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Silencing EGFR-upregulated expression of CD55 and CD59 activates the complement system and sensitizes lung cancer to checkpoint blockade

Nature Cancer volume 3, pages 1192–1210 (2022)Cite this article

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Abstract

The complement system is a critical immune component, yet its role in tumor immune evasion and CD8+ T cell activation is not clearly defined. Here, we demonstrate that epidermal growth factor receptor (EGFR)/Wnt signaling induces β-catenin-mediated long noncoding RNA (lncRNA) LINC00973 expression to sponge CD55-targeting miR-216b and CD59-targeting miR-150. The consequently upregulated CD55/CD59 expression suppresses the complement system and cytokine secretion required for CD8+ T cell activation. CD55/CD59-neutralizing antibody treatment or mutation of the LINC00973 promoter activates the complement and CD8+ T cells, inhibiting tumor growth. Importantly, combined anti-CD55/CD59 and anti-programmed death 1 (anti-PD-1) antibody treatments elicit a synergistic tumor-inhibiting effect. In addition, CD55/CD59 levels are inversely correlated with infiltration of M1 macrophages and CD8+ T cells in human lung cancer specimens and predict patient outcome. These findings underscore the critical role of EGFR/Wnt/β-catenin-upregulated CD55/CD59 expression in inhibiting the complement and CD8+ T cell activation for tumor immune evasion and immune checkpoint blockade resistance and identify a potential combination therapy to overcome these effects.

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Fig. 1: EGFR activation increases the expression of CD55 and CD59.
Fig. 2: EGFR upregulates CD55/CD59 by inhibiting miR-216b/miR-150.
Fig. 3: EGFR upregulates LINC00973 to sponge miR-216b and miR-150.
Fig. 4: EGFR/Wnt-activated β-catenin enhances LINC00973 expression.
Fig. 5: EGFR-β-catenin-LINC00973-miR-216b/150 axis inhibits complement.
Fig. 6: EGFR/β-catenin-enhanced CD55/CD59 inhibits immune cell function.
Fig. 7: CD55/CD59 promote tumor growth by inhibiting complement.
Fig. 8: Complement activation eliminates PD-1 blockade resistance.

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Data availability

All data generated or analyzed during the present study, including source data, can be found in the article, Extended data or Supplementary information. The RNA-seq data of H1395 cells are available in the Sequence Read Archive database (accession number: PRJNA847056). The sequence of human LINC00973 (accession number: ON462020) and mouse MLINC00973 (accession number: ON462021) are available in the Genebank database. The RNA-seq data of human lung adenocarcinoma and normal lung tissues were derived from the Gene Expression Profiling Interactive Analysis datasets: http://gepia.cancer-pku.cn/detail.php?clicktag=degenes. Source data for Figs. 1–8 and Extended Data Figs. 1–10 are provided as Source data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

This study was supported by the National Key R&D Program of China (grant nos. 2020YFA0803300 to Z.L. and 2020AAA0109500 to J.H. and Y.G.), the National Natural Science Foundation of China (grant nos. 82188102 to Z.L. and J.H., 82030074 to Z.L., 82122053 to Y.G. and 32100574 to F.S), the Zhejiang Natural Science Foundation-Key Project (grant no. LD21H160003 to Z.L.), the Zhejiang University Research Fund (grant no. 188020*194221901/029 to Z.L.), the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang (grant no. 2019R01001 to Z.L.), the Beijing Municipal Science & Technology Commission (grant no. Z191100006619115 to J.H.), R&D Program of Beijing Municipal Education Commission (grant no. KJZD20191002302 to J.H.), CAMS Initiative for Innovative Medicine (grant no. 2021-I2M-1-012, 2021-I2M-1-015 to J.H.), Aiyou Foundation (grant no. KY201701 to J.H.), Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (grant no. 2021-PT310-001 to Y.G.), Key-Area Research and Development Program of Guangdong Province (grant no. 2021B0101420005 to Y.G.) and the Natural Science Foundation of Shandong Province (grant no. ZR2020QH191 to F.S.). Z.L. is the Kuancheng Wang Distinguished Chair.

