SR-4835 promotes ICD induction in breast cancer cells
4T1 cells treated with SR-4835 were assessed for cell death. The results showed a reduction in 4T1 cell viability (Figure 1A) and an increase in apoptosis (Figure 1B) upon SR-4835 treatment in a dose-dependent manner. Similar results were obtained in human breast cancer cells with differences in hormone epidermal growth factor receptor 2 (HER-2), estrogen receptors (ER), and progesterone receptor (PR) expression, including MCF-7, T47D, MDA-MB-231 and MDA-MB-468 cells (Figure 1C and 1D). Based on previous research, not every cell death modality is immunogenic and causes anti-tumor effects (19, 30, 31) , with in vitro ICD identification primarily depending on the detection of damage-associated molecular patterns (DAMPs) (32). Therefore, we assessed 4T1 cells with respect to the role of plasma on the viability of cells and the effects of DAMP signaling, including ATP secretion and CRT translocation.
The surface exposure of CRT regulates the immunogenicity of dying cancer cells (33) . Although present on the ER membrane, CRT sends an ‘eat me’ DAMP signal on the cell surface that triggers the APC-mediated identification, engulfment, and processing of tumor cells (34) . After one day of SR-4835 treatment, the presence of CRT on the surface was measured. Anti-CRT antibodies were used to label intact cells, which were then stained with secondary fluorescent antibodies and evaluated by flow cytometry. We observed a dose-dependent increase in CRT levels on the surfaces of 4T1, T47D and MDA-MB-231 cells, indicating that the immunogenicity of these cells increased due to the SR-4835 treatment (Figure 1E-1F). An important molecule for metabolism is ATP, which is secreted by cells during the process of ICD (35) . ATP secretion even involves pathways that intersect with CRT externalization (35). ATP is involved in ICD once it reaches the extracellular space and acts as a ‘find me’ DAMP signal to promote the engagement and activation of APCs (36) . To assess this DAMP secreted by SR-4835-exposed cells, the cell culture supernatant was collected 10 minutes post-treatment and assessed for extracellular ATP levels, which were observed to increase (Figure 1G and 1H). Furthermore, the secretion of HMGB1 by SR-4835-treated 4T1 cells also increased (Figure 1I and 1J). Taken together, these results suggest that ICD is induced by SR-4835 in breast cancer cells.
SR-4835 induces endoplasmic reticulum (ER) stress through metabolic changes
The ER plays a major role in intracellular signaling pathways that induce ICD (37). The ER stress response begins after initiation eIF2α, a eukaryotic translation factor, becomes phosphorylated, and this protein has also been put forward as an ICD marker (38). Thus, we assessed the level of eIF2α phosphorylation in SR-4835-treated 4T1 cells and observed enhanced eIF2α phosphorylation at serine 51 (Figure 2A). In addition to eIF2α, inositol-requiring enzyme 1 (IRE1), another ER protein acting as a sensor of ER stress, mediates the unfolded protein response (UPR) (39) . Similar to the activation of eIF2α, an increase in the phosphorylation of IRE1 was observed in SR-4835-treated 4T1 cells (Figure 2A). In addition, we also observed p-PERK and BIP upregulation upon SR-4835 treatment (Figure 2A), and the upregulation of p-eIF2α, IRE1, p-PERK, and BIP levels was also observed in T47D and MDA-MB-231 cells after SR-4835 treatment (Figure 2B and 2C). Thus, our findings suggest SR-4835 initiates the ER stress response, which is associated with ICD in breast cancer cells.
ER processes rely on sources of extrinsic energy provided through glycolysis or oxidative phosphorylation (40) . Therefore, we explored if changes in glucose metabolism have a role in extracellular ATP increase. Glucose uptake is the first important step in glucose metabolism, and glucose uptake was observed to be significantly decreased in 4T1, T47D and MDA-MB-231 cells after SR-4835 treatment (Figure 2D-2F). This decrease may be due to the dysregulation of several metabolic steps, such as a decrease in glucose transport. Therefore, we subsequently evaluated the levels of glucose transporters, in these treated cells and observed a significant decrease in GLUT3 protein levels (Figure 2G-2I). Therefore, the induction of ER stress due to SR-4835 occurs possible via ATP secretion, which results from reduced glucose transporter levels, ultimately causing reduced glucose uptake.
SR-4835 enhances DCs function in 4T1 cells
Because DCs play an important role in recognizing ICD-associated DAMPs followed by tumor antigen uptake and presentation (41) , we examined the phagocytosis of SR-4835-treated tumor cells by DCs. We cultured SR-4835-treated 4T1 cells with mouse bone marrow-derived DCs (BMDCs) and observed efficient DC phagocytosis of the treated tumor cells (Figure 3A), which caused enhanced maturation of DCs, as shown by MHCII, CD86, CD80 and CD83 surface expression (Figure 3B-3E). Furthermore, the coculture supernatant exhibited increased IL-1β, TNF-α, IL-6 and IL-12p70 levels (Figure 3F-3I). Thus, our findings indicated that SR-4835 promotes DC activation in cancer cells.
