Cytokine Signaling in the Microenvironment May Aid in Tumor Development and Progression

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Dr. Kim O’Neill, Department of Microbiology and Molecular Biology

Studies have indicated that in breast tumors a high number of tumor associated macrophages (TAM’s) is indicative of a poor prognosis. TAM’s in the breast tumor microenvironment generally have a compromised immune response and focus their energy toward tissue repair which leads to angiogenesis, tumor growth, and metastasis. These macrophages have been termed M2 macrophages. Conversely, studies also indicate that in the microenvironment of some colon cancers, M1 phenotypic macrophages are present and that a high number of TAM’s is associated with a good prognosis.

This study is centered on better understanding the effects of the tumor microenvironment on macrophages. Specifically, we investigated the effect that co- incubation with both breast and colon cancer cell lines had on the ability of macrophages to engulf latex microspheres (beads). We supposed that incubation with breast cancer cells would down-regulate overall macrophage engulfment of microspheres, while colon cancer would have minimal effect. Our data indicates that both breast and colon cancer down-regulate phagocytic capabilities of macrophages, lowering both their ability to engulf microspheres and the aggressiveness at which they do so.

The microenvironment of most tumors is filled with a large population of macrophages. In breast cancer, studies have found that macrophages can account for >50% of the tumor mass. Their presence within the tumor microenvironment, in some cases, has been proven to increase metastasis, angiogenesis, and immunosuppression. However, colorectal cancer has been seen in some cases to exhibit decreased tumor growth and proliferation when a larger number of macrophages are associated with the tumor. Previous studies have suggested that cytokines and chemokines released by the tumor cells in the tumor microenvironment influence the polarization of the macrophages present. The M1 phenotype will often lead to aggressive destruction of the tumor, while an M2 phenotype will in fact aid tumor survival. M2 macrophages have been suggested to have a reduced phagocytic function. In this study, we investigated overall phagocytosis of microspheres by U-937 derived macrophages when exposed to nutrient-depleted media from both breast and colon cancer cell lines as well as when exposed to nutrient-depleted media containing cancer cells themselves. U-937’s were stained with Efluor 670 dye for identification and then stimulated using phorbol 12-myristate 13-acetate (PMA). Macrophages were then incubated for various time periods with cancer cells seeded in 6- well plates. Engulfment was measured by adding fluorescent latex microspheres to samples and assayed using a flow cytometer. Overall engulfment was measured as the percent of macrophages in a sample that engulfed microspheres while aggressiveness was measured as the amount of beads that macrophages were seen to phagocytose. Controls indicate that 5-10% of cancer cells non-specifically bind microspheres and that approximately 20% of unstimulated U937’s engulf beads, whereas positive controls indicate that 80% of PMA stimulated U937’s engulf microspheres. Further results show that incubation in spent media from both breast and colon cell lines did not significantly alter overall phagocytosis or aggressiveness. However, when incubated with either colon or breast cancer cell lines in spent media, there is as high as a 30% reduction in overall engulfment, indicating a shift toward an M2 phenotype. When macrophages are co- cultured with non-cancerous lymphocytes, the overall engulfment stays consistent with positive controls. Also, highly aggressive macrophages engulfed significantly less aggressively when exposed to tumor microenvironments as compared to positive controls.

This study suggests that the microenvironment influences the polarization of macrophages via cell to cell contact and that both overall engulfment and aggressiveness of macrophages are affected. It also suggests that the microenvironments of both breast and colon cancer are equally able to polarize macrophages toward M2.

This MEG funding helped support the following presentations given by Dr. O’Neill’s laboratory in 2010-11. (Undergraduates in bold)

2010

  • Poster presentation at the annual intermountain branch meeting of the American Society for Microbiology, April 10th, Brigham Young University, Provo. Utah. Entitled, ‘The resistance Profile of Human Papillomavirus Type 16 to Chemical Disinfectants.’ J. Meyers, E. Ryndock, M.J. Conway, K.L. O’Neill, C. Meyers, and R.R. Robison.
  • Poster presentation at the annual intermountain branch meeting of the American Society for Microbiology, April 10th, Brigham Young University, Provo. Utah. Entitled, ‘ Improved recovery of spores from coated carriers used in the AOAC Sproicidal Activity of Disinfectants (Method II) spore Enumeration Method , by addition of a surfactant to the rinse solution. T. Bills, Q. C. Shepherd, A. J. Blam, E. A. Moore, J. Gardner, B. Schaalje, K. L. O’Neill and R.R. Robison.
  • Poster presentation at the annual intermountain branch meeting of the American Society for Microbiology, April 10th, Brigham Young University, Provo. Utah. Entitled, ‘Migration and Polarization of Macrophages May Be Simultaneously Affected by Macrophages Inhibition Factor (MIF).’ B.S. Hendriksen, R.A. Enz, R.R. Robison, and K. l. O’Neill.
  • Poster presentation at the annual intermountain branch meeting of the American Society for Microbiology, April 10th, Brigham Young University, Provo. Utah. Entitled, ‘The Potential of TK1 as a Molecular Target for Cancer Treatment.Dagoberto Estevez, Robert Whitehurst, Jaden D. Evans, Lafe T. Peavler, Melissa Tovar, Richard A. Robison, and Kim L. O’Neill.
  • Poster presentation at the annual intermountain branch meeting of the American Society for Microbiology, April 10th, Brigham Young University, Provo. Utah. Entitled, ‘Could Macrophage Phagocytosis be Regulated by the Tumor Microenvironment?’ D. Griffin, B. Hansen, B.R. Hendricks, R. Robison, and K.L. O’Neill.
  • Poster presentation at the annual intermountain branch meeting of the American Society for Microbiology, April 10th, Brigham Young University, Provo. Utah. Entitled, ‘Oxidative Stress Promotes Increased Antioxidant Uptake in a Cellular Model.’ Jorge A. Chauca, Andrew R. Garrett, Andres D. Martinez, Evita Giraldez, Jason Loong, Jon Whitaker, José M. Peña, Richard A.Robison, and Kim L. O’Neill.
  • Poster presentation at the annual intermountain branch meeting of the American Society for Microbiology, April 10th, Brigham Young University, Provo. Utah. Entitled, ‘Immunolabeling Transmission Electron Microscopy: an effective method for examining location of thymidine kinase1 in human lymphoma cells.’ M. Hardy, M. Tovar, M. Standing, R. Robison, and K.L. O’Neill.
  • Undergraduate poster competition presentation at the 101st annual meeting of the American Association for Cancer Research, April 17th, Washington DC., entitled, ‘Can drinking tea prevent cancer?’ J. K. Loong, E. Giraldez, J. Whitaker, R. A. Robison, K. L. O’Neill.
  • Undergraduate poster competition presentation at the 101st annual meeting of the American Association for Cancer Research, April 17th, Washington DC., entitled, ‘Antioxidant studies on the Peruvian cherimoya fruit give an insight into the healthy lifestyle of the Incas.’ G. Gupta Elera, J. Whitaker, J. Chauca, R. Robison, and Kim O’Neill.
  • Undergraduate poster competition presentation at the 101st annual meeting of the American Association for Cancer Research, April 17th, Washington DC., ‘Changes in Antioxidant capacities of isolated radish components during growth. J. Whitaker, E. Giraldez, J. Loong, J. Wagstaff, R Robison and K.L. O’Neill.
  • Undergraduate poster competition presentation at the 101st annual meeting of the American Association for Cancer Research, April 17th, Washington DC., ‘Inhibition of M1 Macrophages May Aid the Conversion Process to M2, which May Aid Tumor Survival.’ R.A. Enz, C. Knechtel, A.D. Enz, B. Swanson, E. Giraldez, R. Robison, and K.L. O’Neill.
  • Poster presentation at the 101st annual meeting of the American Association for Cancer Research, April 17-21, Washington DC., entitled, ‘Can drinking tea prevent cancer?’ J. K. Loong, E. Giraldez, J. Whitaker, R. A. Robison, K. L. O’Neill.
  • Poster presentation at the 101st annual meeting of the American Association for Cancer Research, April 17-21, Washington DC., entitled, ‘Antioxidant studies on the Peruvian cherimoya fruit give an insight into the healthy lifestyle of the Incas.’ G. Gupta Elera, J. Whitaker, J. Chauca, R. Robison, and Kim O’Neill.
  • Poster presentation at the 101st annual meeting of the American Association for Cancer Research, April 17-21, Washington DC., ‘Changes in Antioxidant capacities of isolated radish components during growth. J. Whitaker, E. Giraldez, J. Loong, J. Wagstaff, R Robison and K.L. O’Neill.
  • Poster presentation at the 101st annual meeting of the American Association for Cancer Research, April 17-21, Washington DC., ‘Inhibition of M1 Macrophages May Aid the Conversion Process to M2, which May Aid Tumor Survival.’ R.A. Enz, C. Knechtel, A.D. Enz, B. Swanson, E. Giraldez, R. Robison, and K.L. O’Neill.
  • Oral presentation at the annual conference of the American Association for the Advancement of Science (AAAS) Pacific Division on June 15, 2010: entitled, ‘Localization of Thymidine Kinase 1 in Human Lymphoma Cells through Immunogold Labeling Transmission Electron Microscopy.’ Moragn Hardy and K.L. O’Neill.
  • Poster presentation at the annual conference of the American Association for the Advancement of Science (AAAS) Pacific Division on June 15, 2010: entitled, ‘Can drinking tea prevent cancer?’ J. Loong, R. Robison and K. l. O’Neill
  • Oral Presentation at the 35th Annual Biological Sciences Undergraduate Research Conference Saturday, April 24, 2010, Santa Clara University. California, entitled, ‘Release of MIF by Aggressive Adenocarcinoma cell lines May Be an Initial Step in the Polarization of Macrophages.’ B.S. Hendriksen, R.A. Enz, C. Knechtel, A.D. Enz, B. Swanson, R. Robison, and K.L. O’Neill.
  • Oral Presentation at the 35th Annual Biological Sciences Undergraduate Research Conference Saturday, April 24, 2010, Santa Clara University. California, entitled, ‘A Novel Target for Cancer Immunotherapy.’ D. E. Estevez, R. Whitehurst, J. D. Evans, M. Tovar, R. A. Robison, and K. L. O’Neill.
  • Gunnell, M.K., C.D. Carter, W.S. Jonas, B.A. Satterfield, E.A. Moore, K.L. O’Neill, and R.A. Robison. A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies. 8th Annual ASM Biodefense and Emerging Disease Research Meeting. February 21-24, 2010. Baltimore, MD.
  • Gunnell, M.K., C.D. Carter, W.S. Jonas, B.A. Satterfield, E.A. Moore, K.L. O’Neill, and R.A. Robison. A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies. 3rd Annual Biothreat Agenst Workshop. March 15, 2010. Charlotte, NC.
  • Bills, T.M., Q.C. Shepherd, A.J. Blam, E.A. Moore, J. Gardner, B. Schaalje, K.L. O’Neill, and R.A. Robison. Improved recovery of spores from coated carriers used in the AOAC Sporicidal Activity of Disinfectants (Method II) spore enumeration method, by the addition of a surfactant to the rinse solution. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #39, p.12, April 10, 2010. Provo, UT. (Note: T.M. Bills won first place in the student poster presentation competition.)
  • Bills, T.M., Q.C. Shepherd, A.J. Blam, E.A. Moore, J. Gardner, B. Schaalje, K.L. O’Neill, and R.A. Robison. Improved recovery of spores from coated carriers used in the AOAC Sporicidal Activity of Disinfectants (Method II) spore enumeration method, by addition of a surfactant to the rinse solution. General Meeting of the American Society for Microbiology, Session 329/Q, Abstract # Q-3097/176, p. 218. May 27, 2010. San Diego, CA.
  • Meyers, J.M., E. Ryndock, M.J. Conway, K.L. O’Neill, C.M. Meyers, and R.A. Robison. Susceptibility of native and synthetic HPV16 virions to clinical disinfectants. General Meeting of the American Society for Microbiology, Session 329/Q, Abstract # Q-3111/190, p. 218. May 27, 2010. San Diego, CA.

2011

  • Presented a poster at the 6th Annual Undergraduate Student Caucus and Poster Competition, American Association for Cancer Research (AACR), Orlando, Florida, 2 April, 2011 ‘Localization of several potential biomarkers in various types of cancer’. Ballantines F Alegre, Melissa M Alegre, Morgan S Hardy,Richard A Robison, Kim L O’Neill.
  • Presented a poster at the 6th Annual Undergraduate Student Caucus and Poster Competition, American Association for Cancer Research (AACR), Orlando, Florida, 2 April, 2011 ‘Assessing the therapeutic and diagnostic potential of human thymidine kinase 1 in leukemia.” Dagoberto Estevez, Robert A. Whitehurst, Daniel W. Sharp, Richard A. Robison, and Kim L. O’Neill.
  • Presented a poster at the 6th Annual Undergraduate Student Caucus and Poster Competition, American Association for Cancer Research (AACR), Orlando, Florida, 2 April, 2011 ‘Microsphere engulfment by macrophages when exposed to breast and colon tumor microenvironments’ David Griffin, Ryan Quinton, Burke Hendricks, Brock Hansen, Paul Montoya, Atif El Naggar, Richard Robison, and Kim L. O’Neill.
  • Presented a poster at the 6th Annual Undergraduate Student Caucus and Poster Competition, American Association for Cancer Research (AACR), Orlando, Florida, 2 April, 2011 ‘Measuring TK1 autoantibodies in the serum of cancer and non-cancer patients’ Wesley D LaPorte, Taylor Abegg-Lawrence, Melissa M. Alegre, Richard A. Robison, and Kim L. O’Neill.
  • Presented a poster at the 6th Annual Undergraduate Student Caucus and Poster Competition, American Association for Cancer Research (AACR), Orlando, Florida, 2 April, 2011 ‘Visualization of Thymidine Kinase 1 on the surface of cancer cell lines: a potential diagnostic marker and therapeutic target.’ Robert A. Whitehurst, Dagoberto Estevez, Daniel W. Sharp, Melissa Alegre, Richard A. Robison, and Kim L. O’Neill.
  • Presented a poster at the 6th Annual Undergraduate Student Caucus and Poster Competition, American Association for Cancer Research (AACR), Orlando, Florida, 2 April, 2011 ‘‘Thymidine kinase 1, a novel biomarker specific to the plasma membrane of cancerous cell lines.’ Daniel W. Sharp, Dagoberto Estevez, Robert A. Whitehurst, Melissa M. Alegre, Brit L. Germann, Joshua W. Foster, Richard A. Robison and Kim L. O’Neill.
  • Presented a poster at the 6th Annual Undergraduate Student Caucus and Poster Competition, American Association for Cancer Research (AACR), Orlando, Florida, 2 April, 2011 ‘Macrophage Aggressiveness in the Tumor Microenvironment.’ Ryan Quinton, David Griffin, Melissa Alegre, Richard A. Robison, and Kim L. O’Neill.
  • Presented a poster at the 6th Annual Undergraduate Student Caucus and Poster Competition, American Association for Cancer Research (AACR), Orlando, FLorida, 2 April, 2011 ‘Raji antioxidant uptake of exotic fruit juices increases following pre-exposure to oxidative stress.’ Andrés D. Martínez, Andrew R. Garrett, Timothy C. Michaelis, José M. Peña, Richard A. Robison and Kim L. O’Neill.
  • Presented a poster at the 6th Annual Undergraduate Student Caucus and Poster Competition, American Association for Cancer Research (AACR), Orlando, Florida, 2 April, 2011 ‘The Organic Panic’. Jacob R. Jensen, Andrew R. Garrett, Ryan D. Kraus, Kylie Measom, Matthew Gillam, Richard A. Robison, and Kim L. O’Neill.
  • Presented a poster at the American Association of Cancer Research Meeting 102nd annual meeting held April 2-6, 2011 in Orlando, Florida. “Assessing the therapeutic and diagnostic potential of human thymidine kinase 1 in leukemia.” Dagoberto Estevez, Robert A. Whitehurst, Daniel W. Sharp, Richard A. Robison, and Kim L. O’Neill.
  • Presented a poster at the 102nd Annual meeting of the American Association for Cancer Research, April 2-6, 2011, Orlando, Florida. Entitled “Microsphere engulfment by macrophages when exposed to breast and colon tumor microenvironments” David Griffin, Ryan Quinton, Burke Hendricks, Brock Hansen, Paul Montoya, Atif El Naggar, Richard Robison, and Kim L. O’Neill.
  • Presented a poster at the 102nd Annual meeting of the American Association for Cancer Research, April 2-6, 2011, Orlando, Florida. Entitled “Measuring TK1 autoantibodies in the serum of cancer and non-cancer patients” Wesley D LaPorte, Taylor Abegg-Lawrence, Melissa M. Alegre, Richard A. Robison, and Kim L. O’Neill.
  • Presented a poster at the American Association of Cancer Research Meeting 102nd annual meeting held April 2-6, 2011 in Orlando, Florida “Visualization of Thymidine Kinase 1 on the surface of cancer cell lines: a potential diagnostic marker and therapeutic target” Robert A. Whitehurst, Dagoberto Estevez, Daniel W. Sharp, Melissa Alegre, Richard A. Robison, and Kim L. O’Neill.
  • Presented a poster at the American Association of Cancer Research Meeting 102nd annual meeting held April 2-6, 2011 in Orlando, Florida. ‘Thymidine kinase 1, a novel biomarker specific to the plasma membrane of cancerous cell lines.’ Daniel W. Sharp, Dagoberto Estevez, Robert A. Whitehurst, Melissa M. Alegre, Brit L. Germann, Joshua W. Foster, Richard A. Robison and Kim L. O’Neill.
  • Presented a poster at the American Association of Cancer Research Meeting 102nd annual meeting held April 2-6, 2011 in Orlando, Florida. ‘Localization of Several Potential Biomarkers in Various Types of Cancer. Ballantines F Alegre, Melissa M Alegre, Morgan S Hardy, Richard A Robison, Kim L O’Neill.
  • Presented a poster at the American Association of Cancer Research Meeting 102nd annual meeting held April 2-6, 2011 in Orlando, Florida. ‘Macrophage Aggressiveness in the Tumor Microenvironment.” Ryan Quinton, David Griffin, Melissa Alegre, Richard A. Robison, and Kim L. O’Neill.
  • Presented a poster at the American Association of Cancer Research Meeting 102nd annual meeting held April 2-6, 2011 in Orlando, Florida. ‘The Organic Panic’. Jacob R. Jensen, Andrew R. Garrett, Ryan D. Kraus, Kylie Measom, Matthew Gillam, Richard A. Robison, and Kim L. O’Neill.
  • Gave an oral presentation at the American Society for Microbiology, Intermountain Branch meeting, April 9, 2011 in Ogden Utah. ‘Power juices: The role of exotic, anti-oxidant fruit juices in preventing cancer. Timothy C. Michaelis, Andres D. Martinez, Andrew R. Garrett, Jose M. Pena, Richard A. Robison and Kim L. O’Neill.
  • Gave an oral presentation at the American Society for Microbiology, Intermountain Branch meeting, April 9, 2011 in Ogden Utah. ‘Antioxidant Levels in Blueberries: Are Organic Better Than Conventional? Ryan D. Kraus, Jacob R. Jensen, Andrew R. Garrett, Richard A. Robison, and Kim L. O’Neill.
    *This presentation won first-place award at the conference.
  • Gave an oral presentation at the American Society for Microbiology, Intermountain Branch meeting, April 9, 2011 in Ogden Utah. ‘Antioxidant Levels in Blueberries: Are Organic Better Than Conventional? Ryan D. Kraus, Jacob R. Jensen, Andrew R. Garrett, Richard A. Robison, and Kim L. O’Neill.
  • Gave an oral presentation at the American Society for Microbiology, Intermountain Branch meeting, April 9, 2011 in Ogden Utah. ‘Cancer Patients Possess Lower Serum Antioxidant Levels than Age Matched Healthy Subjects’ Adam Grooms and Kim L. O’Neill.
  • Gave an oral presentation at the American Society for Microbiology, Intermountain Branch meeting, April 9, 2011 in Ogden Utah. ‘Macrophage Phagocytic Activity in the Tumor Microenvironment of Multiple Cancer Cell Lines.’ Ryan J. Quinton and Kim L. O’Neill.

This MEG funding helped support the following publications given by Dr. O’Neill’s laboratory in 2010-11. (Undergraduates in bold)

  • C.J. Capua, N.P. Hopson, M.L.Stewart, G.R. Johnson, K.L. O’Neill, G. B. Schaalje, G.M. Booth. 2009. Cytotoxicity of Atriplex confertifolia. Pharmaceutical Biology Journal of Toxicology, Volume 2010 , Article ID 976548, 7 pages
  • A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F. B. A. Satterfield,1 A. F. Stewart,1 C. S. Lew, D. O. Pickett, M. N. Cohen, E. A. Moore, P. F. Luedtke, K. L. O’Neill1 and Richard A. Robison. (2010) Journal of Medical Microbiology (2010), 59, 55–64
  • A R. Garrett, Byron K. Murray, Richard A. Robison, and Kim L. O’Neill, 2010. Measuring Antioxidant Capacity Using the ORAC and TOSC Assays. Methods in Molecular Biology 2010; 594:251-62, Advanced Protocols in Oxidative Stress II, Humana Press Ch 17.
  • Satterfield, B.A., A.F. Steward, C.S. Lew, D.O. Pickett, M.N. Cohen, E.A. Moore, P.F. Luedtke, K.L. O’Neill, and R.A. Robison. 2010. A quadraplexed real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F. Journal of Medical Microbiology 59:55-64. Note: This paper was featured in the ‘Highlights of Hot papers in Recent SGM Journals” section of Microbiology Today, February, 2010, p. 60.
  • Gaytri Gupta Elera, B.Sc.; Andrew R Garrett, B.Sc; Martinez Martinez, B.Sc; Richard A Robison, Ph.D.; Kim O’Neill, 2010. The antioxidant properties of the Cherimoya (Annona cherimola) fruit. 2011. Food Research International Volume 44, Issue 7, August 2011, Pages 2205-2209.
  • March, Jordon; Cohen, Marissa; Lindsey, James; Millar, D; O’Neill, Kim; Schaalje, G.; Robison, Richard, 2011 The disinfectant susceptibility of virulent and attenuated Bacillus anthracis spores varies with disinfectant type. Applied Microbiology.
  • Gupta-Elera, Gaytri; Garrett, Andrew R.; Robison, Richard A.; O’Neill, Kim L.2011 The role of oxidative stress in prostate cancer. European Journal of Cancer Prevention., POST AUTHOR CORRECTIONS, 19 August 2011 doi: 10.1097/CEJ.0b013e32834a8002

Patents

U.S Patent Application No. 61/291,351 For “Compositions and Methods with Antibodies specific for Nucleotide Salvage Pathway Enzymes of Cancer Patients. Filed December 30th 2009. (Pending) Inventors: Kim Leslie O’Neill Robert Alan Whitehurst Jaden Duss Evans Daniel William Sharp and Melissa Marie Alegre