c-MYC overexpression induces choroid plexus papillomas through a T-cell mediated inflammatory mechanism

Abstract

Choroid plexus tumours (CPTs) account for 2-5% of brain tumours in children. They can spread along the neuraxis and can recur after treatment. Little is known about the molecular mechanisms underlying their formation and only few high fidelity mouse models of p53-deficient malignant CPTs are available.We show here that c-MYC overexpression in the choroid plexus epithelium induces T-cell inflammation-dependent choroid plexus papillomas in a mouse model. We demonstrate that c-MYC is expressed in a substantial proportion of human choroid plexus tumours and that this subgroup of tumours is characterised by an inflammatory transcriptome and significant inflammatory infiltrates. In compound mutant mice, overexpression of c-MYC in an immunodeficient background led to a decreased incidence of CPP and reduced tumour bulk. Finally, reduced tumour size was also observed upon T-cell depletion in CPP-bearing mice. Our data raise the possibility that benign choroid plexus tumours expressing c-MYC could be amenable to medical therapy with anti-inflammatory drugs.

Keywords: C-MYC; Choroid plexus tumours; Inflammation; Mouse models.

Fig. 1

CPT develop at high penetrance in NestinCre;STOPFloxc-MYC mice. a Schematic of generation of double transgenic NestinCre;STOPfloxc-MYC mice. b CPT arising in a double transgenic mouse, with a tumour seen within the lateral ventricle (left) as compared to normal choroid plexus in the fourth ventricle (centre). c A large proportion (84.2%) of compound mice developed CPT (pie graph on the left); the majority of them (81.3%) were CPP, whist the rest (18.8%) were ACPP, (graph on the right). d GFP immunohistochemistry showing strong and even expression in the CPT as compared to control CP (inset). e-h H&E of CPT. CPP shows mild stratification of cells albeit with largely preserved papillary architecture and with mild cellular and nuclear pleomorphism (e low magnification; g high magnification) while ACPP showed moderate cellular and nuclear pleomorphism with more blunting of papillary architecture (f low magnification and h high magnification). In addition ACPPs showed significantly higher Ki-67 labelling as compared to CPPs (i CPP; j ACPP). k Ki67 quantification (Bar graph representing average cells/HPF as mean ± SEM, n = 3 in each cohort, * P < 0.05). Scale bar = 1 mm (b), 250 μm (d, e, f) and 125 μm (g, h, i, j). Abbreviations: CP – choroid plexus; CPT-choroid plexus tumour; ACPP – atypical choroid plexus tumour; CPP – choroid plexus papilloma; NSPCs – neural stem progenitor cells; H&E – haematoxylin and eosin; GFP – Green Fluorescent Protein, indicative of c-MYC expression

Fig. 2

Increased proliferation and decreased apoptosis in CP cells overexpressing c-MYC. a, b Slightly enlarged CP was observed in NestinCre;STOPfloxc-MYC mice at P7 as compared to controls. No histological atypia was noted. c-f Patchy hyperplasia with mild nuclear atypia was seen in NestinCre;STOPfloxc-MYC mice at P15 in comparison to controls. The hyperplastic areas showed GFP positivity in NestinCre;STOPfloxc-MYC mice, in keeping with activation of c-MYC construct. g, h At 3 months, widespread CP hyperplasia with mild nuclear atypia was observed in c-MYC overexpressing mice. i-k A trend toward increased proliferation was observed in the CP of NestinCre;STOPfloxc-MYC mice at E12.5, E16.5, P7 and P15, a finding which reached statistical significance from 3 months onwards (n = 6 in each cohort for 3 months and 6 months; n = 4 in each cohort for 9 months). l Immunolabelling for transthyretin (TTR) confirmed the epithelial nature of the majority of the cultured cells. m GFP expression upon transduction with adeno-Cre (A-Cre) confirmed construct activation. n-p Increased proliferation on Edu immunoassay and q-s decreased apoptosis on cCasp3 immunoassay upon c-MYC overexpression by A-Cre induced construct activation (cMYC^OverCP) as compared to control CP cells. t Soft agar colony formation assay demonstrating cMYC^overCP cultures forming colonies as compared to control CP. A negative (no cells) and positive control (DAOY, medulloblastoma cell line) are included on the left; bottom panel shows high magnification images. t Quantification of the finding (n = 3). k, m, p, s, u– Bar graph representing Mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001. Scale bar is 125 μm (l, n, o, q, r)

