1. Chen Yan
Vivek Shukla
May 9th, 2016

2. ž Introduction to Metastatic Breast Cancer
1. How does this cancer spread?
2. Symptoms
3. Treatment Options
4. Current Study Methods
ž Origin of Synthesis
1. Contributing Papers
2. Our Research Proposal
ž Methods and Expectations

3. ž Classified as Stage 4 Cancer
ž The cancer originates in the breast but is
capable of moving to various parts of the
body such as
1. Lungs
2. Liver
3. Bones
4. Brain
ž Creates complications in those regions

4. ž Brain
1. Head aches
2. Vision disturbances
3. Seizures
4. Behavioral/
Personality changes
ž Bone
1. Increased pain
2. Weaker and more
fracture prone
ž Liver
1. Increased digestive
enzymes
2. pH imbalance
3. Itchy skin
4. Nausea and
vomiting
ž Lung
1. Chest Pain
2. Trouble Breathing

5. ž Utilize circulatory
and lymphatic system
to travel to different
organs.
ž Cells growing at an
abnormal rate are
capable of invading
surrounding healthy
tissues and
spreading.

6. 1. Invasion of normal healthy
tissue
2. Intravasation – entering through
walls of circulatory and lymph
system.
3. Transport/Circulation – use
circulatory and lymph system to
travel.
4. Arrest and Extravasation –
cancer cells stop and move
outward towards tissues.
5. Growth – can form smaller
tumors known as
micrometastases
6. Angiogenesis – harness linkage
to new blood vessels to promote
growth

7. ž HER2 targeted therapy– Human Epidermal
Growth Factor – drugs to decrease expression
of this factor. (Herceptin)
ž Radiation Therapy
ž Surgical removal of tumorous regions
ž Endocrine Therapy: Drugs that target
hormone receptors and control levels of
progesterone and estrogen. (Aromatase
Inhibitors).

8. 1. “Selective Events in the Metastatic
Process Defined by Analysis of the
Sequential Dissemination of
Subpopulations of a Mouse Mammary
Tumor”
2. “TWIST, a master regulator of
morphogenesis, plays an important role in
Tumor Metastasis”
3. “Regulation of the expression of E-
Cadherin on Human Cancer Cells by
Linolenic Acid (GLA)”

9. ž In our mouse model, we wish to study the
selective events of metastasis, the effect of
TWIST in breast cancer metastasis along with
potential therapeutic effects of Linolenic
Acid (GLA) that regulate E-cadherin.

10. ž MMTV-PyMT: Commonly used mouse model
for metastatic breast cancer where a mouse
mammary tumor virus is used to induce
cancer.
ž Genetically Altered Mice: (Knockout Mice)

11. ž Our cell line has to be first be established as
a working cell line by growing it in culture.
ž Media content:
1. DMEM
2. 5% Fetal Calf Serum and 5% Newborn Calf
Serum
3. NEAA
4. 2 mM L-glutamine
5. Streptomycin

12. ž Strain: BALB/c Mice
ž Injection of a known titer of viable cancer
cells into the fat pat of the mouse model.
ž Injected into the Fat Pad

13. How can we
assess the
spread of our
tumor cell line
in the model?

14. ž Metastatic process is a sequence of steps
(invasion, intravasation, transport, arrest,
extravasation, and growth).
ž Inefficient.
ž Both random and selective events are
responsible for this inefficiency.
ž Selective:
-cells isolated from spontaneous metastases more
metastatic than original parent tumor.
-genetically stable metastatic and nonmetastatic
sublines have been characterized.
¡ nonmetastatic lines unable to complete one or
more steps in the metastatic cascade, whereas
metastatic lines can complete all steps involved.

15. ž random events eliminate majority of tumor cells,
irrespective of metastatic potential
¡ if any population of cancer cells entering the metastatic
cascade goes sequentially through five randomly
traumatic steps associated with invasion, etc, each of
which kills 90% of the cells, then only 0.001% of the
initial cellular input from the primary cancer will form
metastases.
ž one can define any step that more efficiently
(additional log kill, 2 selective steps would decrease
100-fold) eliminates cells of nonmetastatic lines than
metastatic lines as a selective step.
ž If host immune functions are important for the
elimination of potentially metastatic cells, the
selective events best targets for therapeutic
intervention.

16. ž Knowledge of which steps in the metastatic
cascade are selective is limited. Deficiencies
in quantitatively analyze clonogenicity of
potentially metastatic cells at various points
in the metastatic cascade.

