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Tumor – Immunobiology of tumor origin development and treatment

Tumor mortality has been steadily rising, but recently, with declining mortality from colon, lung, breast, and prostate cancers, stagnation has occurred. Every 3. get sick, and every 5. dies of a tumor. Tumor cells evade growth control mechanisms, antigens appear on the cell surface, which could theoretically serve as a target antitumor therapies . Based on experimental and clinical data that the organism can reject tumor the theory of immune surveillance has been established.

Tumor origin and development

Vascularization represents the boundary between tumor formation and development. Normally, the body maintains a balance between dying and cell renewal, so that the number and architecture in an organ remain the same.

A cell that avoids the mechanisms of regulation begins to divide autonomously and purposelessly, resulting in a neoplasm or tumor. Benign tumors grow well confined to surrounding tissue, while malignant ones infiltrate healthy tissue, spread by lymphoma, and blood . The altered cell goes through several stages before it becomes a metastatic cancer:

Genetically modified cell -> hyperplasia -> dysplasia -> carcinoma in situ -> invasive cancer

Genetic background

Accumulated mutations in genes essential for cell proliferation and control lead to the formation of defective proteins.

Oncogenes

Mutated forms of normal genes (protooncogenes) whose products stimulate cell growth. Protooncogenes encode proteins, which bring growth signals into the cell’s interior, and their mutation can lead to excessive proliferation, ie. excessive production of these proteins.

– Sarcomas and gliomas produce platelet-derived growth factors
– Creating altered growth stimulators
– Creation of aberrant receptors that flood the cells with growth signals even when they are not activated.

The most famous oncogene is  race ,,  and the ras protein is stuck in the on state. 

Tumor suppression genes

These genes inhibit cell division through signals that are passed on to the nucleus, and their mutation can lead to malignancy. The most famous example is gen RB  (tumor retinoblastoma), which encodes the protein pRB (DNA duplication brake). Its mutation produces a non-inactive protein, so the cell can constantly multiply. Gene p53  with its protein through DNA binding and p21 protein synthesis stops the division of damaged DNA.

A gene for checking and repairing DNA

They control the genetic material and through their proteins stop entering the next phase of division if the Dna has not doubled properly. They can prevent or induce apoptosis, and by mutating them, cells can avoid apoptosis.

Gene expression in a tumor cell is disrupted quantitatively and qualitatively.

Tumor growth

Sleeping tumor cells can survive in the body for decades before they establish blood circulation and their malignant phenotype

Angiogenesis

In order for a tumor to grow it must establish its own blood supply. Cells on the surface of the tumor are constantly growing and travel to the middle of the node where they necrotize. Although they have a common ancestor, the cells are genetically unstable, and over time they increasingly differ in size, shape, growth rate, antigenic properties, sensitivity to external factors and metastatic potential.

Some cells secrete a TAF factor (  tumor angiogenesis factor , it is also secreted by macrophages), which stimulates the ingrowth of new blood vessels and infiltration into the surrounding tissue.

Metastasis

Metastases are biologically rare but clinically common. The cell first breaks away from the primary tumor, infiltrates the surrounding tissue, enters a blood or lymph vessel (or hollow organ), travels through them, grabs the wall, implants, proliferates, and establishes new blood circulation. Every step involves the body’s defense system.

Tumor antigens

Tumor antigens are peptides formed by processing tumor proteins into T lymphocytes represented by MHC I or MHC II molecules.

1.  PROOF

– Methods in vivo
a) Methods of immunization

b) adoptive immunity transfer

c) passive transmission of immunity

d) neutralization test

e) skin test delayed hypersensitivity
 
–  Methods  in vitro
 
a) interaction of lymphocytes and tumors
 
b) antigen detection by antibodies

 – Genetic approach

In vivo methods

a) Immunization method

The best and oldest method for detecting tumor transplant antigens. If a member of a pure strain is immunized with a tumor induced in that strain, the secondary tumor graft may be rejected if it contains tumor transplant antigens. Immunization is achieved by ligation or surgical removal of the tumor, or by administration of small amounts of living or dead tumor cells. The virulence of tumor cells can be reduced while maintaining antigenicity by ionizing radiation, successive freezing and thawing, cytostatics, iodoacetate.

