Cancer, inflammation, and immunity crosstalk

Transcription

1 Contact Technical Support Cancer, inflammation, and immunity crosstalk Jesse Liang, Ph.D.

2 Legal disclaimer QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at or can be requested from QIAGEN Technical Services or your local distributor. 2

3 Inflammation and cancer 1. Sources of inflammation: Environmental triggers pollution, infection Self obesity, aging Cancer-related drivers tumors are frequently heavily infiltrated by macrophages and neutrophils Therapy-related chemotherapy, radiotherapy 2. Inflammation can be both tumor-suppressive and tumor-promoting. 3. Inflammation is a part of cancer.

4 Examples of inflammation-associated cancers Elinav, E. et al. (2013) Inflammation-induced cancer: crosstalk between tumours, immune cells and microorganisms. Nat. Rev. Cancer 13,

5 Inflammation enables the core events of tumorigenesis & tumor development Core events inflammation: 1. Activates major inflammatory pathways NFκB and STAT3 cell survival and proliferation 2. Increases rate of DNA damage Via ROS/RNS, cytokines, microbiota, directly increasing cellular susceptibility to mutagenesis 3. Promotes angiogenesis 4. Promotes cancer metastasis Enhances EMT and the survival of circulating metastatic cells 5. Selects and shapes cancer The induction of genomic instability is central to the role of inflammation in neoplastic transformation.

6 The roles of microbiota The best evidence for causal bacterial involvement in inflammation-induced cancer comes from infections with Helicobacter pylori in gastric cancer. Members of the microbiota are crucially involved in tumor-promoting inflammation. Alterations in microbiota composition may function as a prime driver of intestinal tumorigenesis. For example, intestinal dysbiosis has been associated with colorectal cancer. 6

7 The roles of inflammasomes Inflammasome - an intracellular multiprotein complex of the innate immune system, consisting of sensor proteins of the NODlike receptor (NLR) family, adaptor proteins and the proinflammatory serine protease caspase 1. The function of the inflammasome is to cleave the cytokines pro-interleukin-1β and pro-interleukin-18 into their biologically active forms. The inhibition of inflammasomes or neutralization of their products, mainly interleukin 1β (IL-1β) and IL-18, have profound effects on carcinogenesis and tumor progression. (2012 Nat. Immunol. 13, 343.) Inflammasome-derived interleukin-18 (IL-18) is necessary for tumor surveillance at the intestinal mucosal surface. Decreased levels of IL-18 decrease the antitumor immune response mediated by interferon-γ (IFNγ). Inflammasome deficiency also leads to aberrations in microbial ecology, which results in the outgrowth of bacterial strains with potentially tumorigenic activity. Minireview: Inflammasomes: keeping the gut microbiota in check

8 How is anti-cancer immunity induced? Step 1. Tumor cells express tumor-associated antigens on MHC class I. Step 2. Cytotoxic T cells recognize tumorassociated antigens and kill tumor cells. Step 3. Dendritic cells (DCs), which are important antigen presenting cells (APCs), take up and process antigen from dead or dying tumor cells, present the tumor-associated antigens on MHC class I and II, and stimulate T cells and other immune cells.

9 What does cancer do to immunity? Cancer cells are able to modulate their microenvironment and interfere with every step of the inflammatory response in order to gain benefit. Tumor-derived soluble factors Tumor cells produce immunosuppressive factors: IL-10, TGF-β, VEGF, prostaglandins, etc. For example, VEGF is a suppressor of dendritic cell function and maturation. Cancer influences antigen-presenting cells by secretion of TGF-β, IL-6, IL-10, etc. Cellular components Regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and invariant natural killer T cells (inkts).

10 Cancer immunoediting immune selection Some cancers are more immunogenic than the others. Immuno-silent cancers easily escape. Strongly immunogenic tumors would fail to develop to the point that they become clinically significant. In this way, the immune system may exert a selective pressure for cancer-causing mutations (such as a Ras point mutation or complete loss of p53 or Rb) that are largely immunologically silent a process that is termed immunoediting. In other words, immunoediting is an immune selection process ranging from immune surveillance to immune escape.

