Upfront dose-reduced chemotherapy synergizes with immunotherapy to optimize chemoimmunotherapy in squamous cell lung carcinoma

Abstract

Background: The survival benefits of combining chemotherapy (at the maximum tolerated dose, MTD) with concurrent immunotherapy, collectively referred to as chemoimmunotherapy, for the treatment of squamous cell lung carcinoma (SQCLC) have been confirmed in recent clinical trials. Nevertheless, optimization of chemoimmunotherapy in order to enhance the efficacy of immune checkpoint inhibitors (ICIs) in SQCLC remains to be explored.

Methods: Cell lines, syngeneic immunocompetent mouse models, and patients' peripheral blood mononuclear cells were used in order to comprehensively explore how to enhance ectopic lymphoid-like structures (ELSs) and upregulate the therapeutic targets of anti-programmed death 1 (PD-1)/anti-PD-1 ligand (PD-L1) monoclonal antibodies (mAbs), thus rendering SQCLC more sensitive to ICIs. In addition, molecular mechanisms underlying optimization were characterized.

Results: Low-dose chemotherapy contributed to an enhanced antigen exposure via the phosphatidylinositol 3-kinase/Akt/transcription factor nuclear factor kappa B signaling pathway. Improved antigen uptake and presentation by activated dendritic cells (DCs) was observed, thus invoking specific T cell responses leading to systemic immune responses and immunological memory. In turn, enhanced antitumor ELSs and PD-1/PD-L1 expression was observed in vivo. Moreover, upfront metronomic (low-dose and frequent administration) chemotherapy extended the time window of the immunostimulatory effect and effectively synergized with anti-PD-1/PD-L1 mAbs. A possible mechanism underlying this synergy is the increase of activated type I macrophages, DCs, and cytotoxic CD8+ T cells, as well as the maintenance of intestinal gut microbiota diversity and composition. In contrast, when combining routine MTD chemotherapy with ICIs, the effects appeared to be additive rather than synergistic.

Conclusions: We first attempted to optimize chemoimmunotherapy for SQCLC by investigating different combinatorial modes. Compared with the MTD chemotherapy used in current clinical practice, upfront metronomic chemotherapy performed better with subsequent anti-PD-1/PD-L1 mAb treatment. This combination approach is worth investigating in other types of tumors, followed by translation into the clinic in the future.

Keywords: combination; drug therapy; immunotherapy; lung neoplasms; lymphocytes; translational medical research; tumor-infiltrating.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Low-dose chemotherapy may induce ICD via the PI3K/Akt/NF-κB signaling pathway in SQCLC cell lines. (A and B) RNA sequencing analysis to evaluate immune-related gene expression following low-dose chemotherapeutic treatment (CDDP 4 µM, GEM 20 µM, PTX 1 µM, DTX 1 µM) for 24 hours. (C–F) Percentage of SQCLC cells expressing cell surface PD-L1, as revealed by FACS, after incubation with the indicated chemotherapy agents or controls (negative and positive) for 24 hours, with or without pretreatment with 1–3 µM BAY 11–7082 (BAY), an NF-κB inhibitor, for 30 min–3 hours. (G–I) Percentage of (G) H226, (H) H520, and (I) SK-MES cells expressing cytoplasmic HMGB-1 by FACS, after incubation with the indicated chemotherapy agents or negative control for 24 hours, with or without concurrent treatment with 0.2 µM BAY. (J–L) Percentage of (J) KLN-205, (K) H226, and (L) SK-MES cells expressing cell surface PD-L1 by FACS, after incubation with the indicated chemotherapy agents or controls for 24 hours, with or without concurrent treatment with 10 µM LY294002, a PI3K inhibitor, or 10 µM MK-2206, an Akt inhibitor. (M) Percentage of KLN-205 cells expressing cell surface MHC-class I. (N) Percentage of H226 cells expressing cell surface HLA-A/B/C, after incubation with the indicated chemotherapy agents or controls for 24 hours, with or without concurrent treatment with 10 µM LY294002 or 10 µM MK-2206. Data are presented as the mean±SD; *p

