Abstract P1-06-04: Spatial, temporal and therapy dependent heterogeneity in transport phenotype of multiple liver metastases in murine breast cancer model
Authors: Nguyen Thao , ting Liu Yan , Ziemys Arturas , Haifa Shen , Yokoi Kenji 
Published: March 2022

Introduction: Inter- and intra-tumor heterogeneities in response of tumors to systemic anti-cancer therapy are the major source of therapeutic resistance. Cancer cells from bulk tumors and cancer cell lines are not clonal but are composed of subclones, creating heterogeneous tumor microenvironment. Furthermore, metastasis can evolve during the disease progression as well as in response to systemic therapies. These spatial and temporal heterogeneities in tumors can affect blood perfusion and vascular permeability which determine the transport of various masses and chemotherapeutic (we refer to as “transport phenotype”). Insufficient drug transport to the tumor in vivo can generate transport-based therapeutic resistance even for agents with proven efficacy against the target cells in vitro. Our goal is to understand spatial and temporal heterogeneity in the transport of mass/anti-cancer drugs to metastatic tumors to leverage novel strategies for improving therapeutic efficacy using mouse tumor models. The study of bulk tumors will provide a mixture of different functions from heterogeneous sets of various clones. We sought to establish single cell-derived clones from the parental murine breast cancer cell line to dissect and simplify tumor heterogeneity. Methods: Single cells were isolated via aliquoting 4T1-luc-tomato red cell suspension (polyclonal) in single-cell concentration into 96 well plate. To evaluate the phenotype of the clonal and parental cells, we performed clonogenic assay, proliferation and growth inhibition assay using pegylated liposomal doxorubicin (PLD), and angiogenic protein production assay using membrane array. Among the established clones, we selected 2 clones and parental cells for further experiments. Experimental liver metastases were created by injecting cell suspension into the spleen of BALB/c mice. Then, the window chamber was implanted above the liver of anesthetized mice and fluorescently labeled dextran tracers with different molecular weights were intravenously (iv) injected. To determine transport phenotype, circulation and diffusion of tracers inside/around individual metastasis were imaged and quantified through the window using Intravital Microscopy (IVM) at various time points. In another set of experiment, PLD was iv injected, and change in the transport phenotype was monitored. Results: While parental cells formed heterogeneous colonies, clonal cells created each cell line dependent uniform colonies in petri dish. Also, parental and selected two clonal cells exhibited different growth, response to PLD, and protein production in vitro. IVM imaging of individual liver metastasis over time using different cell lines revealed that relative fluorescent intensity of tracers transported inside/around tumor compared to that in the uninvolved tumor-free liver was more heterogeneous among parental cell derived tumors compared to clonal cells. Interestingly, these “transport phenotypes” evolved and increased their heterogeneity as tumors progressed regardless of cell type. Furthermore, transport phenotype was modulated from one to another after PLD therapy, creating possible mechanisms of transport based on drug resistance. Discussion: These data suggest that transport phenotype cannot be determined solely by genotype of tumors but modulated as a function of tumor growth and therapy response to become more heterogeneous. The intrinsic heterogeneity of transport phenotype and its acquired transformation suggests a more complicated scenario of tumor drug-resistance mechanism. The clinical implication of our findings is that the transport of therapeutics to the tumor and its efficacy may not be predicted by DNA-based assay and transport-based therapeutics resistance will eventually occur in every tumor type.

Citation Format: Thao Nguyen, Yan ting Liu, Arturas Ziemys, Shen Haifa, Kenji Yokoi. Spatial, temporal and therapy dependent heterogeneity in transport phenotype of multiple liver metastases in murine breast cancer model [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-04.