Chen Yan Vivek Shukla

May 9th, 2016

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

� Classified as Stage 4 Cancer � 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 � Creates complications in those regions

� Brain 1.  Head aches 2.  Vision disturbances 3.  Seizures 4.  Behavioral/

Personality changes � Bone 1.  Increased pain 2.  Weaker and more

fracture prone

� Liver 1.  Increased digestive

enzymes 2.  pH imbalance 3.  Itchy skin 4.  Nausea and

vomiting � Lung 1.  Chest Pain 2.  Trouble Breathing

� Utilize circulatory and lymphatic system to travel to different organs.

� Cells growing at an abnormal rate are capable of invading surrounding healthy tissues and spreading.

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

� HER2 targeted therapy– Human Epidermal Growth Factor – drugs to decrease expression of this factor. (Herceptin)

� Radiation Therapy � Surgical removal of tumorous regions � Endocrine Therapy: Drugs that target

hormone receptors and control levels of progesterone and estrogen. (Aromatase Inhibitors).

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)”

� 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.

� MMTV-PyMT: Commonly used mouse model for metastatic breast cancer where a mouse mammary tumor virus is used to induce cancer.

� Genetically Altered Mice: (Knockout Mice)

� Our cell line has to be first be established as a working cell line by growing it in culture.

� Media content: 1.  DMEM 2.  5% Fetal Calf Serum and 5% Newborn Calf

Serum 3.  NEAA 4.  2 mM L-glutamine 5.  Streptomycin

� Strain: BALB/c Mice � Injection of a known titer of viable cancer

cells into the fat pat of the mouse model. � Injected into the Fat Pad

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

� Metastatic process is a sequence of steps (invasion, intravasation, transport, arrest, extravasation, and growth).

�  Inefficient. � Both random and selective events are

responsible for this inefficiency. �  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.

�  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.

�  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.

�  If host immune functions are important for the elimination of potentially metastatic cells, the selective events best targets for therapeutic intervention.

� 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.

� Bioassays used to estimate # of tumor cells, but insensitive and not quantitative. ¡  Require multiple animals to test in 1 tumor-

bearing animal.

� Suzuki. Enzymatically dissociated lungs with protease, plated the cell onto irradiated feeder layers. Colonies counted.

� Background lawn made it impossible.

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

�  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. �  4T1 spontaneously metastasizes to both lung and liver

(formation of visible nodules in these organs) �  66cl4 spontaneously metastasizes to lung. �  67NR, 168FARN, 4TO7 highly tumorigenic but rarely

metastasize spontaneously. �  Tumor cells injected into mammary fatpad.

SELECTIVE STEPS OF METASTASIS

a portion of the cell suspension was removed and an equal volume ofDME containing 10% calf serum (DME-10) added (10).Livers were minced and an enzyme solution containing collagenase

type I (Sigma) and hyaluronidase (Sigma), each at a concentration of 1mg/ml, in a final volume of 5 ml was added. After a 15-min incubationat 37°C,5 ml of DME-10 were added and samples were mechanicallydispersed for 5 sequential 1-min periods. Between bursts, a portion ofthe cell suspension was removed and an equivalent volume of DME-10added (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-airatmosphere at 37°C.The colonies were fixed with Carnoy's solution,stained with crystal violet, and counted, and total colony-forming cellsper organ calculated.