17. ž Bioassays used to estimate # of tumor cells,
but insensitive and not quantitative.
¡ Require multiple animals to test in 1 tumor-
bearing animal.
ž Suzuki. Enzymatically dissociated lungs with
protease, plated the cell onto irradiated
feeder layers. Colonies counted.
ž Background lawn made it impossible.

18. ž Use tumor cells with drug-resistance
markers, simultaneously quantitate and
identify tumor cell colonies developing in the
presence of selective media.

19. ž Tumor subpopulation lines 66, 67, 168, and 410.4 isolated
from a single spontaneously arising mammary tumor from a
BALB/cfC3H mouse.
¡ 66cl4 a thioguanine-, ouabain-resistant variant of line 66.
¡ 168FARN a diamino-purine, geneticin-resistant variant clone of
line 168, transfecting plasmid containing neomycin resistance
gene into 168FAR.
¡ 67NR: transfecting line 67, geneticin-resistant.
¡ 4TO7: thioguanine-, ouabain-resistant variant of 410.4.
¡ 4T1 thioguanine-resistant variant selected from 410.4 without
mutagen treatment.
ž 4T1 spontaneously metastasizes to both lung and liver
(formation of visible nodules in these organs)
ž 66cl4 spontaneously metastasizes to lung.
ž 67NR, 168FARN, 4TO7 highly tumorigenic but rarely
metastasize spontaneously.
ž Tumor cells injected into mammary fatpad.

