1. Introduction
Worldwide more than 1 million women are diagnosed annually with a gynecological cancer. Non-specific symptoms (such as in ovary cancer) and disparities in accessibility to health services (such as in cervical cancer) explain the differences in gynecological cancer outcome globally. Most gynecological cancer types are characterized by an accompanying stroma that not only supports malignant cells growth but is also largely responsible for the high resistance of the cancer to conventional and targeted therapies [
1,
2].
Epithelial ovarian cancer (EOC) is one of the most common gynecological cancers, with one of the highest mortality rates in women close to cervical and uterine cancer mortality rates [
3]. Globally, EOC is the seventh most common malignancy diagnosed among women accounting for more than two hundred thousand deaths globally [
4]. More than 300,000 women were estimated to have been diagnosed with EOC worldwide in 2020 [
4]. Due to the late onset of symptoms and the absence of early screening and detection modalities, EOC is usually diagnosed at an advanced stage and approximately 75% of women present with advanced stage disease (stage III or IV) [
5]. Whilst patients initially respond to chemotherapy, 80% of them rapidly develop chemoresistance and the recurrence is pretty high [
6,
7]. Exfoliated individual cells or spheroids of ovarian cancer cells disseminate through the peritoneal cavity colonizing the mesenterium, the omentum and the diaphragm and also the external layers of organs such as intestine and spleen [
8,
9].
Ovarian cancer shows a remarkable resistance to available therapies. Current management of ovarian cancer at presentation consists of cytoreductive surgery followed by chemotherapy that includes platinum and taxanes. However, almost 30% of patients have primary platinum resistance, 80% of patients will rapidly become refractory to the treatment and almost all patients will ultimately succumb of their disease [
7]. Interestingly, recent studies have shown that higher stroma proportion at the tumor at initial diagnosis of ovarian carcinoma, is associated with eventual emergence of platinum chemoresistance [
10]. With current 5-year survival rates under 50%, and 15% of women with ovarian cancer dying within few months of diagnosis, there is an urgent need for novel treatments for this deadly disease.
Many treatment options are being assessed in recurrent EOC settings including targeted therapy with the anti-VEGF antibody bevacizumab or poly (ADP ribose) polymerase inhibitor (PARPi) therapy that have shown some efficacy to extend progression-free survival rates but not overall survival [
11,
12]. Bevacizumab, combined with platinum/ taxane-based chemotherapy has been recommended by the National Comprehensive Cancer Network (NCCN) guidelines as a first-line treatment for EOC [
13]. Interestingly, the combination of the anti-PD1 and anti-CTLA4 check point inhibitors showed promising results in platinum resistant ovarian cancer at the six months-interim analyses with an overall response rate (ORR) of 34% (doubling the results of nivolumab monotherapy [
14,
15]. In November 2022, mirvetuximab soravtansine (a conjugated antibody targeting the folate receptor α to inhibit microtubules) was granted an accelerated approval by the FDA for the treatment of patients with folate receptor α positive, platinum-resistant EOC who have received 1-3 prior systemic treatment regimens; the median duration of response being less than 7 months [
16]. The efficacy of the different and novel targeted therapies reduces with each recurrence and even the more advanced targeted medicines are still far from providing reliable therapeutics for this deadly disease.
Oncolytic viruses are a state of the art therapeutic strategy for cancer treatment. Oncolytic adenoviruses (OAdV) can be engineered with tumor specific promoters (TSP) and transcriptionally targeted to selectively attack and kill target cells [
17,
18]. In previous studies we have shown that hybrid TSPs can be designed to target the cancer stromal cells compartment in addition to the malignant cell compartment [
18]; moreover, TSPs can be engineered with the addition of tumor microenvironment responsive motifs [
19]. In the present study we extend the studies of AR2011, a stroma targeted, tumor microenvironment responsive OAdV, by showing its lytic capacity on fresh explants obtained from different gynecological cancers (ovarian, uterus and cervical cancer). We also show its lytic capacity on human ovarian cancer cells obtained from peritoneal ascites combined with cisplatin. Finally, we describe the
in vivo efficacy in different murine models of a AR2011-derived version armed with cytokines’ genes.
