Research ArticleCANCER IMMUNOLOGY

Hypofractionated EGFR tyrosine kinase inhibitor limits tumor relapse through triggering innate and adaptive immunity

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Science Immunology  09 Aug 2019:
Vol. 4, Issue 38, eaav6473
DOI: 10.1126/sciimmunol.aav6473
  • Fig. 1 EGFR TKI treatment notably suppressed the tumor growth both in vitro and in vivo.

    (A) The sensitivity of tumor cells to afatinib was measured by the Cell Counting Kit-8 assay. TUBO and NOP23 cells grown in 96-well plates were treated with different doses of afatinib as indicated for 48 hours, and then viability reagent was added to measure the cell viability. (B) TUBO (left) and NOP23 (right) cells were treated for 3 hours with increasing concentrations (0, 0.016, 0.08, 0.4, and 2 μM) of afatinib and then cell lysates from different treatments were used to detect total and phosphorylated protein levels with Western blot assay. (C) F1 Neu-Tg mice (BALB/c × FVB Neu Tg mice) (n = 5 or 6 per group) were transplanted with 5 × 105 TUBO cells and treated with afatinib (25 mg/kg, daily) by gavage for 12 days between days 12 and 23. The stippled portion of the graph is the duration of the HyperTKI treatment. (D) NeuOT-I/OT-II Tg mice (n = 4 per group) were transplanted with 1 × 106 NOP23 cells and treated with afatinib (12.5 mg/kg, daily) by gavage for 14 days between days 16 and 29. (E) BALB/c mice (n = 4 per group) were transplanted with 5 × 105 TUBO cells and treated with afatinib (25 mg/kg, daily) by gavage for 8 days between days 16 and 23. (F) C57BL/6 mice (n = 3 or 4 per group) were transplanted with 3 × 106 NOP23 cells and treated with afatinib (10 mg/kg, daily) by gavage for 10 days between days 19 and 28. Tumor growth was monitored twice a week. Data are representative of two or three experiments. (A and C to F) Data are shown as mean ± SEM. ****P < 0.0001.

  • Fig. 2 HypoTKI treatment could markedly limit tumor relapse.

    (A) F1 Neu-Tg mice (BALB/c × FVB Neu Tg mice) (n = 5 or 7 per group) were transplanted with 5 × 105 TUBO cells and treated with afatinib (100 mg/kg, three times) by gavage at indicated time points (arrowheads). (B) NeuOT-I/OT-II Tg mice (n = 4 per group) were transplanted with 1 × 106 NOP23 cells and treated with afatinib (100 mg/kg, twice). (C) BALB/c mice (n = 4 per group) were transplanted with 5 × 105 TUBO cells and treated with afatinib (100 mg/kg, twice). (D) C57BL/6 mice (n = 5 per group) were transplanted with 3 × 106 NOP23 cells and treated with afatinib (25 mg/kg, twice). (E) TUBO tumor–bearing BALB/c mice (n = 4 or 5/ per group) were treated with gefitinib (400 mg/kg, twice or 80 mg/kg, daily for 8 days) by gavage. (F) TUBO tumor–bearing BALB/c mice (n = 5–8 per group) were treated with osimertinib (50 mg/kg, twice or 5 mg/kg, daily for 20 days) by gavage. The stippled portion of the graph is the duration of the HyperTKI treatment, and arrowheads indicate the HypoTKI treatment. Tumor growth was monitored twice a week. Data are representative of two or three experiments. (A to F) Data are shown as mean ± SEM. **P < 0.01 and ****P < 0.0001.

  • Fig. 3 Host adaptive immunity is essential for HypoTKI to limit tumor relapse.