Example: in a mouse, the tumor is caused by chemical agents, it is immunized surgically, and after transplantation, the tumor is rejected, thus proving the presence of TSTA

b) Adaptive immunity transfer

Immunity is transmitted by injecting sensitized lymphocytes into the syngeneic organism, and immunization is successful if the tumor has transplant tumor antigens.

c) Passive transmission of immunity

Serum immunization is usually not successful, but with rare inhibition (RNA viruses, leukemias), growth is more common (tumors caused by DNA viruses, chemical or physical agents)

d) Neutralization test

The antitumor activity of sensitized lymphocytes is determined. Lymphocytes are mixed in a certain ratio with live tumor cells, injected into the syngeneic host and the dynamics of tumor growth is monitored. By changing the ratio, immunity can be determined approximately quantitatively.

e) Delayed hypersensitivity skin test

Various extracts of living or dead tumor cells are initiated into the skin and the appearance of redness or swelling characterized by abundant infiltration of inflammatory cells, especially lymphocytes and macrophages, is monitored. The method can be used to assess specific sensitivity to the tumor, in patients and in the immunized organism. It is synonymous with cellular hypersensitivity and the test detects cell-mediated immunity.

In vitro methods

A faster, simpler method, performed under defined conditions, better determines quantitative relationships and is suitable for studying human tumors due to the instability of ethical barriers, but for proven antigens it is not certain whether they are transplantable and what their importance is. The method is based on studying the interactions of tumor cells with sensitized lymphocytes and / or with specific antibodies.

Interaction of lymphocytes and tumors (antigens)

Lymphocytes and tumor cells are mixed, and lymphocyte proliferation (3H-thymidine) and cytokine secretion are monitored. The direct effect of cytotoxic T CD8 + lymphocytes was observed by trypan blue staining and measurement of dead tumor cells, measurement of radioactivity released after membrane damage of subsequently labeled cells, counting living tumor cells adhering to the medium and determining the ability of tumor cells to form colonies.

Detection of tumor antigens using antibodies

The cytotoxic effect of antibodies on the tumor or by binding them to the tumor is observed.

Genetic approach

In bacteria it amplifies cDNA formed by mold mRNA isolated from tumors. The isolated DNA is transferred to a eukaryotic cell that expresses a high concentration of MHC-I molecules, which allows the preparation of larger amounts of protein for analysis.

Properties of tumor antigens

tumor antigens

Most are similar to the normal cells from which the tumor originated (species-specific tissue matching antigens) and are mostly glycoproteins. In spontaneous tumors, in contrast to those experimentally induced, antigens are more difficult to detect, less frequent, and do not elicit a transplant rejection reaction.

There are two types of antigens:

–  Tumor specific antigens , TSA – are not found on any normal cell and are really specific

–  Tumor-associated antigens , TAA – are not new to the host because they are found on embryonic tissue cells, on cells during viral infection and on normal cells in lower concentrations

–  Transplant tumor antigens , TATA – include TSA or TAA

They can be located:

And)  on the surface – similar to tissue matching antigens, serve as a theoretical target of antibodies or sensitized lymphocytes, which can cause cell lysis

b)  in the interior – interesting for studying malignant transformation and as markers of tumors

c) in the environment – after necrosis or spontaneous release, responsible for the production of blocking factors, which cause immune growth enhancement. They also serve in the diagnosis and assessment of tumor Peter (afp in hepatoma).

Most of the research was performed on animal experimental models, especially mice, while a smaller part on spontaneously formed tumors.

TSA (tumor specific antigens)

They most often belong to the Heat-shock-protein family ( HSP  ), which is a large number of soluble cellular proteins, which are classified on a molecular basis  Peter. They are also found in normal tissue and are not immunologically active, but HSP tumors cause a specific tumor immunoreaction because a peptide is inserted into it, which binds to progenitor cells (macrophage or dendritic cell), enters the remodeling process for endogenous and exogenous antigens, and is proposed on the surface of the presentation cell within MHC-I or MHC-II.

1. Caused by chemical agents

They have unique and unrepeatable antigens. By ingestion or rubbing into the skin of carcinogens (polycyclic hydrocarbons, aromatic amines, azo dyes and nitroso compounds). They have strong individual antigens that act as transplantable and are specific for one particular solid tumor.

