Gemcitabine and celecoxib synergistically promote antitumor efficacy of αPD-1 by triggering immunogenic cell death

doi:10.21203/rs.3.rs-2723644/v1

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Gemcitabine and celecoxib synergistically promote antitumor efficacy of αPD-1 by triggering immunogenic cell death

https://doi.org/10.21203/rs.3.rs-2723644/v1

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There is emerging evidence that immunogenic chemotherapy is not only cytotoxic toward tumor cells but also ameliorates the immunosuppressive tumor microenvironment by inducing immunogenic cell death (ICD) to achieve long-lasting antitumor efficacy. However, comprehensive analysis of ICD inducers is lacking in lung cancer. We investigated the ability of five chemotherapeutic agents to trigger ICD. And further study of how gemcitabine (GEM) activates antitumor immunity and synergistic enhancement of antitumor immunochemotherapy. Herein, we observed that GEM induced characteristics of ICD. Moreover, we demonstrated that celecoxib could enhance ICD by attenuation of indoleamine 2,3-dioxygenase 1 (IDO-1) expression and augmentation of ROS-based endoplasmic reticulum stress. In the present study, we found the combination of GEM, celecoxib and anti-PD-1 monoclonal antibody (aPD-1) exhibited potent antitumor activity and long-term antitumor efficacy in immunocompetent mice by synergistic anti-tumor activity and recruitment of tumor infiltrating lymphocytes. These results support a combination of GEM, celecoxib and aPD-1 as a potential treatment regimen for patients with lung cancer.

immunosuppressive tumor microenvironment

immunogenic cell death

immunochemotherapy

lung cancer

Immunogenic cell death (ICD) is characterized as a particular form of cell death that is triggered by certain chemotherapeutic agents, radiotherapy and photodynamic therapy(1). A feature of ICD is the presence of damage-associated molecular patterns (DAMPs), including calreticulin (CRT), high mobility group box-1 (HMGB1), adenosine triphosphate (ATP), heat-shock proteins (HSP70 and HSP90) and type-1 interferons (IFNs). Translocation of CRT to the cell surface acts as an effective “eat me” signal, interacting with CD91 receptors on phagocytes in the immunogenic tumor medium and efficiently activating phagocytosis of the dying cells(2). HMGB1 is a powerful cytokine that can attract various immune cells and induce maturation of dendritic cells (DCs)(3). ATP, an effective “find me” signal that can also function as an adjuvant of the antitumor immune response, is sensed by purinergic P2RX7 receptors on DCs(4). Recently, Legrand and colleagues proposed that ICD should be defined as regulated cell death that results from successful dialog between the dying cells and an appropriately disposed immune system(5).

Immunogenetic chemotherapy (IC) is a special type of chemotherapy that utilizes drugs that can cause ICD(6). One such drug is doxorubicin (DOX), which is a known inducer of ICD that enables transformation of immune “cold” tumors to “hot” tumors(7). In addition, some non-ICD inducers are able to induce ICD with the assistance of other agents. For example, the acknowledged non-ICD inducer cisplatin can induce ICD when co-administered with digoxin(8). Mechanistically, the tumor microenvironment, including that of lung and breast cancer, is characterized by high expression of cyclooxygenase-2 (COX-2) and its product, prostaglandin E2 (PGE2)(9, 10). Furthermore, PGE2 can dampen the function of T cells and Th lymphocytes by inducing production of immunosuppressive cytokines and molecules such as IDO1(11, 12). The overexpression of indoleamine 2,3-dioxygenase 1 (IDO1) can contribute to this detrimental effect by metabolizing the essential amino acid tryptophan into tyrosine, which is an effective T cell metabolic inhibitor that inhibits T cell proliferation(13).As expected, the COX-2 inhibitor celecoxib enhanced the efficacy of a breast cancer vaccine(14). Accumulating data has inspired us to investigate the treatment of lung cancer using a combination of gemcitabine (GEM) and celecoxib.