20. at 37°C,5 ml of DME-10 were added and samples were mechanically
dispersed for 5 sequential 1-min periods. Between bursts, a portion of
the cell suspension was removed and an equivalent volume of DME-10
added (10).
All cell suspensions were plated in DME-10 containing the appro
priate selective drug and incubated for 10 to 14 days in 10% CCh-air
atmosphere at 37°C.The colonies were fixed with Carnoy's solution,
stained with crystal violet, and counted, and total colony-forming cells
per organ calculated.
RESULTS
Sequential dissemination of tumor cells from a mammary
fatpad was evaluated using both metastatic (4T1 and 66cl4; Fig.
1) and nonmetastatic (67NR, 168FARN, and 4TO7; Fig. 2)
subpopulations of a mouse mammary tumor. Line 4T1 ap
peared to metastasize primarily, but not exclusively, by a hem-
atogenous route because clonogenic tumor cells were recovered
significantly more frequently (P < 0.001, x2 analysis) from the
blood (38 of 58 mice) than from draining lymph nodes (12 of
58 mice). Figure A depicts 1 of 2 experiments in which the
sequential spread of 4T1 from a mammary fatpad was moni
tored. Lungs were seeded early (clonogenic 4T1 cells were first
detected at day 7, and 5 of 5 mice had clonogeneic 4T1 cells in
the lung by day 14) and the lung tumor burden increased on
subsequent assay days with an apparent doubling time of 30 h.
(The apparent doubling time is an underestimate because it not
only measures the growth rate of occult métastasesbut also
reflects continuous seeding of tumor cells from the primary
tumor.) Livers were seeded later, in that 4T1 cells were first
detected at day 21 and incidence did not reach 100% until day
28. Seven mice were necropsied at day 31; all had visible lung
nodules (43 ±7; mean ±SEM) and 5 had visible liver nodules
(2.0 ±0.8). In a second experiment, lungs were again seeded
early (66% incidence by day 9) and the lung tumor burden
increased on subsequent assay days. Clonogenic 4T1 cells could
be detected in the liver within days after detection in the lungs.
Ten mice from the second experiment were sacrificed for ne
cropsy between days 34 and 40. All 10 had visible nodules on
the surface of the lungs (49 ±6.5) and 5 mice had a visible
nodule on the surface of the liver.
Clonogenic tumor cells were recovered from both the blood
(27 of 82 mice) and lymph nodes (31 of 59 mice) of mice
bearing line 66cl4 tumors in a mammary gland, x2 analysis
suggests that line 4T1 more often metastasized hematogenously
than did line 66cl4 (P < 0.02) and that line 66cl4 more often
metastasized via the draining lymph node than did line 4T1 (P
< 0.01). Fig. IB depicts 1 of 2 experiments in which the
sequential spread of 66cl4 from a mammary fatpad was moni
tored. Clonogenic cells were detected in lungs of 66cl4 tumor
bearing mice on day 14 and all mice had detectable tumor cells
in the lungs by day 28. After day 28, expansion of the tumor
cell population in the lung proceeded at an exponential rate
with an apparent doubling time of 39 h. In addition, clonogenic
cells were recovered from livers on day 35 in this experiment,
but all 8 mice sacrificed for necropsy at day 50 had visible
were found.
The nonmetastatic line 67NR appeared to be unable to
intravasate. Clonogenic cells were not recovered from any of
the tissues (blood, lymph nodes, lungs, or liver) sampled from
a total of 44 mice over a period of 7 weeks for 2 experiments,
with one exception in which 3 clonogenic cells were recovered
from a draining lymph node of one animal on day 7 in one
experiment (Fig. 2A). Livers were dissociated and plated in
selective media in one experiment (no clonogenic cells were
detected) but not in the other. None of the mice sacrificed at
days 49-50 for necropsy in the 2 experiments had visible
métastasesor detectable clonogenic cells in any organ. The size
I0e A. 4T1 I0. B. 66cl4
co
10s
io2
IO1
IO»
io*
10s
IO1
IO'
I
I
I
I
I
t*t i -7°
DAYS POST INJECTION
Fig. 1. Recovery of clonogenic tumor cells from mice bearing metastatic
primary tumors. A, mice were given injections of 1 x 10s 4T1 tumor cells in the
mammary fatpad. At multiple intervals, groups of mice were bled and then
sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node
in the mammary fatpad was removed through day 19, after which the size of the
tumor made it difficult to find the enveloped lymph node. The draining brachial
lymph nodes were collected throughout the experiment. Lungs were digested
whether or not métastaseswere observed. B, mice were given injections of 1 x
10s 66cl4 tumor cells in the mammary fatpad. At multiple intervals, groups of
mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed.
The draining lymph node in the mammary fatpad was removed through day 17,
after which the size of the tumor made it difficult to find the enveloped lymph
node. The draining brachial lymph nodes were collected throughout the experi
ž Line 4T1 appear to
metastasize primarily
by hematogenous route.
(clonogenic tumor cells
recovered more
frequently from the
blood (38 of 58) than
from draining lymph
nodes (12 of 58)).
ž Lungs seeded early,
liver seeded later.
ž After necropsy, all had
lung nodules, 5 of 7
liver nodules
spersed for 5 sequential 1-min periods. Between bursts, a portion of
e cell suspension was removed and an equivalent volume of DME-10
ded (10).
All cell suspensions were plated in DME-10 containing the appro
ate selective drug and incubated for 10 to 14 days in 10% CCh-air
mosphere at 37°C.The colonies were fixed with Carnoy's solution,
ined with crystal violet, and counted, and total colony-forming cells
r organ calculated.
ESULTS
Sequential dissemination of tumor cells from a mammary
pad was evaluated using both metastatic (4T1 and 66cl4; Fig.
and nonmetastatic (67NR, 168FARN, and 4TO7; Fig. 2)
bpopulations of a mouse mammary tumor. Line 4T1 ap
ared to metastasize primarily, but not exclusively, by a hem-
ogenous route because clonogenic tumor cells were recovered
gnificantly more frequently (P < 0.001, x2 analysis) from the
ood (38 of 58 mice) than from draining lymph nodes (12 of
mice). Figure A depicts 1 of 2 experiments in which the
quential spread of 4T1 from a mammary fatpad was moni
ed. Lungs were seeded early (clonogenic 4T1 cells were first
tected at day 7, and 5 of 5 mice had clonogeneic 4T1 cells in
e lung by day 14) and the lung tumor burden increased on
bsequent assay days with an apparent doubling time of 30 h.
he apparent doubling time is an underestimate because it not
ly measures the growth rate of occult métastasesbut also
lects continuous seeding of tumor cells from the primary
mor.) Livers were seeded later, in that 4T1 cells were first
tected at day 21 and incidence did not reach 100% until day
. Seven mice were necropsied at day 31; all had visible lung
dules (43 ±7; mean ±SEM) and 5 had visible liver nodules
0 ±0.8). In a second experiment, lungs were again seeded
rly (66% incidence by day 9) and the lung tumor burden
creased on subsequent assay days. Clonogenic 4T1 cells could
detected in the liver within days after detection in the lungs.
n mice from the second experiment were sacrificed for ne
opsy between days 34 and 40. All 10 had visible nodules on
e surface of the lungs (49 ±6.5) and 5 mice had a visible
dule on the surface of the liver.
Clonogenic tumor cells were recovered from both the blood
7 of 82 mice) and lymph nodes (31 of 59 mice) of mice
aring line 66cl4 tumors in a mammary gland, x2 analysis
ggests that line 4T1 more often metastasized hematogenously
an did line 66cl4 (P < 0.02) and that line 66cl4 more often
etastasized via the draining lymph node than did line 4T1 (P
0.01). Fig. IB depicts 1 of 2 experiments in which the
quential spread of 66cl4 from a mammary fatpad was moni
ed. Clonogenic cells were detected in lungs of 66cl4 tumor
aring mice on day 14 and all mice had detectable tumor cells
the lungs by day 28. After day 28, expansion of the tumor
l population in the lung proceeded at an exponential rate
th an apparent doubling time of 39 h. In addition, clonogenic
ls were recovered from livers on day 35 in this experiment,
t all 8 mice sacrificed for necropsy at day 50 had visible
dules in the lung (26 ±3) but not liver nodules. Metastatic
The nonmetastatic line 67NR appeared to be unable to
intravasate. Clonogenic cells were not recovered from any of
the tissues (blood, lymph nodes, lungs, or liver) sampled from
a total of 44 mice over a period of 7 weeks for 2 experiments,
with one exception in which 3 clonogenic cells were recovered
from a draining lymph node of one animal on day 7 in one
experiment (Fig. 2A). Livers were dissociated and plated in
selective media in one experiment (no clonogenic cells were
detected) but not in the other. None of the mice sacrificed at
days 49-50 for necropsy in the 2 experiments had visible
métastasesor detectable clonogenic cells in any organ. The size
I0e A. 4T1 I0. B. 66cl4
co
10s
io2
IO1
IO»
io*
10s
IO1
IO'
I
I
I
I
I
t*t i -7°
DAYS POST INJECTION
Fig. 1. Recovery of clonogenic tumor cells from mice bearing metastatic
primary tumors. A, mice were given injections of 1 x 10s 4T1 tumor cells in the
mammary fatpad. At multiple intervals, groups of mice were bled and then
sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node
in the mammary fatpad was removed through day 19, after which the size of the
tumor made it difficult to find the enveloped lymph node. The draining brachial
lymph nodes were collected throughout the experiment. Lungs were digested
whether or not métastaseswere observed. B, mice were given injections of 1 x
10s 66cl4 tumor cells in the mammary fatpad. At multiple intervals, groups of
mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed.
The draining lymph node in the mammary fatpad was removed through day 17,
after which the size of the tumor made it difficult to find the enveloped lymph
node. The draining brachial lymph nodes were collected throughout the experi
ments. The tissues were processed as detailed in "Materials and Methods." The
data were plotted as geometric means for each tissue. Symbols, which tissues were

