Exploring the Causal Relationship between Gut Microbiome and Programmed Cell Death Protein-1/Programmed Cell Death Ligand-1: Mediating Effects of Serum Lipid and Amino Acid Metabolic Biomarkers in a Two-step Mendelian Randomization Study
-
Yan Kang
,
Baocen Xu
,
Siyu Wang
,
Shaozhi Xi
Abstract
Programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) plays a pivotal role in tumor immune evasion. The efficacy of these treatments is limited by variable patient responses and adverse effects. It is necessary for a deeper understanding of the underlying biological mechanisms.
This study used a 2-step Mendelian randomization (MR) approach to investigate causal relationships among gut microbiota, lipid and amino acid metabolic traits, and PD-1/PD-L1. The summary statistics for 412 traits of the gut microbiome (N=7738), 249 traits of serum metabolites (N=115 078), and 2 traits of PD-1/PD-L1 (N=3301) were derived from publicly genome-wide association studies. The primary method employed for MR was inverse-variance weighted regression. We conducted a series of sensitivity analyses to evaluate the reliability of the causal estimates. Subsequently, mediation analysis was undertaken to elucidate the pathway from gut microbiome to PD-L1, mediated by serum metabolic markers.
Our analyses identified 28 gut microbial traits significantly affecting PD-L1 and 14 affecting PD-1, 8 of which remained consistently linked to PD-L1 after sensitivity analysis. Furthermore, 13 serum lipid and amino acid metabolic traits exhibited significant causal effects on PD-L1, with 6 remaining robust post analysis. Notably, Bacteroides dorei demonstrated a causal effect on PD-L1, mediated 9.6% by the metabolic biomarker phenylalanine.
These findings highlight the intricate interplay among gut microbiome, metabolic biomarkers, and immune regulation. They suggest novel therapeutic targets for cancer treatment that emphasize the value of microbiome and metabolic biomarkers in improving immunotherapy outcomes and promoting personalized medicine.
1. Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74:229–63.
2. Shiravand Y, Khodadadi F, Kashani SMA, Afshar S, Assar S, Ghahremani MH, et al. Immune checkpoint inhibitors in cancer therapy. Curr Oncol. 2022;29:3044–60.
3. Liu Q, Guan Y, Li S. Programmed death receptor 1/programmed death ligand 1 in urological cancers: the all-around warrior in immunotherapy. Mol Cancer. 2024;23:183.
4. Morad G, Helmink BA, Sharma P, Wargo JA, et al. Hallmarks of response, resistance, and toxicity to immune checkpoint blockade. Cell. 2021;184:5309–37.
5. Bagchi S, Yuan R, Engleman EG. Immune checkpoint inhibitors for the treatment of cancer: clinical impact and mechanisms of response and resistance. Annu Rev Pathol. 2021;16:223–49.
6. Yang J, Chen M, Li R, Zhang Y, Liu Y, Wang J, et al. A responsive cocktail nano-strategy breaking the immune-excluded state enhances immunotherapy for triple-negative breast cancer. Nanoscale. 2025;17:4610–23.
7. Baruch EN, Youngster I, Ben-Betzalel G, Ortenberg R, Lahat A, Katz L, et al. Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients. Science. 2021;371:602–9.
8. Gunjur A, Shao Y, Rozday T, Rhee K, Simeone P, O’Donnell JS, et al. A gut microbial signature for combination immune checkpoint blockade across cancer types. Nat Med. 2024;30:797–809.
9. Glitza IC, Seo YD, Spencer CN, Reuben A, Andrews MC, Peng W, et al. Biomarker-stratified phase Ib microbiome modulation in melanoma. Cancer Discov. 2024;14:1161–75.
10. Peng Z, Cheng S, Kou Y, Wang Z, Jin R, Hu H, et al. Gut microbiome is associated with clinical response to anti-PD-1/PD-L1 immunotherapy in gastrointestinal cancer. Cancer Immunol Res. 2020;8:1251–61.
11. Skrivankova VW, Richmond RC, Woolf BAR, Davies NM, Swanson SA, VanderWeele TJ, et al. STROBE-MR statement. JAMA. 2021;326:1614–21.
12. Lopera-Maya EA, Kurilshikov A, van der Graaf A, Hu S, Andreu-Sánchez S, Chen L, et al. Effect of host genetics on the gut microbiome. Nat Genet. 2022;54:143–51.
