The H1299 and A549 human lung cancer cell lines and the T47D human breast cancer cell line were obtained from the American Type Culture Collection (Manassas, Virginia) and cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum (FBS). The PC9 human lung cancer cell line was a gift from Dr C.-H. Yang (Graduate Institute of Oncology, Cancer Research Center, National Taiwan University). The CL141 and CL1-5 human lung cancer cell lines, derived from clinical patients, were established in our laboratory22 23 and maintained in Roswell Park Memorial Institute (RPMI) medium containing 10% FBS. Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors. Isolation of PBMC in the buffy coats of fresh whole blood samples was performed by density gradient centrifugation on Ficoll-Paque Premium (GE Healthcare). Human PBMCs and Jurkat leukemia T cell line were cultured in RPMI medium containing 10% heat-inactivated FBS.

The IκB kinase (IKK) inhibitor BAY 11-7082 was acquired from MCE (MedChemExpress). Cell viability was analyzed by sulforhodamine B assay according to the manufacturer’s instructions. Further details about immunoblot analysis, real-time reverse transcriptase PCR, flow cytometry analysis, RNA interference and luciferase reporter assay are fully provided in the online supplemental materials.

CL1-5 cells (1×10⁶) were subcutaneously injected into the right flanks of 6-week-old nude mice, followed by intraperitoneal pemetrexed (50 mg/kg) every 4 days for two cycles. Tumors were collected 1 week after the final treatment. The isolated tumors were cut into small pieces, which were lysed with Radioimmunoprecipitation assay buffer (RIPA buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS) containing complete protease inhibitor cocktail (Roche). Protein samples were subjected to immunoblotting with antibodies against PD-L1 (#13684, Cell Signaling) and anti-β-actin (Sigma-Aldrich), the latter being the internal control.

CL1-5, CL141 and H1299 lung cancer cells were pretreated with chemotherapeutic drugs for 24 hours. These cells were subsequently cocultured with Jurkat T-cells or PBMCs at the cancer cell to T cell ratio of 5:1 (CL1-5 cells) or 10:1 (CL141 cells) for additional 48 hours in the presence of 1× T Cell Stimulation Cocktail (eBioscience). Coculture mediums were kept containing 1× T Cell Stimulation Cocktail and chemotherapeutic drugs. For blockade of the PD-1/PD-L1 pathway, 10 µg/mL of anti-PD-L1 antibody or control mouse IgG was added to the coculture medium. The culture supernatants were harvested at 48 hours after coculture and the concentrations of IL-2 were assessed by IL-2 Human ELISA Kit (Catalog # 88-7025-88, Thermo Scientific).

For intracellular cytokine staining (ICS), monensin (3 µM) was added into coculture media for blocking cytokine secretion. Human Jurkat T-cells or PMBCs were harvested and washed with ice-cold PBS. Cells were then stained with specific antibodies for surface markers, CD45 (Biolegend, #368511) and CD69 (Biolegend, #310903), in cell staining buffer (Biolegend, #420201) for 30 min on ice in accordance with the manufacturer’s protocols. The intracellular staining of IL-2 (Biolegend, #500306) was performed with Cyto-Fast Fix/Perm buffer set (Biolegend, #426803). Afterwards, cells were washed twice with ice-cold PBS/FBS, resuspended, then analyzed by Fluorescence-activated cell sorting (FACS) analysis using a FACS Calibur system. FACS data analysis was performed using FlowJo software (Tree Star, Ashland, Oregon).

CL141 lung cancer cells stably expressing NucLight red fluorescence protein (RFP) (#4476, Essen Bioscience) were seeded into 96-well plates and treated with anti-PD-L1 antibody (10 µg/mL) and/or pemetrexed (100 nM) for 24 hours. Cells were subsequently coincubated with activated Jurkat T-cells at the cancer cell to T cell ratio of 1:5. The fluorescence signals were detected by a SpectraMax iD3 fluorescence spectrometer (Molecular Devices).

