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. 2020 Jul 6;15(7):e0235356. doi: 10.1371/journal.pone.0235356

Real-time monitoring of oncolytic VSV properties in a novel in vitro microphysiological system containing 3D multicellular tumor spheroids

Kyoung Jin Lee 1,2,#, Sang Woo Lee 3,#, Ha-Na Woo 1,2, Hae Mi Cho 1,2, Dae Bong Yu 1,4, Soo Yeon Jeong 3, Chul Hyun Joo 1,2, Gi Seok Jeong 3,5,*, Heuiran Lee 1,2,*
Editor: Salvatore V Pizzo6
PMCID: PMC7337297  PMID: 32628693

Abstract

As a new class of cancer therapeutic agents, oncolytic viruses (OVs) have gained much attention not only due to their ability to selectively replicate in and lyse tumor cells, but also for their potential to stimulate antitumor immune responses. As a result, there is an increasing need for in vitro modeling systems capable of recapitulating the 3D physiological tumor microenvironment. Here, we investigated the potential of our recently developed microphysiological system (MPS), featuring a vessel-like channel to reflect the in vivo tumor microenvironment and serving as culture spaces for 3D multicellular tumor spheroids (MCTSs). The MCTSs consist of cancer A549 cells, stromal MRC5 cells, endothelial HUVECs, as well as the extracellular matrix. 3D MCTSs residing in the MPS were infected with oncolytic VSV expressing GFP (oVSV-GFP). Post-infection, GFP signal intensity increased only in A549 cells of the MPS. On the other hand, HUVECs were susceptible to virus infection under 2D culture and IFN-β secretion was quite delayed in HUVECs. These results thus demonstrate that OV antitumoral characteristics can be readily monitored in the MPS and that its behavior therein somewhat differs compared to its activity in 2D system. In conclusion, we present the first application of the MPS, an in vitro model that was developed to better reflect in vivo conditions. Its various advantages suggest the 3D MCTS-integrated MPS can serve as a first line monitoring system to validate oncolytic virus efficacy.

Introduction

Oncolytic viruses (OVs) selectively infect and destroy tumor cells, sparing the normal cells and minimizing normal tissue damage [1]. These characteristics of OVs have enabled the growth of oncolytic virotherapy combined with immunotherapy [2]; consequently, oncolytic virotherapy has become one of the burgeoning fields of cancer immunotherapy [3]. To improve antitumor therapeutic efficacy, native features of OVs have been optimized by genetically modifying the viruses in various ways by inserting immune-stimulating genes and by removing cytotoxic viral genes [4]. For example, Talimogene Laherparepvec (T-VEC) acquired the first FDA approval as an oncolytic virus therapy with the recent completion of phase III clinical trials.

Two dimensional (2D) in vitro cell culture systems are routinely applied to determine the characteristics of OVs [5]. However, 2D systems do not reflect the 3D physiological microenvironments where the tumors reside [6]. Tumor xenograft models including mouse [7] and rat [8] are widely used to evaluate the tumor killing efficacy of OVs, but are still not able to accurately predict the outcome of human clinical trials [9]. OVs have dual therapeutic effects that depend on onco-selective cell lysis and the induction of antitumor immune responses [10]. Yet, in vivo tumor xenograft models do not show induced immune responses following OV administration. Therefore, well-defined in vitro models that imitate the in vivo cancer microenvironment conditions would be more beneficial as a first line study platform. Among several types of 3D-cultured spheroid models [11], 3D multicellular tumor spheroids (3D MCTSs) show suitable in vivo environments for evaluating the properties of onco-selective infection of OVs [12]. Another approach is applying a microphysiological system (MPS) to simulate blood vessel-like structures [13,14]. If the MPS and 3D MCTSs are properly combined, it would serve as a much better in vitro tumor model for evaluating the efficacy of OVs because it mimics the actual physiological conditions of the tumor tissue, including the fluid dynamics and cell-to-cell interactions in the TME.

Recently, we successfully invented a model MPS combined with 3D MCTSs and demonstrated that the microfluidic device could be adopted for the development and screening of anticancer chemical drugs by the formation of the same 3D MCTSs under similar conditions [15]. Here, we report a novel approach to investigate the antitumor activities of oncolytic vesicular stomatitis virus (oVSV) through employing our newly developed MPS. By employing oVSV-expressing GFP (oVSV-GFP), the data provide evidence that the antitumoral characteristics of oVSV can be readily monitored in the 3D MCTS-integrated MPS and that its behavior somewhat differs in this condition compared to that in a conventional 2D system. The 3D MCTS-integrated MPS thus can serve as the first line evaluation system for the onco-selective infection of OVs.

Materials and methods

Cell cultures, fluorescence labeling, and fluorescence analysis

Human lung cancer A549 cells (ATCC, Manassas, VA) and human lung fibroblast MRC5 (ATCC) were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA) containing 10% FBS (Biowest, Riverside, MO) and 1% penicillin/streptomycin (Thermo Fisher Scientific). HUVECs were maintained in endothelial cell medium (Sciencell Research Laboratories, Carlsbad, CA).

According to the manufacturer’s protocol, cell components were fluorescently labeled in 3D MCTSs using green PKH67GL or red PKH26 (Sigma-Aldrich; ST. Louis, MO). Briefly, the cell pellet was suspended in Diluent C solution, and the dye solution was prepared with a PKH ethanolic dye solution. The suspended cells and the prepared dye solution were rapidly mixed. After incubation, 1% bovine serum albumin (BSA) was added to stop the staining. After centrifugation, the cells were resuspended in complete medium, centrifuged and washed to ensure the removal of any unbound dye. After washing, the fluorescent dye-stained cells were used in experiments to confirm their position and migration.

Fluorescence images were developed using a fluorescence microscope (EVOS) and confocal microscope (Zeiss LSM780, Carl Zeiss AG, Oberkochen, Germany). The confocal images were analyzed using ZEN Microscope Software (Carl Zeiss AG, Oberkochen, Germany).

