A549 (CLL-185™) and H157 (CRL-5802™) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 containing 10% FBS. We acknowledge that the H157 cell line has been demonstrated to be contaminated with the squamous cell lung carcinoma cell line, NCI-H1264 (https://web.expasy.org/cellosaurus/CVCL_0463). However, we hereby confirm that this possible contamination is unlikely to affect the outcomes of our study. The radioresistant A549 and H157 subline cells were developed by fractionated radiation treatment for several months. Following a selection procedure as described in a previous study (10), two radioresistant subline cells, A549R26-1 and H157R24-1, were selected and used in the entire experiment. The THP-1 cell line (ATCC^® TIB-202™) was also obtained from ATCC. The cells were cultured in RPMI-1640 containing 10% FBS and maintained in a humidified 5% CO₂ environment at 37°C.

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors following the approval of the Human Subject Protection Review Board of University of Rochester Medical Center (Rochester, NY, USA). The healthy volunteers provided consent on an institutional review board (IRB) approved study prior to donating their blood. Primary NK cells were purified from these PBMCs using a NK cell isolation kit (130-092-657; Miltenyi Biotec, Cambridge, MA, USA) according to the manufacturer's instructions. Following isolation, the cells were maintained in IL-2-containing NK cell medium. The purity of the isolated cells (CD56⁺CD3⁻) was confirmed by flow cytometric analysis following staining with anti-CD56-PE (12-0267-41; e-Bioscience, San Diego, CA, USA) and anti-CD3-Cy7 (300429; BioLegend, San Diego, CA, USA) antibodies (Abs). These primary NK cells were cultured in α-MEM containing sodium bicarbonate (M4655; Sigma, St. Louis, MO, USA), IL-2 (100 U/ml) (200-02; Peprotech, Rocky Hills, NJ, USA), inositol (0.2 mM), 2-mercaptoethanol (0.1 mM), folic acid (0.02 mM), 12.5% horse serum (Sigma) and 12.5% FBS (HyClone, Logan, UT, USA).

The THP-1 cells were differentiated by exposure to 100 ng/ml phorbol myristate acetate (PMA) for 24 h followed by the addition of human recombinant IL-4 (rhIL-4, 20 ng/ml, 200-04; Peprotech) as previously described (16,17). THP-1 CM was obtained from the PMA/IL-4-treated THP-1 culture 48 h after the addition of fresh medium. For the immunofluorescence (IF) staining of the PMA- and PMA/IL-4-treated THP-1 cells, the cells were plated in a chamber slide and stained with FITC-CD206 Ab (321103; BioLegend, Dedham, MA, USA). Following the mounting procedure using mounting solution including DAPI (Ventashield, H-1200; Vector Laboratory, Burlington, ON, Canada), fluorescence images were obtained on an F-microscope (Axioskop 40, XE120-XL-0255; Zeiss, Goettingen, Germany).

For testing the migration of THP-1 cells to tumor cells, the THP-1 cells were placed in the upper chamber, while tumor cells (A549R26-1 and H157R24-1, 5×10⁴) were placed in the lower chamber of Transwell plates (Corning, 8 µm pore size, 24-well plates, no serum-containing media). Migrated macrophages at the end of 24 h of incubation were visualized by staining with a crystal violet (C-0775; Sigma) solution and counted under an inverted microscope (Axiovert 40; Zeiss). For experiments blocking IL-6 or chemo-kine (C-C motif) ligand2 (CCL)2/CCL5 actions, neutralizing Abs of IL-6 (0.1 µg/ml), CCL2 (2 µg/ml, MAB279) (both from R&D Systems, Minneapolis, MN, USA) and/or CCL5 (5 µg/ml, ab9679; Abcam, Cambridge, MA) were used (species matched control IgG was used as controls). Three independent experiments (in triplicate) were carried out and quantification was obtained from average numbers of positively stained cells in 3 phases.