For the CT26 tumor model, BALB/c mice aged 6–8 weeks were inoculated subcutaneously with murine CT26 (2×10⁵) colon cancer cells. For the LL2 tumor model, C57BL/6 mice aged 6–8 weeks were inoculated subcutaneously with murine LL2 (2×10⁵) lung cancer cells. Tumor volumes were measured at 4 or 5 days after inoculation using the formula: L × D²/2, where L and D are the long and short dimensions, respectively, of the tumor. Mice were injected intraperitoneally every 4 days with 3 mg/kg of antimouse PD-L1 antibody (10F.9G2, Bio X cell) or control rat IgG2b (LTF-2, Bio X cell). Mice were also injected intraperitoneally with 100 mg/kg of pemetrexed every 2 days for 2 weeks. Tumors were harvested on day 20 and fixed in formaldehyde for the immunohistochemistry (IHC) staining.

To assess the activation and/or infiltration of CD4⁺ and CD8⁺ TILs surrounding tumor tissues, tumors treated by monotherapy or combination therapy were harvest and analyzed by IHC staining. Details of analysis methods are fully provided in the online supplemental materials.

Using the limited dilution method, the parental CL1-5 lung cancer cells were seeded in the 96-well plate with an average of one cell per well. After 20 days of culture, subclones were expanded in RPMI-1640 medium supplemented with 10% FBS. Twenty single-cell colonies were selected as CL1-5 subclones and then treated with a sublethal dose of pemetrexed (50 nM). After 48 hours of treatment, the levels of PD-L1 protein were detected by immunoblotting. In treatment with pemetrexed (PEM), the CL1-5 subclones exhibiting elevated PD-L1 protein levels were defined as pemetrexed responders (PEM-R) while the subclones exhibiting no significant PD-L1 upregulation were defined as non-responders (PEM-NR).

For RNA sequencing and gene expression analysis, total RNA was extracted from pemetrexed-treated CL1-5 subclones. rRNA was depleted by the rRNA depletion method. Library preparation was followed by the manual protocol and paired-end reads with 150 bps were sequenced by Illumina HiSeq 4000 (Illumina). Sequencing quality and adaptor contamination were further evaluated by FASTQC (v0.11.7). Raw reads were cleaned by Trimmomatic (v0.33) for removal of adaptor sequences and lower quality bases (average quality score of 4-bases wide sliding window <20). Trimmed paired-end reads were aligned to human reference genome GRC37/hg19 using STAR2 (STAR-2.6.0c). Based on ensemble GRC37 gene annotation, gene expression was quantified by raw read counts using the htseq-count algorithm.

Before identifying differentially expressed genes between experiment groups, several preprocess steps were applied to avoid system errors. The read throughput difference was first adjusted by the trimmed mean of M-values algorithm of edgeR (R package). Next, count per million (CPM) was as gene expression unit. Genes with CPM<1 and not detected in at least two samples were excluded. Finally, logarithm 2 transformation and quantile normalization were applied before data analysis. For statistical analysis, the Log2 ratio of gene expression between the experiment and the corresponding control group was calculated. A two-sample t-test was used to identify differentially expressed genes. All tests were two-tailed and p values<0.05 were considered significant.

Cells were trypsinized and resuspended in PBS containing 10 µM CM-H2DCFDA (#C6827; Invitrogen). After incubation at 37°C for 30 min in the dark, intracellular ROS levels were assessed by flow cytometry using a FACS Calibur system.

Data are presented as the mean±SD; statistical significance was assessed via GraphPad Prism Software V.7.0; groups were compared using the t-test, with p<0.05 considered significant.

