Tumorbiology:Effect of environmental

factors,Cancer immunology

Neogrády Zsuzsa, 2018

Effect of environmental factors on tumorformation http://news.mit.edu/2016/diet-influences-colon-cancer-0302

1.

Drs. Yamagiwa and Ichikawa: “Experimental studyon the pathogenesis of epithelial tumors”, 1915

coal-tarpainting inrabbit ear:squamouscarcinomformation

Effect of environmental factors on tumorformation

• General pathomechanism: carcinogen → damaged genetic material→ tumor formation

I.) Chemical carcinogensII.) Physical carcinogens: radiation, (mechanical effects)III.) Biological carcinogens: oncogenic bacteria,

virus, parasitic worms, (chronic inflammation,aging)

I.) Chemical carcinogens: mode ofaction

• Carcinogens can act:1. Directly2. Indirectly: carcinogenicity develops following structural changes inthe organism such as by activity of CYP enzymes, glutation-S-transferase.

• Carcinogen chemical or its metabolite: electrophile → binds to theelectron-rich molecules of the cell (mostly DNA) → DNA damage →changes in cell division and differentiation, etc → tumor formation(One certain carcinogen acts mostly on one certain organ! )

carcinogen

I.) Chemical carcinogens

• Carcinogen can be initiator or promoter1. Initiator: DNA of the cells will be primarily modified (mutated)2. Promoters are the chemicals which cannot cause DNA damage butact on cells after application of initiators

• If for some time there is no promoter action, the primarily producedDNA modification will be reversed and the tumor formation failsfollowing the next promoter action. Human carcinogens:https://www.cancer.org/cancer/cancer-causes/general-info/known-and-probable-human-carcinogens.htmlra

I.) Classification of chemicalcarcinogens1. Initiators:• Alkylating agents (immunsuppressive drugs and cytostatics)• Polycyclic aromatic hydrocarbons: metabolic activation is needed (tobacco, charcoal-

broiled meat, smoked meat and fish)• Aromatic amines and azo dyes: metabolic activation is needed• Mycotoxin (Aflatoxin B1)• Nitrosamines and –amides: intestinal microbes produce from nitrate and nitrite (source

of nitrate and nitrite: preservatives, cured meat, drinking water)• Asbestos, silicate, talcum, vinylchloride monomer (PVC), metals: chromium, nickel,

arsenic2. Promoters:• Mostly produced in the body (hormones, free radicals, hydrogen peroxide, bile acids etc.)

II. Radiation

• The carcinogenic effect of radiation has been proved in eachSpecies both in vivo and in vitro• Consequence: double and simple DNA strand breaks, damaged bases, covalent crosslinks inside

and between the DNA strands (pyrimidin dimer), DNA-protein crosslinks, protein-proteincrosslinks

• Types of radiation:1. Sun light UVB (280-320 nm) → mutagenic, UVC (200-280 nm) is mutagenic also, butatmospheric osone can protect partly against this radiation. Consequence of DNA damage:mutation of the nuclear DNA, mutations in tumorsuppressor genes, enzyme inactivation, etc.2. Ionizing radiation (very low wavelenght): electromagnetic CT, PET (X-ray, gamma-ray) andparticle radiation (proton, electron, alpha)

• Consequence in the cells: H2O → H+ + OH-, followed by DNA damage and mutation• Ionising radiation sources: radioactive nucleus, (Hiroshima, Csernobil), cosmic radiation• PET=Positron emission tomograph (x-ray), CT=computed tomograph (gamma-ray)

II. Radiation: electromagnetic spectrum → lowwavelenght UV, X-ray, gamma ray → carcinogen

https://hu.wikipedia.org/wiki/Elektrom%C3%A1gneses_sug%C3%A1rz%C3%A1s

III. Biological carcinogens

• Virus: chicken Rous sarcoma virus (1911), hepatitis B, C virus, Bittnermouse mammary tumor virus (MMTV), Human papilloma (HPV) virus,Epstein-Barr virus, HIV type 1, Human T-cell lymphotrophic virus

• Bacteria: Helicobacter pylori• Helminths: Opisthorchis viverrini, Clonorchis sinensis,

Spirocerca lupi (canine esophageal carcinom)• Consequence: virus can be integrated in the genom,

Changes gene expression: mutations and epigeneticeffects

III. Biological carcinogens: role of MMTV= Bittner virusin the development of MMT (mouse mammary tumor)

• (Bittner,J J, 1936) "Some Possible Effects of Nursing on the Mammary Gland TumorIncidence inMice". Science. 84(2172):162.

doi:10.1126/science.84.2172.162. PMID 17793252• MMT cells were cultured in vitro or injected in newborn mouse(2500 or 7500 cells/animal IP). Bittnervirus could be detected both in the cell cultureand developed abdominal tumor(after 80 days) (arrow).(Gálfi and Neogrády, 1995)(Gálfi P, Neogrády S, Kutas F: Vet. Res. Commun. 1991;15(4):261-9. The inhibitory action of sodium butyrate on thegrowth of KB, MMT and RPMI cells.)

