Mentored Professional Enrichment Experience
Applicant:
Michael Halyko
Name of Project/Experience:
Preclinical Evaluation of SPARC in Combination with Chemotherapy for Treatment of
Neuroblastoma (NB) using Subcutaneous NB Xenografted Mice
Location where Project/Experience will take place:
University of Chicago, Department of Pediatric Medicine, Section of
Hematology/Oncology, Chicago, IL 60637
Mentor Name and Contact Information:
Dr. Susan Cohn, University of Chicago, KCBD Room 5100, 900 E. 57th St., Chicago, IL
60637, Phone: 773-702-2571, (scohn@peds.bsd.uchicago.edu)
RATIONALE
Neuroblastoma (NB) is the most common extracranial solid tumor of childhood,
representing over 8% of all childhood tumors, and is the most common tumor in infants
(Esiaschvili, 2009). The clinical behavior of NB reflects its biological heterogeneity,
with outcomes ranging between complete spontaneous regression and differentiation to a
very aggressive disease with rapid growth and metastasis. Although outcomes in patients
with NB have improved, the increase in 5-year survival rates witnessed between 1975
and 2005 have been primarily due to increased cure rates among patients with more
benign forms of the disease, and the rates among children with high-risk NB have shown
only modest improvements despite the more intense therapies being provided, indicating
a need for more effective treatments of aggressive tumors (Maris 2010). A strong
correlation between high-risk tumors and angiogenesis has been reported, which suggests
that blood vessels may be a therapeutic target for clinical treatment of aggressive NB
(Meitar, 1996). Furthermore, preclinical studies supporting this hypothesis have
demonstrated that NB tumor growth can be significantly impaired following treatment
with anti-angiogenic agents (Katzenstein 1999; Chesler 2007).
Studies have demonstrated that Schwann cells in the NB tumor stroma release
angiogenesis inhibitors, with the most potent being Secreted Protein Acidic and Rich in
Cysteine (SPARC). Additionally, it has been shown that NB tumors with abundant
Schwann cells in the tumor stroma have a more benign clinical behavior and better
prognosis, making SPARC a potent target molecule for study as a potential therapeutic
(Chlenski, Cohn 2010). SPARC is a matricellular glycoprotein that mediates the
interaction of cells with their microenvironment. It has been shown that during the
extensive matrix remodeling of neoplastic disease, SPARC is expressed in cancerassociated stroma and in some malignant cell types, affecting tumor development,
invasion, metastases, angiogenesis, and inflammation (Chlenski, Cohn 2010). The
specific action of SPARC in tumorigenesis varies by cell-type, likely due to its diverse
function in a given microenvironment. In some types of cancer, including melanoma,
osteosarcoma, and glioma, high levels of SPARC expression have been shown to
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correlate with disease progression and poor prognosis (Chlenski, Cohn 2010). In
contrast, a decrease in SPARC expression is seen in other types of cancers, including
breast, prostate, small cell lung cancer, and neuroblastoma (Chlenski, Cohn 2010). In
short, the role of SPARC in regulating the microenvironment of tumors is complex, and
SPARC-induced changes can suppress or promote progression of neoplasms depending
upon the specific requirements of the cell-matrix and tumor-stroma interactions. In some
cancers, treatment with SPARC has led to anti-tumor effects, while in other cancers,
targeting SPARC for inactivation has proven effective (Chlenski, Cohn 2010).
In preclinical models of NB, SPARC has been demonstrated as a potent
angiogenesis inhibitor with anti-tumorigenic effects that may be related to an ability to
induce tumor cell apoptosis and promote the assembly of stroma that is non-permissive
for tumor-cell growth, in addition to its anti-angiogenic effects (Chlenski 2006; Chlenski
2007). Recently, it was shown that FSEC, a peptide corresponding to the C-terminal loop
of the follistatin domain of the SPARC protein, is a potent inhibitor of angiogenesis in
vitro and in vivo, as well as an inhibitor of tumorigenesis in a preclinical model of NB
(Chlenski A, Guerrero L 2010). Additionally, FSEC induced normalization of blood
vessels in NB animal models, which may enhance drug delivery to the tumor tissue
(Chlenski, Guerrero 2010). In support of this, a recent study by Shusterman, et al.,
suggests that the combination of angiogenesis inhibitors with conventional chemotherapy
yields better results than either therapy alone without additive toxicity (Shusterman,
2005). In our current study, we propose to test combination therapy with SPARC and
chemotherapy similar to that performed by Shusterman. We hypothesize that
combination therapy with SPARC and conventional chemotherapy will yield greater
tumor reduction and remission than with either drug alone in preclinical models of NB.
GOALS
Our goal is to determine the efficacy of SPARC peptide used in concert with
chemotherapy (cyclophosphamide) in the treatment of NB in a preclinical model. My
personal goals include becoming more familiar with factors involved in angiogenesis and
tumorigenesis, learning new techniques necessary to investigate these questions, and to
learn more about current research on cancer treatment and development, especially
regarding NB.
METHODS
Pharmaceutical grade cyclophosphamide powder will be weighed in a balance,
shielded on all sides. It will be reconsituted at 20 mg/ml in water, filter sterilized, stored
dark at 40 Celcius and used within 1 week. Nude mice will be purchased from Harlan or
Charles River and their skin will be scrubbed with Betadine followed by 70% isopropyl
alcohol. They will receive a subcutaneous injection of 1x107 SMS-KCNR NB cells.