3. Discussion
The potential of ovarian cancer cells to disseminate and metastasize into the peritoneal cavity is governed by, among others, the ECM composition. The outcome of oncogenic events in epithelial cells can be significantly modified by the nature of surrounding cancer associated fibroblasts and endothelial cells. Stromal cells could be implicated in the acquisition of a chemoresistant phenotype. An extensive infiltrative pattern with desmoplasia is one of the major features favouring metastases [
24]. In a recent study aimed to identify novel molecular subtypes of ovarian cancer by gene expression profiling a poor prognosis subtype was defined by a reactive stroma gene expression signature, correlating with extensive desmoplasia [
25]. In previous studies we have shown the therapeutic efficacy of a novel stroma-targeted oncolytic adenovirus, AR2011, on preclinical models of ovarian cancer including fresh human explants. We extend the previous data by showing here that AR2011 was able to replicate and lyse fresh explants obtained from additional gynaecologic cancers; moreover, it was able to replicate and eliminate
in vitro in combination with mainstay chemotherapeutic agents, ovarian cancer cells obtained from ascites of advanced stage disease; AR2011 armed to express hCD40L and h4-1BBL showed an extended
in vivo efficacy on human tumors established in the flanks of nude mice; moreover, the armed OAdV was able to arrest and even completely eliminate intraperitoneally disseminated human tumors. In preliminary studies, the armed OAdV expressing murine cytokines was able to exert a limited but significant abscopal effect in a syngeneic murine model.
Chemotherapy resistance is the major limitation of current treatments for ovarian cancer. Since most patients are treated with neoadjuvant and adjuvant chemotherapy, the development of OAdV aimed at reversing resistance and sensitizing cells to chemotherapy agents can be a major advance in the field. Different
in vitro and
in vivo approaches have been used combining non-replicative viruses expressing sensitizing agents to chemotherapy compounds. As an example, it was shown that ovarian cancer cells can be sensitized
in vitro and
in vivo to cisplatin by adenoviral expression of the manganese superoxide dismutase gene [
26]. Previous studies have also shown that myxoma virus can be combined
in vitro and in preclinical models with chemotherapy agents to treat mice with syngeneic ovarian tumors [
27]. It has been also shown that paclitaxel resistance can increase oncolytic virus lytic effect through a mechanism that involves upregulation of viral receptors [
26]. Previous studies from our group showed that AR2011 can replicate and lyse fresh explants of solid metastases arising from human ovarian cancer heavily pretreated with chemotherapeutic agents [
18]. Here, we extend the data by showing that AR2011 can synergize
in vitro with cisplatin to eliminate ovarian cancer cells obtained from patients’ ascites, even in those cases where patients have been previously treated with neoadjuvant chemotherapy.
A major limitation for adenovirus use in preclinical studies is the absence of syngeneic models that can recapitulate a human
scenario, in particular the secondary immune response that follows initial administration of an OAdV. In the absence of a full syngeneic ovarian cancer model in rodents [
28] most of the studies with OAdV are being performed in nude mice with the limitation of the absence of the full immune response. The lack of cross reactivity of hCD40L with murine models posed an additional limitation to assess the immune response associated with arming AR2011 with human CD40L. Interestingly, CD40-L has been shown to sensitize epithelial ovarian cancer cells to cisplatin treatment clearly indicating that activation of the CD40 intracellular pathway in cancer cells can be of relevance beyond CD40 effect in establishing the adaptive immune response [
29]. Despite the limitation with human CD40-L, it was interesting to note that AR2011(404) demonstrated a slightly higher
in vivo efficacy than AR2011 in nude mice studies. We cannot rule out that this enhanced
in vivo effect of AR2011(404) could be related to 4-1BBL expression. Although with markedly reduced affinity it was shown that human 4-1BBL can bind murine 4-1BBL receptor [
30]. This cytokine has been shown to expand T cells and NK cells and it is likely that NK activation could explain the slightly improved activity of AR2011(404)
in vivo in the nude mice model. Activation of the 4-1BB pathway in T cells restoration of effector functions has been hampered by the liver toxicity induced by soluble agonists [
31]. Therefore, it was compelling to express 4-1BBL locally under the regulation of the extremely specific hTERT promoter. Interestingly, activation of 4-1BB with agonists has been used in combination with other immune check points agents such as PD-1 and TIM-3 in murine models of ovarian cancer with remarkable efficacy [
32]. In this context it was interesting to see in preliminary studies, that expression of murine CD40-L and 4-1BBL in the CT26 model was able to induce not only a clear anti-tumor immune mediated response, locally, but also a modest but significant abscopal effect. Both the local and the abscopal effects were observed upon administration of AR2011(m404), while AR2011(H3) lacking cytokines expression was unable to induce any antitumor effect in this syngeneic model, not local neither systemic.
