Three-dimensional electron microscopy uncovers mitochondrial network remodeling in residual triple negative breast cancer persisting after conventional chemotherapy treatments

ABSTRACT Mitochondria are hubs of metabolism and signaling, playing crucial roles in tumorigenesis, therapeutic resistance, and metastasis in many types of cancer. Various laboratory models of cancer demonstrate the extraordinary dynamics of mitochondrial structure, but little is known about the full extent of the complexity of the mitochondrial network, nor its regulation upon exposure to therapeutic stressors. We previously demonstrated the importance of mitochondrial structure and oxidative phosphorylation in the survival of chemotherapy-refractory triple negative breast cancer (TNBC) cells. As TNBC is a highly aggressive breast cancer subtype with few targeted therapy options, conventional chemotherapies remain the backbone of TNBC treatment. Unfortunately, approximately 45% of TNBC patients retain substantial residual tumor burden following chemotherapy, associated with abysmal prognoses. Herein we present the first three-dimensional analysis of mitochondrial networks in human tumor tissues. Use of two experimentally tractable orthotopic patient-derived xenograft (PDX) models TNBC enabled us to conduct longitudinal analyses to construct mitochondrial networks in treatment-naïve and residual tumors persisting after exposure to a variety of conventional chemotherapies. Further, we modeled lipid droplet (LD) structures and their physical contacts with mitochondria. In total, we reconstructed 3,750 mitochondria and 800 LDs in three dimensions using serial block-face scanning electron microscopy (SBF-SEM), providing unprecedented insights into the complexity and intra-tumoral heterogeneity of mitochondria in TNBC. Both carboplatin (CRB) and docetaxel (DTX) chemotherapies produced residual tumors that harbored mitochondria with significantly increased areas, volumes, and perimeters in both PDX models. Additionally, treatment with the conventional combinations DTX plus CRB or Adriamycin plus cyclophosphamide (AC), led to reduced mitochondrial branching and elongation. In contrast, DTX or CRB alone elicited model-specific changes in mitochondrial complexity. Further, the extensive intra-tumoral heterogeneity of mitochondrial structure in untreated PDX tumors significantly decreased in residual tumors. Analyses of LDs revealed significant and consistent elevation of the number and physical proximity of MLCs in residual tumors, congruent with our previous studies providing evidence for transcriptomic and proteomic rewiring of lipid metabolism in residual TNBC. These results highlight the potential for structure-based monitoring of chemotherapeutic metabolic reprogramming and suggest unique molecular mechanisms that may underlie chemoresistance in TNBC. Furthermore, our findings provide novel insights into a new type of intratumoral heterogeneity, that of mitochondrial intratumoral heterogeneity, which complements our understanding of the genomic, epigenomic, transcriptomic, and proteomic complexity of TNBC.