When tumors are 100 – 200 mm3, cyclophosphamide will be given as 3
intraperitoneal (IP) injections (150 ul each) of 150 mg/kg/dose over 5 days (to be given
on Mon., Wed., Fri.). Tumors should regress following the 3rd treatment with
cyclophosphamide. If they do not fully regress by the Monday following the 3rd
treatment, another round will be administered. If side effects of the drug are seen, then
treatment will stop. SPARC peptide, scrambled peptide, or vehicle control will be given 5
days/week for 2 weeks starting on the Monday following the last cyclophosphamide
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treatment. 150 ul of peptide at a dose of 0.3 mg/mouse/day or of PBS vehicle control will
be given by IP injection.
Tumor size will be measured 3 times per week. All animals will be euthanized
when tumors treated with cyclophosphamide alone recur and reach a size of 2 cm3 using
CO2 followed by cervical dislocation and tumors/organs removed for molecular,
pathological, and immunohistochemical evaluation.
ANALYSIS
The data will be analyzed using a variety of techniques. We will be looking for
signs of angiogenesis by performing histological examination of H&E and immunostained sections. Vessels will be counted using fluorescent microscopy. Cell
proliferation rates will be examined using Ki67 immunostaining. Microscopic analysis of
blood vessel architecture will be performed using fluorescent microscopy visualizing
pericytes with anti-α-SMA antibody and visualizing endothelial cells using anti-CD31
antibody. A TUNEL assay will be used to look for signs of apoptosis. In addition to
looking for signs of angiogenesis, we will also be looking for an immune response by
staining for macrophages and other immune cells and quantifying the response. Masson's
trichrome staining will be used to evaluate collagen maturation. Finally, we will be
looking for fibroblast activation using immunofluorescence and western blot with antiSMA antibodies.
SUPPORT
1. Do you request support funds? Yes
2. Would you be able to participate if a scholarship is not available? Yes
Please note that we do not need an exhaustive or extensive list of literature references.
References
Chesler, L., Goldenberg, D. D., Seales, I. T., Satchi-Fainaro, R., Grimmer, M., Collins, R., Struett, C., et al. (2007). Malignant
progression and blockade of angiogenesis in a murine transgenic model of neuroblastoma. Cancer research, 67(19), 9435-42.
doi:10.1158/0008-5472.CAN-07-1316
Chlenski, A, Guerrero, L. J., Yang, Q., Tian, Y., Peddinti, R., Salwen, H. R., & Cohn, S. L. (2007). SPARC enhances tumor stroma
formation and prevents fibroblast activation. Oncogene, 26(31), 4513-22. doi:10.1038/sj.onc.1210247
Chlenski, Alexandre, & Cohn, S. L. (2010). Modulation of matrix remodeling by SPARC in neoplastic progression. Seminars in cell
& developmental biology, 21(1), 55-65. doi:10.1016/j.semcdb.2009.11.018
Chlenski, Alexandre, Guerrero, L. J., Peddinti, R., Spitz, J. A., Leonhardt, P. T., Yang, Q., Tian, Y., et al. (2010). Anti-angiogenic
SPARC peptides inhibit progression of neuroblastoma tumors. Molecular cancer, 9, 138. doi:10.1186/1476-4598-9-138
Chlenski, Alexandre, Liu, S., Guerrero, L. J., Yang, Q., Tian, Y., Salwen, H. R., Zage, P., et al. (2006). SPARC expression is
associated with impaired tumor growth, inhibited angiogenesis and changes in the extracellular matrix. International journal of
cancer. Journal international du cancer, 118(2), 310-6. doi:10.1002/ijc.21357
Esiashvili, N., Anderson, C., & Katzenstein, H. M. (2009). Neuroblastoma. Current problems in cancer, 33(6), 333-60.
doi:10.1016/j.currproblcancer.2009.12.001
Katzenstein, H M, Rademaker, A. W., Senger, C., Salwen, H. R., Nguyen, N. N., Thorner, P. S., Litsas, L., et al. (1999). Effectiveness
of the angiogenesis inhibitor TNP-470 in reducing the growth of human neuroblastoma in nude mice inversely correlates with tumor
burden. Clinical cancer research: an official journal of the American Association for Cancer Research, 5(12), 4273-8. Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/10632370
Maris, J. M. (2010). Recent advances in neuroblastoma. The New England journal of medicine, 362(23), 2202-11.
doi:10.1056/NEJMra0804577
Meitar, D., Crawford, S. E., Rademaker, A. W., & Cohn, S. L. (1996). Tumor angiogenesis correlates with metastatic disease, N-myc
amplification, and poor outcome in human neuroblastoma. Journal of clinical oncology: official journal of the American Society of
Clinical Oncology, 14(2), 405-14. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8636750
Shusterman, S., & Maris, J. M. (2005). Prospects for therapeutic inhibition of neuroblastoma angiogenesis. Cancer letters, 228(1-2),
171-9. doi:10.1016/j.canlet.2005.01.049
Tai, I. T., & Tang, M. J. (2008). SPARC in cancer biology: its role in cancer progression and potential for therapy. Drug resistance
updates: reviews and commentaries in antimicrobial and anticancer chemotherapy, 11(6), 231-46. doi:10.1016/j.drup.2008.08.005
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