13. Carter AR, Sanderson E, Hammerton G, Richmond RC, Smith GD, Heron J, et al. Mendelian randomisation for mediation analysis. Eur J Epidemiol. 2021;36:465–78.
14. Sun BB, Maranville JC, Peters JE, Stacey D, Staley JR, Blackshaw J, et al. Genomic atlas of the human plasma proteome. Nature. 2018;558:73–9.
15. Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews MC, Karpinets TV, et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma. Science. 2018;359:97–103.
16. Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, et al. Gut microbiome influences efficacy of PD-1-based immunotherapy. Science. 2018;359:91–7.
17. Zhang M, Bzura A, Baitei EY, Al-Mutairi F, Al-Sagheer F, Mbarek H, et al. Gut microbiota rheostat forecasts responsiveness to PD-L1 and VEGF blockade. Nat Commun. 2024;15:7187.
18. Zhang H, Liu J, Yuan W, Wang Y, Wu J, Chen Z, et al. Ammonia-induced lysosomal and mitochondrial damage causes CD8+ T cell death. Nat Cell Biol. 2024;26:1892–902.
19. Tang K, Zhang H, Deng J, Wang Y, Liu J, Chen Z, et al. Ammonia detoxification promotes CD8+ T cell memory development. Nat Immunol. 2023;24:162–73.
20. Huang D, Chen Y, Li C, Zhang X, Wang J, Liu Y, et al. Salivary microbiome variations associated with immunotherapy efficacy in NSCLC. mSystems. 2025;10:e01115–24.
21. Bakir MA, Sakamoto M, Kitahara M, Benno Y. Bacteroides dorei sp. nov., isolated from human faeces. Int J Syst Evol Microbiol. 2006;56:1639–43.
22. He S, Lu S, Yang T, Song L, Xiao Y, Huang Y, et al. Bacteroides dorei-derived bile acid alleviates influenza virus infection. Cell Commun Signal. 2025;23:382.
23. He S, Song L, Xiao Y, Huang Y, Lu S, Yang T, et al. Genomic and probiotic properties of Bacteroides dorei RX2020. Nutrients. 2025;17.
24. Song L, Huang Y, Liu G, He S, Xiao Y, Lu S, et al. Bacteroides dorei ameliorates influenza virus infection in mice. Front Immunol. 2022;12:828887.
25. Su Y, Shadike Q, Wang M, Liu Y, Abudureheman A, Yusufu M, et al. Low abundance of Bacteroides correlates with phenylalanine levels. Transl Pediatr. 2021;10:2521–32.
26. Cong S, Wang L, Meng Y, Liu X, Zhang Y, Li J, et al. Saussurea involucrata regulates gut microbiota in arthritis rats. Phytother Res. 2022;37:1242–59.
27. Tan M, Cao G, Wang R, Zhang Y, Liu J, Li X, et al. Phenylalanine nanostructures sensitize breast tumour to checkpoint blockade. Nat Nanotechnol. 2024;19:1903–13.
28. May P, Winter C, Hubrecht I, Seidel C, Hentschel M, Schumann C, et al. Metabolic phenotype predicts atezolizumab outcomes in SCLC. Transl Lung Cancer Res. 2025;14:3836–46.
29. Liu Y, Ping Y, Zhang L, Wang J, Chen X, Huang D, et al. L-phenylalanine predicts response to anti-PD-1 therapy in NSCLC. MedComm. 2025;6:e70100.
30. Plaza-Díaz J, Álvarez-Mercado AI, Ruiz-Marín CM, Reina-Pérez I, Pérez-Muñoz ME, Gomez-Llorente C, et al. Breast and gut microbiota dysbiosis and breast cancer risk. BMC Cancer. 2019;19:495.
31. García-Vega ÁS, Corrales-Agudelo V, Reyes A, Mancabelli L, Milani C, Ventura M, et al. Diet quality and gut microbiota in a nonwestern population. Nutrients. 2020;12:2938.
32. Daillère R, Derosa L, Bonvalet M, Segata N, Routy B, Kroemer G, et al. Gut microbiota to boost anticancer immunotherapy. Oncoimmunology. 2020;9:1774298.
33. Farhadi Rad H, Tahmasebi H, Javani S, Zare M, Ebrahimi M, Akbari A, et al. Microbiota and cytokine modulation in anticancer immunity. Biomedicines. 2024;12.
34. Byrd DA, Gomez MF, Hogue SR, Wang Y, Rhoades KP, Figueiredo JC, et al. Colon tissue microbiome and bile acids in colorectal adenoma. Cancer Med. 2025;14:e71048.
