Enhancement of oncolytic flavi- and alphavirus replication in resistant human pancreatic adenocarcinoma cell lines using ruxolitinib

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Introduction

Despite significant improvements in survival rates for many types of tumors, pancreatic cancer remains highly lethal. As a rule, patients with this diagnosis survive no more than 5 years due to late diagnosis and high drug resistance. While early surgical intervention can significantly prolong survival in patients with localized tumors, the prognosis for patients with metastases is generally poor. In clinical practice, a combination of surgery and adjuvant therapy is often used, which includes radiation therapy, gemcitabine chemotherapy, and the mFOLFIRINOX protocol (combination therapy with fluorouracil, folinic acid, irinotecan, and oxaliplatin), which, in the latter case, increases overall survival to 54 months for patients without metastatic disease [1]. However, the clinical response in most patients remains limited, a phenomenon attributed, in particular, to high stromal density, an immunosuppressive microenvironment, and the preservation of antiviral signaling cascades in tumor cells. In this regard, alongside conventional treatment methods, the use of new classes of cancer immunotherapy–oncolytic viruses (OVs)–appears promising. OVs possess the innate ability to infect and lyse tumor cells without harming normal cells [2]. A number of OV have demonstrated efficacy against pancreatic cancer in studies. Various variants of adenoviruses, herpes simplex virus, reovirus, parvovirus, and vaccinia virus are currently in clinical trials [3]. However, despite active development of new treatment methods, no breakthrough results in the treatment of pancreatic cancer have been observed yet.

Cancer cells often have defects in their ability to either produce or respond to interferons (IFNs), which makes them more sensitive to antiviral drugs than healthy cells [4]. Virus-induced IFNs are crucial for the development of antitumor immunity and for overcoming the tumor’s immunosuppressive microenvironment. At the same time, IFNs also trigger antiviral defense mechanisms that limit the replication of anticancer drugs [5]. Overcoming the tumor’s antiviral response–in particular, interferon defense–is considered a key mechanism for enhancing the efficacy of anticancer drugs. Furthermore, patients with higher expression of STAT1–one of the key proteins of the immune response–have a poorer prognosis [6]. In light of this, the development of new approaches appears necessary, one of which is combination virotherapy. Selective JAK1/2 kinase inhibitors, particularly ruxolitinib, are capable of enhancing OV replication, as demonstrated in studies using the vesicular stomatitis virus and its recombinant derivatives in resistant pancreatic cancer cell lines [7, 8].

The tumor microenvironment of pancreatic cancer, characterized by a high degree of immunosuppression, can be significantly remodeled by introducing a virus into it. The oncolytic properties of the 17D vaccine strain of the yellow fever virus (YFV 17D)–which is characterized by safety and immunogenicity proven by many years of (70 years) of use in vaccine prophylaxis, were previously investigated in vitro using human pancreatic carcinoma cells MIA Paca-2 and human pancreatic ductal carcinoma cells PANC-1 [9] and in vivo, including using the syngeneic mouse model of pancreatic cancer Pan02. It was demonstrated that YFV 17D, despite its extremely weak replication in Pan02 cells in vitro, nevertheless exhibited an effective immune-mediated effect against transplanted tumors in vivo [10].

Another promising platform with the potential to overcome the repressive microenvironment of pancreatic cancer is the Sindbis virus (SINV). A genetically engineered variant of SINV that expresses interleukin-12 and an agonistic antibody against OX40 alters the expression profile of T cells, promoting their differentiation into effector T cells with enhanced antitumor activity and the ability to infiltrate the tumor. In the UN-KC-6141 pancreatic cancer model (C57BL/6 mice), the SV.IL12 variant, in combination with αOX40, significantly slowed tumor growth and increased the median survival of animals receiving this combination. Administration of a genetically engineered variant of SINV to SCID mice with orthotopically growing human pancreatic tumor cells led to tumor lysis [11].

However, despite the success of these oncolytic viruses in antitumor therapy, researchers often face the problem of oncolytic virus sensitivity to IFN. In such cases, the effectiveness of virotherapy depends significantly on the preservation of the interferon response in target cells. SINV is sensitive to IFN and, without genetic modifications, can be used only in tumors with defective interferon pathways [12]. A similar situation is observed with YFV 17D—in particular, the PANC-1 pancreatic ductal carcinoma cell line exhibited higher resistance to it than the MIA Paca-2 pancreatic carcinoma cell line, which may be associated with increased expression of IRF2 and IRF7 in the cell line, which regulate the IFN-α and -γ genes [9].