Finally, the immunogenic potential of SR-4835 was assessed in a vaccine setting. Immunocompetent BALB/c mice were injected with 4T1 tumor cells treated with SR-4835 in vitro. Ten days later, the mice were challenged again by injecting live tumor cells, and the mice immunized with SR-4835-treated dead tumor cells exhibited enhanced tumor-free survival compared with those injected with tumor cells that were freeze-thawed (Figure 3J). Taken together, these results suggest that SR-4835 promotes ICD induction in this model.
αPD-1 and SR-4835 combination therapy elicits 4T1 tumor rejection.
We next assessed if αPD-1 therapy is synergized by CDK12/13 inhibition in vivo. To this end, mice were randomized and inoculated with 4T1 tumor cells, and the growth of tumors was assessed every two days (Figure 4A). Tumor growth did not decrease significantly in the 4T1 model by SR-4835 treatment alone, although the median survival was indeed extended. Moreover, there was no effect of anti-PD-1 alone on the survival or growth of the tumor. However, SR-4835 and anti-PD-1 combined led to significant regression of tumors, with complete regression in 50% of tumors observed during the time of the study. On day 8, a remarkable difference in tumor size was observed after treatment, and the survival increased significantly in the combination and control treatment groups (Figure 4B-4E).
Efficacy of the combination treatment depends on CD8+ T cells.
Next, we explored the role of CD8+ T cells as the crucial T-cell component mediating the synergistic effect of combined SR-4835 and αPD-1 on durable tumor regression. CD8+ T-cell ablation was performed in mice bearing 4T1 tumors by depleting antibodies prior to and when combination treatment was done (Figure 5A) and was confirmed by peripheral CD8+ depletion after five days through FACS. No tumor rejection was observed after combination treatment when the cells were depleted of CD8+ T cells (Figure 5B and 5C). Ten days post-treatment, the combination group showed an increase in mean tumor volume together with the depletion of CD8 compared with the tumor volume observed in the non-depleted group. CD8 depletion also significantly decreased survival and had few damaging effects compared with the control in terms of survival and the growth of tumors. This result suggests that CD8+ T-cell-mediated immunity at baseline levels serve to limit the growth of 4T1 under basal conditions. Thus, the synergy of SR-4835 with αPD-1 was dependent upon adaptive immunity mediated by CD8+ T cells in this model.
SR-4835 and anti-PD-1 treatment enhance DC activation and intertumoral CD8+ T cells.
To assess the ability of SR-4835 to in inhibit or facilitate an increase in T cell responses mediated by anti-PD-1, we investigated infiltration and activation of T cells in the tumor. To this end, BALB/c mice with established 4T1 tumors were treated as before with SR-4835 and anti-PD-1. Exactly two weeks after treatment initiation, flow cytometry analysis of tumors was performed after harvesting tissues. Compared with the SR-4835 and anti-PD-1 treatments alone, the number of CD4+ and CD8+ T cells that infiltrated the tumors were increased in combination-treated cells (Figure 6A-6C). Moreover, the expression of the T cell activation marker was higher in tumor-infiltrating T cells compared with that observed for the controls, with the combination treatment group exhibiting the highest proportion (Figure 6D and 6E). To address whether T cell function was enhanced by combination treatment, the dissociated tumors were subjected to intracellular cytokine staining for isolated tumor-infiltrating cells. Compared with the SR-4835 and anti-PD-1 monotherapies, we observed enhanced expression of IFN-γ in CD8+ T cells in the combination treatment (Figure 6F), which also increased the production of granzyme-B (GzB) by CD8+ T cells that infiltrated tumors (Figure 6G). Thus, the above data demonstrate that the combination treatment of SR-4835 and anti-PD-1 antibody increased the functionally active T cell numbers in the tumors.
Because SR-4835 induces tumor cell death, we hypothesized that this activity would lead to the activation of local APCs, thereby enhancing antitumor responses. We indeed observed that combination treatment using SR-4835 and anti-PD-1 enhanced the 4T1 tumor-infiltrating CD11c+ DC cell numbers, which also had increased levels of the activation markers CD80, MHC class II (MHCII), and CD86 in comparison to the cells in the monotherapy groups (Figure 6H-6K). Therefore, these results suggest that therapy combining SR-4835 and anti-PD-1 increases the activation and function of T cells and APC in the tumor microenvironment compared with the single treatments.