Cancer Cells Impair the Clearance of Oncolytic Virus M1 by Macrophage in a Non-Contact-Dependent Manner

Despite emerging advances in cancer treatment during the
past decades, the increasing incidence of morbidity and mortality
has made cancer the leading cause of death worldwide.


Introduction
Despite emerging advances in cancer treatment during the past decades, the increasing incidence of morbidity and mortality has made cancer the leading cause of death worldwide. Oncolytic viruses (OVs) have been used as an innovative biotherapeutic agent with the ability to specifically replicate in and kill cancer cells without harming normal cells [1]. In addition to direct cell killing by the virus, it has been demonstrated that a virus-induced immune response plays an important part in OV therapy [2,3].
Since OVs can destroy cancer cells via a mechanism distinct from conventional chemotherapy and radiotherapy, these viruses may be ideal for treating cancers that are nonresponsive to conventional treatment. To date, more than 10 species of OVs has been tested in pre-clinical and clinical trials and the milestone in this field is that the US Food and Drug Administration (FDA) approved talimogene laherparepvec (T-Vec), which is derived from HSV for treatment of melanoma patients, making it the first oncolytic virus approved for treating patients [4,5]. In spite of the evidence for oncolytic viruses killing cancer cells in vitro is unambiguous, the penetrance of oncolytic activity depends on various critical cellular barriers. For instance, the innate antiviral immunity in both normal tissue, especially the mononuclear phagocyte system, and tumor microenvironment.
As a key innate immunocyte, macrophage has a great threat in blocking the therapeutic dissemination of the oncolytic virus, which limits the efficacy of OVs as an anticancer agent. Some reports showed pharmaceutical inhibition or systemic depletion of macrophages could increase the administration efficacy and duration of OVs infection in tumor site [6,7]. However, macrophages are a class of immune cells with extremely strong plasticity and macrophages of different phenotypes may have distinct effects on the virus. In some models of viral infection, M2-polarized macrophages promoted viral replication. For example, the massive replication of rhinovirus in chronic asthma is related to the inhibition of antiviral immunity by interleukin 10 (IL-10) secreted by M2 macrophages [8]. Linked to the similarity between Tumor-associated macrophages and M2 macrophages in the majority of tumor models [9,10], macrophages in the tumor microenvironment is not necessarily an obstacle to OVs.
Our team has previously identified a strain of Getah-like alphavirus (M1), isolated from culicine mosquitoes in the Hainan province of China, as a naturally occurring oncolytic virus that targets zinc-finger antiviral protein (ZAP)-deficient cancer cells [11,12].
The genome of M1 is a positive single-strand RNA, which encodes several structural and nonstructural proteins. Importantly, we found that M1 causes prolonged and severe endoplasmic reticulum (ER) stress that in turn induced apoptosis in tumor cells and was nonpathogenic in nonhuman primates after multiple rounds of repeated i.v. injections [13]. After that, we have identified multiple signaling pathways associated with tumor cells killing effect of M1 and a series of related small molecule compounds as synergist [14][15][16][17][18]. However, a significant portion of cells in tumor mass is nontumorous [19], little is known about how these cells, especially the phagocytic macrophages usually occupying a large proportion of the tumor mass, affect antitumor effects of the M1 virus.
Tumor-associated macrophage (TAM) was found to be a distinct phenotype compared with macrophages in normal tissue.
Considering the tumor tropism of oncolytic virus M1, it is intriguing to examine if TAM has different responsiveness upon M1 infection.
In this study, we investigated the infection and replication characteristics of M1 virus in macrophages and found that tumor-derived factors dampen the antiviral activity of Raw264.7 macrophages to M1 infection. These results imply that immuno-compromised of host innate immunity could be utilized by oncolytic M1 virus and also provided new insight into the tumor selectivity of M1 virus.

Cell Lines and Viruses
Cell lines were all purchased from American Type Culture Collection. Raw 264.7, 4T1, B16F10 were cultured at 37°C under 5% CO 2 in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100μg/ml streptomycin. THP-1 cells were cultured in RPMI 1640 supplemented with 10% FBS and stimulated with 50ng/ ml Phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, USA) for 72h to differentiate as macrophages. All cell lines tested are negative for mycoplasma contamination. Production of Alphavirus M1 in this study was described previously [11]. M1-GFP is a

RNA Interference
Specific and nontargeting siRNAs were synthesized by Ribobio

Flow Cytometry
Cells were harvested and washed twice with cold PBS solution, immediately analyzed by flow cytometry (Becton-Dickinson, USA).