In this case, the use of ruxolitinib in combination with OVs may be an effective strategy for enhancing OV replication in resistant cell lines.

The aim of this study is to investigate the effect of ruxolitinib (a JAK1/2 kinase inhibitor) on the replicative activity of certain oncolytic flaviviruses (using YFV 17D as an example) and alphaviruses (using SINV K1487 as an example) in human pancreatic adenocarcinoma cell lines AsPС-1, BxPC-3, and Capan-2, which are characterized by a high degree of resistance to viral oncolysis.

Materials and methods

Cell lines and culturing conditions

Passaged Vero cell culture (derived from the American Type Culture Collection–ATCC) was used for virus titration by the plaque assay; the human pancreatic adenocarcinoma cell lines AsPC-1, Capan-2, and BxPC-3 (purchased from PrimeBioMed LLC) were used to determine viral susceptibility.

The cells were cultured in DMEM growth medium containing 4.5 g/L glucose (PanEco, Cat. No.: C420p), supplemented with 2 mM L-glutamine (PanEco, Cat. No. F032), the antibiotics penicillin (250 IU/mL) and streptomycin (200 μg/mL) (PanEco, Cat. No. A065p), and 10% fetal bovine serum (FBS) (Gibco #2412072) in a 5% CO₂ atmosphere at 37 °C.

Virus strains

This study used the attenuated vaccine strain YFV 17D (family Flaviviridae) and the native strain SINV K1487 (GenBank AC No. PZ244961.1) (family Togaviridae) from the collection of the M.P. Chumakov Federal Research Center for Infectious Diseases and Radiation Protection of the Russian Academy of Sciences. The SINV isolate was isolated from mosquitoes of the genus Culex, passaged once in mouse brain, and lyophilized in 1970 at the Poliomyelitis Institute. Currently, work with the virus has been resumed to study its oncolytic potential. The virus was passaged twice in Vero cell culture, after which its genome was fully sequenced and deposited in the GenBank database (AC No. PZ244961.1) under the strain designation K1487 (LKE_1). Virus titers were assessed using the plaque assay on Vero cells, and the concentration of viral RNA in the culture medium was determined using real-time quantitative polymerase chain reaction (PCR).

A Study of the Effect of Ruxolitinib on Viral Replication in Human Pancreatic Adenocarcinoma Cell Cultures

Human pancreatic adenocarcinoma cells (AsPC-1, BxPC-3, and Capan-2) were cultured in 12-well plates until they reached 80–90% confluence. The culture medium was then removed, and 400 μL of DMEM medium containing ruxolitinib (BLDpharm, China) at a final concentration ranging from 0.1 to 5 μM was added to each well, according to the experimental protocol. Control wells were filled with 400 μL of DMEM. The cells were incubated for 3 hours at 37 °C and 5% CO₂.

Afterward, 400 μL of viral suspension was added to each well (except for the control wells, to which an equivalent volume of DMEM was added) to achieve a multiplicity of infection (MOI) of 1.0. The plates were incubated for 1 hour at 37 °C and 5% CO₂. The inoculum was then removed, the wells were washed once with 1 mL of DMEM, and finally, 2 mL of fresh DMEM containing ruxolitinib at concentrations ranging from 0.1 to 5 μM was added to each well, in accordance with the experimental protocol (except for the control wells, to which an equivalent volume of DMEM was added). The cells were then incubated for 7 days under standard culture conditions. Two wells were used for each experimental condition. Culture medium samples of 50 μL were collected from the two wells daily from day 3 through day 7 to assess viral titers using the plaque assay and quantitative real-time PCR. The experiment was repeated in at least 3 independent replicates for each virus and cell line, respectively.