Antitumor therapy becomes more successful when it can induce an immunogenic form of tumor cell death (ICD) that elicits a strong immune response.24 25 Recently, monotherapy of the frontline NSCLC chemotherapeutics, such as pemetrexed, carboplatin and paclitaxel (PTX), have been shown to induce ICD in syngeneic mouse models with various degrees while, interestingly, only the folate antimetabolite pemetrexed show the greater potential to synergize with PD-1/PD-L1 blockade.26 Although pemetrexed exerts the strongest immunogenic effects on tumor and immune cells, we noticed that pemetrexed also induced substantial levels of PD-L1 upregulation.26 Knowing that higher PD-L1 expression on tumor cells associated with better responses to ICB therapy,27 we attempted to assess the potential PD-L1 priming ability of pemetrexed and other frontline NSCLC chemotherapeutics in NSCLC cell lines. The half-maximal inhibitory concentration (IC₅₀) of pemetrexed (PEM), 5-FU, cisplatin, and PTX were initially determined in the commonly used NSCLC cell lines CL1-5, CL141 and H129922 23 (online supplemental table S1). To avoid triggering massive tumor cell death, these NSCLC cell lines were treated with the corresponding sublethal doses of chemotherapeutics for 72 hours and examined for their PD-L1 levels. Interestingly, we found that protein levels of PD-L1 were elevated by pemetrexed or 5-FU in a dose-dependent manner (figure 1A), while treatment with cisplatin or PTX did not increase PD-L1 expression (online supplemental figure S1A). We further confirmed that upregulation of PD-L1 (encoded by CD274) by either pemetrexed or 5-FU was initiated at the transcriptional level (figure 1B). To assess whether the pemetrexed and 5-FU-induced PD-L1 gene upregulation produces functionally and clinically targetable PD-L1 proteins, we detected the membrane-bound PD-L1 protein by anti-PD-L1 flow cytometry antibodies. NSCLC cells treated with pemetrexed or 5-FU displayed a significant increase in PD-L1 levels on the cell surface (figure 1C,D), whereas cisplatin and PTX did not give such PD-L1 priming effects (online supplemental figure S1C,D). To further confirm this indication, we assessed the pemetrexed-mediated PD-L1 priming effect in human xenograft tumors. Human CL1-5 lung cancer cells were implanted into nude mice to form subcutaneous tumors followed by the injection of pemetrexed. We found that mice treated with pemetrexed displayed a substantial increase in PD-L1 levels in the CL1-5 xenograft tumor (figure 1E), suggesting that pemetrexed may stimulate certain signaling(s) in NSCLC tumors that drive the upregulation of PD-L1. Collectively, these results suggest that the sublethal dose of antimetabolic chemotherapeutics, such as pemetrexed and 5-FU, is capable of inducing PD-L1 expression in NSCLC cells whether in vitro or in vivo.

