2015 mammary tumor immunotherapy presentation

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 system 1 . 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 responses 2 . When PS is blocked with AnnexinV, a protein that specifically binds and blocks PS, immunogenicity of apoptoHc cells increased 3 . PS is also expressed on the surface of tumor cells suggesHng that it may play a role in tumor immune evasion 4 . 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 2way ANOVA and student ttest. 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):4968. 2. Schujers K, Reutelingsperger C. PhosphaHdylserine targeHng for diagnosis and treatment of human diseases.Apoptosis. 2010 Sep; 15(9):107282. 3. Yan X, Doffek K, Yin C, Krein M, Phillips M, Sugg SL, Johnson B, Shilyansky J. AnnexinV promotes anHtumor immunity and inhibits neuroblastoma growth in vivo. Cancer Immunol Immunother. 2012 Nov;61(11):191727. 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):76985. Review. Control AnV Untreated Annexin V * 0 20 40 60 Groups Average Number of Pulmonary Mets Pulmonary Metastases Untreated Annexin V * Untreated Annexin V * 0 100000 200000 300000 400000 500000 Day 22 Lung Luminescence Groups Photons Untreated Annexin V * Untreated Annexin V ** 0 200 400 600 800 Culture of Lung Metastases Groups Colony Forming Units Untreated Annexin V ** 5 10 15 20 0.0 0.2 0.4 0.6 Days After Tumor Inoculation Tumor Size (cm 3 ) Effect of Annexin V on Tumor Growth Untreated Annexin V *

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Page 1: 2015 Mammary tumor immunotherapy Presentation

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.

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