Skip to main content


Log in

Endothelial Rap1B mediates T-cell exclusion to promote tumor growth: a novel mechanism underlying vascular immunosuppression

  • Original Paper
  • Published:
Angiogenesis Aims and scope Submit manuscript


Overcoming vascular immunosuppression: lack of endothelial cell (EC) responsiveness to inflammatory stimuli in the proangiogenic environment of tumors, is essential for successful cancer immunotherapy. The mechanisms through which Vascular Endothelial Growth Factor A(VEGF-A) modulates tumor EC response to exclude T-cells are not well understood. Here, we demonstrate that EC-specific deletion of small GTPase Rap1B, previously implicated in normal angiogenesis, restricts tumor growth in endothelial-specific Rap1B-knockout (Rap1BiΔEC) mice. EC-specific Rap1B deletion inhibits angiogenesis, but also leads to an altered tumor microenvironment with increased recruitment of leukocytes and increased activity of tumor CD8+ T-cells. Depletion of CD8+ T-cells restored tumor growth in Rap1BiΔEC mice. Mechanistically, global transcriptome and functional analyses indicated upregulation of signaling by a tumor cytokine, TNF-α, and increased NF-κB transcription in Rap1B-deficient ECs. Rap1B-deficiency led to elevated proinflammatory chemokine and Cell Adhesion Molecules (CAMs) expression in TNF-α stimulated ECs. Importantly, CAM expression was elevated in tumor ECs from Rap1BiΔEC mice. Significantly, Rap1B deletion prevented VEGF-A-induced immunosuppressive downregulation of CAM expression, demonstrating that Rap1B is essential for VEGF-A-suppressive signaling. Thus, our studies identify a novel endothelial-endogenous mechanism underlying VEGF-A-dependent desensitization of EC to proinflammatory stimuli. Significantly, they identify EC Rap1B as a potential novel vascular target in cancer immunotherapy.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others


  1. Hendry SA, Farnsworth RH, Solomon B, Achen MG, Stacker SA, Fox SB (2016) The role of the tumor vasculature in the host immune response: implications for therapeutic strategies targeting the tumor microenvironment. Front Immunol.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Fridman WH, PagèsSaut̀s-Fridman FC, Galon J (2012) The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 12(4):298–306.

    Article  CAS  PubMed  Google Scholar 

  3. Ager A, Watson HA, Wehenkel SC, Mohammed RN (2016) Homing to solid cancers: a vascular checkpoint in adoptive cell therapy using CAR T-cells. Biochem Soc Trans 44(2):377–385.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ley K, Laudanna C, Cybulsky MI, Nourshargh S (2007) Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 7(9):678–689.

    Article  CAS  PubMed  Google Scholar 

  5. Joyce JA, Fearon DT (2015) T cell exclusion, immune privilege, and the tumor microenvironment. Science 348(6230):74–80.

    Article  CAS  PubMed  Google Scholar 

  6. Griffioen AW, Damen CA, Martinotti S, Blijham GH, Groenewegen G (1996) Endothelial intercellular adhesion molecule-1 expression is suppressed in human malignancies: the role of angiogenic factors. Can Res 56(5):1111–1117

    CAS  Google Scholar 

  7. Piali L, Fichtd A, Terpe HJ, Imhof BA, Gisler RH (1995) Endothelial vascular cell adhesion molecule 1 expression is suppressed by melanoma and carcinoma. J Exp Med 181(2):811–816.

    Article  CAS  PubMed  Google Scholar 

  8. Huang H, Langenkamp E, Georganaki M, Loskog A, Fuchs PF, Dieterich LC, Kreuger J, Dimberg A (2015) VEGF suppresses T-lymphocyte infiltration in the tumor microenvironment through inhibition of NF-κB-induced endothelial activation. FASEB J 29(1):227–238.

    Article  CAS  PubMed  Google Scholar 

  9. Dirkx AEM, Oude Egbrink MGA, Castermans K, Van Der Schaft DWJ, Thijssen VLJL, Dings RPM, Kwee L, Mayo KH, Wagstaff J, Bouma-ter Steege JCA, Griffioen AW (2006) Anti-angiogenesis therapy can overcome endothelial cell anergy and promote leukocyte-endothelium interactions and infiltration in tumors. FASEB J 20(6):621–630.