NK cell cytotoxicity against tumor cells was analyzed using a lactate dehydrogenase (LDH) release assay as previously described (10,14,18-22). The cancer cells (2,500 to 5,000) were plated and on the following day, NK cells were added at various ratios (1:1, 1:5 and 1:15, target cells:effector cells) (all samples in triplicate). Following 4 h of co-culture, an aliquot of 50 µl medium was used in the LDH cytotoxic assay using the LDH cytotoxic assay kit (88954; Thermo Fisher Scientific, Waltham, MA, USA). The experimental LDH release was corrected by the subtraction of the spontaneous release of effector cells at corresponding dilutions. %Cytotoxicity = (experimental value − effector cell spontaneous control − target cell spontaneous control)/(target cell maximum control − target cell spontaneous control) ×100. In examining the effects of neutralizing Abs on NK cell cytotoxicity, PD-L1 Ab (10 µg/ml, 16-5982-82; Invitrogen, Carlsbad, CA, USA) and U0126 (10 µM, 9903; Cell Signaling Technology, Danvers, MA, USA) were used and species matched IgG and vehicle was used for control, respectively.

The luciferase-tagged A549P and A549R26-1 cells (1×10⁶) that were tagged by transfection with luciferase plasmid (pcDNA3.1-luc, V790-20; Addgene, Cambridge, MA, USA) using PolyFect Transfection reagent (301105; Qiagen, Toronto, ON, Canada). The luciferase-tagged cells were selected and maintained by the addition of G418 disulfate (400 µg/ml; Sigma) to the culture. The cells were orthotopically injected (1×10⁶ cells in media with Matrigel, 1:1 ratio in volume) into 8-week old female nude mice (NCI) (total 12 mice, average body weight 20.5 g). Tumor development and volumes were monitored once a week by the In Vivo Imaging System (IVIS). The mice were divided into 2 groups (n=6 per group). When luminescence reached to 5×10⁵ to 1×10⁶ radiance (p/sec/cm²/sr), mice of test group (lung site) were irradiated at 5 Gy for 5 consecutive days (23), while the remainder of the body was shielded from IR using lead blocks. Tumor size in the irradiated group and control group was monitored twice a week by IVIS for 3 weeks. At the end of the experiments, the mice were sacrificed and tumors were obtained. At the time of sacrifice, the average body weights of the mice bearing A549P-cell-derived and A549R26-1 cell-derived tumors were 28.5 and 28 g, respectively. All animal experiments were performed under the supervision and guidelines of the University of Rochester Medical Center Animal Care and Use Committee. The facilities and programs for animal care at the University of Rochester comply with state and federal laws and NIH policies. The entire Animal Resource program is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Mice were housed in the University of Rochester Vivarium, which is in the Medical Center and supervised by the Department of Laboratory Animal Medicine. Mice were kept at a room temperature of 64–79°F, a humidity range of 30–70%, and were allowed free access to food and uncontaminated drinking water. We used university tumor burden guidelines as our humane endpoints. The mice did not experience any stress, discomfort or pain during the whole procedure of the experiment.

The tissues obtained were fixed in 10% (v/v) formaldehyde in PBS, embedded in paraffin, and cut into 5-µm-thick sections. Tumor tissue sections were deparaffinized in xylene solution, rehydrated and immunostained with the IHC kit (sc2018; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Tumor tissues were stained with Abs for F4/80 (1:1,000, ab6640; Abcam), IL-6 (1:1,000, MAB206; R&D Systems, Minneapolis, MN, USA), CCL2 (1:1,000, ab9669) and CCL5 (1:1,000, ab9679; both from Abcam). After staining, tissues were counterstained with hematoxylin (H3401; Vector Laboratories). The average numbers of positively stained cells were obtained from the careful counting of 3 different randomly selected fields under a microscope (Axiostar Plus; Zeiss).

IL-6 levels in the supernatants of THP-1 cells, PMA-treated and PMA/IL-4-treated cells, was determined using the human IL-6 ELISA kit according to the manufacturer's instructions (550799; BD Biosciences, Franklin Lakes, NJ, USA). The secreted IL-6 level (24 h of incubation) was normalized to the cell number.

