Cancer Immunology, Immunotherapy

, Volume 63, Issue 6, pp 587–599 | Cite as

Salmonella-mediated tumor regression involves targeting of tumor myeloid suppressor cells causing a shift to M1-like phenotype and reduction in suppressive capacity

  • Suneesh Kaimala
  • Yassir A. Mohamed
  • Nancy Nader
  • Jincy Issac
  • Eyad Elkord
  • Salem Chouaib
  • Maria J. Fernandez-Cabezudo
  • Basel K. al-Ramadi
Original Article


The effectiveness of attenuated Salmonella in inhibiting tumor growth has been demonstrated in many therapeutic models, but the precise mechanisms remain incompletely understood. In this study, we show that the anti-tumor capacity of Salmonella depends on a functional MyD88-TLR pathway and is independent of adaptive immune responses. Since myeloid suppressor cells play a critical role in tumor growth, we investigated the consequences of Salmonella treatment on myeloid cell recruitment, phenotypic characteristics, and functional activation in spleen and tumor tissue of B16.F1 melanoma-bearing mice. Salmonella treatment led to increased accumulation of splenic and intratumoral CD11b+Gr-1+ myeloid cells, exhibiting significantly increased expression of various activation markers such as MHC class II, costimulatory molecules, and Sca-1/Ly6A proteins. Gene expression analysis showed that Salmonella treatment induced expression of iNOS, arginase-1 (ARG1), and IFN-γ in the spleen, but down-regulated IL-4 and TGF-β. Within the tumor, expression of iNOS, IFN-γ, and S100A9 was markedly increased, but ARG1, IL-4, TGF-β, and VEGF were inhibited. Functionally, splenic CD11b+ cells maintained their suppressive capacity following Salmonella treatment, but intratumoral myeloid cells had significantly reduced suppressive capacity. Our findings demonstrate that administration of attenuated Salmonella leads to phenotypic and functional maturation of intratumoral myeloid cells making them less suppressive and hence enhancing the host’s anti-tumor immune response. Modalities that inhibit myeloid suppressor cells may be useful adjuncts in cancer immunotherapy.


Myeloid suppressor cells Salmonella Tumor immunity Macrophages 



We thank Dr. Toby Eisenstein (Temple University School of Medicine, Philadelphia, USA) for critical review of the manuscript. We are grateful to Dr. Samir Attoub (Department of Pharmacology, College of Medicine & Health Sciences, United Arab Emirates University) for providing us with the NMRI/nude mice. We also thank Arshad Khan for animal care and husbandry. This work was funded by grants from the Terry Fox Fund for Cancer Research and the UAE University-NRF (to B. K. al-Ramadi).

Conflict of interest

The authors declare no competing interests.

Supplementary material

262_2014_1543_MOESM1_ESM.pdf (648 kb)
Supplementary material 1 (PDF 648 kb)