Figure 2

Low-dose chemotherapy induces HMGB-1 release by SQCLC cells, thereby promoting DC maturation (A–D) and inducing systemic immune responses as well as immunological memory in vivo (E, F). KLN 205-Luc cells were incubated with 4 µM CDDP, 20 µM GEM, 1 µM DTX, or 1 µM PTX for 30 hours, after which the conditioned media were dialyzed at 4°C for 24 hours using a 10,000-MWCO dialysis tubing to remove any residual chemotherapeutic drug. Immature murine bone marrow-derived dendritic cells (BMDCs) cultured in 6-well plates (1×106 cells/mL) were then cultured in unconditioned media (negative control), media containing LPS (10 µg/mL, positive control), or different conditioned media, and were harvested 24 hours later. The maturation state of BMDCs was evaluated by measuring the cell surface co-expression of CD11c+ CD11b+ CD80+, CD11c+ CD11b+ CD86+, and CD11c+ CD11b+ MHC class II+ by FACS. (A) percentage of BMDCs expressing the above-described combinations of three different cell maturation biomarkers after the different stimuli, as revealed by FACS. (B) Proportion of total mature BMDCs in each group after the different stimuli. (C) HMGB-1 levels in the media after treatment with different chemotherapeutic agents for 30 hours, as assessed by ELISA. (D) Immature BMDCs were stimulated with 50 ng/mL or 100 ng/mL recombinant HMGB-1 for 24 hours, and FACS analysis of CD11c+ CD11b+ CD80+, CD11c+ CD11b+ CD86+, CD11c+ CD11b+ MHC class II+ expression was carried out. (E, F) Murine KLN-205-Luc cells were pretreated with 1 µM CDDP, 15 µM CDDP for 72 hours or with PBS as the negative control. These pretreated cells (1×106) were subcutaneously injected into the right flanks of mice. Splenocytes were harvested after 7 days, and IFN-γ secretion was evaluated using ELISPOT, according to the kit manufacturer’s protocol. (G) The aforementioned mice were rechallenged with 2×105 live KLN-205 cells in the opposite flank, and tumor growth was monitored using vernier calipers and BLI. Data are presented as mean±SD. No significance, *p<0.05; **p<0.01; ***p<0.001. BLI, bioluminescence imaging; CDDP, cisplatin; DCs, dendritic cells; DTX, docetaxel; ELISPOT, enzyme-linked immunospot; FACS, fluorescence-activated cell sorting; GEM, gemcitabine; HMGB-1, high mobility group box-1; IFN-γ, interferon-gamma; LPS, lipopolysaccharide; Luc, luciferase; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; PTX, paclitaxel.

Figure 3

High-dose SQCLC monochemotherapy induce greater immunosuppression compared with low-dose regimens in vivo. (A) Chemotherapy treatment scheme. Syngeneic mouse models were established as mentioned above. When tumors became palpable, mice were randomly divided into nine groups receiving an intraperitoneal injection of low-dose CDDP 2.8 mg/kg, high-dose CDDP 8.4 mg/kg, low-dose GEM 60 mg/kg, high-dose GEM 240 mg/kg, low-dose PTX 11 mg/kg, high-dose PTX 33 mg/kg, low-dose DTX 11 mg/kg, high-dose DTX 33 mg/kg, or vehicle. (B) After 30 hours, tumors were harvested for FACS analysis. (C, D) Patients with reatment-naïve SQCLC received either MTD albumin-PTX (at day 0)+nivolumab (routine administration) or low-dose albumin-PTX (1/2 MTD at day 0 and day 7)+nivolumab (routine administration). Before treatment (day 0), baseline PBMCs were evaluated by FACS. At day 7 (before second low-dose albumin-PTX), PBMCs were again assessed by FACS. Cell fractions (cytotoxic T cells and Treg cells) before and after treatment were then compared. Data are presented as mean±SD. N.S., no significance, *p

Figure 4

Low-dose SQCLC monochemotherapy elicits an active TIME in vivo. (A) Chemotherapy treatment scheme. The syngeneic mouse models were established as previously described. When the tumors were palpable, mice were randomly divided into five groups and then received an intraperitoneal injection of CDDP 2.8 mg/kg, GEM 80 mg/kg, DTX 11 mg/kg, PTX 11 mg/kg, or vehicle. (B) After 24 hours, the tumor and spleen tissues were harvested for FACS analysis. Data are presented as mean±SD. N.S., no significance, *p