Author information

Author notes

  1. These authors contributed equally: Fei Shao, Yibo Gao, Wei Wang.

Authors and Affiliations

  1. Department of Thoracic Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Fei Shao, Yibo Gao, Wei Wang, Xiao Geng & Jie He

  2. The Affiliated Hospital of Qingdao University, Qingdao University, and Qingdao Cancer Institute, Qingdao, China

    Fei Shao & Jing Fang

  3. Laboratory of Translational Medicine, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Fei Shao & Yibo Gao

  4. State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Yibo Gao & Jie He

  5. Central Laboratory, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen, China

    Yibo Gao

  6. Department of Medical Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Wei Wang

  7. Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China

    Haiyan He, Liwei Xiao, Dong Guo & Zhimin Lu

  8. Department of Neuro-Oncology, Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Yan Xia & Zhimin Lu

  9. Zhejiang University Cancer Center, Zhejiang University, Hangzhou, China

    Zhimin Lu

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Contributions

Z.L. conceived and designed the study and wrote the manuscript. Z.L., J.H., Y.G. and F.S. acquired the funding support and supervised the study. F.S. performed most experiments. F.S., Y.G. and W.W. provided support for generating various knock-in mutant cell lines and stably transfected cell lines. X.G. and D.G. provided support for IHC staining. H.H. and L.X. were involved in the studies of mice. Y.X. and D.G. performed statistical analysis of the data. J.H. provided the NSCLC samples and technical support. Z.L., J.H., Y.G., J.F. and F.S. reviewed and edited the manuscript.

Corresponding authors

Correspondence to Jie He or Zhimin Lu.

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Nature Cancer thanks Maite Huarte, Lubka Roumenina and Tuomas Tammela for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 EGFR activation increases the expression of CD55 and CD59.

a, H322M cells were treated with or without EGF (100 ng/ml) for 24 h. Immunoblotting analyses were performed with the indicated antibodies. b-e, H322M cells with or without expressing shRNAs against CD55 and/or CD59 were treated with or without EGF (100 ng/ml) for 24 h. These cells were incubated with DMEM medium (10% FBS) supplemented with or without 25% v/v human serum and 25 μg/ml lepirudin for 3 h. b, Representative results of the expression of C3b and C5b–9 on the tumor cell surface determined by flow cytometry with the indicated antibodies. c, The expression of C3b and C5b–9 on the tumor cell surface was determined by flow cytometry with the indicated antibodies. d, The amount of anaphylatoxin (C3a and C5a) in the supernatant was determined by ELISA. e, The indicated cells were cocultured with human PBMCs. The expression of the indicated cytokines in the medium was detected. f, H1395 cells were treated with or without EGF (100 ng/ml) for 12 h. The relative expression levels of CFH, CFB, C3 and C5 mRNAs were measured using qPCR. g, H1395 cells were treated with or without EGF (100 ng/ml) for 12 h. The expression levels of factor H, factor B, C3 and C5 were detected in the cell culture supernatant. h, H1395 cells were treated with or without EGF (100 ng/ml) for 24 h and then incubated with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25 μg/ml lepirudin for 3 h. These cells were then cocultured with human PBMCs. The expression levels of granzyme B and perforin in CD8+ T cells were detected by flow cytometry with the corresponding antibodies. The representative results were shown. c, d, e, f, g, The data are presented as the mean ± s.d. Data were analyzed by two-sided unpaired Student’s t-test. n = 6 biologically independent replicates (c, d, f, g). n = 7 human donors (e). HS, human serum.

Source data

Extended Data Fig. 2 EGFR upregulates CD55/CD59 by inhibiting miR-216b/miR-150.