Diagnoses at a late stage of most ovarian cancers makes it imperative to develop innovative therapeutic approaches to tackle the disease. Both RNA and DNA oncolytic viruses have been used in the few clinical studies in ovarian cancer; all the clinical studies did not proceed after phase 1, or are recruiting patients for phase 1. Measless viruses expressing the carcinoembryonic antigen (CEA) were used in a phase I trial administered intraperitoneally in advanced stage cases [
33]. The treatment was well tolerated and resulted in dose-dependent biological activity in a cohort of heavily pre-treated recurrent ovarian cancer patients [
33]. Vaccinia virus and Reovirus reolysin reached also phase 1 trials but definitive reports are still lacking [
34]. Oncolytic adenoviruses have been used also in few clinical trials in ovarian cancer with unclear effects. After initial studies with the 55K-E1B-deleted dl1520 oncolytic adenovirus halted after phase 1 with no clear benefit [
35], few other groups reached phase 1 trials stage with modified oncolytic adenoviruses. Ad-delta24-RGD is an oncolytic adenovirus modified in the knob fiber domain to express an RGD moiety able to retarget the virus to integrins in a viral receptor-independent manner; this OAdV was well tolerated in a phase 1 trial after intraperitoneal administration and showed promising clinical activity [
36]. A variant of this OAdV expressing GM-CSF used in a compassionate mode in very few ovarian cancer patients was well tolerated and appeared to induce an anti-tumor immune response [
37]. Combination of this OAdV with a daily low dose of cyclophosphamide was also attempted in a phase 1 trial that involved few ovarian cancer patients with promising results [
38]. Few other trials are still under patients’ recruitment [
39]. We provide here novel data on the stroma targeted AR2011 OAdV that has been shown to kill ovarian cancer cells obtained from liquid ascites corresponding to patients pre-treated or not with chemotherapy. AR2011 could be combined with mainstay chemotherapy and was able to eliminate cells that seem to be refractory to chemotherapy. Moreover, AR2011 could be armed with transcriptionally targeted cytokines to be expressed only in tissues overexpressing the hTERT gene. The hTERT proximal promoter has been already used to drive specific adenoviral replication in malignant tissues and oncolytic cell death [
40]. OAdV driven by hTERT have entered clinical trials stages in different cancer cell types.
4. Materials and Methods
4.1. Cell lines and cell culture
The human ovarian cancer cell lines SKOV-3 and OV-4, A549 lung carcinoma cells, human embryonic retinoblasts 911 and the murine colon carcinoma cells CT26, were already described [
18] . The human ovarian cancer cells PA-1 (CRL-1572), and cervical cancer cells (HeLa, CCL-2; SiHa, HTB-35 and Ca Ski, CRL-1550) were obtained from the ATCC (Manassas, VA, USA). HEK293 cells were purchased from Microbix (Toronto, Canada). All the cell lines were grown in the recommended medium supplemented with 15% of fetal bovine serum (Natocor, Cordoba, Argentina), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and maintained in a 37 °C atmosphere containing 5% CO
2.
4.2. Construction and Production of the oncolytic adenoviruses
The main features of AR2011 have been already described [
9]. AR2011(404) was derived from AR2011 and expressed either human (h) or murine (m) CD40-L and 4-1BBL. For AR2011(h404) construction, a 2.8 Kb
BglII fragment containing the hTERT promoter followed by the sequence encoding for hCD40L, an internal ribosome entry site (IRES) from encephalomyocarditis virus and h4-1BBL, was cloned into the
BglII site of pshuttle 2kbHREF512ΔRb in the 3’-5’ orientation. A similar design was used for cloning a 2.9 Kb fragment in AR2011(m404) that included the murine version of each cytokine under hTERT. Both CD40L and 4-1BBL are the full length membrane bound forms and have a deletion in the cleavage site FEMQK (in hCD40L) and FEMQR (in mCD40L). All the DNAs were synthetized at Genscript (NJ, USA) following our own design. All the constructs were confirmed by automatic sequencing at Macrogen (Seoul, Korea).
