Summary LE 1 MIN04
Hallmarks of cancer
- Resisting cell death
- Inducing angiogenesis
- Enabling replicative immortality
- Activating invasion metastasis
- Evading growth suppressors
- Sustaining proliferative signaling
- Reprogramming energy metabolism
- Immune evasion, inhibit B and T cells
Melanoma = most aggressive skin cancer
- A asymmetry
- B border
- C color
- D diameter
Chemotherapy = rapid damage to dividing cells
Targeted therapy = target specific pathway
- Mutation is BRAF, without signal from outside it will grow
o BRAFi, MEKi (BRAF works in non epithelial cells)
- Side effects:
o Inhibition signaling proteins in other cells
o Hyperkeratosis
Immunotherapy = stimulation of the immune system
- Non-antigen specific:
o Non-specific stimulation → IFN, IL-2
o Immune checkpoint inhibition (ICI)
▪ Anti-CTLA-4 → CTLA4 (on T-cell) binds 2nd signal (with APC) so T-cell downregulation
▪ Anti-PD1 → PD1 (on T-cell) binds PD-L1 (on tumor cell) producing inhibitory signal
o T-VEC → invades and replicates in tumour cells selectively, lyse tumour cells
- Antigen-specific therapy:
o Dendritic cell vaccination
o Adoptive T cell transfer
Primary cell cultures → directly isolated from an organism
- Lifespan = limited
- Retain characteristics of normal cells/tissue
Cell line culture → permanently established cell culture (immortalized)
- Lifespan = unlimited
- Derived from primary cell culture
- Mel624, human melanoma cell line, epithelial-like cells, adherent
Subculture → cell passaging
- Because of: exhaustion medium, cell density too high, increasing cell numbers
,Suspension cells → free-floating single cells or clumps of a few cells
Adherent cells → single layer of cells (monolayer)
Unhealthy cells → blebbing = irregular bulbe
- Necrosis – cell swelling, leakage of contents
- Apoptosis – cell shrinkage, apoptotic bodies
Biological contaminations
- Bacteria, yeast, molds, viruses, mycoplasm
Growth curve cells
- Lag phase, log phase, stationary phase, death phase
Culture environment
- Temperature (RT)
- CO2 concentration (5%)
- Humidity in air
- pH (7.4), phenal red indicator
o Acid: orange → yellow
o Base: orange → pink/purple
- Osmotic pressure
- Concentration nutrients, proteins, hormones
Basal media
- DMEM → high glucose
- IMDM → HEPES buffered
- Additions
o FBS (fetal bovine serum), source of growth and adhesion factors, hormones, lipids, minerals
o AA, anti biotic anti miotic
o Glutamine, essential amino acid
GMSO classification
- ML-I → ML-IV (minimum containment levels)
Cell counting → 16 squares, 50-150 cells
Cancer cells = undifferentiated, characteristics of stem cells, not perform normal function
- The less differentiated the more malignant the cancer
- Neoplasm = new growth
Inherited mutations → cell cycle, DNA repair, apoptosis
- Fail DNA repair → activation oncogenes, inactivation genes apoptosis, inactivation TSG
Somatic mutations → riskfactors
- Environmental mutagens
- Hormones
- Immunosuppression
- Oncogenic virus infections
, DNA viruses
- Papillomavirus, carcinoma of uterine cervix
- Hepatitis B virus, liver cancer
- Epstein-Bar virus, Burkitt’s lymphoma
RNA viruses
- HIV-1, immunodeficiency
- HHV-8, herpes
- Kaposi’s sarcoma
Proto-oncogenes = code for proteins in cell division (growth factors, membrane receptors, signaling
ras gene)
- Oncogenes = abnormally active protein = gain of function (missense mutation)
- Wnt signaling pathway, gene transcription
Tumor suppressor genes = code for proteins that prevent uncontrolled cell division by blocking key
steps (p53 gene, prevent cell entering S phase, repair DNA, cause apoptosis)
- Loss of function (nonsense/frameshift)
Knudson’s two-hit hypothesis
- Need 2 hits, or germ cell mutation and 1 hit
Cell cycle
- G0 → G1 → S (inhibited by p53, Rb) → G2 → M (mitosis)
- Inhibition removed by cyclin-dependent kinases (CDK)
Tumor immune evasion = inactivation T cells with CTLA-4 and PD1
- Downregulation MHC class I, TGF-β and IL-10
- Downregulation co-stimulatory molecules
- Loss tumor antigens
- Production suppressive cytokines
- Upregulation inhibitory receptors (PD-L1/2), blocks T cell function
