Abstract 1243: The pro-metastatic phenotype of chemoresistant breast cancer is stable and transcriptionally defined

Abstract Chemotherapy remains the most used systemic treatment option for triple-negative breast cancer. Lineage tracing studies demonstrated that the treatment does not significantly alter the clonal composition of primary breast tumors. Nevertheless, the specific transcriptional phenotype(s) conferring chemoresistance at primary tumor (PT) level, as well as at metastatic level, are still largely undetermined, specifically at single-clone resolution.To tackle such unmet clinical issues, we leveraged our recently published in vivo single-cell lineage tracing approach under therapeutic pressure, using two chemotherapeutic agents: adriamycin and cyclophosphamide (A+C). Upon administration of A+C we obtained stable disease, as confirmed by histopathological and IHC analyses. Chemoresistance was formally demonstrated by ex vivo IC50.By using single-cell lineage tracing on treated and untreated matched PT-lung metastases, we found that chemotherapy shifted clonal hierarchy, with few high-fit clones outcompeting the others, at primary level. Despite this, we noticed the striking increase of pro-metastatic clone frequencies in the chemoresistant model, thus suggesting that chemotherapy selects for few highly-aggressive clones. At metastatic level we found general drop of clonality.Using scRNA-seq, we observed that chemotherapy shapes distinct chemoresistant patterns in the PT and metastases. We then explored the transcriptomics features of the most fit clones within treated tumors, so-called dominant: they were characterized by enrichment of distinctive cancer hallmarks with respect of the whole chemoresistant tumor, including tumor-promoting inflammation and sustained angiogenesis.Chemoresistant/pro-metastatic clones were characterized by the expression of genes that were prognostic for poor survival across patients. Importantly, we observed that the chemoresistant/pro-metastatic phenotype was exacerbated by re-challenge with the same treatment but also stable across multiple re-transplantations. Among the of chemoresistant/pro-metastatic genes, PAEP appeared as the top marker. This was corroborated by GRN analyses, where we observed that the network around PAEP was extremely perturbed by therapeutic pressure. We tested the pro-metastatic effect of PAEP expression in a chemoresistant scenario. We orthotopically injected MDA-MB-231 cells silenced for PAEP expression via shRNA into recipient NSG mice and we performed neoadjuvant and adjuvant chemotherapy. Upon PAEP silencing, we observed slight increase of chemosensitivity at primary tumor level. Strikingly, chemoresistant metastases formation was significantly impaired by PAEP downregulation, as compared to controls.Overall, our data demonstrate that the pro-metastatic phenotype of chemoresistant breast cancer is stable, transcriptionally defined and driven by the expression of PAEP. Citation Format: Alberto Dalmasso, Andrea Cossa, Ilaria Servidio, Guido Campani, Elisa Bugani, Francesco Antonio Tucci, Lucrezia Stabellini, Salvatore Pece, Pier Giuseppe Pelicci. The pro-metastatic phenotype of chemoresistant breast cancer is stable and transcriptionally defined [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1243.