I2 - Multi-probe Metabolic Fluorescence Microscopy Captures Tumor-Immune Interplay

Introduction: : Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer, with the worst recurrence free survival – it lacks molecular targets, making chemotherapy the main option for treatment, but recurrence rates are still high due to dysregulated metabolism [1]. Immunotherapy has been a hot topic as of late, but the characteristic heterogeneity of TNBC also limits its efficacy to immune checkpoint inhibitors to 20-30% [2]. Being able to image the tumor microenvironment that fosters this inhospitality to immune cells and how each cell is fueling its function will help us identify metabolic signatures that can point us towards immune efficacy or immune evasion. Because tumor cells and immune cells take up similar nutrients for metabolism, we wanted to study their interplay in a controlled environment before moving to a more complex in vivo environment. We designed a tumor-immune coculture experiment where we (1) first validated the dynamic range of our probes when characterizing the metabolism of cytotoxic T lymphocytes (CTLs) from quiescence to activation, (2) then introduced 4T1 TNBC cells in close proximity to the CTLs using a direct coculture format, and (3) finally isolated the effect of cell-cell contact by observing only the effect of secreted factors in an indirect coculture format. We developed an optical imaging tool that can identify distinct populations of cells depending on their condition using three metabolic biomarkers – 2-NBDG for glucose uptake, TMRE for mitochondrial membrane potential, and CTL diameter for cell size.

Materials and

Methods: : In parallel, we cultured 4T1 TNBC cells and CD8+ cytotoxic T cells (or CTLs) (Fig. 1). The cells were plated either alone in monoculture or together in coculture at a 1:1 ratio (D7). We were interested in isolating the effect of direct cell-cell contact when the cells are in proximity with one another, versus when they share the same environment but not in contact. Therefore, we cocultured in either a direct format or in an indirect format using a Transwell permeable membrane. After 24 hours, we separated the cells to be stained with our probes, either 2-NBDG or TMRE, then imaged with confocal microscopy (D8). It is widely established that quiescent T cells upregulate their glycolysis once activated, during which the T cells can use pyruvate for biosynthesis to increase in size [3]. Once they are activated, T cells rely on both glycolysis and oxidative phosphorylation for survival [4]. With that being said, we wanted to validate the dynamic range of our probes by first zooming into the phenotypical changes that occur as CTLs go from quiescence to activation. We characterized the metabolic changes of the CTLs by staining with 2-NBDG or TMRE and imaging on D2, D4, D5, D6, and D7. For all imaging of all studies, we took images of both brightfield and fluorescence. We used the brightfield image to measure the size of the immune cells, which we would then segment using ImageJ and measure the corresponding fluorescence per cell.

Results, Conclusions, and Discussions:: We report distinct CTL populations during activation states. 2-NBDG quickly increases, while there is a gradual increase in TMRE with subsequent activations, suggesting a shift towards oxidative phosphorylation (Fig. 2A-B). We characterize “low” and “high” signals and show population shifts from low diameter/low 2-NBDG (55%) to high diameter/high 2-NBDG (90%) (Fig. 2C). Most activated CTLs also show higher mitochondrial metabolism by moving from low diameter/low TMRE (95%) to high diameter/high TMRE (80%) (Fig. 2D). However, both probes see smaller intermediate populations within the activated state that are not distinguishable in size.

We hypothesized that direct coculture of CTLs with 4T1s will result in fatigued CTLs; whereas in indirect coculture, we hypothesized that secreted factors would induce metabolic changes to prepare for opposition [5,6]. The direct coculture (Fig. 3A-B) demonstrates CTLs sustaining 2-NBDG signal and dropping significantly in TMRE. When splitting CTL populations by “low” and “high” signal, most direct coculture CTLs (65%) maintain their glucose uptake but decrease diameter significantly, and we see an increased “tired” population (35%) with low diameter/low glucose uptake (Fig. 3C). Most CTLs in direct coculture (65%) show decreased diameter and mitochondrial activity from monoculture (80%), but there remains a population that maintains TMRE signal (35%) (Fig. 3D). In indirect coculture, we see most CTLs (75%) maintain 2-NBDG and, interestingly, significantly increase their size (Fig. 3G). Like direct, a low diameter/low 2-NBDG “tired” population appears (25%) (Fig. 3G). For TMRE, we again see diameter increase and a concomitant increase in mitochondrial activity (70%) (Fig. 3H), suggesting CTLs are still using oxidative phosphorylation and are possibly primed by tumor-secreted factors, in contrast to direct coculture where TMRE is reduced.

Using three metabolic endpoints, we observed that activated CTLs upregulate glycolysis and biomass production, followed by increased oxidative phosphorylation that fuels survival. In contact with 4T1s, we pinpoint populations of “tired” CTLs with decreased metabolic activity and size. Without contact with 4T1s, we observe CTL metabolic reprogramming and a slight increase in size. These biomarkers not only inform us of the cells’ activation state, but also their metabolism to quantify the influence of cell-cell contact on CTL behavior.

Acknowledgements and/or References (Optional):: [1] Wang, T. et al. (2018) Cell metabolism. [2] Ribeiro, R. et al. (2022) Front. Mol. Biosci. [3] Pearce, E. (2010) Curr Opin Immunol. [4] Chang, C. et al. (2013) Cell. [5] Jiang, Y. et al. (2015) CDD. [6] Valbon, S. et al. (2016) Cytokine.