Servicenavigation

Project Details

Back

Projekt Print View

Role of Interleukin-22 in breast and lung cancer metastasis

Subject Area Hematology, Oncology
Term from 2019 to 2024
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 426715005
 
Final Report Year 2025

Final Report Abstract

III Zusammenfassung 1. Allgemeinverständliche Darstellung der wichtigsten wissenschaftlichen Fortschritte und ggf. ihrer Anwendungsaspekte. Interleukin-22 (IL-22) plays a complex role in cancer biology, particularly in the metastasis of breast and lung cancers. In the current project, we summarized the current understanding of the role of interleukins as the messengers of the immune system in anti-cancer immunity and cancer development and progression alongside the latest developments utilizing their therapeutic potential (Briukhovetska, Dörr, et al. 2021). In the current project, we elucidated details of newly discovered IL-22-regulated CD155 expression on tumor cells and its role in cancer progression. Our first aim was to confirm these effects in additional mouse models on the C57BL/6 background. Using newly generated IL-22-deficient C57BL/6 mice, we successfully confirmed the effects of IL-22 on cancer progression across multiple mouse models. Although the E0771 breast cancer model yielded an insufficient number of lung metastases in a spontaneous metastasis model (data not shown), we confirmed that when injected intravenously, E0771 cells form fewer metastases in the lungs of IL-22-deficient animals in the forced metastasis model (Fig. 2A). It is important to note, that we detected IL-22-mediated upregulation of CD155 on E0771 cells that formed lung metastases in vivo (Fig. 2B). Together with our collaboration partners, we demonstrated that lung cancer LLC cells also form fewer spontaneous metastases to the lung when injected subcutaneously in C57BL/6 IL-22-deficient mice, similar to the results observed with 4T1 and LCCL1 (Line-1) cells (Fig. 2C). Furthermore, MC38 colon carcinoma cells metastasize less to the lung when injected intravenously in the absence of IL-22 (Fig. 2D). Here we confirmed that IL-22 deficiency in C57BL/6 mice leads to reduced lung metastasis across multiple cancer models, highlighting the role of IL-22 regulation in cancer progression. Similar to 4T1 cells, we used CRISPR/CAS9 to knock out IL-22RA1, a receptor for IL-22, in LCCL1 (Line-1) cells. We confirmed that IL-22RA1 on tumor cells mediated the effects of IL-22 on the ability of LCCL1 (Line-1) cells to metastasize to the lung in vivo (Fig. 2E). However, we were unable to generate a complete knockout of IL-22RA1 in E0771 cells, which is complicated by the aneuploid karyotype of cancer cell lines. Our initial experiments relied exclusively on transplantable models. To strengthen and validate the observed phenotype, we proposed employing the MMTV-PyVT breast cancer model (Jax, Stock 022974). This model exhibits high tumor penetrance via the specific expression of the mouse mammary tumor virus, and in 80–90% of cases, the tumors spontaneously metastasize to the lung. We planned to cross IL-22-KO mice on a C57BL/6 background with Campus Innenstadt Seite 8 von 15 Abteilung für Klinische Pharmakologie MMTV-PyVT mice to generate MMTV-PyVT-IL-22KO and WT cohorts. We intended to evaluate the incidence of primary tumors and metastatic spread, including macroscopic quantification and subsequent histological examination of the lungs for metastatic lesions. However, additional data were published soon after the beginning of the current project [1] that provided data relevant to the role of IL-22 in lung metastases in the MMTV-PyVT model, thus making the proposed experiment redundant. Building on these insights, we pursued the 4T1 orthotopic breast cancer approach to test whether our observations would hold under conditions that mirror the native tumor environment more closely (Fig. 2F). This alternative approach corroborated our original findings, reinforcing the proposed role of IL-22 in the