In the present study, we hypothesized that combination of IC agents and aPD-1 might achieve more efficacious antitumor immunotherapy.Firstly, we have demonstrated that GEM is an inducer of ICD. We subsequently examined the synergistic anti-tumor efficacy of combined therapy with GEM and celecoxib in vitro and in vivo. Furthermore, we tested the effects of the combination therapy on treatment with anti-PD-1 monoclonal antibody. Our findings suggested a favorable anti-tumor effect of the combination of GEM and celecoxib with aPD-1. Mechanistically, we demonstrated that celecoxib inhibited IDO1 expression by suppressing the PI3K/AKT signaling pathway, thus enhancing the induction of ICD by GEM in vitro. In sum, our cumulative results should facilitate the development of IC strategies that will be superior alternatives for cancer therapy.

Cell lines and cell culture

The murine lung cancer cell lines (LLC and CMT-64) derived from C57 bl/6j were purchased from Jennio Biotech Co., Ltd. (Guangzhou, Guangdong, China). LLC cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/mL). CMT-64 cells were cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin (100 U/mL).

Western blot (WB)

Pretreated CMT-64 and LLC cells were washed with phosphate-buffered saline (PBS) and lysed in RIPA buffer containing 1% proteinase and phosphatase inhibitor. Total protein was extracted from the lysates and quantified using a BCA kit (Beyotime, Shanghai, China). A 50 µg protein sample was used for the immunoblotting assay. After incubation with primary antibodies (GAPDH: Proteintech 60004-1-Ig; COX-2: CST 12282; IDO1: CST 86630; AKT: CST 4685; phospho-AKT: CST 4060; PI3K: CST 4257; phospho-PI3K: CST 4228; Bip: CST 3177; CHOP: CST 2895; Bcl-2: BioworldBS70205; CRT: CST 12238; HSP90: Proteintech 60318-1-Ig) the immunoreaction was visualized using an ECL kit.

Apoptosis assay in vitro

Cells (LLC and CMT-64) were cultured in a 6-well plate and treated with drugs [dimethyl sulfoxide (DMSO), GEM: 2 µg/mL, celecoxib: 40 µM, combined GEM and celecoxib (GC)] for 24 h. Cells floating in the supernatants and cells on the culture plate were collected and then washed with PBS. The cells were incubated with the prepared working solution (Annexin-V-FITC and PI) in the dark for 15 min and then analyzed by flow cytometry.

Determination of reactive oxygen species (ROS)

The levels of ROS in cells were determined using a probe (2,7-dichloro-dihydro-fluorescein diacetate, DCFH-DA). According to the manufacturer’s instructions, treated-cells were collected and washed with culture medium. The cells were incubated with prepared working solution in the dark for 20–30 min and then analyzed by flow cytometry.

Extraction of mouse bone marrow-derived dendritic cells

DCs were generated from female C57 bl/6j mice and cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin (100 U/mL), IL-4 (5 ng/mL) and GM-CSF (10 ng/mL).

DC phagocytosis

Briefly, mouse-derived lung cancer cells (LLC and CMT-64) were stained with CytoTraceTM Red Fluorescent Probe (YEASEN, 40717ES50) and DCs were stained with CellTracker Green CMFDA (YEASEN, 40721ES50). The pretreated cells were co-cultured with DCs to investigate phagocytosis during incubation for 24 h. The collected cells were analyzed by flow cytometry.