21. mg/ml, in a final volume of 5 ml was added. After a 15-min incubation
t 37°C,5 ml of DME-10 were added and samples were mechanically
ispersed for 5 sequential 1-min periods. Between bursts, a portion of
he cell suspension was removed and an equivalent volume of DME-10
dded (10).
All cell suspensions were plated in DME-10 containing the appro
riate selective drug and incubated for 10 to 14 days in 10% CCh-air
mosphere at 37°C.The colonies were fixed with Carnoy's solution,
ained with crystal violet, and counted, and total colony-forming cells
er organ calculated.
RESULTS
Sequential dissemination of tumor cells from a mammary
atpad was evaluated using both metastatic (4T1 and 66cl4; Fig.
) and nonmetastatic (67NR, 168FARN, and 4TO7; Fig. 2)
ubpopulations of a mouse mammary tumor. Line 4T1 ap
eared to metastasize primarily, but not exclusively, by a hem-
togenous route because clonogenic tumor cells were recovered
ignificantly more frequently (P < 0.001, x2 analysis) from the
lood (38 of 58 mice) than from draining lymph nodes (12 of
8 mice). Figure A depicts 1 of 2 experiments in which the
equential spread of 4T1 from a mammary fatpad was moni
ored. Lungs were seeded early (clonogenic 4T1 cells were first
etected at day 7, and 5 of 5 mice had clonogeneic 4T1 cells in
he lung by day 14) and the lung tumor burden increased on
ubsequent assay days with an apparent doubling time of 30 h.
The apparent doubling time is an underestimate because it not
nly measures the growth rate of occult métastasesbut also
eflects continuous seeding of tumor cells from the primary
umor.) Livers were seeded later, in that 4T1 cells were first
etected at day 21 and incidence did not reach 100% until day
8. Seven mice were necropsied at day 31; all had visible lung
odules (43 ±7; mean ±SEM) and 5 had visible liver nodules
2.0 ±0.8). In a second experiment, lungs were again seeded
arly (66% incidence by day 9) and the lung tumor burden
ncreased on subsequent assay days. Clonogenic 4T1 cells could
e detected in the liver within days after detection in the lungs.
en mice from the second experiment were sacrificed for ne
ropsy between days 34 and 40. All 10 had visible nodules on
he surface of the lungs (49 ±6.5) and 5 mice had a visible
odule on the surface of the liver.
Clonogenic tumor cells were recovered from both the blood
27 of 82 mice) and lymph nodes (31 of 59 mice) of mice
earing line 66cl4 tumors in a mammary gland, x2 analysis
uggests that line 4T1 more often metastasized hematogenously
han did line 66cl4 (P < 0.02) and that line 66cl4 more often
metastasized via the draining lymph node than did line 4T1 (P
0.01). Fig. IB depicts 1 of 2 experiments in which the
equential spread of 66cl4 from a mammary fatpad was moni
ored. Clonogenic cells were detected in lungs of 66cl4 tumor
earing mice on day 14 and all mice had detectable tumor cells
n the lungs by day 28. After day 28, expansion of the tumor
ell population in the lung proceeded at an exponential rate
ith an apparent doubling time of 39 h. In addition, clonogenic
ells were recovered from livers on day 35 in this experiment,
All 7 had visible nodules in the lungs (6 ±2); no liver nodules
were found.
The nonmetastatic line 67NR appeared to be unable to
intravasate. Clonogenic cells were not recovered from any of
the tissues (blood, lymph nodes, lungs, or liver) sampled from
a total of 44 mice over a period of 7 weeks for 2 experiments,
with one exception in which 3 clonogenic cells were recovered
from a draining lymph node of one animal on day 7 in one
experiment (Fig. 2A). Livers were dissociated and plated in
selective media in one experiment (no clonogenic cells were
detected) but not in the other. None of the mice sacrificed at
days 49-50 for necropsy in the 2 experiments had visible
métastasesor detectable clonogenic cells in any organ. The size
I0e A. 4T1 I0. B. 66cl4
co
10s
io2
IO1
IO»
io*
10s
IO1
IO'
I
I
I
I
I
t*t i -7°
DAYS POST INJECTION
Fig. 1. Recovery of clonogenic tumor cells from mice bearing metastatic
primary tumors. A, mice were given injections of 1 x 10s 4T1 tumor cells in the
mammary fatpad. At multiple intervals, groups of mice were bled and then
sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node
in the mammary fatpad was removed through day 19, after which the size of the
tumor made it difficult to find the enveloped lymph node. The draining brachial
lymph nodes were collected throughout the experiment. Lungs were digested
whether or not métastaseswere observed. B, mice were given injections of 1 x
10s 66cl4 tumor cells in the mammary fatpad. At multiple intervals, groups of
mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed.
The draining lymph node in the mammary fatpad was removed through day 17,
after which the size of the tumor made it difficult to find the enveloped lymph
ž Line 66cl4 recover
clonogenic tumor
cells from both blood
(27 of 82) and lymph
nodes (31 of 59).
ž metastasize more via
the draining lymph
node and less
hematogenously than
line 4T1.
ž After necropsy, all
had lung nodules but
no liver nodules.

