1. Anne Philips
Lab Meeting

2. ID8 Cells
“Development of a Syngeneic Mouse Model for events related to ovarian cancer”
Katherine F. Roby et. al Carinogenesis vol21 no4
Mouse ovarian surface epithelial cells (MOSEC) obtained from virgin mature mice by mild trypsinization of mouse ovaries
Repeatedly passaged in vitro
After ~20 passages -
- cells changed morphology
- lost contact inhibition of growth
- exhibited tumor forming potential
ip injections of late passage cells into syngeneic mice -
- growth of tumor implants throughout the abdominal cavity
- production of hemorrhagic ascitic fluid.
10 clonal lines established from these late passage cells

3. Each cell line was injected i.p. into C57BL6 mice (5X106 cells, n=6)
Animals were killed (days to death) and tumor load was measured and scored on a 0. +, ++, +++ scale for 11 different abdominal organs and tissues

4. Advantages
cells had the ability to form extensive tumors within the peritoneal cavity similar to those seen with women with Stage III and IV cancer
produce tumors in mice with intact immune systems
development of cancer in mice occurred over a longer time with these clonal cell lines compared to parent cells.
slower development of disease is useful for the detection and manipulation of the molecular processes and the immune interactions in ovarian cancer development and progression.

5. Generation of a Syngeneic Mouse Model to Study the Effects of Vascular Endothelial Growth Factor in Ovarian Carcinoma
Lin Zhang, American Journal of Pathology, Vol. 161, No. 6, December 2002
VEGF promotes growth, proliferation, survival and/or migration of tumor cells
Increased levels of tumor VEGF have been reported in invasive ovarian carcinoma compared to benign tumors
Serum levels of VEGF are 10-fold higher in patients with advanced ovarian cancer compared
to healthy controls
Importantly, increased serum and/or tumor levels of VEGF have been associated with
poor clinical outcome
Aim
Develop a syngeneic mouse model of ovarian carcinoma to investigate the multifaceted functions of VEGF.

6. Protocol
The murine VEGF164 cDNA was inserted in the murine stem cell retroviral vector MigR1 upstream of enhanced GFP, from which it was separated by an internal
ribosome entry site
Retroviral vector containing GFP alone or VEGF164/GFP was transfected into BOSC23 (a murine kidney 293T-derived packaging cell line)
ID8 cells were infected with the retrovirus
7x106 VEGF/GFP transfected cells, GFP-transfected cells or WT cells injected ip into C57BL6 mice.
5x106 VEGF/GFP-transfected cells was injected subcutaneously into the flank of 8-week-old C57BL6 mice and tumor size was measured weekly.
Animals were followed for survival or were sacrificed 8 weeks after inoculation to evaluate tumor growth

7. Results
A. Animals bearing VEGF/GFP intraperitoneal tumors exhibited 12.9-fold higher ascites levels and 2.6-fold higher serum levels of VEGF compared to animals bearing control GFP tumors 2 weeks after inoculation of cells
B. animals injected with VEGF/GFP-positive cells displayed a median survival of 8
weeks, whereas control animals displayed a median survival of 16 weeks

8. VEGF/GFP-transfected ID8 cells give rise to markedly larger flank tumors (left flank) compared to control GFP-transfected ID8 cells injected to the contralateral (right) flank

9. A–D: Flank tumors resected from both sides of the same mouse.
(A and B), few blood vessels grow into the tumor; whereas in tumors from VEGF/GFP-transfected ID8 cells
(C and D), prominent blood vessels growing into the tumor from nearby normal tissue are observed.