Figure 5

Combined with anti-PD-1 mAb, sequential low-dose CDDP exerts greater synergistic antitumor effects than MTD CDDP. (A) Treatment scheme. Upfront low-dose CDDP (2.8 mg/kg, Q7D), MTD CDDP (8.4 mg/kg, Q14D), low-dose or MTD CDDP combined with an anti-PD-1 mAb, anti-PD-1 mAb alone, and vehicle control were injected intraperitoneally (I.P.). (B) Tumor growth curves during treatment. (C, D) Tumor weight and specimens after treatment. (E) Body weight changes during treatment. (F) Tumor growth monitored by BLI. (G–L) Single-cell tumor suspensions were used for FACS to evaluate intratumoral CD45+ CD3+ cells, CD45+ CD3+ CD8+ cells, CD45+ CD3+ CD8+ PD-1+ cells, CD11c+ CD83+ cells, and CD11c+ CD86+ cells. (M–Q) ALT and AST in serum, RBCs, HGB, and PLT in whole blood after treatment. Data are presented as mean±SD. N.S., no significance, *p<0.05; **p<0.01; ***p<0.001. ALT, alanine transaminase; AST, aspartate aminotransferase; BLI, bioluminescence imaging; CDDP, cisplatin; FACS, fluorescence-activated cell sorting; HGB, hemoglobin; mAb, monoclonal antibody; MTD, maximum tolerated dose; PD-1, programmed death 1; PLT, platelet; RBC, red blood cell.

Figure 6

Combined with anti-PD-1 mAb, upfront metronomic DTX exerts greater tumor inhibitory effects than routine MTD DTX. (A) Treatment scheme. Upfront low-dose metronomic DTX (10 mg/kg, Q4D), routine MTD DTX (33 mg/kg, Q7D), equivalent to the standard dose administered to patients, low-dose or MTD DTX combined with an anti-PD-1 mAb, anti-PD-1 mAb alone, or vehicle control were injected intraperitoneally (I.P.) in mice. (B) Body weight changes during treatment. (C) Tumor growth curves during treatment. (D, E) Tumor specimens and weight after treatment. (F) Tumor growth monitored by BLI. (G–L) After treatment, single-cell tumor suspensions were used for FACS. (G) CD11b+ F4/80+ cells (TAMs). (H) CD11b+ F4/80+ MHC-II+cells (type I TAMs). (I) CD11b+ F4/80+ CD206+ cells (type II TAMs). (J) CD11c+ CD83+ cells (activated DCs). (K) CD11c+ CD86+ cells (activated DCs). (L) CD11c+ MHC-II+ cells (activated DCs). (M, N) Mice harboring established OVA-expressing KLN-205 tumors were treated with low-dose+anti-PD-1 mAb, high-dose DTX+anti-PD-1 mAb, or vehicle control by I.P. injection. After 3-week treatment, homolateral tumor-draining lymph nodes were prepared into single-cell suspensions, and subjected to ELISPOT assays. Data are presented as mean±SD. N.S., no significance, *p<0.05; **p<0.01; ***p<0.001. BLI, bioluminescence imaging; DCs, dendritic cells; DTX, docetaxel; ELISPOT, enzyme-linked immunospot; FACS, fluorescence-activated cell sorting; Luc, luciferase; mAb, monoclonal antibody; MHC, major histocompatibility complex; MTD, maximum tolerated dose; PD-1, programmed death 1; TAMs, type I tumor-associated macrophages.

Figure 7

Macrophages, CD8+ T cells, and gut microbiota may contribute to the synergistic antitumor effects of DTX plus anti-PD-1 mAb. (A) Schematic of dosing schedule. Macrophage depletion (clodronate, neutral clodronate liposomes, 100 µL per mouse, Q3D, intraperitonially (I.P.)) and CD8+ T cell depletion (in vivo anti-CD8α mAb) treatments were conducted 5 days prior to initiation of the experiment. The treatment groups included DTX (10 mg/kg, Q3D, I.P.) combined with anti-PD-1 mAb (10 mg/kg, Q3D, I.P.), DTX (10 mg/kg, Q3D, I.P.) combined with anti-PD-L1 mAb (10 mg/kg, Q3D, I.P.), or negative control (vehicle, Q3D, I.P.). (B) Tumor growth monitored by BLI. (C) Tumors dissected from the SQCLC models after treatment. (D) Tumor growth curves during treatment, monitored using vernier calipers. (E) Tumor weight after treatment. (F) Intestinal gut microbiota diversity after treatment. (G) Microbial composition after treatment. Data are presented as mean±SD. N.S., no significance, *p<0.05; **p<0.01; ***p<0.001. BLI, bioluminescence imaging; DTX, docetaxel; Luc, luciferase; mAb, monoclonal antibody; PD-1, programmed death 1; PD-L1, programmed death-ligand 1; SQCLC, squamous cell lung carcinoma.