22. SELECTIVE STEPS OF METASTASIS
coJ
<fLU0oO
io*0§^^.0
IOi:oIIA.
67NR_i/vi
i 'i iiZ
IO 3050<
.»«.»..a iiUJ
1 i i !
B. 168FARN
io2
IO1
,Ãil
u
ioV
I01
Kf
C. 4TO7
V
IO
.«.,.f 30
t
so IO 30
•¿ rmt
DAYS POST INJECTION
Fig. 2. Recovery of clonogenic tumor cells from mice bearing nonmetastatic primary tumors. A, mice were given injections of 1 x 10* 67NR tumor cells in the
mammary fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes and lungs removed. B, mice were given injections of 1 x 10*
I68FARN tumor cells in the mammary fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes and lungs were removed. The
draining lymph node in the mammary fatpad was removed through day 21, after which the size of primary tumor made it difficult to find the lymph node. The
draining brachial lymph nodes were collected throughout the experiment except on day 50. C, mice were given injections of 1 x IO54TO7 tumor cells in the mammary
fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node in the mammary
fatpad was removed through day 19, after which the size of the primary tumor made it difficult to find the lymph node. The draining brachial lymph nodes were
collected throughout the experiment. The tissues were processed as detailed in "Materials and Methods." The data were expressed as geometric means for each tissue.
The symbols indicate which tissues were sampled and the day on which sampling was done: .. all tissues for that day; *, blood and lymph nodes only.
of the primary 67NR tumors at the time of necropsy (6.6 ±1.5
g and 11.2 ±4.6 g in the 2 experiments) far exceeded the size
of either 66cl4 (2.6 ±0.3 g and 2.3 ±0.8 g at necropsy) or 4T1
(1.9 ±0.2 g and 1.7 ±0.5 g at necropsy) primary tumors, when
metastatic nodules were detectable in animals with the latter 2
tumor lines.
The nonmetastatic line 168FARN cells spread through the
lymphatics, since clonogenic cells were recovered from draining
lymph nodes in the mammary gland (Fig. 2H). In a second
100
80
LEGEND:
4TO7 ' •¿
66cl4 ' »
4T1 * *
67NR «• »
IMFARN 0--0
ž The nonmetastatic line
67NR appear unable to
intravasate.
ž Clonogenic cells not
recovered from any
(blood, lymph nodes,
lungs, liver) samples
(only 3 from a draining
lymph node on day 7).
ž None of the necropsied
mice hand visible
metastasis.
ž Size of primary tumor
bigger than 66cl$ or
4T1.

