1. Inflammatory Cytokines Promote Melanoma Proliferation
and Migration
Mawusi Kamassa, M.S., Maxwell Schwam, Omama Ahmad, Yassmin Hegazy, Zachariah Burns, Elizabeth Brandon, Ph.D.
Department of Biological Sciences, Mississippi College, Clinton, MS
References
Summary
Background
Experimental and clinical data indicate that obesity increases the risk for
colorectal, esophageal, pancreatic, endometrial, renal cell carcinoma, and post-
menopausal breast cancer. We found that melanoma grows much faster in
obese mice than in normal body weight mice. Several metabolic, endocrine, and
immune system factors are positively correlated with a poor prognosis and
shorter disease-free survival times in obesity associated cancers. The paracrine
signaling between tumor cells and stromal cells is a hallmark of cancer that has
only recently become appreciated as a catalyst for mutagenesis, immune
evasion, and metastasis. Although some mechanistic knowledge has been
obtained from a “one growth factor, one effect” approach, this must be paired
with a systems analysis to gain an understanding of the interactions between
stromal cells and melanoma cells. Our data reveals that the inflammatory
cytokines, resistin and IL-6, promote melanoma proliferation (6.1x105 cells/day
and 1.0x106 cells/day v. 5.5x105 cells/day for controls, p=0.05). There is still a
wide gap in our knowledge of how obesity promotes melanoma metastasis.
Individual inflammatory cytokines had no effect on melanoma migration, but co-
culturing cells with macrophages significantly increases migration. Preliminary
data also suggests that co-culture with adipocytes increases melanoma
migration. The influence of inflammation on tumors is well known, but the
mechanisms by which fat cells affect melanomas is unknown. These results
suggest that there could be many stromal cells that affect melanoma growth and
metastasis in obese individuals.
• Physiologic concentrations of resistin and IL-6 stimulate
melanoma cell proliferation.
• Macrophages significantly increase melanoma cell
migration while individual cytokines have no
effect.
• Previous results from our lab revealed a role for the
adipokine leptin in melanoma tumor growth in
obese mice. These results further our
understanding of the mechanisms by which
obesity increases melanoma growth by indentifying
a link between inflammation melanoma cell
proliferation and migration.
• Cytokines and Growth Factors
When treating with an individual cytokine or growth factor, the concentrations were
as follows: resistin and IL-6 both at 100 ng/mL and 10 ng/mL and IL-4 was used at
10 ng/mL. Cytokines were recombinant mouse proteins purchased from R & D
Systems (Minneapolis, MN).
• Proliferation
24 well plates were plated with 20,000 cells/well in 1 mL of RPMI w/ 10% serum.
After an overnight incubation, the medium was aspirated and 1 mL of RPMI w/
0.5% serum was added. After 24 hours to synchronize, medium was aspirated
and 1 mL of treatment medium was added. Medium containing 10% serum was
added as positive control. 24 hours later, cells for the first time point were
aspirated and then trypsinized with 200ul trypsin for 10 minutes. Cells were
resuspended and counted using a Cellometer Auto T4 (Nexcelcom, Lawrence,
MA). Cells reserved for the 48 hour time point were fresh medium. These cells
were collected and counted the same as the 24 hour cells.
• Mueller MM, Fusenig NE. Friends or foes- bipolar effects of the tumour stroma in cancer. Nature 2004.
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• Lee, Yi-Chen, et al. Resistin expression in breast cancer tissue as a marker of prognosis and hormone
therapy stratification. Gynecologic Oncology 125.3 (2012): 742-750.
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monocytes. Journal of Clinical Endocrinology & Metabolism 86.2 (2001): 783-791.
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modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature
394:897
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enhances activation and proliferation of human circulating T lymphocytes. Cell. Immunol. 199:15.
• Brandon E., Gu J., Cantwell L., Zhi H., Wallace G., Hall J.E.(2009) Obesity promotes melanoma tumor:
growth: role of leptin. Cancer Biology & Therapy 8:19, 1871-1879.
Obesity is a serious health risk in America, approximately 32% of
adults are obese, 65% are overweight and 18.8% of 6 to 11 year old
children are obese. There is a strong, positive correlation between BMI
and the occurrence of several types of cancer, such as colon, gastric,
breast, ovarian, uterine, endometrial, renal cell carcinoma, prostate, and
melanoma. The increasing incidences of these cancers is well correlated
to the increasing trend in obesity in the last 30 years (Calle, 2003). Few
studies have demonstrated a direct cause and effect relationship
between obesity and cancer and the epidemiology of cancer suggests
that the response to obesity is not the same for all tumors. Consequently,
the mechanisms by which obesity may influence carcinogenesis are
poorly understood. What is clear from these studies is that when obese
patients get cancer, they have a poorer prognosis and shorter survival
times. Metastatic disease significantly increases mortality and there is
accumulating experimental and clinical evidence that obesity increases
metastasis.
Results from experiments in which mouse models of obesity were
used to test the hypothesis that obesity is associated with increased
melanoma growth identified a positive correlation between obesity and
melanoma growth. Tumor growth was partly dependent on the adipokine
leptin. The ratio of tumor weight to body weight was significantly lower in
obese leptin-deficient mice (ob/ob) compared to the ratio in obese leptin
replete animals (Brandon, 2009). Tumors from the obese mice had
significant necrosis, which is a potent stimulator of macrophage
infiltration and inflammation. Obesity causes inflammation in adipose
tissue and many tumors exhibit large numbers of immune cells in the
tumor stroma. Inflammation promotes the growth and metastasis of
many kinds of cancers, but its role in obesity-associated melanoma is
unknown.