Oncolytic VSV expressing GFP (oVSV-GFP) preparation and single growth curve analysis

The VSV full-length plasmid with GFP tagging, pVSV Venus-VSVG (Addgene, Watertown, MA), was the template to generate oVSV-GFP. It was generated as previously described [16]. Briefly, 293T cells were infected with vaccinia virus encoding T7 polymerase (ATCC, vTF7-3) at 10 MOI. After 1 h, the vaccinia virus was washed, and the cells were transfected with 1.1 μg of pVSV Venus-VSVG, 0.6 μg of pN, 1.4 μg of pP, and 0.9 μg of pL by using 32 μl of PEImax (Polyscience; Germany). After 48 h, filtered culture media were treated with new 293T cells. oVSV-GFP present in the supernatant were further passaged in 293T cells to amplify the viral titer. A549 cells were used to quantify the infectious titer of oVSV-GFP virus stock by TCID50. Cells treated with oVSV-GFP for 1 h, at 1 multiplicity of infection (MOI) representing one infectious virus per cell, were used in single growth curve analysis. Fresh growth media was added after washing and the supernatants were collected at designated time points. Virus titers were also determined using the supernatants by TCID50.

Generation of the microphysiological system (MPS)

i) Fabrication of the microfluidic culture device and passive powerless micropump

The manufacturing procedure of the polydimethylsiloxane (PDMS; Sylgard® 184, Dow Chemical Co., MI)-based microfluidic device was described in our previous study [15]. A passive micropump with a siphon effect was established as well. This pump provided continuous medium without mechanical energy. A polyethylene (PE) tube was linked between the device and the micropump. Eluate was drained into the outlet reservoir via a PE tube filled with yarn fiber. After connecting the pump to the microfluidic device, the external structure of the MPS was completed.

ii) Generation of the tumor microenvironment (TME)-like structure with 3D MCTSs

The generation of 3D MCTS with ECM in a microfluidic device was established in our previous studies [17]. The internal device was coated with type I collagen solution (collagen I; BD Bioscience, San Jose, CA) to simulate an ECM. A549 and MRC5 cells (2 x 106 cells/mL) were suspended in 2 mg/mL type I collagen solution. Each cell line was injected into the microfluidic device and centrifuged to trap the cancer cells in the wells. Afterwards, the microfluidic device was washed with a collagen solution. HUVECs (1 x 106 cells/mL) were then seeded in a channel to generate a vessel-like environment. The number of cells remaining per chip was identified as an average of 1 x 105 cells of each line. The microfluidic device was connected to the passive micropump to supply continuous medium, so the internal components of the microphysiological system (MPS) were completed. For LDLR expression studies, MPS was prepared only using HUVEC cells while all other procedures remained the same.

oVSV-GFP infection of the MPS and virus growth kinetics

At 24 h after preparing MPS, a microtip containing oVSV-GFP at 5 MOI based on the total number of cells per MPS (3 x 105 cells) was inserted into the inlet hole of the MPS. The height difference between the microtip containing fluid and chip was used to introduce viruses into MPS channels via pressure and flow. After 1 h, residual viruses were removed, and the infected MPS was washed. The MPS was then connected with the passive micropump to supply oxygen and nutrients. The drained eluate from the MPSs were collected at 24, 48 and 72 h to measure infectious viral particles (IPs) by TCID50.

Cell death assay

For propidium iodide (PI) analysis, cells were stained with PI and DAPI solution in a 2D culture and examined under a fluorescence microscope at ×200 magnification. To evaluate antitumor effects by oVSV-GFP in MPS, cellular mortality was analyzed by fluorescence staining using a live/dead kit according to the manufacturer’s protocol. Morphologies and fluorescence images of 3D MCTSs in the MPS were examined using a fluorescence microscope at 24, 48 and 72 h after oVSV-GFP infection.

RT-quantitative PCR (qPCR)

QIAamp viral RNA mini kits (QIAGEN) were used to recover viral RNA genome from eluates. Reverse transcription was performed using SuperScript III Reverse Transcriptase (Life Technologies). qPCR was performed using IQ SYBR Green Supermix (Bio-Rad; Hercules, CA) in a real-time thermal cycler (CFX96 Real-time system; Bio-Rad). The following sequences were selected: VSV-G, 5’-CCCGGTACCTTTTTCTTTATCATAGG GT -3’, 5’-CCCGTTAACTTACTTTCCAAGTCGGTT-3’.

Susceptibility analysis of cell lines against oVSV infection in a 2D culture system

Cells were plated and infected with serially diluted oVSV-GFP for 1 h at 37°C. After 5–6 days, the cells were stained with 2% crystal violet in 50% methanol. The lowest MOI of each cell line for cytotoxicity was calculated by TCID50. Based on the lowest MOI of A549, the relative susceptibility of each cell line against oVSV-GFP was calculated (n = 4).

Quantification of secreted human interferon-beta (IFN-β)

Cells in 2D culture were infected with oVSV-GFP at 1 MOI for 1 h, then washed and given fresh media. Culture media was collected at 6 h and 14 h, then stored at -80°C prior to analysis. The concentration of IFN-β was measured using a human IFN-β ELISA kit (PBL Assay Science, Piscataway, NJ).

Immunofluorescence staining for detecting the expression of LDLR

After fixing with 4% paraformaldehyde (Sigma-Aldrich) and subsequently washing with PBS, 3D MCTSs were incubated with permeabilizing solution using PBS containing 0.1% Triton X-100 (Sigma-Aldrich) and blocked with 2% BSA (Sigma-Aldrich) in 0.1% Tween 20 (Sigma-Aldrich). The 3D MCTSs were incubated with a fluorophore-conjugated antibody against low-density lipoprotein receptor (LDLR; Bioss, Woburn, MA) in 1% BSA in PBS overnight at 4°C, washed with PBS and stained with DAPI (Invitrogen).

Statistical analysis

The area and fluorescence intensity of the MCTSs were determined by ImageJ software (1.46 ver, NIH). Quantitative data are presented as the mean ± standard deviation (SD). Group differences were assessed by paired t-tests or one-way and two-way ANOVAs followed by Bonferroni’s test using GraphPad Instat (GraphPad Software, La Jolla, CA). Statistical significance was set at p > 0.05(ns), p < 0.05(*), p < 0.01(**) and p < 0.001(***).