a, A luciferase reporter gene driven by the promoters of CD55 and CD59 was cotransfected with a vector expressing EGFR into 293 T cells. The cells were treated with or without EGF (100 ng/ml) for 8 h. The luciferase activity was measured. Relative luciferase activity was normalized to that in the group treated without EGF. b, H1395 (b) or H322M (c) cells were treated with or without EGF (100 ng/ml) for 12 h. The relative RNA expression levels of the transcribed introns of CD55 and CD59 genes were measured using qPCR. d-f, The indicated cells expressing one of the two different small interfering RNAs (siRNAs) against Dicer (d) were then treated with or without EGF (100 ng/ml) for 24 h (e, f). Immunoblotting (e) and qPCR (f) analyses were performed. Immunoblotting analyses were performed with the indicated antibodies (d, e). g, The miRNA code prediction algorithm (http://www.mircode.org/index.php)-predicted miRNAs and their target sequences on CD55 and CD59 mRNAs are displayed. h, i, H322M cells expressing luciferase reporter genes fused with or without the WT 3’ UTRs or the corresponding mutants (MUT) of the CD55 and CD59 genes were transfected with miR-Control, miR-150-mimics, or miR-216b-mimics (h) or with miR-Control, miR-150-inhibitor, or miR-216b-inhibitor (i). Relative luciferase activity normalized to that in the miR-Control group in the cells expressing luciferase reporter genes without fusion of the 3’ UTRs is shown. j-m, H322M cells expressing miR-Control, miR-150-mimics or miR-216b-mimics (j, k) or expressing miR-Control, miR-150-inhibitor or miR-216b-inhibitor (l, m) were treated with or without EGF (100 ng/ml) for 12 h (j, l) or 24 h (k, m). qPCR (j, l) and immunoblotting (k, m) analyses were performed. Immunoblotting analyses were performed with the indicated antibodies (k, m). n, o, H1395 and H322M cells were treated with or without EGF (100 ng/ml) for 24 h. Immunoblotting analyses were performed (n) and quantified (o). Immunoblotting analyses were performed with the indicated antibodies (n). a-c, f, h-j, l, o, The data are presented as the mean ± s.d. Data were analyzed by two-sided unpaired Student’s t-test. n = 6 (a, c, f, h-j, l) or 4 (b) or 3 (o) biologically independent replicates.

Source data

Extended Data Fig. 3 EGFR upregulates LINC00973 to sponge miR-216b and miR-150.

a, b, Diagram of the binding sites of miR-216b and miR-150 on lncRNA LINC00973. c, The 5’ and 3’ sequences of LINC00973 were obtained using RACE. PCR products amplified from the 5’ and 3’ sequences of LINC00973 and the known region of the LINC00973 sequence from the Ensembl Genome Browser were separated on an agarose gel. The sizes of the sequences are indicated based on the sequencing results of the PCR products. Representative gel electrophoreses are reflective of n = 3-4 biologically independent experiments yielding similar results. d, The PCR products obtained using the primers of LINC00973 in RACE were sequenced. This new identified full-length sequence of LINC00973 is aligned with the de novo RNA-seq-assembled transcript sequence of LINC00973 in H1395 cells and the Ensembl-annotated transcript sequence of human LINC00973. e, The de novo assembly of the reads that are mapped to the genomic location of LINC00973 from RNA-seq data (SRA accession number: PRJNA847056) of H1395 cells. f, The LINC00973 expression levels were determined using GEPIA. Box plot elements are defined as: box extending 25th to 75th percentile; whiskers extending minimum to maximum values; and median indicated by traversing line. n = 483 tumor tissues; n = 347 normal tissues. LUAD, lung adenocarcinoma; TPM, Transcripts Per Million; T, tumor; N, normal; num, number of samples. g, Total cell lysate and cytosolic and nuclear fractions of H322M cells were prepared. RNA was purified. The relative expression levels of ACTB (a cytosol control), RNU6-1 (a nuclear control) and LINC00973 RNAs were measured using qPCR. h, H322M cells were treated with or without EGF (100 ng/ml) as indicated. qPCR was performed. i, Diagram of RIP by an anti-GFP antibody for the detection of miRNAs endogenously associated with LINC00973. j, l, m, MS2-GFP was cotransfected with MS12 or the indicated MS12-lncRNA into H322M cells. GFP-RIP and subsequent microRNA qRT-PCR were performed to detect lncRNA-associated microRNAs. k, A schematic showing the mutations of the binding sites of miR-216b and miR-150 on LINC00973. n-p, H322M cells were transfected with the indicated biotin-conjugated lncRNA followed by a streptavidin pulldown assay and subsequent microRNA qRT-PCR to detect associated endogenous microRNAs. g, h, j, l-p, The data are presented as the mean ± s.d. Data were analyzed by two-sided unpaired Student’s t-test. n = 6 biologically independent replicates.