For the recombination steps, the pshuttle containing the sequences encoding for the human cytokines was linearized with
PmeI and co-transformed with pvK500C F5/3 in BJ5183 cells to obtain the viral plasmid corresponding to AR2011(h404). To obtain AR2011(m404), the pshuttle encoding the sequences for the murine cytokines was recombined with pVK500C F5/3 where the entire hexon protein was replaced by the hexon protein of hAdV3. For hexon exchange, an adenovirus 5 backbone without hexon named pARΔHexon was prepared as described [
41]. A 6.9 Kb fragment containing the entire hexon of hAdV3 and flanking regions from hexon 5 was released with
SfiI from pAd5H3 / GL [
41] and recombined with pVKΔHexon linearized with
AsisSI. As a control of AR2011(m404), we constructed AR2011(H3) modified in the hexon protein following a similar procedure. The different recombined adenoviral genomes were linearized with
PacI and transfected in 911 cells. The rescued adenoviruses were used to infect HEK293 cells to produce the viral stocks [
42]. All the constructs were confirmed by restriction pattern and automatic sequencing.
4.3. In vitro cytotoxicity assay
For determination of virus-mediated cytotoxicity, 1 x 10
4 cells were seeded in 24 well - tissue culture plates and infected with the oncolytic adenoviruses at the indicated MOI [
42]. Hypoxic and normoxic conditions as well as addition of TNFα to recreate an inflammatory environment was already described [
19]. After 6 days, cell viability was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS assay; Promega, Madison, WI).
Fresh human explants were obtained at the Hospital Municipal de Oncología Marie Curie, Buenos Aires, Argentina, following institutional review board approval. Written informed consent was obtained from each patient. The declaration of Helsinki was followed in all the protocols. Samples were kept in RPMI medium on ice (Invitrogen, Carlsbad, CA). Time from harvest to slicing was kept at an absolute minimum (<2 hours). Between 6 to 10 slices, 1-2 mm depth, were placed in 24-well plates followed by the addition of the virus at 500 MOI in 500 μl of DMEM/F12 including 2% v/v FBS, 1% antibiotics and 1% L-glutamine [
43]. Infections were allowed to proceed for 5 hours where 3-5 slices were harvested for E4 quantification. In the remaining slices the medium was replaced with fresh DMEM/F12 containing 10 % FBS until the end of the experiment at 72 hours. For assessment of E4 levels as a surrogate of viral particles, DNA was obtained from tissue slices and qPCR for E4 was performed as described [
18].
4.4. Isolation of malignant cells from ovarian cancer liquid ascites
Samples obtained from the Hospital Marie Curie were centrifuged at 1500 RPM for 10 minutes to clear the ascites from cells. Cells were incubated in DMEM/F12 supplemented with 15% of FBS, brought to confluence and stored in liquid nitrogen until use. Each cells’ isolate was named as OC-LA followed by the respective number.
4.5. In vitro lytic assays in combination with cisplatin
Malignant cells obtained from liquid ascites were seeded in 96 well tissue culture plates (5 x 103 per well) and incubated for 48 hours with cisplatin at a final concentration of 2.5 µg/ml for 48 hours. After medium removal, cells were trypsinized, quantified and replated in the presence of AR2011 at 100 MOI for another 96 hours. When only cisplatin was used, cells were plated in the presence of cisplatin at 2.5 µg/ml for 48 hours followed by medium addition without virus for another 96 hours; for the control with virus alone, cells were plated only in medium for 48 hours followed by incubation in the presence of AR2011 (MOI 100) for another 96 hours as described above. At the end of the experiments cell viability was assessed with the MTS system.