Flow cytometry

Spleen cells were extracted from treated mice and red blood cells were removed using an erythrocyte lysis buffer (Biosharp, Shanghai, China). The spleen cells were then probed with the following primary antibodies: Pharmingen™ PE hamster anti-mouse CD11c (557401), purchased from BD Biosciences; CD86/B7-2 (GL-1) rat mAb (FITC conjugate), bought from CST (99879); APC anti-mouse I-A/I-E (107613), Brilliant Violet 421™ anti-mouse/human CD11b (101235), PerCP/Cyanine5.5 anti-mouse Ly-6G/Ly-6C (Gr-1) (108427), Brilliant Violet 421™ anti-mouse CD3ε (100341), PE anti-mouse CD8a (100707), PE/Cyanine7 anti-mouse IFN-γ (505825), FITC anti-mouse CD4 (100405), Alexa Fluor® 647 anti-mouse FOXP3 (126407), PE anti-mouse CD62L (104407) and PerCP/Cyanine5.5 anti-mouse/human CD44 (103031), purchased from BioLegend. For staining of intracellular FOXP3 and IFN-γ, Fixation Buffer (420801) and Intracellular Staining Permeabilization Wash Buffer (10×) (421002) were purchased from BioLegend. The specific experimental procedures were the same as those used in a previous study. Data were acquired on a flow cytometer (Beckman CytoFLEX) and analyzed using the FlowJo application.

Immunohistochemistry (IHC)

Tissue specimens were collected and fixed with 10% formalin solution. The specimens were processed using an automatic tissue dehydrator and embedded in paraffin using an embedding machine (Tissue-Tek). The tissue sections (4 µm) were stained with hematoxylin and eosin (H&E) or IHC to further analyze proteins of interest. The specific procedures were as follows: After dewaxing in xylene and hydration with an alcohol gradient (100% for 5 min, 95% for 5 min, 85% for 5 min and 75% for 5 min), endogenous peroxidase activity was blocked by incubation with 2% H₂O₂, and then antigen retrieval was conducted by microwaving in 10 mM citrate buffer for 15 min at 95°C. Sections were then incubated with primary antibodies (COX-2: 1:500, CST 12282, PD-1: 1:200, CST 84651, IDO1: 1:200, Proteintech 13268-1-AP, and CD8: 1:100, Affinity AF5126) overnight at 4°C. Streptavidin-linked secondary antibody (LSAB + Kit, Dako) was applied for 30 min, followed by streptavidin peroxidase for 15 min. After incubation for 5 min in the substrate developer, counterstaining with hematoxylin was conducted for 3 min.

Mouse cytokine ELISA assay

Mouse cytokine (IL-1β, IFN-γ, HMGB1) ELISA kits were used to estimate levels of these cytokines in supernatants of samples from in vivo or in vitro experiments. Initially, the collected supernatants were centrifuged to remove impurities and incubated on the enzyme plate for 90 min. The plate was washed with buffer (5×) and incubated with biotin-labeled antibodies at 37°C for 60 min. Subsequently, horseradish peroxidase enzyme conjugates were added and incubated in the dark for 30 min after washing. Finally, the reaction was terminated and the absorbance at 450 nm was recorded on a microplate reader.

Tumor preventive vaccine experiment

All animal experiments were approved by the Animal Care and Use Committee of Southern Medical University. The welfare of live experimental animals was maintained in strict accordance with the Ministry of Science and Technology (2006) No. 398 “Guidelines for the Treatment of Experimental Animals” and the project code was LAEC-2020-049. 6-7-week-old C57 bl/6j mice were bred in a specific pathogen-free (SPF) animal experiment center. LLC cells were treated with PBS, GEM or DOX, and then the flanks of C57 bl/6j mice were inoculated subcutaneously with 1 × 10⁶ dying cells. The PBS-treated cells were frozen and thawed as the control group and DOX-treated cells acted as the positive group. After 7 days, 5 × 10⁵ LLC cells were injected into the contralateral armpit and monitored every three days. Prevention was considered to have failed if a palpable subcutaneous tumor could be detected.