Total RNA (1 µg) from the cells was subjected to reverse transcription using Superscript III transcriptase (Invitrogen). qPCR was conducted using the appropriate primers and a Bio-Rad CFX96 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with SYBR-Green to determine the mRNA expression levels of the genes of interest. Primers used were as follows: human IL-6 forward, 5′-AAGCCAGAGCTG TGCAGATGAGTA-3′ and reverse, 5′-TGTCCTGCAGCCACTGGTTC-3′; human CCL2 forward, 5′-CAGCCAGATGCAATCAATGC-3′ and reverse, 5′-GCACTGAGATCTTCCTATTGGTGAA-3′; human CCL5 forward, 5′-GCTGTCATCCTCATTGCTACTG-3′ and reverse, 5′-TGGTGTAGAAATACTCCTTGATGTG-3′; human PD-L1 forward, 5′-TATGGTGGTGCCGACTACAA-3′ and reverse, 5′-TGGCTCCCAGAATTACC AAG-3′; human ULB1 forward, 5′-GCGTTCCTTCTGTGC CTC-3′ and reverse, 5′-GGCCTTGAACTTCACACCAC-3′; human ULBP2 forward, 5′-CCCTGGGGAAGAAACTAAATGTC-3′ and reverse, 5′-ACTGAACTGCCAAGATCCACTGC-3′; human ULBP3 forward, 5′-AGATGCCTGGGGAAAACAACTG-3′ and reverse, 5′-GTATCCATCGGCTTCACACTCAC-3′; human MICA forward, 5′-ATCTTCCCTTTTGCACCTCC-3′ and reverse, 5′-AACCCTGACTGCACAGATCC-3′; human MICB forward, 5′-CTGCTGTTTCTGGCC GTC-3′ and reverse, 5′-ACAGATCCATCCTGGGACAG-3′; human GAPDH forward, 5′-CTCCTCCACCTTTGACGCTG-3′ and reverse, 5′-TCCTCTTGTGCTCTTGCTGG-3′. RT-PCR was performed with the following reaction conditions: 95°C 3 min followed by 39 cycles of, 95°C, 10 sec, 55°C, 30 sec, 72°C for 30 sec. Quantification was analyzed by the 2^−ΔΔCq method by normalized by the GAPDH level based on the study by Livak and Schmittgen (24). For inhibitor experiments, Janus kinase (JAK) inhibitor 1 (5 µM; CAS457081-03-7; Calbiochem, San Diego, CA, USA), AG490 (50 µM, T3434; Sigma), Stattic (10 µM; CAS19983-44-9; Calbiochem), LY294002 (5 µM; 440202; Sigma), U0126 (10 µM; 9903; Cell Signaling Technology), SB203580 (10 µM; 559387), Bay11-7082 (5 µM; B5556), rapamycin (100 nM; R8781) (all from Sigma) that inhibit the JAK, JAK/signal transducer and activator of transcription 3 (Stat3), Stat3, PI3K/Akt, MEK/extracellular-signal-regulated kinase (Erk), mitogen-activated protein kinase (MAPK), nuclear factor (NF)-κB and mammalian target of rapamycin (mTOR) pathways, respectively, were added to the radioresistant lung tumor cells.

The cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml aprotinin and 0.2 mM PMSF). After protein determination by BCA method, proteins (20–40 µg) were separated on a 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Following the blocking procedure (blocking using 5% milk in PBS, 30 min at room temperature), membranes were incubated with primary Abs (1:1,000) and HRP-conjugated secondary Abs (goat anti-rabbit-HRP, 170-6515; goat anti-mouse-HRP, 170-6516) (1:5,000), and visualized in Imager (both from Bio-Rad Laboratories, Inc.) using the enhanced chemiluminescent (ECL) solution (34095; Thermo Fisher Scientific). The Abs used were PD-L1 (MAB1086; R&D Systems), p-Erk (4695; Cell Signaling Technology), Erk (ab54230), IL-6 (ab6672) (both from Abcam) and GAPDH (2118S; Cell Signaling Technology). For quantification of the bands, the software of the Chemi Doc™XRS⁺ imaging system (Bio-Rad Laboratories, Inc.) was used.