4.6. Assessment of CD40L and 4-1BBL expression
For assessment of hCD40-L expression by flow cytometry, A549 and SKOV-3 cells were infected with AR2011(h404). At the end, cells were harvested in 0.5 mM EDTA, washed and resuspended in PBS containing 0.5 % BSA at a concentration of 5x106 cells/ml followed by incubation with phycoerythrin (PE) - conjugated hCD40L monoclonal antibody (eBioscience, CA, USA). A PE-conjugated mouse IgG1 kappa isotype (clone P3.6.2.8.1, eBioscience, CA, USA) was used as a matched control antibody (eBioscience, CA, USA). The cells were washed again and resuspended in 0.4% paraformaldehyde in PBS prior to analysis with a FACS Calibur flow cytometer (Becton Dickinson, Oxford, United Kinngdom). Ten thousand cells were analysed in each case.
For western blot assessment of h4-1BBL in cell lines, SKOV3 and A549 cells were seeded at 1 x 105/well in a 6 multiplate well plate. The next day cells were infected with AR2011(h404) and incubated at 37 °C for 30 hours. Cells were then harvested, and total protein extracts were prepared in lysis buffer containing 10 mM Tris (pH 7.5), 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS and a protease inhibitor cocktail. Total protein extracts were separated in 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, CA, USA). The membranes were probed with anti-human 4-1BBL antibody (ab68185, Abcam, MA, USA) and anti–β-actin antibody (A4700; Sigma, USA). HRP-Goat anti Rabbit (111035144, Jackson, NE, USA) and HRP Goat anti Mouse (115035003: Jackson, NE, USA) were used as secondary antibodies. Enhanced chemiluminescence (ECL) reagents were used to detect the signals following the manufacturer’s instructions (Amersham, USA). Antibody signals were digitized by Image Quant LAS 4000 (GE-Cytiva MA, USA). Anti β-actin antibody (A4700; Sigma, USA) and anti-αTubulin Ab (12g10 DSHB) were used as a loading control. HRP-Goat anti Rabbit (111035144: Jackson NE, USA and HRP Goat anti Mouse (115035003: Jackson NE, USA) were used as secondary antibodies. Enhanced chemiluminescence reagents were used to detect the signals following manufacturer’s instructions (Amersham, USA). Antibody signals were digitized by Image Quant LAS 4000 (GE-Cytiva MA, USA).
For assessment of hCD40-L and h4-1BBL expression in tumors, the tumor samples extracts were prepared using lysis buffer containing 50mM Tris (pH 7.3), 150 mM NaCl and 0.1% Tween 20 plus Halt protease inhibitor cocktail (8775, Thermo, IL USA). One hundred μg of protein extract was separated in 12% SDS-PAGE and transferred into nitrocellulose membranes. The membranes were probed with anti-h4-1BBL (ab68185, Abcam, MA, USA), and anti-hCD40L (ab2391, Abcam, MA, USA). Anti β-actin antibody (A4700; Sigma, USA) and anti-α Tubulin (12g10 DSHB, IA, USA) were used as a loading control. HRP-Goat anti Rabbit (111035144, Jackson NE, USA) and HRP Goat anti Mouse (115035003, Jackson NE, USA) were used as secondary antibodies. Enhanced chemiluminescence reagents were used to detect the signals following manufacturer’s instructions (Amersham, USA). Antibody signals were digitized by Image Quant LAS 4000 (GE-Cytiva MA, USA).
4.7. In vivo studies with nude mice.
Six to eight weeks-old female nude mice were obtained from the animal facility of the University of La Plata. After an acclimation period in Instituto Leloir animal facility, mice were injected with 4.5 x 106 SKOV3 cells in the flank. Tumor volumes were followed with caliper measurement every 2-3 days. Once tumors reached an average volume of 100 mm3, mice were injected intratumorally with 1 x 1010 v.p. of AR2011 or AR2011(h404) in 30 μl (as well as an equivalent volume of PBS for the control group). Viral or PBS administration was repeated 2 and 4 days later. Mice were followed with daily observations on general health until the control group (PBS) needed to be sacrificed due to animal distress following the approved protocol of the Institutional Animal Care and Use Committee of Instituto Leloir. At the end of the study remaining tumors were excised and weighed. For the in vivo assessment of hCD40L and h4-1BBL expression, mice were injected with 4.5 x 106 SKOV3 cells in the flank. When tumors reached 100 mm3 mice were administered i.t. with 5 x 1010 v.p. of AR2011, AR2011(h404) or PBS. Seventy-two hours later mice were sacrificed, the tumor area was removed, and a protein extract was prepared for western blot analyses as described above.