    (A) NOD-SCID mice (n = 3 or 4 per group) were transplanted with 3 × 105 TUBO cells and treated with afatinib (100 mg/kg, twice) by gavage at indicated time points (arrowheads). (B) BALB/c Rag1−/− mice (n = 4 or 5 per group) were transplanted with 5 × 105 TUBO cells and treated with afatinib (100 mg/kg, twice). (C) BALB/c Rag1−/− mice (n = 5 or 6 per group) were transplanted with 5 × 105 TUBO cells and treated with afatinib (25 mg/kg, daily). The stippled portion of the graph is the duration of the HyperTKI treatment. Tumor growth was monitored twice a week. (D) BALB/c mice (n = 4 or 5 per group) were transplanted with 5 × 105 TUBO cells and treated with afatinib (100 mg/kg, twice); anti-CD4/CD8 or anti-CD20 antibodies were injected four times at 3-day intervals during the treatment. Tumor growth was monitored twice a week. (E and F) F1 Neu-Tg mice (BALB/c × FVB Neu Tg mice, n = 4 or 6 per group) were transplanted with 1 × 106 TUBO-HA cells and treated with HypoTKI (afatinib, 100 mg/kg, once) or HyperTKI (afatinib, 25 mg/kg, daily for 5 days) by gavage. Six days after the first treatment, intratumoral CD3+ T cells (E) and HA tetramer+ CD8+ T cells (F) were detected by flow cytometry. (G) BALB/c mice (n = 4 or 5 per group) were transplanted with 1 × 106 TUBO cells. Tumor-bearing mice were treated with HypoTKI by gavage twice; FTY720 (1 mg/kg) was injected 5 days before the first HypoTKI treatment and then injected (0.5 mg/kg) every other day during the experiment. Tumor growth was monitored twice a week. (H) Naïve and HypoTKI-cured tumor-free BALB/c mice (n = 5 per group) were rechallenged subcutaneously with 2.5 × 106 TUBO cells on the opposite side from the primary tumor 40 days after complete rejection and then tumor growth was monitored. (I) NOP23 tumor–bearing C57BL/6 or Batf3−/− mice (n = 4 or 7 per group) were treated with HypoTKI (afatinib, 25 mg/kg, twice) and then tumor growth was monitored. Data are representative of two or three experiments. (A to I) Data are shown as mean ± SEM. n.s., no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 4 Type I IFN signaling is required for HypoTKI to enhance antitumor-specific T cell responses.

    (A) BALB/c mice (n = 4 per group) were transplanted with 5 × 105 TUBO cells and treated with afatinib (100 mg/kg, once or 25 mg/kg, daily). Three days later, tumor tissues were collected for protein extraction. IFN-β protein level was measured by ELISA. (B) BALB/c mice (n = 4 per group) were transplanted with 1 × 106 TUBO cells and treated with HypoTKI (afatinib, 100 mg/kg) or HyperTKI (afatinib, 25 mg/kg, daily). Three days later, tumor tissues were collected for protein extraction. CXCL10 protein level was measured by Bio-Rad multiplex. (C) C57BL/6 and Ifnαr1−/− mice (n = 4 or 5 per group) were injected subcutaneously with 3 × 106 NOP23 cells and treated twice with afatinib (25 mg/kg, twice) in 6-day intervals (arrowheads). Tumor growth was monitored twice a week. (D) NOP23 tumor–bearing C57BL/6 or Ifnαr1−/− mice (n = 6 or 7 per group) were treated with afatinib (25 mg/kg, twice) by gavage. Ten days after the initial treatment, lymphocytes from the spleens were isolated and stimulated with NOP23 tumor cells irradiated with 40 Gy. IFN-γ–producing cells were detected by ELISpot assay. Data are representative of two or three experiments. (A to D) Data are shown as mean ± SEM. *P < 0.05 and ***P < 0.001.

  • Fig. 5 Myd88 signaling is required for HypoTKI to enhance antitumor-specific T cell responses.