Results

In vitro microphysiological system harboring 3D MCTS for the evaluation of oVSV-GFP activity

To successfully establish an appropriate in vitro tumor model reflecting the in vivo TME, we employed our recently developed MPS [15] (Fig 1). Oxygen, nutrients, and components simulating the TME were constantly provided through a vessel-like channel in the integrated MPS using a passive micropump (Fig 1A). The internal components forming the TME-like environment in the MPS were ECM (type I collagen matrix) and 29 3D MCTSs located in 29 wells (Fig 1B). In particular, 3D MCTS consisting of human cancer A549 cells (green, Fig 1C) and human fibroblast MRC5 cells mimic the structure of in vivo tumor tissues. (red, Fig 1C). As expected, cells observed in the 3D overlap region of MCTS appeared yellow in the merged image. The fluorescence image suggests the successful lining of HUVEC cells in the MPS (Fig 1D, red).

Fig 1. The structure of the microphysiological system (MPS).

Fig 1

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(A) Schematic representation of the external structure of the MPS. (B) Schematic of the internal components of the MPS. In the well, collagen type I matrix was coated as ECM, and 3D MCTSs were generated with human cancer A549 cells and human fibroblast MRC5 cells. In the vessel-like channel, HUVECs were coated, and continuous medium was provided to the 3D MCTSs. (C) Fluorescent images of a labeled 3D MCTS in the MPS (A549, green; MRC5, red). (D) Images of HUVECs stained with red in the MPS; scale bars: 275 μm.

Effective replication of oVSV-GFP in A549 cells

To evaluate the efficacy of oncolytic virotherapy, we employed oVSV encoding GFP (Fig 2A). By one-step growth curve analysis, A549 human cancer cell susceptibility to oVSV-GFP was determined (Fig 2B). The viruses rapidly replicated, reaching a plateau at approximately 25 h p.i. (6.6±4.2X107 TCID50/ml). Unlike untreated cells (Fig 2C), infected cells showed irreversible cytopathic effects and strong GFP signaling under fluorescence microscopy (Fig 2D). The PI staining results indicated that A549 cancer cells are highly susceptible to oVSV-GFP replication, resulting in cancer cell death and subsequent progeny virus release.

Fig 2. Growth properties and cytopathic effects of oVSV-GFP.

Fig 2

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(A) Schematic drawing of oVSV-GFP. GFP was inserted in front of gene N of viral genome. (B) Single growth curve analysis of oVSV-GFP in A549 cells. The supernatants were collected from infected A549 cells at the indicated time points (0, 5, 10, 15, 25h and 50 h p.i.), and the virus titration was calculated by TCID50 assay. (C) The characteristics of mock-treated cells at specified times. (D) Cytopathic effects of oVSV-GFP in infected cells. GFP-positive cells were identified starting at 5 h p. i., and PI-positive cells were easily identified at 25 h and 50 h. The mean and SE data of 3 independent experiments; scale bars: 200 μm.

Differential replication of oVSV-GFP in the 3D MCTS-integrated MPS

Viral infection with oVSV-GFP at 5 MOI (determination based on the total number of cells in MPS) was achieved through natural flow and subsequent MPS washing (Fig 3A). GFP signaling in the fluorescence images of 3D MCTSs was evident at 24 h p.i., then gradually declined (Fig 3B and 3C). Using the eluates released from the MPS every 24 h, progeny virus production was quantified by TCID50 (Fig 3D) and VSV-G gene (Fig 3E) specific RNA genome amplification was measured by RT-qPCR. Similar to the change in fluorescence intensity, progeny virus production was the highest at 24 h (1.1±0.4X106), and then decreased at 48 h (3.2±1.3X104) and 72 h (4.8±3.1X104). The peaking of production at 24 h followed by decline was also observed in RNA genome analysis of the virus.

Fig 3. Effective replication of oVSV-GFP in the MPS.

Fig 3

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(A) Schematic representation of virus infection. As described in M&M, viruses at 5 MOI were introduced into MPS. The residual viruses were removed by washing, and the infected MPS was connected to a passive micropump. (B) Fluorescence images of a 3D MCTS infected by oVSV-GFP. (C) At the indicated time points, a fluorescence intensity graph of oVSV-GFP was obtained from 3D MCTS (tumor spheroid; n = 8). (D) Quantitative analysis (n = 3) of progeny virus production by TCID50 assay using the eluates at the indicated time points. (E) Viral RNA genome copies from the eluates were calculated by RT-qPCR targeting VSV-G gene (n = 3). In addition to the results in C and D, the viral genome of oVSV-GFP at 24 h was the highest. The mean and SE data of 3 independent experiments; scale bars: 200 μm. *p < 0.05, *** p < 0.001.

The replication ability of oVSV-GFP in different cells consisting of the 3D MCTSs in the MPS was examined by labeling A549 and MRC5 cells with a red fluorescence tracker. At every time point p.i., GFP signals were clearly observed in red A549 cells. This is indicated in the yellow merged images (Fig 4A). In contrast, red MRC5 cells did not turn yellow following oVSV-GFP replication (Fig 4B). Though the tumor spheroids consisted of both cells, the data indicates that GFP signal following oVSV replication was restricted in A549 cells. To further investigate the characteristics of the cells in MCTSs after oVSV-GFP treatment, the tumor spheroids unlabeled with fluorescent dye were analyzed with PI staining (Fig 4C). The PI-positive dead cell region perfectly overlaps with the GFP-positive region. This indicated that the GFP-positive A549 cells, but not the MRC5 cells or HUVECs, were dead. Finally, we observed that the size of infected MCTSs gradually decreased when that of uninfected control MCTSs increased (Fig 4D and 4E). Taken together, these results demonstrate that oVSV-GFP selectively replicates in A549 human cancer cells and eventually induces cell death in infected cells in the MPS.

Fig 4. oVSV-GFP selectively kills A549 cells following virus replication in the MPS.

Fig 4

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(A) Infection patterns of the MPS generated by A549 cells marked with a red fluorescence tracker at the indicated time points (tumor spheroid; n = 8). The locations of the GFP signal are similar to those of labeled A549 cells. (B) Infection patterns of the MPS generated by MRC5 cells marked with a red fluorescence tracker at the indicated time points (tumor spheroid; n = 8). The locations of the GFP signal did not overlap with those of labeled MRC5 cells (indicated with white arrows). (C) PI staining with 3D MCTSs infected with oVSV-GFP at the designated time points (tumor spheroid; n = 8). The locations of the GFP signal perfectly overlaped with those of dead-staining cells. (D) Light microscopy images of 3D MCTSs infected with oVSV-GFP (tumor spheroid; n = 10). (E) Growth kinetics of the MCTSs at the indicated time points (tumor spheroid; n = 10). Scale bars: 400 μm. ** p < 0.01.

