Introduction
Gallbladder carcinoma (GBC) remains the most common biliary tract malignancy characterized by its high lethality, aggressive nature, and dismal prognosis [
1]. As standard radio- and chemotherapy are insufficient treatments, surgical resection is the only potential curative approach. However, few patients qualify for surgery, leading to a 5% overall 5-year survival rate [
2]. Thus, novel therapeutic strategies are needed.
Oncolytic viruses that selectively infect and kill tumors exhibit modest clinical success [
3]. Myxoma virus (MYXV), a rabbit-specific poxvirus, exhibits narrow host tropism likely as a consequence of protective induced-interferon (IFN) responses in other species [
4]. MYXV can infect and kill over 70% of tested human tumor cell lines by exploiting the same cellular defects such as IFN-mediated mutations [
5].
Akt, a serine/threonine kinase important in balancing cell survival, proliferation, and cell death, is dysregulated in many human cancers [
6]. Endogenous phosphorylated Akt (p-Akt) levels highly correlate to permissiveness for MYXV infection. Tumor cell lines exhibiting high p-Akt are susceptible to MYXV and defined as type I cells; those with low but detectable p-Akt that increase following MYXV infection are type II; and those with undetectable p-Akt that generally resist MYXV are type III [
7]. Rapamycin (Rap), a macrocyclic lactone, increases the oncolytic potential of MYXV by elevating endogenous p-Akt in the context of MYXV infection. Additionally, Rap is an immunosuppressant that modifies host innate or adaptive cellular immunity, further facilitating MYXV infection [
8]. Combined MYXV + Rap therapy has successfully treated glioma, medulloblastoma, and other tumors [
9,
10]. Whether combined therapy can target GBC, however, remains unknown.
Hyaluronan (HA), a large glycosaminoglycan (GAG) [
11], is a chief extracellular matrix (ECM) component that contributes significantly to cell proliferation and migration. HA is natively a large polymer but degrades into low-molecular-weight HA under inflammation [
12‐
14]. All CD44 isoforms contain an HA-binding site in their extracellular domain and serve as the major HA cell-surface receptors [
15]. HA–CD44 binding stimulates a number of signaling pathways. Among them, firstly, HA activates PI3K/Akt/mTOR signaling [
16], which also elevates p-Akt; in the second place, HA induces matrix metalloproteinase-9 (MMP-9, gelatinase B) expression [
17]. MMP-9 preferentially degrades denatured collagens and native collagen type IV, a main component of ECM and basal membranes. ECM structures present a barrier to therapeutic molecules and virus particle diffusion within tissues, which may affect the effectiveness of virotherapy [
18].
In the present study, we showed that Rap enhanced MYXV-mediated GBC oncolysis in vitro, but not in vivo. Furthermore, we demonstrated that collagen IV was a critical factor hindering intratumoral MYXV distribution and it limited MYXV-mediated anti-tumor effects in vivo. Finally, HA-induced Akt activation and MMP-9 production significantly improved host survival following MYXV + HA therapy.
Materials and methods
Cell lines
Three human gallbladder cancer cell lines were used: GBC-SD (Cell Bank of the Chinese Academy of Sciences, Shanghai, China); NOZ (Health Science Research Resources Bank, Osaka, Japan); and SGC-996 (Academy of Life Science, Tongji University, Shanghai, China). CV-1 (monkey kidney), NIH3T3 (murine fibroblast) and U251 (human giloma) cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences. GBC-SD, NOZ, and NIH3T3 cells were cultured in DMEM (Gibco BRL, Carlsbad, CA, USA) containing 15% FBS (HyClone, Logan, UT, USA). SGC-996, CV-1 and U251 cells were cultured in RPMI medium 1640 (Gibco BRL) with 15% FBS at 37°C and 5% CO2.
Virus
The MYXV construct for transfection studies, vMyx-gfp, contains a green fluorescent protein (GFP) cassette driven by a synthetic vaccinia virus early/late promoter [
19]. Control UV-inactivated MYXV (termed “dead virus,” or DV) was irradiated for 2 h.
Reagents
Rat anti-CD44 mAb (clone 020, isotype IgG2b) (CMB-TECH, Inc., San Francisco, CA) blocked HA by recognizing the HA-binding region common among all CD44 isoforms. Low-molecular-weight HA (LMW-HA) fragments were purchased from RD (Minneapolis, MN, USA). Rap was obtained from Wyeth Pharmaceuticals, Inc. (Collegeville, PA, USA).