Virus titration using the plaque assay method

Virus titers were determined by using the plaque assay method with a 1.25% methylcellulose coating in Vero cell cultures on 24-well plastic plates. Vero cells were seeded onto 24-well plates and incubated for 24 hours at 37 °C in a CO₂ incubator. Tenfold dilutions of viral samples were prepared in Earl’s medium 199 and applied to the cells at a volume of 200 μL per well. The plates were incubated in a CO₂ incubator for 1 hour. Then, 1 mL of a 1.25% methylcellulose cover solution was added to each well.

Plate counts were performed on day 4 (for SINV K1487) and day 6 (for YFV 17D) after infection. The virus titer was expressed as the number of plaque-forming units (PFU) per 1 mL of virus-containing material. The titer was calculated using the following formula:

ΣN / (d × n × V) = PFU/mL,

where: N – the number of plates in a single well for the corresponding dilution; n – the number of wells per dilution; V – the sample volume (inoculation dose), mL; d – the dilution (expressed as tenths).

Determination of Viral RNA Concentration in Culture Medium Using Real-Time Polymerase Chain Reaction

A 50-μL sample of the culture medium was collected, and cellular debris was removed by centrifugation at 3,000 rpm for 5 minutes. Viral RNA was isolated from the resulting supernatant using the Ribo-Prep reagent kit (AmpliSens, Russia), and reverse transcription (cDNA synthesis) was performed using the Reverta-L kit (AmpliSens, Russia). For real-time PCR of SINV K1487, the following gene-specific primers were used: 5’-CCAAGAGCCTGCCCCTATTC-3’ (forward), 5’-GCTATTGCTTGCCGGTGGAG-3’ (reverse), and the TaqMan probe 5’-FAM-AGGCGGGCCATCTTCTTTAGCCTTG-BHQ1-3’. Amplification was performed using the PCR 5× qPCRmix-HS kit (Eurogen, Russia). Vaccine K1487, with a known titer of 1 × 10⁷ PFU/mL, was used as a standard; its RNA was isolated along with the samples and used to prepare dilutions (from 10¹ to 10⁷) for constructing a calibration curve and determining the RNA concentration in the samples. For PCR of YFV 17D, the AmpliSens Yellow Fever Virus-FL reagent kit was used; YFV 17D with a known titer of 1 × 10⁷ PFU/mL was used as a standard, whose RNA was isolated along with the samples and used to prepare dilutions (from 10¹ to 10⁷) for constructing a calibration curve and determining the RNA concentration in the samples. Quantitative real-time PCR was performed on a CFX96 amplifier (Bio-Rad, USA) using the FAM and HEX detection channels.

Microscopy (assessment of cytopathic effects)

Visual inspection and assessment of cytopathic effect (CPE) based on the condition of the cell monolayer were performed using an OLYMPUS CKX53 inverted biological microscope equipped with a digital camera and ADFImageCapture software.

Statistical analysis

Each value represents the average result of three independent biological experiments (n = 3). Statistical analysis of the data was performed using GraphPad Prism 8.0.1 with a two-way analysis of variance (ANOVA).

Results

Human pancreatic adenocarcinoma cells (AsPC-1, BxPC-3, and Capan-2) were infected with YFV 17D and SINV K1487 at doses corresponding to an MOI of 1.0, in the presence of 0.1 to 5 μM ruxolitinib in the culture medium. It should be noted that BxPC-3 and Capan-2 cells are resistant to viral oncolysis, and from days 1 through 7 of the experiment, no visible cytopathic effect (CPE) caused by YFV-17D and SINV K1487 was observed. AsPC-1 cells were more sensitive to the viruses under study; however, CPE was observed only by day 6, and more than 50% of the monolayer remained intact on day 7.

In AsPC-1 cell culture, the infectious titer of YFV 17D reached 10⁵ PFU/mL from day 3 through day 7. Treatment of AsPC-1 cells with ruxolitinib 3 hours before infection and subsequent cultivation in medium containing ruxolitinib led to a two-order-of-magnitude increase in the infectious titer of YFV 17D — from 10⁵ PFU/mL on day 3 to 10⁷ PFU/mL by day 7 (Fig. 1 a). A ruxtolitinib concentration of 1 μM proved effective, and increasing it to 5 μM did not further enhance the effect. AsPС-1 cells were more permissive to SINV K1487, and by day 3, the infectious titer was 10⁷ PFU/mL; however, by day 7, the viral infectious titer had decreased to 10⁴ PFU/mL. A concentration of 0.5 μM ruxolitinib was sufficient to produce a virus titer of 10⁷ PFU/mL in AsPC-1 cells up to day 7 (Fig. 1 b).

