Assessment of hypoxia and oxidative-related changes in a lung-derived brain metastasis model by [64Cu][Cu(ATSM)] PET and proteomic studies

Abstract

Background: Brain metastases (BM) are the most frequent malignant brain tumors. The aim of this study was to characterize the tumor microenvironment (TME) of BM and particularly hypoxia and redox state, known to play a role in tumor growth and treatment resistance with multimodal PET and MRI imaging, immunohistochemical and proteomic approaches in a human lung cancer (H2030-BrM3)-derived BM model in rats.

Results: First, in vitro studies confirmed that H2030-BrM3 cells respond to hypoxia with increasing expression of HIF-1, HIF-2 and their target genes. Proteomic analyses revealed, among expression changes, proteins associated with metabolism, oxidative stress, metal response and hypoxia signaling in particular in cortical BM. [⁶⁴Cu][Cu(ATSM)] PET revealed a significant uptake by cortical BM (p < 0.01), while no uptake is observed in striatal BM 23 days after tumor implantation. Pimonidazole, HIF-1α, HIF-2α, CA-IX as well as GFAP, CTR1 and DMT1 immunostainings are positive in both BM.

Conclusion: Overall, [⁶⁴Cu][Cu(ATSM)] imaging and proteomic results showed the presence of hypoxia and protein expression changes linked to hypoxia and oxidative stress in BM, which are more pronounced in cortical BM compared to striatal BM. Moreover, it emphasized the interest of [⁶⁴Cu][Cu(ATSM)] PET to characterize TME of BM and depict inter-metastasis heterogeneity that could be useful to guide treatments.

Keywords: Brain metastasis; Copper-64; Cu-ATSM; HIF; Hypoxia; Imaging; Lung cancer; Oxidative stress; PET; Preclinical; Proteomic; Rodent.

Fig. 1

Effect of hypoxia on the expression of a HIF-1α, HIF-2α and b several HIF-target genes in H2030-BrM3 cells. a Immunocytology for HIF-1α and HIF-2α evaluated after 3 h or 24 h in hypoxic or normoxic condition. b Gene expression of HIF-target genes evaluated by real-time RT-PCR (TUBB3, VEGF-A, SLC2A1, CCDN1, SERPINE1 and CA-IX) in H2030-BrM3 cells submitted or not to hypoxia (1% O2) during 40 h. Median ± IQR (Interquartile Range), n = 4 independent experiments, * p < 0.05, one sample t-test vs theorical value of 1

Fig. 2

Global proteomic analyses in H2030-BrM3 lung-derived brain metastasis (BM) vs healthy brain tissues. a Volcano plots of quantified proteins between BM vs healthy brain tissues. Significant differentially expressed proteins are shown in red, cut-off: fold change (FC) = log2(1.2) (FC = 2.3) and p < 0.01, Bonferroni test. b GO enrichments of differentially expressed proteins on biological processes (BP). **FDR 1%, *FDR 5%. n = 5 for BM and n = 3 for healthy brain tissues

Fig. 3

Proteomic analyses in cortical and striatal H2030-BrM3 lung-derived BM vs their respective healthy tissues. a Venn diagrams (Venn) of differentially expressed proteins identified from BM versus healthy tissues, from both cortical and striatal parts. b, c GO enrichment, using a Bonferroni’s method, in Kegg (b, GO-Kegg) and BP (c, GO-BP). Functions are shown in red for those preferentially enriched in cortical BM, blue in striatal BM and gray for unspecific (both in cortical and striatal BM). n = 5 for cortical and striatal BM, n = 3 for healthy brain tissues

Fig. 4

In vivo uptake of [¹⁸F]FDG and [⁶⁴Cu][Cu(ATSM)] in the H2030-BrM3 BM model. a Timeline of experimental protocol. b Representative images of anatomical T2w MRI, [¹⁸F]FDG PET (1-h post-injection) and [⁶⁴Cu][Cu(ATSM)] (4-h post-injection), white arrows represent the intratumoral hemorrhage. c MRI and [⁶⁴Cu][Cu(ATSM)]PET, in the three planes (coronal, sagittal and transversal sections) acquired at D23. d Quantification of [¹⁸F]FDG, [⁶⁴Cu][Cu(ATSM)] uptake into cortical BM and striatal BM (at 1-h and 4-h post-injection, respectively). Mean ± SD, n = 12 rats for cortical BM and n = 8 rats for striatal BM for [¹⁸F]FDG; n = 13 rats for cortical BM and n = 8 rats for striatal BM for [⁶⁴Cu][Cu(ATSM)]. *p < 0.05 and **p < 0.01, one sample t-test vs theorical value of 1 and Mann–Whitney for comparison rSUV between cortical BM and striatal BM

Fig. 5

Immunohistological studies in cortical and striatal BM for hypoxia-related proteins, copper transporters and astrogliosis (red) with Hoechst 33,342 nuclear counterstaining (blue). Representative images of pimonidazole, CA-IX, HIF-1a, HIF-2a, DMT1, CTR1 and GFAP immunostaining in BM and healthy brain tissue