(P 412) LEIOMYOSARCOMA POLYANEUPLOID CANCER CELLS DEVELOP IN RESPONSE TO CHEMOTHERAPY AND CONTRIBUTE TO CHEMORESISTANCE

Objective: Polyaneuploid cancer cells (PACCs) are large, treatment resistant cancer cells identified in different cancer types, sometimes called “giant cells.” PACCs have stem cell-like phenotypes and appear to play a role in treatment resistance that leads to poor patient outcomes. The PACC phenotype is a transient state that cancer cells can adopt to protect themselves from stress. PACCs give rise to non-PACC progeny that maintain resistance to chemotherapy. While PACCs are documented in many cancer types, they have not yet been documented in sarcoma. We identified PACCs for the first time in cell culture from leiomyosarcoma (LMS) patient tumors. The current study characterizes LMS PACCs in vitro, in vivo, and in patient samples. This effort may identify precision-based approaches to therapeutically target LMS PACCs.

Methods: LMS PACCs in vitro: LMS01, LMS03, LMS04, and LMS05 cells were treated with doxorubicin, docetaxel, or gemcitabine for 7 days followed by 7 days without chemotherapy to enrich for PACCs. PACC progeny were then generated by culturing the enriched LMS PACCs for 1-2 weeks without chemotherapy. Cells were imaged before treatment (parent cells) and after 7 days of chemotherapy treatment followed by 7 days without drug (PACCs). Cell size, DNA content, and in vitro response to chemotherapy were measured and compared between parent, PACC, and PACC progeny.
LMS PACCs in vivo: In vivo PACC formation was assessed by xenograft. LMS tumors were grown in mice, followed by treatment with doxorubicin. Tumors were harvested 4-5 days post-treatment and imaged for PACC formation by staining with DAPI (DNA) and CD47 (tumor cell marker).
LMS PACCs in human patients: LMS human patient microarrays were stained with H&E and analyzed by a pathologist to determine the percent of samples with giant cells and the percent with abnormal nuclei.
LMS PACC response to p53 expression: LMS PACC cells were transfected to express elephant p53 or mCherry with or without doxorubicin and apoptosis was quantified.

Results: LMS cells treated in vitro and in vivo with chemotherapy (doxorubicin, gemcitabine, or docetaxel) led to the development of large cells with atypical nuclei as the predominant cellular phenotype (Figures 1 and 4). In vitro, these LMS PACCs were more resistant to chemotherapy (Figure 2) and repopulated the culture with non-PACC LMS progeny cells potentially through a budding process called neosis. These LMS PACC progeny cells maintained a significantly decreased (p < 0.0001) response to chemotherapy compared to parent cells (Figure 3). PACCs were identified in clinical samples from patients with LMS (Figure 4). 29% of LMS samples contained abnormally large tumor cells and 53% contained abnormal nuclei. LMS PACCs transfected with functional TP53 undergo rapid apoptosis upon gene expression. Additionally, combining functional TP53 expression with low doses of doxorubicin increases the apoptotic response of LMS cells, potentially by targeting PACCs.

Conclusion: We offer the first description and in-depth characterization of LMS PACCs. These large, non-dividing cells are chemoresistant and may contribute to the lack of clinical response often seen in LMS. Current research efforts are focused on correlating LMS PACCs in clinical samples with initial response to therapy, as well as long-term patient outcomes. Identifying therapeutic approaches to target and kill LMS PACCs may improve patient response to chemotherapy and clinical outcome. Given the very high rate of TP53 involvement in LMS, TP53 loss may contribute to the ability of LMS PACCs to form abnormal, multiple nuclei. Ongoing investigations may support the development of effective TP53 based therapeutics to target PACCs and to improve outcomes for LMS patients.