MYC and Twist1 cooperate to drive metastasis by eliciting crosstalk between cancer and innate immunity

  1. Renumathy Dhanasekaran
  2. Virginie Baylot
  3. Minsoon Kim
  4. Sibu Kuruvilla
  5. David I Bellovin
  6. Nia Adeniji
  7. Anand Rajan KD
  8. Ian Lai
  9. Meital Gabay
  10. Ling Tong
  11. Maya Krishnan
  12. Jangho Park
  13. Theodore Hu
  14. Mustafa A Barbhuiya
  15. Andrew J Gentles
  16. Kasthuri Kannan
  17. Phuoc T Tran
  18. Dean W Felsher  Is a corresponding author
  1. Stanford University, United States
  2. University of Iowa Hospitals and Clinics, United States
  3. Johns Hopkins University School of Medicine, United States
  4. Stanford University School of Medicine, United States
  5. NYU Langone Medical Center, United States
9 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
Twist1 induces spontaneous metastatic progression of MYC driven HCC in vivo.

(a) Mouse model of MYC induced HCC where MYC is under the control of a tetracycline responsive element (TRE) which contain the tetracycline-controlled transactivator protein (tTA) driven by the …

Figure 1—figure supplement 1
Twist1drives metastasis ofMYC-induced HCC by non-cell-autonomous mechanisms.

(a) Representative images from liver and lung (1X and 10X) H and E from liver and lungs of mice with Twist1-transgenic mice demonstrating lack of pathology. (b) Representative images from …

Figure 2 with 1 supplement
MYC and Twist1 cooperate to remodel the tumor immune microenvironment.

(a) Principal component analysis (PCA) showed that MYC- (n = 5) and MYC/Twist1-HCC (n = 5) overall had distinct, non-overlapping expression profiles. Volcano plot shows comparative analysis of …

Figure 2—figure supplement 1
Tumor Microenvironment changes in MYC/Twist1 HCC.

(a) The top biological processes upregulated in genes differentially expressed in MYC/Twist1- (n = 5) versus MYC-HCC (n = 5) in transgenic mouse primary tumor tissue. (b) Representative images from …

Figure 3 with 1 supplement
Coordinate expression of both MYC and Twist1 are necessary for inducing metastasis.

(a) Schematic of the experiment to extract tumor associated macrophages from the primary tumors of MYC- (n = 5) and MYC/Twist1-HCC (n = 5). Conditioned media from macrophages extracted from MYC-HCC …

Figure 3—figure supplement 1
MYC and Twist1 cooperate to induce recruitment of tumor associated macrophages.

(a) Expression of MYC upon Dox treatment of MYC/Twist-HCC cells stably transfected with MYC or Twist1 or control vector, quantified by qPCR. (b) Expression of Twist1 upon Dox treatment of MYC/Twist-H…

Figure 4 with 1 supplement
Tumor associated macrophages are required for Twist1 to induce metastasis of MYC-HCC.

(a) Experimental scheme- MYC and MYC/Twist1 cells were implanted orthotopically and metastatic burden in liver and lung assessed after 4 weeks. (b) Representative BLI imaging, gross organ …

Figure 4—figure supplement 1
Effect of Macrophage depletion on MYC/Twist1 HCC metastasis.

(a) Conditional mRNA expression of MYC and Twist1 in primary cell lines derived from MYC-HCC(n = 5, with three technical replicates each) and MYC/Twist1-HCC (n = 5, with three technical replicates …

Figure 5 with 1 supplement
MYC and Twist1 reprogram the cytokinome to induce macrophage recruitment and polarization.

(a) Experimental scheme- conditioned media (CM) from MYC or MYC/Twist1 cells was used to treat non polarized macrophages for 48 hr. Following that, macrophage migration or polarization was assessed. …

Figure 5—figure supplement 1
Mechanisms of MYC and Twist1 cooperation.

(a) Expression of M1 marker genes in macrophages treated with conditioned media from MYC-HCC (n = 3, with three technical replicates each) or MYC/Twist1-HCC (n = 3, with three technical replicates …

Figure 6 with 1 supplement
MYC and TWIST1 require both Ccl2 and Il13 to promote metastasis.

(a) Experimental scheme- Mice orthotopically transplanted with MYC-HCC cells were treated either with PBS or Il13 or Ccl2 or Ccl2+Il13 recombinant cytokines for 4 weeks. (b) MYC expressing single …

Figure 6—figure supplement 1
MYC and Twist1 induce macrophage polarization and angiogenesis via Il13.

(a) Representative images from immunofluorescence staining for Cd206 in liver primary tumor reveals polarization to M2-like phenotype in MYC-HCC bearing mice treated with Il13 and Ccl2+Il13 but not …

Figure 7 with 1 supplement
Combined inhibition of Ccl2 and Il13 has a synergistic effect on prevention of HCC metastasis.

(a) Experimental scheme- Mice orthotopically transplanted with MYC/Twist1-HCC cells were treated either with control (ctrl) antibody or anti-Ccl2 antibody (ab) or anti-Il13 ab or anti-Ccl2+Il13 abs …

Figure 7—figure supplement 1
Combined inhibition of Ccl2 and Il13 inhibits macrophage polarization.

(a) Representative images from Cd206 staining of liver primary tumor sections (10X) of orthotopic transplanted MYC/Twist1-HCC bearing mice treated with control (ctrl) ab or anti-Ccl2 ab or anti-Il13 …

Figure 8 with 1 supplement
MYC and TWIST1 predict poor prognosis, TAM infiltration and pro-TAM cytokines in 33 human cancers.