Viral replication assays
For single-step growth analysis, MYXV at a multiplicity of infection (MOI) of 5 was added to a 95% confluent cell monolayer. After 1 h adsorption, inoculum was removed, and each well was washed 3× with 1× PBS. Supplemented DMEM was added before incubation (37°C). Cells were collected by cell scraping at 1, 4, 8, 12, and 24 h post-infection. Following a 5-min spin (1500 rpm), cells were resuspended in 100 μL of hypotonic swelling buffer. To release virus, each Eppendorf tube underwent 3 freeze–thaw (−80°C and 37°C, respectively) cycles. Lysed cells were sonicated for 1 min and centrifuged (1500 rpm) for 5 min to disaggregate virus complexes.
For multi-step growth analysis, cells were infected (MOI = 0.01) and collected at 12, 24, 48, 72, and 96 h, and infectious virus was titrated in CV-1 cells [
20]. Serial virus dilutions (10
−2 to 10
−8) in serum-supplemented DMEM were added to CV-1 cells. After viruses adsorbed (1 h), un-adsorbed virus was removed, and DMEM was added to each well. Infection proceeded for 48 h. Titers (FFU/mL) were calculated as the number of foci × dilution × 2. Foci were counted from each well containing <100 foci under the fluorescent microscope (Leica); average titers were calculated from counts obtained from at least two wells.
Cell viability assays
Cell viability was determined by the water-soluble tetrazolium (WST)-1 method using the WST-1 cell proliferation and cytotoxicity assay kit (Beyotime, Shanghai, China). Briefly, 5 × 103 cells were seeded in 200 μL/well culture medium in 96-well plates for 24 h and treated with Rap or HA for 72 h. After incubation with WST-1 reagent for 2 h at 37°C, absorbance (450 nm) was measured using an automated microplate reader (Bio-Rad 5 Model 550, Bio-Rad, Hercules, CA, USA). Cell viability percentage = mean optical density (OD) of one experimental group/mean OD of the control × 100%.
Western blotting
Western blot examined protein expression using antibodies against MYXV M-T7 and Serp-1 (Biogen, Cambridge, MA); host p-Akt (Thr308) and Akt (Cell Signaling Technology, MA, USA); and host collagen I and IV (abcam, Cambridge, UK). β-Actin was used as the control. Crude membranes were prepared in lysis buffer (Hepes [10 mM], pH 7.4; NaCl [38 mM]; PMSF [25 μg/mL]; leupeptin [1 μg/mL]; and aprotinin [1 μg/mL]) and centrifuged at 33000 rpm for 1 h, and the pellet was resuspended. Tumor tissues were collected after virus infection, washed with 1× PBS, and subsequently lysed with lysis buffer containing protease inhibitors. Proteins were quantified using the Bradford protein assay (Beyotime, CHN), separated by 10–12% SDS-PAGE, and transferred to PVDF membranes (Millipore, Billerica, MA, USA), which were blocked with 5% non-fat dry milk and incubated with primary antibodies. Proteins were visualized by the ChemiDoc™ XRS image system (Bio-Rad) using the appropriate secondary antibodies conjugated to horseradish peroxidase.
Real-time PCR
Total RNA was extracted using Trizol (Gibco BRL) according to the manufacturer’s instructions. After quantification, complementary DNA (cDNA) was synthesized from 2 μg of total RNA using a Takara RNA PCR kit (Takara Bio Inc., Dalian, China). Primers were designed by Primer Premier software version 5.0 (PREMIER Biosoft, Palo Alto, CA, USA) and synthesized by Sangon Biotech (Shanghai, China). The following sequences were selected: MMP-9, CGGACCAAGGATACAGTTTGTT (forward) + GCGGTACATAGGGTACATGAGC (reverse); CD44, GAAGATTTGGACAGGACAGGAC (forward) + CGTGTGTGGGTAATGAGAGGTA (reverse). PCR program: initial denaturation at 95°C for 5 min, 40 cycles of 94°C for 20 s and 61°C for 20 s for annealing extension. β-Actin was used as the control.
Viral diffusion assays
BD Biocoat inserts for 24-well plates were pre-coated with collagen IV on 3-μm membranes (BD Biosciences, San Diego, CA, USA). Briefly, GBCs were plated at the base (1.5 × 105 cells/well). After 24 h incubation, inserts containing vMyx-gfp (MOI = 5) were placed on top. After 24 h, MYXV diffusion was analyzed by calculating the area of fluorescent foci/field in the base using Image-Pro Plus 6.0 software (Media Cybernetics Inc., Washington, USA).
