Ectopic Ifnl3 expression significantly inhibits MB49 tumor progression
We used the subcutaneous MB49 bladder cancer mouse model to evaluate the in vivo antitumor effect of IFN-λ3. Given the low endogenous expression of IFN-λ3 in the MB49 cell line, we ectopically overexpressed Ifnl3 in MB49 cells (MB49-Ifnl3OE) and confirmed the altered expression by RT-qPCR and ELISA (Fig. 1a-b). We also confirmed high IFN-λ3 expression in MB49-Ifnl3OE tumor tissues by ELISA assay and immunohistochemistry (IHC) staining (Figure S1a-b). Although MB49-Ifnl3OE cells did not change tumor cell proliferation when compared to MB49 cells transfected with empty vector (EV), as indicated by Cell counting kit-8 (CCK-8) and colony-formation in vitro assays (Fig. 1c and Figure S1c-d), ectopic Ifnl3 expression significantly reduced MB49 tumor growth and weight in immune-competent C57BL/6 mice (Fig. 1d–f). H&E staining of sections, analyzed by Nuclear Phenotype on the HALO software platform (Indica Labs, USA), revealed increased immune cell infiltration in MB49-Ifnl3OE tumors (Fig. 1g). These results demonstrated that IFN-λ3 induced a potent antitumor response against bladder cancer.
Ectopic Ifnl3 expression promotes immune activation in MB49 -Ifnl3 OE tumors
We compared RNA-Seq transcriptome analysis of MB49-Ifnl3OE and MB49-EV tumors 23 days after subcutaneous tumor cells injection to investigate the potential mechanisms underlying the in vivo antitumor effect of IFN-λ3. As expected, most upregulated genes were associated with Types Ⅰ and III interferon responses, JAK-STAT signaling and lymphocyte activation in MB49-Ifnl3OE tumors (Fig. 2b-d and Figure S2c). Further analysis revealed significant upregulation of many genes associated with cytokine-cytokine receptor interactions, chemokine signaling pathways, and phagocytosis in MB49-Ifnl3OE tumors (Figure S2a-b and S2d-e). Lymphocyte exhaustion genes such as Cd274 (PD-L1) and Pdcd1 (PD-1) were also significantly upregulated at the detection time (Fig. 2a). Moreover, gene expressions of selected markers were confirmed by RT-qPCR in a larger group of specimens, including those used for RNA-Seq (Figure S3a). The expressions of T-cell maturation and activation makers, including Ifng, Il15, and Il18, were increased in MB49-Ifnl3OE tumors. Furthermore, the expressions of antitumoral Mφ markers (Cd86, Tnfsf10, and Nos2) and T cell-associated chemokines, such as Cxcl9 and Cxcl10, were also increased in MB49-Ifnl3OE tumors. However, the pro-tumoral Mφ marker Arg1 expression was comparable in MB49-Ifnl3OE and MB49-EV tumors. These data indicated that the effector T cell and antitumoral Mφ phenotypes were positively correlated with antitumor effects of IFN-λ3.
Ectopic Ifnl3 expression led to tumor cytotoxic T and myeloid cell infiltration
We used two complementary approaches, multicolor flow cytometry of cells harvested from the tumors, draining lymph nodes, and spleens, and IHC and multiplex immunofluorescence (mIF) of cells within the tumor, to assess the tumor-infiltrating cell phenotypes in the tumor microenvironment (TME). The flow cytometry gating strategy is illustrated in Figure S4. Compared with the MB49-EV group, ectopic Ifnl3 expression induced a significant increase in the proportion of tumor-infiltrating CD45+ leukocytes and NK1.1+ CD3– NK, CD3+ NK1.1– T, total CD4+ T, CD4+ T-bet+ Th1, CD4+ Foxp3+ regulatory T, and CD8+ effector T (Teff) cells. However, the Teff to regulatory T cell ratio and cytotoxic or proliferative NK cells were comparable in MB49-Ifnl3OE and MB49-EV tumors (Fig. 3a, Figure S5c-d). Among T cells, proliferation (Ki-67+) and activation (GZMB+) markers were significantly enhanced in CD4+ and CD8+ T cells in MB49-Ifnl3OE tumors (Fig. 3a). Detailed analysis of the immune cells in draining lymph nodes on the tumor side and the spleen showed markedly higher CD3+ T cells, total CD4+ T cells, CD4+ T-bet+ Th1 cells, and CD8+ Teffs in mice with MB49-Ifnl3OE tumors than in the MB49-EV mice (Figure S5a-b). Intriguingly, only CD8+ T cells in the draining lymph node, but not in the spleen, of mice with MB49-Ifnl3OE tumors showed increased Ki-67 expression and Teff to regulatory T cell ratio (Figure S5b and S5d), indicating that IFN-λ3 induced a systemic antitumor immune response.