(a) Disease-free survival in pan-cancer TCGA cohort of 9502 patients from 33 cancers stratified by median MYC+TWIST1 expression (first KM curve) and median MYC+TWIST1+ CCL2+IL13 (second KM curve). (b

Figure 8—figure supplement 1
M2 macrophages have a prognostic role in human cancers including liver cancer.

(a) M2 macrophage infiltration in 33 different cancers from the pan-cancer TCGA cohort based on transcriptome deconvolution analysis using CIBERSORT. (b) CIBERSORT analysis of human HCC TCGA RNAseq …

MYC and Twist1 Drive Metastasis via CCL2/IL13 mediated macrophage activation.

Graphical representation showing that MYC and Twist1 elicit a cytokinome including CCL2/IL13 which mediate the crosstalk between cancer cells and macrophages. Therapeutic blockade of the cytokines …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
AntibodyMYC (Rabbit, Monoclonal)EpitomicsRRID:AB_11000313IHC (1:150), WB (1:1000)
AntibodyF4/80 (Rat, Monoclonal)ThermoFisherRRID: AB_10376289IHC (1:150)
AntibodyTwist1 (Mouse, Monoclonal)AbcamRRID:AB_883294WB (1:500)
AntibodyPhospho-Histone 3 (Rabbit, Polyclonal)Cell Signaling TechnologyRRID:AB_331535IHC (1:100)
AntibodyCleaved Caspase3 (Rabbit, Polyclonal)Cell Signaling TechnologyRRID:AB_2341188IHC (1:100)
AntibodyNeutrophil (Rat, Monoclonal)AbcamRRID:AB_881409IHC (1:100)
AntibodyCD4 (Mouse, Monoclonal)AbcamRRID:AB_2686917IHC (1:1000)
AntibodyGlutamine Synthetase (Rabbit, Monoclonal)AbcamRRID:AB_446132IHC (1:200)
AntibodyCD31 (Rabbit, Polyclonal)AbcamRRID:AB_726362IHC(1:100)
AntibodyCD206 (Mouse, Monoclonal)R and DRRID:AB_2745540ICC (1:100)
AntibodyCCL2 (Mouse, Monoclonal)BioXCellRRID:AB_10950302In vivo treatment (10 mg/kg body weight three times per week)
AntibodyIl-4 (Mouse, Monoclonal)GenentechPropreitaryIn vivo treatment (10 mg/kg body weight three times per week)
AntibodyIL-13 (Mouse, Monoclonal)GenentechPropreitaryIn vivo treatment (10 mg/kg body weight three times per week)
Genetic Reagent (M. musculus)Twist1-tetO7-lucPMID: 22654667Twist1 Transgenic mouse modelFelsher Lab
Genetic Reagent (M. musculus)LAP-tTAPMID: 15475948LAP-tTA mouseFelsher Lab
Genetic Reagent (M. musculus)TetO-MYCPMID: 10488335MYC Transgenic mouseFelsher Lab
Genetic Reagent (M. musculus)Nod Scid-Gamma (NSG) miceJackson LaboratoryMGI:3577020Felsher Lab
CytokineCCL2PeprotechRRID:AB_147738In vivo treatment (500 ng/mouse)
CytokineIL13PeprotechRRID:AB_147840In vivo treatment (250 ng/mouse)
CytokineIL4PeprotechRRID:AB_147635In vivo treatment (250 ng/mouse)
DrugClodronate liposomesLiposomaCP-005–005In vivo treatment (6.5 μl/g body weight/mouse)
Sequence-based reagentMYCStanford PAN facilityN/Af-CTGCGACGAGGAGGAGAACT r-GGCAGCAGCTCGAATTTCTT
Sequence-based reagentUBCStanford PAN facilityN/Af-AGCCCAGTGTTACCACCAAG r-ACCCAAGAACAAGCACAAGG
Sequence-based reagentTwist1Stanford PAN facilityN/Af-GGACAAGCTGAGCAAGATTCA r-CGGAGAAGGCGTAGCTGAG
Sequence-based reagentCD206Stanford PAN facilityN/Af-CTCTGTTCAGCTATTGGACGC r-TGGCACTCCCAAACATAATTTGA
Sequence-based reagentArginase-1Stanford PAN facilityN/Af-CTCCAAGCCAAAGTCCTTAGAG r-AGGAGCTGTCATTAGGGACATC
Sequence-based reagentCCR2Stanford PAN facilityN/Af-GTGTACATAGCAACAAGCCTCAAAG r-CCCCCACATAGGGATCATGA
Sequence-based reagentINFaRStanford PAN facilityN/Af-CTTCCACAGGATCACTGTGTACCT r-TTCTGCTCTGACCACCTCCC
Sequence-based reagentiNOSStanford PAN facilityN/Af-ACACCGACCCGTCCACAGTAT r-CAGAGGGGTAGGCTTGTCTC
Sequence-based reagentCX3CR1Stanford PAN facilityN/Af-TACCTTGAGGTTAGTGAACGTCA r-CGCTCTCGTTTTCCCCATAATC

Additional files

Supplementary file 1

List of genes (220 up and 294 down) that were differentially expressed between the MYC-HCC and MYC/Twist1-HCC.

https://cdn.elifesciences.org/articles/50731/elife-50731-supp1-v1.xlsx
Supplementary file 2

The top biological processes, and associated genes, upregulated in MYC/Twist1-HCC versus MYC-HCC.

https://cdn.elifesciences.org/articles/50731/elife-50731-supp2-v1.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/50731/elife-50731-transrepform-v1.pdf

Download links