Different susceptibilities of endothelial cells to oVSV in the MPS environment

To examine whether the physiological environment of the MPS influences the susceptibility of the 3 different cells residing in the MPS, each cell line was infected with oVSV-GFP at 1 MOI in a conventional 2D culture system, and the relative sensitivity to oVSV was analyzed (Fig 5A). In 2D, A549 cells were highly susceptible to oVSV while MRC5 cells were relatively resistant, similar to the MPS model. However, HUVECs were very sensitive to oVSV in 2D only. The influence of IFN-β was investigated by quantifying IFN-β secretion from the culture medium of each cell line following oVSV-GFP treatment in 2D culture using ELISA (Fig 5B). A549 cells constantly secreted IFN-β at very low levels, while MRC5 cells rapidly secreted IFN-β at high concentrations, reflecting the susceptibility to oVSV. In the case of HUVECs, IFN-β secretion showed a delayed pattern; in the early phase of infection (6 h p.i), its level was very low, which was even lower than that for A549 cells. These results indicate that the delayed IFN-β secretion in the infected HUVECs could have a correlation with their susceptibility to oVSV in the 2D system. The effect of fluid flow on the expression of low density lipoprotein receptor (LDLR), the main receptor of oVSV, was further investigated in HUVECs after virus treatment (Fig 5C). In MPS consisting of HUVECs alone, substantial GFP signals were observed regardless of fluidic flow compared to MPS with all three cell lines (Fig 4). Similarly, LDLR expression was also detected. Thus, the data suggest the susceptibility of HUVEC cells in MPS without tumor spheroid and the consistent LDLR expression levels regardless of fluidic flow.

Fig 5. The fluidic flow in the MPS influences the susceptibility of HUVECs to oVSV.

Fig 5

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(A) Relative susceptibility of three cell lines to oVSV without continuous fluidic flow conditions 2D culture system. (B) Comparison of IFN-β secretion in the culture medium of each cell line after oVSV-GFP treatment in 2D culture. (C) LDLR expression and GFP signal monitoring after 8 h oVSV-GFP infection in the MPS consisting of HUVEC cells alone with or without fluidic flow. The mean and SE data of 3 independent experiments; scale bars: 275 μm. *p < 0.05, *** p < 0.001.

Discussion

To establish an advanced in vitro tumor model reflecting the in vivo TME for better evaluating the antitumor efficacy and safety of oncolytic viruses, we employed our recently developed MPS and examined its potential [15]. A durable 3D structure was formed with HUVEC lining, 3D MCTS composed of cancer A549 cells, and fibroblast MRC5 cells. When connected to a passive micropump, the MPS channel supports continuous media supply. oVSV expressing GFP can be readily applied to this 3D MCTS, and the virus replication pattern can be visualized under fluorescence microscopy in real time. The present study indicates that oVSV preferentially infects cancer cells and that the morphology of the 3D MCTS collapses as the infected cells die. Based on these advantages, as an in vitro tumor model, the MPS offers a convenient monitoring method for validating the efficacy of oncolytic viruses.

VSV is considered a particularly promising OV due to its effective antitumor activity in preclinical models and has the following advantages [18]: i) it has a natural tropism for malignant cells because of its high sensitivity to type I IFN, thus protecting normal cells from destruction [19]; ii) virtually no preexisting immunity against VSV exists in the general population, and the rare natural infections are asymptomatic; iii) it has a rapid replication cycle and high titers and can thus rapidly destroy cancer cells; and iv) it induces substantial immunogenicity to stimulate antitumor immunity. Recently, the Russell group developed a genetically modified oVSV expressing IFN-β and sodium iodide symporter and examined this oVSV in phase I clinical trials for patients with refractory multiple myeloma, acute myeloid leukemia, or T-cell lymphoma [20].

Unlike A549 or MRC cells, HUVEC cells showed a difference in viral sensitivity in 2D and 3D culture conditions. HUVEC cells, which were sensitive to oVSV-GFP in the 2D culture system, were resistant in the 3D MPS. Since the viral sensitivity is presumed to be due to the difference in IFN signaling in each infected cell line [21,22], IFN-β secretion was measured by ELISA. The IFN-β secretion pattern correlated with the oVSV sensitivity of each cell line in 2D. Uninfected cells can be efficiently protected from secondary infections through the paracrine signaling of IFN secreted by nearby infected cells [23,24]. Previous in vivo studies also showed that tumor cells are infected and killed by VSV while normal endothelial cells are protected [25,26]. Therefore, the low susceptibility of HUVECs to oVSV in the MPS may be explained by the protective effect of IFN secretion by neighboring cells against viral infection.

LDLR is a major surface receptor of mammalian cells for VSV infection through endocytosis [27], and LDLR activity plays an important role in determining the degree of infection of VSV [28]. The present study clearly demonstrates consistent LDLR expression levels in HUVEC cells regardless of fluidic flow and may suggest that differences in HUVEC sensitivity between MPS and 2D systems are not related to LDLR expression on the cell surface. The effect of fluidic flow on LDLR activity has been also illustrated in previous studies showing a decrease of LDLR activity in vascular endothelial cells in vitro in the presence of microscopic or low degree laminar shear stress (0.1 ~ 0.5 dyne/cm2) [29]. To identify the shear stress that affects LDLR activity, we previously demonstrated that laminar flow occurs in the microchannels of the microfluidic, with the resulting laminar shear stress in a microchannel to be 0.2 ~ 0.3 dyne/cm2 [17]. It is thus possible to conclude that the laminar flow in our microfluidic system, which reflects the in vivo TME, affects HUVEC LDLR activity in the microchannel which results in low LDLR activity. Further research is still needed to elucidate the effect of shear stress on the viral sensitivity of endothelial cells in detail.

To apply the 3D microfluidic MPS in vitro and assess the therapeutic potential of oncolytic virotherapy, quantitative analysis is essential. In our MPS, the virus replication pattern can be monitored by tracing the fluorescence signal in real time. Noninvasive repeated monitoring of the MCTSs residing in each well is possible under conventional fluorescence microscopy. The fluorescent area can be quantified using proper software, and then the data can be further analyzed for evaluating the oncolytic efficacy of OVs. Analysis via immunocytochemistry is also possible. Additional quantitative data can be obtained utilizing eluate released from MPS, including viral growth kinetics or total viral genomic quantification, where TCID50 or RT-qPCR is performed. Furthermore, the 3D MCTSs residing in the MPS can be used to analyze total protein or RNA expression patterns upon extracting the MCTS pellets from the device.

