Low-dose IFN-γ induces tumor cell stemness in tumor microenvironment of non-small cell lung cancer

<jats:title>Abstract</jats:title><jats:p>Interferon-γ (IFN-γ) is conventionally recognized as an inflammatory cytokine that play a central role in antitumor immunity. Clinically, although has been used clinically to treat a variety of malignancies, low-level IFN-γ in the tumor microenvironment (TME) increases the risk of tumor metastasis during immunotherapy. Accumulating evidence has suggested that IFN-γ can induce cancer progression. The mechanisms underlying the controversial role of IFN-γ regulating tumor development remain unclear. Herein, we firstly revealed a dose-dependent effect of IFN-γ in inducing tumor stemness to accelerate cancer progression in patients with a variety of cancer types. Mechanically, low-level IFN-γ endowed cancer stem-like properties via the intercellular adhesion molecule-1 (ICAM1)-PI3K-Akt-Notch1 axis, whereas high-level IFN-γ activated the JAK1-STAT1-caspase pathway to induce apoptosis in non-small cell lung cancer (NSCLC). Inhibition of ICAM1 abrogated the stem-like properties of NSCLC cells induced by the low dose of IFN-γ both<jats:italic>in vitro</jats:italic>and<jats:italic>in vivo</jats:italic>. Our study first defines the role of low-level IFN-γ in conferring tumor stemness and clearly elucidate the distinct signaling pathways activated by IFN-γ in a dose-dependent manner, providing new insights into cancer treatment, particularly patients with low-level IFN-γ expression in the TME.</jats:p>

section_content: Interferon (IFN)-γ is widely considered to be a crucial antitumor cytokine that is mainly produced by activated T cells, natural killer (NK) and NKT cells (Sadanaga et al, 1999; Tannenbaum & Hamilton, 2000). After binding to the heterodimeric IFNGR1/IFNGR2 receptor complex, IFN- initiates the activation cascade of downstream signaling events, particularly classical JAK-STAT signaling, and the transcription of multiple IFN-γ-inducible genes, both of which induce cell cycle arrest and apoptosis in tumor cells (Platanias, 2005). However, IFN-γ signaling has paradoxically been reported to promote carcinogenesis and metastasis (Muller-Hermelink et al, 2008; Murohashi & Hoang, 1991) through inducing inflammatory responses (Xiao et al, 2009), immunosuppression or other unknown mechanisms (Katz et al, 2008; Ostrand-Rosenberg & Sinha, 2009). 

Sustained low-level IFN- boosted the development of several types of tumor in mouse models (He et al, 2005; Kelly et al, 1991). Clinically, although IFN- has been used in many anti-cancer clinical trials, low-level IFN- generated at the tumor site has been shown to increase the risk of tumor metastasis during immunotherapy (Gong et al, 2008). Unfortunately, the molecular mechanisms underlying IFN- mediating cancer progression were not fully characterized in these previous studies. Notably, recent studies have described a close relationship between IFN- and cancer stem cells (CSCs). IFN- are capable of inducing CSCs dormancy (Liu et al, 2017) and metastatic CSCs generation (Chen et al, 2011). These studies imply that IFN- may involve in tumor progression through conferring tumor stemness, but the exact mechanism was not fully understood. 

CSCs have been reported to play key roles in tumor initiation, metastasis, recurrence and multidrug resistance in various cancer types (Batlle & Clevers, 2017). 

CSCs are a small subset of self-renewed, pluripotent, immune-privileged, high-tumorigenic and long-living malignant cells. Persistent activation of highly conserved signaling pathways, such as the Notch, Hedgehog, and/or Wnt pathways, partially determines the stem-like properties and tumorigenicity of CSCs (Takebe et al, 2015). In addition, epithelial-to-mesenchymal transition (EMT), an essential developmental process that is often activated during cancer invasion and metastasis (Mani et al, 2008), was also verified as a characteristic of CSCs. Recently, a new concept of "CSC immunology" has been established indicating that the features of CSCs are dependent on a specialized immune-niche. Many immune factors produced in tumor niches, such as IL-6, IL-8 and TGF-, endow tumor cells with stem-like properties to accelerate tumor progression (Plaks et al, 2015; Qiao et al, 2017). 