We further assessed various immune cell markers expressed in the tumors using IHC, mIF, or the ELISA assay. We observed a three to fivefold increase in NCR1+ NK, CD4+ T, and CD8+ T cells in MB49-Ifnl3OE tumors compared to the MB49-EV group (Fig. 3b-c). Moreover, in myeloid cells, ectopic Ifnl3 expression resulted in a pronounced increase in F4/80+ Mφs infiltrate, and a smaller increase in CD11c+ F4/80– dendritic cell (DC) infiltrate. We questioned whether a Mφ phenotype transformation occurred in MB49-Ifnl3OE tumors. To address this, we first assessed the expression of known pro- and antitumoral Mφ genes in tumor tissues from both groups (Figure S3a-b). Our data revealed increased expression of antitumoral Mφ genes, including Nos2, Tnfsf10, Tnfa, Cd80, Cd86, Cxcl9, and Cxcl10, and some parallel expression of pro-tumoral Mφ genes (Arg1) in MB49-Ifnl3OE tumors. Additionally, we found that the number of iNOS+F4/80+ Mφs increased while that of Arg1+F4/80+ Mφs decreased (Fig. 3d-e), following the increase in CXCL9+ and CXCL10+ cells in the tumor region (Fig. 3f-g), indicating an effective reprogramming of Mφs toward antitumor functions. Moreover, protein levels of IFN-λ3, CXCL9, and CXCL10 were associated with each other in tumor tissues (Figure S6), suggesting that ectopic Ifnl3 expression increased CXCL9 and CXCL10 in the TME, contributing to increased tumor infiltration by T and NK cells. Furthermore, CD14+ monocytes isolated from human peripheral blood mononuclear cells were differentiated into resting Mφs in vitro (Figure S7a). We confirmed that IFN-λ1 could stimulate these cells to acquire antitumoral phenotypes, including CXCL9, CXCL10, CD80, and CD169 expression (Figure S7b-c) and CXCL10 secretion into the supernatant (Figure S7d). Taken together, these results indicated that ectopic Ifnl3 expression in tumors promoted T-cell infiltration and activation, an influx of myeloid cells, and antitumoral Mφ polarization.
The antitumor effect of IFN-λ3 in MB49 bladder tumors relies on T cells and Mφs
As CD8+ T cells within the TME are strongly associated with the T cell-based antitumor immune response following immunotherapy35, we hypothesized that tumor-infiltrating CD8+ T cells play a crucial role in mediating Ifnl3-driven antitumor immunity. To test this hypothesis, we compared MB49-Ifnl3OE and MB49-EV tumor inhibition rates in immune-deficient nude mice (lacking T and B cells) and immune-competent C57BL/6 mice. Although MB49-Ifnl3OE tumors in C57BL/6 mice exhibited greater suppression than in nude mice (Fig. 4a), a significant delay in tumor growth was observed in MB49-Ifnl3OE compared to MB49-EV tumors in nude mice (Fig. 4b-c), indicating a lymphocyte-independent antitumor component and function in MB49-Ifnl3OE tumors.
Consistent with immune-competent mice, we observed an increase in total F4/80+ and iNOS+F4/80+ Mφs and a decrease in Arg1+F4/80+ Mφs in MB49-Ifnl3OE tumors (Fig. 4d-e). Notably, a phagocytic Mφ subset, identified by the co-expression of the surface markers Ly6C and the MHC class Ⅱ molecule IA/IE34, was highly enriched in MB49-Ifnl3OE tumors (Fig. 4f-j). However, we observed relatively balanced infiltrate distributions of NCR1+ NK and CD11c+ DC cells in MB49-Ifnl3OE and MB49-EV tumors in nude mice, while Ly6G+ neutrophils were significantly reduced at the detecting point in MB49-Ifnl3OE tumors (Figure S8a-f). These data indicated that Mφs play a role in the antitumor effect of IFN-λ3 in an immune-deficient MB49 bladder tumor model.
