the electrochemical mediator. We then decoupled the morphology signal in figure 2(g, j) from the corresponding complex signal in figure 2(f, i) according to the previously described work 26 (see SI text for details). The SECM current image acquired with PAP (figure 2f, i) is converted to a kinetic map shown in figure 2 (h, k), where the color bar quantifies the apparent heterogeneous rate constant (kf). Comparing the kinetic constants extracted for both patterned cell spheroid areas, the maximum kf value of 3.88 x 10-2 cm s-1 obtained over MCF-7 cell spheroids was 4.1 times larger than that of 9.7 x 10-3 cm s-1 over NHDF cell spheroids, which can typically represent the pure electrochemical information of PAP, and thus reflecting the ALP activity. To date, no reports have shown that 3D cell aggregate SECM images can be correspondingly coupled with multiplex gene expression analysis. Thus, to extend our



technique for downstream biological assay in given spheroid, we further explored the mRNA expression level of 17 genes (figure 2l, including GAPDH) in tumor and stroma 3D cluster. Immediately after the SECM measurements, the single spheroids were retrieved from the substrate and then subjected to mRNA analysis by real-time PCR (details are described in SI text and video S1).39, 40 The expression level of each gene was calculated by the -ΔΔCt method (As showed in figure 2l). We determined that 5 genes (SOX2, ALP, MUC-1, EPCAM, ESR1) were up-regulated (>2-fold changes) whereas 4 genes (FN1, ACTA2, VIM, TM4SF1) out of 17 total genes were down-regulated in MCF-7 micro-tumor compared with the fibroblast clumps. The gene expression plot shows that the mRNA level of ALP in tumor spheroids is 8 folds higher than that in fibroblast, which is coincident with the electrochemical evaluation. Notably,

we only used three given spheroids for

each type, with their electrochemical information of ALP activity have been deciphered, to accomplish this analysis instead of using large amount of 3D culture cells (figure 2e). The pluripotency relevant transcriptional factor, Sox2, was significantly upregulated by nearly 16 folds in tumor than that it was in stroma counterpart. Besides, in figure 2l, we also found that the two epithelial markers EPCAM (a transmembrane glycoprotein) and MUC-1 (polymorphic epithelial mucin) were remarkably enriched (~5.2 and ~6.9 fold, respectively) in 3D tumor. Consistent with expectation, compared to fibroblast, the estrogen receptor gene ESR1 was highly expressed (2.8 fold increased) in breast tumor cluster as MCF-7 cells have served as a model for the study of estrogen response. On the contrary, we confirmed the increased expression level of the mesenchymal markers


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such as FN1 (~2 folds), α-SMA (encoded by ACTA2, ~2.6 folds), VIM (~5.5 folds), and TM4SF1 (~5.6 folds) in fibroblast clump. These delightful results may indicate that compared with fibroblast, the MCF-7 cell spheroids are originated from epithelium with a certain degree of potential stemness. Other genes, such as TGFB1, ALDH1A2, CD133, and SNCG are remotely up-regulated in tumor whereas N-cadherin gene CDH2, CXCL12 (SDF1), CD44, and PDGFR are slightly enhanced in fibroblast spheroids (as shown in figure 2l). Taken together, we are able to dissect gene expression pattern within a single cell spheroid that has been previously scanned by electrochemical microscopy. This capability allows us not only to validate the obtained SECM results with standard biological assay but also to study relevant pathways that link with specific membrane proteins, which can be measured electrochemically. In this study, we have demonstrated a systematic analysis of different cell spheroids by leveraging a top-removable microfluidic chip, SECM platform, and downstream quantitative PCR assay on the single spheroid. This methodology has solved the lack of comprehensive and in-depth analysis in current research. Our strategy earns several cardinal virtues, including on-chip heterogeneous cell culturing, on-site SECM imaging, differentiation of target reaction from the background, and downstream molecular biological assay for corresponding 3D cell aggregates. The results of SECM measurement indicated that the tumor cell spheroids exhibited a significantly higher ALP activity than stromal fibroblasts cell clumps. Consequent qPCR assay confirmed that the ALP level was also higher in tumor cell clump. In addition, the consequence also showed



that together with ALP, the Sox2 is up-regulated in MCF-7 cell compared with fibroblast, implying the stemness of tumor spheroids. To our knowledge, this approach is the first demonstration for deciphering heterotypical cell aggregates on a single microfluidic device using label-free SECM and seamless gene expression profiling. We also suggest that this paradigmatic workflow could be integrated with RNA-seq technology due to its ability to gain transcriptional landscape. Therefore, we envision that this systematic analysis strategy will find various applications for biological and clinical research, wherein studying for high-throughput and label-free cellular imaging and corresponding regulation by gene expression quantification are both required. Acknowledgement We thank Prof. Lei Su, Prof. Liping Xu, and Prof. Yongqiang Wen, in School of Chemistry and Biological Engineering, University of Science and Technology Beijing for fruitful discussion. We acknowledge funding support from National Natural Science Foundation of China (21775011, 21675011, 21727815) and Fundamental Research Funds for the Central Universities (FRF-TP-17-001A2).