Intercellular adhesion molecule 1 (ICAM1), a transmembrane molecule and member of the immunoglobulin superfamily of proteins expressed by leukocytes, endothelial cells and epithelial cells, is involved in many important processes, including leukocyte endothelial transmigration, cell signaling, cell-cell interaction, cell polarity and tissue stability (Duperray et al, 1997; Kanters et al, 2008; Lawson & Wolf, 2009; Millan et al, 2006). ICAM-1 can be upregulated by lipopolysaccharide and some inflammatory cytokines, such as IFN- and TNF- (Chen et al, 2000; Rothlein et al, 1988). The protumor role of ICAM1 has been identified in various cancers (Huang et al, 2017; Liu et al, 2013; Min et al, 2017; Tsai et al, 2015), which has been recently used as a target of engineered chimeric antigen receptor T cell therapy in advanced thyroid tumors (Min et al, 2017) and a marker of CSCs in hepatocellular carcinoma (HCC) and esophageal squamous cell carcinoma (ESCC) (Liu et al, 2013; Tsai et al, 2015). In lung cancer, ICAM1 is highly expressed in CSCs (Levina et al, 2008) and responsible for the cancer initiation and metastasis (Lin et al, 2006). These studies support a potential role of ICAM1 in modulating tumor stemness and progression. 

In this study, we firstly found a dose-dependent effect of IFN- on tumor
section_name: Introduction

section_name: Results

section_content: IFN-γ is known as a representative antitumor cytokine in the tumor microenvironment (TME) (Tannenbaum & Hamilton, 2000). We firstly detected the source of IFN- in 5 fresh tumor tissues from patients with NSCLC by flow cytometry. CD3 + CD8 + T cells, CD3 + CD4 + T cells, NK cells and NKT cells showed high production of IFN- while CD3 + CD4 + Foxp + Treg cells showed low production (Fig. S1A, B ). Next, we explored the actual distribution pattern of IFN- in 86 NSCLC tissues by immunohistochemistry (IHC) staining. Samples were grouped into 'low (-or +)' and 'high (++ or +++)' according to the IHC staining score (Fig. 1A ). Interestingly, we

found that low-level IFN- expression accounted for the majority in NSCLC tissues (Fig. 1b ). Moreover, low-level IFN- expression was strongly correlated with the tumor node metastasis (TNM) stage, brain metastasis, chemoresistance (Table 1 ) as well as shorter overall survival (OS) and progression free survival (PFS) times (Fig. 1C-E ). These results suggest that low-level IFN-γ in the TME may play a role in NSCLC progression. 

CSCs have been reported to participate in tumor initiation, metastasis, recurrence and multidrug resistance (Batlle & Clevers, 2017), serving as a key mechanism of tumor progression. CD133 has been identified as a marker of CSCs in NSCLC (Roy et al, 2017; Zakaria et al, 2017). As CSCs are also characterized by enhanced EMT (Mani et al, 2008), Vimentin, an EMT marker, was used as another CSC marker in our study. By immunofluorescence (IF) staining, we intriguingly found much higher expression of CD133 and Vimentin on tumor cells in IFN--low TME than that in IFN--high TME in NSCLC tissues (Fig. 1F, G ). A significantly negative correlation between IFN- and CD133 or Vimentin was observed by IHC staining analysis in 86 NSCLC tissues (Fig. 1H, I ). In addition, a negative correlation between IFNG and PROM1 was also observed in lung adenocarcinoma collected from TCGA (Fig. S2A ). To further accurately quantify the level of IFN- promoting tumor stemness, we performed enzyme-linked immunosorbent assay (ELISA) to detect the concentration of IFN- in tumor interstitial fluid (TIF) isolated from fresh 9 NSCLC tissues. Accordingly, cancer stem properties were evaluated by the percentage of CD133 + stem cells in CD326 + tumor cells and the expression of stemness related genes. Intriguingly, we found patients with relative low-level (around 0. 137ng/mL) IFN- in TIF showed increased cancer stem properties, and patient #4 with 0. 110 ng/mL IFN- showed the highest percentage CD133 + stem cells and highest expression of most stemness related genes. However, patients with high-level IFN- in TIF showed decreased stem-like properties (Fig. 1J ). We also found similar results in tumor tissues from ESCC, CRC and HCC patients, whose low-level border of IFN- correlated with highest stem-like properties was 0. 072 ng/mL, 0. 152 ng/mL and 0. 118 ng/mL respectively (Fig. S2B-D ). Therefore, we preliminarily surmise that the low-level IFN- may serve as a key determinant inducing tumor stemness.
section_name: Low-level IFN- expression is closely associated with poor prognosis and increased expression of CSC markers in patients with NSCLC.

