Visualization of PD-L1-positive and PD-1-positive immune cell contact in the breast cancer microenvironment

Functioning of the PD-1/PD-L1 immune checkpoint in the microenvironment of breast cancer may lead to the tumor escape from the immune response. However, it is unknown how often PD-L1 binds to PD-1 in breast cancer patients, which PD-L1-positive cells are predominantly involved in the interaction, and what prognostic significance it has. The objective of the study was to assess the frequency of co-location of PD-1/PD-L1- positive cells in the microenvironment of breast cancer as well as to determine the population of these cells. Material and Methods. The study included 25 patients with invasive breast carcinoma. Interaction between cells carrying the PD-1 receptor and the PD-L1 ligand in the tumor microenvironment were visualized using multiplex TSA (tyramide signal amplification)-modified immunohistochemistry. Participation of M1 macrophages (CD68+CD163-CD3-CKAE1/3-), M2 macrophages (CD68+/-CD163+CD3-CKAE1/3-), lymphocytes (CD68- CD163-CD3+CKAE1/3-) and other immune cells in these interactions was assessed. Results. Half of the breast cancer patients included in the study had interactions of immune cells of the microenvironment, one of which carried PD-1, and the other carried PD-L1. The contact of cells carrying PD-1 and PD-L1 was associated with the level of TILs and the ratio of PD-1+/ PD-L1+ cells in the tumor microenvironment. The PD-1/PD-L1 interaction was found with similar frequency in PD-L1 positive and negative patients. In the cell contacts, macrophages acted as PD-L1+ cells in the vast majority of cases. Lymphocytes were PD-1-positive cells rather than PD-L1- carrying cells. In addition, it was found that metastasis-free survival was not associated with the presence or absence of co-localized cells carrying PD-1 and PD-L1 in the tumor microenvironment. Conclusion. Co-location of immune cells carrying PD-1 and PD-L1 occurs in breast cancer. M1 and M2 macrophages, CD3+ lymphocytes and other immune cells are involved in these interactions. However, further studies are needed to establish the prognostic significance of these contacts.

1. Okazaki T., Honjo T. The PD-1-PD-L pathway in immunological tolerance. Trends Immunol. 2006; 27(4): 195–201. doi: 10.1016/j.it.2006.02.001.

2. Han Y., Liu D., Li L. PD-1/PD-L1 pathway: current researches in cancer. Am J Cancer Res. 2020; 10(3): 727–42.

3. Lu D., Ni Z., Liu X., Feng S., Dong X., Shi X., Zhai J., Mai S., Jiang J., Wang Z., Wu H., Cai K. Beyond T Cells: Understanding the Role of PD-1/PD-L1 in Tumor-Associated Macrophages. J Immunol Res. 2019. doi: 10.1155/2019/1919082.

4. Singhal S., Stadanlick J., Annunziata M.J., Rao A.S., Bhojnagarwala P.S., O’Brien S., Moon E.K., Cantu E., Danet-Desnoyers G., Ra H.J., Litzky L., Akimova T., Beier U.H., Hancock W.W., Albelda S.M., Eruslanov E.B. Human tumor-associated monocytes/macrophages and their regulation of T cell responses in early-stage lung cancer. Sci Transl Med. 2019; 11(479). doi: 10.1126/scitranslmed.aat1500.

5. Zhu Z., Zhang H., Chen B., Liu X., Zhang S., Zong Z., Gao M. PD-L1-Mediated Immunosuppression in Glioblastoma Is Associated With the Infiltration and M2-Polarization of Tumor-Associated Macrophages. Front Immunol. 2020; 11. doi: 10.3389/fimmu.2020.588552.

6. Horn L., Spigel D.R., Vokes E.E., Holgado E., Ready N., Steins M., PoddubskayaE.,BorghaeiH.,FelipE.,Paz-AresL.,PluzanskiA.,ReckampK.L., Burgio M.A., Kohlhäeufl M., Waterhouse D., Barlesi F., Antonia S., Arrieta O., Fayette J., Crinò L., Rizvi N., Reck M., Hellmann M.D., Geese W.J., Li A., Blackwood-Chirchir A., Healey D., Brahmer J., Eberhardt W.E.E. Nivolumab Versus Docetaxel in Previously Treated Patients With Advanced Non-Small-Cell Lung Cancer: Two-Year Outcomes From Two Randomized, Open-Label, Phase III Trials (CheckMate 017 and CheckMate 057). J Clin Oncol. 2017; 35(35): 3924–33. doi: 10.1200/JCO.2017.74.3062.

7. Schmid P., Adams S., Rugo H.S., Schneeweiss A., Barrios C.H., Iwata H., Diéras V., Hegg R., Im S.A., Shaw Wright G., Henschel V., Molinero L., Chui S.Y., Funke R., Husain A., Winer E.P., Loi S., Emens L.A.; IMpassion130 Trial Investigators. Atezolizumab and Nab-Paclitaxel in Advanced Triple-Negative Breast Cancer. N Engl J Med. 2018; 379(22): 2108–21. doi: 10.1056/NEJMoa1809615.

8. Kang Y.K., Boku N., Satoh T., Ryu M.H., Chao Y., Kato K., Chung H.C., Chen J.S., Muro K., Kang W.K., Yeh K.H., Yoshikawa T., Oh S.C., Bai L.Y., Tamura T., Lee K.W., Hamamoto Y., Kim J.G., Chin K., Oh D.Y., Minashi K., Cho J.Y., Tsuda M., Chen L.T. Nivolumab in patients with advanced gastric or gastro-oesophageal junction cancer refractory to, or intolerant of, at least two previous chemotherapy regimens (ONO-4538-12, ATTRACTION-2): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2017; 390(10111): 2461–71. doi: 10.1016/S0140-6736(17)31827-5.

