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Laboratory Strategies for Investigating Disease By Charles J. Dimitroff, Ph.D. Associate Professor of Dermatology Associate Director for Laboratory Research Department of Dermatology Brigham and Women’s Hospital Harvard Medical School Faculty, Ph.D. Program in Immunology The Graduate School of Arts and Sciences Division of Medical Sciences, Harvard University Outline 1.) Establishing a Disease’s Threat to Human Health • Does a disease impact a human’s lifespan or quality of life? Outline 1.) Establishing a Disease’s Threat to Human Health • Does a disease impact a human’s lifespan or quality of life? 2.) Identifying Gaps in Our Knowledge of Disease • Biomarkers of disease development/progression • Mechanism(s) of disease development/progression Outline 1.) Establishing a Disease’s Threat to Human Health • Does a disease impact a human’s lifespan or quality of life? 2.) Identifying Gaps in Our Knowledge of Disease • Biomarkers of disease development/progression • Mechanism(s) of disease development/progression 3.) Approaches to Studying Disease • Exploring tissue, cellular, molecular and genetic features of disease Outline 1.) Establishing a Disease’s Threat to Human Health • Does a disease impact a human’s lifespan or quality of life? 2.) Identifying Gaps in Our Knowledge of Disease • Biomarkers of disease development/progression • Mechanism(s) of disease development/progression 3.) Approaches to Studying Disease • Exploring tissue, cellular, molecular and genetic features of disease 4.) Seeking Funding to Conduct for Studies on Disease 1.) Establishing a Disease’s Threat to Human Health Examples of Diseases: Autoimmune Diseases (Rheumatoid Arthritis, Lupus, Psoriasis, MS) Neuro-degenerative/-developmental Disorders (Alzheimer's, Muscular dystrophy, ALS) Drug Addiction/Cognitive/Depression Disorders Infectious Diseases (viral/fungal/bacterial/parasitic)/Immune Cell Function Cardiovascular Disease 1.) Establishing a Disease’s Threat to Human Health Examples of Diseases: Autoimmune Diseases (Rheumatoid Arthritis, Lupus, Psoriasis, MS) Neuro-degenerative/-developmental Disorders (Alzheimer's, Muscular dystrophy, ALS) Drug Addiction/Cognitive/Depression Disorders Infectious Diseases (viral/fungal/bacterial/parasitic)/Immune Cell Function Cardiovascular Disease Cancer Lifetime Risk: Nearly 1 in 2 men get cancer Nearly 1 in 3 women get cancer Nearly 1 in 4 will die from cancer 1.) Establishing a Disease’s Threat to Human Health Examples of Diseases: Autoimmune Diseases (Rheumatoid Arthritis, Lupus, Psoriasis, MS) Neuro-degenerative/-developmental Disorders (Alzheimer's, Muscular dystrophy, ALS) Drug Addiction/Cognitive/Depression Disorders Infectious Diseases (viral/fungal/bacterial/parasitic)/Immune Cell Function Cardiovascular Disease Cancer Metastasis 2.) Identifying Gaps in Our Knowledge of Disease Normal (or benign) vs. Cancer Non-metastatic vs. Metastatic Transformation (Carcinogenesis) Intravasation/Extravasation Invasion Survive/Grow in Distant Tissue Tumor Angiogenesis Tumor Angiogenesis Immune Evasion Immune Evasion 2.) Identifying Gaps in Our Knowledge of Cancer Cancer Discovery/Basic Science Biomarkers of Cancer/Metastasis Cancer-host genes/proteins as markers of early disease or progression Functional Factors in Cancer/Metastasis Cancer-host genes/proteins as causal factors in progression pathways Cancer Therapeutics Cancer Treatment Anti-Cancer/-Metastatic Drugs and Immune Boosting Agents 3.) Approaches to Studying Disease • Exploring tissue, cellular, molecular and genetic features of disease 3.) Approaches to Studying Disease Experimental Contexts: In vitro (Latin: “In glass”) Studies on cells or molecules outside their biological context • Proteins in solution; Cells in artificial culture medium 3.) Approaches to Studying Disease Experimental Contexts: In vitro (Latin: “In glass”) Studies on cells or molecules outside their biological context • Proteins in solution; Cells in artificial culture medium In situ (Latin: “On site”) Studies on cells or molecules within tissues (live or fixed) H&E S100 Gal-1hFc DAPI Merged Laboratory Methods: Immunohistochemistry/-fluorescence; Fluorescence In situ Hybridization 3.) Approaches to Studying Disease Experimental Contexts: In vitro (Latin: “In glass”) Studies on cells or molecules outside their biological context • Proteins in solution; Cells in artificial culture medium. In situ (Latin: “On site”) Studies on cells or molecules within tissues (live or fixed) H&E S100 Gal-1hFc DAPI Merged Laboratory Methods: Immunohistochemistry/-fluorescence; Fluorescence In situ Hybridization In vivo (Latin: “Within the living”) Studies on biological/pathobiological pathways in living organisms • Humans; Mice; Rats; Rabbits; Fish; Dogs; Flies; Worms 3.) Approaches to Studying Cancer A. Correlative