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Extended Data Fig. 4 EGFR increases LINC00973 to sponge miR-216b and miR-150.

a, H1395 and H322M cells expressing luciferase reporter genes with or without fusion with the first 200 5’ nucleotides of LINC00973 containing with or without the mutations of the binding sequences for miR-216b or miR-150 were transfected with miR-Control, miR-150-mimics or miR-216b-mimics. The relative luciferase activity is shown. b, c, H1395 and H322M cells were transfected with miR-Control, miR-150-mimics or miR-216b-mimics. The relative expression levels of LINC00973 RNA were measured using qPCR (b). RIP using an antibody against Ago2 and subsequent lncRNA qRT-PCR to detect the indicated microRNA-associated lncRNAs were performed (c). d, e, H1395 and H322M cells were transfected with the indicated lncRNA. The relative expression levels of LINC00973, lncRNA-225205 RNAs (d), miR-216b, or miR-150 miRNAs (e) were measured using qPCR. f, g, H1395 and H322M cells were treated with or without EGF (100 ng/ml) for 12 h (f) or the indicated time (g). Total RNA was extracted from the cells. The copy numbers of LINC00973, miR-216b and miR-150 in the indicated cells were quantified using qPCR. The stoichiometry of the LINC00973 binding to miR-216b and miR-150 was calculated. Representative stoichiometry relationships are reflective of n = 3-4 biologically independent experiments yielding similar results. (g). h, i, H1395 and H322M cells were stably transfected with or without LINC00973 shRNAs. The cells were treated with or without EGF (100 ng/ml) for 12 (h) or 24 h (i). qPCR (h) and immunoblotting analyses (i) were performed. j, k, A schematic showing the miR-216- and miR-150-binding elements (miR-216b/150-BE) on LINC00973. Genomic DNA was extracted from parental H1395 (j) and H322M (k) cells and two individual clones of H1395 (j) and H322M (k) cells with knock-in mutations of miR-216b/150-BE. PCR products were amplified from the indicated DNA fragment and separated on an agarose gel. Representative gel electrophoreses are reflective of n = 3-4 biologically independent experiments yielding similar results. The sequencing data of parental H1395 (j) and H322M (k) and two individual clones of H1395 (j) and H322M (k) cells with knock-in mutations of miR-216b/150-BE are shown. Red line with and without an arrow indicates the sgRNA-targeting sequence and PAM, respectively. Mutated nucleotides are indicated by blue arrows. The positions of miR-216b/150-BE with or without mutated nucleotides are indicated by solid red box. Silent mutations of the indicated nucleotides were introduced into the sequence to avoid repeat cutting by Cas9. l, The relative mRNA (l) and protein (m) levels of CD55 and CD59 mRNAs in the indicated cells were measured using qPCR and immunoblotting analyses, respectively. a-f, h, l, The data are presented as the mean ± s.d. Data were analyzed by two-sided unpaired Student’s t-test. n = 6 biologically independent replicates. WT, wild type; C1, clone 1; C2, clone 2.

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Extended Data Fig. 5 EGFR/Wnt-activated β-catenin enhances LINC00973 expression.