11 Cancer immunoediting 3E theory Phase 1: Recognition, infiltration, recruitment Elimination Phase 2: Induce tumor death Phase 3: Promote more tumor death Phase 4: Destroy all tumor cells Equilibrium: Tumor cells which have survived the elimination phase enter the equilibrium phase Escape: Tumor cells which have acquired resistance to equilibrium enter the escape phase

12 Cancer immunoediting process Jeremy B. Swann and Mark J. Smyth (2007) Immune surveillance of tumors. JCI 117,

13 Tumor escape immunosuppression Tumor-derived soluble factors: Tumor cells produce immunosuppressive factors: IL-10, TGF-β, VEGF, prostaglandins, etc. Cellular components: Regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and invariant natural killer T cells (inkts).

14 How do cancer cells become resistant to elimination? Paper 1: These results demonstrate that the strong immunogenicity of an unedited tumor can be ascribed to expression of highly antigenic mutant proteins. Outgrowth of tumor cells that lack these strong antigens through a T cell-dependent immunoselection process represents one mechanism of cancer immunoediting. (2012 Nature, 482, 400.)

15 How do cancer cells become resistant to elimination? Paper 2: By comparing the development of sarcomas in immunecompetent mice to that in mice with broad immunodeficiency or specific antigenic tolerance, we show that recognition of tumorspecific antigens by lymphocytes is critical for immunoediting. Furthermore, primary sarcomas were edited to become less immunogenic through the selective outgrowth of cells that were able to escape T cell attack. Loss of tumor antigen expression or presentation on MHC (Major Histocompatibility Complex) class I was necessary and sufficient for this immunoediting process to occur. These results highlight the importance of tumor-specific-antigen expression in immune surveillance, and potentially, immunotherapy. (2012 Nature 482, 405.)

16 Sample & Assay Technologies Conclusion T cell recognition of tumor antigens is key to cancer immunoediting A clinical case of cancer immunoediting: Most glioblastomas at relapse are negative for EGFRvIII expression. This is a relevant and direct example of cancer immunoediting...

17 Anti-cancer immunotherapies (non-specific) 1. Non-specific immune stimulation: * Stimulate T cells IL-2 IFNα * Activate dendritic cells TLR7 agonist imiquimod * Inhibit/deplete Treg cells anti-cd25 antibody low-dose cyclophosphamide 17

18 Anti-cancer immunotherapies (non-specific) 2. Immune checkpoint blockade Block CTLA4-B7 and PD1-PDL1 interactions The CTLA4-blocking antibody ipilimumab (Bristol-Myers Squibb) was approved by the FDA for treating melanoma in March Merck s KEYTRUDA (pembrolizumab) (also known as MK-3475) is the first anti-pd-1 therapy approved in the United States and received FDA s breakthrough therapy designation for advanced melanoma in September

19 Anti-cancer immunotherapies (specific) 3. Adoptive Cell Transfer Two main approaches are being explored: * T cells that reside in the tumor are cultured and expanded ex vivo in the presence of IL-2. When enough of these polyclonal T cells are obtained, they are re-infused into the patient. * Isolated peripheral blood T cells are transfected to express tumor-antigen-specific TCRs (T cell receptors) and then re-administered to the patient. This strategy is advantageous in that enough T cells can be obtained for infusion in all patients, but a potential drawback is that the TCRs that are transfected into the T cells have a limited antigen-specificity repertoire.

20 Anti-cancer immunotherapies (specific) 4. Cellular Vaccines Tumor cell or tumor antigen vaccination Extracted, irradiated tumor cells are re-administered to the patient. Adjuvants such as TLR agonists or GM-CSF are required. APC vaccination APCs are extracted from a patient s bloodstream, cultured and activated with cytokines or adjuvants, loaded with tumor antigen ex vivo, and re-administered to the patient. Sipuleucel- T (made by Dendreon) was approved by the FDA for prostate cancer treatment in April DC vaccination DCs are cultured from peripheral blood monocytes in the presence of IL-4 and GM-CSF, and activated and loaded with tumor antigen ex vivo, then re-administered to the patient.