35. Mokhashi O, Chakladar J, Li WT, McClellan J, Sarkar S, Amatya VJ, et al. Obesity-related microbial dysbiosis and tumour progression. Access Microbiol. 2025;7:e000846.
36. Moseeb HM, Aizaz MM, Aiza K, Rahman S, Ahmed R, Khan MA, et al. From obesity to cancer: gut microbiome mechanisms. Oncoscience. 2025;12:175–88.
37. Soni S, Mittal P, Lo JH, Zhang Y, Patel S, Wang J, et al. Age-diet interactions influence tumor microbiome and microenvironment. Neoplasia. 2025;70:101245.
2. Shiravand Y, Khodadadi F, Kashani SMA, Afshar S, Assar S, Ghahremani MH, et al. Immune checkpoint inhibitors in cancer therapy. Curr Oncol. 2022;29:3044–60.
3. Liu Q, Guan Y, Li S. Programmed death receptor 1/programmed death ligand 1 in urological cancers: the all-around warrior in immunotherapy. Mol Cancer. 2024;23:183.
4. Morad G, Helmink BA, Sharma P, Wargo JA, et al. Hallmarks of response, resistance, and toxicity to immune checkpoint blockade. Cell. 2021;184:5309–37.
5. Bagchi S, Yuan R, Engleman EG. Immune checkpoint inhibitors for the treatment of cancer: clinical impact and mechanisms of response and resistance. Annu Rev Pathol. 2021;16:223–49.
6. Yang J, Chen M, Li R, Zhang Y, Liu Y, Wang J, et al. A responsive cocktail nano-strategy breaking the immune-excluded state enhances immunotherapy for triple-negative breast cancer. Nanoscale. 2025;17:4610–23.
7. Baruch EN, Youngster I, Ben-Betzalel G, Ortenberg R, Lahat A, Katz L, et al. Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients. Science. 2021;371:602–9.
8. Gunjur A, Shao Y, Rozday T, Rhee K, Simeone P, O’Donnell JS, et al. A gut microbial signature for combination immune checkpoint blockade across cancer types. Nat Med. 2024;30:797–809.
9. Glitza IC, Seo YD, Spencer CN, Reuben A, Andrews MC, Peng W, et al. Biomarker-stratified phase Ib microbiome modulation in melanoma. Cancer Discov. 2024;14:1161–75.
10. Peng Z, Cheng S, Kou Y, Wang Z, Jin R, Hu H, et al. Gut microbiome is associated with clinical response to anti-PD-1/PD-L1 immunotherapy in gastrointestinal cancer. Cancer Immunol Res. 2020;8:1251–61.
11. Skrivankova VW, Richmond RC, Woolf BAR, Davies NM, Swanson SA, VanderWeele TJ, et al. STROBE-MR statement. JAMA. 2021;326:1614–21.
12. Lopera-Maya EA, Kurilshikov A, van der Graaf A, Hu S, Andreu-Sánchez S, Chen L, et al. Effect of host genetics on the gut microbiome. Nat Genet. 2022;54:143–51.
13. Carter AR, Sanderson E, Hammerton G, Richmond RC, Smith GD, Heron J, et al. Mendelian randomisation for mediation analysis. Eur J Epidemiol. 2021;36:465–78.
14. Sun BB, Maranville JC, Peters JE, Stacey D, Staley JR, Blackshaw J, et al. Genomic atlas of the human plasma proteome. Nature. 2018;558:73–9.
15. Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews MC, Karpinets TV, et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma. Science. 2018;359:97–103.
16. Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, et al. Gut microbiome influences efficacy of PD-1-based immunotherapy. Science. 2018;359:91–7.
17. Zhang M, Bzura A, Baitei EY, Al-Mutairi F, Al-Sagheer F, Mbarek H, et al. Gut microbiota rheostat forecasts responsiveness to PD-L1 and VEGF blockade. Nat Commun. 2024;15:7187.
18. Zhang H, Liu J, Yuan W, Wang Y, Wu J, Chen Z, et al. Ammonia-induced lysosomal and mitochondrial damage causes CD8+ T cell death. Nat Cell Biol. 2024;26:1892–902.
19. Tang K, Zhang H, Deng J, Wang Y, Liu J, Chen Z, et al. Ammonia detoxification promotes CD8+ T cell memory development. Nat Immunol. 2023;24:162–73.