The chemoimmunotherapy combination of pemetrexed, cis/carboplatin and immune checkpoint inhibitors (anti-PD-1/anti-PD-L1) has recently been approved as first-line treatment in advanced NSCLCs.19–21 However, which chemotherapeutics in the chemoimmunotherapy combination exert beneficial effects and the underlying antitumor mechanism(s) still remain obscure. T-cell activation is a key indicator of ICB therapy, which can be assessed by monitoring well-known surface markers (eg, CD69) and cytokine molecules (eg, IL-2, IFN-γ) enriched in activated T cells in human cytotoxic T lymphocyte (CTL) or Jurkat leukemia T cell coculture systems.28 To evaluate whether treatment of NSCLC cells with frontline NSCLC chemotherapeutics can modulate T-cell activation, we measured the levels of secreted IL-2 and IFN-γ in human Jurkat T-cells or PBMCs, which include CD3⁺ T-lymphocytes, incubated either alone or in coculture with NSCLC cells. The ELISA showed that activated Jurkat T-cells or PBMCs secreted high amounts of IL-2 and IFN-γ into the coculture medium, whereas the ability of the T cells to produce these antitumor cytokines was substantially suppressed when cocultured with CL1-5 or CL141 cells (figure 2A–D). This is consistent with previous findings that tumor cells may hijack the immune checkpoints to inhibit or exhaust T-cell activities through the PD-1/PD-L1 pathway.29 Indeed, CL1-5 and CL141 cells pretreated with pemetrexed or 5-FU displayed greater abilities to inhibit Jurkat T-cell or CTL-mediated cytokine secretion (figure 2A–D), supporting the idea that these antimetabolic chemotherapeutics are capable of inducing PD-L1 expression in tumor cells and the following T-cell suppression. In contrast, there were no detectable changes in IL-2 secretion in T cells when NSCLC cells were pretreated with chemotherapeutics (ie, cisplatin and PTX) showing no effects on PD-L1 upregulation (online supplemental figure S2A). The ICS assays further confirmed that NSCLC cells pretreated with pemetrexed or 5-FU greatly suppressed the levels of CD69 and intracellular IL-2 in human Jurkat T-cells (figure 2E–G and online supplemental figure S2C,D). To further test whether pemetrexed in combination with ICB therapy could exert antitumor activities through T-cell activation, we evaluated effects of the combined treatment of pemetrexed and anti-PD-1 antibody on T-cell activation in the NSCLC/T-cell coculture system. We found that addition of anti-PD-1 antibody or anti-PD-L1 antibody in pemetrexed-treated coculture system greatly reinvigorates the exhausted T cells by blocking the PD-1/PD-L1 interactions (online supplemental figure S2B). The effects of the combined treatment of pemetrexed and anti-PD-L1 antibody on the tumor-killing activity of T cells were also tested. CL141 cells stably expressing a nuclear RFP, NucLight RFP, were cocultured with activated Jurkat T-cells in the presence of pemetrexed and/or anti-PD-L1 antibody. By measuring the RFP signals, which represent cell viability, we found that sublethal dose of pemetrexed only caused partial suppression of tumor cell viability (figure 2H). However, the combined treatment of pemetrexed and anti-PD-L1 antibody resulted in the synergistic inhibition of tumor cell growth (figure 2H). These findings indicate that while pemetrexed could only exert minor cytotoxic effects on tumor cells, pemetrexed-induced PD-L1 upregulation plus the subsequent PD-1/PD-L1 blockade could sensitize tumor cells to the T cell-mediated tumor cell killing. Altogether, our data suggest that pemetrexed or 5-FU-induced PD-L1 upregulation in NSCLC cells can modulate T-cell activation when combined with PD-1/PD-L1 blockade, while addition of cisplatin or PTX did not have such immunomodulatory effects.

The observed immunomodulatory effects of pemetrexed in combination with PD-1/PD-L1 blockade in the in vitro system prompted us to evaluate this chemoimmunotherapy combination in immunocompetent syngeneic mouse tumor models. We chose the commonly used murine Lewis lung carcinoma (LL2) and colorectal cancer cell (CT26) lines and confirmed that both cell lines respond to the treatment with pemetrexed or 5-FU by membrane-bound PD-L1 protein upregulation (online supplemental figure S3A). To test the effects of pemetrexed on increasing the efficacy of ICB therapy in the mouse tumor models, we cocultured mouse LL2 or CT26 cancer cell lines with primary splenocytes derived from the mouse spleen and measured the levels of secreted IL-2 and IFN-γ in the coculture system. Similar to the effects in human NSCLC/T cell coculture systems, we found that pemetrexed treatment significantly suppresses the splenocyte-mediated cytokine secretion (figure 3A,B). Notably, the addition of anti-PD-L1 antibody in pemetrexed-treated coculture system greatly induced splenocyte activation (figure 3A,B), confirming that the effects of the pemetrexed/anti-PD-L1 antibody combinational therapy can also be observed in the mouse tumor models. Moreover, the number of CD3⁺ T-lymphocytes, which contains CD4⁺ helper and CD8⁺ cytotoxic T-cells, in the splenocyte population was greatly increased in the combinational treatment of pemetrexed and anti-PD-L1 antibody (online supplemental figure S3B). The effects of the pemetrexed/anti-PD-L1 antibody combinational therapy on tumor growth were further evaluated by monitoring the tumor sizes and the surrounding tumor microenvironment in mouse syngeneic tumor models. Although treatment with pemetrexed or anti-PD-L1 antibody showed significant tumor growth hindrance in both mouse models (ie, LL2 and CT26), the combined treatment exerted far more superior tumor suppressive effects (figure 3C,D). The tumors were harvested and assessed for PD-L1 expression and the recruitment and/or activation of CD4⁺ and CD8⁺ TILs. The IHC staining in tumor specimens showed substantial PD-L1 signals in tumors receiving pemetrexed (online supplemental figure 3C,D). IHC staining of CD4 in tumor specimens showed that pemetrexed treatment induced a significant increase in the number of CD4⁺ TILs in tumor areas (figure 3E), suggesting that the tumor cytotoxic effects of pemetrexed may trigger the activation and/or recruitment of CD4⁺ T helper cells to the tumors. Through the IHC staining, we also found that the anti-PD-L1 antibody triggered the activation and/or infiltration of CD8⁺ T cells into tumors (figure 3E), consistent with the concept of ICB therapy. Interestingly, the combined treatment of pemetrexed and anti-PD-L1 antibody synergistically amplified the numbers of both CD4⁺ and CD8⁺ TILs even more in the tumor surroundings (figure 3E), indicating that the immunosuppressive tumor microenvironment has been altered through this combinational treatment. To further confirm this idea, we detected the expression levels of various antitumor cytokines, tumor necrosis factor (TNF)-α, IFN-γ and IL-2, that have been shown to lose their expressions in exhausted TILs30 31 and found that this combinational treatment induced much higher levels of IL-2, TNF-α and IFN-γ in tumor areas than individual drug treatments (figure 3F).

