This is the final poster I presented at the conclusion of the KEYS 2017 program. Abstract: Glioblastoma is a lethal brain cancer that is resistant to many treatments. It is observed that cells with both olfactomedin1 and latrophilin2 proteins have enhanced metastatic abilities– cancer spreading. Using the Duolink kit, the steps are: fix cells to coverslips, apply two sets of primary antibodies, apply probes, then image. The probes attach to primary antibodies and hybridize if they are within a certain distance, a circular bridge forms then is amplified and lit up. Strong signal colorations validate the relationship since the kit only amplifies connections between sets of both proteins in close proximity. Future goals consist of furthering investigation on the details of the relationship and reducing invasion. Slowing down the progression of Glioblastoma through limiting spreading makes less harmful treatment options available.

1. The Effect of Oflactomedin1 and Latrophilin2 in Glioblastoma Metastasis
Tingting Thompson1, Beca Gardner2, Dr. Raymond Runyan2, Martha Nuñez2, Gianna Bortoli2
1KEYS Intern, 2Department of Cellular and Molecular Medicine
Introduction
Figure 4.1 Nucleus stain with Hoechst Figure 4.2 LPHN2 stain with LPHN2 primary
antibody
Figure 4.3 Overlap stain of nucleus and LPHN2
protein
epithelial cell
primary epithelial
cancer cell
metastatic cell endothelial cell basement membrane
Methods
Results
Figure 5.1 OLFM1 Experimental: T-ARB with 1˚+2˚ Figure 5.2 OLFM1 Control: T-ARB with 2˚ only
Since the staining signal
continued to show the
same strength in both
the experimental and
control samples, we
failed to find the correct
method of primary
antibody attachment.
Introduction
Varying conditions: 6 wells
1. Fixation methods
2. Antigen retrieval buffers (ARB)
Experimental vs. control:
1˚+ 2˚ v 2˚ only
Figure 3.2
Staining: Round Two
Tray with six varying well
conditions to expose OLFM1
antigen. 1˚ means primary
antibody, ø had no retrieval
buffer, top labels were the
different fixation methods:
methanol vs. tannic
Test 1 failure for OLFM1 primary
antibody caused need to further
explore how to expose the
OLFM1 antigen for the primary
antibody to attach to.
Factors tested: 6 wells
1. Coverslip vs. Non-Coverslip
2. Cell density- 25,000 v 75,000
3. Primary antibodies
Testing
Figure 3.1
Staining: Round One
Trays contain six varying
wells that were designed to
compare different factors for
imaging
Glioblastoma (GB) is a lethal brain cancer known for its aggressiveness. There are several factors that
account for its aggressiveness, one of them is metastasis, the ability to spread quickly. Within the GB cancer
cell line 62 (GB62), two proteins– olfactomedin1 (OLFM1) and latrophilin2 (LPHN2)– were observed to
enhance metastatic abilities.
Previous work in the lab determined that cells with both proteins present were observed to be more
invasive compared to those with only one or none. Further investigation of the relationship between the two
proteins and GB is needed to confirm the hypothesized connection. Identifying how the proteins work to
intensify the aggressiveness of the cancer through metastasis is a major objective that will allow for
explorations on how to disable the proteins causing cancer.
There are several possibilities that could occur from suppressing the OLFM1 or its receptor that may
significantly limit the cancer spread (1). If cancer progression could be delayed, less harmful alternative
treatment options may become more available to the patients.
LPHN2 and OLFM1 assist in the invasive late stage of Epithelial-Mesenchymal transition (EMT)– a
process often used in embryonic development and wound mending. EMT is the transition from epithelial
cells into mesenchymal cells which is similar to Glioblastoma Invasion. Previous work identified GB62 as one
of the most invasive cell lines and one that responded to OLFM1 by becoming more aggressive (1). Cancers
have different subtypes that often vary in aggressive characteristics such as invasiveness– cell line 62 was the
most promising to study due to its response to OLFM1 in an earlier study.
