Diapositiva 1 - Area Biotecnologie

Definizione (Willis)
UNA NEOPLASIA E’ UNA MASSA ANOMALA DI TESSUTO LA CUI CRESCITA
ECCESSIVA E’ SCOORDINATA RISPETTO A QUELLA DEL TESSUTO
NORMALE E PERSISTE NELLA SUA ECCESSIVITA’ ANCHE DOPO LA
CESSAZIONE DEGLI STIMOLI CHE L’HANNO PROVOCATA.
Altre caratteristiche:
- il tumore cresce a spese dell’ospite  le cellule neoplastiche competono con le cellule dei
tessuti normali per l’approvvigionamento energetico e nutritivo
- il tumore è virtualmente autonomo
BASI MOLECOLARI DEL CANCRO
Principi fondamentali
- ALLA BASE DEL PROCESSO DI CANCEROGENESI CI SONO DANNI GENETICI NON
LETALI (MUTAZIONI). La massa tumorale deriva dall’espansione clonale di una singola
cellula progenitrice che ha subito un danno genetico  i tumori sono monoclonali.
- I bersagli principali del danno genetico sono costituiti da tre classi di geni che normalmente
regolano importanti funzioni cellulari:
a) Proto-oncogeni  promuovono la crescita (oncogeni dominanti)
b) Geni oncosoppressori  inibiscono la crescita (oncogeni recessivi)
c) Geni che regolano la morte cellulare programmata o apoptosi
Numerose evidenze sperimentali dimostrano che nessun oncogene è in grado di
trasformare completamente una cellula in vitro da solo.
OGNI TUMORE UMANO FINORA STUDIATO MOSTRA ALTERAZIONI GENETICHE
MULTIPLE CHE CONSISTONO NELL’ATTIVAZIONE DI DIVERSI ONCOGENI E NELLA
PERDITA DI DUE O PIU’ GENI ONCOSOPPRESSORI.
- Il processo di cancerogenesi è un processo a tappe successive sia a livello fenotipico
che genetico. Il processo attraverso il quale i tumori acquisiscono gradualmente le loro
caratteristiche fenotipiche (crescita eccessiva, invasività locale, capacità di dare metastasi,
ecc) è chiamato progressione neoplastica.
NELL’AMBITO DELLA PROGRESSIONE NEOPLASTICA UNA TAPPA FONDAMENTALE
E’ RAPPRESENTATA DALLA CAPACITA’ DEL TUMORE DI DOTARSI DI UNA RICCA
VASCOLARIZZAZIONE MEDIANTE UN PROCESSO DI NEOANGIOGENESI.
LE METASTASI
ALLA BASE DEL PROCESSO DI
METASTATIZZAZIONE
CI
SONO
ALCUNE PROPRIETA’ DELLE CELLULE
NEOPLASTICHE CHE PERMETTONO
LORO DI MIGRARE, ANCORARSI A
MOLECOLE
DELLA
MATRICE
EXTRACELLULARE, INDURRE LA LISI
DELLE MEMBRANE BASALI, ECC:
Terapia dei tumori
Chirurgia:
Radioterapia
Chemioterapia
The limitations of clinical chemotherapy and radiotherapy have been ascribed primarily to mechanisms that
mediate drug resistance at the cellular level. Functional gene mutations or other changes that affect the expression
of genes encoding proteins that influence the uptake, metabolism, and export of drugs from a single cell are
important determinants of drug resistance, as are epigenetic changes that can lead to transient drug resistance.
However, the mechanisms that involve the tumor microenvironment also mediate resistance of solid tumors
to therapy. For an anticancer drug to kill a high proportion of cancer cells in a solid tumor, it must be distributed
throughout the tumor vasculature, cross vessel walls, and traverse the tumor tissue. However, the distribution of
many drugs within tumors is heterogeneous, such that only a proportion of the target tumor cells is exposed to a
potentially lethal concentration of the cytotoxic agent.
The tumor microenvironment is characterized not only by marked gradients in drug concentration but also by
gradients in the rate of cell proliferation and by regions of hypoxia and acidity ( Fig. 1 ) all of which can influence
tumor cell sensitivity to drug treatment.