metastatic process. Combining the knowledge gained from the published research with our orthotopic model results, we have secured a more comprehensive understanding of the relevant mechanisms at play, paving the way for further investigation into the therapeutic implications of targeting IL-22 in breast cancer. Figure 2: Interleukin-22 promotes lung cancer spread in various syngeneic models of spontaneous and forced metastasis of lung, breast, and colon cancer. A. Number of E0771 Campus Innenstadt Seite 9 von 15 Abteilung für Klinische Pharmakologie metastases in the lungs (n = 22 and 23). Representative flow cytometry plots of the lung cells. The numbers are the frequency of the parent gate. Frequency of E0771-GFP+ cells among live cells (n = 19 and 21). B. Representative histogram and MFI of CD155 staining of GFP + tumor cells. Linear regression analysis of CD155 MFI and frequency of E0771-GFP + tumor cells. C. Schematic overview of the experiment (flank injection of LLC cells for spontaneous lung metastasis), representative pictures, and number of macroscopic metastases. D. Schematic overview of the experiment (i.v. injection of MC38 cells for forced lung metastasis), representative pictures and number of macroscopic metastases, H&E staining, and metastatic foci percentage of Il22+/+ and Il22−/− mice. E. Number of metastases and colonies of the Line-1 (LCCL1) control (#1, #2, #3) or Il22ra1− (#4, #5, #6) clones (n = 6 per clone). F. Syngeneic 4T1 model of orthotopic mammary pad injection in WT and Il22-/- animals. Subcutaneous tumor growth, macroscopic metastases in the lungs, and colonies in clonogenic metastasis assay. A, B, E, F from [3], C and D from [4]. In the second work package, we elucidated the role of IL-22-mediated CD155 (Pvr) upregulation on metastasis development. According to the proposal, we generated LCCL1 (Line-1) and 4T1 cells deficient in expression of CD155. Although CD155 is known for its intrinsic function regulating adhesion, migration, and survival of cells, we detected no deficiencies in the ability of tumor cells to proliferate and migrate in vitro (data not shown). The differences in the lung metastases in wildtype and IL-22 deficient animals could also not be fully explained by seeding and proliferation of cells, as detected by EdU incorporation assay, during the initial 7 days after intravenous injection of tumor cells (Fig. 3A). However, CD155 deficiency significantly diminished the ability of 4T1 and LCCL1 (Line-1) cells to form metastases in the lung in spontaneous and forced models of metastasis (Fig. 3B). Overexpression of CD155 independent of its native regulation restored the capacity of these cells to metastasize in wild-type and also in IL-22-deficient mice (Fig. 3C). Here, we focused on the extrinsic role of CD155 in the regulation of antitumor immunity. The latest developments demonstrated that CD155 does not only bind to CD96 but is a part of the regulatory network that also includes TIGIT and CD226 (DNAM-1) expressed on the surface of NK and T cells [5, 6]. Ablation of NK cells in our model completely abolished the lung's antitumor immunity following intravenous injection of 4T1 cells, confirming their critical role but failing to elucidate CD155’s specific contribution (data not shown). However, we determined that IL-22 did not influence the number of NK cells in the lung but their ability to produce IFNg in response to tumor cell dissemination, and, hence, mount proper antitumor response (Fig. 3F). Considering the absence of IL-22RA1 on hematopoietic cells including NK cells, this is an indirect effect mediated through regulation by CD155 on tumor cells. We interrogated binding partners of CD155 on NK and T cells and found that NK cells in the lungs of IL-22-deficient animals retained higher expression of co-stimulatory receptor CD226 (DNAM-1) (Fig. 3D). This receptor is essential to the activation of NK cells and is typically internalized and degraded upon excessive stimulation. However, we found no primary