Cell culture Tumor

Tumor development in the abdomen of 80-day old miceinoculated with MMT cells

(Gálfi and Neogrády, 1995)

MMTV: Reverse-transcribing RNA viruses(retroviruses) use the enzyme toreverse-transcribe their RNAgenomes into DNA, which isthen integrated into thehost genome and replicatedalong with it.

Tumor development in the abdomen of 80-day old miceinoculated with MMT cells

Immunological background of tumor formation http://slideplayer.com/slide/4766949/

2.

https://www.slideshare.net/MUBOSScz/immunology-vi-labcellimmunol

lymphocytes (CD4: helper,CD8: cytotoxic T lymphocyteslymphocytes

CD=cluster ofdifferentiation

Immunological background of tumor formation.Introduction

Antigenpresentation ofdendritic cells

Activation of Tand Blymphocytes,immuneresponse

Immuneresponse(inflammatorycytokines)

Inhibition of apoptosis,stimulation of cell division

Inhibition of anti-tumoractions: cytotoxicity of Tcells, antibody-dependent cytotoxicity

Immunological background of tumor formation: 1st step ofimmune response: antigen presentation by dendritic cells

https://courses.lumenlearning.com/suny-ap2/chapter/the-adaptive-immune-response-t-lymphocytes-and-their-functional-types/

MHC=majorhistocompatibilitycomplex

https://courses.lumenlearning.com/suny-ap2/chapter/the-adaptive-immune-response-t-lymphocytes-and-their-functional-types/

Immunological background of tumor formation: 2nd step ofimmune response: role of helper T and cytotoxic T cells

https://courses.lumenlearning.com/suny-ap2/chapter/the-adaptive-immune-response-t-lymphocytes-and-their-functional-types/

(a) CD4 is associated with helperT cells. An extracellularpathogen is processed andpresented in the binding cleftof a class II MHC molecule,and this interaction isstrengthened by the CD4molecule.

(b) CD8 is associated withcytotoxic T cells. An intra-cellular pathogen is presentedby a class I MHC molecule,and CD8 interacts with it.

https://www.ncbi.nlm.nih.gov/pubmed/1105061

Congenitally athymic nude mice (nu/nu) are presented as a model forthe study of cell-mediated immunologic deficiencies

Classification of tumor antigens• Tumor-Specific Antigens (TSA), which are present only on tumor cells and

not on any other cell (specific vaccines)• Tumor-Associated Antigens (TAA), which are present on some tumor cells

and also some normal cells (weak immune response against vaccines).• Overexpressed or Aberrantly Expressed Cellular Proteins

• Tumor Antigens Produced by Oncogenic Viruses• Oncofetal Antigens

• alphafetoprotein: hepatocellular cancer• Altered Cell Surface Glycolipids and Glycoproteins

• Her2=human epidermal growth factor receptor 2: breast cancer• EGFR=epithelial growth factor receptor• Tyrosinase: melanoma

• Cell Type-Specific Differentiation Antigens• PSA=prostate specific antigen: prostate cancer• CEA=carcinoembryonic antigen: adenocarcinomas of colon, breast, lung, and other epithelial

cancers• MUC1=a transmembrane type I molecule: almost in all human epithelial adenocarcinomas• 1A10 and SB2: in the serum of dogs: canine mammary carcinoma• CA-125=Cancer antigen 125: epithelial ovarian cancers. CA-125 has not found application in

veterinary medicine

Failure of host defenses against tumor antigen• Although many tumors are eliminated by the immune system (and thus are never

detected), others continue to grow despite the presence of TAAs. Reasons ofdeficient host response to the TAA, including the following:

• Specific immunologic tolerance to TAAs in a process that involves antigen-presenting cells: no appropriate antigen presentation occurs in the tumor cells!!