Histology and immunohistochemistry
Tissues were immediately washed twice with physiologic salt solution followed by fixation in 4% paraformaldehyde for 24 h. After paraffin-embedding, 5-μm serial sections were cut, deparaffinized in xylene, and rehydrated in graded alcohols, followed by 3 rinses with 1× PBS. Antigen retrieval was performed in 10 mmol/L citrate buffer (pH 6.0) at 98°C for 10 min, and the sections cooled to room temperature (20 min). Sections were incubated in 1% H2O2 for 15 min to block endogenous peroxidase and incubated with 1:100 rabbit polyclonal anti-collagen IV at 4°C overnight. The corresponding biotinylated goat anti-rabbit IgG (Vector, BA-1000) (1:200) was added for 30 min, washed 3× in PBS, and incubated at room temperature in ABC complex (Vectastain ABC kit, Vector Cat# PK-6100) for 30 min. Staining was detected with DAB peroxide substrate solution for 5 min, followed by briefly rinsing in distilled water. Slides were dehydrated in graded ethanol, cleared in xylene, and mounted with Permount medium after counterstaining with Gill’s hematoxylin solution for 3 min. Control sections were incubated with the antibody preincubated with a blocking peptide. Sections omitting primary antibodies were used as negative controls.
Immunohistochemical scoring system
Immunostained sections were scored by 2 pathologists with no knowledge of experimental details using a semi-quantitative histologic scoring (H-Score) method [
21]; contradictory scores were re-evaluated until consensus was reached. Briefly, immunostaining intensity was scored as follows: 0 = none; 1 = weak; 2 = moderate; and 3 = intense compared to strong staining intensity of intratumoral macrophages. The designated H-Score value was obtained by multiplying each intensity (I) with the corresponding percentage of positive areas (PC) [H-Score = ∑(I × PC)]. Final score values ranged from 0–300.
Transwell invasion assay
Cell migration was evaluated using BD Matrigel Matrix Thin Layer 24-well plates (BD Biosciences). Sub-confluent cells were serum-starved for 24 h before the experiment. Cells were harvested by trypsin/EDTA, washed, resuspended in FBS-free media at a 106 cells/mL density, and transferred (100 μL) onto the matrigel. Lower chambers were filled with 600 μL of media containing 20% FBS, and the plates were incubated at 37°C for 24 h. Transwells were removed, stained with 1% crystal violet, and non-migrating cells were scraped off with a cotton swab. Six fields/Transwell were photographed using an inverted microscope (200×).
Hydraulic conductivity assay
Tumor-bearing mice were anesthetized by breathing diethyl ether. Evans blue solution (0.04%) was infused into tumor centers with 28G needle connected to a reservoir via 0.52-mm tubing. Infusion pressure (P
inf) was defined by the reservoir height relative to the needle tip. Flow rate (Q) measured the velocity of the bubble inside the tube. Hydraulic conductivity was based on Darcy’s law for unidirectional flow in an infinite region around a spherical fluid cavity: hydraulic conductivity = Q/(4πa
0 P
inf), where a
0 was the initial fluid-cavity radius that approximately equaled the 28G needle radius (0.18 mm). Here, all hydraulic conductivity was measured under 50 cm H
2O (P
inf = P
50cm H2O), and all the measurements were repeated 5× in different tumors [
22].
Gelatin zymogram analysis
Gelatinolytic activity was visualized on zymograms as described [
23]. Briefly, protein samples (50 μg) were separated on 10% SDS-PAGE containing 1 mg/mL gelatin. Gels were then washed with 50 mM Tris–HCl (pH 7.5) and 2.5% Triton X-100 buffer for 30 min; washed with above buffer plus 5 mM CaCl
2, and 1 μM ZnCl
2 for 30 min; and incubated with above buffer plus 10 mM CaCl
2, and 200 mM NaCl for 24 h (for supernatant) or 48 h (for membrane and tissue extracts) at 37°C. Zymograms were stained with 0.5% Coomassie blue.
In vivo studies in CD-1 nude mice bearing gallbladder cancer cells
Female CD-1 nude mice (age: 5 weeks; weight: 20–25 g) were obtained from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China) and housed at 3–5/cage on a 12-h light/dark schedule at 22 ± 1°C and 50 ± 5% relative humidity. All procedures followed the Ethics Committee guidelines of Xinhua Hospital, School of Medicine, Shanghai Jiaotong University.
