Tumor PD-L1 expression is upregulated after radionuclide or radiotracer stimulation in vitro
First, we validated the radionuclide-induced PD-L1 upregulation in multiple tumor cell lines. For quick reference, Fig. 1A lists the radionuclides used in this article. 18F, 99mTc, 177Lu, 64Cu and 131I were compared on multiple tumor cell lines (melanoma, breast and colorectal cancer cells) in the immunofluorescence assay (Fig. 1B), which revealed that different radionuclides upregulated PD-L1 expression to different degrees. This stimulation was also embodied prominently through the flow cytometric analysis. As shown in Fig. S1, the proportions of PD-L1-positive cells in the CT26, MC38, 4T1 and B16F10 tumor cells were significantly increased after co-incubation with radionuclides. Taking 18F for instance, the percentages of PD-L1 positive cells increased from 23.3% to 96.5%, 54.3% to 98.7%, 21.8% to 60.6%, and 61.4% to 96.2% in the CT26, MC38, 4T1 and B16F10 tumor cells after 24 h, respectively. Heat maps generated from reverse transcription-quantitative real-time PCR (RT-qPCR) analysis (Fig. 1C) revealed that all the radionuclides increased the expression of PD-L1 mRNA on tumor cells. For example, after 8 h co-incubation, 18F increased PD-L1 mRNA on MC38 and CT26 cells by 53-fold and 17-fold, respectively. For radiotherapeutic isotopes, 64Cu increased PD-L1 mRNA on MC38 cell by 28-fold, and 177Lu increased PD-L1 mRNA on CT26 cell by 22-fold.
The expression of PD-L1 was elevated to a greater extent by a higher dose of radiotracer, which was further confirmed by flow cytometric analysis in Fig. 1D, clearly indicating that PD-L1 was upregulated in a dose-dependent manner. The expression levels of PD-L1 mRNA and protein in MC38 and CT26 cell lines after stimulation with 2-[18F]FDG were further evaluated by Western blot (WB) (Fig. 1E). As expected, PD-L1 expression was significantly increased in response to radionuclides.
Differentially expressed genes (DEGs) and potential mechanisms of radionuclide-induced PD-L1 upregulation
Transcriptomic analysis and WB study were performed to explore the potential mechanism of PD-L1 upregulation stimulated by radionuclides. From the volcano plot (Fig. 1F), there were a total of 2002 DEGs which had changed in 2-[18F]FDG-treated MC38 cells compared to the control group, with 1223 upregulated genes and 779 downregulated genes (|log2(FC)| > 1.0, P-value < 0.05). For 2-[18F]FDG-treated CT26 tumor cells, the changed number was 2167 (1357 upregulated genes and 810 downregulated genes).
A total of 21725 genes and 21144 genes were identified in 2-[18F]FDG treated MC38 cells and CT26 cells, respectively. As shown in Fig. 1G, Fos, Stat3, Nfkbia, Nfkbib, Nfkbie and Cd274 (PD-L1) genes in 2-[18F]FDG treated MC38 cells were significantly upregulated compared with the untreated cells. Note that Nfkbia, Nfkbib and Nfkbie genes belong to the NF-kappa-B (NF-κB) inhibitor family, which has been reported to upregulate PD-L1 transcription in tumor cells, such as ovarian cancer, gastric carcinoma and lung cancer19-21. As verified in previous studies22, 23, the IκBα kinases (IKK) is a key regulator of the NF-κB pathway and TANK-binding kinase 1 (TBK1) is closely related to the phosphorylation of IRF3.
We also showed the similar results that the radionuclide-induced PD-L1 upregulation was positively correlated with phosphorylated NF-κB P65 (p-NF-κB P65) and phosphorylated IRF3 (p-IRF-3) in radionuclide-treated MC38 cells (Fig. 1H). Intriguingly, we found that the PD-L1 upregulation in radionuclide-treated MC38 cells could be blocked by the inhibitors of IKK or TBK1. All these data suggested that the activation and phosphorylation of NF-κB and IRF3 have contributed to promoting PD-L1 expression in radiation-induced MC38 murine colon carcinoma cells.