Many factors contribute to metastasis and they are interrelated.
Angiogenesis, the extracellular matrix, interactions between tumor cells
and stromal cells, and inflammation are all associated with metastasis
(Mueller, 2004). Obesity is also associated with inflammation of adipose
tissue and this may explain the poor prognosis of many obese cancer
patients. Leptin production increases with BMI and stimulates expression
of several inflammatory cytokines: the endogenous antagonist of the IL-1
receptor, IL-1Ra (Gabay, 2001), TNF-α, IL-6, and IL-12 (reviewed in
Mantzoros, 2005). Leptin also promotes the proinflammatory T helper
cell type 1 (Th1) differentiation (Lord, 1998; Martin-Romero, 2000).
Resistin levels also rise in obesity and it is also associated with
inflammation. Macrophages secrete resistin, which acts via positive
feedback to increase the secretion of more resistin, TNF-α and IL-6.
Some data indicate that resistin is correlated with breast, prostate, and
colorectal cancers and high resistin levels in breast tumors is correlated
with poor prognosis and survival (Lee, 2012). Therefore, a major goal of
our research is to discern the mechanisms by which inflammation affects
melanoma migration and invasion in vitro in order to identify points of
intervention that may aid in slowing metastasis in vivo.
• Cell Culture: B16F10 mouse melanoma cancer cells were grown in an
incubator at 37°C with 5% CO2 in RPMI with 10% fetal bovine serum (FBS), 4.5
g/L glucose, 2 mM sodium pyruvate, 4 mM L-glutamine, 1.5 g/L sodium
bicarbonate, and 1% penicillin/streptomycin—hereafter referred to as normal
RPMI. RAW 264.7 cells were cultured using the same conditions. These cells
differentiated when exposed to melanoma cells.
• Migration: 80,000 cells were plated on inserts (8 µm pores, Millicell) and put
in 700 ul of RPMI w/ 10% serum in 24-well plates. For the co-culture
experiments, 150,000 cells were plated into the bottoms of 24-well plates and
allowed to attach overnight. After 24 hours, inserts were removed from wells and
medium was carefully pipetted out. Inserts were then place in 700 µL of
treatment medium or in wells containing cells. Cells were incubated for 24 hours.
Inserts were removed from plates, fixed in methanol, and stained with
hematoxylin and eosin. Cells were removed from the proximal side of the insert
with wetted cotton swabs and then migrated cells were counted on an inverted
Nikon Eclipse Ti microscope with the 10x objective. Images were taken on the
20x objective with a Nikon DS-Fi2 camera.
Day 1: 80,000 cells were plated in inserts and 150,000 macrophages were plated in 24-well
plates. Day 2: Inserts containing melanoma cells were transferred to wells containing
macrophages and the cells were incubated overnight in regular RPMI. Day 3: Inserts were
removed and stained. A. The graph represents the means of 2 independent experiments. A
Student’s T-test was used to compare control and co-cultured cells. Error bars = SEM. B. and
C. are representative images taken of the inserts.
0.00E+00
5.00E+05
1.00E+06
1.50E+06
2.00E+06
2.50E+06
24 48
CellConcentration(cells/mL)
Cell Proliferation
RPMI 10% serum
Resistin 10 ng/mL
IL-6 10ng/mL
IL-6 100ng/ml
P=0.05
Cytokine induced proliferation of melanoma cells. Melanoma cells were seeded in 24-well plates
(20K/well). Synchronized cells were treated with mouse resistin or IL-6 in regular RPMI. Cells were
trypsinized and counted at 24 and 48 hrs with a T4 automated cell counter. Graphs show the means
of 4 independent experiments for each treatment. Student’s T-test used to compare growth rates of
treated cells to RPMI 10% serum at 48 hrs. Error bars = SEM.
We would like to thank Dr. Stephanie Carmicle for the RAW 264.7 mouse macrophages.
This work was supported by the Mississippi INBRE, funded by an Institutional
Development Award (IDeA) from the National Institute of General Medical Sciences of
the National Institutes of Health under grant number P20GM103476.
Acknowledgments
0
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NumberofCells/Insert
Culture Conditions
Macrophage-stimulated Melanoma Cell Migration
Melanoma Alone
With Macrophages
P=0.03
640
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690
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730
NumberofCells/Insert
Culture Conditions
Melanoma-stimulated Macrophage Migration
Macrophages Alone
With Melanoma
0
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350
NumberofCells/Insert
Treatment Conditions
Melanoma Cell Migration with Individual Cytokines
Control
Resistin 10 ng/mL
IL-6 10 ng/mL
IL-4 10 ng/mL
Figure 3
60,000 cells/insert were plated and incubated overnight in regular RPMI. Cytokines were prepared
in regular RPMI and added to the lower chambers. The medium in the upper chamber was replaced
with fresh regular RPMI and the inserts were incubated for 24 hours. The graph represents the
means of 3 independent experiments. ANOVA was used to compare treatments with control cells.
Error bars = SEM.
Figure 4
B. Melanoma Alone C. Melanoma with Macrophages
A.
B. Macrophages Alone C. Macrophages with Melanoma
A.
Day 1: 80,000 macrophages were plated in inserts and 150,000 melanoma cells were
plated in 24-well plates. Day 2: Inserts containing macrophages were transferred to wells
containing melanoma cells and the cells were incubated overnight in regular RPMI. Day 3:
Inserts were removed and stained. A. The graph shows data from one preliminary
experiment. B. and C. are representative images taken of the inserts.
Abstract: P1919
Abstract Methods
Figure 1
Figure 2