section_content: To clarify the role of low-level IFN- in inducing tumor stemness, NSCLC cell lines, A549 and H460 cells, were treated with different doses of recombinant human IFN- for 1-6 day(s). As expected, the apoptosis rate of the cells progressively increased with an increasing IFN- dose and time (Fig. 2A ). Strikingly, we observed that the frequency of Annexin V -CD133 + cells increased over time after treatment with a relatively low dose of IFN- (≤0. 2ng/mL). In consistent with the optimal IFN- concentration correlated with highest stem-like properties in NSCLC TIF (0. 110 ng/mL), the optimal concertation of IFN- inducing CD133 + cells in vitro was 0. 1 ng/mL. However, the percentage of CD133 + cells turned to decrease over time after treated with a relatively high dose of IFN- (≥100 ng/mL) (Fig. 2B ). Therefore, we speculated that low-dose and long-term IFN- stimulation might enhance the stem-like properties of NSCLC cells. To validate this conception, we next sorted the Annexin V -NSCLC cells treated with either low-dose (0. 1 ng/mL) or high-dose (100 ng/mL) IFN- to examine their sphere-formation ability. Significantly increased sphere number was observed in the low-dose IFN- treatment group compared with the control and the high-dose IFN- treatment groups (Fig. 2C ). Similar results were also found in the expression of CSC markers (Fig. 2D ) and CSC signature genes (Fig. 2E ). Since high tumorigenicity is a key characteristic of CSCs, we additionally explored the effect of low dose of IFN- on tumor growth in NSCLC xenograft models. A549 cells stably expressing luciferase were injected subcutaneously into BABL/c nude mice and then intratumorally treated with PBS or different doses of IFN-γ. Interestingly, we found that both tumor volumes (Fig. 2F, G ) and the frequency of CD133 + tumor cells in xenografts (Fig. 2H ) were dramatically increased following low-dose (0. 1 g/day) but decreased following high-dose (10 g/day) IFN- administration. Taken together, these results suggest that low dose of IFN- facilitates the stem-like properties of NSCLC cells both in vitro and in vivo.
section_name: Low-dose IFN- augments the stem-like properties of NSCLC cells.

section_content: To understand the molecular mechanism underlying low dose of IFN- inducing the stem-like properties in NSCLC cells, we searched the IFN- signaling downstream genes using the GCBI website (https://www. gcbi. com. cn/) and evaluated the different expression levels of these genes by real-time reverse transcription-PCR (RT-PCR) in A549 and H460 cells treated with or without low dose of IFN-. Most of these genes, including IFNGR1 and IFNGR2, were significantly upregulated (Fig. 3A ). Among them, ICAM1 showed the most obvious gene upregulation in response to low dose of IFN- (Fig. 3A ), which was further confirmed by flow cytometry (Fig. 3B ). After neutralization with anti-IFN- antibody, ICAM1(Fig. S3A ) expression was efficiently blocked, as well as IFNGR1 and IFNGR2 (Fig. S3B, C ). ICAM1 is a transmembrane molecule that stabilizes cell-cell interactions and enhances transendothelial transmigration (Kanters et al, 2008; Millan et al, 2006). In NSCLC cells, we observed an obvious upregulation of ICAM1 in Calu-3 cells (a highly metastatic NSCLC cell line) and CSCs generated by non-adhesive culture system (Fig. S4 ), suggesting a potential role of ICAM1 in mediating NSCLC stemness and metastasis. Moreover, IF colocalization analysis demonstrated that ICAM1 was co-expressed with CD133 and Vimentin in A549 cells, which was dramatically increased by low-dose IFN- (Fig. 3C ). Based on these results, we hypothesized that ICAM1 might be essential for low-dose IFN- inducing the stem-like properties in NSCLC cells. 

To test this hypothesis, silibinin, a pharmacological inhibitor of ICAM1, was added to A549 and H460 cells following treatment with low dose of IFN-. 

Interestingly, we found silibinin partially abrogated the sphere-formation ability (Fig. 3D ) and the expression of CSC markers (Fig. 3E ) and CSC signature genes (Fig. 3F )

in NSCLC cells treated with low dose of IFN-. Next, we stably knocked down ICAM1 expression in A549 and H460 cells with shICAM1s viruses (Fig. 3H ). As Next, we explored the signaling pathways involved in the stem-like properties driven by low dose of IFN-. We chose several CSC-relevant signaling activation inhibitors, including PI3K, Akt, Notch1, STAT3, p38 and ERK1/2 inhibitors, to pretreat A549 and H460 cells following low-dose IFN- treatment. Intriguingly, the RT-PCR results

showed that none of the inhibitors had an obvious effect on ICAM1 expression compared with DMSO, but PF04691502, AZD5363 and LY3039478 partially reversed the elevated stem-like properties of A549 and H460 cells (Fig. 4A, B ). 