    NOP23 tumor–bearing mice were treated twice with afatinib (25 mg/kg) by gavage in 6-day intervals (arrowheads) and then tumor growth was monitored. (A) Tumor growth in WT and STINGmut/mut mice (n = 5 per group). (B) Tumor growth in Trif−/− mice (n = 3 or 5 per group). (C) Tumor growth in WT and Myd88−/− mice (n = 5 or 7 per group). (D and E) BMDCs from WT or Myd88−/− mice were cocultured with 0.2 μM afatinib or phosphate-buffered saline (PBS)–treated NOP23 tumor cells for 24 hours; supernatants from cell cultures were assayed for IFN-β level by ELISA (D). BMDCs were collected, and the mRNA level of CXCL10 was quantified by real-time polymerase chain reaction assay (E). BMDCs were collected for surface staining. CD11c+ cells were gated for CD86 expression detection. Mean fluorescent intensity analysis is shown (F). (G) NOP23 tumor–bearing C57BL/6 or Myd88−/− mice (n = 4 per group) were treated with afatinib (25 mg/kg, twice) by gavage. Ten days after the initial treatment, lymphocytes from the spleens were isolated and stimulated with NOP23 tumor cells irradiated with 40 Gy. IFN-γ–producing cells were detected by ELISpot assay. Data are representative of two or three experiments. (A to G) Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 6 Concurrent PD-L1 blockade synergizes with HypoTKI to control advanced large tumor and limit tumor relapse.

    (A) EGFR-expressing A431 cells were treated with PBS, afatinib (0.08 μM), IFN-γ, or afatinib plus IFN-γ for 24 hours, and then PD-L1 expression was detected by flow cytometry. (B) BALB/c mice were transplanted with 5 × 105 TUBO cells and treated with afatinib (100 mg/kg) by gavage on day 16. Five days after treatment, tumors were removed and digested into single-cell suspensions, and then the PD-L1 expression on myeloid cells and tumor cells was detected by flow cytometry. (C to H) F1 Neu-Tg mice (BALB/c × FVB Neu Tg mice) (n = 5 or 8 pooled from two experiments) were inoculated with 5 × 105 TUBO cells and treated with afatinib (100 mg/kg) twice as indicated (C), and 200 μg of anti–PD-L1 or rat immunoglobulin (Ig) was injected at different start time points. Days 0 and 3 after the first afatinib treatment were considered an early combination; day 7 was considered a late combination (C). Antibodies were given entirely three times as indicated (C). Tumor growth was monitored twice a week. (D to H) Individual tumor growth curves and (I) survival curves are shown.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/38/eaav6473/DC1

    Fig. S1. HyperTKI could not prevent tumor relapse.

    Fig. S2. HypoTKI is more potent in suppressing Her2 downstream AKT signaling, inducing apoptosis, and suppressing tumor cell proliferation than HyperTKI in vivo.

    Fig. S3. HypoTKI is more potent than HyperTKI in controlling tumor growth with fewer side effects.

    Fig. S4. Characterization of immune cell profile in tumor tissues after EGFR TKI treatment.

    Fig. S5 HypoTKI could enhance tumor-specific T cell responses.

    Fig. S6. EGFR TKI induces dsDNA, RNA, and LDH release from tumor cells.

    Fig. S7 Anti–PD-L1 synergizes with HypoTKI to control advanced large tumor and limit tumor relapse.

    Fig. S8. HypoTKI combined with anti–PD-L1 causes less toxicity than HyperTKI combined with anti–PD-L1.

    Fig. S9. Schematic of proposed mechanism for tumor control by EGFR TKI and PD-L1 blockade.

    Table S1. Key resources.

    Table S2. Raw data (Excel).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. HyperTKI could not prevent tumor relapse.
    • Fig. S2. HypoTKI is more potent in suppressing Her2 downstream AKT signaling, inducing apoptosis, and suppressing tumor cell proliferation than HyperTKI in vivo.
    • Fig. S3. HypoTKI is more potent than HyperTKI in controlling tumor growth with fewer side effects.
    • Fig. S4. Characterization of immune cell profile in tumor tissues after EGFR TKI treatment.
    • Fig. S5 HypoTKI could enhance tumor-specific T cell responses.
    • Fig. S6. EGFR TKI induces dsDNA, RNA, and LDH release from tumor cells.
    • Fig. S7 Anti–PD-L1 synergizes with HypoTKI to control advanced large tumor and limit tumor relapse.
    • Fig. S8. HypoTKI combined with anti–PD-L1 causes less toxicity than HyperTKI combined with anti–PD-L1.
    • Fig. S9. Schematic of proposed mechanism for tumor control by EGFR TKI and PD-L1 blockade.
    • Table S1. Key resources.

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    Other Supplementary Material for this manuscript includes the following:

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