Analysis (Biomarker/Target Identification) Normal vs. Cancer vs. Metastasis Molecular Expression Gene/Genome Protein/Proteome Glycome, Lipidome & Protein Modifications Glycans Lipids Sulfation Phosphorylation 3.) Approaches to Studying Cancer A. Correlative Analysis (Biomarker/Target Identification) Normal vs. Cancer vs. Metastasis Comparative Analysis of Host/Cancer Genes/Genome, Proteins, Lipids & Protein Modifications • Gene expression (Relative gene levels/mutations) Laboratory Methods: Hybridization DNA microarrays, Whole genome/exome sequencing, qPCR, Real-time RT-qPCR, Nanostring Technology, RNA sequencing, SNP analysis 3.) Approaches to Studying Cancer A. Correlative Analysis (Biomarker/Target Identification) Normal vs. Cancer vs. Metastasis Comparative Analysis of Host/Cancer Genes/Genome, Proteins, Lipids & Protein-Modifications • Gene expression (Relative gene levels/mutations) Laboratory Methods: Hybridization DNA microarrays, Whole exome sequencing, qPCR, Real-time RT-qPCR, Nanostring Technology, RNA sequencing, SNP analysis • Protein expression (Relative protein levels/variants) Laboratory Methods: Western blot, 2D-electrophoresis, flow cytometry, Immunobeads, Immunohistochemistry/-fluorescence, High-performance liquid chromatography, Mass spectrometry, Immuno/lectin affinity chromatography, ELISAs, SDS-PAGE 3.) Approaches to Studying Cancer A. Correlative Analysis (Biomarker/Target Identification) Normal vs. Cancer vs. Metastasis Comparative Analysis of Host/Cancer Genes/Genome, Proteins, Lipids & Protein-Modifications • Gene expression (Relative gene levels/mutations) Laboratory Methods: Hybridization DNA microarrays, Whole exome sequencing, qPCR, Real-time RT-qPCR, Nanostring Technology, RNA sequencing, SNP analysis • Protein expression (Relative protein levels/variants) Laboratory Methods: Western blot, 2D-electrophoresis, flow cytometry, Immunobeads, Immunohistochemistry/-fluorescence, High-performance liquid chromatography, Mass spectrometry, Immuno/lectin affinity chromatography, ELISAs, SDS-PAGE • Lipid expression (Relative lipid levels/variants) Laboratory methods: Organic extractions; Thin-layer chromatography, HPLC, MS 3.) Approaches to Studying Cancer A. Correlative Analysis (Biomarker/Target Identification) Normal vs. Cancer vs. Metastasis Comparative Analysis of Host/Cancer Genes/Genome, Proteins, Lipids & Protein-Modifications • Gene expression (Relative gene levels/mutations) Laboratory Methods: Hybridization DNA microarrays, Whole exome sequencing, qPCR, Real-time RT-qPCR, Nanostring Technology, RNA sequencing, SNP analysis • Protein expression (Relative protein levels/variants) Laboratory Methods: Western blot, 2D-electrophoresis, flow cytometry, Immunobeads, Immunohistochemistry/-fluorescence, High-performance liquid chromatography, Mass spectrometry, Immuno/lectin affinity chromatography, ELISAs, SDS-PAGE • Lipid expression (Relative lipid levels/variants) Laboratory methods: Organic extractions; Thin-layer chromatography, HPLC, MS • Protein-Modifications (Relative modification levels) Laboratory Methods: Western blot, flow cytometry, Immuno-beads, Immunohistochemistry/-fluorescence , HPLC, MS, Immuno/lectin affinity chromatography 3.) Approaches to Studying Cancer B. Molecular Function Assessments (Establish a Pro/Anti-tumor Role) X Pro-Invasion Malignant Transformation Pro-Adhesion P A X Normal Melanocyte Pro-Angiogenic A Pro-Immune Evasion Melanoma Cell Y Z X Genes “A” • Proteins “X” • Lipids “Y” • Protein Modifications “Z” Hypothesis-driven Research Examples: 1.) Gene “A” triggers melanomagenesis. 2.) Protein “X” or Modification “Z” causes intravascular adhesion and metastasis. 3.) Cancer cell invasion is dependent on Lipid “Y” expression. 3.) Approaches to Studying Cancer B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role) 1.) Turn on genes (Gain of Function) - Elevate gene and corresponding protein, lipid or protein-modification levels 2.) Turn off genes (Loss of Function) - Silence gene expression, ablate the gene or replace with drug-resistance gene 3.) Approaches to Studying Cancer B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role) 1.) Turn on genes (Gain of Function) - Elevate gene and corresponding protein, lipid or protein-modification levels 2.) Turn off genes (Loss of Function) - Silence gene expression, ablate the gene or replace with drug-resistance gene Laboratory Methods: • Overexpress gene expression in host/cancer cells - Transfection (plasmid vector)/ transduction (virus) of cDNA • Silence gene expression in host/cancer cells - Transfection (plasmid)/ transduction (virus) of cDNA - mRNA interference (destruction of mRNA species) - short-hairpin interfering RNAs - small-interfering RNAs or microRNAs - Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas9 - Targeted gene resection 3.) Approaches to Studying Cancer