association with the expression of inhibitory molecules TIGIT or CD96 (Fig. 3E). Furthermore, we tested the functionality of such interactions using antibodies in vivo (Fig. 3G). In the final working package, we investigated the role of IL-22 production in cancer metastasis, focusing on its source and effects on tumor progression. CD4+ T cells primarily produce IL-22, which influences metastatic spread through immunosuppressive effects. To study this, we used IL-22 and IL-17 reporter mice on a C57BL/6 background, enabling simultaneous Campus Innenstadt Seite 10 von 15 Abteilung für Klinische Pharmakologie detection of IL-22 (sgBFP) and IL-17 (GFP) [7]. We injected E0771 tumor cells intravenously and, after three to four weeks, analyzed tissues, including tumors, spleen, lymph nodes, and lungs, using flow cytometry with various surface markers to identify IL-22-producing cell populations (Fig. 4A). Campus Innenstadt Seite 11 von 15 Abteilung für Klinische Pharmakologie Campus Innenstadt Seite 12 von 15 Abteilung für Klinische Pharmakologie Figure 3: IL-22-mediated CD155 (Pvr) overexpression suppresses NK cell immunity to enable lung metastases. A. Intravenous mouse model of 4T1-GFP breast cancer cell proliferation. Mice were co-injected with EdU four hours before preparation and sacrificed at 12-, 24-, 48- hour, and 7-day time points. Representative contour plots of the lung cells. Numbers represent the frequency of the parent gate. Total numbers of 4T1-GFP+ and % of EdU+ cells in the lungs as reported per mouse. B. Numbers of metastases in the lung and colonies in metastasis assay of Line-1 (LCCL1) and 4T1 Pvr− (CD155 knockout) cells after i.v. injection. C. Numbers of metastases in the lung and colonies in metastasis assay of Line-1 (LCCL1) and 4T1 Pvr+ (CD155 overexpression) cells after i.v. injection. D. CD226 staining on CD8+ T, CD4+ T, NKT, and NK cells in the lungs of wt and IL-22KO mice bearing 4T1 tumors. E. CD96 and TIGIT staining on CD8+ T, CD4+ T, NKT, and NK cells in the lungs of wt and IL-22KO mice bearing 4T1 tumors. F. Frequency of IFNγ+ NK cells in the lungs of wt and IL-22KO mice bearing 4T1 tumors. G. Intravenous mouse model of 4T1 control or Pvr+ (CD155 overexpressing) metastasis in Il22−/− mice. Animals received injections of anti-CD226 blocking antibody (420.1, 200 μg per mouse) , anti-TIGIT agonist antibody (1G9, 250 μg per mouse), anti-CD96 blocking antibody (3.3, 250 μg per mouse). All from [3]. We performed a spatial analysis of tumor-immune interactions by fixing, clearing, and visualizing precision-cut lung slices. This approach reconstructed the topography of IL-22- producing cells relative to tumor cells (Fig. 4B). Counterstaining with specific antibodies validated the identified IL-22+ cell populations. To further characterize the impact of IL-22, we generated T cell-specific IL-22 knockouts by crossbreeding IL-22-floxed mice with CD4-Cre mice. We quantified tumor metastasis using macroscopic counts and flow cytometry (Fig. 4C). Moreover, we adoptively transferred CD4+ T cells from wildtype and IL-22-deficient mice into Rag-/-IL-22-/- mice that lack mature T cells and IL-22 production to see whether it would be sufficient to restore the metastatic phenotype (Fig. 4D). The experiments identified CD4+ T cells as the primary source of IL-22 in both primary tumors and metastatic sites. T cell-specific IL-22 deletion significantly reduced metastatic burden without altering primary tumor growth. Spatial imaging showed IL-22-producing cells clustering near metastatic foci, and their absence disrupted tumor-immune interactions. The study also revealed a role for IL-1 signaling in enhancing effects of IL-22, with ongoing experiments investigating this further. These findings underscore the critical role of IL-22 in promoting metastasis through its interactions with tumor cells and the immune microenvironment. Targeting the IL-22-CD155 axis presents a promising therapeutic strategy to reduce metastasis in cancers such as breast