  1. 1.
    Gabrilovich DI, Ostrand-Rosenberg S, Bronte V (2012) Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol 12(4):253–268PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Mantovani A, Sozzani S, Locati M, Allavena P, Sica A (2002) Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 23(11):549–555PubMedCrossRefGoogle Scholar
  3. 3.
    Poschke I, Kiessling R (2012) On the armament and appearances of human myeloid-derived suppressor cells. Clin Immunol 144(3):250–268PubMedCrossRefGoogle Scholar
  4. 4.
    Qian BZ, Pollard JW (2010) Macrophage diversity enhances tumor progression and metastasis. Cell 141(1):39–51PubMedCrossRefGoogle Scholar
  5. 5.
    Wynn TA, Chawla A, Pollard JW (2013) Macrophage biology in development, homeostasis and disease. Nature 496(7446):445–455PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Steidl C, Lee T, Shah SP, Farinha P, Han G, Nayar T, Delaney A, Jones SJ, Iqbal J, Weisenburger DD, Bast MA, Rosenwald A, Muller-Hermelink HK, Rimsza LM, Campo E, Delabie J, Braziel RM, Cook JR, Tubbs RR, Jaffe ES, Lenz G, Connors JM, Staudt LM, Chan WC, Gascoyne RD (2010) Tumor-associated macrophages and survival in classic Hodgkin’s lymphoma. N Engl J Med 362(10):875–885PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Zabuawala T, Taffany DA, Sharma SM, Merchant A, Adair B, Srinivasan R, Rosol TJ, Fernandez S, Huang K, Leone G, Ostrowski MC (2010) An ets2-driven transcriptional program in tumor-associated macrophages promotes tumor metastasis. Cancer Res 70(4):1323–1333PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    al-Ramadi BK, Brodkin MA, Mosser DM, Eisenstein TK (1991) Immunosuppression induced by attenuated Salmonella. Evidence for mediation by macrophage precursors. J Immunol 146(8):2737–2746PubMedGoogle Scholar
  9. 9.
    al-Ramadi BK, Chen YW, Meissler JJ Jr, Eisenstein TK (1991) Immunosuppression induced by attenuated Salmonella. Reversal by IL-4. J Immunol 147(6):1954–1961PubMedGoogle Scholar
  10. 10.
    Sander LE, Sackett SD, Dierssen U, Beraza N, Linke RP, Muller M, Blander JM, Tacke F, Trautwein C (2010) Hepatic acute-phase proteins control innate immune responses during infection by promoting myeloid-derived suppressor cell function. J Exp Med 207(7):1453–1464PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Van Ginderachter JA, Beschin A, De Baetselier P, Raes G (2010) Myeloid-derived suppressor cells in parasitic infections. Eur J Immunol 40(11):2976–2985PubMedCrossRefGoogle Scholar
  12. 12.
    Balkwill F, Charles KA, Mantovani A (2005) Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 7(3):211–217PubMedCrossRefGoogle Scholar
  13. 13.
    Saccani A, Schioppa T, Porta C, Biswas SK, Nebuloni M, Vago L, Bottazzi B, Colombo MP, Mantovani A, Sica A (2006) p50 nuclear factor-kappaB overexpression in tumor-associated macrophages inhibits M1 inflammatory responses and antitumor resistance. Cancer Res 66(23):11432–11440PubMedCrossRefGoogle Scholar
  14. 14.
    Mazzieri R, Pucci F, Moi D, Zonari E, Ranghetti A, Berti A, Politi LS, Gentner B, Brown JL, Naldini L, De Palma M (2011) Targeting the ANG2/TIE2 axis inhibits tumor growth and metastasis by impairing angiogenesis and disabling rebounds of proangiogenic myeloid cells. Cancer Cell 19(4):512–526PubMedCrossRefGoogle Scholar
  15. 15.
    Du R, Lu KV, Petritsch C, Liu P, Ganss R, Passegue E, Song H, Vandenberg S, Johnson RS, Werb Z, Bergers G (2008) HIF1alpha induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion. Cancer Cell 13(3):206–220PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Corzo CA, Condamine T, Lu L, Cotter MJ, Youn JI, Cheng P, Cho HI, Celis E, Quiceno DG, Padhya T, McCaffrey TV, McCaffrey JC, Gabrilovich DI (2010) HIF-1alpha regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment. J Exp Med 207(11):2439–2453PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Hix LM, Karavitis J, Khan MW, Shi YH, Khazaie K, Zhang M (2013) Tumor STAT1 transcription factor activity enhances breast tumor growth and immune suppression mediated by myeloid-derived suppressor cells. J Biol Chem 288(17):11676–11688PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Sonda N, Simonato F, Peranzoni E, Cali B, Bortoluzzi S, Bisognin A, Wang E, Marincola FM, Naldini L, Gentner B, Trautwein C, Sackett SD, Zanovello P, Molon B, Bronte V (2013) miR-142-3p prevents macrophage differentiation during cancer-induced myelopoiesis. Immunity 38(6):1236–1249PubMedCrossRefGoogle Scholar