Mouse ectopic model of lung cancer

To investigate the inhibitory efficacy of a combination of GEM, celecoxib and aPD-1 (GCP) in mice bearing a lung cancer xenograft, and further evaluate whether there was any long-lasting antitumor immunity. 35 6-7-week-old C57 bl/6j mice were divided into five groups (PBS, GEM, aPD-1, GC and GCP). LLC cells (5 × 10⁵) were subcutaneously inoculated into the armpit or the leg. Therapeutic drugs (GEM: 25 mg/kg, aPD-1: 100 µg/mouse) as described above were injected via the tail vein when the tumor volume reached approximately 100–150 mm³. Celecoxib (50 mg/kg) was administered by gavage. Tumor volumes were monitored using an electronic vernier caliper every 2 days. Tumor volume was calculated as follows: volume (mm³) = length × width × width/2.

Cell depletion assay

Cell depletion was conducted to investigate the specific cell type involved in anti-tumor immunity. For removal of CD4 + and CD8 + T cells, anti-CD4 (BE0003, 100 µg/mouse) and anti-CD8 (BE0061, 100 µg/mouse) were injected via the tail vein 1 day before commencement of chemotherapy and then every 2 days thereafter, as shown in Fig. 5A. The DMSO group was used as a control group and tumor growth was monitored every 2 days. Depletion of CD4 + and CD8 + T cells was determined by flow cytometry.

Lung metastasis model of lung cancer

Subcutaneous tumors were induced in 6- 8-week-old C57 bl/6j mice and treated three times with GCP. Frozen and thawed LLC cells were injected subcutaneously into the armpit as the control (PBS) group. Next, parental LLC cells (2 × 10⁵) were injected via the tail vein. The mice were anesthetized and euthanized 30 days post-injection. Subsequently, the lungs were obtained and stained with H&E. Representative results were photographed and recorded using an optical microscope.

Statistical analysis

Data analyses were performed using SPSS 22.0 and data expressed as mean ± SE. Statistical significance was assessed by two-tailed unpaired Student t-test. P values < 0.05 were considered to be statistically significant.

Gemcitabine is an inducer of ICD

To determine whether a chemotherapy drug could induce immunogenic cell death, WB, ELISA, DC phagocytosis and activation assays were performed. As indicated from the WB results (Fig. 1A), GEM and DOX (positive control) enhanced expression of CRT and HSP90. There were no differences in the levels of HMGB1 between the DMSO, DOX and GEM groups from the ELISA data (Fig. 1B), but it shows an increasing trend. Because activation of DCs is a feature of ICD, we tested DC activation and rates of phagocytosis post treatment with chemotherapeutic drugs. As illustrated in Figs. 1C, 1D, S1G and S1H, almost all groups exhibited enhanced phagocytosis and more activated DCs (CD11c + MHC-II+) with the exception of the DTX group. The effects of DTX (negative control), DOX (positive control) and GEM (our ICD inducer) on expression of CRT and HSP90 were also explored. Impressively, GEM enhanced expression of CRT and HSP90 in a time- and concentration-dependent manner within a certain concentration and time range (Figs. 1E, 1F and S1C–F). Interestingly, in LLC and CMT-64 cells pretreated with GEM, we found high expression of CRT, ATP and some chemokines (CCL5, CXCL3 and CXCL10) (Figures S1A and S1B), which are known to have important roles in recruitment of infiltrating T lymphocytes (TILs)[24].

Consequently, we found that three mice in the PBS group developed palpable subcutaneous tumors by day 4, and all eight mice had obvious tumors on day 7infirmed fromin vivo prophylactic vaccine experiments (Fig. 1G). Comparatively, GEM protected most of the mice (6/8) from reinvasion of tumor cells (Fig. 1H) during 28 days. In addition, analysis of immune cells in the spleen found no significant differences in the numbers of CD3 + CD4 + or CD3 + CD8 + T cells among the groups (Figs. 1I and 1J), but there were more CD3 + IFN-+ T cells in the GEM and DOX groups (Fig. 1K). Consistent with our in vitro data, GEM and DOX activated DCs (Fig. 1L). Moreover, we found that the proportion of memory T cells in the GEM and DOX groups was significantly increased compared with the PBS group (Fig. 1M). These results suggested that GEM acted as an inducer of ICD via stimulation of DAMPs and release of chemokines, leading to the attraction and activation of immune cells against tumors.