and lung. Campus Innenstadt Seite 13 von 15 Abteilung für Klinische Pharmakologie Figure 4: CD4+ T cells as the major source of IL-22 at the metastatic lesion. A. Intravenous model of E0771 lung metastasis in Foxp3-mRFPxIl17a-GFPxIl22-sgBFP reporter mice. Gating strategy to identify CD4+, CD8+, and double-negative (DN) T cells, γδ T cells, CD3+NK1.1+, and CD3−NK1.1+ cells in the lungs. Breakdown of CD45+IL-22+ cells by cell type as defined by the mean frequency of reported experiments. B. Representative 3D render of the z stack confocal images of metastatic foci and normal tissue of precision-cut lung slices from reporter mice injected with E0771 cells i.v. Il17aGFP is depicted in green, Il22sgBFP in blue, CD4 PerCP-Cy5.5 staining in magenta, and TO-PRO-3 nuclear staining in red. Correlation between Il17aGFP and Il22sgBFP median fluorescence intensity (MFI) in the reporter cells in the metastatic foci. The number of reporter cells per field of view in the metastatic foci and normal tissue. Normalized CD4 PerCP-Cy5.5 MFI in Il22sgBFP+ cells gated from the previous graph. C. Intravenous Campus Innenstadt Seite 14 von 15 Abteilung für Klinische Pharmakologie mouse model of E0771-GFP lung metastasis in Il22floxCd4cre mice. Representative dot plots, numbers of macroscopic metastases, and frequency of E0771-GFP cells in the lungs of WT and Il22floxCd4cre mice. D. Model of metastasis in Rag1−/−Il22−/− that received WT or Il22−/− CD4+ T cells i.p. (2 × 106 per mouse) 28 days before i.v. tumor injection. Representative dot plots, numbers of macroscopic metastases, and frequency of E0771-GFP cells in the lungs. 2. „Überraschungen“ im Projektverlauf und bei den Ergebnissen. Unexpected findings during the project provided new insights and highlighted the complexity of IL-22’s role in metastasis. One surprising result was the discovery that IL-22, while not directly affecting the number of NK cells in the lungs, significantly influenced their functionality by modulating CD226 expression through CD155 on tumor cells. This indirect mechanism of immune suppression was not anticipated and revealed a nuanced interaction within the IL-22- CD155-NK cell-IFNg axis. Another unexpected outcome involved the role of CD155 itself. While initially assumed to primarily regulate adhesion and migration, its capacity to suppress NK cell immunity through receptor interactions emerged as a critical factor in metastasis. Overexpression of CD155 restored metastatic potential even in IL-22-deficient settings, emphasizing its pivotal role beyond tumor cell-intrinsic properties. Finally, spatial imaging of IL-22-producing T cells revealed their clustering near metastatic foci, which provided unexpected clarity on tumor-immune dynamics and suggested previously unrecognized mechanisms of IL-22-driven immune evasion. 1. Katara, G.K., et al., Interleukin-22 promotes development of malignant lesions in a mouse model of spontaneous breast cancer. Mol Oncol, 2020. 14(1): p. 211-224. 2. Giannou, A.D., et al., Tissue resident iNKT17 cells facilitate cancer cell extravasation in liver metastasis via interleukin-22. Immunity, 2023. 56(1): p. 125-142 e12. 3. Briukhovetska, D., et al., T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis. Immunity, 2023. 56(1): p. 143-161 e11. 4. Giannou, A.D., et al., Tissue resident iNKT17 cells facilitate cancer cell extravasation in liver metastasis via interleukin-22. Immunity, 2023. 56(1): p. 125-142.e12. 5. Gao, J., et al., CD155, an onco-immunologic molecule in human tumors. Cancer Sci, 2017. 108(10): p. 1934- 1938. 6. Chauvin, J.M. and H.M. Zarour, TIGIT in cancer immunotherapy. J Immunother Cancer, 2020. 8(2). 7. Perez, L.G., et al., TGF-beta signaling in Th17 cells promotes IL-22 production and colitis-associated colon cancer. Nat Commun, 2020. 11(1): p. 2608. 3. Hinweise auf mögliche Erfolgsberichte in den Publikumsmedien

Publications

 
 

Additional Information

© 2026 DFG Disclaimer / Copyright / Privacy Policy

Textvergrößerung und Kontrastanpassung