Although this 3D MCTS-integrated MPS has several valuable advantages for evaluating the potential of oncolytic virotherapy, some limitations exist, such as a lack of immune response-related cells. Immune cells play a key in switching from hot to cold tumor conditions, attenuating cold tumor properties driving the interactions among tumor cells, other resident cells, and oncolytic viruses, particularly when cancer destruction occurs [30,31]. Since the direct killing of tumor cells by OVs and subsequent killing by activated immune cells are important points for cancer removal [32], the lack of immune cells in the system limits the current study. Subsequent studies can explore how to apply immune cells to this system and its effects in evaluating oncolytic virotherapy.

In conclusion, we present the first application of an MPS-harboring 3D MCTS as an in vitro model for the validation of the antitumor effects of an oVSV. The 3D MCTS-integrated MPS was developed to better reflect the in vivo environment and is an improved in vitro tumor model system compared to the 3D MCTS system because it simultaneously incorporates 3D MCTSs and a microfluidic device. Due to these advantages, the 3D MCTS-integrated MPS can serve as the first line monitoring system for successfully validating the efficacies of OVs, which may prove to be a valuable model system for future evaluations of various anticancer therapies beyond OVs.

Acknowledgments

We thank Paula Khim for her important advice on editing a draft of this manuscript.

Data Availability

All relevant data are within the manuscript.

Funding Statement

This work was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF, http://www.nrf.re.kr) granted funded by the Korea government (NRF-2016R1A2B4014912 to CH Joo; NRF-2019R1A2C2005244 to GS Jeong; NRF-2019R1C1C1007468 to KJ Lee). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Salvatore V Pizzo

6 Mar 2020

PONE-D-19-34362

Real-time monitoring of oncolytic VSV properties in a novel in vitro microphysiological system containing 3D multicellular tumor spheroids

PLOS ONE

Dear Dr. Lee,

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Reviewer #1: No

Reviewer #2: Partly

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The authors previously developed a microphysiological system (MPS) which features a vessel-like channel that can be lined with endothelial cells and a total of 29 side chambers which can be populated with various cell types. In the current study they generated spheroids in the side channels by infusing A549 cancer cells and MRC5 fibroblasts, then lined the vessel-like channels with HUVECs. They infused VSV-GFP into the vessel-like channels and were able to show that the virus infected only the A549 cells, not the MRC5 cells in the spheroids. This result was expected since the MRC5 cells were resistant to the virus in 2D culture and the A549 cells are susceptible. Interestingly the HUVECs did not get infected in the MPS, but were susceptible to virus infection in 2D culture. The authors speculate based their analysis of IFNb release from VSV infected A549, MRC5 and HUVEC cells, and the protection of those cells from VSV infection upon IFNb exposure, that the release of IFNb by virus-exposed MRC5 cells in the MPS is able to render the HUVEC resistant to infection.

The MPS system colonized with multicellular spheroids does offer an interesting in vitro test system for the study of oncolytic viruses that might well catch on as an alternative to mouse xenograft models if it were widely available. The ability to form multicellular tumor spheroids in side chambers accessible only via a vessel-like channel is appealing and allows for detailed analyses of the dynamical aspects of virus interaction with microvessels, tumor cells and fibroblasts in a controllable system. However, there are significant weaknesses with the current manuscript which, in the opinion of this reviewer, should be addressed prior to publication:

1. The authors state that they perfuse their system with VSV-GFP using a virus moi (multiplicity of infection) of 5.0. But what does that mean? First, they do not give details of the cell substrate on which they titrate their virus stock. Second, it is not clear how they determine the "moi" for the MPS when they do not know how many cells are present in the MPS that they are perfusing. This aspect of the paper is confusing and needs to be clarified.

2. The vessel-like channels in the MPS are supposedly lined by HUVEC cells but there are no images provided to confirm this, nor to show whether the HUVEC lining is indeed sufficiently comprehensive to form vessel-like channels. This is a critical aspect of the system and should be addressed with additional data. Without a direct visualization of the HUVEC lining cells, and an assessment of their GFP expression post virus infusion, the claim that the HUVEC cells resident in the MPS are resistant to virus infection remains unproven.

3. The authors state that there are 29 side chambers in their MPS but do not indicate how they captured data from all of these chambers and do not provide any statistical backup for the conclusions they have drawn.

4. The hypothesis that HUVEC cells lining the MPS channels are rendered resistant to VSV-GFP by exposure to IFNb from MRC5 cells could easily be tested by rerunning the experiment using A549-only spheroids that do not contain MRC5 cells . Data from such an experiment should be included in the manuscript.

Reviewer #2: Authors have investigated the potential of their recently developed microphysiological system (MPS) that mimics the in vivo tumor microenvironment and serves as culture space for 3D multicellular tumor spheroids (MCTS). This system was used as a model to check the oncolytic potential of Vesicular Stomatitis virus and concluded that this system can be used as a first line of monitoring system to validate any oncolytic virus efficacy. It is a helpful model system for evaluation of anticancer therapies including virotherapy.

It is a good piece of work; however it was not presented very well. Methodology and results need to be written clearly. At many places the clarity is missing. Experiment description in the legends needs lot of improvement.

Comment 1: In Figure 1C, A549 cells stained green and MRC5 red. But why is it bright yellow at the interface of these two cell populations? Is it due to overlap?

Comment 2: Reference to the “effective replication of oncolytic VSV expressing GFP in A549cells” under result section, related Fig 2C, oVSV effects were shown at 5h and 50h. It would be better, if 25h data of GFP expression and PI staining is also incorporated in the image panel. In this experiment, CPE/GFP expression/cell death was measured up to 50h post infection; but the negative control (mock infected) was not included. How was it confirmed without a negative control that the noted cell death was virus induced?

Comment 3: In Figure 3E, quantification of viral genome copies was done to analyse oVSV-GFP replication in MCTS eluates. How was it done? Was it the amplification of VSV –G gene by quantitative PCR? Please clarify in the text and legend. In this experiment as well, negative control was missing.