Celecoxib enhances the cellular immunogenicity induced by gemcitabine.

Although GEM can promote the expression of DAMPs in dying cells, it also increases the expression of PGE2, which is an inhibitory DAMP that can suppress the ICD effect induced by GEM[26]. As shown in Fig. 2A, although there was no significant difference between the celecoxib and DMSO groups, there was a tenuous trend toward decreased levels of PGE2 in the celecoxib group. Meanwhile, a ROS probe (DCFH-DA) was used to track the levels of ROS induced by GEM or celecoxib. Unexpectedly, celecoxib enhanced ROS production stimulated by GEM (Figs. 2B, 2C, S2A and S2B). Additionally, analysis of the apoptosis state by flow cytometry was consistent with the previous notion that celecoxib could increase the antitumor effect of chemotherapeutic agents (Figs. 2D, 2E, S2C and S2D)(15). Both GEM and celecoxib accelerated the apoptosis of cancer cells, but addition of celecoxib to the GEM group increased tumor suppression efficiency.

Next,the major pathways identified by KEGG pathway analysis of GEM-treated cells included cell growth and death, signal transduction, folding, sorting and degradation, and the immune system (Fig. 2F). Subsequently, we used WB to examine the expression of ER stress-related proteins (Bip and CHOP), anti-apoptotic protein (Bcl-2) and other proteins (COX-2, PI3K/AKT signaling pathway) (Figs. 2G and S2E). Treatment of cells (CMT-64 and LLC) with GEM and celecoxib promoted the production of Bip and CHOP. In addition, COX-2 would be inhibited by the addition of celecoxib. Decreased levels of P-PI3K and P-AKT implied that the PI3K/AKT signaling pathway was inactivated by GEM and celecoxib. The Bcl-2 expression was down-regulated after treatment with GEM, celecoxib alone or the combination. Taking the results together, GEM- or celecoxib-treated cells may be subject to apoptosis and ER stress mediated by the PI3K/AKT signaling pathway.

Combination therapy with GEM and celecoxib enhances activation of DCs.

While the combination of GEM and celecoxib is a powerful stimulant of ROS-production and tumor-suppression, we wondered whether addition of celecoxib could increase ICD induced by GEM. Both GEM and celecoxib treatment increased activation of DCs compared to DMSO, and the GC group exhibited greater numbers of mature DCs compared to the other groups (Figs. 3A, 3C and 3E). Similar to the results obtained from the aforementioned assay, the phagocytic activity of DCs was enhanced by treatment with GEM or celecoxib compared to DMSO, and the best activity was observed in the GC group (Figs. 3B, 3D and 3F). In contrast to previous research(16), which found that CRT expression was dependent on ROS-based ER stress, we observed no significant difference between the GEM and GC groups in the expression of CRT despite higher levels of ROS in the GC group compared to the GEM group. Similar results were observed for HSP90 (Fig. 3G). As shown in Fig. 3G, celecoxib counteracted the stimulatory effect of GEM, with lower expression of IDO1 in the GC group compared to the GEM group. Consistent with this viewpoint, the addition of celecoxib dramatically enhanced the level of ROS compared to GEM alone. The effective immunogenicity induced by GC may therefore be partially due to ROS-based ER stress.

Combination therapy induces tumor cell immunogenicity by inhibiting the PI3K/AKT signaling pathway.