  19. 19.
    Forbes NS (2010) Engineering the perfect (bacterial) cancer therapy. Nat Rev Cancer 10(11):785–794PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Lee CH (2012) Engineering bacteria toward tumor targeting for cancer treatment: current state and perspectives. Appl Microbiol Biotechnol 93(2):517–523PubMedCrossRefGoogle Scholar
  21. 21.
    Eisenstein TK, Bushnell B, Meissler JJ Jr, Dalal N, Schafer R, Havas HF (1995) Immunotherapy of a plasmacytoma with attenuated Salmonella. Med Oncol 12(2):103–108PubMedCrossRefGoogle Scholar
  22. 22.
    Pawelek JM, Low KB, Bermudes D (1997) Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res 57:4537–4544PubMedGoogle Scholar
  23. 23.
    al-Ramadi BK, Fernandez-Cabezudo MJ, El-Hasasna H, Al-Salam S, Bashir G, Chouaib S (2009) Potent anti-tumor activity of systemically-administered IL2-expressing Salmonella correlates with decreased angiogenesis and enhanced tumor apoptosis. Clin Immunol 130(1):89–97PubMedCrossRefGoogle Scholar
  24. 24.
    al-Ramadi BK, Fernandez-Cabezudo MJ, El-Hasasna H, Al-Salam S, Attoub S, Xu D, Chouaib S (2008) Attenuated bacteria as effectors in cancer immunotherapy. Ann N Y Acad Sci 1138:351–357PubMedCrossRefGoogle Scholar
  25. 25.
    Richter-Dahlfors A, Buchan AMJ, Finlay BB (1997) Murine Salmonellosis studied by confocal microscopy: Salmonella typhimurium resides intracellularly inside macrophages and exerts a cytotoxic effect on phagocytes in vivo. J Exp Med 186:569–580PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    al-Ramadi BK, Adeghate E, Mustafa N, Ponery AS, Fernandez-Cabezudo MJ (2002) Cytokine expression by attenuated intracellular bacteria regulates the immune response to infection: the Salmonella model. Mol Immunol 38(12–13):931–940PubMedCrossRefGoogle Scholar
  27. 27.
    Fernandez-Cabezudo MJ, El-Kharrag R, Torab F, Bashir G, George JA, El-Taji H, al-Ramadi BK (2013) Intravenous administration of manuka honey inhibits tumor growth and improves host survival when used in combination with chemotherapy in a melanoma mouse model. PLoS One 8(2):e55993PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    al-Ramadi BK, Bashir G, Rizvi TA, Fernandez-Cabezudo MJ (2004) Poor survival but high immunogenicity of IL-2-expressing Salmonella typhimurium in inherently resistant mice. Microbes Infect 6(4):350–359PubMedCrossRefGoogle Scholar
  29. 29.
    Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sakagami M, Nakanishi K, Akira S (1998) Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9(1):143–150PubMedCrossRefGoogle Scholar
  30. 30.
    Xu J, Foy TM, Laman JD, Elliott EA, Dunn JJ, Waldschmidt TJ, Elsemore J, Noelle RJ, Flavell RA (1994) Mice deficient for the CD40 ligand. Immunity 1:423–431PubMedCrossRefGoogle Scholar
  31. 31.
    Issac JM, Sarawathiamma D, Al-Ketbi MI, Azimullah S, Al-Ojali SM, Mohamed YA, Flavell RA, Fernandez-Cabezudo MJ, al-Ramadi BK (2013) Differential outcome of infection with attenuated Salmonella in MyD88-deficient mice is dependent on the route of administration. Immunobiology 218(1):52–63PubMedCrossRefGoogle Scholar
  32. 32.
    al-Ramadi BK, Fernandez-Cabezudo MJ, Ullah A, El-Hasasna H, Flavell RA (2006) CD154 is essential for protective immunity in experimental salmonella infection: evidence for a dual role in innate and adaptive immune responses. J Immunol 176(1):496–506PubMedCrossRefGoogle Scholar
  33. 33.
    Fernandez-Cabezudo MJ, Mechkarska M, Azimullah S, al-Ramadi BK (2009) Modulation of macrophage proinflammatory functions by cytokine-expressing Salmonella vectors. Clin Immunol 130(1):51–60PubMedCrossRefGoogle Scholar
  34. 34.
    Dolcetti L, Peranzoni E, Ugel S, Marigo I, Fernandez Gomez A, Mesa C, Geilich M, Winkels G, Traggiai E, Casati A, Grassi F, Bronte V (2010) Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF. Eur J Immunol 40(1):22–35PubMedCrossRefGoogle Scholar
  35. 35.
    Ehrchen JM, Sunderkotter C, Foell D, Vogl T, Roth J (2009) The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer. J Leukoc Biol 86(3):557–566PubMedCrossRefGoogle Scholar
  36. 36.