23. SELECTIVE STEPS OF METASTASIS
coJ
<fLU0oO
io*0§^^.0
IOi:oIIA.
67NR_i/vi
i 'i iiZ
IO 3050<
.»«.»..a iiUJ
1 i i !
B. 168FARN
io2
IO1
,Ãil
u
ioV
I01
Kf
C. 4TO7
V
IO
.«.,.f 30
t
so IO 30
•¿ rmt
DAYS POST INJECTION
Fig. 2. Recovery of clonogenic tumor cells from mice bearing nonmetastatic primary tumors. A, mice were given injections of 1 x 10* 67NR tumor cells in the
mammary fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes and lungs removed. B, mice were given injections of 1 x 10*
I68FARN tumor cells in the mammary fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes and lungs were removed. The
draining lymph node in the mammary fatpad was removed through day 21, after which the size of primary tumor made it difficult to find the lymph node. The
draining brachial lymph nodes were collected throughout the experiment except on day 50. C, mice were given injections of 1 x IO54TO7 tumor cells in the mammary
fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node in the mammary
fatpad was removed through day 19, after which the size of the primary tumor made it difficult to find the lymph node. The draining brachial lymph nodes were
collected throughout the experiment. The tissues were processed as detailed in "Materials and Methods." The data were expressed as geometric means for each tissue.
The symbols indicate which tissues were sampled and the day on which sampling was done: .. all tissues for that day; *, blood and lymph nodes only.
of the primary 67NR tumors at the time of necropsy (6.6 ±1.5
g and 11.2 ±4.6 g in the 2 experiments) far exceeded the size
of either 66cl4 (2.6 ±0.3 g and 2.3 ±0.8 g at necropsy) or 4T1
(1.9 ±0.2 g and 1.7 ±0.5 g at necropsy) primary tumors, when
metastatic nodules were detectable in animals with the latter 2
tumor lines.
The nonmetastatic line 168FARN cells spread through the
lymphatics, since clonogenic cells were recovered from draining
lymph nodes in the mammary gland (Fig. 2H). In a second
100
80
LEGEND:
4TO7 ' •¿
66cl4 ' »
4T1 * *
67NR «• »
IMFARN 0--0
ž Line 168FARN spread
through lymphatics.
(clonogenic cells
recovered from
draining lymph
nodes).
ž No clonogenic cells
recovered in blood.
ž No visible nodules in
lungs.
ž Large primary tumors.
SELECTIVE STEPS OF METASTASIS
coJ
<fLU0oO
io*0§^^.0
IOi:oIIA.
67NR_i/vi
i 'i iiZ
IO 3050<
.»«.»..a iiUJ
1 i i !
B. 168FARN
io2
IO1
,Ãil
u
ioV
I01
Kf
C. 4TO7
V
IO
.«.,.f 30
t
so IO 30
•¿ rmt
DAYS POST INJECTION
Fig. 2. Recovery of clonogenic tumor cells from mice bearing nonmetastatic primary tumors. A, mice were given injections of 1 x 10* 67NR tumor cells in the
mammary fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes and lungs removed. B, mice were given injections of 1 x 10*
I68FARN tumor cells in the mammary fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes and lungs were removed. The
draining lymph node in the mammary fatpad was removed through day 21, after which the size of primary tumor made it difficult to find the lymph node. The
draining brachial lymph nodes were collected throughout the experiment except on day 50. C, mice were given injections of 1 x IO54TO7 tumor cells in the mammary
fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node in the mammary
fatpad was removed through day 19, after which the size of the primary tumor made it difficult to find the lymph node. The draining brachial lymph nodes were
collected throughout the experiment. The tissues were processed as detailed in "Materials and Methods." The data were expressed as geometric means for each tissue.
The symbols indicate which tissues were sampled and the day on which sampling was done: .. all tissues for that day; *, blood and lymph nodes only.
of the primary 67NR tumors at the time of necropsy (6.6 ±1.5
g and 11.2 ±4.6 g in the 2 experiments) far exceeded the size
of either 66cl4 (2.6 ±0.3 g and 2.3 ±0.8 g at necropsy) or 4T1
(1.9 ±0.2 g and 1.7 ±0.5 g at necropsy) primary tumors, when
metastatic nodules were detectable in animals with the latter 2
tumor lines.
The nonmetastatic line 168FARN cells spread through the
lymphatics, since clonogenic cells were recovered from draining
100
80
LEGEND:
4TO7 ' •¿
66cl4 ' »
4T1 * *
67NR «• »
IMFARN 0--0

