(G-247) Tumor Heterogeneity in Hypoxia-induced Metabolic Rewiring

Introduction: Rewiring of cellular metabolism in response to the tumor microenvironment (TME) is a hallmark of cancer[1]. Metabolic rewiring promotes heterogeneity of metabolic phenotypes intratumorally, between cancer types, and among patients, which contributes to differential drug sensitivities and limits the efficacy of existing cancer therapies[2]. It is thus crucial to evaluate the heterogeneous landscape of cancer metabolism to develop new therapies that overcomes such therapeutic resistance. However, the existing metabolic profiling approaches such as mass spectrometry and next-generation sequencing are often resource-intensive and difficult to capture the dynamic rewiring mechanisms. Limited oxygen availability (i.e. hypoxia) in the TME is among the main driving forces of metabolic rewiring and heterogeneity in cancer cells[3]. In our previous study, we have identified two cancer cell types with distinct rewiring behaviors using an in vitro microtumor model that drives differential metabolic rewiring of cancer cells through physical limits of oxygen delivery as seen in TME. Here, we have equipped each cell type with genetically encoded fluorescent sensors that detect the activation of hypoxia-inducible factors (HIFs) and investigate their response to different severity of hypoxia.

Materials and Methods: Cancer cell types with distinct response to hypoxia were identified (Fig. 1A) and engineered to express UnaG, a fluorescent protein which does not rely on molecular oxygen for its fluorescent maturation, by HIF activation via lentivirus transduction (Fig. 1B). Transduced cancer cells were isolated based on tCD19 expression (constitutively expressed) with fluorescence-activated cell sorting. Isolated cells were cultured in different oxygen levels (21%, 4%, 2%, or 1% oxygen) and their response was quantified. Differences in the response to hypoxia was further illustrated by examining the change in each cancer cell type’s mitochondrial activity using TMRM (mitochondrial membrane potential) and CM-H2DCFDA (reactive oxygen species; ROS) staining.

Results, Conclusions, and Discussions: Prostate cancer cells (LNCaP) respond to a more severe level of hypoxia compared to ovarian cancer cells (SKOV3), 2% vs. 4% oxygen, respectively (Fig. 1C). Interestingly, in severe hypoxia (i.e. < 2% oxygen), the magnitude of rewiring, represented by the UnaG fluorescence intensity, is higher for prostate cancer cells compared to ovarian cancer cells. Overall, this suggests that each cancer cell type has a specific threshold of hypoxia that is needed to induce rewiring, and each cancer cell type has an intrinsic limit of rewiring, even in severe hypoxia. It is reported that the activation of HIF-1 is bound to the mitochondrial function, and mitochondria act as oxygen sensors. Therefore, we hypothesized that the observed differences in the rewiring behaviors were related to their change in mitochondrial function in response to hypoxia. As noted by the change in TMRM staining, mitochondria become more active under severe hypoxia for prostate cancer cells but more inactive for ovarian cancer cells (Fig. 1D). Furthermore, prostate cancer cells increased their mitochondrial production of ROS (CM-H2DCFDA) in response to hypoxia, while the ovarian cancer cells did not show a significant response. Overall, this suggests that mitochondria may contribute to the hypoxia-induced rewiring mechanisms.

Our engineered cells provided real-time visual insight of intertumoral heterogeneity related to cancer cells’ intrinsic response to hypoxia. Our study illustrated cell-specific rewiring behaviors and mechanisms that change dynamically based on the severity of hypoxia. Furthermore, we demonstrated that the difference in their response to hypoxia is also related to the changes in their mitochondrial activity and ROS production.

Acknowledgements (Optional): This work has been supported by an NIH NCI grant R01CA220012, STOP CANCER Marni Levine Memorial Research Career Development Award, and USC Viterbi School of Engineering.

References (Optional): :
[1] Hanahan, D., et al. Cancer Discov. 2022 12(1): 31–46. [2] Frezza, C., Br J Cancer. 2020 122,133-135. [3] Kim & DeBerardinis, Cell Metab. 2019 30, 434-446.