The data are presented as the means ± SEM. Differences in mean values between 2 groups were analyzed by a two-tailed Student's t-test. In comparing multiple groups, we used ANOVA with a post hoc (Tukey-Kramer) test. A value of P≤0.05 was considered to indicate a statistically significant difference.

We developed radioresistant A549 (A549R26) and H157 (H157R24) cells by treating parental A549 (A549P) and H157 (H157P) cells with fractionated radiation treatment (total 26 and 24 Gy treatment for A549 cells and H157 cells, respectively). From plating these cells at a low density, several colonies were selected and expanded and the subline cells that exhibited the most significant radioresistance (A549R26-1 and H157R24-1) were selected and used in further experiments. The in vitro and in vivo results confirming the radioresistance of these cells were shown our previous study (10).

THP-1 cells are a human monocyte cell line often used for macrophage differentiation. As M2 macrophages (TAM) are activated by IL-4 produced by CD4⁺ T-cells, PMA/IL-4-treated THP-1 cells are often used as a TAM source (17,25). Following the differentiation induction process of THP-1 cells by PMA, IL-4 was subsequently added. As previously demonstrated by Xu et al (25), we found that the PMA/IL-4-treated THP-1 cells became larger and adherent, and were positively stained with the M2 type macrophage marker, CD206 (26,27) (Fig. 1A). These PMA/IL-4-treated THP-1 cells were used as the TAM source in the entire experiment. We first examined the migration of these PMA/IL-4-treated THP-1 cells to A549P vs. A549R26-1 and H157P vs. H157R24-1 cells. In migration assays, we found that the A549R26-1 and H157R24-1 cells recruited more THP-1 cells than the parental cells (Fig. 1B).

In our previous study, we developed orthotopic xenografts in nude mice by injecting A549R26-1 and A549P cells and confirmed the radioresistance of tumors derived from A549R26-1 cells compared to those derived from A549P cells [radioresistance was evaluated by tumor regression difference upon radiation treatment, data shown in our previous study (10)]. We examined macrophage infiltration to the tumor tissues of A549R26-1-xenografts and A549P-xenografts by IHC staining. We detected higher numbers of mouse macrophage marker F4/80-positively stained cells in the tumors derived from A549R26-1 cells than in tumors derived from A549P cells (Fig. 1C), suggesting that higher numbers of macrophages infiltrated into radioresistant tumors than radiosensitive tumors.

In a previously published in vitro and in vivo study, we demonstrated that IL-6 signaling was elevated in parental lung cancer cells following radiation treatment compared to non-irradiated cells and such elevated IL-6 signaling was important in attracting macrophages to lung tumors (15). Consistent with this result, in this study, we detected higher IL-6 levels in the A549R26-1 and H157R24-1 cells than in the respective parental cells (Fig. 2A, left panel, mRNA level; right panel, protein level), and in tumor tissues derived from A549R26-1 cells than in those derived from A549P cells (Fig. 2B).

In a previous study, we revealed CCL2 and CCL5 molecules as the most critical IL-6 downstream chemokines that are important in macrophage infiltration to tumors (15). Thus, in this study, we examined whether CCL2 and CCL5 expression was also higher in radioresistant cells than in parental cells. We observed higher levels of CCL2 and CCL5 in radio-resistant cells (Fig. 2C) than in parental cells, and consistently detected higher numbers of CCL2/CCL5-positively stained cells in the radioresistant tumors than in the radiation-sensitive tumors (Fig. 2D). To confirm the IL-6 regulation of CCL2/CCL5 molecules, we added a neutralizing Ab to IL-6 and examined whether CCL2/CCL5 levels decreased. We observed a decreased CCL2/CCL5 expression in the radioresistant cells when IL-6 Ab was added to the culture (Fig. 2E), confirming the IL-6 regulation of CCL2/CCL5 molecules in radioresistant cells. We then examined whether macrophage infiltration to radioresistant cells decreases when neutralizing Abs to IL-6, CCL2 and/or CCL5 were added to the culture. We observed that the migration of the THP-1 cells to radioresistant cells was inhibited when Abs to IL-6, CCL2 and/or CCL5 were added to the lower chamber of Transwell plates containing tumor cells; however, the THP-1 cell migration to parental cells was not affected (Fig. 2F), confirming the IL-6-CCL2/CCL5 signaling role in recruiting the macrophages into radioresistant cells.