Comment4: What was the rationale behind using different MOIs, 1 and 5 for infections in 2D cultures and MPS?

Comment 5: From the methodology section given under ‘quantification of secreted human interferon beta (IFNβ) after VSV-GFP infection’ it is clear that IFNβ response was measured in all three types of cells following oVSV-GFP infection in 2D cultures and eluates collected from infected MPS by ELISA. But the related result and Figure/ figure legend are confusing. Please check the result and the figure related to this experiment and write more in detail.

Comment 6: To check the differential replication of oVSV-GFP in the 3D MCTS integrated MPS, A549cells, MRC-5 cells were labeled with fluorescence tracker. Figure 4A and 4B, both the cells; A549 and MRC5 were stained red. How were these two cell populations separated to check the expression oVSV-GFP through red and green merge? Are 4A and 4B images from the same spheroid? Clarify and also mention in the text and legends. In the figure 1, A549 cells were stained green.

Comment 7: There is a lot of room for improvement in discussion section. One sentence in the discussion from lines 326 to 329 is the exact repetition of a sentence under result section from lines 281 to 284. Improve the discussion.

Comment 8: The intensity of GFP expression in A549cells does indicate the active replication of oVSV-GFP virus but not the anti tumor activity. Experiment of Live/Dead cell staining following virus infection does not commend the anti tumor activity of oncolytic virus used. An anti tumor/ anti proliferative marker may be used to confirm the anti tumor activity of the virus in MCTSs. Following infection, spheroids can be checked for the expression of aforementioned markers.

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PLoS One. 2020 Jul 6;15(7):e0235356. doi: 10.1371/journal.pone.0235356.r002

Author response to Decision Letter 0

9 Apr 2020

Dear Dr. Pizzo,

Thank you for your positive consideration of our manuscript by Lee et al. entitled, “Real-time monitoring of oncolytic VSV properties in a novel in vitro microphysiological system containing 3D multicellular tumor spheroids” (Manuscript ID PONE-D-19-34362)

The manuscript has been thoroughly revised to reflect the reviewers’ comments, including additional experiments. Here, we re-submit our revised manuscript while addressing all the issues raised on a point-by-point basis. Please find all the responses in this letter, which also provides identifying page and line numbers when referring to specific parts of the manuscript for your convenience.

Once again, we sincerely appreciate your thoughtful consideration and look forward to hearing from you soon.

Best regards,

Heuiran Lee, Ph.D.

Reviewer #1: The authors previously developed a microphysiological system (MPS) which features a vessel-like channel that can be lined with endothelial cells and a total of 29 side chambers which can be populated with various cell types. In the current study they generated spheroids in the side channels by infusing A549 cancer cells and MRC5 fibroblasts, then lined the vessel-like channels with HUVECs. They infused VSV-GFP into the vessel-like channels and were able to show that the virus infected only the A549 cells, not the MRC5 cells in the spheroids. This result was expected since the MRC5 cells were resistant to the virus in 2D culture and the A549 cells are susceptible. Interestingly the HUVECs did not get infected in the MPS, but were susceptible to virus infection in 2D culture. The authors speculate based their analysis of IFNb release from VSV infected A549, MRC5 and HUVEC cells, and the protection of those cells from VSV infection upon IFNb exposure, that the release of IFNb by virus-exposed MRC5 cells in the MPS is able to render the HUVEC resistant to infection.

The MPS system colonized with multicellular spheroids does offer an interesting in vitro test system for the study of oncolytic viruses that might well catch on as an alternative to mouse xenograft models if it were widely available. The ability to form multicellular tumor spheroids in side chambers accessible only via a vessel-like channel is appealing and allows for detailed analyses of the dynamical aspects of virus interaction with microvessels, tumor cells and fibroblasts in a controllable system. However, there are significant weaknesses with the current manuscript which, in the opinion of this reviewer, should be addressed prior to publication:

Ans:

We thank you for your thoughtful consideration of our manuscript. Your comments and concerns raised are addressed below on a point-by-point basis. The manuscript has been thoroughly revised, including additional experiments.

Rev-1 Q1: The authors state that they perfuse their system with VSV-GFP using a virus moi (multiplicity of infection) of 5.0. But what does that mean? First, they do not give details of the cell substrate on which they titrate their virus stock. Second, it is not clear how they determine the "moi" for the MPS when they do not know how many cells are present in the MPS that they are perfusing. This aspect of the paper is confusing and needs to be clarified.

Ans: Thank you for your careful reading of our work. In order to ensure the consistent infection of oVSV-GFP in this study, the stock virus titer was quantified by TCID50 indicating how many infectious virus particles are present. This information is included in the revised M&M (page 6, lines 120-122). 1 MOI represents 1 infectious virus particle per cell as described in the updated M&M section (page 6, lines 122-123).

In MCTSs, MOI was determined based on the total number of cells (3 x 105 cells/chip) in MPS where 1 x 105 cells were identified identically from A549, MRC5 and HUVEC cells, respectively. Under this infection condition, consistent results were achieved in MPS. The above information is now found in the M&M section (page 7, 138-143; page 7-8, 147-151), the results (page 11, lines 234-235), and the legend (page 12, lines 258-260) of our updated manuscript.

Rev-1 Q2: The vessel-like channels in the MPS are supposedly lined by HUVEC cells but there are no images provided to confirm this, nor to show whether the HUVEC lining is indeed sufficiently comprehensive to form vessel-like channels. This is a critical aspect of the system and should be addressed with additional data. Without a direct visualization of the HUVEC lining cells, and an assessment of their GFP expression post virus infusion, the claim that the HUVEC cells resident in the MPS are resistant to virus infection remains unproven.

Ans: The data directly visualizing HUVEC lining cells were not included because the characteristics of MPS designed in-house, including vessel-like tubular structure with HUVEC cells, had recently been published (Fibroblast-associated tumor microenvironment induces vascular structure-networked tumouroid. Scientific reports. 2018; 8:2365) and this tubular structure was consistently observed when performing this study. However, we agree that the data showing HUVEC linings is necessary to strengthen our conclusions and appreciate your valuable suggestion. We therefore performed an additional experiment to generate MCTSs in MPS where HUVEC cells were fluorescently labeled with red PKH26 as described in the M&M. HUVEC lining in channels of MPS was clearly observed under a fluorescence microscope and the fluorescence image along with the LM image were included in the revised Figures as Fig. 1D.