RESULTS

Sequential dissemination of tumor cells from a mammaryfatpad 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 appeared to metastasize primarily, but not exclusively, by a hem-atogenous route because clonogenic tumor cells were recoveredsignificantly more frequently (P < 0.001, x2 analysis) from theblood (38 of 58 mice) than from draining lymph nodes (12 of58 mice). Figure \A depicts 1 of 2 experiments in which thesequential spread of 4T1 from a mammary fatpad was monitored. Lungs were seeded early (clonogenic 4T1 cells were firstdetected at day 7, and 5 of 5 mice had clonogeneic 4T1 cells inthe lung by day 14) and the lung tumor burden increased onsubsequent assay days with an apparent doubling time of 30 h.(The apparent doubling time is an underestimate because it notonly measures the growth rate of occult métastasesbut alsoreflects continuous seeding of tumor cells from the primarytumor.) Livers were seeded later, in that 4T1 cells were firstdetected at day 21 and incidence did not reach 100% until day28. Seven mice were necropsied at day 31; all had visible lungnodules (43 ±7; mean ±SEM) and 5 had visible liver nodules(2.0 ±0.8). In a second experiment, lungs were again seededearly (66% incidence by day 9) and the lung tumor burdenincreased on subsequent assay days. Clonogenic 4T1 cells couldbe detected in the liver within days after detection in the lungs.Ten mice from the second experiment were sacrificed for necropsy between days 34 and 40. All 10 had visible nodules onthe surface of the lungs (49 ±6.5) and 5 mice had a visiblenodule 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 micebearing line 66cl4 tumors in a mammary gland, x2 analysissuggests that line 4T1 more often metastasized hematogenouslythan did line 66cl4 (P < 0.02) and that line 66cl4 more oftenmetastasized via the draining lymph node than did line 4T1 (P< 0.01). Fig. IB depicts 1 of 2 experiments in which thesequential spread of 66cl4 from a mammary fatpad was monitored. Clonogenic cells were detected in lungs of 66cl4 tumorbearing mice on day 14 and all mice had detectable tumor cellsin the lungs by day 28. After day 28, expansion of the tumorcell population in the lung proceeded at an exponential ratewith an apparent doubling time of 39 h. In addition, clonogeniccells were recovered from livers on day 35 in this experiment,but all 8 mice sacrificed for necropsy at day 50 had visiblenodules in the lung (26 ±3) but not liver nodules. Metastaticnodules have never been observed in livers of mice with 66cl4primary tumors. In a second experiment with 66cl4, clonogenic

cells were again detected in lungs on day 14, and the apparentdoubling time in the lungs after day 28 was 36 h. Seven micefrom this experiment were sacrificed for necropsy at day 50.All 7 had visible nodules in the lungs (6 ±2); no liver noduleswere found.The nonmetastatic line 67NR appeared to be unable to

intravasate. Clonogenic cells were not recovered from any ofthe tissues (blood, lymph nodes, lungs, or liver) sampled froma total of 44 mice over a period of 7 weeks for 2 experiments,with one exception in which 3 clonogenic cells were recoveredfrom a draining lymph node of one animal on day 7 in oneexperiment (Fig. 2A). Livers were dissociated and plated inselective media in one experiment (no clonogenic cells weredetected) but not in the other. None of the mice sacrificed atdays 49-50 for necropsy in the 2 experiments had visiblemétastasesor detectable clonogenic cells in any organ. The size

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Fig. 1. Recovery of clonogenic tumor cells from mice bearing metastaticprimary tumors. A, mice were given injections of 1 x 10s 4T1 tumor cells in themammary fatpad. At multiple intervals, groups of mice were bled and thensacrificed, and lymph nodes, lungs, and livers removed. The draining lymph nodein the mammary fatpad was removed through day 19, after which the size of thetumor made it difficult to find the enveloped lymph node. The draining brachiallymph nodes were collected throughout the experiment. Lungs were digestedwhether or not métastaseswere observed. B, mice were given injections of 1 x10s 66cl4 tumor cells in the mammary fatpad. At multiple intervals, groups ofmice 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 lymphnode. The draining brachial lymph nodes were collected throughout the experiments. The tissues were processed as detailed in "Materials and Methods." Thedata were plotted as geometric means for each tissue. Symbols, which tissues weresampled and the day on which sampling was done: t, all tissue for that day; *,blood and lymph nodes only.

1400

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�  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)).

�  Lungs seeded early, liver seeded later.

�  After necropsy, all had lung nodules, 5 of 7 liver nodules

SELECTIVE STEPS OF METASTASIS

a portion of the cell suspension was removed and an equal volume ofDME containing 10% calf serum (DME-10) added (10).Livers were minced and an enzyme solution containing collagenase

type I (Sigma) and hyaluronidase (Sigma), each at a concentration of 1mg/ml, in a final volume of 5 ml was added. After a 15-min incubationat 37°C,5 ml of DME-10 were added and samples were mechanicallydispersed for 5 sequential 1-min periods. Between bursts, a portion ofthe cell suspension was removed and an equivalent volume of DME-10added (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-airatmosphere at 37°C.The colonies were fixed with Carnoy's solution,stained with crystal violet, and counted, and total colony-forming cellsper organ calculated.