Xenograft tumor models were established by subcutaneously injecting GBC-SD, SGC-996, or U251 cells (1 × 107 cells/0.1 mL) into the right flank. Nine days later, vMyx-gfp or DV (1 × 107 PFU) was intravenously injected every other day for a total of 3 injections. For testing Rap, mice were randomly divided (n = 5/group): (a) DV, (b) vMyx-gfp, (c) vMyx-gfp + Rap (5 mg/kg/d injected intraperitoneally beginning 5 days after tumor implantation, continuing 5 times/week for 2 weeks). For testing HA, mice were randomly divided (n = 5/group): (a) DV, (b) vMyx-gfp, (c) vMyx-gfp + Rap, (d) vMyx-gfp + HA (200 μg/mL injected intratumorally at multiple points every other day for 2 weeks beginning 9 days after tumor implantation), (e) vMyx-gfp + HA + anti-CD44 (100 μg/mL). Tumor areas were measured every 3 days. On day 13 after injection, mice were imaged with the Xenogen IVIS Spectrum system to record GFP-labeled virus in tumors, which were then removed for histological examination. For survival studies, animals were followed until sacrifice was required or the experiment was terminated. To examine vMyx-gfp distribution, frozen tumor tissues were cut into 5-μm serial sections, and GFP expression was imaged using a fluorescence microscope. GFP signal intensity was analyzed with ImagePro software to quantify both GFP and total tumor area.
Statistical analysis
Statistical Analysis Software (SAS Institute, Inc.) analyzed all statistics. Survival curves were generated by the Kaplan-Meier method. All reported P values were two-sided and considered to be statistically significant at P < 0.05. All experiments were performed at least 3 times.
Discussion
Gallbladder cancer is an aggressive disease with dismal clinical outcome [
1,
2]. Oncolytic virotherapy is an innovative alternative to conventional therapies [
3], and MYXV not only has an extremely narrow host-species tropism but also can selectively infect and kill many human tumor cells utilizing dysregulated signaling pathways [
24]. For example, MYXV treatment of human gliomas (U87 or U251) implanted into immunocompromised mice progressively decreased tumor size, increased host survival, and even completely cured the disease [
25,
26].
Rap dramatically increases permissiveness of certain type II human tumor cell lines to MYXV [
9]. Increased MYXV replication in cells is concomitant with global effects on mTOR signaling and correlates with increased Akt kinase activation [
10]. Rap also enhances MYXV oncolysis
in vivo in a murine xenograft human medulloblastoma model [
10]. Here, we demonstrated that two GBC cell lines, GBC-SD and SGC-996, are type II cells (Figure
1). Furthermore, Rap significantly increased p-Akt levels and improved MYXV oncolytic efficiency
in vitro (Figure
1A). Notably, MYXV-mediated oncolysis of GBC-SD cells was comparable to that of U251 glioma cells in the presence of Rap at 100 ng/mL (73.8% vs. 73.3%). However, in contradictions to previous studies in glioma tumors [
9], MYXV + Rap neither significantly reduced tumor area in GBC-SD xenografts nor prolonged host survival compared to MYXV alone (Figure
1B-F).
To explain the discrepancy between MYXV therapy for gliomas and GBCs
in vivo as well as how MYXV + Rap effectively killed GBCs
in vitro but not
in vivo, we hypothesized that tumor-associated ECM may be different between the 2 tumor types. Previous clinical and animal model studies indicate that intratumoral spread of replicating adenovirus
in vivo can be surprisingly poor compared to viral spread in comparable cell types
in vitro[
27], which may render the virus unable to disseminate within the growing tumor for any clinical benefit [
28,
29]. Brown et al. found that extracellular collagen hindered diffusive therapeutic-molecule penetration within tumors and that matrix modification alleviated this barrier [
18]. Administering collagenase or trypsin to glioma xenografts enhanced infectious adenoviral spread [
30]. Furthermore, an MMP-8–expressing adenovirus construct, which effectively degraded collagen I, improved viral spread and oncolysis [
31]. The role of tumor-associated collagen in MYXV intratumoral spread, however, has not yet been investigated. We found 2 GBC tumors expressed more collagen IV than U251 glioma tumors
in vivo (Figure
3A-D). The same outcome was observed when comparing clinical samples from 10 GBC and 5 glioma patients (Figure
3E,F). Thus, increased collagen IV in both xenografts and solid tumors suggested the universality of enhanced collagen distribution in GBC-associated ECM. Functionally, collagen IV significantly blocked MYXV diffusion in diffusion assays, which was restored by collagenase treatment (Figure
3G,H). No binding was detected between collagen IV and MYXV, suggesting that collagen IV likely serves as a physical barrier to prevent viral passage through the membrane and, by inference, within GBC tissues. Thus, the abundant collagen IV distribution within GBCs may account for the poor intratumoral viral spread and suboptimal effect of MYXV + Rap
in vivo.