 

Fig. 1. Changes in infectious titers of the yellow fever virus strain 17D (YFV 17D) (a) and Sindbis virus strain K1487 (SINV K1487) (b) in the culture medium of the human pancreatic adenocarcinoma cell line AsPC-1 from 3 to 7 days post-infection.

Рис. 1. Изменение инфекционных титров вируса желтой лихорадки штамм 17D (ВЖЛ 17D) (a) и вируса Синдбис штамм К1487 (ВС) (б) в культуральной среде аденокарциномы поджелудочной железы человека AsPC-1 c 3-х по 7-е сутки после инфицирования.

 

A similar effect of increased viral replication in the presence of ruxolitinib was also observed in other human pancreatic adenocarcinoma cells, BxPC-3 (Fig. 2). Infectious titers on day 3 were low (10⁴ PFU/mL); for YFV 17D, the titer remained at the same level until day 7, while for SINV K1487, a drop in titer to 10² PFU/mL was observed. Thus, treatment of BxPC-3 cells with ruxolitinib correlates with an increase in the titers of the viruses under study. For YFV 17D, the addition of ruxolitinib at a dose of 5 μM succeeded in increasing the viral titer to 10⁷ PFU/mL by day 7 (Fig. 2 a), however, no statistically significant difference was observed between the groups treated with 1 and 5 μM ruxolitinib, confirming that a 1 μM dose of ruxolitinib is sufficient. For SINV K1487, only when 5 μM ruxolitinib was added was a two-order-of-magnitude increase in viral titer (up to 10⁶ PFU/mL) observed (Fig. 2 b).

 

Fig. 2. Changes in infectious titers of the yellow fever virus strain 17D (YFV 17D) (a) and Sindbis virus strain K1487 (SINV K1487) (b) in the culture medium of the human pancreatic adenocarcinoma cell line BxPC-3 from 3 to 7 days post-infection.

Рис. 2. Изменение инфекционных титров вируса желтой лихорадки штамм 17D (ВЖЛ 17D) (a) и вируса Синдбис штамм К1487 (ВС) (б) в культуральной среде аденокарциномы поджелудочной железы человека BxPC-3 c 3-х по 7-е сутки после инфицирования.

 

Capan-2 cells are characterized as among the most resistant to antiviral therapy among the studied pancreatic cancer cell lines. The YFV 17D titer in Capan-2 cells on day 3 was 10³ PFU/mL, with a subsequent decrease to 10³ PFU/mL by day 7 (Fig. 3 a). Virus K1487 proved incapable of effective replication in Capan-2 cells: the virus was detected for up to 3 days at levels of less than 10² PFU/mL, which was significantly lower than the inoculation dose (10⁶ PFU/mL). Starting on day 4, infectious SINV K1487 was no longer detected in the Capan-2 culture. In the case of YFV 17D, the addition of 1 μM ruxolitinib helped maintain the titer: by day 7, its titer remained the same as on day 3. A similar effect was observed for virus strain K1487 upon the addition of 5 μM ruxolitinib (Fig. 3 b).

 

Fig. 3. Changes in infectious titers of the yellow fever virus strain 17D (YFV 17D) (a) and Sindbis virus strain K1487 (SINV K1487) (b) in the culture medium of the human pancreatic adenocarcinoma cell line Capan-2 from 3 to 7 days post-infection.

Рис. 3. Изменение инфекционных титров вируса желтой лихорадки штамм 17D (ВЖЛ 17D) (a) и вируса Синдбис штамм К1487 (ВС) (б) в культуральной среде аденокарциномы поджелудочной железы человека Capan-2 c 3-х по 7-е сутки после инфицирования.

 

The results of PCR analysis of samples for the amount of RNA from the viruses under study in human pancreatic adenocarcinoma cell cultures (AsPС-1, BxPC-3, Capan-2) on days 3–7 of the experiment are presented in Tables 1 and 2; however, these results were less representative than the infectious titer values. There was only a statistically significant increase in RNA levels (copies/mL) by one order of magnitude in the groups treated with ruxolitinib compared to the groups treated with the virus alone; however, no statistically significant differences were observed between groups with different concentrations (0.1–5.0 μM) or sampling times.