RESULTS

Sequential dissemination of tumor cells from a mammaryfatpad 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 appeared to metastasize primarily, but not exclusively, by a hem-atogenous route because clonogenic tumor cells were recoveredsignificantly more frequently (P < 0.001, x2 analysis) from theblood (38 of 58 mice) than from draining lymph nodes (12 of58 mice). Figure \A depicts 1 of 2 experiments in which thesequential spread of 4T1 from a mammary fatpad was monitored. Lungs were seeded early (clonogenic 4T1 cells were firstdetected at day 7, and 5 of 5 mice had clonogeneic 4T1 cells inthe lung by day 14) and the lung tumor burden increased onsubsequent assay days with an apparent doubling time of 30 h.(The apparent doubling time is an underestimate because it notonly measures the growth rate of occult métastasesbut alsoreflects continuous seeding of tumor cells from the primarytumor.) Livers were seeded later, in that 4T1 cells were firstdetected at day 21 and incidence did not reach 100% until day28. Seven mice were necropsied at day 31; all had visible lungnodules (43 ±7; mean ±SEM) and 5 had visible liver nodules(2.0 ±0.8). In a second experiment, lungs were again seededearly (66% incidence by day 9) and the lung tumor burdenincreased on subsequent assay days. Clonogenic 4T1 cells couldbe detected in the liver within days after detection in the lungs.Ten mice from the second experiment were sacrificed for necropsy between days 34 and 40. All 10 had visible nodules onthe surface of the lungs (49 ±6.5) and 5 mice had a visiblenodule 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 micebearing line 66cl4 tumors in a mammary gland, x2 analysissuggests that line 4T1 more often metastasized hematogenouslythan did line 66cl4 (P < 0.02) and that line 66cl4 more oftenmetastasized via the draining lymph node than did line 4T1 (P< 0.01). Fig. IB depicts 1 of 2 experiments in which thesequential spread of 66cl4 from a mammary fatpad was monitored. Clonogenic cells were detected in lungs of 66cl4 tumorbearing mice on day 14 and all mice had detectable tumor cellsin the lungs by day 28. After day 28, expansion of the tumorcell population in the lung proceeded at an exponential ratewith an apparent doubling time of 39 h. In addition, clonogeniccells were recovered from livers on day 35 in this experiment,but all 8 mice sacrificed for necropsy at day 50 had visiblenodules in the lung (26 ±3) but not liver nodules. Metastaticnodules have never been observed in livers of mice with 66cl4primary tumors. In a second experiment with 66cl4, clonogenic

cells were again detected in lungs on day 14, and the apparentdoubling time in the lungs after day 28 was 36 h. Seven micefrom this experiment were sacrificed for necropsy at day 50.All 7 had visible nodules in the lungs (6 ±2); no liver noduleswere found.The nonmetastatic line 67NR appeared to be unable to

intravasate. Clonogenic cells were not recovered from any ofthe tissues (blood, lymph nodes, lungs, or liver) sampled froma total of 44 mice over a period of 7 weeks for 2 experiments,with one exception in which 3 clonogenic cells were recoveredfrom a draining lymph node of one animal on day 7 in oneexperiment (Fig. 2A). Livers were dissociated and plated inselective media in one experiment (no clonogenic cells weredetected) but not in the other. None of the mice sacrificed atdays 49-50 for necropsy in the 2 experiments had visiblemétastasesor detectable clonogenic cells in any organ. The size

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Fig. 1. Recovery of clonogenic tumor cells from mice bearing metastaticprimary tumors. A, mice were given injections of 1 x 10s 4T1 tumor cells in themammary fatpad. At multiple intervals, groups of mice were bled and thensacrificed, and lymph nodes, lungs, and livers removed. The draining lymph nodein the mammary fatpad was removed through day 19, after which the size of thetumor made it difficult to find the enveloped lymph node. The draining brachiallymph nodes were collected throughout the experiment. Lungs were digestedwhether or not métastaseswere observed. B, mice were given injections of 1 x10s 66cl4 tumor cells in the mammary fatpad. At multiple intervals, groups ofmice 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 lymphnode. The draining brachial lymph nodes were collected throughout the experiments. The tissues were processed as detailed in "Materials and Methods." Thedata were plotted as geometric means for each tissue. Symbols, which tissues weresampled and the day on which sampling was done: t, all tissue for that day; *,blood and lymph nodes only.