2. Challenged by physical means

They have unique and unrepeatable antigens. They occur after the action of ionizing radiation, chronic mechanical or thermal stimuli or after implantation in the tissue of inert sheets of cellophane or metal, which interrupt intercellular communication. Antigens are less immunogenic.

3. Caused by viruses

Tumor antigens characteristic of the virus are expressed, along with which, in addition to oncogenic DNA or RNA virus, antigens of an incidental virus can also be expressed. Viruses cause lysis of the host cell and in a productive infection the viral genome takes over genetic control over metabolism and creates its own ingredients, which bind to each other into viruses. With integrated infection, malignant cell transformation can occur.

Proviruses act as part of indigenous genetic material and it may be a long time before a normal cell transforms. Virus proteins (antigens) react with proteins that are essential for cellular regulation (products of suppression genes) and remove them. Some of the DNA viruses are: papovirus, adenovirus and herepsvirus. RNA viruses (retroviruses) incorporate their genome into the cell and thus complementary DNA synthesis to RNA virus occurs with the enzyme reversible transcriptase (RNA-dependent DNA polymerase) encoded by viral RNA.

Examples of RNA viruses are HIV and HTLV (human T cell leukemia virus).
Because antigens are determined by the viral genome, they are the same for all antigens caused by the same virus.

TAA

Also present in normal cells in smaller amounts.

1. Oncofetal tumor antigens

Macromolecules in high concentrations present in the embryonic period, while in an adult only in traces. They are weakly immunogenic and their recurrence may or may not be associated with tumors. Reappearance is caused by activation of embryonic genes (dedifferentiation caused by neoplastic transformation). The best known are carcinoembryonic antigen and alpha-fetoprotein and TL antigen, a differentiating thymic antigen present on cells in murine leukemia.

2. Oncogenic proteins

Antigens encoded by cellular oncogenes. They are normally found in small amounts encoded by proto-oncogenes. The receptor for the growth factor Neu is found in very small amounts, and in breast tumors in large quantities and can stimulate an immune response. Some oncogenic proteins are different from normal and can function as TAA.

3. Tumor antigens of histological type

Antigens common to most tumors of the same histological type and characteristic are human tumors.

Division of antigens based on structure and genetic origin

1.  Products of mutated onogens and suppression genes
From the cytoplasm, tumors are presented on the surface with MHC-I and MHC-II, but are not the target of antitumor lymphocytes in patients.

2.  products of other mutated genes
They used to be considered TSTAs, and are present in tumors caused by chemical or physical means because they cause a random mutation of any gene.

3.  overexpression of normal gene products
Normal proteins that are present in small concentrations, and in tumors in high ones. – – Tyrosinase – an enzyme present only in melanocytes, and a larger amount triggers an immune response.

4.  Expression of repressed products of normal genes
Proteins are present only in a certain period of embryonic development, and reappear in malignant transformation, and in the wrong tissue and at the wrong time it causes an immunoreaction.

a) MAGE – melanoma antigens

b) CT-group of cancer-testis antigen

5. Altered glycolipid and glycoprotein antigens
Present in human and experimental tumors and targets are tumor immunotherapy and diagnostic markers. These tumors express altered gangliosides, blood group antigens, and mucins.

6. Tissue-specific differentiation antigens
Consequence of deregulation of gene expression (TL antigen)

7. Antigens encoded by oncogenetic viruses

8. Oncofetal antigens 

Human tumor antigens

They have been studied in vitro and in vivo

And) In vitro 
– does not allow the inference of the ability to induce immune rejection of tumors.

b) In vivo
– skin test of late hypersensitivity to extracts of own tumor cells. Tumor nodule formation is reduced if lymphocytes are added to the injected cells.