Fig. 3

c-MYC is expressed in a proportion of human CPTs. a Unsupervised hierarchical clustering analysis of 356 genes, the expression of which significantly correlated with c-MYC expression in 40 CPTs from Merino et al. dataset, showing clustering of CPTs based on c-MYC expression. The heat map colour for each tumour is defined by the Pearson correlation coefficient between the log-ratio expression profiles of genes. b-g Representative histology of the analysis of an independent (Brain UK) cohort of human CPT: papilloma, WHO grade I (b CPP), atypical papilloma WHO grade II (c ACPP) and carcinoma, WHO grade III (d CPC). e-f c-MYC immunohistochemistry showing mostly diffuse expression (e) and in some cases focal expression (f). Nuclear expression of c-MYC in more than 10% was considered as positive. g Graphical representation of cases positive for c-MYC, overall and among different histological subtypes. 18/42 (43%) of all cases were c-MYC positive; 4/6 (67%) CPC, 6/12 (50%) ACPP and 8/24 (33%) CPP were c-MYC positive. Scale bar = 125 μm (b, c, d, e, f)

Fig. 4

Deregulation of inflammatory pathways in NestinCre;STOPfloxc-MYC CPT. a Normalized c-MYC expression values (CPM) of 3 murine control and 3 CPT samples. Median and interquartile range are depicted. b IPA analysis on differentially expressed genes (n = 245) between murine CPT and control samples. Positive z-score is associated with enrichment in the CPT context. c Unsupervised hierarchical clustering analysis and relative expression of c-MYC-correlated genes in the murine context (n = 2290), across control and CPT samples. d Unsupervised hierarchical clustering analysis and relative expression of murine orthologs of c-MYC-correlated genes in the human context (n = 356), across control and CPT samples

Fig. 5

Characterisation of the inflammation in c-MYC+ CPT and c-MYC^Over CPT. a An increased T-lymphocytic infiltrate on CD3 immunohistochemistry in c-MYC+ tumours in relation to c-MYC- is noted (quantification showed on bar graph on the right; Mean ± SEM; n = 4 in each cohort; * P < 0.05). b Subtyping of T-lymphocytes showed predominant increase in CD4 subtype population (quantification showed on bar graph on the right; Mean ± SEM; n = 11 for c-MYC+ and n = 10 for c-MYC-; * P < 0.05). c Pronounced macrophage infiltration was also observed among c-MYC+ on CD68 immunohistochemistry (quantification showed on bar graph on the right; Mean ± SEM; n = 4 in each cohort; * P < 0.05). d, e Comparative analysis of the CPT (CPP) developing in the c-MYC overexpressing mouse model as compared to normal CP showed T-lymphocytic (d) and macrophagic (e) infiltration in the tumour parenchyma. Quantification is shown on bar graph on the right; d Mean ± SEM; n = 6 in CPT and n = 7 in control; *** P < 0.001 and e Mean ± SEM; n = 7 in CPT and n = 3 in control; ** P < 0.01. Scale bar = 125 μm (a, b, c, d, e)

Fig. 6

CD3 depletion in NestinCre; STOPFloxc-MYC mice reduces proliferation and bulk of CPT. a A significant reduction in tumour incidence was noted in the triple compound mutant (NestinCre; STOPFlox-c-MYC;NOD-SCID) as compared to double compound mutant (NestinCre; STOPFlox-c-MYC). NODSCID mouse group confirmed no tumour development without c-MYC overexpression in an immunocompromised background. b, c Tumour area reduction, as assessed by Definiens® image analysis software in the triple compound mutant (b right) as compared to double compound mutant (b left). Representative tumour histology (b upper panel) and tumour area measured on Definiens (b lower panel, no colour). c Quantification bar graph with mean area ± SEM; n = 6 for double transgenic and n = 5 for triple transgenic; * P < 0.05. d Flow peaks showing reduction in CD3+ cells in blood following anti-CD3 injection (pre-treatment blue, post treatment red peaks). e Reduction in CD3+ population was noted post injection in the anti-CD3 treated group in comparison to pre injections. No significant changes where observed in the isotype treated group. Common Leukocyte Antigen - CD45+ CD3+ cells were selected; Circles = pre-injection levels, Squares = post injection levels after 4 weeks, each representing one mouse. Mean ± SEM represented; * P < 0.05. f Similar results were observed in spleen where a significant reduction of CD3+ cells was noted in post injection anti-CD3 cohort in comparison to isotype control; Mean ± SEM represented; ** P < 0.01. g H&E of CPT assessed by Definiens® image analysis software showed a reduced tumour area in the anti-CD3 injected cohort in comparison to isotype injected tumours. Representative tumour histology (g, upper panel) and tumour area measured on Definiens (G, lower panel, red) and quantification in (h) bar graph with mean area ± SEM; n = 9 for anti-CD3 treated and n = 8 for isotype control; * P < 0.05. i, j Reduced number of CD3+ cells (arrow head) was observed in anti-CD3 treated group (right panel) in comparison to isotype control (left panel). j Quantification bar graph of Ki67+ and CD3+ cells with mean cells/HPF ± SEM; n = 9 for anti-CD3 treated and n = 8 for isotype control; * P < 0.05; *** P < 0.001. Scale bar = 400 (G top) 200 μm (g bottom) and 50 μm (i)