1400

on May 3, 2016. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

SELECTIVE STEPS OF METASTASIS

a portion of the cell suspension was removed and an equal volume ofDME containing 10% calf serum (DME-10) added (10).Livers were minced and an enzyme solution containing collagenase

type I (Sigma) and hyaluronidase (Sigma), each at a concentration of 1mg/ml, in a final volume of 5 ml was added. After a 15-min incubationat 37°C,5 ml of DME-10 were added and samples were mechanicallydispersed for 5 sequential 1-min periods. Between bursts, a portion ofthe cell suspension was removed and an equivalent volume of DME-10added (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-airatmosphere at 37°C.The colonies were fixed with Carnoy's solution,stained with crystal violet, and counted, and total colony-forming cellsper organ calculated.

RESULTS

Sequential dissemination of tumor cells from a mammaryfatpad 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 appeared to metastasize primarily, but not exclusively, by a hem-atogenous route because clonogenic tumor cells were recoveredsignificantly more frequently (P < 0.001, x2 analysis) from theblood (38 of 58 mice) than from draining lymph nodes (12 of58 mice). Figure \A depicts 1 of 2 experiments in which thesequential spread of 4T1 from a mammary fatpad was monitored. Lungs were seeded early (clonogenic 4T1 cells were firstdetected at day 7, and 5 of 5 mice had clonogeneic 4T1 cells inthe lung by day 14) and the lung tumor burden increased onsubsequent assay days with an apparent doubling time of 30 h.(The apparent doubling time is an underestimate because it notonly measures the growth rate of occult métastasesbut alsoreflects continuous seeding of tumor cells from the primarytumor.) Livers were seeded later, in that 4T1 cells were firstdetected at day 21 and incidence did not reach 100% until day28. Seven mice were necropsied at day 31; all had visible lungnodules (43 ±7; mean ±SEM) and 5 had visible liver nodules(2.0 ±0.8). In a second experiment, lungs were again seededearly (66% incidence by day 9) and the lung tumor burdenincreased on subsequent assay days. Clonogenic 4T1 cells couldbe detected in the liver within days after detection in the lungs.Ten mice from the second experiment were sacrificed for necropsy between days 34 and 40. All 10 had visible nodules onthe surface of the lungs (49 ±6.5) and 5 mice had a visiblenodule 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 micebearing line 66cl4 tumors in a mammary gland, x2 analysissuggests that line 4T1 more often metastasized hematogenouslythan did line 66cl4 (P < 0.02) and that line 66cl4 more oftenmetastasized via the draining lymph node than did line 4T1 (P< 0.01). Fig. IB depicts 1 of 2 experiments in which thesequential spread of 66cl4 from a mammary fatpad was monitored. Clonogenic cells were detected in lungs of 66cl4 tumorbearing mice on day 14 and all mice had detectable tumor cellsin the lungs by day 28. After day 28, expansion of the tumorcell population in the lung proceeded at an exponential ratewith an apparent doubling time of 39 h. In addition, clonogeniccells were recovered from livers on day 35 in this experiment,but all 8 mice sacrificed for necropsy at day 50 had visiblenodules in the lung (26 ±3) but not liver nodules. Metastaticnodules have never been observed in livers of mice with 66cl4primary tumors. In a second experiment with 66cl4, clonogenic

cells were again detected in lungs on day 14, and the apparentdoubling time in the lungs after day 28 was 36 h. Seven micefrom this experiment were sacrificed for necropsy at day 50.All 7 had visible nodules in the lungs (6 ±2); no liver noduleswere found.The nonmetastatic line 67NR appeared to be unable to

intravasate. Clonogenic cells were not recovered from any ofthe tissues (blood, lymph nodes, lungs, or liver) sampled froma total of 44 mice over a period of 7 weeks for 2 experiments,with one exception in which 3 clonogenic cells were recoveredfrom a draining lymph node of one animal on day 7 in oneexperiment (Fig. 2A). Livers were dissociated and plated inselective media in one experiment (no clonogenic cells weredetected) but not in the other. None of the mice sacrificed atdays 49-50 for necropsy in the 2 experiments had visiblemétastasesor detectable clonogenic cells in any organ. The size