Antigens found in humans are classified into three groups:

1. Antigens characteristic of histological origin
–  colon, lungs, skin, thyroid
– Patients with malignant melanoma show reactivity against melanoma of other patients, but not reactive to other tumors

2. Viral antigens
–  Epstein-Barr virus -> Burkitt’s lymphoma, nasopharyngeal carcinoma, infectious mononucleosis
–  Simple hrpes virus type 2 -> cervical cancer
–  type C -> leukemia
– Heliobacter pylori (bac.) -> stomach cancer

3. Oncofetal antigens
–  glycoproteins, which are secretory products and are used in the diagnosis and prognosis of the disease, but are not involved in tumor rejection
– alpha – fetoprotein  – the main serum protein of embryonic age present in liver blackness, hepatitis, liver tumors, chronic hepatitis, ulcerative colitis
– carcinoembryonic antigen – Colon cancer and rectum

Display tumor markers  and connections with authorities:

tumors dividing antigens

Immunoreaction to tumor

Tumor cells are genetically unstable and subject to apoptosis, and apoptotic vesicles contain antigens, which activate lymphocytes via predation cells.

Immune resistance to tumor

The host immune system plays an essential role in tumor formation. Tumors, especially of the hematopoietic system, are more common in animals with a weakened immune system by radiation, immunosuppressive agents, antilymphocyte serum or neonatal thymectomy. Abundant infiltration of the tumor by lymphocytes, macrophages, and plasma cells is evidence of an immune response to the tumor.

A person develops concomitant immunity to a tumor because injecting the cells of that tumor into another place the body shows resistance. Nonspecific and specific immunoreactions (cellular and humoral) are involved in the response to tumors. The cellular response involves lymphocytes, NK cells, macrophages, monocytes, Petertocytes, eosinophils, and other polymorphonuclear leukocytes. Different antibodies participate in the humoral.

Cellular immunity

Cytotoxic T lymphocytes

Upon direct contact, they can destroy the cell by a mechanism via the antigen proposed in MHC-I, but many tumors weakly express MHC-I molecules, thus limiting the cytotoxic effect of CD8 + lymphocytes. Sensitized lymphocytes from an immunized donor or a person with a progressive tumor, which release cytokines in contact with the antigen, also have the ability to kill, but tumors, unlike bacteria and viruses, do not cause cytokines and chemokines required for progenitor cell-specific T lymphocytes. .

NK stations

nk cells

They can kill tumor cells without prior sensitization, and the carbohydrate components of glycoproteins embedded in the membrane of target cells are involved in the recognition process.

After binding to the cell, it releases soluble factors, which cause the cell to lyse. It then separates and connects to another station. Their activity is stimulated by interferons, and inhibition by antigen and tumor complexes.

Macrophages and Monocytes

Participants in the afferent and efferent part of the immunoreaction. Immunity can be passively transmitted through peritoneal macrophages, as well as their ability to kill tumor cells in vivo by inserting the contents of their lysosomes. Their anti-tumor activity is based on lytic enzymes and metabolites of the reactive oxygen species and nitrous dioxide.

Their toxicity can be specific or nonspecific. They also carry receptors for fc-antibodies. They become nonspecific cytotoxic after treatment of animals with nonspecific reticuloendtothelium (BCG) and they selectively recognize and destroy cells with malignant features and can be isolated from tumors.

Humoral immunity

Sensitivity to antibodies is weak and varied. In some cases, immunity can be transmitted by the serum of immunized animals passively by antibodies, but more often there is growth enhancement, after enhancements.

Antibody-dependent cellular cytotoxicity

Antibodies after binding to the tumor make it susceptible to lysis mediated by macrophages and NK cells.

Complement-dependent cytotoxicity of antibodies

It is not present, except in leukemias, especially in those enchanted RNA viruses. Such antibodies sometimes appear in the early stages of the disease (melanoma) or after surgical excision.

Blocking factors

Often found in the serum of hosts with progressively growing tumors, which inhibit specific cytotoxic T lymphocytes in antitumor activity, and these are antibodies, immunocomplexes, but also the tumor antigens themselves. Passive transmission of serum in vivo induces immune enhancement of tumor growth.

Immune monitoring of tumor cells

The immune system constantly monitors the body and destroys malignant cells to preserve antigenic and genetic integrity. The main purpose of the transplant reaction would be to protect the organism from tumors. According to some opinions, this system has a role in the elimination of tumor cells at the very beginning, and not when the tumor is already clinically noticeable.

Avoiding tumors by immune defense

1. Poor immunogenicity / MHC-1 deficiency 

Lack of MHC-I, beta2-microglobulin and other parts of the antigen processing mechanism (proteasome) inhibit the activity of cytotoxic CD8 + lymphocytes. Patients with a tumor that does not express MHC-I have a poorer prognosis. A weak immune response can stimulate tumor growth because a small amount of sensitized splenocytes or antibodies to the tumor inoculum accelerates its growth.