24. SELECTIVE STEPS OF METASTASIS
coJ
<fLU0oO
io*0§^^.0
IOi:oIIA.
67NR_i/vi
i 'i iiZ
IO 3050<
.»«.»..a iiUJ
1 i i !
B. 168FARN
io2
IO1
,Ãil
u
ioV
I01
Kf
C. 4TO7
V
IO
.«.,.f 30
t
so IO 30
•¿ rmt
DAYS POST INJECTION
Fig. 2. Recovery of clonogenic tumor cells from mice bearing nonmetastatic primary tumors. A, mice were given injections of 1 x 10* 67NR tumor cells in the
mammary fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes and lungs removed. B, mice were given injections of 1 x 10*
I68FARN tumor cells in the mammary fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes and lungs were removed. The
draining lymph node in the mammary fatpad was removed through day 21, after which the size of primary tumor made it difficult to find the lymph node. The
draining brachial lymph nodes were collected throughout the experiment except on day 50. C, mice were given injections of 1 x IO54TO7 tumor cells in the mammary
fatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node in the mammary
fatpad was removed through day 19, after which the size of the primary tumor made it difficult to find the lymph node. The draining brachial lymph nodes were
collected throughout the experiment. The tissues were processed as detailed in "Materials and Methods." The data were expressed as geometric means for each tissue.
The symbols indicate which tissues were sampled and the day on which sampling was done: .. all tissues for that day; *, blood and lymph nodes only.
of the primary 67NR tumors at the time of necropsy (6.6 ±1.5
g and 11.2 ±4.6 g in the 2 experiments) far exceeded the size
of either 66cl4 (2.6 ±0.3 g and 2.3 ±0.8 g at necropsy) or 4T1
(1.9 ±0.2 g and 1.7 ±0.5 g at necropsy) primary tumors, when
metastatic nodules were detectable in animals with the latter 2
tumor lines.
The nonmetastatic line 168FARN cells spread through the
lymphatics, since clonogenic cells were recovered from draining
100
80
LEGEND:
4TO7 ' •¿
66cl4 ' »
4T1 * *
67NR «• »
IMFARN 0--0
ž 4TO7 able to complete
all steps except the
final one.
ž Spread via the blood to
lungs and occasionally
to livers, but did not
establish progressively
growing metastatic
nodules.
ž Necropsy, visible
nodules absent but
clonogenic cells were
recovered in lungs.

25. ž Aggregation and initial arrest of [125I]IUrd-
labeled cells (identify tumor cells) similar for
metastatic and nonmetastatic clones, but
clearance was much more rapid for
nonmetastatic clone than for a metastatic
one.

26. ž Intravasation appears to be an important
selective step. (67NR appears unable to
leave the primary site).
ž cells more highly efficient at colonizing lungs
following iv injection were similar in abilities
to spontaneously metastasize from im
tumors.

27. ž We have established an experimental model
that allow the validation of many phenotypes
implicated in metastasis.
ž has value in analysis of tumor cell
interactions.
¢ Demonstrate clonal dominance and interactions
affecting responses to chemotherapeutic drugs.
¢ Nonmetastatic subpopulations derived from mouse
mammary tumor used in our study may metastasize in
the presence of some metastatic subpopulations.