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Fig. 1. Recovery of clonogenic tumor cells from mice bearing metastaticprimary tumors. A, mice were given injections of 1 x 10s 4T1 tumor cells in themammary fatpad. At multiple intervals, groups of mice were bled and thensacrificed, and lymph nodes, lungs, and livers removed. The draining lymph nodein the mammary fatpad was removed through day 19, after which the size of thetumor made it difficult to find the enveloped lymph node. The draining brachiallymph nodes were collected throughout the experiment. Lungs were digestedwhether or not métastaseswere observed. B, mice were given injections of 1 x10s 66cl4 tumor cells in the mammary fatpad. At multiple intervals, groups ofmice 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 lymphnode. The draining brachial lymph nodes were collected throughout the experiments. The tissues were processed as detailed in "Materials and Methods." Thedata were plotted as geometric means for each tissue. Symbols, which tissues weresampled and the day on which sampling was done: t, all tissue for that day; *,blood and lymph nodes only.

1400

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� Line 66cl4 recover clonogenic tumor cells from both blood (27 of 82) and lymph nodes (31 of 59).

� metastasize more via the draining lymph node and less hematogenously than line 4T1.

� After necropsy, all had lung nodules but no liver nodules.

SELECTIVE STEPS OF METASTASIS

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DAYS POST INJECTIONFig. 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. Thedraining 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. Thedraining 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 mammaryfatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node in the mammaryfatpad 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 werecollected 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 ±1.5g and 11.2 ±4.6 g in the 2 experiments) far exceeded the sizeof 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, whenmetastatic nodules were detectable in animals with the latter 2tumor lines.The nonmetastatic line 168FARN cells spread through the

lymphatics, since clonogenic cells were recovered from draininglymph nodes in the mammary gland (Fig. 2H). In a secondexperiment, low numbers of clonogenic 168FARN cells wereoccasionally recovered from lungs (1 animal of 6 at necropsyon day 51 had 43 clonogenic cells) and livers (none of the 6animals at necropsy had detectable cells in the liver, but 2 of 5mice on day 21 had 13 and 65 clonogenic cells, and 2 of 5 miceon day 28 had 8 and 15 clonogenic cells in the liver). Althoughclonogenic 168FARN cells could be recovered from draininglymph nodes, clonogenic cells were not recovered in eitherexperiment from the blood of any of the 61 mice bearing line168FARN tumors. Visible nodules were not observed on lungsat necropsy, and clonogenic cells were recovered in only oneanimal as noted above despite the presence of very large primarytumors (9.3 ±1.1 g and 8.6 ±2.2 g at necropsy for the 2experiments).The nonmetastatic line 4TO7 appeared to be able to complete

all steps of metastasis except the final one. Line 4TO7 spreadvia the blood to lungs and occasionally to livers, but did notestablish progressively growing metastatic nodules. Fig. 2Cdepicts one of 2 experiments in which the sequential spread of4TO7 from a mammary fatpad was monitored. Clonogeniccells were recovered from the lungs at day 19. At necropsy,visible nodules were absent but clonogenic 4TO7 cells wererecovered from lungs in 4 of 5 mice (primary tumor weight of1.35 ±0.32 g on day 28) with a geometric mean of 32 clonogeniccells detected.In a second experiment with 4TO7, clonogenic cells were

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Fig. 3. Sensitivity of the mammary tumor lines to mechanical disruption.Single cell suspensions prepared from monolayer culture were subjected to thelung digest method. Following incubation with elastase and collagenase IV for 60min at 4V. medium containing 10% calf serum was added and the cell suspensionswere disrupted by 4 30-s and 3 1-min bursts in the Stomacher lab blender.Beginning with the second 30-s disruption, triplicate samples were diluted andplated in the appropriate selective medium after each manipulation.

on May 3, 2016. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

�  The nonmetastatic line 67NR appear unable to intravasate.