To test the therapeutic effect platinum-based chemotherapy has when combined with pemetrexed and ICB therapy in NSCLC treatment, we investigated whether the addition of cisplatin into the combinational treatment of pemetrexed and anti-PD-L1 antibody would exhibit an enhanced effect on the suppression of lung tumor growth in vivo. Although monotherapy with pemetrexed, cisplatin, or anti-PD-L1 antibody all showed significant tumor growth inhibition in the LL2 mouse lung tumor model, the combined treatment of pemetrexed and anti-PD-L1 antibody exerted far more superior tumor suppressive effects (figure 3G). Interestingly, there seems to be no advantage of cisplatin in the combination therapy of pemetrexed and anti-PD-L1 antibody for LL2 lung cancer treatment (figure 3G). Thus, our results indicate that the combination of antimetabolic chemotherapeutics (eg, pemetrexed) with PD-1/PD-L1 blockade (eg, anti-PD-L1 antibody) could further enhance the antitumor immune responses and restore exhausted T-cell activities, thereby priming a favorable tumor microenvironment for cancer immunotherapy.

Increasing evidence indicates that the intratumoral heterogeneity is a critical factor that leads to chemoresistance.32 We speculate that the tumor heterogeneity caused different response rates to chemotherapeutics, which may also account for the failure of ICB therapy.33 34 To investigate whether NSCLC cells respond to pemetrexed treatment differently and generate drug-resistant subpopulations, we selected CL1-5-derived subclones that displayed different pemetrexed responses and classified them into two groups, PEM-R and non-responder (PEM-NR) (figure 4A,B). Interestingly, we found that the PEM-NR cell clones expressed extremely low levels of PD-L1 at both the mRNA and protein levels, and had little response, if any, to the pemetrexed-induced PD-L1 upregulation (figure 4A,B). This suggests that pemetrexed might fail to stimulate certain signaling(s) in this subpopulation that lost the ability to drive the transcriptional activation of PD-L1 gene (ie, CD274). To dissect the molecular mechanisms by which pemetrexed may render distinct regulations on PD-L1 expression depending on different cellular contexts, we performed RNA-sequencing (RNA-seq) for each CL1-5 subclone (PEM-R1, R2, NR1, or NR2) and compared transcriptome profiles between the PEM-R and PEM-NR groups. An estimate of 337 genes, which matched our criteria for upregulation in the PEM-R group and downregulation in the PEM-NR group, were identified (online supplemental figure S4A). Gene ontology enrichment analysis further identified that the NF-κB regulatory pathway and its related genes have significant changes in their expressions in response to pemetrexed treatment (figure 4C,D). This encouraged us to investigate the potential regulation of pemetrexed in the NF-κB signaling pathway.

