A melanoma brain metastasis CTC signature and CTC:B cell clusters associate with secondary liver metastasis: a melanoma brain-liver metastasis axis.

Melanoma brain metastasis (MBM) is linked to dismal prognosis, low overall survival, and is detected in up to 80% of patients at autopsy. Circulating tumor cells (CTCs) are the smallest functional units of cancer and precursors of fatal metastasis. We previously employed an unbiased multilevel approach to discover a unique ribosomal protein large/small subunits (RPL/RPS) CTC gene signature associated with MBM. Here, we hypothesized that CTC-driven MBM secondary metastasis ("metastasis of metastasis" per clinical scenarios), has targeted organ specificity for liver. We injected parallel cohorts of immunodeficient and newly-developed humanized NBSGW (HuNBSGW) mice with cells from CTC-derived MBM to identify secondary metastatic patterns. We found the presence of a melanoma brain-liver metastasis axis in humanized NBSGW mice. Further, RNA-Seq analyses of tissues showed a significant upregulation of the RPL/RPS CTC gene signature linked to metastatic spread to liver. Additional RNA-Seq of CTCs from HuNBSGW blood revealed extensive CTC clustering with human B cells in these mice. CTC:B cell clusters were also upregulated in blood of primary melanoma patients, and maintained either in CTC-driven MBM or MBM CTC-derived cells promoting liver metastasis. CTC-generated tumor tissues were interrogated at single-cell gene and protein expression levels (10x Genomics Xenium and HALO spatial biology platforms, respectively). Collectively, our findings suggest that heterotypic CTC:B cell interactions can be critical at multiple stages of metastasis.