�  Clonogenic cells not recovered from any (blood, lymph nodes, lungs, liver) samples (only 3 from a draining lymph node on day 7).

�  None of the necropsied mice hand visible metastasis.

�  Size of primary tumor bigger than 66cl$ or 4T1.

SELECTIVE STEPS OF METASTASIS

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DAYS POST INJECTIONFig. 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. Thedraining 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. Thedraining 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 mammaryfatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node in the mammaryfatpad 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 werecollected 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 ±1.5g and 11.2 ±4.6 g in the 2 experiments) far exceeded the sizeof 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, whenmetastatic nodules were detectable in animals with the latter 2tumor lines.The nonmetastatic line 168FARN cells spread through the

lymphatics, since clonogenic cells were recovered from draininglymph nodes in the mammary gland (Fig. 2H). In a secondexperiment, low numbers of clonogenic 168FARN cells wereoccasionally recovered from lungs (1 animal of 6 at necropsyon day 51 had 43 clonogenic cells) and livers (none of the 6animals at necropsy had detectable cells in the liver, but 2 of 5mice on day 21 had 13 and 65 clonogenic cells, and 2 of 5 miceon day 28 had 8 and 15 clonogenic cells in the liver). Althoughclonogenic 168FARN cells could be recovered from draininglymph nodes, clonogenic cells were not recovered in eitherexperiment from the blood of any of the 61 mice bearing line168FARN tumors. Visible nodules were not observed on lungsat necropsy, and clonogenic cells were recovered in only oneanimal as noted above despite the presence of very large primarytumors (9.3 ±1.1 g and 8.6 ±2.2 g at necropsy for the 2experiments).The nonmetastatic line 4TO7 appeared to be able to complete

all steps of metastasis except the final one. Line 4TO7 spreadvia the blood to lungs and occasionally to livers, but did notestablish progressively growing metastatic nodules. Fig. 2Cdepicts one of 2 experiments in which the sequential spread of4TO7 from a mammary fatpad was monitored. Clonogeniccells were recovered from the lungs at day 19. At necropsy,visible nodules were absent but clonogenic 4TO7 cells wererecovered from lungs in 4 of 5 mice (primary tumor weight of1.35 ±0.32 g on day 28) with a geometric mean of 32 clonogeniccells detected.In a second experiment with 4TO7, clonogenic cells were

recovered from the lungs on day 7 after tumor cells were1401

100

80

60

40

20

LEGEND:4TO7 ' •¿ 66cl4 ' »4T1 * *67NR «• »IMFARN 0--0

1.0 2.0 30 4.0 5.0

CUMULATIVE TIME INSTOMACHER BLENDER (MINUTES)

Fig. 3. Sensitivity of the mammary tumor lines to mechanical disruption.Single cell suspensions prepared from monolayer culture were subjected to thelung digest method. Following incubation with elastase and collagenase IV for 60min at 4V. medium containing 10% calf serum was added and the cell suspensionswere disrupted by 4 30-s and 3 1-min bursts in the Stomacher lab blender.Beginning with the second 30-s disruption, triplicate samples were diluted andplated in the appropriate selective medium after each manipulation.

on May 3, 2016. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

� Line 168FARN spread through lymphatics. (clonogenic cells recovered from draining lymph nodes).

� No clonogenic cells recovered in blood.

� No visible nodules in lungs.

� Large primary tumors.

SELECTIVE STEPS OF METASTASIS

coJ\<fLU0oO

io*0§^^.0

IOi:oIIA.

67NR_i/vi

i 'i iiZIO 3050<

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B. 168FARN

io2

IO1

,Ã il

u

ioV

I01

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C. 4TO7

VIO.«.,.f 30

tso IO 30•¿ rmt

DAYS POST INJECTIONFig. 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. Thedraining 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. Thedraining 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 mammaryfatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node in the mammaryfatpad 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 werecollected 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 ±1.5g and 11.2 ±4.6 g in the 2 experiments) far exceeded the sizeof 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, whenmetastatic nodules were detectable in animals with the latter 2tumor lines.The nonmetastatic line 168FARN cells spread through the