NF-κB activation is tightly controlled by IKK-mediated phosphorylation of IκB, which normally sequestrates NF-κB in the cytosol.35 On the phosphorylation by IKK, IκB is degraded through proteasome-mediated mechanisms, thereby releasing NF-κB and enabling it to enter the nucleus for its transactivation function.35 36 We first investigated the effects of pemetrexed on NF-κB signaling by monitoring the phosphorylation status of IκB-α. Interestingly, we observed that the phosphorylated IκB-α can only be detected in pemetrexed-responsive CL1-5 subclones but not in non-responsive subclones (figure 4E and online supplemental figure S4B). The pemetrexed-induced IκB-α phosphorylation was accompanied by a significant loss of IκB-α protein and the phosphorylation of NF-κB p65; both events facilitate NF-κB to enter the nucleus and bind to the promoters of target genes.37 These data strongly suggest that pemetrexed triggers the activation of NF-κB signaling, and thereby transactivates PD-L1 gene expression. Supporting the idea, luciferase reporter experiments in CL1-5 subclones demonstrated that pemetrexed induced the transactivation activity of NF-κB in a dose-dependent manner, which is only detectable in pemetrexed-responsive CL1-5 subclones (figure 4F and online supplemental figure S4C). The mRNA expression levels of PD-L1 in each group of CL1-5 subclones also displayed the same patterns with corresponding protein expression levels when cells responded to pemetrexed treatment (figure 4G and online supplemental figure S4D). To further confirm that the NF-κB signaling pathway acts downstream of pemetrexed to transactivate PD-L1 expression, we treated the pemetrexed-responsive CL1-5 subclones with an IKK inhibitor, BAY11-7082, to suppress the phosphorylation of IκB-α. As expected, the treatment of BAY11-7082 significantly repressed the pemetrexed-induced PD-L1 expression at both the mRNA and protein levels (figure 4H,J and online supplemental figure S4E). Moreover, silencing the major subunit of NF-κB p65 (encoded by RelA) by siRNA oligonucleotides (siRelA) could also recapitulate the effects of BAY11-7082 on the pemetrexed-responsive CL1-5 subclones; as we found that the knockdown of p65 greatly suppressed the ability of pemetrexed to induce PD-L1 upregulation (figure 4I,K and online supplemental figure S4F). Taken together, we demonstrated that pemetrexed induces PD-L1 expression through the engagement of NF-κB-mediated transactivation activity in NSCLC cells.

We next sought to trace the direct target(s) of pemetrexed that is required for its PD-L1 priming activity. Since the JAK1/2 kinase family has been reported to mediate PD-L1 expression on extracellular stimulation in tumor cells,38 we first investigated the effects of ruxolitinib (a JAK inhibitor) on the expression of PD-L1 in pemetrexed-treated CL1-5 and CL141 cells. Our results showed that ruxolitinib did not have any effect on pemetrexed-dependent upregulation of PD-L1 (online supplemental figure S5), suggesting that pemetrexed might regulate PD-L1 expression through a different target(s). It is also known that pemetrexed and 5-FU can inhibit DNA biosynthesis processes by suppressing the activity of TS, an enzyme that catalyzes the transformation of deoxyuridine monophosphate to deoxythymidine monophosphate for the maintenance of DNA replication and repair, thereby inducing DNA damage in tumor cells through TS inhibition.17 39 Although there is no evidence showing TS being involved in gene regulation, we tried to evaluate the possibility that TS might be the direct target of pemetrexed for regulating PD-L1 gene expression. We performed RNAi silencing approaches to knock down the endogenous TS expression in CL1-5 and CL141 cells. We found that both mRNA and protein expression of PD-L1 significantly elevated on the knockdown of TS (figure 5A,C), suggesting that TS is capable of regulating PD-L1 gene expression. Essentially, knockdown of TS greatly elevated membrane-bound PD-L1 levels in CL1-5 and CL141 cells (figure 5B), thereby enhancing the ability of tumor cells to suppress Jurkat T-cell or CTL activation (figure 5G). Thus, these results suggest that pemetrexed may upregulate PD-L1 expression through the inactivation of the TS-dependent DNA synthesis pathway. The elevated PD-L1 levels on the cell surface of NSCLC cells, in turn, diminish immune responses by suppressing T-cell activation (eg, IL-2 secretion).