a, A schematic showing three potential LEF/TCF-binding elements (TBEs; CTTTG(A/T)(A/T)) in the promoter region of LINC00973. b, c, Genomic DNA was extracted from parental H1395 (b) and H322M (c) cells and two individual clones of H1395 (b) and H322M (c) cells with knock-in mutations (AT to GC) in three TBEs. PCR products were amplified from the indicated DNA fragments and separated on an agarose gel. Representative gel electrophoreses are reflective of n = 3-4 biologically independent experiments yielding similar results. The sequencing data of parental H1395 (b) and H322M (c) cells and two individual clones of H1395 (b) and H322M (c) cells with knock-in mutations (AT to GC) in three TBEs are shown. Red line with and without an arrow indicates the sgRNA-targeting sequence and PAM, respectively. Mutated nucleotides are indicated by black arrows. Three TBEs with or without mutated nucleotides are indicated by a solid red box. Silent mutations of the indicated nucleotides were introduced into the sequence to avoid repeat cutting by Cas9. d, Parental H1395 and H322M cells and the indicated clones with LINC00973 TBE knock-in mutations (AT to GC) were stimulated with or without EGF for 12 h. qPCR was performed. e, f, H322M cells with or without β-catenin shRNA expression, reconstituted rβ-catenin expression, or expression of miR-216b inhibitor (e) or miR-150 inhibitor (f) were treated with or without EGF for 24 h. Immunoblotting analyses were performed with the indicated antibodies. g, h, H322M cells expressing with or without LINC00973 shRNA#1 or LINC00973 shRNA#2 in the presence or absence of miR-216b inhibitor (g) or miR-150 inhibitor (h) were treated with or without EGF for 24 h. Immunoblotting analyses were performed with the indicated antibodies. i, Parental H1395 and H322M cells and the indicated clones with LINC00973 TBE knock-in mutations were stimulated with or without EGF for 24 h. Immunoblotting analyses were performed with the indicated antibodies. j, k, Parental H1395 or H322M cells with or without LINC00973 TBE2 knock-in mutation and/or expression of an inhibitor of miR-216b (j) or miR-150 (k) were treated with or without EGF (100 ng/ml) for 24 h. Immunoblotting analyses were performed with the indicated antibodies. l-n, H1395 or H322M cells with or without expression of EGFR L858R (l-n), TBE2 knock-in mutation, β-catenin shRNA, rβ-catenin, LINC00973 shRNA (l), miR-216b mimics (m), or miR-150 mimics (n) were analyzed. Immunoblotting analyses were performed with the indicated antibodies. o, r, H1395 cells were treated with or without Wnt-7B or Wnt-5A (200 ng/ml) for 12 h. qPCR was performed. p, Relative mRNA levels of the indicated Fzd receptors, LRP5, and LRP6 in H1395 cells were measured using qPCR. q, Frizzled 6 shRNA was expressed in H1395 cells. Immunoblotting analyses were performed with the indicated antibodies. d, o, p, r, The data are presented as the mean ± s.d. Data were analyzed by two-sided unpaired Student’s t-test (d, o, r). n = 6 (d, o) or 4 (p, r) biologically independent replicates. b-d, i-k, o, WT, wild type; C1, clone 1; C2, clone 2.

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Extended Data Fig. 6 EGFR-β-catenin-LINC00973-miR-216b/150 axis inhibits complement.

a-c, H322M cells expressing with or without β-catenin shRNA or LINC00973 TBE2 knock-in mutations were stimulated with or without EGF for 24 h. The expression of C3b and C5b–9 on the tumor cell surface was determined by flow cytometry (a). Anaphylatoxin (C3a and C5a) in the supernatant was determined by ELISA (b). The indicated cells were cocultured with human PBMCs. The expression of the indicated cytokines was examined by ELISA (c). d-f, H322M (d-f) or H1395 (d, e) cells expressing the indicated miR or shRNAs against CD55 and CD59 were treated with or without EGF (100 ng/ml) for 24 h. The expression of C3b and C5b–9 on the tumor cell surface was determined by flow cytometry (d). Anaphylatoxin (C3a and C5a) in the supernatant was determined by ELISA (e). The indicated cells were cocultured with human PBMCs. The expression of the indicated cytokines was examined by ELISA (f). g-l, H1395 or L858R-expressing H1395 cells with or without expression of β-catenin shRNA (g-i), LINC00973 TBE2 knock-in mutations (g-l), shRNAs against CD55 and CD59, or miR-216b/miR-150 inhibitors (j-l) were cultured with human serum for examination of expression of C3b and C5b–9 by flow cytometry (g, j), C3a and C5a (h, k), and the indicated cytokines after coculturing with human PBMCs (i, l) by ELISA. a-l, The data are presented as the mean ± s.d. Data were analyzed by two-sided unpaired Student’s t-test. n = 6 biologically independent replicates (a, b, d, e, g, h, j, k) or 7 human donors (c, f, i, l). C1, clone 1; C2, clone 2.