2. Immunoselection

Immune attack-sensitive tumor cells decay, and resistant cells, which have been phenotypically modified by induction of an immunoreaction (tumor correction), survive.

3. Antigen modulation

Reversible disappearance of antigen from the surface while there are antibodies (TL antigen in leukemia) or Peterking of antigen by glycocalyx.

4. Lack of costimulator / MHC-II

One signal for TH lymphocyte activation comes from the antigen receptor fused to the antigen exposed to MHC-II, and the other signal through the interaction of CD80 (B7) on the progenitor cell and CD28 molecule on T lymphocyte, which stimulates IL-2 production and T lymphocyte proliferation. T cells can be inhibited via CTLA-4, which like CD28 belongs to the same molecular family and are expressed on CD8 + lymphocytes. Poor activation can lead to clone anergy. The tumor can lose other cells essential for lymphocyte adhesion and lead to a shift of TH1 to TH2. The cytokine microenvironment can create a hostile environment for lymphocytes and prevent their division (TGF-beta).

5. Antigen release / smoke curtain

The action of cytotoxic cells and antibodies requires a certain local density of antigens, but if they are constantly released, a so-called “smoke curtain” is created.

6. Sneaking theory

Tumor cells are initially too small and later too large. The tumor is faster than the effector arc of the immune system.

7. Immune nonreactivity

a) tolerance to vertically transmitted oncogenic viruses (specifically acquired immunotolerance)

b) Tolerance due to inappropriate overdose of tumor antigen
 
c) Nonspecific natural, iatrogenic or tumor-induced tolerance (VEGF – vascular endothelial growth factor or TGF-beta which inhibits the stimulation of T lymphocytes with IL-2).
 
Tumors are more common in children due to its underdevelopment and in the elderly due to exhaustion. Suppressive mechanisms are also involved in the immunoreaction, and increasing the number of CD25 + lymphocytes leads to suppression of the antitumor response.

8. Theory of blocking factors

The serum of a progressive tumor carrier can block cellular cytotoxicity in vitro, whereas the serum of a regressive tumor organism does not have this effect.

Tumor immunotherapy

Immunotherapy is based on stimulating the patient’s immune response to the tumor, but is not successful in therapy, but in prophylaxis it is.

Passive immunotherapy

Transfer of specific antibodies prepared in another organism of the same or different species. There is a risk of growth enhancement, poor entry of antibodies into the tumor node and their binding to normal cells (same antigen, binding to the fc-receptor), and they may be antigenic. Antibodies can also enter a tumor cell (toxin), a cytostatic or a radioactive isotope.
 
Successes have been achieved in the treatment of B-lymphoma with monoclinic antibodies against the antigen CD20 / 22/52, in breast cancer against HER2 / Neu (herceptin) and in leukemia with B and T anti-idiotypic antibodies, and active vaccination against idiotypes of B lymphoma can be performed. antibodies neutralize growth factors and angiogenesis , block growth factor receptors.

Adaptive immunotherapy

Transmission of sensitized lymphocytes can protect, cause regression, or slow tumor growth.

Specific active immunotherapy

Own tumor cells are killed by radiation, heat, successive freezing and thawing and together with the adjuvant are injected under the skin (in melanoma due to common antigens). A promising approach is the manipulation of the costimulatory signal using transfection with the genome that determines the B7 ligand. It is possible to create a spectrum of melanoma cell lines ratified with B7.
 
In the experimental phase are cellular vaccines, which are divided into 3 types:
 
  1. dendritic cells
  2. atologic tumor cells
  3. hybridomas of tumor cells and progenitor cells
 
Attempts are being made to antigenic peptide which would be administered as a vaccine. Vaccination against the idiotypes of non-Hodgkin’s lymphoma and multiple myeloma has been successful.

Nonspecific active immunotherapy

The use of non-specific stimulants is also successful. FOR stimulation of a nonspecific reticuloendothelial system such as zymosan, glucan, levamisole, BCG, Corynebacterium parvum, Bordotella pertussis. The number and activity of immunocompetent cells, which activate cytokine-secreting macrophages and express MHC-II molecules and B7 costimulatory molecules, increases.