• Suppression of immune response by chemical, physical, or viral agents (eg, helperT-cell destruction by HIV)

• Suppression of the immune response by cytotoxic drugs or radiation• Suppression of the immune response by the tumor itself:

• Tumor does not produce: HLA (human leukocyte-antigen complex), LMP-2 andLMP-7 proteases, and /or TAP-1 and TAP-2 transporters. These factors are neededfor complet antigen presentation

• Tumor does not produce: MHC (major histocompatibility gen complex)• Dendritic cells of tumor have not appropriate maturation• Concentration of Trp and Arg is low in the surrounding of tumors, however these

amino acids are needed for immune response• High concentration of immunsuppressor mediators such as IL-10, TGF and low

concentration of CD8 (tumor killing function) supporting mediators such as IL- 7,-IL-12, IL-15

• Immunologically protecting stroma around the tumor

Immunological background of tumor formation: 3rd step of immune response:perforin and granzyme produced by the CTL (cytotoxic T lymphocytes) and NK (naturalkiller cells) can kill tumor cells. However, tumor-specific T cells do not allow to enforce

antitumor action of CTLs and NKs https://www.slideshare.net/gamaldawood/the-immune-response1 https://www.westburg.eu/immunotherapy-for-cancer/active-immunotherapy/nk-cell-mediated-cytotoxicity

NK (natural killercells produce alsoperforin andgranzyme

Immune response in cancer: specific role ofdendritic cells, 1st step

• Dendritic cells: specialized antigen presenting cells• To produce active, tumor-killing CTL (cytotoxic T-lymphocytes) are

needed: interaction of naive CD8 cells and mature dendritic cells,further other co-stimulating and co-inhibiting stimules

• However in tumors unmatured dendritic cells can be found →decreased activity of CTL

• Decreased activity of CTL → tumor progression

Function of dendritic cells

https://www.nature.com/articles/4402243/figures/1

Immune response in cancer: specific role of Treg(regulatory T cells), 2nd and 3rd steps

• Changes of different stimulation markers in tumors → activation of PD-1 element of T cells by unmatured tumor dendritic cells → T cells →differentiation in Treg → apoptosis of T cells and inhibition of CTL(cytotoxic T lymphocytes)

• Further explanation of the decreased activity of CTL in the tumors:• Unmatured (tumor) dendritic cells → much IL-10 and TGF →

differentiation in Treg• Treg → further increase of IL-10 and TGF synthesis → decreased

synthesis/function of CTL• Treg concentration in tumor is high → decreased tumor elimination

→ tumor progression

Function of Treg

https://www.nature.com/articles/nri2580

Immune response in cancer: tumor specific TLs (CD8+

cells) has no cytotoxic action, 3rd step• Tumor TLs (CD8+ T lymphocytes) do not produce interferon, perforin

and granzyme (these molecules have antitumor activity)• So, in tumors the CTL/CD8 „cell-killing” function will not occur. The

reason of that are the followings:• Differentiation of T cells into Treg• Immunosuppressive mediators• Existing inhibitory surface receptors on the CD8+ cells• Few Trp and Arg in tumors. Decrease of Trp concentration is due to increased

activity of IDO (indolamine 2,3 dioxygenase) - Trp-metabolizing enzyme - inthe tumor cells. Decrease of Arg concentration is due to increased arginaseactivity in the tumor cells.

Summary: during elimination phase the effector cells of immune system, CTL and NKcells recognize (by the help of dendritic and CD4+ T cells) and kill (by the help ofCTL/CD8 +) the tumor cells. Left: Elimination= tumor regression, right: Escape=

inhibition of antitumor activity=tumor progression)https://hu.wikipedia.org/wiki/Immunonkol%C3%B3gia#/media/File:Tumor_microenvironment.jpg

CTLHelperlymphocyte

Immune system destroyestumor

Tumor escapes from immunecontrol

Antitumor immunotherapy=immunoncology:„active immunization”

• Tumor cells: weak immune response, because they do not expressHLA (human leukocyte antigen complex) which are needed forantigen presentation and in the tumor cells can not be found realantigen presenting cells

• Active immunization by tumor specific antigens (TSA), tumorassociated antigens (TAA) or by the tumor cells

• Weak antitumor effect and as side effect: autoimmunity

Antitumor immunotherapy=immunoncology:„passive immunization”• Passive immunization → AST method: in vitro production and injection of tumor

antigen-specific CD8+ cells in patients with cancer• Consequence: tumor cells carrying the specific antigen will be killed, but tumor

cells carrying another antigen will survive, so complet recovery will not occur• Lymphodepletion + AST method: systhemic cytostatic treatment, or total body

irradiation (killing existing T and B lymphocytes), AST, followed by lymphotrophicinterleukin 2 (IL-2) treatment → production of healthy lymphocytes → very goodmethod!