    Article  CAS  PubMed  Google Scholar 

  10. Shrimali RK, Yu Z, Theoret MR, Chinnasamy D, Restifo NP, Rosenberg SA (2010) Antiangiogenic agents can increase lymphocyte infiltration into tumor and enhance the effectiveness of adoptive immunotherapy of cancer. Can Res 70(15):6171–6180.

    Article  CAS  Google Scholar 

  11. Boettner B, Van Aelst L (2009) Control of cell adhesion dynamics by Rap1 signaling. Curr Opin Cell Biol 21(5):684–693

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chrzanowska-Wodnicka M, Kraus AE, Gale D, White GC, Vansluys J (2008) Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice. Blood 111(5):2647–2656.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yan J, Li F, Ingram DA, Quilliam LA (2008) Rap1a is a key regulator of fibroblast growth factor 2-induced angiogenesis and together with Rap1b controls human endothelial cell functions. Mol Cell Biol 28(18):5803–5810

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Carmona G, Gottig S, Orlandi A, Scheele J, Bauerle T, Jugold M, Kiessling F, Henschler R, Zeiher AM, Dimmeler S, Chavakis E (2009) Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis. Blood 113(2):488–497.

    Article  CAS  PubMed  Google Scholar 

  15. Chrzanowska-Wodnicka M (2010) Regulation of angiogenesis by a small GTPase Rap1. Vascul Pharmacol 53(1–2):1–10.

    Article  CAS  PubMed  Google Scholar 

  16. Lakshmikanthan S, Sobczak M, Li Calzi S, Shaw L, Grant MB, Chrzanowska-Wodnicka M (2018) Rap1B promotes VEGF-induced endothelial permeability and is required for dynamic regulation of the endothelial barrier. J Cell Sci.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Xin G, Khatun A, Topchyan P, Zander R, Volberding PJ, Chen Y, Shen J, Fu C, Jiang A, See WA, Cui W (2020) Pathogen-boosted adoptive cell transfer therapy induces endogenous antitumor immunity through antigen spreading. Cancer Immunol Res 8(1):7–18.

    Article  CAS  PubMed  Google Scholar 

  18. Picelli S, Faridani OR, Björklund ÅK, Winberg G, Sagasser S, Sandberg R (2014) Full-length RNA-seq from single cells using smart-seq2. Nat Protoc 9(1):171–181.

    Article  CAS  PubMed  Google Scholar 

  19. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Method 14(4):417–419.

    Article  CAS  Google Scholar 

  20. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M (1999) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 27(1):29–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fabregat A, Jupe S, Matthews L, Sidiropoulos K, Gillespie M, Garapati P, Haw R, Jassal B, Korninger F, May B, Milacic M, Roca CD, Rothfels K, Sevilla C, Shamovsky V, Shorser S, Varusai T, Viteri G, Weiser J, Wu G, Stein L, Hermjakob H, D’Eustachio P (2018) The reactome pathway knowledgebase. Nucleic Acids Res 46(D1):D649–D655.

    Article  CAS  PubMed  Google Scholar 

  23. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdóttir H, Tamayo P, Mesirov JP (2011) Molecular signatures database (MSigDB) 3.0. Bioinformatics 27(12):1739–1740.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102(43):15545–15550.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc: Ser B (Methodol) 57(1):289–300

    Google Scholar 

  26. Gu SX, Blokhin IO, Wilson KM, Dhanesha N, Doddapattar P, Grumbach IM, Chauhan AK, Lentz SR (2016) Protein methionine oxidation augments reperfusion injury in acute ischemic stroke. JCI Insight.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Seigner J, Junker-Samek M, Plaza A, D’Urso G, Masullo M, Piacente S, Holper-Schichl YM, de Martin R (2019) A symphytum officinale root extract exerts anti-inflammatory properties by affecting two distinct steps of nf-κb signaling. Front Pharmacol 10:289–289.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wilhelmsen K, Farrar K, Hellman J (2013) Quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions. J V Exp: JoVE 78:e50677–e50677.

    Article  CAS  Google Scholar 

  29. Mansour AA, Raucci F, Sevim M, Saviano A, Begum J, Zhi Z, Pezhman L, Tull S, Maione F, Iqbal AJ (2022) Galectin-9 supports primary T cell transendothelial migration in a glycan and integrin dependent manner. Biomed Pharmacother 151:113171.

    Article  CAS  PubMed  Google Scholar 

  30. Graham VA, Marzo AL, Tough DF (2007) A role for CD44 in T cell development and function during direct competition between CD44+ and CD44 cells. Eur J Immunol 37(4):925–934.