We then examined whether the susceptibility of radioresistant lung cancer cells to NK cell cytotoxicity could be further decreased upon treatment with THP-1 CM. We obtained THP-1 CM from the PMA/IL-4-treated THP-1 culture 48 h after the addition of fresh media. To monitor NK cell cytotoxicity to tumor cells, the LDH release-based NK cytotoxicity test (10,14,18–22) was applied. For the NK cell source, primary NK cells that were isolated from the peripheral blood mononuclear cells (PBMCs) of healthy donors via the magnetic beads isolation method were used in all the experiments. Following the isolation of primary NK cells, the purity (>90%) of CD56⁺CD3⁻ NK cells was confirmed by flow cytometric analyses as shown in a previous study (28).

In NK cell cytotoxicity tests, consistent with our previous study (10), we observed decreased susceptibilities of radioresistant lung cancer cells to NK cell cytotoxicity compared with the parental cells (Fig. 3A). Furthermore, we observed that radioresistant cells exhibited a significantly higher resistance to NK cell cytotoxicity following treatment with THP-1 CM than the control medium-treated cells (Fig. 3A, left panel, the data with A549 cell set; right panel, with H157 cell set), suggesting that the infiltration of macrophages to radioresistant lung cancer cells may promote the development of the additional resistance radioresistance of lung cancer cells to NK cell cytotoxicity.

The importance of the PD-L1/PD-1 immune checkpoint in regulating the NK cell-mediated immune function to cancer cells has been demonstrated in previous studies (29), as well as in our previous studies (10,14,22). Thus, in this study, we examined whether the treatment of radioresistant lung cancer cells with THP-1 CM alters the PD-L1 level in tumor cells. As shown in Fig. 3B, THP-1 CM treatment triggered the upregulation of PD-L1 in radioresistant lung cancer cells, suggesting that this modulation may be associated with the THP-1CM effect we observed. When PD-L1 Ab was added to the tumor cell/NK cell co-culture, a decreased susceptibility of THP-1 CM-treated radioresistant lung cancer cells to NK cell cytotoxicity was observed (Fig. 3C), suggesting that the upregulation of PD-L1 is important in developing the additional resistance of THP-1 CM-treated radioresistant cells to NK cell cytotoxicity.

The NKG2D ligand/NKG2D interaction has been reported as important in activating the NK cell-mediated immune function (13,31,32), and we also confirmed the importance of this interaction in modulating the NK cell action in our previous studies (10,14,22). We found that THP-1 CM treatment also triggered the downregulation of NKG2D ligands (ULBP1, ULBP2, ULBP3, MICA and MICB) in radioresistant lung cancer cells (Fig. 3D), suggesting that this modulation may also be associated with the THP-1 CM effect.

The immunosuppressive effects exerted by THP-1CM treatment may be due to the released cytokines by macrophages, such as IL-6 and IL-10 (33,34). We also detected high secreted IL-6 levels in the PMA/IL-4-treated THP-1 cells (Fig. 4A) (we observed a higher IL-6 secretion in PMA/IL-4-treated THP-1 cells than in PMA alone-treated cells). We then examined whether the presence of IL-6 in THP-1 CM plays a critical role in exerting the THP-1CM effect in alteration of the PD-L1/NKG2D ligands in THP-1 CM-treated radioresistant lung cancer cells. IL-6 Ab was added to THP-1 CM when treating the radioresistant cells, and the PD-L1/NKG2D ligand levels in those cells were analyzed. We found that the THP-1 CM effects in altering the PD-L1/NKG2D ligand levels in radioresistant lung cancer cells were abolished upon the addition of IL-6 Ab; thus, decreased PD-L1 (Fig. 4B) and recovered NKG2D ligand levels (Fig. 4C) were observed. We then examined the effect of IL-6 Ab on the susceptibility of THP-1 CM-treated radioresistant cells to NK cell cytotoxicity. As shown in Fig. 4D, we observed an enhanced susceptibility of THP-1 CM-treated radioresistant lung cancer cells to NK cell cytotoxicity following the addition of IL-6 Ab into the co-culture. These data suggest an important role of IL-6 in triggering the THP-1 CM effect.