For intraperitoneally injected tumors, mice were injected with 6 x 10
6 SKOV3 cells. Five days later, mice were injected i.p. with 5 x10
10 vp in 400 μl of AR2011, AR2011(h404) or PBS. Injection was repeated 2 and 4 days later. Three mice per group were sacrificed 3 days after the last viral administration for assessment of E4 levels as a surrogate marker of viral particles [
18]. Mice were followed as described above. The remaining mice were sacrificed at day 54 of the first viral administration following institutional guidelines and remaining i.p. tumors were photographed
in situ, excised, weighed and a sample used for assessment of E4 levels.
4.8. In vivo studies with syngeneic models
Balb/c mice (6-8 week old male) were obtained from the animal facility of the University of La Plata. After an acclimation period in Instituto Leloir animal facility, mice were injected in both flanks with 5 x 105 syngeneic CT26 colorectal carcinoma cells. The tumorigenic inoculum (with 100% tumor take) was selected from a pilot study where mice were injected with 3 x 105, 5 x 105 and 1 x 106 cells in 100 μl of PBS (data not shown). Once tumors reached a volume of 75-100 mm3, mice were injected in the left tumor with 7.5 x 1010 vp of either AR2011(H3) or AR2011(m404) in a final volume of 50 μl PBS. Control mice were injected with 50 μl PBS. Tumors were measured bi-weekly in two dimensions with a caliper. The mice were followed until they need to be sacrificed due to a tumor size that exceeded 2000 mm3. In a second type of experiment Balb/c mice were injected in the left flank with 5 x 105 CT26 cells. Once tumors reached an average volume of 100 mm3 mice were injected in the contralateral right flank with 50 μl containing 5 x 105 CT26 cells pre-infected overnight with 3 x 104 MOI of either AR2011(H3) or AR2011(m404). Control CT26 cells were pretreated with PBS. Mice tumors were assessed with digital calipers and the volume was obtained with the following formula: volume=0.52 x (width)2 x length.
4.9. Statistical analysis
For figures 1B to E, 2B, 4A to C, 5C and D the statistical difference between groups was determined by a t-test and the F test (H. J. Motulsky, GraphPad Statistics Guide.
http://www.graphpad.com/guides/prism/7/statistics/index.htm). The survival curve in
Figure 5E was analyzed with Log-rank (Mantel-Cox) test. A
P-value of <0.05 was considered statistically significant. Data analysis was performed with the GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA). Bliss independence model [
21] was used to analyze drug combination data. The Bliss method compares the observed combination response (Y(O)) with the predicted combination response (Y(P)), which was obtained based on the assumption that there is no effect from drug-drug interactions. Typically, the combination effect is declared synergistic if Y(O) is greater than Y(P).
4.10. Ethics statement
All the experiments were approved by the Institutional Animal Care and Use Committee of the Fundación Instituto Leloir (Protocol #69OP). The Fundación Instituto Leloir has an approved Animal Welfare Assurance as a foreign institution with the Office of Laboratory Animal Welfare (NIH), Number A5168-01. All the patients at the Hospital Maria Curie signed an informed consent for samples’ use for research. All the samples reached Instituto Leloir in an anonymized code. The study was approved by the Ethics Committee of Hospital Maria Curie and by the Bioethics Committee of Fundación Instituto Leloir that also approved the use of human samples.
4.11. Patents
Part of the present data is included in the US patent application No.: 16/797,291 whose inventors are D.T.C., O.L.P. and M.V.L.
Author Contributions
Conceptualization O.L.P. and M.V.L..; Methodology M.V.L., E.G.A.C., A.A.; M.G; A.N.C; C.M.; I.B; G.D. R; C.R; Data Analysis O.L.P., M.V.L., E.G.A.C., A.A.; Resources O.L.P., M.V.L., N.C., D.T.C.; Data Curation O.L.P., M.V.L.; Writing-original draft preparation O.L.P., M.V.L.; Writing-review and editing O.L.P., M.V.L.