Fig. 1 . The tumor microenvironment in relation to blood vessels. A ) Diagrammatic representation of tumor cells and the extracellular matrix (ECM)
surrounding a capillary. B ) Schematic representation of the gradient of oxygen concentration ( p O 2 : dashed line ) and of pH ( dotted line ) in relation to
the nearest tumor blood vessel. The relationship of p O 2 and pH with distance from the nearest blood vessel is similar to that reported by Vaupel
Tumor Vasculature and Blood Flow
Tumor responsiveness to chemotherapy is influenced both directly and indirectly by the
vasculature, which is abnormal in solid tumors. The vasculature influences the
sensitivity of the tumor to drugs because anticancer drugs gain access to tumors via
the blood and because the limited supply of nutrients in tumors leads to metabolic
changes (including hypoxia) and to gradients of cell proliferation that influence drug
sensitivity.
In normal tissues, fluid is removed through a network of lymphatic vessels as well as
through the veins. Solid tumors may lack or have fewer functional lymph vessels than
normal tissues which contributes to the increased interstitial fl uid pressure within
them .Increased interstitial fluid pressure inhibits the distribution of larger molecules by
convection and compresses blood vessels such that blood is diverted away from the
center of the tumor toward the periphery
Effetto ossigeno e radioterapia
Thomlinson & Gray (1955) key paper
Conclusioni schematizzate
del lavoro
di Thomlinson e Gray:
-ogni centro tumorale con raggio
maggiore di 200 m presenta un
centro necrotico
-nessuna necrosi è presente in
tumori
con raggio minore di 160 
-Comunque grande sia il raggio del
centro necrotico, lo spessore
esterno
composto di cellule tumorali attive
non è mai maggiore di 180 m ed
è
sempre compreso tra 100 m e
180 m.
L’area delle cellule tumorali attive appare, in sezione,
come un anello
Conclusioni ottenute da Thomlinson e Gray dallo studio di
sezioni istologiche di carcinoma bronchiale umano.
Diffusione dell’O2 da un capillare
Conclusioni del lavoro di
Thomlinson e Gray:
•Le cellule tumorali proliferano e
crescono attivamente soltanto se
sono
in prossimità di una sorgente di
ossigeno o nutrienti (stroma).
150 m
Conclusioni ottenute da Thomlinson e Gray dallo studio di
sezioni istologiche di carcinoma bronchiale umano.
Fino a 150 m dal capillare le
cellule sono ossigenate; a
distanze maggiori l’ossigeno è
consumato e le cellule diventano
necrotiche.
•Le cellule ipossiche formano
uno strato (di una o due cellule)
nel quale la concentrazione di
ossigeno è alta abbastanza da
permettere la loro attività e bassa
abbastanza da renderle
relativamente protette dagli effetti
dei raggi x.
Presenza di Ossigeno e Radioterapia:
• protettori: agenti che riducono l’effetto di una determinata dose di radiazione
• sensibilizzanti: agenti che aumentano l’efficacia di una determinata dose di radiazio
l’ossigeno è un potente “sensibilizzante”.
In presenza di O2 tutti i sistemi biologici sono molto più sensibili alla
radiazione
.
Tumor Hypoxia
The limited vasculature of tumors leads tumoral cells in hypoxia
Cells in hypoxic regions may be viable, but they are often adjacent to regions of
necrosis.
If cells close to blood vessels are killed by treatment, the nutrient supply to previously
hypoxic cells may improve, allowing those cells to survive and regenerate the tumor.
The presence of hypoxia in tumors is known to lead to the activation
of genes that are associated with angiogenesis and cell survival, and this effect is
mediated by the transcription factor hypoxiainducible factor 1.