Hallmarks of cancer
- Resisting cell death
- Inducing angiogenesis
- Enabling replicative immortality
- Activating invasion metastasis
- Evading growth suppressors
- Sustaining proliferative signaling
- Reprogramming energy metabolism
- Immune evasion, inhibit B and T cells
Melanoma = most aggressive skin cancer
- A asymmetry
- B border
- C color
- D diameter
Chemotherapy = rapid damage to dividing cells
Targeted therapy = target specific pathway
- Mutation is BRAF, without signal from outside it will grow
o BRAFi, MEKi (BRAF works in non epithelial cells)
- Side effects:
o Inhibition signaling proteins in other cells
o Hyperkeratosis
Immunotherapy = stimulation of the immune system
- Non-antigen specific:
o Non-specific stimulation → IFN, IL-2
o Immune checkpoint inhibition (ICI)
▪ Anti-CTLA-4 → CTLA4 (on T-cell) binds 2nd signal (with APC) so T-cell downregulation
▪ Anti-PD1 → PD1 (on T-cell) binds PD-L1 (on tumor cell) producing inhibitory signal
o T-VEC → invades and replicates in tumour cells selectively, lyse tumour cells
- Antigen-specific therapy:
o Dendritic cell vaccination
o Adoptive T cell transfer
Primary cell cultures → directly isolated from an organism
- Lifespan = limited
- Retain characteristics of normal cells/tissue
Cell line culture → permanently established cell culture (immortalized)
- Lifespan = unlimited
- Derived from primary cell culture
- Mel624, human melanoma cell line, epithelial-like cells, adherent
Subculture → cell passaging
- Because of: exhaustion medium, cell density too high, increasing cell numbers
,Suspension cells → free-floating single cells or clumps of a few cells
Adherent cells → single layer of cells (monolayer)
Unhealthy cells → blebbing = irregular bulbe
- Necrosis – cell swelling, leakage of contents
- Apoptosis – cell shrinkage, apoptotic bodies
Biological contaminations
- Bacteria, yeast, molds, viruses, mycoplasm
Growth curve cells
- Lag phase, log phase, stationary phase, death phase
Culture environment
- Temperature (RT)
- CO2 concentration (5%)
- Humidity in air
- pH (7.4), phenal red indicator
o Acid: orange → yellow
o Base: orange → pink/purple
- Osmotic pressure
- Concentration nutrients, proteins, hormones
Basal media
- DMEM → high glucose
- IMDM → HEPES buffered
- Additions
o FBS (fetal bovine serum), source of growth and adhesion factors, hormones, lipids, minerals
o AA, anti biotic anti miotic
o Glutamine, essential amino acid
GMSO classification
- ML-I → ML-IV (minimum containment levels)
Cell counting → 16 squares, 50-150 cells
Cancer cells = undifferentiated, characteristics of stem cells, not perform normal function
- The less differentiated the more malignant the cancer
- Neoplasm = new growth
Inherited mutations → cell cycle, DNA repair, apoptosis
- Fail DNA repair → activation oncogenes, inactivation genes apoptosis, inactivation TSG
Somatic mutations → riskfactors
- Environmental mutagens
- Hormones
- Immunosuppression
- Oncogenic virus infections
, DNA viruses
- Papillomavirus, carcinoma of uterine cervix
- Hepatitis B virus, liver cancer
- Epstein-Bar virus, Burkitt’s lymphoma
RNA viruses
- HIV-1, immunodeficiency
- HHV-8, herpes
- Kaposi’s sarcoma
Proto-oncogenes = code for proteins in cell division (growth factors, membrane receptors, signaling
ras gene)
- Oncogenes = abnormally active protein = gain of function (missense mutation)
- Wnt signaling pathway, gene transcription
Tumor suppressor genes = code for proteins that prevent uncontrolled cell division by blocking key
steps (p53 gene, prevent cell entering S phase, repair DNA, cause apoptosis)
- Loss of function (nonsense/frameshift)
Knudson’s two-hit hypothesis
- Need 2 hits, or germ cell mutation and 1 hit
Cell cycle
- G0 → G1 → S (inhibited by p53, Rb) → G2 → M (mitosis)
- Inhibition removed by cyclin-dependent kinases (CDK)
Tumor immune evasion = inactivation T cells with CTLA-4 and PD1
- Downregulation MHC class I, TGF-β and IL-10
- Downregulation co-stimulatory molecules
- Loss tumor antigens
- Production suppressive cytokines
- Upregulation inhibitory receptors (PD-L1/2), blocks T cell function