Figure 1.
In vitro lytic activity of AR2011. (A) lytic effect of different MOIs of AR2011 on various human cervical cancer cell lines. (B to E) AR2011 replication on fresh explants obtained from human ovarian, cervix and uterus cancer samples and normal uterus. For further details and statistical analysis, see materials and methods. Statistical significance, *p<0.05, **p<0.01, ***p<0.001.
Figure 1.
In vitro lytic activity of AR2011. (A) lytic effect of different MOIs of AR2011 on various human cervical cancer cell lines. (B to E) AR2011 replication on fresh explants obtained from human ovarian, cervix and uterus cancer samples and normal uterus. For further details and statistical analysis, see materials and methods. Statistical significance, *p<0.05, **p<0.01, ***p<0.001.
Figure 2.
In vitro lytic activity of AR2011 on malignant cells obtained from human ovarian cancer ascites. (A) Lytic effect of AR2011 at different MOIs on three different samples of OC-AF. (B) lytic effect of AR2011 at MOI 100 on different samples of OC-AF combined or not with cisplatin. *p<0.05, **p<0.01, ***p<0.001. For further details, see materials and methods.
Figure 2.
In vitro lytic activity of AR2011 on malignant cells obtained from human ovarian cancer ascites. (A) Lytic effect of AR2011 at different MOIs on three different samples of OC-AF. (B) lytic effect of AR2011 at MOI 100 on different samples of OC-AF combined or not with cisplatin. *p<0.05, **p<0.01, ***p<0.001. For further details, see materials and methods.
Figure 3.
Cytokines expression and lytic activity of AR2011(h404). (A) Scheme of AR2011(h404) genome structure showing that the cytokines cassette under hTERT regulation was cloned in the 3’-5’ orientation in the AR2011(h404) genome. (B) Cell surface expression of hCD40L in SKOV-3 (left) and A549 (right) cells: Light grey: Isotype control, Grey: AR2011(h404) MOI 100; Dark grey: AR2011(h404) MOI 1000. (C) Western Blot for the detection of h4-1BBL expression. The blot shows that both cell lines expressed endogenous 4-1BBL. Data for cytokines’ expression was obtained 30 hr after infection since after 48 hr cells were completely lysed by the OAdVs. (D) In vitro lytic activity of AR2011(h404) in different human malignant cell lines. Percent of surviving cells was assessed by the MTS assay. AR2011 was used as a comparator.
Figure 3.
Cytokines expression and lytic activity of AR2011(h404). (A) Scheme of AR2011(h404) genome structure showing that the cytokines cassette under hTERT regulation was cloned in the 3’-5’ orientation in the AR2011(h404) genome. (B) Cell surface expression of hCD40L in SKOV-3 (left) and A549 (right) cells: Light grey: Isotype control, Grey: AR2011(h404) MOI 100; Dark grey: AR2011(h404) MOI 1000. (C) Western Blot for the detection of h4-1BBL expression. The blot shows that both cell lines expressed endogenous 4-1BBL. Data for cytokines’ expression was obtained 30 hr after infection since after 48 hr cells were completely lysed by the OAdVs. (D) In vitro lytic activity of AR2011(h404) in different human malignant cell lines. Percent of surviving cells was assessed by the MTS assay. AR2011 was used as a comparator.
Figure 4.
In vivo efficacy of the OAdVs. (A) Nude mice harboring established SKOV-3 tumors were injected with PBS, AR2011 or AR2011(h404) at the indicated times (see arrows). Tumor growth was followed with digital calipers. At day 56 the experiment was finalized. (B) Mice harboring intraperitoneal 5 days-old SKOV-3 tumors were i.p. injected either with PBS, AR2011 or AR2011(h404) as described in Methods. At the end of the study tumors were removed, photographed, and weighted. (C) Levels of viral E4 in tumor samples as a surrogate marker of virion numbers. **p<0.01, ***p<0.001. For statistical analysis, see materials and methods.
Figure 4.