21 We are entering a new era for anticancer immunotherapy! * The recent clinical success * The unexpected finding of a positive interaction between immunotherapy and chemotherapy inducing immunogenic cell death depleting suppressive immune cells disrupting inhibitory pathways sensitizing tumor cells to T cell-induced cell death

22 RT 2 Profiler PCR Array introduction 84 pathwayspecific genes of interest Total RNA extraction cdna mix with master mix & load the plate run real-time PCR data analysis

23 Gene expression at the mrna level PCR arrays Apoptosis, autophagy, necrosis Cancer pathway finder Angiogenesis Tumor metastasis EMT (epithelial-mesenchymal transition) DNA damage Oxidative stress Cancer stem cells Telomeres and telomerase Cellular senescence p53 signaling PI3K-AKT signaling Myc targets Cell cycle Growth factors mtor signaling Cancer drug targets Breast cancer Prostate cancer Lung cancer Liver cancer

24 Gene expression at the mrna level PCR arrays Inflammatory cytokines and receptors Cancer inflammation and immunity crosstalk Chemokines and receptors Common cytokines Cytokines and chemokines TNF signaling pathway Antibacterial response Antiviral response Inflammatory response and autoimmunity Toll-like receptors (TLRs) Innate and adaptive immune responses Inflammasomes NFκB signaling NFκB targets IL-6/STAT3 signaling T helper cell differentiation Th1 and Th2 responses Th17 response Interferons and receptors MAPK signaling TGFβ/BMP signaling profiler-pcr-arrays

25 Cancer Inflammation & Immunity Crosstalk RT 2 Profiler PCR Array Immune & Inflammatory Responses: Immunostimulatory Factors: IFNG, IL2, IL12A, IL12B, IL15, TNF. Immunosuppressive Factors: CD274 (PD-L1), CSF2 (GM-CSF), CTLA4, CXCL12 (SDF1), CXCL5, IDO1 (IDO), IL10, IL13, IL4, IL8, MIF, NOS2 (inos), PDCD1 (PD1), PTGS2 (COX2), TGFB1, VEGFA. Pro-Inflammatory Genes: CCL2 (MCP-1), CCL20 (MIP-3A), IFNG, IL1A, IL1B, IL2, IL6, IL12A, IL12B, IL17A, IL23A, PTGS2 (COX2), TLR4, TNF, VEGFA. Anti-Inflammatory Genes: IL4, IL10, IL13, TGFB1. Enzymatic Modulators of Inflammation & Immunity: AICDA (AID), GZMA, GZMB, IDO1 (IDO), NOS2 (inos), PTGS2 (COX2). Antigen Presentation: HLA-A, HLA-B, HLA-C, MICA, MICB. Chemokines: CCL2 (MCP-1), CCL4 (MIP-1B), CCL5 (RANTES), CCL18 (PARC), CCL20 (MIP-3A), CCL21, CCL22 (MDC), CCL28, CXCL1, CXCL2, CXCL5, CXCL9 (MIG), CXCL10 (IP-10), CXCL11 (I-TAC, IP-9), CXCL12 (SDF1). Chemokine Receptors: ACKR3 (CXCR7), CCR1, CCR2, CCR4, CCR7, CCR9, CCR10, CXCR1 (IL8RA), CXCR2 (IL8RB), CXCR3, CXCR4, CXCR5. Interleukins: IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL12A, IL12B, IL13, IL15, IL17A, IL23A. Other Cytokines: KITLG (SCF), MIF, SPP1, TNF, TNFSF10 (TRAIL). Growth Factors & Receptors: CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), EGF, EGFR, IGF1, TGFB1, VEGFA. Signal Transduction: Interferon Signaling: GBP1, IFNG, IL6, IRF1. Interferon-Responsive Genes: CCL2 (MCP-1), CCL5 (RANTES), CXCL9 (MIG), CXCL10 (IP-10), GBP1, IRF1, MYD88, STAT1, TLR3, TNFSF10 (TRAIL). NFκB Targets: BCL2L1 (BCL-XL), CCL2 (MCP-1), CCL5 (RANTES), CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), IFNG, IL8, TNF. STAT Targets: CCL2 (MCP-1), CCL4 (MIP-1B), CCL5 (RANTES), CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), CXCL9 (MIG), CXCL10 (IP- 10), CXCL11 (I-TAC, IP-9), CXCL12 (SDF1), IL1B, IL6, IL8, IL10, IL17A, IL23A, MYC. Toll-Like Receptor Signaling: TLR2, TLR3, TLR4, MYD88. Transcription Factors: FOXP3, HIF1A, IRF1, MYC, NFKB1, STAT1, STAT3, TP53 (p53). Apoptosis: Pro-Apoptotic: FASLG (TNFSF6), TNF, TNFSF10 (TRAIL), TP53 (p53). Anti-Apoptotic: BCL2, BCL2L1 (BCL-XL), MYC, STAT3.

