The mechanisms underlying lung metastases in bladder cancer (BLCA) remain poorly understood. Cancer-associated fibroblasts (CAFs) are key modulators of the metastatic microenvironment, but how they are activated and contribute to BLCA lung metastases remains unclear. Single-nucleus RNA sequencing was conducted on metastatic lung lesions from patients with BLCA to characterize the tumor microenvironment. Functional and molecular biology experiments, including co-culture assays, luciferase reporter assays, chromatin immunoprecipitation and in vivo lung metastasis models, were performed to explore the mechanisms by which tumor-derived factors and CAFs-secreted exosomes contribute to metastasis. The results revealed that CAFs were enriched in metastatic lung lesions and activated by tumor-derived WNT7A via the Wnt/β-catenin pathway. These activated CAFs promoted BLCA cell proliferation, stemness and migration through the exosomal delivery of miR-1910-5p, which directly suppressed CTDNEP1 expression and activated MYC signaling. Mechanistically, RBMX was identified as a regulator of miR-1910-5p packaging into CAFs-derived exosomes. In vivo, inhibition of CAFs-derived exosomes secretion reduced lung metastasis, highlighting their critical role in metastasis formation. In conclusion, this study uncovers a novel reciprocal activation loop between tumor cells and CAFs in BLCA lung metastases, where tumor-secreted WNT7A activates resident pulmonary CAFs, which in turn enhance tumor malignancy through exosomal delivery of miR-1910-5p. This exosome-mediated crosstalk promotes metastatic progression via the CTDNEP1/MYC signaling pathway. These findings provide potential therapeutic targets for mitigating metastatic progression in BLCA.

Circulating Cancer-Associated Fibroblasts (cCAFs) have been identified in circulating tumor cell clusters from all stages of disease progression in breast cancer patients. We have shown that CAFs promote lung metastases in the mouse tail vein model when they are clustered with triple negative breast cancer (TNBC) MDA-MB231 cells. Following on this observation, we saw that MDA-MB231-luciferase labeled cells persist at higher levels when present in CAF23/MDA-MB231 co-clusters compared to MDA-MB231 mono-clusters within the first 3 days after tail vein injection. This prompted us to investigate whether CAFs aid cancer cell extravasation from capillary venules into the lung parenchyma, which would impart better survival and faster seeding of metastases. Ex vivo lung extravasation assays showed that within the first 8-24 h after tail vein injection, more cells from CAF23/MDA-MB231 co-clusters extravasated than cells from MDA-MB231 mono-clusters. Using in vitro endothelial binding assays, we determined that CAF/TNBC co-clusters bind to HUVEC endothelial cells better than TNBC mono-clusters. Single-Cell RNA sequencing identified several genes in the fibrinolysis pathway whose expression increases in TNBC cells when they are clustered with CAFs. One of these genes is PLAU, which encodes the urokinase-type plasminogen activator, uPA. siRNA knockdown of PLAU decreased in vitro TNBC-endothelial cell interactions and ex vivo extravasation of MDA-MB231 mono-clusters, revealing a potential role for CAF-induced factors expressed in TNBC cells, such as uPA/PLAU, in breast cancer cell extravasation. Our data helps to define the role of CAFs in breast cancer extravasation and highlights the importance of our previous work showing that CAFs promote tumor cell dissemination and metastasis.

Lung adenocarcinoma is a highly malignant and life-threatening disease, with brain metastases representing a major cause of mortality in patients with lung cancer. Although extensive research has focused on interpreting single-cell data from lung cancer brain metastases, most studies emphasize the immune microenvironment, leaving the molecular mechanisms and pathways involving epithelial cells under explored. In this study, we investigated epithelial cells using single-cell data derived from lung cancer brain metastases (comprising six lung cancer and six lung-to-brain metastasis samples) and identified a significant enrichment of the protein-folding pathway in brain metastases, with SEC61G emerging as a particularly prominent contributor. Subsequent in silico replication in clinical samples and public cohort confirmed that SEC61G can serve as a biomarker for brain metastases in lung cancer. These results provide novel insights into the molecular mechanisms driving brain metastases in lung cancer and underscore the potential of SEC61G as both a therapeutic target and a diagnostic marker. Our findings advance understanding of the metastatic process in lung adenocarcinoma and provide a foundation for developing targeted therapies aimed at improving patient outcomes.