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Extended Data Fig. 7 EGFR/β-catenin-enhanced CD55/CD59 inhibits immune cell function.

a-d, H322M cells with or without the expression of nanoluciferase (a, c), β-catenin shRNA, LINC00973 TBE2 knock-in mutations (a, b), miR-216b mimics, miR-150 mimics, miR-216b-inhibitor, miR-150-inhibitor, or shRNAs targeting CD55 and CD59 (c, d) were stimulated with or without EGF for 24 h and then cocultured with human PBMCs. CARIA assays for specific lysis were performed using the nanoluciferase-release method (a, c). MTS assays were performed (b, d). e-h, H1395 and EGFR L858R-expressing H1395 cells with or without expression of nanoluciferase (e, g), β-catenin shRNA (e, f), LINC00973 TBE2 knock-in mutations (e-h), shRNAs against CD55 and CD59, or miR-216b/miR-150 inhibitors (g, h) were cocultured with human PBMCs. CARIA assays for specific lysis were performed using the nanoluciferase-release method (e, g). MTS assays were performed (f, h). i, Normalized phagocytosis rates of H1395 and H322M cells by M0, M1 and M2 macrophages. j-m, H322M (j, k) and EGFR L858R-expressing H1395 cells (l, m) with or without expression of β-catenin shRNA (j, l), LINC00973 TBE2 knock-in mutations (j, l, m), miR-216b mimics, miR-150 mimics, miR-216b-inhibitor, miR-150-inhibitor, or shRNAs targeting CD55 and CD59 (k, m) were stimulated with or without EGF (100 ng/ml) for 24 h. Normalized phagocytosis rates of the tumor cells were determined (j-m). Flow cytometry with an anti-CD11b antibody was performed. FITC, fluorescein isothiocyanate (l, left panel). n-p, H1395 and EGFR L858R-expressing H1395 cells (n, o) or H322M cells (p) with or without expression of β-catenin shRNA, LINC00973 TBE2 knock-in mutations (n, p), miR-216b-mimics and miR-150-mimics (o), or CD55/CD59 shRNAs (p) were cocultured with human PBMCs. The expression of granzyme B and perforin in CD8+ T cells was detected by flow cytometry (n, o). The indicated cells were cocultured with human PBMCs in medium with or without anti-CD8 monoclonal antibody for 30 h. MTS assays were performed (p). a-p, The data are presented as the mean ± s.d. Data were analyzed by two-sided unpaired Student’s t-test. n = 7 (a-h, n-p) or 4 (i) or 6 (j-m) human donors. C1, clone 1; C2, clone 2.

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Extended Data Fig. 8 CD55/CD59 promote tumor growth by inhibiting complement.

a, The 5’ and 3’ sequences of MLINC00973 were obtained using RACE. PCR products amplified from the 5’ and 3’ sequences of MLINC00973 and the known region of the MLINC00973 sequence from the National Center for Biotechnology Information database were separated on an agarose gel. The sizes of the sequences are indicated based on the sequencing results of the PCR products. Representative gel electrophoreses are reflective of n = 3-4 biologically independent experiments yielding similar results. M, marker; L1, lane 1. b, The whole sequence of MLINC00973 was obtained using RACE. The PCR products obtained using the primers of the 5’ and 3’ sequences of MLINC00973 were sequenced. c, Diagram of the LEF/TCF-binding elements (TBE; CTTTG(A/T)(A/T)) in MLINC00973 promoter. d, Genomic DNA was extracted from two individual clones of LA795 cells with knock-in mutations (AT to GC or AA to GG, as indicated) in two TBEs. PCR products were amplified from the indicated DNA fragment and separated on an agarose gel. Representative gel electrophoreses are reflective of n = 3-4 biologically independent experiments yielding similar results. C1, clone 1; C2, clone 2. The sequences of the parental cells and two individual knock-in mutation clones are shown. Red line with an arrow indicates the sgRNA-targeting sequence. Red line without an arrow indicates the protospacer adjacent motif (PAM). Two TBEs with or without mutated nucleotides are indicated by solid red box. Silent mutations of the indicated nucleotides were introduced into the sequence to avoid repeat cutting by Cas9. e, The relative RNA levels of MLINC00973 in LA795 cells with or without MLINC00973 TBE knock-in mutation or EGFR L858R expression were measured using qPCR. The data are presented as the mean ± s.d. Data were analyzed by two-sided unpaired Student’s t-test; n = 6 biologically independent replicates. C1, clone 1; C2, clone 2. f, Diagram showing the binding sites of mmu-miR-216b and mmu-miR-150 on MLINC00973. g, h, LA795 cells with or without EGFR L858R expression were stably transfected with or without miR-Control, mmu-miR-216b or mmu-miR-150 (g), β-catenin shRNA, rβ-catenin, or the mmu-miR-216b/mmu-miR-150 sponges (h). Immunoblotting analyses were performed with the indicated antibodies. C1, clone 1; C2, clone 2. i, LA795 cells with or without MLINC00973 TBE knock-in mutation were transfected with EGFR L858R and/or mmu-miR-216b/mmu-miR-150 sponges. Immunoblotting analyses were performed with the indicated antibodies. C1, clone 1; C2, clone 2.