Accumulating evidence suggests that certain types of chemotherapeutic agents induce tumor cell death and an inflammatory response, which subsequently activates adaptive antitumor immunity(17). We have found that phospho-PI3K and phospho-AKT protein levels declined after treatment of cells with GEM and celecoxib, accompanied by increased apoptosis and ER stress. Activation of the PI3K/AKT pathway impaired apoptosis and ER stress caused by GEM and celecoxib (Figs. 4A-4D and S3A-S3D). Moreover, activation of DCs was clearly suppressed (Figs. 4F, 4G, S3F and S3G). Consequently, further analysis of the PI3K/AKT pathway, ER stress, DAMPs and IDO1 was conducted. The addition of SC-79,an AKT agonist, had no significant effect on the expression of DAMPs, indicating that there was no relationship between the enhanced ICD induced by celecoxib and the protein levels of DAMPs (Figs. 4H and S3H). The ER stress-related proteinsshowed a downward trend after addition of SC-79, implying that ER stress partially contributed to the enhanced ICD effect (Figs. 4E and S3E). Furthermore, a similar result was obtained in respect of IDO1, suggesting that DCs were effectively activated due to lack of immunosuppression by IDO1 (Figs. 4H and S3H).Overall, we have demonstrated that the enhanced ICD induced by celecoxib was a consequence of increased ER stress and downregulation of IDO1.

CD8 + T cells play a vital role in the anti-tumor effect elicited by GEM and celecoxib combination therapy.

Previous studies found that enhanced activation and antigen presentation by DCs promoted adaptive immunity and that CD8 + T cells were a prerequisite(18). Therefore, we used anti-CD4 and anti-CD8 monoclonal antibodies (aCD4 and aCD8) to determine the specific cell type. As shown in Figs. 5A–5I, concurrent administration of GEM and celecoxib significantly delayed tumor growth. Addition of aCD8 restored growth of the subcutaneous xenograft, but there was no obvious difference between the GC group and the GC + aCD4 group. A similar trend was observed for the combination of DOX and celecoxib. The results demonstrated that application of aCD4/aCD8 effectively decreased the numbers of CD4+/CD8 + T cells (Figs. 5J and 5K), and the number of mature DCs was increased by combinations of GEM and celecoxib or DOX and celecoxib (Figs. 5L and 5M). Overall, these results determined that CD8 + T cells played a crucial role in the antitumor immunity.

Combination therapy with checkpoint inhibitors contributes to anti-tumor immunity.

As a previous study reported, reactivation of T cells contributes to immune checkpoint blockade (ICB) therapy(19). In the aforementioned research results, combination therapy with GC enhanced the number of CD8 + T cells. As observed in Figs. 6A and 6B, aPD-1 alone did not significantly delay tumor growth compared to PBS, while there was obvious suppression in the GEM, GC and GCP groups. The best anti-tumor effect was obtained by treatment with GCP, which indicated that combination therapy with GC and aPD-1 synergistically destroyed the tumor. In addition, GCP combination therapy significantly prolonged survival compared with the PBS group (Fig. 6C). We subsequently used IHC to determine several proteins (Fig. 6D), Compared with the PBS group, there was a trend toward reduced expression of COX-2 and IDO1 in the GCP group, which was consistent with the in vitro data. Impressively, levels of CD8 were significantly increased in the GCP group compared with the PBS group, indicating the viability of ICB therapy.

Subsequently, we found there were no differences in IL-2 levels among the groups, but there was a trend toward reduced IL-10 levels in GEM- and GCP-treated mice, which may indicate reduced numbers of M2 macrophages (Figs. 6E and 6F). And there were no significant differences of CD4 + T cells or CD8 + IFN-γ + cells among the groups. The GCP group showed a decrease trend in the number of CD8 + T cells compared with the PBS group, which is in line with our abovementioned results. Meanwhile, we found that the number of myeloid-derived suppressor cells (MDSCs)was significantly reduced in the GCP group compared to the PBS group (Figs. 6G–6J).

Co-administration of GEM, celecoxib and aPD-1 results in long-lasting antitumor immunity and systemic antitumor immunity on distal metastasis.