9. Keenan T.E., Tolaney S.M. Role of Immunotherapy in Triple-Negative Breast Cancer. J Natl Compr Canc Netw. 2020; 18(4): 479–89. doi: 10.6004/jnccn.2020.7554.

10. Kamakura D., Asano R., Kawai H., Yasunaga M. Mechanism of action of a T cell-dependent bispecific antibody as a breakthrough immunotherapy against refractory colorectal cancer with an oncogenic mutation. Cancer Immunol Immunother. 2021; 70(1): 177–88. doi: 10.1007/s00262-020-02667-9.

11. Yamaguchi K., Tsuchihashi K., Tsuji K., Kito Y., Tanoue K., Ohmura H., Ito M., Isobe T., Ariyama H., Kusaba H., Akashi K., Baba E. Prominent PD-L1-positive M2 macrophage infiltration in gastric cancer with hyper-progression after anti-PD-1 therapy: A case report. Medicine (Baltimore). 2021; 100(19). doi: 10.1097/MD.0000000000025773.

12. Salgado R., Denkert C., Demaria S., Sirtaine N., Klauschen F., Pruneri G., Wienert S., Van den Eynden G., Baehner F.L., Penault-Llorca F., Perez E.A., Thompson E.A., Symmans W.F., Richardson A.L., Brock J., Criscitiello C., Bailey H., Ignatiadis M., Floris G., Sparano J., Kos Z., Nielsen T., Rimm D.L., Allison K.H., Reis-Filho J.S., Loibl S., Sotiriou C., Viale G., Badve S., Adams S., Willard-Gallo K., Loi S.; International TILs Working Group 2014. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol. 2015; 26(2): 259–71. doi: 10.1093/annonc/mdu450.

13. Larijani B., Miles J., Ward S.G., Parker P.J. Quantification of biomarker functionality predicts patient outcomes. Br J Cancer. 2021; 124(10): 1618–20. doi: 10.1038/s41416-021-01291-3.

14. Sánchez-Magraner L., Miles J., Baker C.L., Applebee C.J., Lee D.J., Elsheikh S., Lashin S., Withers K., Watts A.G., Parry R., Edmead C., Lopez J.I., Mehta R., Italiano A., Ward S.G., Parker P.J., Larijani B. High PD-1/PD-L1 Checkpoint Interaction Infers Tumor Selection and Therapeutic Sensitivity to Anti-PD-1/PD-L1 Treatment. Cancer Res. 2020; 80(19): 4244–57. doi: 10.1158/0008-5472.CAN-20-1117.

15. Cirqueira M.B., Mendonça C.R., Noll M., Soares L.R., de Paula Carneiro Cysneiros M.A., Paulinelli R.R., Moreira M.A.R., Freitas-Junior R. Prognostic Role of PD-L1 Expression in Invasive Breast Cancer: A Systematic Review and Meta-Analysis. Cancers (Basel). 2021; 13(23): 6090. doi: 10.3390/cancers13236090.

16. Huang W., Ran R., Shao B., Li H. Prognostic and clinicopathological value of PD-L1 expression in primary breast cancer: a meta-analysis. Breast Cancer Res Treat. 2019; 178(1): 17–33. doi: 10.1007/s10549-019-05371-0.

17. Yunna C., Mengru H., Lei W., Weidong C. Macrophage M1/M2 polarization. Eur J Pharmacol. 2020; 877. doi: 10.1016/j.ejphar.2020.173090.

18. Peranzoni E., Lemoine J., Vimeux L., Feuillet V., Barrin S., Kantari-Mimoun C., Bercovici N., Guérin M., Biton J., Ouakrim H., Régnier F., Lupo A., Alifano M., Damotte D., Donnadieu E. Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment. Proc Natl Acad Sci U S A. 2018; 115(17): 4041–50. doi: 10.1073/pnas.1720948115.

19. Hartley G.P., Chow L., Ammons D.T., Wheat W.H., Dow S.W. Programmed Cell Death Ligand 1 (PD-L1) Signaling Regulates Macrophage Proliferation and Activation. Cancer Immunol Res. 2018; 6(10): 1260–73. doi: 10.1158/2326-6066.CIR-17-0537.

20. Sánchez-Magraner L., Gumuzio J., Miles J., Quimi N., Martínez Del Prado P., Abad-Villar M.T., Pikabea F., Ortega L., Etxezarraga C., Martín-Algarra S., Lozano M.D., Saiz-Camin M., Egurrola-Izquierdo M., Barredo-Santamaría I., Saiz-López A., Gomez-Mediavilla J., Segues-Merino N., Juaristi-Abaunz M.A., Urruticoechea A., Geraedts E.J., van Elst K., Claessens N.J.M., Italiano A., Applebee C.J., Del Castillo S., Evans C., Aguirre F., Parker P.J., Calleja V. Functional Engagement of the PD-1/ PD-L1 Complex But Not PD-L1 Expression Is Highly Predictive of Patient Response to Immunotherapy in Non-Small-Cell Lung Cancer. J Clin Oncol. 2023. doi: 10.1200/JCO.22.01748.