 

Table 1. The amount of YFV 17D RNA in the culture medium of infected (MOI=1.0) human pancreatic adenocarcinoma cells (AsPC-1, BxPC-3, Capan-2) on the 3rd-7th day of the experiment, copies/mL

Таблица 1. Количество РНК ВЖЛ 17D в культуральной среде инфицированных (MOI = 1,0) клеток аденокарциномы поджелудочной железы человека (AsPС-1, BxPC-3, Capan-2) на 3–7-е сутки эксперимента, копий/мл

Days Сутки	AsPС-1 + YFV 17D (MOI = 1.0) in the presence of ruxolitinib / AsPС-1 + ВЖЛ 17D (MOI = 1,0) в присутствии руксолитиниба
	0	0.1 µМ / 0,1 мкМ	0.5 µМ / 0,5 мкМ	1.0 µМ / 1,0 мкМ	5.0 µМ / 5,0 мкМ
3	1 × 106	1 × 106	9 × 105	9 × 105	9 × 105
4	9 × 105	1 × 106	3 × 106	3 × 106	5 × 106
5	9 × 105	9 × 105	5 × 105	8 × 106	9 × 106
6	8 × 105	1 × 106	5 × 106	2 × 107	4 × 107
7	8 × 105	9 × 105	6 × 106	6 × 107	7 × 107
Days Сутки	BxPC-3 + YFV 17D (MOI = 1.0) in the presence of ruxolitinib / BxPC-3 + ВЖЛ 17D (MOI = 1,0) в присутствии руксолитиниба
	0	0.1 µМ / 0,1 мкМ	0.5 µМ / 0,5 мкМ	1.0 µМ / 1,0 мкМ	5.0 µМ / 5,0 мкМ
3	3 × 105	3 × 105	3 × 105	3 × 105	7 × 105
4	3 × 105	4 × 105	6 × 105	3 × 106	1 × 106
5	2 × 105	6 × 105	8 × 105	5 × 106	6 × 106
6	3 × 105	6 × 105	2 × 106	1 × 107	1 × 107
7	2 × 105	5 × 105	2 × 106	5 × 107	3 × 107
Days Сутки	Capan-2 + YFV 17D (MOI = 1.0) in the presence of ruxolitinib / Capan-2 + ВЖЛ 17D (MOI = 1,0) в присутствии руксолитиниба:
	0	0.1 µМ / 0,1 мкМ	0.5 µМ / 0,5 мкМ	1.0 µМ / 1,0 мкМ	5.0 µМ / 5,0 мкМ
3	3 × 105	2 × 105	3 × 105	1 × 105	2 × 105
4	7 × 105	6 × 105	8 × 105	7 × 105	8 × 105
5	6 × 105	6 × 105	7 × 105	6 × 105	7 × 105
6	8 × 105	8 × 105	1 × 106	1 × 106	9 × 105
7	7 × 105	6 × 105	7 × 105	6 × 105	1 × 106

Note. Each value represents the average of three independent biological experiments (n = 3). A complete description of the statistical analysis is provided in the «Statistical Analysis» section.

Примечание. Каждое значение представляет собой среднее из трех независимых биологических экспериментов (n = 3). Полное описание статистического анализа приведено в разделе «Статистический анализ».

 

Table 2. The amount of SINV K1487 RNA in the culture medium of infected (MOI = 1.0) human pancreatic adenocarcinoma cells (AsPC-1, BxPC-3, Capan-2) on the 3rd–7th day of the experiment, copies/mL

Таблица 2. Количество РНК ВС К1487 в культуральной среде инфицированных (MOI = 1,0) клеток аденокарциномы поджелудочной железы человека (AsPС-1, BxPC-3, Capan-2) на 3–7-е сутки эксперимента, копий/мл