Similar results were also observed in the flow cytometry and western blot analyses, which demonstrated that PF04691502, AZD5363 and LY3039478 did not affect ICAM1 expression but dramatically blocked the increased expression of CSC markers compared with DMSO in A549 and H460 cells treated with low-dose IFN- (Fig. 4C, D). Therefore, we hypothesized that the PI3K-Akt and Notch1 pathways might act as Upon activation, the Notch1 receptor is cleaved and subsequently releases the Notch1 intracellular domain (N1ICD), which migrates into the nucleus and forms a complex with the nuclear proteins of the RBP-J family. RBP-J acts as a transcriptional activator to activate the expression of target genes after forming a complex with N1ICD (Ohtsuka et al, 1999). We next used the PROMO website to predicate putative RBP-J binding sites in the CD133 promoter region and identified an RBP-J binding site in the -156 to -145 region (Fig. 4G ). Chromatin immunoprecipitation assay (ChIP) assays were performed to validate whether the RBP-J-NICD complex bound to the putative site. As shown in Fig. 4H and I, RBP-J and NICD bound directly to the putative RBP-J-binding site in the CD133 promoter region in A549 and H460 cells, which showed an increase in shScramble cells treated with low-dose IFN- but a decrease in shICAM#1 and shICAM1#3 cells treated with low-dose IFN- compared with shScramble cells. These data suggest that Notch1 directly regulates CD133 transcription in NSCLC cells.
section_name: ICAM1 is crucial for the increased stem-like properties of NSCLC cells induced by low-dose IFN-

section_content: Above data revealed that low dose of IFN- promoted the stem-like properties of NSCLC cells through activation of the ICAM1-PI3K-Akt-Notch1 axis. However, high dose of IFN- was found to induce apoptosis and decrease the stem-like properties of NSCLC cells, as shown in Fig. 2, which prompted our interest in the signaling pathways involved in the high-dose IFN- treatment system. The JAK1-STAT1-caspase pathway has been previously described as the key downstream pathway of IFN--mediated apoptosis (Platanias, 2005), and our results indicated that the ICAM1-PI3K-Akt-Notch1 axis was the key signaling cascade in IFN--mediated stem-like properties. Therefore, we next examined the activation of the JAK1-STAT1-caspase pathway and ICAM1-PI3K-Akt-Notch1 axis in NSCLC cells treated with either low-dose or high-dose IFN-, respectively. The results showed that cells treated with high-dose IFN- exhibited much higher activation of JAK1, STAT1, caspase3 and caspase7 than those treated with low-dose IFN- (Fig. 4J ). Surprisingly, we further observed a significant decrease in the activation of Akt and Notch1 in cells treated with high-dose IFN- compared to those treated with low-dose of IFN- although both high-dose and low-dose IFN- were able to induce high levels of ICAM1 expression (Fig. 4J ). Thus, high-dose IFN- promoted apoptosis of NSCLC cells through the JAK1-STAT1-caspase pathway, whereas high-dose IFN--induced ICAM1 was insufficient to endow the stem-like properties of NSCLC cells through the PI3K-Akt-Notch1 signaling pathway. 

Next, we wondered whether ICAM1 played a role in high-dose IFN--mediated apoptosis. After treatment with different doses of IFN-, we found that ICAM1 knockdown had no influence on apoptosis in A549 and H460 cells (Fig. 4K ), while activation of JAK1, STAT1, caspase3 and caspase7 was increased with the increase of IFN- dose (Fig. 4L ). Moreover, inhibition of JAK1 (Ruxolitinib), STAT1

(Fludarabine) and caspase (Z-VAD-FMK) significantly attenuated high-dose IFN--mediated apoptosis (Fig. 4M ). Western blot analysis showed that phospho-JAK1 expression was only inhibited by Ruxolitinib and phosoho-STAT1 expression was inhibited by both Ruxolitinib and Fludarabine, while caspase 3 and 7

were inhibited by Ruxolitinib, Fludarabine and Z-VAD-FMK (Fig. 4N ). These data indicate that high-dose IFN- promotes apoptosis of NSCLC cells by activating the JAK1-STAT1-caspase signaling pathway in an ICAM1-independent manner.
section_name: High-dose IFN- contributes to apoptosis through activation of the JAK1-STAT1-caspase signaling pathway in NSCLC cells.

section_content: To identify whether ICAM1 played a role in low-dose IFN--induced tumor growth, A549 cells stably expressing luciferase were injected subcutaneously into BALB/c nude mice. The mice were treated intratumorally with PBS or low-dose IFN-γ, silibinin was orally administered. We found that the enhanced tumor growth induced by low-dose IFN- was efficiently blocked by silibinin (Fig. S5A, B ). 

Silibinin also dramatically reduced the high frequency of ICAM1 + CD133 + tumor cells in xenografts treated with low-dose IFN- (Fig. S5C ). 