DEGs were mapped into the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database to further explain the individual function analysis. Herein, transcriptome analysis using the RNA-seq technology was applied to compare DEGs between 2-[18F]FDG and saline-treated MC38 cells. As shown in Fig. 1I, many receptor-interaction signaling pathways and metabolic pathways were significantly enhanced, including the cytokine-cytokine receptor interaction, the NOD-like receptor signaling pathway, necroptosis, TNF signaling pathway, IL-17 signaling pathway, and the Jak-STAT signaling pathway. According to the literature reports, the NF-κB signaling pathway is one of the major NOD-like receptor signaling pathways, that Nod1 and Nod2 stimulation induces NF-κB activation24. Besides, TNF (also known as TNF-α) could induce the expression of NF-κB target genes and trigger the activation of NF-κB signaling pathway, which indirectly upregulated PD-L1 expression25,26. These results suggest that multiple inflammatory signaling pathways and metabolic process participate in 2-[18F]FDG induced MC38 cells.
Radiotracers cause PD-L1 upregulation in tumor tissue
With the help of the IHC technique, PD-L1 expression in tumor tissue was compared between different groups. We first investigated the biodistribution profile of 2-[18F]FDG and 131I-αPD-L1 in CT26 and MC38 tumor types at different time points using a small-animal PET scanner. The signals in the tumor sites were well delineated from that of other tissues, indicating the high affinity of the radiotracers to tumor lesion (Fig. 2A). Strikingly, as shown in Fig. 2B,C, PD-L1 levels in the tumor region with radiotracers uptake were more strongly positive compared to control samples.
IFN-γ and active CD4+/CD8+ T cells in TIME play important roles in mediating antitumor immunity. For this reason, we used 2-[18F]FDG as a stimulus to observe the responses of TIME and determine the optimized immunotherapy time window for the administration of αPD-L1 mAb. From the IHC in Fig. 2D,E, we could see that 2-[18F]FDG gradually upregulated the expression of IFN-γ, and enhanced the infiltration of CD4+ and CD8+ T cells over time. Prior studies have demonstrated that the expression of PD-L1 has been an inclusion criterion for selecting patients of non-small cell lung cancer (NSCLC) for anti-PD-L1 treatment27,28. To date, PD-1/PD-L1 ICBs have shown promise in advanced NSCLC without driver oncogene mutations, but wider use is restricted to the low objective response rate29,30. In this study, we established NSCLC-PDX models to clarify this T2T immunoediting effect further. After 2-[18F]FDG PET imaging (Fig. 3A,B), the PDXs were divided into high and low 2-[18F]FDG uptake (denoted as PDXH-FDG and PDXL-FDG) groups to evaluate the in vivo biological behavior of radiotracers and predict the PD-L1 response of NSCLC to the radionuclide. Flow cytometry analysis revealed that the PD-L1 positive population in PDXL-FDG (24.9 ± 3.5%) was lower than that in PDXH-FDG (76.9 ± 4.9%) at 4 h p.i. (Fig. 3C,D). For PDXL-FDG tumor, PD-L1 expression increased from 16.2 ± 2.3% (injected with saline) to 24.9 ± 3.5% (injected with 2-[18F]FDG) at 4 h p.i., *p ≤ 0.05). For PDXH-FDG tumor, this uptrend was even more noticeable (from 40.1 ± 6.5% to 76.9 ± 4.9%, **p ≤ 0.01). That is probably means improved PD-L1 expression in 2-[18F]FDG groups was associated with the stimulation of 18F. Consistent with flow cytometry results, PD-L1 IHC showed more prominent expression in post-tracer PDXH-FDG tumor biopsies (Fig. 3E). Hence, as we have observed, the T2T effect of radionuclides could reasonably explain the PD-L1 upregulation in the tumor.
2-[18F]FDG causes increased αPD-L1 uptake in tumor
Intuitively, the PD-L1 upregulation caused by radiotracer would increase αPD-L1 mAb uptake in the tumor. We confirmed this with a fluorescent αPD-L1 probe (Fig. 4A). The flow cytometric analysis showed that 2-[18F]FDG groups had high uptake of fluorescent probe Cy5.5-αPD-L1 which further increased over time, presumably due to the upregulated PD-L1 levels in CT26 and MC38 tumor cells. In striking contrast, much lower Cy5.5-αPD-L1 uptakes were observed in the control tumors without 2-[18F]FDG treatment. Representative histograms of the PD-L1 expression after radionuclides stimulation were shown in Fig. 4B.