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Extended Data Fig. 9 CD55/CD59 accelerate tumor growth by suppressing complement.

a, Immunofluorescence analyses were performed with the indicated antibodies. Representative immunofluorescence images are reflective of n = 3-4 biologically independent experiments yielding similar results. b-e, LA795 cells expressing GFP and with or without expression of EGFR L858R in the presence or absence of MLINC00973 knock-in TBE4 mutant or CD55/CD59 shRNA expression were subcutaneously injected into immunocompetent syngeneic “615” mice. The tumors were resected 23 days after the injection. Minced tumor tissues were homogenized. Anaphylatoxin (C3a and C5a) release (b) and cytokine protein levels (c) in the tumor microenvironment (TME) was determined by ELISA. Representative flow cytometry plots demonstrate TAM phagocytosis in the indicated tumors. The numbers indicate the frequency of phagocytosis events normalized to all TAMs (d). Representative results show granzyme B and perforin expression in tumor-infiltrating CD8+ T cells (e). f-n, LA795 cells expressing GFP and with or without expression of EGFR L858R in the presence or absence of MLINC00973 knock-in TBE4 mutant or mmu-miR-216b/mmu-miR-150 sponges were subcutaneously injected into immunocompetent syngeneic “615” mice. The tumors were resected 23 days after the injection. f, The tumor volumes and weights were measured. The longitudinal analyses of tumor volume were performed; n = 6 mice (left). Data were analyzed by two-sided unpaired Student’s t-test; n = 6 mice (right). g, The survival rates of mice were analyzed by the Kaplan–Meier plot. P values were calculated using a log-rank test (two-sided). n = 10 mice. h, IHC and ISH analyses of tumors were performed with the indicated antibodies and probes. Representative images are shown. The regions in red boxes are shown at higher magnification. The significance of the differences in the expression between the groups was analyzed by nonparametric tests. i, Single-cell suspensions were obtained from minced tumor pieces. Tumor cells expressing GFP were isolated by flow cytometry. The expression of C3b and C5b–9 on the surface of the tumor cells was determined by flow cytometry. j-l, Minced tumor tissues were homogenized and adjusted to 10 mg/ml. Anaphylatoxin (C3a and C5a) release (j) and cytokine protein levels (k) in TME were determined by ELISA. A CARIA assay for specific lysis was performed using the nanoluciferase-release method (l). m, Frequency of phagocytosis events normalized to all TAMs in the indicated tumors was determined. n, TUNEL analyses of the indicated tumor samples from mice were performed. Apoptotic cells (green) were stained (left) and quantified (right). o-q, Four weeks after “615” mouce-tail vein injection of AAV8 virus expressing shControl or C3 shRNA and C5 shRNA, the mice were euthanized. Minced mouse liver tissue were collected. The relative expression levels of mRNAs (o) and protein levels (p) of C3 and C5 were measured using qPCR and ELISA, respectively. Peripheral blood was collected from the mice and probed for C3 and C5 protein levels by ELISA (q). b-c, h-q, The data are presented as the mean ± s.d. Data were analyzed by two-sided unpaired Student’s t-test. n = 9 (b, j, m) or 6 (c, h, k, o-q) 8 (i) or 10 (l, n) mice. C1, clone 1; C2, clone 2.