To confirm the role of IL-6 in influencing the susceptibility of radioresistant cells to NK cell cytotoxicity, hrIL-6 was added to the mixture of tumor cells/NK cells. As shown in Fig. 4E, we observed an increased resistance of the radioresistant lung cancer cells to NK cell cytotoxicity, confirming the role of IL-6 in promoting the additional resistance of radioresistant lung cancer cells to NK cell cytotoxicity.

To reveal the signaling pathway that is important in triggering the THP-1 CM effect (in altering the PD-L1/NKG2D ligand levels), we added inhibitors of candidate signaling pathways to the THP-1 CM-treated radioresistant cells and examined which signaling pathway can abolish the THP-1 CM effect. For the candidate signaling pathways, we selected the pathways that have been previously reported to be involved in PD-L1 regulation in cancer cells, including JAK1/2 (35,36), Stat3 (37,38), Akt-mTOR (39), NF-κB (40), MAPK/Erk kinase (MEK), Erk (41,42), PI3K/Akt (39,43) and MAPK (44). We first analyzed the PD-L1 level in the inhibitor-treated cells and observed the most significant reduction in PD-L1 level when the MEK/Erk inhibitor was added into THP-1 CM (Fig. 5A). These results were consistent with those of our previous study, demonstrating MEK/Erk signaling as the critical signaling pathway for mediating the PD-L1/NKG2D ligand level alterations in radioresistant lung cancer cells (10). The reduced PD-L1 protein level in THP-1 CM-treated radioresistant lung cancer cells upon treatment with the MEK/Erk inhibitor is shown in Fig. 5B.

We investigated whether the MEK/Erk signaling inhibition also affects the NKG2D ligand levels in THP-1 CM-treated radioresistant lung cancer cells. We found that inhibitor treatment recovered the decreased 5 NKG2D ligand levels (ULBP1, ULBP2, ULBP3, MICA and MICB) in the THP-1 CM-treated radioresistant lung cancer cells (Fig. 5C), indicating that the inhibition of the MEK/Erk signaling was effective not only in lowering the PD-L1 level, but also in recovering the NKG2D ligands levels in THP-1 CM-treated radioresistant cells.

To investigate whether the activation of MEK/Erk signaling in THP-1 CM-treated radioresistant cells was due to IL-6 signaling, we added IL-6 Ab and examined whether the activation of this signaling can be suppressed. As shown in Fig. 5D, we detected reduced p-Erk levels when IL-6 Ab was added to the THP-1 CM when treating radioresistant lung cancer cells.

We then examined whether the MEK/Erk signaling inhibition enhances the susceptibility of the THP-1 CM-treated radioresistant lung cancer cells to NK cell cytotoxicity. As shown in Fig. 6A (left panel, A549R26-1 data; right panel, H157R24-1 cells data), the addition of MEK/Erk signaling inhibitor enhanced the susceptibility of THP-1 CM-treated radioresistant lung cancer cells to NK cell cytotoxicity and the effect was similar to the effect shown by PD-L1 Ab (Fig. 6B), suggesting that the strategy of altering the PD-L1/NKG2D levels by these inhibitors may be an alternative way of recovering the THP-1 CM-induced resistance of radioresistant lung cancer cells to NK cell cytotoxicity. The schematic diagram shown in Fig. 6C summarizes our results.