In Apart from the high sphere-formation ability and high tumorigenicity, CSCs can also form a hierarchy of stem-like and differentiated tumor cells to initiate metastatic growth (Plaks et al, 2015). Therefore, we investigated the effect of low-dose IFN- on the metastatic growth of NSCLC cells and the role of ICAM1 in this process. Before intravenous injection into the lateral tail vein of NOD-SCID mice, luciferase-shScramble-infected, luciferase-shICAM1#1-infected and luciferase-shICAM1#3-infected cells were pretreated with PBS or low-dose IFN- for 6 days in vitro. Metastasis was evaluated by luminescence imaging at day 0, 6, 12 and 18 after injection. We observed that luciferase-shScramble-infected cells pretreated with low-dose IFN- generated larger lung metastatic nodules than luciferase-shScramble-infected cells pretreated with PBS, whereas luciferase-shICAM1#1-and shICAM1#3-infected cells pretreated with low-dose IFN- did not show significant difference compared with those pretreated with PBS (Fig. 5H, I ). In the PBS-pretreated groups, mice injected with luciferase-shICAM1#1and shICAM1#3-infected cells had smaller lung metastatic nodules than those injected with luciferase-shScramble-infected cells (Fig. 5H, I ). Consistently, similar results were also obtained in the frequency of green fluorescent protein (GFP) + NSCLC cells (Fig. 5J ) and the percentage of metastatic cancerous areas in the lungs of mice (Fig. 5K ). Together, these results suggest that low-dose IFN- greatly enhances lung metastasis of NSCLC cell in vivo, which is mediated by ICAM1.
section_name: ICAM1 knockdown reverses tumor growth induced by low-dose IFN- in xenograft model of NSCLC.

section_content: Subsequently, we detected the expression level of ICAM1 in clinical samples. By flow cytometry analysis, we observed higher ICAM1 expression by tumor cells from tumor tissues than epithelial cells from adjacent normal tissues in patients with NSCLC (Fig. 6A ). Moreover, a dramatically elevated frequency of ICAM1 + cells in CD133 + tumor cells was observed compared with CD133 -tumor cells (Fig. 6B ). Data from the TCGA database revealed a close association of ICAM1 with CSC related genes at the mRNA level in 1016 NSCLC tissues (Fig. S6A-G ). Consistently, the IHC staining results demonstrated that patients with high-level ICAM1 expression had higher CD133 (Fig. 6C ) and Vimentin (Fig. 6D ) expression than those with low-level ICAM1 expression. Strongly positive correlation of ICAM1 with CD133 (Fig. 6E )

and Vimentin (Fig. 6F ) was also observed in these tissues. Moreover, co-expression of ICAM1 with CD133 and Vimentin was found in NSCLC tissues, and the co-expression levels in chemoresistant patients were significantly higher than those in chemosensitive patients (Fig. S6H ). These data suggest that ICAM1 may be a malignant signature of NSCLC cells. 

Based on the IHC staining of ICAM1, a significant correlation was found between ICAM1 expression and TNM stage, recurrence, bone metastasis, epidermal growth factor receptor (EGFR) mutation and chemoresistance (Table 2 ). Patients with high-level ICAM1 had a worse OS (Fig. 6G ) and PFS (Fig. 6H ). Data from TCGA also showed that high-level ICAM1 was strongly associated with a poor PFS in NSCLC patients (Fig. S5I ). These data suggest that ICAM1 is strongly associated with prognosis in NSCLC patients. In addition, we further stratified the 86 NSCLC tissues into four groups according to the expression levels of IFN- and ICAM1

analyzed by IHC staining: IFN- low ICAM1 low, IFN- low ICAM1 high, IFN- high ICAM1 low and IFN- high ICAM1 high. Surprisingly, we found that patients with IFN- high ICAM1 high expression showed a better OS (Fig. 6I ) and PFS (Fig. 6J ) than those with IFN- low ICAM1 high expression, which supported the view that ICAM1 might mediate tumor progression or even stemness only in an IFN--low TME rather than in an IFN--high TME.
section_name: The expression level of ICAM1 is significantly upregulated by CD133 + tumor cells and positively correlated with a poor prognosis in NSCLC patients.

section_content: Emerging evidences has reported that IFN- may involve in tumor progression, but the exact mechanism was not fully understood. In this study, we firstly identified the in certain tumor models (Chen et al, 2011; Liu et al, 2017). However, since it is currently very difficult to precisely control the level of IFN- in immunocompetent mice due to unknown IFN- production in many cells and tissues, we only verified our results in vivo by using immunodeficient mouse models in this study. Moreover, IFN- induces not only apoptosis or the stemness of tumor cells but also strongly upregulates PD-L1 expression on tumor cells, activates immunosuppressive myeloid-derived suppressor cells by inducing iNOS. All these points will be taken into account in in vivo experiments in our further study. 

Clinically, the adoptive transfer of receptor-engineered T cells and immune checkpoints blockade are still limited in treating some solid tumors (Ping et al, 2018; Zou, 2018). It was reported that low-dose IFN- generated at the tumor site increases the risk of tumor metastasis during immunotherapy (Gong et al, 2008; Kelly et al, 1991). A recent study has reported that tumor stemness might cause PD-1/PD-L1 blockade resistance (Zou et al, 2016). In our view, the low-level IFN- generated by dysfunctional T or NK cells in the TME may lead to treatment failure of immunotherapy by inducing tumor stemness. The dose of IFN- delivered to the tumor site should be high when using IFN--based cancer immunotherapy. 