The contents have been also modified in the M&M (page 5, lines 101-102), the results (page 10, lines 205-206), and the legend (page 10, line 213).

Rev-1 Q3: The authors state that there are 29 side chambers in their MPS but do not indicate how they captured data from all of these chambers and do not provide any statistical backup for the conclusions they have drawn.

Ans: We agree with your valuable comment and apologize for not describing our data properly. Detailed information on how data was captured from the chambers and respective statistical significance has been added to the revised manuscript whenever necessary, including conclusions.

Rev-1 Q4: The hypothesis that HUVEC cells lining the MPS channels are rendered resistant to VSV-GFP by exposure to IFNb from MRC5 cells could easily be tested by rerunning the experiment using A549-only spheroids that do not contain MRC5 cells. Data from such an experiment should be included in the manuscript.

Ans: Thank you for thoughtful suggestion. As shown below, the GFP signal from oVSV in HUVEC lining was actually observed even at 8 h p.i. when MPS was prepared with HUVEC cells alone. In contrast, GFP signals were detected exclusively in the tumor spheroid when the virus was introduced into MPS containing all three cell lines.

Since this data is actually part of Fig. 5C, the original Fig. 5C was modified by adding a GFP image panel (the figure below) and corresponding texts, such as M&M (page 7, lines 145-146), results (page 14, lines 294-300) and legend (page 14, lines 304-307) have been also rewritten. In Fig. 5C, the effect of fluid flow on the expression of low density lipoprotein receptor (LDLR), the main receptor of oVSV, was investigated in HUVEC cells after oVSV-GFP treatment. The study was conducted using MPS composed of HUVEC cells alone. In this situation, GFP signals were substantially observed in HUVEC cells compared to MPS with all three cell lines (Fig. 4) and this phenomenon was not dependent on fluidic flow. Consistent LDLR expression levels were also identified regardless of fluidic flow, further suggesting that difference in HUVEC sensitivity between MPS and 2D system is not related to LDLR expression.

Previous studies have shown that IFN-β secreted from primary cells induces anti-viral activities in other nearby cells through paracrine signaling. The amount of IFN-β from MPS eluate was measured. Alhough not statistically significant, The IFN-β level was found to be 5.53 ± 3.94 pg/ml at 14 h p.i. (n=3). We thus believe that with the IFN-β analysis of Fig. 5B, the correlation between the initial IFN-β secretion by neighboring MRC-5 cells and HUVEC's resistance to oVSV in MPS could be suggested.

Related references:

- Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124(4):783–801

- Voigt EA, Swick A, Yin J. Rapid induction and persistence of paracrine-induced cellular antiviral states arrest viral infection spread in A549 cells. Virology. 2.16; 496:59-66

- Kotredes KP, Thomas B, Gamero AM. The protective role of type I interferons in the gastrointestinal tract. Frontiers in immunology. 2017; 8:410

Reviewer #2:

Authors have investigated the potential of their recently developed microphysiological system (MPS) that mimics the in vivo tumor microenvironment and serves as culture space for 3D multicellular tumor spheroids (MCTS). This system was used as a model to check the oncolytic potential of Vesicular Stomatitis virus and concluded that this system can be used as a first line of monitoring system to validate any oncolytic virus efficacy. It is a helpful model system for evaluation of anticancer therapies including virotherapy.

It is a good piece of work; however it was not presented very well. Methodology and results need to be written clearly. At many places the clarity is missing. Experiment description in the legends needs lot of improvement.

Ans: We appreciate your positive consideration and valuable comments on our manuscript. With your comments in mind, we have thoroughly revised the manuscript by resolving all the issues presented below.

Rev-2 Q1: In Figure 1C, A549 cells stained green and MRC5 red. But why is it bright yellow at the interface of these two cell populations? Is it due to overlap?

Ans: Thank you for reading our manuscript carefully. A549 cells were actually fluorescently stained green and MRC5 cells were red in MCTSs (Fig 1C). So, as you think, it appears yellow if you examine merged images in areas where two different types of cells overlap in 3D MCTSs. This information has been added to the revised manuscript (page 10, lines 202-205) to help the reader's understanding.

Rev-2 Q2: Reference to the “effective replication of oncolytic VSV expressing GFP in A549cells” under result section, related Fig 2C, oVSV effects were shown at 5h and 50h. It would be better if 25h data of GFP expression and PI staining is also incorporated in the image panel. In this experiment, CPE/GFP expression/cell death was measured up to 50h post infection; but the negative control (mock infected) was not included. How was it confirmed without a negative control that the noted cell death was virus induced?

Ans: As recommended, the 25H data is also incorporated in the image panel, fore Fig. 2. Unlike the 5H period, green signals indicating active oVSV-GFP replication at 25H where the growth of the virus reaches its peak was easily observed. Upon virus introduction, the proliferation of A549 human cancer cells, which has a doubling time of about one day, was not observed at both 25H and 50H under LM compared to 5H p.i. In addition, we performed a mock-treated negative control to ensure the integrity of A549 cells during the entire experimental time. As expected, untreated negative cells continue to grow to 50H as shown below. There were no signs of cytopathic effect as determined by LM and PI analysis.

Conversely, PI analysis marked cell death was observed when oVSV-GFP was actively replicated (Fig. 2B & the revised Fig. 2D). Taken together, the data indicate that the cytopathic effect is indeed driven by oVSV replication, which is included in the modified Fig. 2. The results (page 10-11, lines 217-222) and legend (page 11, lines 227-230) have been updated accordingly.

Rev-2 Q3: In Figure 3E, quantification of viral genome copies was done to analyse oVSV-GFP replication in MCTS eluates. How was it done? Was it the amplification of VSV –G gene by quantitative PCR? Please clarify in the text and legend. In this experiment as well, negative control was missing.

Ans: We did utilize VSV-G specific primers for PCR reaction in the amplification of the RT-qPCR viral genome. The details can be found in the modified M&M (page 8, lines 162-168). In this experiment, we also performed RT-qPCR using a mock-treated negative control to ensure VSV-G RNA genome-specific reactivity as shown below.