This all leads to the activation of TH helper lymphocytes and increases cellular and humoral immunity. By injecting local BCG into the tumor, its regression and metastasis to the lymph nodes is achieved. Immune status is impaired due to cytostatics and the results are not satisfactory, except in the treatment of melanoma.

Restorative immunotherapy – recovery and stimulation of the immune response

Antagonists of suppressive effects (cyclophosphamide, indomethacin – prostaglandin synthetase inhibitor) are used. Isolation and cloning of genes for various cytokines has led to the possibility of easy production. Interferons, alpha, beta and gamma, IL-1/2/4/5/12, GM-CSF and TNF-alpha are used in therapy, but without much success . Antibodies against 35] CTLA-4 are also used.
 
It is possible to be vaccinated with heat shock proteins, which are released upon cell damage and lead to the presentation of the HSP peptide within the MHC-I on professional presentation cells.

Tumor immunoprophylaxis

– Vaccination against oncogenic viruses successful in experimental animals, but also applied in humans.
–  Clinoran antigenic peptides are products of tumor antigens, and some produce onogens or viruses.
–  Experiments with antiidiotopic antibodies, which mimic the properties of antigens.
– HPV vaccine , hepatitis B vaccine

Explanation of the terms mentioned in the text

Lymphocytes  – central cells in a specific immunoreaction
NK cells – a subtype of lymphocytes that are classified as large granular lymphocytes and is not a clonal organism. 

Petertocytes  – large cells in the connective tissue, especially under the skin and mucous membranes. They bind IgE, which, after binding to the allergen, causes degranulation. They mediate the anaphylactic hypersensitivity reaction.

Macrophages  – professional phagocytes from the bone marrow, travel through the blood as monocytes and roam the tissues like mature macrophages. Participants in a specific and nonspecific immunoreaction. 

Nonspecific immunity  – is innate, the first line of defense that exists without prior encounter with the antigen, and is directed against all antigens. It is based on anatomical, physiological, phagocytic barriers and inflammation. 

Molecule B7  – expressed on presentation cells and provides a costimulatory signal for T lymphocytes, binding to the CD28 molecule, and is also found on macrophages and B lymphocytes. 

BCG  –  Bacillle Calmette Guerin , is an attenuated strain  Myobacterium tuberculosis  which is used for vaccination and as an adjuvant for nonspecific immunostimulation.

Beta2 – microglobulin  – a polypeptide involved in the structure of MHC-I

Energy  – inability to respond to antigens by immunoreaction, and may occur if no other signal is present. 

TH1 lymphocytes  – anti-inflammatory cells that mediate the late hypersensitivity reaction. They secrete cytokines, activate macrophages, and can lyse target cells.

TH2 lymphocytes  – secrete cytokines IL-4/5, and stimulate humoral immunity. 

CTLA-4  – negative signal and inhibits lymphocyte proliferation (CD28 antagonist).

Tumor transplant antigens  – antigens that can cause a tumor transplant rejection reaction 

Transplant reaction  – graft immune rejection reaction 

Transplantation  – the procedure of transplanting tissues and organs from one place to another within the same organism (autotransplantation) or from one individual to another (allotransplantation, to an unrelated individual within the same species, xenotransplantation, from another species) 

Pure strains of animals  – are syngeneic animals created by mating a brother and sister for at least 20 years 

Syngenic  – indicates that two tissues or organisms are genetically identical, e.g. identical twins or pure strains of animals

Inflammation  – stereotypical, standard reaction of the organism to any damage and is the beginning of any specific immunoreaction. 

Cytotoxic T lymphocytes  executive effector lymphocytes that, in direct contact, can kill target cells without the mediation of antibodies and complements. They recognize antigens represented by MHC-I molecules. They are major mediators of cellular immunity, late hypersensitivity, graft and tumor rejection, and killing of one’s own infected cells. 

CD8  – a heterodimeric molecule on cytotoxic lymphocytes, which acts like a receptor that binds to the alpha3 – domain of MHC-I and participates in signal transduction during the activation of T lymphocytes.

Source

Taradi M et al .: Immunology. Medicinska naklada, Zagreb, 2010

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