Days Сутки	AsPС-1 + SINV K1487 (MOI = 1.0) in the presence of ruxolitinib / AsPС-1 + ВС К1487 (MOI = 1,0) в присутствии руксолитиниба
	0	0.1 µМ / 0,1 мкМ	0.5 µМ / 0,5 мкМ	1.0 µМ / 1,0 мкМ	5.0 µМ / 5,0 мкМ
3	5 × 106	2 × 107	1 × 107	1 × 107	2 × 107
4	9 × 106	2 × 107	2 × 107	7 × 107	4 × 107
5	4 × 107	2 × 107	4 × 107	4 × 107	5 × 107
6	6 × 106	1 × 107	3 × 107	2 × 107	5 × 107
7	1 × 106	1 × 107	3 × 107	4 × 107	4 × 107
Days Сутки	BxPC-3 + SINV K1487 (MOI = 1.0) in the presence of ruxolitinib / BxPC-3 + ВС К1487 (MOI = 1,0) в присутствии руксолитиниба
	0	0.1 µМ / 0,1 мкМ	0.5 µМ / 0,5 мкМ	1.0 µМ / 1,0 мкМ	5.0 µМ / 5,0 мкМ
3	9 × 104	1 × 105	9 × 105	2 × 105	8 × 105
4	7 × 104	2 × 105	3 × 105	8 × 105	1 × 106
5	6 × 104	4 × 105	4 × 105	7 × 105	1 × 106
6	6 × 104	3 × 105	4 × 105	7 × 105	1 × 106
7	5 × 104	6 × 104	3 × 105	5 × 105	9 × 105
Days Сутки	Capan-2 + SINV K1487 (MOI = 1.0) in the presence of ruxolitinib / Capan-2 + ВС К1487 (MOI = 1,0) в присутствии руксолитиниба
	0	0.1 µМ / 0,1 мкМ	0.5 µМ / 0,5 мкМ	1.0 µМ / 1,0 мкМ	5.0 µМ / 5,0 мкМ
3	4 × 103	2 × 103	5 × 103	4 × 103	1 × 104
4	2 × 103	2 × 103	2 × 103	5 × 103	8 × 103
5	3 × 103	2 × 103	3 × 103	1 × 104	2 × 103
6	1 × 103	1 × 103	3 × 103	2 × 104	2 × 103
7	1 × 103	1 × 104	3 × 104	2 × 103	3 × 103

Note. Each value represents the average of three independent biological experiments (n = 3). A complete description of the statistical analysis is provided in the «Statistical Analysis» section.

Примечание. Каждое значение представляет собой среднее из трех независимых биологических экспериментов (n = 3). Полное описание статистического анализа приведено в разделе «Статистический анализ».

 

Interestingly, YFV 17D, in the absence of ruxolitinib, on human pancreatic adenocarcinoma cells AsPC-1 and BxPC-3, Capan-2 manifests as a persistent infection (maintenance of viral replicative activity without a pronounced cytopathic effect), in contrast to SINV K1487, in which rapid elimination is observed, which correlates with the intensity of the cell’s interferon-mediated antiviral response, ranging from the most permissive AsPC-1 cells to the more resistant BxPC-3 and Capan-2 cells. However, despite achieving a significant increase in viral replication, we did not observe an enhancement of the CPE of YFV 17D (as measured by monolayer disruption) by ruxolitinib in AsPC-1, BxPC-3, or Capan-2. For SINV K1487, an enhancement of the CPE was observed against AsPC-1 in the presence of 1–5 μM ruxolitinib on day 7 and against BxPC-3 cells in the presence of 5 μM ruxolitinib on day 10.

Discussion

This study investigated the effect of ruxolitinib (a JAK1/2 kinase inhibitor) on the replicative activity of certain oncolytic flaviviruses (using YFV 17D as an example) and alphaviruses (using SINV K1487 as an example) in human pancreatic adenocarcinoma cell lines AsPС-1, BxPC-3, and Capan-2, which are characterized by a high degree of resistance to viral oncolysis. A significant dose-dependent increase in infectious titers (by 2–3 orders of magnitude) of YFV 17D and SINV K1487 was demonstrated in the studied cultures in the presence of various concentrations of ruxolitinib (0.1, 0.5, 1.0, and 5.0 μM). At the same time, a ruxolitinib concentration of 1 μM proved to be the most optimal, and increasing the concentration to 5 μM did not result in a further statistically significant enhancement of the effect.