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Figure Captions: Figure 1. Schematic illustration of the systematic analysis of different cell spheroids with a microfluidic device using SECM and gene expression workflow. (a) Different types of cells are cultured in microfluidic device to generate spheroids. (b). ALP detection using SECM.In this process, PAPP can firstly be catalyzed by ALP, yielding the p-aminophenol (PAP), which was then oxidized into p-quinone imine (PQI) at the tip of Pt microelectrode at +0.3V. (c) The morphology detection at -0.35 V (at which voltage [Ru(NH3)6]3+ was reduced). (d) Spheroids retrieval from the substrate for gene expression analysis by using mouth pipetting. (e) Recovered cell spheroid was directly treated for lysis, reverse transcription and cDNA amplification, for qPCR assay.

Figure 2. The microfluidics and SECM based strategy for systematic analysis of tumor and fibroblast spheroids. (a) The picture of the whole microfluidic device. The microchannels were depicted with different color dye (red, cyan). Scale bar is 1 mm. (b) A micrograph of the whole microfluidic device. Scale bar is 200 μm. (c) to (e) Merged fluorescence and bright-field images of tumor and stromal cell spheroids before (c) and after (d) peeling of the channel chip. Scale bar is 200 μm. (e) The micrograph after we retrieved wanted cell spheroids which have been used for gene expression analysis (Cell tracker staining: MCF-7 in green and NHDF in red). The region marked in white dashed



frame in (d) corresponds with the SECM imaging measurement showed in (f) to (h). (f) The normalized composite signal of ALP and morphology using 4.7 mM PAPP (E = +0.35 V vs Ag). (g) The normalized signal of morphology using 1mM Ruhex (E = -0.35 V vs Ag) (h) The K value represent the extracted ALP profile (the signal of morphology was deducted). Scale bar is 200 μm. The region marked in white dashed arrow in (d) corresponds to the one-line scan measurement showed in (i) to (k). (i) The normalized composite signal of ALP and morphology using 4.7 mM PAPP (E = +0.35 V vs Ag). (j) The normalized signal of morphology using 1 mM Ruhex (E = -0.35 V vs Ag). (k) The K value represent the extracted ALP profile (deduct the signal of morphology). (l) Gene expression analysis in those two types of cell spheroids respectively. GAPDH was used for normalization. Fold change (2-∆∆Ct >1) was chosen as a criterion for significant difference.

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Figure 1. Schematic illustration of the systematic analysis of different cell spheroids with a microfluidic device using SECM and gene expression workflow. (a) Different types of cells are cultured in microfluidic device to generate spheroids. (b). ALP detection using SECM.In this process, PAPP can firstly be catalyzed by ALP, yielding the p-aminophenol (PAP), which was then oxidized into p-quinone imine (PQI) at the tip of Pt microelectrode at +0.3V. (c) The morphology detection at -0.35 V (at which voltage [Ru(NH3)6]3+ was reduced). (d) Spheroids retrieval from the substrate for gene expression analysis by using mouth pipetting. (e) Recovered cell spheroid was directly treated for lysis, reverse transcription and cDNA amplification, for qPCR assay. 83x140mm (300 x 300 DPI)



Figure 2. The microfluidics and SECM based strategy for systematic analysis of tumor and fibroblast spheroids. (a) The picture of the whole microfluidic device. The microchannels were depicted with different color dye (red, cyan). Scale bar is 1 mm. (b) A micrograph of the whole microfluidic device. Scale bar is 200 μm. (c) to (e) Merged fluorescence and bright-field images of tumor and stromal cell spheroids before (c) and after (d) peeling of the channel chip. Scale bar is 200 μm. (e) The micrograph after we retrieved wanted cell spheroids which have been used for gene expression analysis (Cell tracker staining: MCF-7 in green and NHDF in red). The region marked in white dashed frame in (d) corresponds with the SECM imaging measurement showed in (f) to (h). (f) The normalized composite signal of ALP and morphology using 4.7 mM PAPP (E = +0.35 V vs Ag). (g) The normalized signal of morphology using 1mM Ruhex (E = -0.35 V vs Ag) (h) The K value represent the extracted ALP profile (the signal of morphology was deducted). Scale bar is 200 μm. The region marked in white dashed arrow in (d) corresponds to the one-line scan measurement showed in (i) to (k). (i) The normalized composite signal of ALP and morphology using 4.7 mM PAPP (E = +0.35 V vs Ag). (j) The normalized signal of morphology using 1 mM Ruhex (E = -0.35 V vs Ag). (k) The K value represent the extracted ALP profile (deduct the signal of morphology). (l) Gene expression analysis in those two types of cell spheroids respectively. GAPDH was used for normalization. Fold change (2-∆∆Ct >1) was chosen as a criterion for a significant difference. 153x133mm (300 x 300 DPI)


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Systematic Analysis of Different Cell Spheroids with a Microfluidic

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