Expression of these genes may result in the expansion of populations of cells
with altered biochemical pathways that may have a drug-resistant phenotype. For
example, hypoxia selects for cells that have lost sensitivity to p53-mediated
apoptosis and for cells that are deficient in DNA mismatch repair
In the presence of oxygen, many anticancer drugs generate free radicals that
damage DNA. These drugs accept electrons from biologic sources and then
transfer the electrons to oxygen . For example, doxorubicin undergoes chemical
reduction to a semiquinone radical, which in turn reduces oxygen to a superoxide
that may contribute to cytotoxicity . Thus, at low oxygen concentrations
the cytotoxicity of drugs whose activity is mediated by free radicals is decreased
Hypoxic regions of tumors are likely to have a decreased supply of nutrients such as
glucose and essential amino acids. This is because tumor cells often use glycolysis —
the conversion of glucose into lactate to produce ATP — to obtain the energy they
need to survive and proliferate rather than oxidative metabolism, a more effi cient
pathway that leads to production of CO 2 and carbonic acid . Decreased clearance of
these acidic products ofmetabolism leads to low interstitial pH, another characteristic
of solid tumors
Tumor Acidity : The pH in the tumor microenvironment can influence the
cytotoxicity of anticancer drugs. Molecules diffuse passively across the cell
membrane most efficiently in the uncharged form. Because the extracellular pH in
tumors is low and the intracellular pH of tumor cells is neutral to alkaline, weakly
basic drugs that have an acid dissociation constant of 7.5 – 9.5, such as
doxorubicin, mitoxantrone, vincristine, and vinblastine, are protonated and display
decreased cellular uptake
Tumor Cell Proliferation
Nutrient deprivation induces cell cycle arrest, and the rate of proliferation of tumor
cells therefore decreases with increasing distance from tumor blood vessels.
Most chemotherapeutic drugs, including, possibly, biologic agents that target
cell proliferation, are more effective against proliferating thanquiescent cells .
Consequently, slowly proliferating cells at increasing distances from tumor blood
vessels are likely to be resistant to therapy.
Terapia dei tumori
Compounds have to be investigated in vitro
before any in vivo or clinical trials are performed.
Many therapeutics
that demonstrate efficacy
in vitro fail to produce the
same effect in vivo.
Two-dimensional (2-D) cultures are
inadequate or limited models of in vivo
tumor behavior
Although in vitro 2D model has yielded much information , it is ,
nonetheless , unsuitable in representing completely tissues and in vivo
tumors information.
It should be recalled that normal tissues and solid tumors grow in a three
dimensional spatial array and that the cells in these structures are
exposed to non uniform distributions of oxygen and nutrients as well as
other physical and chemical stresses.
Thus , bidimensional growth of cells , in which all of the cells are
equally exposed to oxygen and nutrients , cannot be used to
examine all aspects of biology Thus, cells, nutrients,.
In vitro approaches have been used to
examine how anticancer drugs
penetrate and distribute within tumors.
Solid tumor models in tissue culture
that have been useful in studying drug
distribution include multicellular tumor
spheroids
and multilayered cell cultures .The
penetration of anticancer drugs into
spheroids for doxorubicin ,
methotrexate , vincristine,vinblastine
but better for 5-fluorouracil . A better
model is provided by multilayered cell
cultures, which have a linear geometry
that facilitates the quantification of drug
transport through tumor tissue Studies
with multilayered cell cultures have
confirmed very slow tissue penetration
of drugs that bind avidly to DNA, such
as doxorubicin and mitoxantrone, and
relatively slow penetration of several
other drugs with different modes of
action
Fig. 3 . In vitro models used to study the penetration of anticancer drugs through tumor tissue. Photomicrographs of ( A ) a
multicellular tumor spheroid and ( B ) a multilayered cell culture (MCC) on a permeable membrane support. Proliferating cells
labeled with bromodeoxyuridine ( black ) are located predominantly in the peripheral areas. C ) Schematic representation of
experimental method used to quantify drug penetration. A drug is added on the top of the MCC (blue) and sampled as a funtion of
time in the receiving compartment below the MCC. ( D ) Time-dependent penetration of a drug through an MCC compared with
penetration through the permeable support membrane alone. The y -axis represents the drug concentration in the receiving
compartment as a ratio of that expected when equilibrium has been established.