Accordingly, for adoptive T-cell therapy, a key issue is to ensure the effector function of T cells after reaching the tumor site, particularly when treating advanced solid tumors. 

In this study, we found that ICAM1 merely mediated tumor stemness in the IFN--low rather than the IFN--high TME, and better survival was also observed in IFN- high ICAM1 high patients that that in IFN- low ICAM1 high patients. It may be necessary to evaluate the levels of IFN- in the TME when using ICAM1-targeting therapy, as patients with low-IFN- levels may be more sensitive. However, ICAM1 was expressed on not only tumor cells but also a variety of stroma cells, such as endothelial cells. It cannot be directly used as a specific target. ICAM1 targeting therapy may be applied by using nanoparticles encapsulating ICAM1 small molecular inhibitor, such as silibinin, which can specifically reach tumor sites. 

In
section_name: Discussion

section_name: Materials and Methods

section_content: All patients provided written informed consent according to the consent form of the Ethics Committee of the first affiliated hospital of Zhengzhou University
section_name: Study approval

section_content: The NSCLC cell lines A549, H460 and Calu-3 were purchased from the Chinese Academy of Sciences Cell Repertoire in Shanghai China and maintained in RPMI-1640 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 units/mL of penicillin and 100 μg/mL of streptomycin at 37°C and 5% CO2 in a humidified incubator. The methods of low or high-dose IFN- treatment for 6 days in A549 and H460 cells were as follows:

recombinant human IFN-γ (Peprotech, Rocky Hill, NJ, USA) were added and cultured for 2 days, and re-stimulated at day 3 and 5, totally 6 days' treatment. Inhibition and blockade treatment were as follows: anti-IFN- antibody (R&D system lnc., Minneapolis, MN, USA), inhibitors, including silibinin (Sigma, Santa Clara, CA, USA), PF04691502, AZD5363 and LY3039478 (Selleck Chemicals, Houston, TX, USA) were pretreatment for 1h following IFN- treatment for 2 days, which were replicated at day 3 and 5, totally 6 days' treatment.
section_name: NSCLC cell lines and cell culture

section_content: Fresh tissues were placed on triple-layered 10-μm nylon mesh and spun at <50g for 5min to remove surface liquid. Next, samples were centrifuged at 400g for 10 minutes to isolate the TIF (Eil et al, 2016).
section_name: Isolation of interstitial fluid from fresh tumor tissues

section_content: A549 cells were stably transfected with luciferase expression vector. Stable knockdown of ICAM1 in A549, H460 and A549 stably expressing luciferase cells by short hairpin RNA was achieved using the pGV248-hU6-MCS-Ubiquitin-EGFP-IRES-puromycin vector plasmid purchased from Gene Pharma (Gene Pharma, Shanghai, China). For viral transductions, the lentiviruses were harvested in 293T cells. Then, A549, H460 and A549 stably expressing luciferase cells were infected with shScramble and shICAM1 lentiviruses. 

After 72 h, the transfected cells were sorted by flow cytometry (Beckman MoFlo XDP, Brea, CA, USA) according to the expression of GFP. The expression of ICAM1 was validated by RT-PCR and western blot analysis. All inserted sequences were verified by DNA sequencing.
section_name: Transfected cell lines

section_content: A549 and H460 cells treated with low or high-dose IFN-γ were sorted. A549 and H460 cells pretreated with DMSO, silibinin (100 M), PF04691502, AZD5363 and LY3039478 or knocked down ICAM1 with shScramble and shICAM1 RNAs following treatment with low-dose IFN- were sorted. A total of 3×10 3 A549 or H460 cells per well were seeded in ultra-low attachment 24-well plates in DMEM/F12 medium (Sigma) containing B27 supplement (Gibco, Invitrogen Carlsbad, CA, USA), EGF (20 ng/mL; Peprotech) and bFGF (20 ng/mL; Peprotech). After 7 days, spheres with a diameter > 75 μm were counted.
section_name: Sphere-formation assay

section_content: To explore the effect of low-dose IFN- on tumor growth in xenograft models, 20 6-week-old female BALB/c nude mice (Vital River Laboratory Animal Technology Co. Ltd, Beijing, China) were randomly divided into five groups (n=5 each) and hypodermically injected with A549 cells stably expressing luciferase (1 × 10 7 cells in 100 μL PBS). After two weeks, 100 to 200 mm 3 tumors were apparent on all mice. 

Each group was intratumorally treated with PBS or different doses of IFN-γ (0. 01, 0. 1, 1, 10 g/day, once per two days) from day 14 to 28. Tumor growth was monitored once per week using an in vivo imaging system (PerkinElmer, IVISLumina Series III, USA). Xenografts were harvested at day 28 to analyze the frequency of CD133 + tumor cells. 