20. Huang D, Chen Y, Li C, Zhang X, Wang J, Liu Y, et al. Salivary microbiome variations associated with immunotherapy efficacy in NSCLC. mSystems. 2025;10:e01115–24.
21. Bakir MA, Sakamoto M, Kitahara M, Benno Y. Bacteroides dorei sp. nov., isolated from human faeces. Int J Syst Evol Microbiol. 2006;56:1639–43.
22. He S, Lu S, Yang T, Song L, Xiao Y, Huang Y, et al. Bacteroides dorei-derived bile acid alleviates influenza virus infection. Cell Commun Signal. 2025;23:382.
23. He S, Song L, Xiao Y, Huang Y, Lu S, Yang T, et al. Genomic and probiotic properties of Bacteroides dorei RX2020. Nutrients. 2025;17.
24. Song L, Huang Y, Liu G, He S, Xiao Y, Lu S, et al. Bacteroides dorei ameliorates influenza virus infection in mice. Front Immunol. 2022;12:828887.
25. Su Y, Shadike Q, Wang M, Liu Y, Abudureheman A, Yusufu M, et al. Low abundance of Bacteroides correlates with phenylalanine levels. Transl Pediatr. 2021;10:2521–32.
26. Cong S, Wang L, Meng Y, Liu X, Zhang Y, Li J, et al. Saussurea involucrata regulates gut microbiota in arthritis rats. Phytother Res. 2022;37:1242–59.
27. Tan M, Cao G, Wang R, Zhang Y, Liu J, Li X, et al. Phenylalanine nanostructures sensitize breast tumour to checkpoint blockade. Nat Nanotechnol. 2024;19:1903–13.
28. May P, Winter C, Hubrecht I, Seidel C, Hentschel M, Schumann C, et al. Metabolic phenotype predicts atezolizumab outcomes in SCLC. Transl Lung Cancer Res. 2025;14:3836–46.
29. Liu Y, Ping Y, Zhang L, Wang J, Chen X, Huang D, et al. L-phenylalanine predicts response to anti-PD-1 therapy in NSCLC. MedComm. 2025;6:e70100.
30. Plaza-Díaz J, Álvarez-Mercado AI, Ruiz-Marín CM, Reina-Pérez I, Pérez-Muñoz ME, Gomez-Llorente C, et al. Breast and gut microbiota dysbiosis and breast cancer risk. BMC Cancer. 2019;19:495.
31. García-Vega ÁS, Corrales-Agudelo V, Reyes A, Mancabelli L, Milani C, Ventura M, et al. Diet quality and gut microbiota in a nonwestern population. Nutrients. 2020;12:2938.
32. Daillère R, Derosa L, Bonvalet M, Segata N, Routy B, Kroemer G, et al. Gut microbiota to boost anticancer immunotherapy. Oncoimmunology. 2020;9:1774298.
33. Farhadi Rad H, Tahmasebi H, Javani S, Zare M, Ebrahimi M, Akbari A, et al. Microbiota and cytokine modulation in anticancer immunity. Biomedicines. 2024;12.
34. Byrd DA, Gomez MF, Hogue SR, Wang Y, Rhoades KP, Figueiredo JC, et al. Colon tissue microbiome and bile acids in colorectal adenoma. Cancer Med. 2025;14:e71048.
35. Mokhashi O, Chakladar J, Li WT, McClellan J, Sarkar S, Amatya VJ, et al. Obesity-related microbial dysbiosis and tumour progression. Access Microbiol. 2025;7:e000846.
36. Moseeb HM, Aizaz MM, Aiza K, Rahman S, Ahmed R, Khan MA, et al. From obesity to cancer: gut microbiome mechanisms. Oncoscience. 2025;12:175–88.
37. Soni S, Mittal P, Lo JH, Zhang Y, Patel S, Wang J, et al. Age-diet interactions influence tumor microbiome and microenvironment. Neoplasia. 2025;70:101245.
| Files | ||
| Issue | Articles in Press | |
| Section | Original Article(s) | |
| Keywords | ||
| Amino acid metabolism Gastrointestinal microbiome Lipid metabolism Mendelian randomization analysis Programmed death-ligand 1 | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Kang Y, Xu B, Wang S, Xi S. Exploring the Causal Relationship between Gut Microbiome and Programmed Cell Death Protein-1/Programmed Cell Death Ligand-1: Mediating Effects of Serum Lipid and Amino Acid Metabolic Biomarkers in a Two-step Mendelian Randomization Study. Iran J Allergy Asthma Immunol. 2026;:1-11.