In vivo efficacy of the OAdVs. (A) Nude mice harboring established SKOV-3 tumors were injected with PBS, AR2011 or AR2011(h404) at the indicated times (see arrows). Tumor growth was followed with digital calipers. At day 56 the experiment was finalized. (B) Mice harboring intraperitoneal 5 days-old SKOV-3 tumors were i.p. injected either with PBS, AR2011 or AR2011(h404) as described in Methods. At the end of the study tumors were removed, photographed, and weighted. (C) Levels of viral E4 in tumor samples as a surrogate marker of virion numbers. **p<0.01, ***p<0.001. For statistical analysis, see materials and methods.
Figure 5.
In vivo studies in syngeneic mice models. (A) and (B) In vitro lytic activity of AR2011(H3) and AR2011(m404) on ID8 and CT26 cells. Surviving cells were assessed with the MTS assay. (C) and (D) Mice harboring established CT26 tumors in both flanks were injected in the left flank either with PBS, AR2011(H3) or AR2011(m404) and followed as described in the text. The arrows indicate the days of AdOV or PBS injection (E) Kaplan-Meier survival curve of mice harbouring established CT26 tumors in the left flank injected in the right flank either with CT-PBS, CT-AR or CT-AR404 cells. Statistical analysis: comparison of CT-PBS vs CT-AR404, p = 0.0017 (**) and CT-AR vs CT-AR404, p = 0.0009 (***) using the Long –Rank (Mantel-Cox) test. For further details, see materials and methods.
Figure 5.
In vivo studies in syngeneic mice models. (A) and (B) In vitro lytic activity of AR2011(H3) and AR2011(m404) on ID8 and CT26 cells. Surviving cells were assessed with the MTS assay. (C) and (D) Mice harboring established CT26 tumors in both flanks were injected in the left flank either with PBS, AR2011(H3) or AR2011(m404) and followed as described in the text. The arrows indicate the days of AdOV or PBS injection (E) Kaplan-Meier survival curve of mice harbouring established CT26 tumors in the left flank injected in the right flank either with CT-PBS, CT-AR or CT-AR404 cells. Statistical analysis: comparison of CT-PBS vs CT-AR404, p = 0.0017 (**) and CT-AR vs CT-AR404, p = 0.0009 (***) using the Long –Rank (Mantel-Cox) test. For further details, see materials and methods.
Table 1.
Characteristics of the human fresh explants.
Table 1.
Characteristics of the human fresh explants.
Sample |
Pathology |
Observation |
1 |
Epithelial ovarian cancer |
First cytoreduction |
2 |
Epithelial ovarian cancer |
Relapse |
3 |
Epithelial ovarian cancer |
Bilateral tumor Neoadjuvant chemotherapy C, P and Bevacizumab |
4 |
Epithelial ovarian cancer |
Neoadjuvant chemotherapy |
5 |
Krukenberg tumor |
First cytoreduction |
6 |
Cervical cancer |
Conization |
7 |
Cervical cancer |
Conization |
8 |
Cervical cancer |
Simple hysterectomy |
9 |
Cervical cancer |
Simple hysterectomy |
10 |
Cervical cancer |
Conization |
11 |
Cervical cancer |
Simple hysterectomy |
12 |
Uterus cancer |
Radical Hysterectomy |
13 |
Uterus cancer |
Normal uterus tissue after radical hysterectomy* |
14 |
Uterus cancer |
Normal uterus tissue obtained after radical hysterectomy* |
15 |
Uterus cancer |
Hysterectomy |
16 |
Uterus cancer |
Hysterectomy |
Table 2.
Analysis of the synergistic interaction of AR2011 and cisplatin combination on OC-AF samples.
Table 2.
Analysis of the synergistic interaction of AR2011 and cisplatin combination on OC-AF samples.
OC-AF |
Y(P)1 |
Y(O)1 |
8 |
0.30 |
0.60 |
10 |
0.71 |
0.81 |
14 |
0.66 |
0.64 |
15 |
0.66 |
0.70 |
18 |
0.81 |
0.77 |
19 |
0.57 |
0.66 |
20 |
0.80 |
0.85 |
21 |
0.88 |
0.87 |
24 |
0.78 |
0.80 |
28 |
0.80 |
0.87 |
30 |
0.88 |
0.83 |
38 |
0.77 |
0.74 |
39 |
0.61 |
0.72 |
40 |
0.90 |
0.92 |