T2T effect sensitizes the TIME to immunotherapy and enhances the immunological memory
To further explore the T2T potential of radiotracers for enhancing immunotherapy, we subsequently investigated the effect of αPD-L1 mAb on MC38 tumor growth delay in cooperation with 2-[18F]FDG. As shown in Fig. 5A and Fig. S2A, tumor models were treated with either 2-[18F]FDG, αPD-L1 mAb, or their combination in specific treatment sequences. In the combination groups, αPD-L1 mAb was tail vein injected into the tumor-bearing mice at different intervals (simultaneous injection, 4-h and 24-h; hereinafter referred as @ 4 h and @ 24 h) after administration of the radiotracer. Fig. 5B,C and Fig. S2B illustrated the tumor volumes, time-dependent tumor growth curves, weight changes and survival curves for each group. In the control groups of αPD-L1 mAb and saline alone, the tumor sizes developed uncontrollably. Also, single-administration of 2-[18F]FDG did not significantly alter MC38 tumor growth. We then compared the therapeutic effect of αPD-L1 mAb which was administered simultaneously, 4 h or 24 h post radiotracer injection. Notably, the 4-h interval turned out to be the most optimal treatment sequence, and administration of 37 MBq 2-[18F]FDG + 400 mg αPD-L1 mAb @ 4 h resulted in the maximum therapeutic efficacy (5/8 of the tumor mice were completely cured), clearly indicating that the T2T antitumor immunotherapy was regulated in a dose and time-dependent manner. As shown in Fig. 5D, we performed 2-[18F]FDG-PET imaging on day 0 and day 90 to provide visualization for evaluating therapeutic effect in the best-performing group. Moreover, the body weights of mice were almost identical for all groups during the therapy period (Fig. 5C) and no obvious side effects were observed in the fully recovered mice (Fig. S3), indicating that the T2T antitumor therapeutic strategy was well tolerated.
To verify the immunological memory of T2T-based immune checkpoint therapy, the effector memory T (TEM) cells (CD8+CD44+CD62L− and CD4+CD44+CD62L−) in the spleen were detected and analyzed (Fig. 5E-G and Fig. S4). As expected, the levels of splenic TEM cells gradually increased between day 1 and day 7 in the best-performing groups (37 MBq 2-[18F]FDG + 400 mg αPD-L1 @ 4 h), which was higher than the saline group. Further, for 2-[18F]FDG-induced immunotherapy, the splenic TEM cells remained at a high level until 60 days after the combined treatment. These results demonstrated that the prevention of tumor recurrence by T2T-based immunotherapy was credited to the activation of immunological memory effect.
Coupling 2-[18F]FDG with anti-PD-L1 antibody reprograms TIME
The impact of radiotracer to PD-1/PD-L1 ICBs is multifaceted. Fig. 6A summarizes the potential mechanisms of T2T-based immunologic responses, which can help us reconsider the role of 2-[18F]FDG in tumor imaging and immunotherapy. Transcriptomics analysis focused on the DEGs during the treatment process. In Fig. 6B, compared with the saline group, the CD274 gene (PD-L1) in the tumor of 2-[18F]FDG or 2-[18F]FDG + αPD-L1 @ 4 h groups was upregulated on day 1. A few days later, this indicator showed a fairly noticeable decline.
As shown in Fig. S5, compared to the saline group and αPD-L1 group, the enhanced change of tumor PD-L1 level following 2-[18F]FDG alone or combined immunotherapy strongly predicted response to radionuclide stimulus. Then this indicator showed a tendency to decrease during the later period. Contrary to the trend of PD-L1, we observed increased level of IFN-γ for radiotracer-induced immunotherapy (Fig. S6A,B). Further results indicated that both the CD4+ Th1 (IFN-γ+CD4+ T cells) and CD8+ cytotoxic T lymphocytes (IFN-γ+CD8+ CTLs) in TIME were enhanced from day 1 to day 7 in the group of 2-[18F]FDG + αPD-L1 mAb @ 4 h, whereas the levels of these indicators were unaltered in the saline group. Meanwhile, tumor samples were harvested for detecting proliferation and apoptosis by immunofluorescence staining of Ki67 and Caspase3. As depicted in Fig. S6C, the dynamic change of PD-L1 expression from day 1 to day 7 in the combination therapy groups were further validated. As expected, at the corresponding time points, the Ki67 indexes were significantly higher in saline groups. While the positive rate of Caspase3 expression in the combination group was significantly higher than that in saline groups.