Is there a relationship between the olfactomedin-1 and latrophilin-2 proteins that could potentially
have significant effects on metastasis–the spreading of cancer? A Duolink kit will test whether there is a
relationship between two identified proteins related to the cell, not what the relationship is, only the
existence of.
Along with the antibody stains, Hoechst, a nucleus stain,
was used to confirm cells. Photos were taken using a light
microscope. During testing, the primary antibody was
evaluated to determine if it worked.
(1) Lencinas, Alejandro et al. “Olfactomedin-1 Activity Identifies a Cell Invasion Checkpoint during Epithelial-Mesenchymal Transition in the Chick Embryonic Heart.” Disease Models & Mechanisms
6.3 (2013): 632–642. PMC. Web. 26 June 2017.
1.1 Carrier, T., and John Allen. "What Is Tumor Progression?" WiseGEEK. Conjecture Corporation, 11 June 2017. Web. 12 July 2017.
1.2 Chiblak, Sara. "Pancreatic Cancer: Current Concepts in Invasion and Metastasis." Research Gate. N.p., Dec. 2011. Web.
1.3 "Mitral Valve Disease and the Cavalier King Charles Spaniel." CavalierHealth.org. N.p., n.d. Web. 12 July 2017.
2.1 Rastogi, Varun. "Diagnostic Procedures for Autoimmune Vesiculobullous Diseases: A Review." JOMFp. N.p., 13 Feb. 2015. Web.
References
I would first like to thank everyone in my lab for putting up with me for a wonderful six weeks! Next, I am extremely thankful to KEYS and KEYS staff for helping me succeed this summer
and giving me the chance to get hands on lab experience. I will cherish the time I have spent with everyone this summer and all the new people I have met. I would also like to give thanks
to my friends– Hannah Nguyen and Olivia Pietz– and family– Carol Thompson, Elizabeth Hurd, and Grace Thompson– for helping me adjust during the program.
Acknowledgements
Due to many circumstances such as cell
contamination and troubleshooting problems, we
were unable to perform the actual Duolink kit.
Although, future steps were concluded for LPHN2.
The LPHN2 primary antibody stained in the green
and properly surrounded the blue nuclei since
LPHN2 is in the membrane. It can also be
concluded that the laminin coating on the
coverslips successfully maintained a large cell
count. Without the protein- protein relationship
assessment from Duolink, support for the
hypothesis is yet to be recorded.
Conclusion
Other methods will need to be tested in order to
create the best environment for determining the
relationship of the proteins. In the end, it is also
possible that there is no relationship at all.
To continue troubleshooting, other factors and
methods may be explored, including:
 Adding extra OLFM1 protein variants if we are
losing too many of the extracellular protein or
if the cells are not secreting enough
 Finding a new primary antibody if the
problem is the antibody itself
 Alternative technique to Duolink would be
Immunoprecipitation
Figure 1.2 An example of EMT in pancreatic cancer– a cancer that does involve epithelial cells. Figure 1.3 The process of EMT. Along side are
characteristics and abilities of the two cell types.
Cancerous
Uncontrolled cell
division
Damaged cells
Well differentiated
cells
Uniform cell
growth
Cancer Progression
Discussion
Successes:
o Coverslip for imaging
o 75,000 cells per well
o LPHN2 primary
antibody
OLFM1 stain did not
work, a possible factor
was inability for the
primary antibody to
find the antigen on the
protein.
Fluorescent
Label
Labeled
Secondary
Antibody
Primary
Antibody
Antigen
Epithelial cells Metastatic cells Endothelial cells basement membrane
Primary epithelial
cancer cell
Carcinoma in situ
primary site
Normal epithelium EMT
Intravasation/
Extravasation
Secondary tumour
distant metastasis
Figure 1.1 Explanation of cancer as regular cells
who have uncontrollable and abnormal growth.