Lung squamous cell carcinoma (LSCC) rarely metastasizes to the parotid gland, with only a handful of cases reported in the literature. Its clinical and pathological presentations closely mimic those of primary parotid squamous cell carcinoma (PSCC). Differentiating solitary metastasis from metachronous primary squamous cell carcinoma (SCC) in patients with a history of smoking and lung cancer presents a significant diagnostic challenge. A 55-year-old man with a history of heavy smoking presented with a solitary parotid mass two years after curative LSCC resection. Excisional biopsy with histological and immunohistochemical analyses confirmed SCC. Based on (1) a clear history of a primary tumor with a known metastatic propensity, (2) the absence of other primary sites on whole-body imaging, (3) histological features (highly consistent with the primary tumor type and no evidence of squamous metaplasia and dysplasia in the ductal or acinar epithelium), and (4) an immunohistochemical profile that effectively ruled out common primary parotid neoplasms, we retained solitary parotid metastasis as LSCC recurrence. However, definitive comparative molecular profiling, which is crucial for distinguishing recurrence from a second primary tumor, could not be performed because of resource limitations. Beyond the diagnostic challenge, this case highlights the complex intersection between rare metastatic sites and the risk of developing a secondary malignancy. In such scenarios, the decision to proceed with systemic therapy without definitive molecular proof of clonal relatedness depends on a robust convergence of clinical context, consistent imaging, and highly suggestive histopathological and immunohistochemical findings. It is imperative that traditional pathology and advanced molecular studies converge to provide not only a diagnosis but also a precise biological understanding of the disease.

Patients with breast cancer brain metastases (BCBM) exhibit dismal prognosis, largely due to the insufficient biological understanding and the scarcity of therapeutics that can penetrate the blood-brain barrier (BBB). This study was focused on Rearranged during transfection (RET) receptor tyrosine kinase that has been implicated in non-small cell lung cancer (NSCLC) and thyroid cancer. Here, we report that RET activation is elevated in patient BCBM samples compared to matched primary tumors (N = 30), and in three brain-tropic breast cancer cell lines compared to the parental lines. High RET pathway activation is associated with worse brain metastasis-free survival in patients with HER2-enriched and triple-negative breast cancer (TNBC). Using TNBC cells with ectopic RET overexpression, we demonstrated that RET strongly promoted their preferential metastasis to the brain in mice with intracardiac injections of tumor cells. Using intracranial tumor implantation of the isogenic lines, we found that RET significantly enhanced the formation and progression of brain tumors in vivo. Moreover, we observed that selective BBB-permeable RET inhibition using Pralsetinib and Selpercatinib, FDA-approved for NSCLC and thyroids cancer, significantly reduced cell viability, enhanced apoptosis, and attenuated migration of brain-tropic breast cancer cells in vitro. Using two mouse studies modeling multi-organ metastases and breast tumor formation in the brain, we observed that RET inhibition significantly prevented the circulating tumor cells from forming brain metastases and suppressed the growth of intracranially implanted tumor cells. Together, our findings demonstrated that RET functions as a novel mediator of BCBM and that RET inhibitors showed promising efficacy for BCBM.

A common problem in phylogeny is to reconstruct the ancestral states of a feature measured at the present time. The classic Fitch-Hartigan and Sankoff algorithms compute the most parsimonious or most likely reconstruction. However, these approaches do not readily extend to structured ancestral reconstruction problems, such as those encountered when inferring the routes of metastases in cancer, deriving the transmission history of viruses, or detecting horizontal gene transfer in phylogenetic networks. We develop a combinatorial optimization approach to ancestral reconstruction problems based on the tree-labeling polytope, a geometric object whose vertices represent the ancestral labelings of a tree. We derive algorithms for three structured ancestral reconstruction problems: parsimonious migration history, softwired small parsimony, and convex recoloring. We apply these algorithms to analyze routes of metastasis in a mouse model of lung adenocarcinoma using lineage-tracing data from thousands of single cells.