M1-GFP infected cells were GFP+ and the infection rates were
calculated relative to the total number of cells analyzed per sample (20,000 events) using CytExpert software.

Statistical Analysis
Statistical analysis and graphs were made using GraphPad Prism (version 6.01, USA). Most of the data were analyzed by the Student t-test or one-way ANOVA with Dunnett's tests for pairwise comparison. Bars show the mean ± SD of at least three independent repeat experiments.

Interferon Pathway is Involved in the Clearance of Oncolytic Virus M1 by Macrophages
To explore whether M1 virus can infect and replicate in macrophage, mouse macrophage Raw264.7 and PMA-induced human macrophages THP-1 were infected with M1-GFP. The presence of green fluorescent protein (GFP) indicating that the M1 virus could enter macrophage and express the reporter gene inserted into the viral genome. As shown in Figure 1A

Tumor Cells can Attenuate the Clearance of Oncolytic Virus M1 by Macrophages in a Non-Contact Dependent Manner
We next sought to explore M1 replication in TAM-like macrophages. Raw264.7 cells pretreated with either self-conditioned medium or breast cancer cell 4T1 conditioned medium (CM) were infected with M1-GFP. To our surprise, Raw264.7 cells pretreated with 4T1CM were seemingly more sensitive to M1 virus infection ( Figure 2A). This suggested that substances secreted by tumor cells

Tumor Cells Co-Culture Pretreatment Did Not Impair Interferon Pathway Activation After Oncolytic Virus M1 Infection
As previously reported [20], tumor-derived exosomes inhibit the production of IFN-β in influenza-infected macrophages, which impaired the clearance activity of macrophage. In order to illustrate the mechanism of impairment, we first examine the type-1 IFN (IFN-α, IFN-β) and type-2 IFN (IFN-γ) mRNA expression before and after M1 infection in Raw264.7 cells under different co-culture pretreatment conditions ( Figure 3A). It was found that compared with the self-cultivation control group, the tumor cell co-culture pretreatment promoted the expression of various types of interfer-on infection. Among them, the increase of type I interferon is more significant, especially the raw264.7 under 4T1 co-culture pretreatment ( Figure 3A

Down-regulation of HPGD in Tumor Co-cultured Raw264.7 is associated with Enhanced M1 Sensitivity
To determine the underlying mechanism of impaired M1 clearance by macrophages, we used Raw264.7 cells co-cultivation pre-treated with themselves as a control group (RAWCC), and co-cultivation pre-treated Raw264.7 cells with cancer cells 4T1 and B16F10 as two different test groups (4T1CC and B16F10CC). All three groups were subjected to transcriptome sequencing by BGISEQ-500 platform. In order to find out the more common    [19], it has inevitable effects on therapeutic efficacy, for example the environment mediated drug resistance protects tumor cells from apoptosis induced by either chemotherapy, radiotherapy or receptor-mediated cell death [24]. For oncolytic virotherapy, the situation might be different because the TME, which commonly considered as "immunosuppressive" influenced by tumor cells, is possibly a suitable habitat for OVs relative to normal tissue. To verify this possibility, we used in vitro non-contact cell co-cultured model to partially simulate the stimulus received by macrophage in TME and identified a phenotypic transition occur in tumor co-cultured Raw264.7 macrophages which are more sensitive to oncolytic virus M1 infection and replication. These results help us to uncover the mechanism of tumor selectivity of the M1 virus from macrophages perspective other than tumor cells and finally increase the response rate of M1 in future clinical trials.

15-hydroxyprostaglandin dehydrogenase (15-PGDH), encoded
by HPGD, is a key enzyme responsible for inactivation of PGE2 in tissue and has a tumor suppressor function in multiple cancer types including colon, lung, and breast cancers [25][26][27]. Therefore it is frequently low expression in tumor cells, and together with the overexpression of cyclooxygenase-2 (COX-2), a higher amount of PGE2 are secreted and persevered in TME, which is often associated with tumor-induced immune suppression [28,29].Through highthroughput transcriptome sequencing, we notice that HPGD was most significantly down-regulated in both tumor co-cultured  [30,31].