The identification of a TNBC liver metastasis gene signature by sequential CTC‐xenograft modeling

Abstract

Triple‐negative breast cancer (TNBC) liver metastasis is associated with poor prognosis and low patient survival. It occurs when tumor cells disseminate from primary tumors, circulate in blood/lymph [circulating tumor cells (CTCs)], and acquire distinct characteristics during disease progression toward the metastatic phenotype. The purpose of this study was to decipher the genomic/transcriptomic properties of TNBC liver metastasis and its recurrence for potential therapeutic targeting. We employed a negative depletion strategy to isolate and interrogate CTCs from the blood of patients with TNBC, and to establish sequential generations of CTC‐derived xenografts (CDXs) through injection of patient CTCs in immunodeficient mice. The isolation and validation of CDX‐derived cell populations [analyses of CTCs were paired with bone marrow‐resident cells (BMRTCs) and liver tissue cells obtained from the same animal] were performed by multiparametric flow cytometry, immune phenotyping, and genomic sequencing of putative CTCs. Comprehensive characterization of gene expression arrays from sequentially generated CDX‐derived cell populations, online gene expression arrays, and TCGA databases were employed to discover a CTC‐driven, liver metastasis‐associated TNBC signature. We discovered a distinct transcriptomic signature of TNBC patient‐isolated CTCs from primary TNBCs, which was consistent throughout sequential CDX modeling. We established a novel TNBC liver metastasis‐specific CDX model that selectively recapitulates CTC biology for four sequential generations of mice. The evaluation of online databases and CDX‐derived populations revealed 597 genes specific to the TNBC liver metastasis signatures. Further investigation of the TNBC liver metastasis signature predicted 16 hub genes, 6 biomarkers with clinically available drugs, and 22 survival genes. The sequential interrogation of CDX‐CTCs is an innovative liquid biopsy‐based approach for the discovery of organ metastasis‐specific signatures of CTCs. This represents the first step for mechanistic and analytical validation in their application as prognostic indicators and therapeutic targets. Targeting CTC drug candidate biomarkers along with combination therapy can improve the clinical outcome of TNBC patients in general and recurrence of liver metastasis in particular.

EMT-Associated Heterogeneity in Circulating Tumor Cells: Sticky Friends on the Road to Metastasis

“…Although some studies use CTCs without prior in vitro expansion, CTC-derived cultures or cell lines are also commonly assessed. MICs have thus been successfully identified in CTCs isolated from different types of cancer including breast, lung or colon cancer [315,316,[330][331][332][333][334]. In some studies, intravenous or intra-femoral injections have been directly performed.…”

Molecular Interplay between Dormant Bone Marrow-Resident Cells (BMRCs) and CTCs in Breast Cancer

“…Having validated that HLA-ABC can identify both PanCK+ and CD44+ breast cancer cells in mouse bone marrow, we depleted doublets (SSC-H v SSC-A) and dead cells (DAPI+), normal blood cell lineages (leukocytes: CD45+, lymphocytes, mesenchymal, endothelial progenitors: CD34+, macrophages, fibroblasts CD90+/CD105+, mesenchymal, hematopoietic stem cells, NK cells: CD73+) by FACS, and implanted the residual CTC-enriched/lineage-negative cells into NOD scid-gamma (NSG) mice by intracardiac injection [ 20 , 28 , 29 ]. Mice were euthanized after 4–8 months of in vivo selection, and target organs (brain, lung, liver, spleen) were evaluated for the presence of tumor cells by H/E staining and IHC using HLA-ABC (to prove human origin) and GCDFP-15/Mammaglobin (MG) cocktail (to prove mammary origin).…”

“…The femur and tibia were isolated, and BM was obtained by flushing the femur and tibia with 1×PBS containing 5 mM of EDTA using 28G×½ needles, followed by centrifugation at 300 × g for 10 min. PBMCs were isolated immediately from the blood and BM for FACS analyses, as previously reported [ 28 , 29 ]. Different organ tissues were also harvested and fixed in 4% paraformaldehyde for downstream analyses.…”

Predicting therapy response by analysis of metastasis founder cells: emerging perspectives for personalized tumor therapy

“…Within the last decades a range of methods have been developed to specifically isolate CTCs [53] and to cultivate these rare cells by optimized in vitro culture conditions such as spheroid and organoid cultures, or by in vivo transplantation into immunodeficient mice [54][55][56][57][58]. To date, the generation of CTC-derived models has been reported for melanoma, breast, colorectal, lung, gastroesophageal, pancreatic, and prostate cancer patients (Table 1) [54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69]. CTC models from metastatic melanoma patients that recapitulate the patient's disease and responses to therapies have been developed, highlighting the power of such models as potential 'avatars' for testing personalized treatments in the future [60].…”