To circumvent this collagen IV barrier, we exploited HA––a non-sulfated, unbranched GAG consisting of repeating disaccharide units––as a potential therapeutic approach. HA binding to CD44 not only affects cell adhesion to the matrix but also stimulates several tumor-specific functions. Also, HA–CD44 interactions increase p-Akt levels [
32]. Recently, it was shown that HA regulated OPN (a transcriptional target of HA) and that the PI3K/Akt/mTOR pathway upregulated OPN [
16]. As expected, we found that the HA–CD44 interaction also mediated and was required for Akt activation in GBC cells. Moreover, considerably enhanced oncolysis by either MYXV + HA or MYXV + Rap was observed in GBCs
in vitro. Despite the less significant tumor inhibitory effect by MYXV + HA compared to MYXV + Rap in SGC-996 cells, the MYXV + HA regimen was still superior than MYXV alone (Figure
5C).
HA–CD44-mediated enhancement of MMP-9 activity was extensively investigated in other tumors [
33‐
36]. HA–CD44 signaling is thought to stimulate FAK and modulate MMP-9 secretion via Ras-ERK 1/2 signaling [
17]. Transcriptional activation of genes containing putative AP-1 and/or NFκB binding sites in their promoter also regulates MMP expression [
37]. In our study, HA enhanced both the pro-enzyme and active form of MMP-9 in GBC tumor cell supernatants as well as membrane-bound MMP-9 in membrane extracts (which may regulate pericellular ECM degradation from the tumor cell surface) in a CD44-dependent fashion
in vitro.
The
in vivo GBC model best reflects the cellular/extracellular environments influencing tumor formation and susceptibility to oncolytic virotherapy. Our immunohistochemistry analysis showed that HA significantly degraded extracellular collagen IV within tumors in a CD44-dependent manner (Figure
6A,B). Increased hydraulic conductivity confirmed that HA reduced intratumoral fluid flow resistance, helping to rationalize how HA promoted MYXV dissemination. MYXV + HA exhibited superior GBC oncolytic efficiency
in vivo compared to MYXV + Rap in immunodeficient mice, both in terms of tumor area and overall host survival (Figure
6F-I). However, MYXV + HA did not completely eliminate GBC tumors. It is possible that HA induces inflammatory mediators, such as IFN via a TLR/MyD88-dependent pathway, which may interfere with MYXV proliferation and diffusion [
38].
HA–CD44 interactions play important roles in tumor invasion and migration [
39]. In our study, we showed that MMP-9 expression rose when HA was above 150, but below 250 ng/mL; in contrast, HA did not increase CD44 expression (Figure
6A-D). The maximal HA safe concentration for GBC-SD and SGC-996 based on the Transwell assay was 250 and 200 ng/mL, respectively. To avoid significantly enhancing tumor-cell migratory capacity, we adopted 200 ng/mL HA
in vitro and
in vivo.
We report for the first time that collagen IV is a critical limiting factor impeding MYXV spread in GBC tissue and reveal the synergistic oncolytic effect of MYXV + HA, which may help develop and optimize GBC therapy. However, some caveats still remain. Firstly, MYXV-induced anti-tumor and anti-viral immunity will undoubtedly affect tumor progression in immunocompetent hosts, which will need to be addressed in future studies, and immunocompetent GBC models will be developed to investigate MYXV + HA synergy within an intact immune system. In the second place, the precise mechanism(s) by which HA elevates MMP-9 and p-Akt expression levels are not yet fully understood. Thirdly, anti-CD44 mAb may inhibit GBC tumor growth by hampering apoptosis or angiogenesis [
40,
41]. Finally, since we based the viral dose on previous reported experience with other tumor types, the ideal MYXV and HA doses against GBC need to be determined.
To conclude, we propose that extracellular tissue collagen IV hinders MYXV dissemination. Moreover, HA–CD44 interaction may elevate oncolytic efficiency not only by activating Akt but also promote viral spread within GBC tissue by degrading collagen IV through MMP-9 secretion, finally converging to enhance the overall MYXV-mediated anti-tumor effect on GBCs in vivo.
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Competing interests
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in the manuscript entitled, “Targeting gallbladder cancer: oncolytic virotherapy with myxoma virus is enhanced by rapamycin in vitro and further improved by hyaluronan in vivo”.
Authors’ contributions
WMZ carried out the molecular experiments and drafted the manuscript. GW participated in the molecular experiments and helped to draft the manuscript. MMZ participated in the molecular and animal experiments. CBF carried out immunoassays. QYY and ZMD carried out viral preparation. LXQ helped to draft the manuscript. MG and FP helped to design the study. YY performed the statistical analysis. QZW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.