Our data suggested that the combination therapy exhibited a synergistic antitumor effect. In our present study, we were pleased to find that although the proportion of tumor recurrence was not significantly reduced in the GCP group, there was a trend toward a decrease (Figures S4A and S4B). This suggested that long-term immunological antitumor memory could be induced by GCP treatment. We then evaluated effects on distal metastasis, as shown in Figures S4C and S4E. The lung was almost full of metastases in the PBS group compared with the GCP group, suggesting that GCP treatment may activate a systemic antitumor immune response.

During recent decades, lung cancer has been the most frequent cause of cancer-related deaths worldwide(20). To further exploit the indications of existing chemotherapeutic agents, it is necessary to study their role in other aspects, including immunity. ICD is a particular mode of cell death observed during tumor immunogenicity, and is different from apoptosis(21). This phenomenon can be stimulated by certain chemotherapeutic agents. In the present study, we investigated the characteristics of ICD induced by GEM, and confirmed the potency of combined GEM, celecoxib and aPD-1 therapy in a mouse model of lung cancer.

As a common chemotherapeutic drug in clinical practice, there is controversy over whether GEM is a qualified ICD inducer. A large number of studies have shown that GEM can act as a qualified ICD-inducer to reject tumor rechallenge in melanoma and breast cancer(22–24). Intriguingly, a recent study deemed GEM to act as a non-ICD inducer, but it is believed that the main reason preventing GEM from becoming a qualified ICD inducer is that GEM induces tumor cells to release PGE2, leading to the polarization of immunostimulatory DAMPs to inhibitory DAMPs. In our present study, GEM was found to induce the production of DAMPs and mice vaccinated with GEM-treated LLC cells were protected from rechallenge with LLC cells.

To the best of our knowledge, IDO1 has an important role in the maturation and function of dendritic cells. It has been reported that PGE2 released from tumor cells can activate β-catenin to induce IDO1 expression by activating the PI3K/AKT signaling pathway(25). PGE2 is an effective inducer of IDO1 expression in antigen presenting cells andcontinuous stimulation of PGE2 can induce IDO1 expression and inhibit anti-tumor immunity(14). In the present study, in order to avoid the inhibitory effect of PGE2 on ICD induced by GEM, celecoxib was added to the treatment regimen. As expected, attenuated expression of COX-2 was observed after the addition of celecoxib, together with decreased expression of IDO1, preventing the weakening of GEM-induced ICD.

A previous study found that more severe ER stress led to higher immunogenic cell death(26, 27). Direct ROS-based ER stress is more effective for induction of ICD than secondary collateral ER stress. In this study, obtained results indicated the augmentation of ROS-based ER stress following addition of celecoxib. Previous research has suggested that simultaneous induction of ER stress could restore and enhance immunogenicity induced by therapeutic agents that are poor ICD inducers, such as etoposide and mitomycin C(2). Consistent with previous reports, we demonstrated potent induction of ICD by combining GEM with celecoxib due to more focused ER stress and higher levels of ROS compared to GEM alone. Furthermore, the decrease of IDO1 expression after incubation with celecoxib was impressive. The findings suggested that GEM-induced ICD could be enhanced by addition of celecoxib through aggravation of ER stress and reduction of IDO1 expression, but not by increasing expression of DAMPs. In agreement with prior studies, which reported that GEM and celecoxib suppressed the PI3K/AKT signaling pathway(28), KEGG pathway analysis indicated that signal transduction was mostly affected in GEM-treated cells. Moreover, we found celecoxib could reduce the production of PGE2 by inhibiting COX-2, further inactivating the PI3K/AKT pathway and eventually leading to decreased IDO1 expression. In subsequent experiments we found that the percentage of apoptosis was decreased, along with reduced DC activation and phagocytosis in the GC group after the addition of SC-79. These results suggested that the combination of GEM and celecoxib promoted cell death by suppressing the PI3K/AKT signaling pathway. Moreover, the combination enhanced ICD by inducing ER stress and inhibiting IDO1 expression.