Furthermore, YFV 17D manifested as a persistent infection without obvious cytopathic effects in the human pancreatic adenocarcinoma cells under study. This is primarily due to a natural characteristic of flaviviruses—the presence of a mechanism for evading the innate immune response, which is mediated by non-structural protein 5 (NS5) [13]. The NS5 protein specifically binds to human STAT2, promoting its polyubiquitination and proteasome-mediated degradation, thereby blocking type I IFN signaling and ISG production. Enhancing the activity of YFV 17D with ruxolitinib could significantly improve the efficacy of therapy for pancreatic cancer that is resistant to standard treatments and is highly immunosuppressive. Such a combined approach to virotherapy will enhance viral replication and activate potent mechanisms of immunogenic cell death even in the absence of an obvious cytopathic effect in culture. A high viral titer means that numerous viral particles, viral proteins, and viral RNA remain in the culture medium and are localized on the cell surface. These pathogen-associated molecular patterns (viral RNA, capsid proteins, etc.) are capable of activating pattern-recognition receptors of the innate immune system on both the tumor cells themselves and on immune cells.

Upon recognition of SINV RNA within a cell, a type 1 interferon response is rapidly activated, leading to the inhibition of viral replication through ISG-induced proteins (OAS, PKR, IFIT, etc.) [12]. This is likely to severely limit its clinical use in oncolytic virotherapy for pancreatic cancer. However, the use of inhibitors such as ruxolitinib or other JAK/STAT signaling pathway inhibitors that reduce the activity of the type 1 interferon response allows alphavirus platforms to be considered a promising basis for oncolytic therapy. With this approach, a JAK/STAT inhibitor will enable active replication of the oncolytic virus, thereby achieving a lytic effect on tumor cells, which will ultimately lead to the release into the tumor microenvironment not only of pathogen-associated molecular patterns but also of tumor-associated antigens, thereby further enhancing the antitumor immune response.

Despite the promising in vitro results obtained, further research is necessary to evaluate the efficacy of this approach in in vivo pancreatic cancer models, including a detailed investigation of the host’s immune response to such therapy.

Conclusion

This study demonstrates the importance of identifying new treatment combinations for pancreatic cancer. The results of the study showed that the use of ruxolitinib, a selective JAK1/2 kinase inhibitor, in combination with oncolytic flaviviruses and alphaviruses in in vitro models helps overcome the main barriers to oncolytic virotherapy associated with the antiviral defense mechanisms of tumor cells. In conclusion, it should be noted that the use of flaviviruses and alphaviruses in combination with ruxolitinib for oncolytic virotherapy of tumors may prove promising and warrants further investigation of in vivo models.

About the authors

Kirill N. Trachuk

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Author for correspondence.
Email: trachuk_kn@chumakovs.su
ORCID iD: 0000-0002-2061-0274

Researcher of the Laboratory of Tick-Borne Encephalitis and Other Viral Encephalitides

Russian Federation, 108819, Moscow

Yulia K. Biryukova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: biryukova_jk@chumakovs.su
ORCID iD: 0000-0002-5804-4001

Ph.D., Leading Researcher of the Laboratory of Tick-Borne Encephalitis and Other Viral Encephalitides

Russian Federation, 108819, Moscow

Vitalii А. Kapranov

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: kapranov_va@chumakovs.su
ORCID iD: 0009-0008-5528-836X

Junior Researcher of the Laboratory of Tick-Borne Encephalitis and Other Viral Encephalitides

Russian Federation, 108819, Moscow

Ekaterina A. Orlova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: orlova_ea@chumakovs.su
ORCID iD: 0009-0009-4175-0493

Junior Researcher of the Laboratory of Tick-Borne Encephalitis and Other Viral Encephalitides

Russian Federation, 108819, Moscow

Alina S. Nazarenko

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: nazarenko_as@chumakovs.su
ORCID iD: 0000-0002-7322-8730

Junior Researcher of the Laboratory of Tick-Borne Encephalitis and Other Viral Encephalitides

Russian Federation, 108819, Moscow

Nadezhda M. Kolyasnikova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: kolyasnikova_nm@chumakovs.su
ORCID iD: 0000-0002-9934-2582

D.M.S, Head of the Laboratory of Tick-Borne Encephalitis and Other Viral Encephalitides

Russian Federation, 108819, Moscow

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