Mφs display enhanced phagocytosis of tumor cells with ectopic Ifnl3 expression
As Mφs can act as phagocytes and directly kill tumor cells, and RNA-Seq data indicated phagocytosis pathway augmentation in MB49-Ifnl3OE tumors (Figure S2e), we tested their phagocytic capacity in in vivo and in vitro experiments. We implanted nude mice with MB49-Ifnl3OE or MB49-EV tumors expressing green fluorescence protein (GFP) to measure in vivo phagocytosis. After the tumors were harvested, infiltrating Mφs were analyzed by flow cytometry (Figure S9a). The results showed increased phagocytosis in Ly6C+IA/IE+ phagocytic Mφs rather than CD11b+ monocyte and Mφ populations in MB49-Ifnl3OE tumors compared to MB49-EV tumors (Fig. 5a-d). Phagocytosis was directly visualized in excised MB49-Ifnl3OE tumor tissue in situ by mIF staining using antibodies to F4/80 and GFP, which the MB49 tumor cells expressed (Fig. 5e-f). The fraction of tumor cell-containing Mφs was significantly higher in MB49-Ifnl3OE than in MB49-EV tumors. To better model the effects of ectopic Ifnl3 expression in MB49 tumor cells on Mφ phagocytosis, a coculture of bone marrow-derived Mφs (BMDMs) and viable tumor cells was developed to conduct the in vitro phagocytosis assay. Flow cytometry showed that more MB49-Ifnl3OE than MB49-EV cells were phagocytized by the BMDMs (Fig. 5g-h). These findings demonstrated that ectopic Ifnl3 expression in MB49 tumors enhanced tumor cell uptake by mononuclear phagocytes.
PD-1/PD-L1 axis blockade leads to better antitumor effects in MB49- Ifnl3 OE tumors
As IFNs are known to contribute to the quality of antitumor immunity and response to immunotherapy7, we evaluated the relationship between ectopic Ifnl3 expression and the PD-1/PD-L1 axis in a mouse model. Using IHC staining, we observed that the MB49-Ifnl3OE tumors had a higher concentration of infiltrating PD-1+ cells than MB49-EV tumors (Figure S10a-b). We next investigated whether blockade of the PD-1/PD-L1 axis could improve IFN- λ antitumor efficacy in MB49 tumors. As shown in Fig. 6a, MB49-Ifnl3OE and MB49-EV tumors were generated in C57BL/6 mice. PD-L1-blocking and IgG control antibodies were injected intraperitoneally four times, once every three days, at a dose of 0.05 mg per mouse. As expected, we detected a significant delay in tumor progression in mice with MB49-Ifnl3OE tumors. While no benefit was detected in MB49-EV tumors treated with anti-PD-L1 antibodies, a further reduction in tumor growth was observed in MB49-Ifnl3OE tumors treated with anti-PD-L1 antibodies (Fig. 6b-c). These findings indicated that blocking the PD-1/PD-L1 axis led to a higher Ifnl3 antitumor efficacy in MB49 tumors.
IFN-λ is associated with innate and adaptive immune responses in bladder cancer
The antitumor effect of ectopic Ifnl3 expression in MB49 tumors, which depends on T and myeloid cells, led us to investigate the relationship between IFN-λ and the innate and adaptive immune response in human bladder cancer. Using data from The Cancer Genome Atlas Urothelial Bladder Carcinoma (TCGA-BLCA), we demonstrated that expression of the IFN-λ genes (IFNL1, IFNL2, and IFNL3) was upregulated in tumor tissues compared to adjacent normal tissues (Fig. 7a). Using the microenvironment cell populations (MCP)-counter algorithm, we defined a human IFN-λ signature that comprised the three IFN-λ isoforms (IFN-λ1, IFN-λ2, and IFN-λ3). Based on the median level of the IFN-λ signature, we divided the 412 patients into high and low IFN-λ signature groups. We identified the tumor infiltrating immune cells (TIICs) from previous studies and calculated their infiltration level using the MCP-counter algorithm in the TCGA-BLCA dataset36,37. Further analysis revealed that the IFN-λ signature was positively associated with the infiltration levels of CD8+ T cells, Th1 cells, NK cells, Mφs, and DCs (Fig. 7b). Gene set enrichment analysis showed that positive regulation of leukocyte activation and phagocytosis pathways was enriched in the high-IFN-λ signature group (Fig. 7c). These results were validated by IHC and mIF staining in a human bladder cancer cohort (n = 15). IHC staining showed that IFN-λ3 is expressed on tumor and stromal cells (Fig. 7d). mIF staining of serial sections was used to evaluate TIIC infiltration, including CD8+ T, CD4+ T, NCR1+ NK, and HLA-DR+ antigen-presenting cells. Consistently, tumors in which IFN-λ3 was detected appeared to have higher TIIC infiltration rates than tumors in which IFN-λ3 was not detected (Fig. 7e-f). These results were confirmed in a cohort of patients with bladder cancer receiving anti-PD-1 neoadjuvant immunotherapy (n = 20). IHC staining data showed that IFN-λ3 detection was higher in responders than in non-responders in this cohort (P < 0.001; Fig. 7g). Furthermore, we evaluated the IFN-λ signature and anti-PD-L1 immunotherapy efficacy in the IMvigor210 cohort38. The IFN-λ signature was positively associated with the immunotherapy efficacy (P = 0.0342; Figure S11). Taken together, these results indicated that IFN-λ is associated with innate and adaptive immune responses and the efficacy of immunotherapy in human bladder cancer.