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J Cancer Metastasis Treat 2019;5:31 I http://dx.doi.org/10.20517/2394-4722.2019.21 Page 4 of 36

obtained her Ph.D. degree from University of Toledo, School of Medicine, and finished her postdoctoral
training with the world renowned scientist Dr. George Stark at Cleveland Clinic, Ohio. Her research
focuses on the discovery of novel regulators of NF-κB, particularly, on the epigenetic regulation of NF-κB
and its role in cancer therapeutics. She won multiple awards at international scientific meetings. Dr. Lu has
published near 50 papers with 2 were highlighted by F1000 Prime. She currently holds 2 provisional patents
regarding NF-κB regulation and serves as the editorial board member of 8 scientific journals.

5. Using a multiplexed immunofluorescence assay to uncover an immunosuppressive mechanism

of tumor-associated macrophages in the pancreatic tumor microenvironment

Anna Juncker-Jensen, Jun Fang, Judy Kuo, Mate Nagy, Qingyan Au, Eric Leones, Flora Sahafi, Raghav
Padmanabhan, Nicholas Hoe, Josette William

NeoGenomics, Aliso Viejo, CA 92656, USA.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an excessive amount of
desmoplastic stroma seeded with inflammatory cells and it is one of the most aggressive forms of cancer
with no current specific therapies. Tumor-associated macrophages (TAMs) are a major component of
the tumor microenvironment (TME), and in most solid cancers increased TAM infiltration is associated
with a poor prognosis. TAMs can be described as classically activated M1 types with pro-inflammatory
antitumor functions, vs. alternatively activated M2 types with immunosuppressive pro-tumor functions.
The immunosuppressive functions of M2 TAMs can be exerted through release of cytokines and growth
factors as well as via direct recruitment of T regulatory cells (Tregs), a subset of lymphocytes responsible
for immune tolerance of the system to the tumor. While the differentiation from M1 to M2 in PDAC has
[1]
been shown to be associated with a worse prognosis , not much is known about PDAC TAM polarization
and its potential correlation to Treg recruitment.

Methods: For this study we have used MultiOmyx, a proprietary, multiplexing assay with similar staining
characteristics as standard IHC stains but with the significant advantage that up to 60 protein biomarkers
[2]
can be interrogated from a single FFPE section . MultiOmyx protein immunofluorescence assays utilize a
pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each round of
staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining
and deactivation.

Results: Using the pan macrophage marker CD68 in combination with either M1 marker HLA-DR or M2
marker CD163 we confirmed the presence of M1 (CD68+HLA-DR+) and M2 (CD68+CD163+) populations
in 9 stage IIB non-metastatic PDAC FFPE samples, the vast majority being of the M2 subtype. Moreover,
we found a positive significant correlation (Pearson’s correlation P < 0.05) between the presence of M2
TAMs and Tregs (CD3+CD4+FoxP3+), but not between M1 TAMs and Tregs. Moreover, in a spatial nearest
neighbor analysis we found M2 type TAMS to be in closer proximity to Tregs compared to M1 type TAMs.

Conclusion: We demonstrate a positive significant correlation between the presence of M2 TAMs
and Tregs in the TME of PDAC, suggesting a possible pathway in which TAM polarization plays an
immunosuppressive function by recruiting Tregs. PDAC is one of the most aggressive forms of cancer
with a 5-year survival rate below 5% and no current specific therapies. An increasing number of studies
show accumulation of immune suppressor cells such as MDSCs and TAMs in PDAC patients. Hopefully,