To evaluate the protumor effect of low-dose IFN- in NOD-SCID mice and the role of ICAM1 during this process, luciferase-shScramble-infected, luciferase-shICAM1#1-infected and luciferase-shICAM1#3-infected cells were injected subcutaneously into 24 5-week-old female NOD-SCID mice (three groups, n=10 each). After 8 days, the tumors volumes reached 100 to 200 mm 3 in all mice. At day 8, each group was randomly dived into two groups (a total of six groups, n=5 each). The mice were further intratumorally treated with PBS or low-dose IFN-γ (0. 1 g/day, once per two days) from day 8 to day 22. Tumor growth was measured at day 8, 15 and 22 using an in vivo imaging system (PerkinElmer, IVISLumina Series III, USA). All the xenografts were harvested at day 22. Flow cytometry analysis was performed to detect the frequency of ICAM1 + CD133 + tumor cells. IHC staining was performed to examine the expression of ICAM1, CD133 and Vimentin. 

In addition, to investigate whether silibinin could reverse the low-dose IFN- induced tumor growth in vivo, 16 5-week-old female BALB/c nude mice (Vital River Laboratory Animal Technology Co. Ltd, Beijing, China) were further randomly divided into four groups (n=5 each). All the groups were hypodermically injected with A549 cells stably expressing luciferase (1 × 10 7 cells in 100 μL PBS). Two weeks later, the tumors in each group reached 100 to 200 mm 3. PBS or low-dose IFN-γ (0. 1 g/day, once per two days) were intratumorally injected from day 14 to 28 concomitantly with oral administration of silibinin (in drinking water, 5 mg/mL, 3-4 mL/mouse/day). Tumor growth was evaluated at day 14, 21 and 28 using an in vivo imaging system (PerkinElmer, IVISLumina Series III, USA). Xenografts were harvested to analyze the frequency of ICAM1 + CD133 + tumor cells by flow cytometry. 

Animal studies were blinded during data measurement.
section_name: Animal xenograft models

section_content: Twenty-four 5-week-old female NOD-SCID mice were randomly divided into six groups (n=5 each). 

Luciferase-shScramble-infected, luciferase-shICAM1#1-infected and luciferase-shICAM1#3-infected cells were pretreated with PBS or low-dose IFN- (0. 1 ng/mL) in vitro for 6 days and then intravenously injected into the lateral tail vein of mice (5 × 10 6 cells in 100 μL PBS). 

Metastasis was evaluated using an in vivo imaging system at day 0, 6, 12 and 18 after Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as endogenous control for normalization.
section_name: In vivo metastatic growth model

section_content: Intracellular staining was performed as previously described (Li et al, 2016). 

Tumor tissues were minced, and the tissue homogenate was then filtered through a
section_name: Flow cytometry

section_content: The protocols used for IHC and IF have been described elsewhere (Qiao et al, 2017). Paraffin-embedded NSCLC tissues or A549 cells treated with or without IFN- were examined for the expression of IFN-, ICAM1, CD133 or Vimentin. For IHC staining, the following primary antibodies were used: rabbit anti-human IFN- (Proteintech Group, Chicago, Illinois, USA, 1:100), rabbit anti-human ICAM1

(Proteintech Group, 1:400), rabbit anti-human CD133 (Thermo Fisher Scientific) or rabbit anti-human Vimentin (Proteintech Group, 1:800). The intensity of the staining was analyzed using the following criteria: 0, negative; 1, low; 2, medium; and 3, high. 

The extent of staining was scored as 0, 0% stained; 1, 1-25% stained; 2, 26-50% stained; and 3, 51-100% stained. Five random fields were analyzed using a light microscope (Olympus, Tokyo, Japan). The final scores were evaluated by multiplying the scores of the intensity by those of the extent. The samples were divided into four staining grades: 0, -; 1-3, +; 4-6, ++; and 7-9, +++. The following criteria were used to quantify the expression levels of IFN- and ICAM1 in NSCLC tissues: low expression, "-or +"; high expression, "++ or +++". For IF staining, the following primary antibodies were used: mouse anti-human IFN- (R&D system, 1:1000), mouse anti-human ICAM1 (Abcam, Cambridge, MA, USA, 1:100), rabbit anti-human CD133 (Thermo Fisher Scientific, 1:1000) or rabbit anti-human Vimentin (Proteintech Group, 1:500). FITC-conjugated anti-rabbit IgG (BioLegend, 1:500) and Cy3-conjugated anti-mouse IgG (BioLegend, 1:500) were used as secondary antibodies. The nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI, Roche, USA, 1:1000). Images were analyzed using a fluorescence microscope (Olympus, IX71, Japan). The IF staining results were quantified using NIH ImageJ software.
section_name: IHC and IF staining

section_content: The protocol used for western blot has been described elsewhere (Qiao et al, 2017). A549 and H460 cells treated with low-dose or high-dose IFN- were sorted. 