B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role) 1.) Turn on genes (Gain of Function) - Elevate gene and corresponding protein, lipid or protein-modification levels 2.) Turn off genes (Loss of Function) - Silence gene expression, ablate the gene or replace with drug-resistance gene Laboratory Methods: • Overexpress gene expression in host/cancer cells - Transfection (plasmid vector)/ transduction (virus) of cDNA • Silence gene expression in host/cancer cells - Transfection (plasmid)/ transduction (virus) of cDNA - mRNA interference (destruction of mRNA species) - short-hairpin interfering RNAs - small-interfering RNAs or microRNAs - Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas9 - Targeted gene resection • Construction of mutant mice (germ line transmission) - Targeted gene disruption (Knock-out) - whole animal or conditional (tissue-specific) - Targeted gene insertion (Knock-in) - inducible or tissue/cell-specific 3.) Approaches to Studying Cancer B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role) 1.) Turn on genes (Gain of Function) - Elevate gene and corresponding protein, lipid or protein-modification levels 2.) Turn off genes (Loss of Function) - Silence gene expression, ablate the gene or replace with drug-resistance gene Laboratory Methods: • Overexpress gene expression in host/cancer cells - Transfection (plasmid vector)/ transduction (virus) of cDNA • Silence gene expression in host/cancer cells - Transfection (plasmid)/ transduction (virus) of cDNA - mRNA interference (destruction of mRNA species) - short-hairpin interfering RNAs - small-interfering RNAs or microRNAs - Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas9 - transcriptional repression (blocks DNA → RNA) • Construction of mutant mice (germ line transmission) - Targeted gene disruption (Knock-out) - whole animal or conditional (tissue-specific) - Targeted gene insertion(Knock-in) – inducible or tissue/cell-specific • Block protein, lipid or protein-modification function in cell assays - Antibodies/Lectins; Dominant negative mutants; Structural variants; Metabolic/enzymatic inhibitors; Chemicals; Structural mimetics/antagonists 3.) Approaches to Studying Cancer B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role) Host/Cancer Cell Virulence In vitro Studies • Cell proliferation/death (apoptosis) • Induce immunoregulatory molecules • Induce invasion/signaling molecules • Alter adhesion/molecules 3.) Approaches to Studying Cancer B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role) Host/Cancer Cell Virulence Role in Metastasis Pathways In vitro Studies • Cell proliferation/death (apoptosis) • Induce immunoregulatory molecules • Induce invasion/signaling molecules • Alter adhesion/molecules • Promote growth/angiogenesis In vivo • Induce invasion/adhesion tumorigenicity • Encourage immune evasion • Promote metastasis (dissemination/seeding/growth) 4.) Seeking Funding to Conduct for Studies on Disease 4.) Seeking Funding to Conduct for Studies on Disease • Hypothesis-driven research 4.) Seeking Funding to Conduct for Studies on Disease • Hypothesis-driven research • Address a public health problem 4.) Seeking Funding to Conduct for Studies on Disease • Hypothesis-driven research • Address a public health problem • Address a gap in our knowledge/Advance the field 4.) Seeking Funding to Conduct for Studies on Disease • Hypothesis-driven research • Address a public health problem • Address a gap in our knowledge/Advance the field • A translational aspect – human/animal studies 4.) Seeking Funding to Conduct for Studies on Disease • Hypothesis-driven research • Address a public health problem • Address a gap in our knowledge/Advance the field • A translational aspect – human/animal studies • Collaborative/Innovative 4.) Seeking Funding to Conduct for Studies on Disease • Hypothesis-driven research • Address a public health problem • Address a gap in our knowledge/Advance the field • A translational aspect – human/animal studies • Collaborative/Innovative • Training/mentorship is lauded Laboratory Research on Cancer in BWH Dermatology Host Immune System Host Microenvironment Melanocyte Melanoma Cell Laboratory Research on Cancer in BWH Dermatology Host Immune System Host/Tumor Factors Host Microenvironment Melanocyte Melanoma Cell Laboratory Research on Cancer in BWH Dermatology Host Immune System Host/Tumor Factors Host Microenvironment Melanocyte Melanoma Cell Tumor Membrane Receptor Laboratory Research on Cancer in BWH Dermatology Host Immune System Host/Tumor Factors Host Microenvironment Melanocyte Tumor Cell Signals Genes Melanoma Cell Tumor Membrane Receptor Laboratory Research on Cancer in BWH Dermatology Host Immune System Host/Tumor Factors Host Microenvironment Melanocyte Tumor Cell Signals Genes Melanoma Cell Tumor Membrane Receptor Become Information Systems Engineers!