In an attempt to design more suitable in vitro systems which take into
consideration the three dimensional arrangement of tissues and solid tumors ,
multicellular tumor spheroids were developed
Spheroids represent quite realistically the three dimensional growth and
organization of avascular solid tumors and, consequently , simulate much
more precisely the cell cell interactions and microenvironmental conditions
found in these tumors
The use of spherical aggregates of cells, or
spheroids, has been introduced byHoltfreter andn
Mosconain in 1944 and 1957, respectively.
Spheroids possess more realistic, three-dimensional
(3D) cell–cell and cell–matrix interactions than 2-D
cell cultures.
Many studies have suggested both correlative and
causal effects on cellular drug response due to
various characteristics of spheroids including
necrotic cores induced by hypoxia, gene
expressions, drug permeability and inhibited
apoptosis.
Scanning electron micrographs of MG 63
osteosarcoma cells grown in MGmonolayer (a) and as three dimensional
tumor spheroids (b ) after 48 h of growth
three-b)
growth.
Establishing spheroid-seeded scaffolds.
A more recent approach for creating realistic tumor models is the use of
biocompatible synthetic polymers such as poly(lactico- glycolide) and poly(ecaprolactone) to engineer the desired 3-D environments for cell growth.(12–14)
Previously established in vitro tumor
models include monolayer cultures,
spheroids and scaffolds with cells.
A novel tumor model is developed by
incorporating spheroids, or 3-D cellular
aggregates, into 3-D scaffolds.
Spheroids remain intact in scaffolds after seeding
The combination of spheroids
consists of 2000 cells and scaffolds
with 500–1000 μm diameter pores .
After 48 h of culture, one hundred
spheroids were pipetted onto each
scaffold ,t he seeded scaffolds were
moved onto poly-HEMAcoated wells of
12-well non-tissue culture treated
plates and 50 μL of culture medium
was added to each scaffold. The
spheroids were left to attach for 4 h in
humidified incubators, and 2 mL of
culture medium was subsequently
added to each well. After 24 h of
culture, the plates were placed on an
orbital shaker.
Microscopic image of a 2-day old spheroid of 2000 cells confirmed
the spherical morphology.
Spheroid diameter gradually decreased over the duration of the
culture (n = 10).
The viability of cells within spheroids cultured for 4 days was
observed using a live stain (calcein-AM). The control represents a
dead spheroid that had been incubated in 70% ethanon for 30 min.
Images of poly(lactic-co-glycolic acid) (PLGA) scaffolds showed that
spheroids were successfully incorporated into the pores of the
scaffold.
One possible component of chemoresistance
is drug penetration.Cellular aggregation as
well as the presence of tortuous in 3-D
scaffold structures may limit the drug
exposure to some cells.
However, drug uptake in SS was efficient and
that doxorubicin was found inside the
spheroids within 3 h of exposure
demonstrating that drug resistance in 3-D
systems cannot merely be
explained by the effects on drug transport.
DOXO green
Combining the two techniques by seeding 3D scaffolds with spheroids instead of
dispersed, monolayer-cultured cells
increases the drug resistance significantly
and the chemoresistance of SS was closer to
the in vivo data than that of MS.
NO DOXO
Utility of spheroid-seeded scaffolds
as a drug screening tool.
The presence of intercellular spaces within the
spheroids were visualized by incubating SS in
PBS containing fluorescein isothiocyanateconjugated 150 kD dextran
Increased drug resistance may be due to increased glycolysis
Since 3-D systems are more susceptible to forming regions of hypoxia, cells compensate for
the need of nutrients by increasing their dependence on the less efficient anaerobic pathways
following glycolysis instead of oxidative phosphorylation. As expected, the highest level of
lactate production was seen in then spheroid-seeded scaffolds.
Importantly, as confirmed by our measurements of the intracellular pH, increased production
of lactate leads to acidosis. Consequently, acidosis can trigger a variety of mechanisms
leading to higher drug resistance. A previous study has shown that inhibiting glycolysis
results in reduced drug resistance.
.