Since we identified that the conventional pemetrexed target TS as well as the NF-κB signaling, both involved in PD-L1 gene expression, we explored whether the TS-dependent DNA synthesis pathway can crosstalk with the NF-κB signaling pathway. Knockdown of TS significantly induced the phosphorylation of IκB-α and NF-κB p65 in CL1-5 and CL141 cells, indicating that TS is capable of cross-regulating the NF-κB activity (figure 5D). To further confirm this idea and verify whether TS acts upstream of the NF-κB pathway to regulate PD-L1 expression, we cosilenced the expression of TS and RelA by specific siRNA oligonucleotides in CL1-5 and CL141 cells. We found that knockdown of TS by TS-siRNA induced PD-L1 upregulation at both the mRNA and protein levels, while the effects of TS-siRNA on PD-L1 expression were abolished by the treatment of RelA-siRNA (figure 5E,F). This confirms that the NF-κB pathway acts downstream of TS for the control of PD-L1 expression.

High level of ROS in tumor cells has been connected to the activation of NF-κB signaling in tumor inflammation.40–42 The intracellular ROS may enhance the phosphorylation of IκB and lead to the proteasomal degradation of IκB, which in turn induces the release and translocation of NF-κB to the nucleus to initiate transcription. Interestingly, the inhibition of TS by TS inhibitors has also been shown to increase the intracellular level of ROS.43 To explore whether pemetrexed-mediated TS inhibition could increase the intracellular level of ROS and result in the activation of NF-κB signaling pathway, we detected intracellular levels of ROS in CL1-5 and CL141 cells on the inhibition of TS. As expected, we found that intracellular levels of ROS were significantly elevated on inhibition of TS by either pemetrexed or TS knockdown (figure 6A,B). Luciferase reporter experiments in CL1-5 and CL141 cells further demonstrated that knockdown of TS induced the transactivation activity of NF-κB, while the effects of TS-knockdown on NF-κB could be rescued when the intracellular ROS was effectively scavenged by the antioxidant, N-acetylcysteine (NAC) (figure 6C). Additionally, we demonstrated that 5-FU, an antimetabolic chemotherapeutic that is also known to target TS and inhibit DNA biosynthesis, has the potential to increase the intracellular level of ROS and result in the activation of NF-κB signaling pathway in NSCLC cells (online supplemental figure 6A-C). These data indicate that antimetabolites, pemetrexed and 5-FU, can induce the activation of NF-κB signaling by regulating the activity of TS and the intracellular level of ROS. To further confirm that this TS−ROS−NF-κB regulatory axis contributes to the upregulation of PD-L1 in NSCLC cells, we checked the PD-L1 level in this scenario. Indeed, knockdown of TS promoted PD-L1 upregulation in CL1-5 and CL141 cells, while the effects can be abolished if the intracellular ROS is reduced effectively by NAC (figure 6D). To further confirm that the TS−ROS−NF-kB regulatory axis is actively involved in the activation of PD-L1 transcription in tumor cells, we selected A549, PC9 human lung cancer and T47D human breast cancer cell lines that are known not to express PD-L144 45 (online supplemental figure S7A), and tested the effects of TS inhibition on these tumor cells by either pemetrexed or TS knockdown. We found that pemetrexed greatly induced the intracellular level of ROS in A549, PC9 and T47D cells (figure 6A). Consistently, knockdown of TS induced the intracellular level of ROS, the transactivation activity of NF-κB, as well as PD-L1 levels in these cancer cell lines while the effects of TS inhibition could be significantly abolished by the antioxidant NAC (figure 6B–D and online supplemental figure S7B). These results strongly support a general function of the TS−ROS−NF-κB regulatory axis in transcriptional regulation of PD-L1 in tumor cells.

In conclusion, our results indicate that pemetrexed can induce PD-L1 upregulation in human NSCLC cells by regulating the TS−ROS−NF-κB regulatory axis and prime a favorable tumor microenvironment for ICB therapy (figure 6E).