lymphatics, since clonogenic cells were recovered from draininglymph nodes in the mammary gland (Fig. 2H). In a secondexperiment, low numbers of clonogenic 168FARN cells wereoccasionally recovered from lungs (1 animal of 6 at necropsyon day 51 had 43 clonogenic cells) and livers (none of the 6animals at necropsy had detectable cells in the liver, but 2 of 5mice on day 21 had 13 and 65 clonogenic cells, and 2 of 5 miceon day 28 had 8 and 15 clonogenic cells in the liver). Althoughclonogenic 168FARN cells could be recovered from draininglymph nodes, clonogenic cells were not recovered in eitherexperiment from the blood of any of the 61 mice bearing line168FARN tumors. Visible nodules were not observed on lungsat necropsy, and clonogenic cells were recovered in only oneanimal as noted above despite the presence of very large primarytumors (9.3 ±1.1 g and 8.6 ±2.2 g at necropsy for the 2experiments).The nonmetastatic line 4TO7 appeared to be able to complete

all steps of metastasis except the final one. Line 4TO7 spreadvia the blood to lungs and occasionally to livers, but did notestablish progressively growing metastatic nodules. Fig. 2Cdepicts one of 2 experiments in which the sequential spread of4TO7 from a mammary fatpad was monitored. Clonogeniccells were recovered from the lungs at day 19. At necropsy,visible nodules were absent but clonogenic 4TO7 cells wererecovered from lungs in 4 of 5 mice (primary tumor weight of1.35 ±0.32 g on day 28) with a geometric mean of 32 clonogeniccells detected.In a second experiment with 4TO7, clonogenic cells were

recovered from the lungs on day 7 after tumor cells were1401

100

80

60

40

20

LEGEND:4TO7 ' •¿ 66cl4 ' »4T1 * *67NR «• »IMFARN 0--0

1.0 2.0 30 4.0 5.0

CUMULATIVE TIME INSTOMACHER BLENDER (MINUTES)

Fig. 3. Sensitivity of the mammary tumor lines to mechanical disruption.Single cell suspensions prepared from monolayer culture were subjected to thelung digest method. Following incubation with elastase and collagenase IV for 60min at 4V. medium containing 10% calf serum was added and the cell suspensionswere disrupted by 4 30-s and 3 1-min bursts in the Stomacher lab blender.Beginning with the second 30-s disruption, triplicate samples were diluted andplated in the appropriate selective medium after each manipulation.

on May 3, 2016. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

SELECTIVE STEPS OF METASTASIS

coJ\<fLU0oO

io*0§^^.0

IOi:oIIA.

67NR_i/vi

i 'i iiZIO 3050<

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B. 168FARN

io2

IO1

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u

ioV

I01

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C. 4TO7

VIO.«.,.f 30

tso IO 30•¿ rmt

DAYS POST INJECTIONFig. 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. Thedraining 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. Thedraining 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 mammaryfatpad. At multiple intervals, groups of mice were bled and then sacrificed, and lymph nodes, lungs, and livers removed. The draining lymph node in the mammaryfatpad 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 werecollected 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 ±1.5g and 11.2 ±4.6 g in the 2 experiments) far exceeded the sizeof 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, whenmetastatic nodules were detectable in animals with the latter 2tumor lines.The nonmetastatic line 168FARN cells spread through the

lymphatics, since clonogenic cells were recovered from draininglymph nodes in the mammary gland (Fig. 2H). In a secondexperiment, low numbers of clonogenic 168FARN cells wereoccasionally recovered from lungs (1 animal of 6 at necropsyon day 51 had 43 clonogenic cells) and livers (none of the 6animals at necropsy had detectable cells in the liver, but 2 of 5mice on day 21 had 13 and 65 clonogenic cells, and 2 of 5 miceon day 28 had 8 and 15 clonogenic cells in the liver). Althoughclonogenic 168FARN cells could be recovered from draininglymph nodes, clonogenic cells were not recovered in eitherexperiment from the blood of any of the 61 mice bearing line168FARN tumors. Visible nodules were not observed on lungsat necropsy, and clonogenic cells were recovered in only oneanimal as noted above despite the presence of very large primarytumors (9.3 ±1.1 g and 8.6 ±2.2 g at necropsy for the 2experiments).The nonmetastatic line 4TO7 appeared to be able to complete