    al-Ramadi BK, Greene JM, Meissler JJ Jr, Eisenstein TK (1992) Immunosuppression induced by attenuated Salmonella: effect of LPS responsiveness on development of suppression. Microb Pathog 12(4):267–278PubMedCrossRefGoogle Scholar
  37. 37.
    al-Ramadi BK, Meissler JJ Jr, Huang D, Eisenstein TK (1992) Immunosuppression induced by nitric oxide and its inhibition by interleukin-4. Eur J Immunol 22(9):2249–2254PubMedCrossRefGoogle Scholar
  38. 38.
    Low KB, Ittensohn M, Le T, Platt J, Sodi S, Amoss M, Ash O, Carmichael E, Chakraborty A, Fischer J, Lin SL, Luo X, Miller SI, Zheng L, King I, Pawelek JM, Bermudes D (1999) Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nat Biotechnol 17(1):37–41PubMedCrossRefGoogle Scholar
  39. 39.
    Kasinskas RW, Forbes NS (2007) Salmonella typhimurium lacking ribose chemoreceptors localize in tumor quiescence and induce apoptosis. Cancer Res 67(7):3201–3209PubMedCrossRefGoogle Scholar
  40. 40.
    Leschner S, Westphal K, Dietrich N, Viegas N, Jablonska J, Lyszkiewicz M, Lienenklaus S, Falk W, Gekara N, Loessner H, Weiss S (2009) Tumor invasion of Salmonella enterica serovar Typhimurium is accompanied by strong hemorrhage promoted by TNF-alpha. PLoS One 4(8):e6692PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Ganai S, Arenas RB, Sauer JP, Bentley B, Forbes NS (2011) In tumors Salmonella migrate away from vasculature toward the transition zone and induce apoptosis. Cancer Gene Ther 18(7):457–466PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Lee CH, Wu CL, Shiau AL (2008) Toll-like receptor 4 mediates an antitumor host response induced by Salmonella choleraesuis. Clin Cancer Res 14(6):1905–1912PubMedCrossRefGoogle Scholar
  43. 43.
    Saccheri F, Pozzi C, Avogadri F, Barozzi S, Faretta M, Fusi P, Rescigno M (2010) Bacteria-induced gap junctions in tumors favor antigen cross-presentation and antitumor immunity. Sci Transl Med 2(44):44ra57PubMedCrossRefGoogle Scholar
  44. 44.
    al-Ramadi BK, Al-Dhaheri MH, Mustafa N, Abouhaidar M, Xu D, Liew FY, Lukic ML, Fernandez-Cabezudo MJ (2001) Influence of vector-encoded cytokines on anti-Salmonella immunity: divergent effects of interleukin-2 and tumor necrosis factor alpha. Infect Immun 69(6):3980–3988PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Dougan G, John V, Palmer S, Mastroeni P (2011) Immunity to salmonellosis. Immunol Rev 240(1):196–210PubMedCrossRefGoogle Scholar
  46. 46.
    Bronte V, Zanovello P (2005) Regulation of immune responses by l-arginine metabolism. Nat Rev Immunol 5(8):641–654PubMedCrossRefGoogle Scholar
  47. 47.
    Gallina G, Dolcetti L, Serafini P, De Santo C, Marigo I, Colombo MP, Basso G, Brombacher F, Borrello I, Zanovello P, Bicciato S, Bronte V (2006) Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells. J Clin Invest 116(10):2777–2790PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Hong EH, Chang SY, Lee BR, Pyun AR, Kim JW, Kweon MN, Ko HJ (2013) Intratumoral injection of attenuated Salmonella vaccine can induce tumor microenvironmental shift from immune suppressive to immunogenic. Vaccine 31(10):1377–1384PubMedCrossRefGoogle Scholar
  49. 49.
    Angiolillo AL, Sgadari C, Taub DD, Liao F, Farber JM, Maheshwari S, Kleinman HK, Reaman GH, Tosato G (1995) Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo. J Exp Med 182(1):155–162PubMedCrossRefGoogle Scholar
  50. 50.
    Sgadari C, Farber JM, Angiolillo AL, Liao F, Teruya-Feldstein J, Burd PR, Yao L, Gupta G, Kanegane C, Tosato G (1997) Mig, the monokine induced by interferon-gamma, promotes tumor necrosis in vivo. Blood 89(8):2635–2643PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Suneesh Kaimala
    • 1
  • Yassir A. Mohamed
    • 1
  • Nancy Nader
    • 1
  • Jincy Issac
    • 1
  • Eyad Elkord
    • 1
  • Salem Chouaib
    • 3
  • Maria J. Fernandez-Cabezudo
    • 2
  • Basel K. al-Ramadi
    • 1
  1. 1.Department of Medical Microbiology and Immunology, College of Medicine and Health SciencesUnited Arab Emirates UniversityAl AinUnited Arab Emirates
  2. 2.Department of Biochemistry, College of Medicine and Health SciencesUnited Arab Emirates UniversityAl AinUnited Arab Emirates
  3. 3.Institut National de la Sante et de la Recherche Medicale, Unite 753Institut Gustave RoussyVillejuifFrance

Personalised recommendations