Since the mock-treated eluate sample has no VSV, no positive signals were detected in real-time thermal cycler regardless of RT reaction. On the contrary, large amounts of viral genomic copies were readily identified when RT reaction was performed prior to qPCR in the eluates from the viral infection group. We clarified this finding by rewriting the results (page 11, lines 238-239, page 12, lines 242) and the legend (page 12-13, lines 263-265).

Rev-2 Q4: What was the rationale behind using different MOIs, 1 and 5 for infections in 2D cultures and MPS?

Ans: As described in the revised M&M (page 6, lines 122-123), the cell lines in 2D culture were infected with oVSV-GFP at 1 MOI to efficiently incorporate viruses into cells. Because viruses could not effectively reach the cells particularly located inside due to three-dimensional architecture of MCTS, MPS was treated with 5 MOI. MOI was determined in MPS (page 7-8. lines 147-151) based on the total number of cells (3 x 105 cells/chip), for consistent results.

Rev-2 Q5: From the methodology section given under ‘quantification of secreted human interferon beta (IFNβ) after VSV-GFP infection’ it is clear that IFNβ response was measured in all three types of cells following oVSV-GFP infection in 2D cultures and eluates collected from infected MPS by ELISA. But the related result and Figure/ figure legend are confusing. Please check the result and the figure related to this experiment and write more in detail.

Ans: To clarify IFN-β secretion was monitored from the culture medium of each cell after oVSV-GFP treatment in 2D culture, The information on 3D eluates has been removed from M&M. We also carefully amended the M&M (page 9, lines 175-179), results (page 13-14, lines 285-290), and legend (page 14, lines 303-304).

Rev-2 Q6: To check the differential replication of oVSV-GFP in the 3D MCTS integrated MPS, A549 cells, MRC5 cells were labeled with fluorescence tracker. Figure 4A and 4B, both the cells; A549 and MRC5 were stained red. How were these two cell populations separated to check the expression oVSV-GFP through red and green merge? Are 4A and 4B images from the same spheroid? Clarify and also mention in the text and legends. In the figure 1, A549 cells were stained green.

Ans: Thank you for the thoughtful comments. Staining cells with a green tracker is not practical because the green signal from cells stained with green cannot be distinguished from GFP signals that appear following oVSV-GFP replication in the infected cells. In other words, when cells were infected with oVSV-GFP, A549 and MRC5 constituting MCTSs could not be stained simultaneously in two colors, green and red.

Therefore, where MCTSs were treated with oVSV-GFP, A549 or MRC5 (Fig. 4, 4A, and 4B, respectively) cells stained with red were utilized. This would cause the red and green to overlap and turn yellow if the virus replicates in the labeled cell, whereas the red and green would not overlap if the virus did not multiply in the labeled cell. The images in Fig. 4A and Fig. 4B are not from the same MCTSs, and we revised the M&M (page 5, lines 101-102), results (page 12, lines 243-248), and legends (page 13, lines 269-274) to clarify this issue.

Unlike Fig 4, we illustrated the mock-treated MCTS in Fig. 1, as we were able to stain A549 and MRC5 cells in two distinct colors. The corresponding content has been rewritten and can also be found in the answer to your first comment.

Rev-2 Q7: There is a lot of room for improvement in discussion section. One sentence in the discussion from lines 326 to 329 is the exact repetition of a sentence under result section from lines 281 to 284. Improve the discussion.

Ans: Yes, we fully agree with your comments and thank you again. Repeated sentences have been rewritten in the revised version of the manuscript (page 13-14, lines 285-290; page 15-16, lines 330-339) and the discussion section has been substantially modified to improve the quality of the manuscript.

Rev-2 Q8: The intensity of GFP expression in A549 cells does indicate the active replication of oVSV-GFP virus but not the anti tumor activity. Experiment of Live/Dead cell staining following virus infection does not commend the anti-tumor activity of oncolytic virus used. An anti tumor/ anti proliferative marker may be used to confirm the anti tumor activity of the virus in MCTSs. Following infection, spheroids can be checked for the expression of aforementioned markers.

Ans: Thank you for the thoughtful opinion. As mentioned, the intensity of GFP expression in A549 cells does not directly indicate anti-tumor activity. However, evidence for the close correlation between oVSV replication and anti-tumor activity by monitoring oVSV-GFP replication, GFP signaling, and cytopathic effects after virus treatment in A549 cells is provided in Fig. 2. Cytopathic effects in A549 cells were determined by LM and PI staining. Anti-tumor activity in MCTSs was investigated by PI staining after oVSV-GFP treatment using tumor spheroids unlabeled with a fluorescent dye (Fig. 4C). The PI-positive dead cell region was perfectively merged with the GFP-positive region, indicating that the GFP-positive A549 cells, but not the MRC5 cells or HUVECs, were dead. Additionally, the size of infected MCTSs gradually decreased, while that of uninfected control MCTSs increased (Fig. 4D, 4E). Therefore, we believe that the results mentioned above have adequately provided evidence to confirm the anti-tumor activity of the virus even without further confirmation using anti-proliferation markers. This content has been added to the results (page 11, lines 219-222 & page 12, lines 248-254) and legend (page 11, lines 227-230 & page 13, lines 274-278).

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Decision Letter 1

Salvatore V Pizzo

15 Jun 2020

Real-time monitoring of oncolytic VSV properties in a novel in vitro microphysiological system containing 3D multicellular tumor spheroids

PONE-D-19-34362R1

Dear Dr. Lee,

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Reviewer #2: Authors have addressed all the questions adequately, also incorporated suggested information and experiments. However, following are couple of things to be considered.

Rev-2 Q4: What was the rationale behind using different MOIs, 1 and 5 for infections in 2D cultures and

MPS?

Ans: As described in the revised M&M (page 6, lines 122-123), the cell lines in 2D culture were infected

with oVSV-GFP at 1 MOI to efficiently incorporate viruses into cells. Because viruses could not effectively reach the cells particularly located inside due to three-dimensional architecture of MCTS, MPS was treated with 5 MOI. MOI was determined in MPS (page 7-8. lines 147-151) based on the total number of cells (3x 105 cells/chip), for consistent results.

Comment 1: The above explanation may be incorporated either in methods or in the relevant results section.

Comment 2: In the manuscript, at some places “in vitro and in vivo” words are written in italics (in vitro/in vivo) and not everywhere. Make it uniform.

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