28. ž Central aim in the study of metastasis:
understand the nature and distinct genetic
and epigenetic changes that program these
individual steps.

29. ž Previous attempts:
ž Microarray analyses
¡ Generated gene expression profiles that are
predictive of metastasis.
¡ Powerful for producing fingerprints, but hard to
elucidate specific contributions of each genes.
ž Experimental animal models
¡ Comparing melanoma cells and their metastatic
derivatives, found RhoC important for pulmonary
metastasis.
¡ Most of these models rely on introduction of
tumor cells directly into systemic circulation.

30. ž Injected cells from four
lines into the mammary
glands of BALB/c mice.
ž Group X: intravasation.
Altered in 168FARN, 4TO7,
and 4T1, but not in 67NR.
ž Group Y: extravasation.
Altered in 4TO7 and 4T1,
but not in 67NR and
168FARN.
ž Group Z: growth into
secondary tumors. Altered
in 4T1, but not in 4TO7,
67NR and 168FARN.

31. ž Twist stood out.
ž 2nd most strongly upregulated in Group X.
ž Known functions as a master regulator of
embryonic morphogenesis.

32. Twist is not required for tumor formation
Twist increases the number of metastatic
nodules
Twist promotes
epithelial-mesenchymal transition
(EMT) and cell migration

33. ž Using gene expression array analysis,
identified genes involved in metastasis. Twist
and Twist-induced EMT are important
components of tumor metastasis.

34. ž E-Cadherin – important calcium dependent
cell to cell adhesion molecule.
ž Research has shown the importance of E-
Cadherin when it comes to suppressing a
tumor.
ž Loss of E-Cadherin has been shown to
increase metastatic effect in cancer cell
lines.
ž ϒ-­‐Linolenic
Acid
has
been
shown
to
improve
expression
of
E-­‐Cadherin.

37. Drawbacks:
ž These results come from various types of cancers
where most of the breast cancer cell lines did
not seem to have produced an effect on E-
Cadherin when given GLA.
ž Used MCF-7, MDA-MB-231, and ZR-751.
We wish to use our cell lines as well as assess the
effect in an in vivo model in addition to in vivo.
ž Western Blotting – (E-Cadherin)
ž Immunostaining – (E-Cadherin)
ž Aggregation Assays
ž Invasions Assay

38. ž ϒ-­‐Linolenic Acid (GLA) has potential for
therapeutic use in our breast cancer mouse
model

39. ž Metastatic Breast Cancer. (2015). National Breast Cancer Foundation. Retrieved from
http://www.nationalbreastcancer.org/metastatic-breast-cancer
ž Metastatic Cancer. (2013, March 10). National Cancer Institute. Retrieved from
http://www.cancer.gov/about-cancer/what-is-cancer/metastatic-fact-sheet
ž How cancer can spread. (2014, October 29). Cancer Research UK. Retrieved from
http://www.cancerresearchuk.org/about-cancer/what-is-cancer/how-cancer-can-spread
ž Hayes, D.F. (2015, May 5). Patient Information: Treatment of metastatic breast cancer (beyond the
basics). UpToDate. Retrieved from
http://www.uptodate.com/contents/treatment-of-metastatic-breast-cancer-beyond-the-basics
ž Russel, T. (2013, November 26). Understanding Breast Cancer through innovative laboratory
models. Aegis. Retrieved from
http://aegiscreative.com/blog/creating-laboratory-models-help-us-understand-breast-cancer/
ž Mouse Models of Breast Cancer Metastasis. (n.d.) Retrieved May 2nd, 2016 from the Metastatic
Breast Cancer Wiki:
https://en.wikipedia.org/wiki/
Mouse_models_of_breast_cancer_metastasis#Genetically_engineered_mice_to_study_metastasis
ž Jenkins, D. (2005, April 8). Bioluminescent human breast cancer cell lines that permit rapid and
sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient
mice. Biomed Central. Retrieved from
http://breast-cancer-research.biomedcentral.com/articles/10.1186/bcr1026
ž Jiang, W, et. al. Regulation of the expression of E-Cadherin on Human Cancer Cells by Linolenic
Acid. Cancer Research. 55; 5043-5048, 1995.
ž Aslakson C; Miller, F. Selective Events in the Metastatic Process Defined by Analysis of
Dissemination of Subpopulations of a Mouse Mammary Tumor. Cancer Research 52, 1399-1405,
March 15, 1992
ž Yang J et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor
metastasis. Cell. 2004 June 25; 117(7): 927-39.