TARGETING OF PHOSPHATIDYLSERINE ON THE SURFACE OF MAMMARY TUMOR CELLS SLOWS TUMOR GROWTH AND LUNG METASTASIS
Liang Huang, Chaobo Yin, Mitchel Kent, Joel Shilyansky, Dept. of Surgery, University of Iowa Children’s Hospital
Purpose Cancer cells employ a variety of molecular mechanisms in order to evade immunosurveillance. One of the major mechanisms by which tumors subvert immune detecHon and destrucHon is by suppression of the host’s immune system1. PhosphaHdylserine(PS) is phospholipid predominantly expressed on the inner leaflet of the cell membrane in living cells. PS is translocate to the external leaflet during apoptosis, promoHng rapid uptake and clearance by phagocytes. ApoptoHc cells, and PS, has been shown to inhibit immune responses2 . When PS is blocked with Annexin-‐V, a protein that specifically binds and blocks PS, immunogenicity of apoptoHc cells increased3. PS is also expressed on the surface of tumor cells suggesHng that it may play a role in tumor immune evasion4. The goal of this study was to determine the effects of blocking tumor PS on tumor growth. Also, there are numerous factors known to influence the metastaHc potenHal of cancer. Annexin V was shown the potenHal to induce tumor immunity. We hypothesize that Annexin V would inhibit tumor growth and a reduce lung metastases. Methods Spontaneously metastasizing murine mammary 4T1–luciferase tumor cells (4T1Luc) were implanted orthotopically in BALB/c mice and tumors developed within 1 week. Modified Annexin V protein was then injected into tumors. Tumor size was measured every other day. To determine the extent of pulmonary metastases, luciferin was administered and mice were imaged using IVIS system on days 15, 22, and 29 a_er inoculaHons. On day 29, lungs were collected for pathological examinaHon or placed in Hssue culture and colony forming units (CFU) were counted. StaHsHcal analysis was performed using 2-‐way ANOVA and student t-‐test.
Results Following treatment with Annexin V, the tumor size was smaller in the treatment group than control group(p=0.024). Live imaging showed less luminescence in the lungs of treated animals than controls, indicaHng reducHon in lung metastases (p=0.037). Pathological examinaHon showed a decrease in the number of metastases (p=0.0217). Lung cultures demonstrated fewer tumor CFUs suggesHng a decrease in tumor load (p=0.008) in Annexin V treated animals.
Conclusion Annexin V has demonstrated efficacy in treatment of established mammary tumors and reduced lung metastases. The results support the potenHal of targeHng tumor phosphaHdylserine for treatment of metastaHc disease.
References 1. Wolchok JD, Chan TA. Cancer: AnHtumour immunity gets a boost.
Nature. 2014 Nov 27;515(7528):496-‐8. 2. Schujers K, Reutelingsperger C. PhosphaHdylserine targeHng for
diagnosis and treatment of human diseases.Apoptosis. 2010 Sep;15(9):1072-‐82.
3. Yan X, Doffek K, Yin C, Krein M, Phillips M, Sugg SL, Johnson B, Shilyansky J. Annexin-‐V promotes anH-‐tumor immunity and inhibits neuroblastoma growth in vivo. Cancer Immunol Immunother. 2012 Nov;61(11):1917-‐27.
4. Graham DK, DeRyckere D, Davies KD, Earp HS. The TAM family: phosphaHdylserine sensing receptor tyrosine kinases gone awry in cancer.Nat Rev Cancer. 2014 Dec;14(12):769-‐85. Review.
Control AnV
Untreat
ed
Annexin
V *
0
20
40
60
Groups
Ave
rag
e N
um
ber
of P
ulm
on
ary
Met
s
Pulmonary Metastases
UntreatedAnnexin V *
Untreat
ed
Annexin
V *
0
100000
200000
300000
400000
500000
Day 22 Lung Luminescence
Groups
Ph
oto
ns
UntreatedAnnexin V *
Untreat
ed
Annexin V
**0
200
400
600
800
Culture of Lung Metastases
Groups
Col
ony
Form
ing
Uni
ts
UntreatedAnnexin V **
5 10 15 200.0
0.2
0.4
0.6
Days After Tumor Inoculation
Tum
or S
ize
(cm
3 )
Effect of Annexin V on Tumor Growth
UntreatedAnnexin V *
B Annexin V A Untreated