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Extended Data Fig. 10 Complement activation eliminates PD-1 blockade resistance.

a, b, H322M cells with or without expressing EGFR L858R in the presence or absence of LINC00973 knock-in TBE2 mutant or CD55/CD59 shRNA expression were subcutaneously injected into humanized NOG mice with restored human complement system. These mice were treated or untreated with anti-PD-1 or anti-CD55/CD59 antibodies. a, e, The tumor volumes and weights were measured 26 days after the injection of tumor cells. The longitudinal analyses of tumor volume were performed (left). b, f, The survival rates of mice were analyzed by the Kaplan–Meier plot. P values were calculated using a log-rank test (two-sided). c, The indicated cells were treated with or without Wnt-7B (200 ng/ml) with or without XAV939 (10 μM) for 12 h. Relative expression levels of CD55 and CD59 mRNAs were measured using qPCR. d, The indicated cells were treated with or without Wnt-7B (200 ng/ml), with or without XAV939 (10 μM) for 24 h. Immunoblotting analyses were performed with the indicated antibodies. e, f, H1395 cells (4 × 106) were subcutaneously injected into humanized NOG mice with restored human complement system. These mice were treated or untreated with an anti-PD-1 antibody and/or XAV939. g, MC38 cells (2 × 106) expressing APC shRNA and with or without CD55 and CD59 shRNAs were subcutaneously injected into C57Bl/6 mice. These mice were treated or untreated with an anti-PD-1 antibody. The tumor volumes and weights were measured 20 days after the injection of tumor cells. The longitudinal analyses of tumor volume were performed (left). h, H1395 cells (4 × 106) were subcutaneously injected into humanized NOG mice with restored human complement system. These mice were treated or untreated with anti-PD-1 antibody or anti-CD55 and anti-CD59 antibodies. The tumor volumes and weights were measured 26 days after the injection of tumor cells. The longitudinal analyses of tumor volume were performed (left). i, j, k, H1395 cells expressing EGFR L858R (4 × 106) were subcutaneously injected into humanized NOG mice with restored human complement system. These mice were treated 5 days per week by oral gavage with vehicle or gefitinib (80 mg/kg) after tumor establishment on day 11. i, The tumor volumes and weights were measured 26 days after the injection of tumor cells. The longitudinal analyses of tumor volume were performed (left). j, k, Immunoblotting (j) and IHC (k) analyses were performed with the indicated antibodies. Representative immunohistochemical staining images are reflective of n = 3-4 biologically independent experiments yielding similar results (k). l-n, Human NSCLC specimens (n = 200 patients) were analyzed by IHC assay with the indicated antibodies and by ISH assay for LINC00973 levels with LNA probes. l, Representative images are shown. The regions in the indicated red boxes are amplified and shown in Fig. 8d. m, Correlation analyses were performed using two-sided Pearson correlation test. The Pearson correlation coefficient was indicated. Note that the scores for some samples overlap. The intensities of red or blue color represent the numbers of human NSCLC specimens (darker red or blue color represents a higher number of human NSCLC specimens). n, Representative images of the expression of the indicated proteins (left panel) and LINC00973 (right panel) are shown. U6 expression was used as a control. The regions in red boxes are shown at higher magnification. The expression levels of the indicated proteins and LINC00973 were scored. n = 200 tumor tissues; n = 200 normal tissues (right panels). a, c, e, g, h, i, n, The data are presented as the mean ± s.d. Data were analyzed by two-sided unpaired Student’s t-test (a (right), c, e (right), g (right), h (right), i (right), n (right)). n = 6 (a, e, g, h, i) or 10 (b, f) or 4 (c) mice. C1, clone 1.

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Supplementary information

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Shao, F., Gao, Y., Wang, W. et al. Silencing EGFR-upregulated expression of CD55 and CD59 activates the complement system and sensitizes lung cancer to checkpoint blockade. Nat Cancer 3, 1192–1210 (2022). https://doi.org/10.1038/s43018-022-00444-4

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