26 PCR arrays are available for 14 species! Human Mouse Rat Rhesus macaque Drosophila Dog Pig Cow Chicken Horse Zebrafish Rabbit CHO (Chinese hamster ovary) C.elegans

27 Custom PCR arrays RNA-seq verification Biomarker discovery CRISPR/RNAi knockdown verification Bioprocessing optimization Biological response investigation Interspecies study (14 species available) 27

28 QIAGEN provides powerful microbiome research tools QIAGEN offers the largest collection of qpcr assays/arrays for microbiome research. QIAGEN developed Microbial DNA qpcr Arrays for the identification of pathogens in clinical specimens in respiratory and fecal swabs. The arrays provide collections of assays to detect bacterial species, virulence factor genes, and antibiotic resistance genes.

29 mirna expression miscript mirna PCR Arrays Focused Array Offerings Species mifinder Human Mouse Rat Other Species Apoptosis New! Human Mouse Rat Brain Cancer Human Mouse Rat Breast Cancer Human Mouse Rat Cancer PathwayFinder Human Mouse Rat Cardiovascular Disease New! Human Mouse Rat Other Species Cancer Stem Cells New! Human Mouse Rat Cell Differentiation & Development Human Mouse Rat Diabetes New! Human Mouse Rat Fibrosis New! Human Mouse Rat Hypoxia Signaling Pathway New! Human Mouse Rat Immunopathology Human Mouse Rat Inflammatory Response and Autoimmunity Human Mouse Rat Liver mifinder New! Human Mouse Rat Neurological Development & Disease Human Mouse Rat Ovarian Cancer Human Mouse Rat Pain: Neuropathic & Inflammatory New! Human Mouse Rat Prostate Cancer New! Human Mouse Rat Serum & Plasma Human Mouse Rat Stem Cells New! Human Mouse Rat T-Cell & B-Cell Activation New! Human Mouse Rat Tumor Suppressor mirnas New! Human Mouse Rat mirna QC Human Mouse Rat Other Species mirnome (mirbase v 20) Human Mouse Rat Other Species

30 Cignal Reporter Arrays or Assays Measure signaling pathway activity in cultured cells NFκB activity assay A transcription factor associated with both inflammation and cancer Inflammation 10-Pathway Reporter Array NFκB, type I interferon, interferon gamma, IL-6, interferon regulation, TGFβ, camp/pka, PKC/Ca 2+, C/EBP, glucocorticoid receptor 108L#geneglobe Cancer 10-Pathway Reporter Array Wnt, Notch, p53, Rb, TGF-β, NFκB, Myc, HIF-1α, ERK, JNK 101L#geneglobe 45-Pathway Reporter Array Cignal 45-Pathway Reporter Array is a novel cell-based assay for monitoring 45 cell signaling pathways in cultured cells in a single experiment. Observe phenotypes and quantitate as a dual-luciferase assay.

31 ELISArrays You can measure 12 cytokines or chemokines for 6 samples at the same time!

32 We also provide services send us your samples RNA extraction DNA extraction Illumina chips All PCR arrays NGS

33 Questions? Thank you for attending today s webinar! Contact QIAGEN Call: BRCsupport@qiagen.com qiawebinars@qiagen.com 33