10. Conclusion
developed a syngeneic model of ovarian carcinoma with stable overexpression of murine VEGF164 in the C57BL6 mouse
the model that was generated exhibits marked similarities with human ovarian carcinoma

11. Ovarian cancer cell–derived migration inhibitory factor enhances tumor growth, progression, and angiogenesis
Hagemann, et al 2007 Mol Cancer Ther:6 (7)
Tumor cell–derived macrophage migration inhibitory factor (MIF)
MIF increases macrophage-mediated ovarian cancer cell invasiveness in vitro
MIF is expressed in borderline and malignant ovarian tumors, and active MIF is found in malignant ascitic fluid
Aim
Look at the significance of ovarian cancer cell–derived MIF for tumor growth, metastasis and angiogenesis

12. Protocol: ID8 cells transfected with:
- pSilencer2.1-U6puro RNAi plasmids for MIF
- control plasmid containing scrambled RNA (mock).
ID8, ID8-MIF RNAi, or ID8-mock cells were injected i.p into female C57Bl/6 mice.
Ascites and solid tumors were collected and and analyzed

13. Results:

14. Results: ID8 parental cells: median survival - 109.5 days
Mock ID8 cells: median survival days
MIF RNAi ID8 cells: median survival -139 days
B. MIF RNAi ID8 tumor bearing mice had a significantly decreased ascites burden compared with parental ID8 or mock RNAi ID8-bearing mice

15. MIF RNAi tumors show a significant reduced
proliferation rate using Ki-67 staining
MIF RNAi tumors show a higher apoptotic rate using TUNEL assay compared with parental or ID8 mock tumors

16. Conclusions
secretion of MIF by ovarian cancer cells regulates the tumor
microenvironment
interaction of MIF with other cytokines, angiogenic factors, and chemokines may act to promote tumor growth, progression

17. “ Re-educating ” tumor-associated macrophages by targeting NF-kB
Thorsten Hagemann , J. Exp. Med. Vol. 205 No
Aim
The nuclear factor kB (NF-kB) signaling pathway is important in cancer related inflammation and malignant progression.
Study the role of NF-kb in maintaining the immunosuppressive TAM phenotype
Block NF-kB activity specifically in macrophages, by targeting IkB kinase, (IKK)ß , the major activator of NF-kB

18. Protocol I
1.Mouse bone marrow – derived macrophages (BMDMs) were infected with a recombinant adenovirus expressing a dominant-negative inhibitor of IKKß (Adv-IKKß DN ).
2.Generated IKKß-null macrophages (IKKßΔ) from IKKß knockout mouse
3.TAMs isolated from established tumors and infected in vitro with the IKKßDN adenovirus (TAM DN ). II
ID8 cells that expressing firefly luciferase were injected i.p. into mice.
Adoptively transferred
- 5 × 106 WT-, IKKßΔ -, or IKKßDN – expressing BMDMs
- TAM DN
Tumor progression was assessed in situ by bioluminescence imaging
on days 0, 7, and 14 after adoptive transfer

19. Results
ID8-Luc were injected i.p. into mice and tumors were allowed to develop.
After 35 d, WT, mock, and IKKßDN BMDMs were adoptively transferred and peritoneal cells were collected after an additional 7 and 14 d.
Representative bioluminescence imaging in vivo 14 d after adoptive transfer of BMDMs

20. ID8-Luc were injected i.p. mice and tumors were allowed to develop.
After 35 d, mock- and IKKßDN -infected TAMs (TAM mock , TAM DN , respectively) were adoptively transferred and peritoneal cells were collected after an additional 7 and 14 d
Representative bioluminescence imaging in vivo 14 d transfer

21. Conclusions:
Targeting IKKß in TAMs reverses their tumor-promoting activity and causes tumor regression in vivo
When NF-kB signaling is inhibited specifically in TAMs, they become cytotoxic to tumor cells and switch to a “ classically ” activated phenotype (M1)

22. Studies utilizing ID8 cells:
Knock-down genes in ID8 cells known to be involved in cancer development and progression
Use modified ID8 cells to study:
tumor formation in mice
gene expression in TAMs
Use ID8 cells in KO mice (eg. GAB2 -/-)
study tumor formation
- evaluate gene expression in TAMs