Prophylactic vaccines are the gold standard for assessment of ICD inducers(29). From our results, GEM-treated LLC cells exhibited potent immunogenicity, inducing strong antitumor immunity that was confirmed by successful protection of immunocompetent mice pretreated with GEM-treated LLC cells from rechallenge with parental LLC cells. Further investigation in Central Memory T (Tcm) cells indicated that GEM-treated cells stimulated immune memory function and prevented tumor cells from reinvasion. In agreement with previous studies(30), our in vivo data further demonstrated that the combination of GEM and celecoxib ameliorated the local immunosuppressive state by increasing the proportions of CD8 + T cells and mature DCs. We wondered whether a synergistic anti-tumor effect could be obtained using a combination of GEM, celecoxib and aPD-1. Consistent with a previous study(31), the addition of aPD-1 clearly contributed to better antitumor efficacy and conferred long-lasting antitumor immunity. Robust antitumor immunotherapeutic efficacy is partially dependent on the enrichment of CD8 + T cells that mediate tumor clearance(1).

In summary, we have identified GEM as a qualified inducer of ICD that is able to ameliorate the immunosuppressive microenvironment in a model of lung cancer. Accumulating data suggest the viability and potential of the combination of GEM, celecoxib and aPD-1 for the treatment of lung cancer. In this regard, the hypothesis requires further investigation to establish clinical efficacy.

DAMPs damage-associated molecular patterns CRT calreticulin

HMGB1 High mobility group box-1 HSP90 heat-shock proteins 90

GEM gemcitabine DCs Dendritic cells

GC Gemcitabine and celecoxib aPD-1 anti-PD-1 monoclonal antibody

TILs Tumor infiltrating lymphocytes DOX Doxorubicin

DTX Docetaxel PEM Pemetrexed

NVB Navelbine

AUTHOR CONTRIBUTIONS

Xiongjie Zhu developed the hypothesis, designed the experiments, performed the experiments and revised the manuscript. Xia Yang, Zhongjian Yu and Ying Li performed the experiments; Rui Cai performed statistical analyses. Min Zhou and Yanfang Zheng supervised the whole study.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

DECLARARTION OF COMPETING INTEREST

There is no conflict of interest between all authors.

ACKNOWLEDGEMENTS

This work is supported by grant from National Natural Science Foundation of China (No. 81974434), grants from Natural Science Foundation of Guangdong Province (No. 2020A0505100038, 2021A1515010793), grant from Scientific Project Foundation of Guangzhou City (No. 201907010037), grant from Affiliated Cancer Hospital & Institute of Guangzhou Medical University (No. 2020-YZ-01), and grant from Clinical Key Specialty Construction Project of Guangzhou Medical University (No. YYPT202017). And we thank International Science Editing ( http://www.internationalscienceediting.com ) for editing this manuscript.

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Scheme 1 is available in Supplementary Files section.

No competing interests reported.

Scheme.png
Scheme 1: Schematic diagram of the specific procedure used to determine antitumor immunity.
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Gemcitabine and celecoxib synergistically promote antitumor efficacy of αPD-1 by triggering immunogenic cell death

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Abstract

Figures

Background

Methods

Cell lines and cell culture

Western blot (WB)

Determination of reactive oxygen species (ROS)

Extraction of mouse bone marrow-derived dendritic cells

DC phagocytosis

Flow cytometry

Immunohistochemistry (IHC)

Mouse cytokine ELISA assay

Tumor preventive vaccine experiment

Mouse ectopic model of lung cancer

Cell depletion assay

Lung metastasis model of lung cancer

Statistical analysis

Results

Gemcitabine is an inducer of ICD

Discussion

Abbreviations

Declarations

References

Scheme 1

Additional Declarations

Supplementary Files

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