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TITLE: IL-6 Receptor Isoforms and Ovarian Cancer
PRINCIPAL INVESTIGATOR: Susan E. Waltz, Ph.D.
CONTRACTING ORGANIZATION: University of Cincinnati
Cincinnati, OH 45221
REPORT DATE: January 2013
TYPE OF REPORT: Final
PREPARED FOR: U.S. Army Medical Research and Materiel
Award Number: W81XWH-09-1-0673



Introduction
The overall goal of the proposed study has been to test the hypothesis that membrane and soluble IL6R play distinct roles in driving tumor progression. Two major Tasks were funded as
outlined in the Statement of Work, which encompasses examining IL6R expression in ovarian patient tumor samples and performing experiments to define the function of IL6R in the tumor
proper and host microenvironment.

Body
The overall goals have been accomplished and the data published related to Task 1 (1). Task 1 entailed the “Identification of IL6R isoforms in patient samples.” In the reported studies,
elevated levels of IL6, total IL6R, differential spliced forms of IL6R, ADAM10 and ADAM17 were found in ovarian tumors. In addition, these studies further demonstrated that both tumor and
host cells contribute to soluble IL6R expression in the tumor microenvironment. Task 2 entailed “In vivo experiments to determine functions of IL6R isoforms and tumor:stroma
relationships. For this task, mouse breeding colonies and genotyping procedures were set up.
A recent paper was also published related to the characterization and general phenotype of these mice (2). Xenograft studies using ovarian cancer cell lines in SCID mice demonstrated that host IL6R may be an important means by which IL6R levels are increased in the tumor microenvironment along with the importance of IL6R in the tumor proper (1). Further studies have been published which also show that overexpression of IL6R and to a more modest extent IL6, in ovarian cancer cells promotes their colonization on the omentum ex vivo and increased have been published which also show that overexpression of IL6R and to a more modest extent
IL6, in ovarian cancer cells promotes their colonization on the omentum ex vivo and increased
their adherence to plastic as well as plates coated with laminin and collagens I and II (3). To test the significance of host derived tumor growth, ovarian tumor cells were next injected
intraperitoneally (IP) into mice deficient in either IL6 or IL6R. In examining tumor adherence to the omentum following IP injection, mice with a complete loss of IL6 or IL6R exhibited less
ovarian cancer cell adherence to the omentum. To obtain information as to the host cell type in which IL6R expression is needed for tumor adherence, ovarian cancer cells were injected into mice with conditional losses of IL6R in either hepatocytes or in myeloid cells (monocytes/granulocytes). Interestingly, tumor adherence was reduced in both hosts with IL6R loss, although IL6R loss in host myeloid cells lead to a greater inhibition of tumor adherence to the omentum compared to loss in IL6R from hepatocytes. At this point, the project was halted
as the initial PI left the institution. A few months later, a new PI was designated (Susan Waltz) and the mouse breeding colonies were re-initiated. Unfortunately, having to regenerate the animal colonies entailed approximately 8-12 months to obtain the complex IL6R-/- mice and the IL6R conditional mutants as the original colonies were sacrificed prior to the designation of a new PI. Over the course of the past year, the focus of the new PI has been to generate the appropriate mouse colonies and cell lines to continue experiments designated in Task 2. The primary goal of these final studies has been to examine the significance and potential mechanisms associated with host IL6R on ovarian cancer growth and metastasis. The following is a synopsis of the final data that has been obtained since the new PI was designated through completion of the funded studies:
have been published which also show that overexpression of IL6R and to a more modest extent IL6, in ovarian cancer cells promotes their colonization on the omentum ex vivo and increased
their adherence to plastic as well as plates coated with laminin and collagens I and II (3). To test the significance of host derived tumor growth, ovarian tumor cells were next injected
intraperitoneally (IP) into mice deficient in either IL6 or IL6R. In examining tumor adherence to the omentum following IP injection, mice with a complete loss of IL6 or IL6R exhibited less
ovarian cancer cell adherence to the omentum. To obtain information as to the host cell type in which IL6R expression is needed for tumor adherence, ovarian cancer cells were injected into mice with conditional losses of IL6R in either hepatocytes or in myeloid cells (monocytes/granulocytes). Interestingly, tumor adherence was reduced in both hosts with IL6R loss, although IL6R loss in host myeloid cells lead to a greater inhibition of tumor adherence to the omentum compared to loss in IL6R from hepatocytes. At this point, the project was halted
as the initial PI left the institution. A few months later, a new PI was designated (Susan Waltz) and the mouse breeding colonies were re-initiated. Unfortunately, having to regenerate the
animal colonies entailed approximately 8-12 months to obtain the complex IL6R-/- mice and the IL6R conditional mutants as the original colonies were sacrificed prior to the designation of a
new PI. Over the course of the past year, the focus of the new PI has been to generate the appropriate mouse colonies and cell lines to continue experiments designated in Task 2. The primary goal of these final studies has been to examine the significance and potential mechanisms associated with host IL6R on ovarian cancer growth and metastasis. The following is a synopsis of the final data that has been obtained since the new PI was designated through completion of the funded studies:

IL6 and IL6R stimulate migration in ovarian overexpressing IL6R exhibited increased migration/invasion within the peritoneal
cavity (Figure 1). To follow up on increased migratory
ability of tumor cells overexpressing IL6R, we next sought to determine whether IL6 signaling regulates tumor cell migration. To accomplish this, ES-2 human ovarian cancer cells, as well as
ES-2 cells overexpressing IL6 or IL6R were plated on tissue culture plates and scratch wounds were made. Over the course of the experiment, photographs of the cultures were taken temporally and the percent migration was measured based on wound closure. As depicted in Figure 2, both IL6 and IL6R overexpression lead to significantly more cell migration compared to control cells. Slight increases, although not significant, were observed following the addition of exogenous IL6 or soluble IL6R. This data suggests that tumor cell derived IL6/IL6R signaling may be an important factor regulating the local migration/invasive ability of ovarian caner cells and that further supplementation of IL6 or IL6R may only marginally be required beyond that present in the tumor cell proper to increase this effect.

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The combination of the specific tumor microenvironment,
ascitic dissemination pathways, and early omentalcolonization may create a situation in which LY75 expression favors the colonization of ovarian tumors. Increased expression of IL6 and IL6Ra in patients with ovarian cancer may enhance LY75 expression in tumor cells and facilitate tumor adherence and colonization of the omentum. Indeed, we found a previously unreported correlation
between IL6Ra and LY75 expression in patients with ovarian cancer in large public databases, providing some clinical significance for our findings. Our study therefore demonstrates a novel mechanism by which IL6 signaling may contribute to tumor progression and suggests an opportunity for reducing the mortality associated with ovarian cancer.

http://www.dtic.mil/dtic/tr/fulltext/u2/a575873.pdf