A549 and H460 cells pretreated with DMSO, silibinin, PF04691502, AZD5363 and LY3039478 or shScramble and shICAM1 RNAs following low-dose IFN- treatment were sorted. A549 and H460 cells pretreated with DMSO, Ruxolitinib, Fludarabine and Z-VAD-FMK following high-dose IFN- treatment were sorted. The primary antibodies used were as follows: anti-ICAM1 (Abcam, 1:100), anti-CD133 (Proteintech Group, 1:1000), anti-Vimentin (Proteintech Group, 1:500), anti-Akt (Cell Signaling Technology, Boston, MA, USA, 1:1000), anti-phospho-Akt (Cell Signaling Technology, 1:1000), anti-Notch1 (Cell Signaling Technology, 1:1000), anti-cleaved-Notch1 (Cell Signaling Technology, 1:1000), anti-JAK1 (Cell Signaling Technology, 1:1000), anti-phospho-JAK1 (Cell Signaling Technology, 1:1000), anti-STAT1 (Cell Signaling Technology, 1:1000), anti-phospho-STAT1 (Cell Signaling Technology, 1:1000), anti-caspase3 (Cell Signaling Technology, 1:1000), anti-cleaved-caspase3 (Cell Signaling Technology, 1:1000), anti-caspase7 (Cell Signaling Technology, 1:1000), anti-cleaved-caspase7 (Cell Signaling Technology, 1:1000) and anti-β-actin (Cell Signaling Technology, 1:4000).
section_name: Western blotting

section_content: The ChIP assay was performed according to the manufacturer's instructions (Cell Signaling Technology). Anti-N1ICD (Cell Signaling Technology 1:200) or anti-RBP-J (Cell Signaling Technology, 1:50) antibody was used to immunoprecipitate the chromatin in A549 and H460 cells transfected with shScramble or with shScramble, shICAM1#1 or shICAM1#3 following low-dose IFN- treatment. 

RT-PCR was performed using primers identified for the RBP-Jκ binding site in the CD133 promoter region as follows: 5′-AGAGACTTCGGACTCGCTCT -3′, 5′-CACAGTGTTGGCCCATTTCC -3′.
section_name: ChIP assay

section_content: The TGCA NSCLC database was accessed via the UCSC Cancer Browser (https://genome-cancer. ucsc. edu). Gene expression based on TCGA RNA-Sequencing data is presented as the mean ± standard error of the mean (SEM) of triplicate determinants. Survival and correlation analyses of NSCLC patients were performed according to the TCGA RNA-Sequencing data and survival information. All analyses were performed using the R Package Survival.
section_name: TCGA database

section_content: Data are shown as the mean ± s. d. and analyzed using Student's t-test or  2 test. 

A paired t-test was used for paired samples. Survival curves were plotted using the Kaplan-Meier method. Statistical analyses were performed using GraphPad Prism 7 software (GraphPad software, La Jolla, CA, USA). A value of P<0. 05 was considered statistically significant.
section_name: Statistical analysis

section_name: The paper explained

section_name: Problem

section_content: In this study, we firstly revealed a dose-dependent effect of IFN- in inducing tumor stemness to accelerate cancer progression in patients with a variety of cancer types. 

Mechanically, low-level IFN- endowed cancer stem-like properties via the (G) Analysis of the CD133 promoter identified an RBP-J-binding site. 

(H, I) ChIP was performed using IgG, anti-NICD or anti-RBP-J antibody, followed by quantitative RT-PCR. 

(J) The expression of ICAM1, phospho-JAK1, phospho-STAT1, cleaved-caspase3, cleaved-caspase7, phospho-Akt, cleaved-Notch1 and their total proteins was tested by western blotting. 

(K, L) Cells were treated with 0, 0. 1 and 100 ng/mL IFN- for 6 days.
section_name: Results

section_name: Acknowledgements

section_content: This study was funded by the National Key Research and Development Program of China ( 2016YFC1303501 ), the National Natural Science Foundation of China (Grant No. 81771781 and 81872410 ) and the National Key R&D Program ( 2018YFC1313400 ).
section_name: Acknowledgements

section_name: Competing interests

section_content: The authors declare that they have no competing interests.
section_name: Competing interests

section_content: Y. Z., M. S. and Y. P. conceived the project. L. Y., F. L., B. Z., J. X. and D. Y. participated in the research design and coordination of the project. M. S. K. Z., S. C., J. Q., S. L. and T. S. performed the experiments. M. S. wrote the manuscript with input from Y. Z., Z. L., Y. P. and N. R. M. M. S. and K. Z. performed data analysis. All the authors read and approved the final manuscript.
section_name: Author contributions