all steps of metastasis except the final one. Line 4TO7 spreadvia the blood to lungs and occasionally to livers, but did notestablish progressively growing metastatic nodules. Fig. 2Cdepicts one of 2 experiments in which the sequential spread of4TO7 from a mammary fatpad was monitored. Clonogeniccells were recovered from the lungs at day 19. At necropsy,visible nodules were absent but clonogenic 4TO7 cells wererecovered from lungs in 4 of 5 mice (primary tumor weight of1.35 ±0.32 g on day 28) with a geometric mean of 32 clonogeniccells detected.In a second experiment with 4TO7, clonogenic cells were

recovered from the lungs on day 7 after tumor cells were1401

100

80

60

40

20

LEGEND:4TO7 ' •¿ 66cl4 ' »4T1 * *67NR «• »IMFARN 0--0

1.0 2.0 30 4.0 5.0

CUMULATIVE TIME INSTOMACHER BLENDER (MINUTES)

Fig. 3. Sensitivity of the mammary tumor lines to mechanical disruption.Single cell suspensions prepared from monolayer culture were subjected to thelung digest method. Following incubation with elastase and collagenase IV for 60min at 4V. medium containing 10% calf serum was added and the cell suspensionswere disrupted by 4 30-s and 3 1-min bursts in the Stomacher lab blender.Beginning with the second 30-s disruption, triplicate samples were diluted andplated in the appropriate selective medium after each manipulation.

on May 3, 2016. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

�  4TO7 able to complete all steps except the final one.

�  Spread via the blood to lungs and occasionally to livers, but did not establish progressively growing metastatic nodules.

�  Necropsy, visible nodules absent but clonogenic cells were recovered in lungs.

� 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.

� Intravasation appears to be an important selective step. (67NR appears unable to leave the primary site).

� cells more highly efficient at colonizing lungs following iv injection were similar in abilities to spontaneously metastasize from im tumors.

� We have established an experimental model that allow the validation of many phenotypes implicated in metastasis.

� 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.

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

� Previous attempts: � Microarray analyses

¡  Generated gene expression profiles that are predictive of metastasis.

¡  Powerful for producing fingerprints, but hard to elucidate specific contributions of each genes.

� 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.

�  Injected cells from four lines into the mammary glands of BALB/c mice.

�  Group X: intravasation. Altered in 168FARN, 4TO7, and 4T1, but not in 67NR.

�  Group Y: extravasation. Altered in 4TO7 and 4T1, but not in 67NR and 168FARN.

�  Group Z: growth into secondary tumors. Altered in 4T1, but not in 4TO7, 67NR and 168FARN.

� Twist stood out. � 2nd most strongly upregulated in Group X. � Known functions as a master regulator of

embryonic morphogenesis.

Twist is not required for tumor formation

Twist increases the number of metastatic nodules

Twist promotes epithelial-mesenchymal transition (EMT) and cell migration

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

� E-Cadherin – important calcium dependent cell to cell adhesion molecule.

� Research has shown the importance of E-Cadherin when it comes to suppressing a tumor.

� Loss of E-Cadherin has been shown to increase metastatic effect in cancer cell lines.

� ϒ-­‐Linolenic  Acid  has  been  shown  to  improve  expression  of  E-­‐Cadherin.  

Drawbacks: � 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.

� 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. � Western Blotting – (E-Cadherin) �  Immunostaining – (E-Cadherin) � Aggregation Assays �  Invasions Assay

� ϒ-­‐Linolenic Acid (GLA) has potential for therapeutic use in our breast cancer mouse model

�  Metastatic Breast Cancer. (2015). National Breast Cancer Foundation. Retrieved from http://www.nationalbreastcancer.org/metastatic-breast-cancer

�  Metastatic Cancer. (2013, March 10). National Cancer Institute. Retrieved from http://www.cancer.gov/about-cancer/what-is-cancer/metastatic-fact-sheet

�  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

�  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

�  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/

�  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

�  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

�  Jiang, W, et. al. Regulation of the expression of E-Cadherin on Human Cancer Cells by Linolenic Acid. Cancer Research. 55; 5043-5048, 1995.

�  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

�  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.