    Article  CAS  PubMed  Google Scholar 

  31. Kelso A, Costelloe EO, Johnson BJ, Groves P, Buttigieg K, Fitzpatrick DR (2002) The genes for perforin, granzymes A-C and IFN-γ are differentially expressed in single CD8+ T cells during primary activation. Int Immunol 14(6):605–613.

    Article  CAS  PubMed  Google Scholar 

  32. Kohli K, Pillarisetty VG, Kim TS (2022) Key chemokines direct migration of immune cells in solid tumors. Cancer Gene Ther 29(1):10–21.

    Article  CAS  PubMed  Google Scholar 

  33. Karin N (2020) CXCR3 ligands in cancer and autoimmunity, chemoattraction of effector t cells, and beyond. Front Immunol 11:976.

    Article  CAS  PubMed Central  Google Scholar 

  34. Lakshmikanthan S, Sobczak M, Chun C, Henschel A, Dargatz J, Ramchandran R, Chrzanowska-Wodnicka M (2011) Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin alphavbeta(3). Blood 118(7):2015–2026.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ferrara N, Adamis AP (2016) Ten years of anti-vascular endothelial growth factor therapy. Nat Rev Drug Discov 15(6):385–403.

    Article  CAS  PubMed  Google Scholar 

  36. Pàez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Viñals F, Inoue M, Bergers G, Hanahan D, Casanovas O (2009) Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 15(3):220–231.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ebos JML, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG, Kerbel RS (2009) Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell 15(3):232–239.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Song Y, Fu Y, Xie Q, Zhu B, Wang J, Zhang B (2020) Anti-angiogenic agents in combination with immune checkpoint inhibitors: a promising strategy for cancer treatment. Front Immunol 11:1956.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ciciola P, Cascetta P, Bianco C, Formisano L, Bianco R (2020) Combining immune checkpoint inhibitors with anti-angiogenic agents. J Clin Med.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Chrzanowska-Wodnicka M (2013) Distinct functions for Rap1 signaling in vascular morphogenesis and dysfunction. Exp Cell Res 319(15):2350–2359.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lakshmikanthan S, Zheng X, Nishijima Y, Sobczak M, Szabo A, Vasquez-Vivar J, Zhang DX, Chrzanowska-Wodnicka M (2015) Rap1 promotes endothelial mechanosensing complex formation, NO release and normal endothelial function. EMBO Rep 16(5):628–637.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Singh B, Kosuru R, Lakshmikanthan S, Sorci-Thomas MG, Zhang DX, Sparapani R, Vasquez-Vivar J, Chrzanowska M (2021) Endothelial Rap1 (Ras-Association Proximate 1) restricts inflammatory signaling to protect from the progression of atherosclerosis. Arterioscler Thromb Vasc Biol 41(2):638–650.

    Article  CAS  PubMed  Google Scholar 

  43. Qian J, Olbrecht S, Boeckx B, Vos H, Laoui D, Etlioglu E, Wauters E, Pomella V, Verbandt S, Busschaert P, Bassez A, Franken A, Bempt MV, Xiong J, Weynand B, van Herck Y, Antoranz A, Bosisio FM, Thienpont B, Floris G, Vergote I, Smeets A, Tejpar S, Lambrechts D (2020) A pan-cancer blueprint of the heterogeneous tumor microenvironment revealed by single-cell profiling. Cell Res 30(9):745–762.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


This work was funded by NIH grant R01HL111582 (M.C.). W.C. is supported by American Cancer Society Research Scholar Grant. RK is supported by the Director’s Fellowship Award at the Versiti Blood Research Institute, Wisconsin.

Author information

Authors and Affiliations



M.C. designed the research, provided funding, analyzed data, and wrote the manuscript; G.P.S. and R.K. performed the experiments, analyzed data, prepared figures, drafted the manuscript; S.L. generated data; S.Z. and R.B analyzed data; Y.C. and G.X. designed the experiments; W.C. designed the experiments and provided funding. All authors reviewed the manuscript.

Corresponding author

Correspondence to Magdalena Chrzanowska.

Ethics declarations

Conflict of interest

The authors declare no potential conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 7829 kb)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sharma, G.P., Kosuru, R., Lakshmikanthan, S. et al. Endothelial Rap1B mediates T-cell exclusion to promote tumor growth: a novel mechanism underlying vascular immunosuppression. Angiogenesis 26, 265–278 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: