Dr. Santos Manes - Simposio Internacional 'Terapias oncológicas avanzadas'
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Los días 15 y 16 de octubre de 2014, la Fundación Ramón Areces y la Real Academia Nacional de Farmacia, en colaboración con la Fundación de la Innovación Bankinter, reunieron en Madrid a algunos de los mayores expertos mundiales en nuevas terapias contra el cáncer. El Simposio Internacional, coordinado por la profesora y académica María José Alonso, analizó el momento actual de la lucha contra esta enfermedad. También fue un punto de encuentro para científicos de los más innovadores institutos de investigación en oncología, quienes debatieron sobre tres grandes temas: la Medicina Personalizada contra el cáncer, los nanomedicamentos en la terapia del cáncer y las terapias basadas en la inmunomodulación.
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Dr. Santos Manes - Simposio Internacional 'Terapias oncológicas avanzadas'
- 1. Cross-communication between immune and endothelial cells: Implications for cancer therapy Santos Mañes Dept Immunology and Oncology CNB/CSIC Ramón Areces Symposium Madrid, 15-16 October 2014
- 2. CANCER: NOT ONLY ONCOGENES AND SUPPRESSORS
- 3. THE TUMOR MICROENVIRONMENT TRIGGERS IMMUNE SUPPRESSION LACK LACK O OFF I MIMMMUUNNOOGGEENNICICITITYY LLOOWW C COO-S-STTIMIMUULLAATTIOIONN ANERGY MEMORY ININHHIBIBITITOORRYY N NEETTWWOORRKKSS TGFß PD-1/PD-L1 CTLA-4 IL-10…. T CELL EXHAUSTION VEGF PlGF… MDSC CTL iDC DC Treg M2-TAM
- 4. CHEMOKINES: CHEMOTACTIC CYTOKINES C C C C X X X C C C C C C C CC (b-chemokines) 24 human (CCL1-28) C X CXC (a-chemokines) 15 human (CXCL1-16) CX3C (d-chemokines) 1 human (CX3CL1) X C C XC (g-chemokines) 2 human (XCL1, 2) The chemokines, a subset of cytokines, were intially characterized by their ability to induce the directed migration of leukocytes. More than 40 human chemokines and 18 chemokine receptors have been discovered so far. Most chemokines are secreted proteins of 67 to 127 amino acids (8-11 kDa); only two chemokines, CXCL16 and CX3CL1, are also membrane-bound. Four major structural chemokine subfamilies are distinguished by the number and arrangement of their N-terminal cysteine residues.
- 5. PROMISCUITY OF THE CHEMOKINE SYSTEM • Tardáguila & Mañ es (2014)
- 6. CHEMOKINES AND LEUKOCYTE TRAFFICKING • Homey et al., Nat. Rev. Immunol. 2:175 (2002)
- 7. CD4+ T CELLS ACCUMULATE CCR5 AT THE IMMUNE SYNAPSE GFP-CCR5 T cell Antigen-dependent Agonist-dependent Pertussis toxin-insensitive Not characteristic of other chemokine receptors • Molon et al., Nat. Immunol. 6:465 (2005)
- 8. CCR5 INDUCES CD4+ T CELL CO-STIMULATION IFNg PROLIFERATION CD4+ CCL5 CCR5 a-CD3
- 9. ADOPTIVE TRANSFER OF CD4+ OR CD8+ T CELLS DOES NOT INHIBIT TUMOR GROWTH OT-I cells: OVA-specific OT1$cells:"OVA& specific " CD8 +"cells" OT2$cells:"OVA& specific " CD4 +"cells" EG7$cells:$OVA&expressing" EL4&derived"cell"line" sc" iv" CCR5$KO$ WT CCR5KO • González-Martín et al., Cancer. Res. 71:5455 (2011) CD8+ cells OT-II cells: OVA-specific CD4+ cells
- 10. TUMOR GROWTH INHIBITION REQUIRES CCR5-MEDIATED CD4+/CD8+ T CELL COOPERATION sc CCR5 KO OT1 infiltration iv OT1 activation (dLN) + + WT CCR5KO
- 11. EFFECTIVE TUMOR INHIBITION REQUIRES CCR5 IN BOTH CD4+ AND CD8+ CELLS Lack of CCR5 in OT2 also results in - Reduced OT1 cell infiltration into tumors - Reduced OT1 cell activation WT CCR5KO iv sc CCR5 KO
- 12. CCR5 IN CD4+ T CELLS ENHANCES CD8+ T CELL CROSS-PRIMING CCL3 CCL4 CCL5 CD40L CD40 CD4+ CD80/86 CD8+ + + OT-IWT OT-IWT OT-IIWT OT-IIKO
- 13. CCR5 INCREASES FORMATION OF TCR NANOCLUSTERS IN CD4+ CELLS Ag OT-II WT OT-II KO Agonist-dependent Antigen-independent
- 14. OT-II WT OT-II KO CCR5 INCREASES THE SENSITIVITY OF * ** ** ** Ag-EXPERIENCED CD4+ T CELLS
- 15. CCR5 TRIGGERS EXPANSION OF EFFECTOR AND MEMORY ANTI-TUMOR T CELLS FVB-Tg(MMTV-neu) WT CCR5KO CpG (ODN1826) (30 μg, 3 times/week)
- 16. NANOPARTICLE-DRIVEN TCR CLUSTERING STIMULATES ANTI-TUMOR IMMUNITY • Perica et al. ACSnano 8:2252 (2014)
- 17. CCR5 DEFICIENCY AFFECTS BREAST CANCER IN HUMANS D32/D32 individuals can be considered “knockouts” for CCR5 D32/D32 genotype occurs in 1% of Caucasians D32/D32 genotype is associated to resistance to HIV-1, compromised survival in West Nile virus-infected patients, and reduced rejection after allograft transplantation D32/D32 individuals can be considered “knockouts” for CCR5 D32/D32 genotype occurs in 1% of Caucasians D32/D32 genotype is associated to resistance to HIV-1, compromised survival in West Nile virus-infected patients, and reduced rejection after allograft transplantation Increased relapse Reduced disease-free survival Reduced lymphocyte infiltration
- 18. BLOOD VESSELS IN TUMORS ARE ABNORMAL Leaky Tortuous Irregular branching Abnormal shunts Poor O2 perfusion Poor drug delivery MDSC M2-TAM INFLAMMATION • modified from Carmeliet & Jain. Nat Rev Drug Discov10: 417 (2011) STATINS
- 19. LOVASTATIN “NORMALIZES” TUMOR VASCULATURE Lovastatin DOES NOT affect tumor growth BLOOD VESSEL FUNCTION • Mira et al. Oncotarget 4: 2288 (2013) BLOOD VESSEL STRUCTURE Pimonidazole staining FVB-Tg(MMTV-neu)
- 20. LOVASTATIN SHAPES THE INFLAMMATORY INFILTRATE TOWARDS AN ANTI-TUMOR PHENOTYPE INCREASED T CELL INFILTRATION SKEW TO M1 MACROPHAGES
- 21. SOD3 MEDIATES SOME LOVASTATIN EFFECTS ON THE TUMOR VASCULATURE CD31 SOD3 DOXORUBICIN PENETRATION ENHANCED T CELL INFILTRATION
- 22. RESTORATION OF SOD3 EXPRESSION IN TUMORS ENHANCES ADOPTIVE T CELL THERAPY • Carmona et al. Submitted EG7 (OVA-expressing) Ad-CDH5p-SOD3 OT-I cells CD3 1 SOD 3
- 23. SOD3 REDUCES CCL2 EXPRESSION AT THE TUMOR-ASSOCIATED ENDOTHELIUM CCL2 SOD 3 ±± C CCCLL22 ± SOD3 in vitro migration Ad-CDH5p-SOD3 CD3 1 CCL 2 Ad-mock
- 24. CHANGING THE TUMOR MICROENVIRONMENT TO FOSTER IMMUNE RESPONSE ENHANCED T CELL INFILTRATION SOD3 INHIBITS CCL2 SOD3 INHIBITS CCL2 EXPRESSION EXPRESSION REDUCED VASCULAR INFLAMMATION SOD3 CCR5 INDUCES CD4+ T CELL CCR5 INDUCES CD4+ T CELL CO-STIMULATION CO-STIMULATION ENHANCED ACTIVATED CROSS-PRESENTATION CTL B zone T zone Medulla Afferent lymphatics HEV INCREASED MEMORY
- 25. Emilia Mira Rosa A. Lacalle Manuel Tardáguila Juan Carlos de Karam Lorena Carmona Jesús Ogando Ana Martín Leal Rosa M. Peregil Former members Alicia González-Martín Antonella Viola (Padova Univ.) Enzo Bronte (Verona Univ.) Joseph Lustgarten (Mayo Clinic Arizona) Balbino Alarcón/Hisse Van Santen (CBM-SO. Madrid) Sergio A. Lira (Mount Sinai, NY) Tim Oury (Pittsburgh Univ.) Gemma Fabriás/Fina Casas (CIAC/CSIC. Barcelona) Iñigo Azcoitia (Univ. Complutense Madrid)
Editor's Notes
- First, I would like to thank the Maria Jose for her kind invitation to this interesting meeting and to have the opportunity to present our results to the audience. In particular, I will present some of our latest results on the crosstalk between immune and endothelial cells.
- Cancer is a disease of our genes. But in recent years, it has become evident that the interaction of transformed cells with the microenvironment is central to cancer promotion and progression. This microenvironment is shaped not only by the tumor cells, but also by fibroblasts, endothelial cells, and infiltrating leukocytes. This creates a specific pro-inflammatory state that supports tumor growth and spread, and suppresses immunity to the tumor cells. Indeed, infiltrating immune cells are the major producers of pro-angiogenic factors in the tumor milieu. Although immune cells are the main support for tumor progression, it is possible to reprogram these cells to fight against tumors.
- As Enzo explained nicely, the tumor microenvironment is essentially immunosuppressive. This is the result of many factors. One is the low immunogenicity of tumor cells because of reduced expression of MHC molecules, or because inhibitory ligands that block natural killer cell function are expressed or shed. Co-stimulation of tumor-specific T cells is also defective; this leads to T cell anergy as well as defects in the generation of long-term responses. Tumors trigger an incredible variety of inhibitory networks in the tumor milieu, whose final aim is to skew immune cell differentiation towards immunosuppressive phenotypes and/or to block effector cell function. Proangiogenic factors, such as vascular endothelial growth factor or placental growth factor, are at high levels in the tumor environment. They are also important compónents of the immunosuppressive ambience. They diréctly affect the differentiation state of some immune cell subtypes. For instance, VEGF inhibits maturation of immature dendritic cells; PlGF skews macrophages to the pro-tumor M2 phenotype. But they also trigger a hyperactivated state in the endothelium. They promote proliferation, migration and branching of endothelial cells, which drives formation of aberrant blood vessels. In such vessels, the transendothelial migration of innate immune cells and other immunosuppressive cells is favored, while infiltration of effector cells is limited.
- Chemokines and their receptors are one of major intercellular communication systems in the tumor environment. Chemokines were initially characterized by their ability to induce directed migration of leukocytes. Indeed, chemokines expressed at the endothelial cell surface determine the arrest and transmigration of specific leukocyte subtypes into inflamed tissues, and maybe into tumors. There are more than 40 chemokines in humans, classified in four groups according to the number and arrangement of their N-terminal cysteine residues. Most chemokines are secreted proteins, with two exceptions that can also be membrane-bound. Although they are secreted, their in vivo function depends on their interaction with glycosaminoglycans, which anchor these proteins to the extracellular matrix and the glycocalyx.
- Chemokines signal through G protein-coupled receptors, and there is generally much promiscuity in ligand/receptor interactions, particularly for those chemokines termed inflammatory (noted here in red).
- Despite this promiscuity, the chemokine system is extremely selective for leukocyte trafficking into inflamed tissues and lymphoid organs. This is achieved by the precise regulation of ligand and receptor expression in specific cell types and tissues. For instance, regulation of CCR7 and its ligands CCL21 and CCL19 enables the encounter between antigen-loaded mature dendritic cells and naïve T lymphocytes in the secondary lymphoid organs, a step that is central to the initiation and strength of immune responses.
- Nonetheless, chemokines do not participate only in the generation of immune responses by controlling leukocyte trafficking. This is the case of the chemokine receptor CCR5 and its ligands CCL3, CCL4 and CCL5. In collaboration with Antonella Viola’s group, we found that CCR5 expressed on T cells accumulates at the immune synapse formed after interaction with an antigen-presenting cell. This accumulation is antigen-dependent; that is, it requires T cell receptor (TCR) engagement. It is also agonist-dependent; the T cell and the APC secrete CCR5 ligands after engagement, and blockade of this secretion or treatment with CCR5 antagonists prevents CCR5 accumulation. Although accumulation requires CCR5 signaling, this signaling is mediated by heterotrimeric Gq proteins. It is thus insensitive to pertussis toxin inhibition, which blocks classical Gi protein-mediated signaling. Finally, it is partially specific; we observed that CXCR4 also accumulates at the synapse, but CCR7 does not.
- In vitro, CCR5 accumulation at the immune synapse cooperates with the TCR to promote T cell costimulation, to the same extent as other classical co-stimulatory receptors such as CD28. Does this co-stimulatory function of CCR5 also happen in vivo?
- To answer this question, we used OT-1/OT-2 mice, which we crossed with CCR5-deficient mice to generate ovalbumin-specific CD4 and CD8 cells that expressed or lacked CCR5. These T cells express a TCR transgenic for OVA and hence recognize and kill the EG7 thymoma that expresses OVA. We generated tumors subcutaneously with EG7 in mice and then adoptively transferred OT1 or OT2 cells that did or did not express CCR5. We found that adoptive transfer of CD4 or CD8 cells at suboptimal doses was unable to stop tumor growth, independently of their CCR5 genotype.
- However, CCR5 was essential for obtaining optimal tumoricídal activity after co-injection of OT1 and OT2 cells. The reduction in tumor size correlated with enhanced OT1 cell infiltration into the tumor parenchyma, and increased expression of IFNgamma, used as an activation marker.
- These results indicated that efficient tumor rejection involves cooperation between CD4 and CD8 cells, and that this cooperation requires CCR5. We next tested whether CCR5 expression was needed in one or both cell types. We performed adoptive transfer experiments with mixed OT1 and OT2 cells of different genotypes, and we found that lack of CCR5 in CD4 or CD8 cells was sufficient to prevent the tumoricídal activity of transferred cells. This suggests that efficient helper-dependent CD8 activation requires CCR5 expression in both CD4 and CD8 cells.
- To make a long story short, we found that CCR5 expression on CD4 cells provides an extra signal that increases CD40L after antigen binding to the TCR. By binding to CD40 on the APC, CD40L is a key molecule in DC maturation. Enhanced CD40L levels on CD4 cells reinforces the APC maturation program, leading to upregulation of costimulatory receptors such as CD80 and CD86. These, in turn, feed back CD4 costimulation positively through CD28, and enhance CD8 crosspriming. In support of this model, we found that activation of wild-type OT1 cells in vitro was significantly higher when they were co-incubated with wild-type OT2 cells, in the presence of APC loaded with both cognate peptides. So, OT1 activation is independent of its own CCR5, but relies on CCR5 expression by the CD4 cells.
- In addition to the CD40L upregulation, CCR5 also affects organization of the TCR. It is thought that the TCR is organized into pre-existing oligomers or nanoclusters. These nanoclusters, whose number and valency is higher in antigen-experienced cells, seems to be important for detection of low antigen concentrations. To test whether CCR5 can regulate TCR oligomer organization, we used OT2 that did or did not express CCR5. They were stimulated with the cognate peptide for 3 days, and then cultured for 7 days with IL-2 to expand the activated cells, but with no antigen. After TCR staining, cell surface replicas of the blast cells were analyzed by electron microscopy. As you can see here, both the number and the valency of the TCR nanoclusters was higher in CCR5-expressing OT2 cells than in the knockouts. This process was dependent on CCR5 stimulation, since adding a CCR5 antagonist prevented formation of these oligomers. And this was antigen-independent, which tells us that it is not consequence of antigen presentation.
- Larger TCR nanoclusters are associated with increased antigen sensitivity. CCR5-expressing cells respond better to low antigen doses than the CCR5-defective cells, as we determined by IFNgamma production and antigen-induced proliferation. This coincides with their ability to induce TCR oligomerization. Increased antigen sensitivity is a characteristic of memory T cells, which correlates with the formation of TCR nanoclusters. Since CCR5 is important for TCR cluster formation in antigen-experienced T cells, we hypothesize that the function of CCR5-deficient memory T cells might be impaired due to their defective production of these nanoclusters.
- To study this question, we used a mouse model that develops breast tumors due to overexpression of the proto-oncogene neu/HER2 in the mammary gland. Lack of CCR5 does not affect the onset of breast tumors in these mice, probably because their immune cells are tolerized to tumor antigens. Nonetheless, there is a pool of low affinity T cells that recognize immunodominant neu peptides. These T cells can be reactivated by injection of Toll-like receptor agonists, such as the TLR9 agonist CpG. We found that repeated CpG injection inhibited tumor growth, but we saw this inhibition only in wild-type mice and not in CCR5 KO hosts. This reduced tumor growth was associated with expansion of neu-reactive CD8 T cells, which also produced more IFNgamma. We also found a modest but clear expansion of the CD4 memory T cell population in the spleen.
- Here I would like to show that the induction of TCR oligomerization using a nanoparticle approach also enhances the anti-tumor response of adoptively transferred T cells. This highlights the importance of increased TCR nanoclustering to enhance the potency of immune responses, not only in cancer but also for vaccination (for instance).
- Do these results have any relevance for human cancer? There is a CCR5 polymorphism termed delta32, which severely affects CCR5 expression. It consists of a 32 bp deletion that alters the reading frame, which leads to a truncated receptor that is not exported to the membrane. Human delta32 homozygotes, around 1% of the Spanish population, can be considered knockouts for CCR5, and heterozygotes show reduced CCR5 expression. Although CCR5 appears to be dispensable in homeostasis, delta32 mutation has been associated with various pathologies, such as resistance to HIV-1 infection –since CCR5 is used as a receptor by some HIV strains—, poor prognosis and compromised survival in patients with West Nile virus infection, and reduced rejection after allograft transplant. These last two effects indicate that d32 polymorphism partially impairs immune responses. We studied the influence of d32 mutation in breast cancer using a cohort of almost 700 patients. We found that d32 polymorphism neither predisposes to nor prevents breast cancer, as was the case in ccr5 KO mice. However, we found that the d32 patients showed a greater propensity to relapse; they also showed reduced disease-free survival in a subset of tumors, and reduced lymphocyte infiltration in their tumors. We do not know if there is a correlation between these observations, but it would be important to analyze in more detail how CCR5 deficiency affects the immune system in these patients, and the possible relevance in their response to treatment.
- In addition to efficient T cell activation, anti-tumor responses rely on the arrival of effector cells to the tumor vicinity. As I mentioned earlier, tumor blood vessels appear to be selective barriers to effector lymphocyte transmigration, while they are very permissive to myeloid cell entry. This selective permeablity might be related to the profound morphological and functional changes in the tumor-associated vasculature. Compared to healthy tissues, tumor blood vessels are irregular, unstable and leaky; most of them are not perfused, which originates large hypoxic areas in the tumor, and decreases the delivery of chemotherapeutic agents. We postulate that inflammation is a major cause of blood vessel abnormalization in tumors. In fact, myeloid cells are a major source of pro-angiogenic factors and cytokines, which trigger this abnormalization. We therefore decided to study how anti-inflammatory drugs affect the tumor vasculature. We chose statins, since as well as reducing cholesterol levels, these compounds are potent immunomodulators with anti-inflammatory activity.
- We treated neu-transgenic mice that had developed spontaneous breast tumors with 10 mg/Kg lovastatin every three days; this is similar to 80 mg/day in humans. This dose does not affect growth of the primary tumor, although lovastatin induces profound changes in its vasculature. At the structural level, we found a decrease in blood vessel diameter compared to vehicle-treated tumors. The discontinuities or gaps in the endothelial layer in vehicle-treated mice contrasted with the continuous, smooth vessels in lovastatin-treated mice. Using a green-labeled lectin, we saw that Lov treatment also improved tumor perfusion, which might be linked to increased vessel stability and maturation. As a result of increased perfusion, the tumor parenchyma showed a reduction in hypoxic areas. This leads to more oxidative metabolism, and increased entry of chemotherapeutic drugs. You can see that here for doxorubicin; when it is combined with lovastatin, it yields similar clinical effects at much lower drug doses.
- In these tumors, lovastatin caused striking changes in the inflammatory infiltrate. On the one hand, Lov treatment increased the number of T lymphocytes, mostly CD8, that infiltrated the tumors, which inverted the ratio between myeloid and lymphoid cells in the tumor parenchyma. On the other hand, Lov treatment skewed the polarization of tumor-associated macrophages towards an M1 phenotype with anti-tumor activity.
- One of the genes that mediates these lovastatin effects on the vasculature is superoxide dismutase 3, also known as extracellular dismutase. This enzyme is a scavenger of the superoxide anion in the extracellular space, which reduces oxidative stress and associated oxidative damage in extracellular elements. SOD3 is expressed highly in the mammary gland, but this is greatly reduced in neu tumors. It is also downregulated in human breast cancers, suggesting that it is is a general phenomenon associated with neoplasia. Lov treatment partially restored SOD3 expression, mainly in areas near blood vessels. Using syngeneic tumor mouse models, we found that SOD3 is essential for mediating the Lov effects on tumor vasculature. Indeed, the increased doxorubicin perfusion into tumors and the enhanced T cell infiltration, both induced by lovastatin, were lost when the tumors were implanted in SOD3-deficient mice.
- To confirm SOD3 involvement in T cell transmigration into tumors, we forced SOD3 expression in the vasculature, using an adenovirus that codes for the enzyme under the control of the VE-cadherin promoter. These viruses were used to infect EG7 tumors generated in syngeneic mice. SOD3 expression in EG7 tumors and the associated vasculature was low, as in the neu tumor model. The adenovirus efficiently increased SOD3 expression near blood vessels. Finally, we performed adoptive transfer of OT1 cells into tumor-bearing mice. The OT-I cells infiltrated tumors infected with SOD3 adenovirus more efficiently than those infected with the control, which correlated with reduced tumor growth.
- In these tumors, we found that SOD3 expression correlated with lower levels of endothelial CCL2, which is a potent chemokine for myeloid cell transmigration. SOD3 overexpression in endothelial cells also enhanced T cell transmigration in vitro, and this effect was reversed when these cells were co-incubated with exogenous CCL2. By inhibiting CCL2 expression, SOD3 appears to change the chemokine pattern on the surface of endothelial cells, thus enabling or promoting T cell transmigration.
- Our results therefore indicate that chemokines are a major intercellular communication system in the tumor microenvironment. Modification of the chemokine pattern in the tumor milieu might be useful for turning a dominant, immunosuppressive environment into an environment in which immunity predominates over the transformed cells. CCR5 and its ligands are central elements in generating potent anti-tumor lymphocyte. They do this by favoring co-stimulation and by enhancing the formation of TCR oligomers, which improves memory cell function and enhances antigen crosspresentation to CD8 lymphocytes. In addition, induction of the anti-oxidant enzyme SOD3 inhibits CCL2 in the tumor-associated blood vessels; this magnifies the infiltration of effector T cells and changes the balance between effector and immunosuppressive cells, leading to immune control of tumor growth. Preliminary data suggest that this enhanced lymphocyte infiltration also affects the endothelium. Lovastatin-induced SOD3 expression did not occur in T cell-deficient mice, thus suggesting positive feedback between immune and endothelial cells. Excessive myeloid cell infiltration, as occurs in established tumors, fosters endothelial cell hyperactivity, blood vessel abnormalization, and more myeloid cell infiltration into the tumor. In contrast, normalization of tumor vasculature fuels lymphocyte infiltration, which attenuates the hyperactivated state of the endothelium and reduces the number of myeloid cells that enter the tumor. SOD3 has additional effects on vasculature function, enabling better perfusion of the tumor parenchyma, which changes the tumor cell metabolism and increases penetration of chemotherapeutic drugs.