• Pathomechanism of lymphodepletion + AST method: killing of Treg, elimination ofsuppressor cells → high number of injected tumor antigen-specific CD8+ cells →accompanied by increased gastrointestinal permeability → absorption andsystemic transport of bacterial lipopolysaccharides (LPS) → binding of LPS to thedendritic cells → increased antigen presentation, the effect will be furtherincreased by the IL-2 treatment also.

Antitumor vaccines: monoclonal antibodyfor treatment of B and T cell lymphomas1. Monoclonal antibody against human B lymphocyte (antibody production inovarial cell culture of chinese hamster). Antibody targets and bound to CD20 cellsurface marker of B lymphocyte.

• Active substance: Rituximab (artificial monoclonal antibody against B cell lymphoma• Rituximab in dog recognizes canine CD20 superficial marker, but will not bound to it.

However, anti-canine CD21 antibody (produced in murine hybridoma cells) is boundspecifically to B lymphocytes of dog.

2. Monoclonal antibody against canine T lymphocyte (T-cell Mab) is used fortreatment of T cell lymphoma (mostly in golden retriever, the boxer, Australianshepherd, Siberian husky). Each existing T lymphocytes will be killed, but fromthe healthy stem cells new, healthy T lymphocytes will develop.

Antitumor vaccines: monoclonal antibodyfor treatment of mammary carcinoma

• 1. Monoclonal antibody targets human epidermal growth factorreceptor type 2 (HER2) on the surface of breast cancer cells (antibodyproduction in ovarial cell culture of chinese hamster), activesubstance: trastuzumab (Herceptin)

• 2. Treatment of canine mammary tumor by herceptin results inallergic reaction. Dog epidermal growth factor receptor type2 (DER-2)has been isolated from the cell surface of canine mammary tumor,antibody targeting DER-2 seems to be a promising step to produceappropriate vaccine in the future.

Antitumor vaccinesfor treatment of canine melanomaCanine melanoma cells have characteristic tyrosinase activity, however this enzymecan be found in healthy cells also (TAA).OnceptR canine melanoma vaccine• Mode of action• Each dose contains plasmid DNA that expresses the gene coding for human

tyrosinase.• Tyrosinase protein is overexpressed on melanoma cells.• Upon injection, the DNA is taken up by muscle cells which then express the

human tyrosinase protein.• The human tyrosinase protein is different enough from the canine tyrosinase

protein that it will stimulate an immune response, yet similar enough to thecanine tyrosinase that the immune response is effective against caninemelanoma cells which express tyrosinase.

Antitumor vaccinesfor treatment of B cell lymphomahttp://www.morphogenesis-inc.com/wp-content/uploads/2015/06/ImmuneFx-VOS-Brochure.pdf

• ImmuneFx™ is an autologous (personalized) cancer vaccine → made fromeach patient’s own cancer cells.

• The patented ImmuneFx™ priming signal (antigen) is a highly immunogenicprotein → normally expressed on the surface of a streptococcal bacterium.Manufacturing process: ImmuneFx™ (=immunogenic protein) → suppliedin vitro to the patient’s own tumor cells (collected at surgery) → theantigen will be expressed on the tumor cells → the cells are then irradiated→ these cells cannot divide when returned to the patient.

• The irradiated vaccine cells are injected into the dermal layer of the skin →large concentration of antigen presenting cells will attack the tumor cells.

Antitumor vaccines for treatment of canineT cell lymphoma (lymphosarcoma=LSA) andfeline vaccine-associated fibrosarcoma1. Canine tumor cells have high telomerase reverse transcriptase

(TERT) activity, absent in the majority of normal dog tissues.Adenovirus expressing TERT injected intradermally inducesintensive immune response in dogs with LSA.

2. One of the messenger chemicals of your cat’s immune system thatis important in the fight against cancer and infection is Interleukin 2(IL-2). The vaccine contains canary pox virus that has been modifiedto produce feline (IL-2) by this way stimulating the cat’s own cancerdefense cells ( number of cytotoxic T lymphocytes and natural killercells will increase).

Cancer is a battle that can only be von if everyoneworks together