Similar to the IHC and immunofluorescence results, tumor PD-L1 expression measured by flow cytometry showed a decreasing trend during the period of day 1 to day 7 in groups containing radiotracer (Fig. 6C,D). ELISA assays were performed to measure the levels of immunostimulatory cytokines in the serum of mice. The combination of 2-[18F]FDG and αPD-L1 mAb increased the production of IFN-γ, TNF-α and IL-6, and maintained for a long period in blood, which might also explain for the unexpected synergistic anticancer efficacy (described in Fig. 6E). Additionally, the flow cytometric results in Fig. S7A,B showed that intratumoral CD4+ Th1 and CD8+ CTLs become exhausted on day 3 and day 7 in the 2-[18F]FDG group. However, the addition of αPD-L1 mAb @ 4 h significantly increased numbers of CD4+ Th1 and CD8+ CTLs compared to the other groups. However, as one type of CD4+ T cells, the immunosuppressive CD4+ FOXP3+ regulatory T cell (Treg) in tumors showed a decrease in 2-[18F]FDG + αPD-L1 mAb @ 4 h group. Specifically, further comparative analysis showed a significant increase of CD4+ Th1/Treg and CD8+ CTLs/Treg ratios in the combination group (Fig. 6F). In the 2-[18F]FDG group, we observed slight increase in CD4+ Th1/Treg and CD8+ CTLs/Treg ratios on day 1. Over time, these ratios seem to be on a downward trend.
Other alterations of immune cells are also notable. M2-like macrophages, M1-like macrophages, myeloid-derived suppressor cells (MDSCs) and dendritic cells (DCs) were detected through flow cytometry (Fig. 6G and Fig. S8). The MC38 tumor-bearing mice that received 37 MBq 2-[18F]FDG + 400 mg αPD-L1 @ 4 h decreased the fraction of M2-like macrophages (CD206+CD11b+F4/80+) in the first few days, implying the reduced immunosuppression. While an opposite tendency was observed in the 2-[18F]FDG group. Similarly, the radionuclide-induced PD-1/PD-L1 immunotherapy generated a 2-3 fold decrease in the fraction of MDSCs (CD45+CD11b+Gr-1+) compared with the saline group or 2-[18F]FDG alone. We also detected pro-inflammatory M1-like macrophages (iNOS+CD11b+F4/80+) and activated DCs (CD80+CD86+) in the TIME. Treatment with 2-[18F]FDG + αPD-L1 @ 4 h led to a significant increase in the fraction of M1-like macrophages, further indicating the repolarization of M2-like macrophages or recruitment of M1-like macrophages. KEGG enrichment analysis was performed to identify the detailed immune activation associated pathways and inflammatory signaling pathways mediated by the therapeutic strategy of 2-[18F]FDG + αPD-L1 @ 4 h. Several representative pathways were shown in Fig. 7A. The most significant differences between the saline and 2-[18F]FDG + αPD-L1 @ 4 h groups were found in the antigen processing and presentation, phagosome, cell adhesion molecules, the NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction and Th17 cell differentiation. Moreover, we went a step further to confirm that the external radiotracers would affect the expression of PD-1, another key component of immune checkpoint blockade (Fig. 7B). Together, these profiles further confirmed that the radionuclide-induced PD-1/PD-L1 immunotherapy could inflame the TIME and activate the immune system.
Coincidentally, consistent with the aforementioned heat map of DEGs (Fig. 1G), subsequent therapeutic trials confirmed that MC38 tumor was more susceptible to 2-[18F]FDG than CT26. We investigated the antitumor efficacy of 2-[18F]FDG plus αPD-L1 mAb in CT26 tumor, another widely used murine colorectal tumor model. Although to a less extent still significant when compared to the MC38 section, the growth of CT26 tumors was greatly suppressed in groups of 18.5 MBq or 37 MBq 2-[18F]FDG + 400 mg αPD-L1 mAb @ 4 h, resulting in prolonged overall survivals (Fig. S9A-C). Dynamic changes of PD-L1 in tumor and cytokine levels in blood were found in the group of combining 2-[18F]FDG with αPD-L1 @ 4 h during the therapy period (Fig. S9D-F). Therapeutically, the activation of CD4+ and CD8+ T cells in the tumor and increased CD4+ Th1/Treg and CD8+ CTLs/Treg levels highlighted the potential of the effective coordination to enhance antitumor immunity (Fig. S9G). Previous studies suggested that the less-immunogenic and microsatellite-instable CT26 model did not respond to irradiation with increased PD-L1 expression31,32. To some extent, this study describes a new method to overcome this setback via radionuclide-induced immunotherapy.