Page 160 - ebook HCC

PROGRAMME AND ABSTRACTSAND ABSTRACTS
GENEV
EASL
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158 PROGRAMME GENEVA, SWITZERLANDA, SWITZERLAND EASL HCC SUMMITHCC SUMMIT 159
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FEBRUARY 13 - 16, 2014Y 13 - 16, 2014
FEBRUAR
Poster Board Number B47 Poster Board Number B48
SURFACE MARKER PROFILING OF HUMAN THE CROSS TALK BETWEEN HEPATIC STELLATE
HEPATOCELLULAR CARCINOMA CELLS USING CELLS AND HCC CELLS OFFSETTS SORAFENIB
HIGH THROUGHPUT FLOW CYTOMETRY EFFECTIVENESS VIA LAMININ-5 INTEGRINS
SCREENING ENGAGEMENT

Kui Chen , Laurie Ailles , John E. Dick , Anand Ghanekar 1 5 Gianluigi Giannelli , Amalia Azzariti , Letizia Porcelli , Anna Elisa Quatrale , Erica Villa 1
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1 Toronto General Research Institute, Ontario Cancer Institute, University Health 1 Emergency and Organ Transplantation, University of Bari, Bari, Italy
Network, Department of Medical Biophysics, Department of Molecular Genetics,
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5 Department of Surgery, University of Toronto, Toronto, Canada
Corresponding author’s e-mail: gianluigi.giannelli@uniba.it
Corresponding author’s e-mail: anand.ghanekar@uhn.ca
Introduction: The mechanisms responsible for resistance to Sorafenib are still unknown.
Introduction: Human hepatocellular carcinoma (HCC) demonstrates significant clinical, Laminin-5 (Ln-5) has recently been reported to be secreted by hepatic stellate cells
phenotypic and genetic heterogeneity. Cell surface markers that can be used to consistently (HSCs), and to enhance the aggressiveness of HCC cells.
identify bulk HCC cells or subpopulations thereof, such as tumor-initiating cells (TICs),
remain poorly defined. Aims: Aim of this study is to investigate the effect of Ln-5 on Sorafeninb effectiveness in
HCC cells.
Aims: We sought to characterize the distribution and frequency of CD antigens expressed
on the surface of primary human HCC cells in an unbiased fashion in order to identify Methodology: HCC cell lines were challenged in vitro with Sorafenib in the presence of
candidate molecules for investigation as markers of bulk HCC cells or HCC TICs. Ln-5 and of HSC conditioned medium.
BASIC POSTER ABSTRACTS had not received any preoperative therapy. We stained the HCC cells with fluorescent Results: Ln-5 and HSC-conditioned medium (CM) strongly reduced the effectiveness of BASIC POSTER ABSTRACTS
Methodology: We obtained fresh human HCC resection specimens from 10 patients who
Sorafenib against the proliferation and apoptosis of different HCC cell lines expressinga3b1
antibodies against 375 unique human CD antigens in 96-well plates. All cells were co-
but not a6b4, the two main Ln-5 receptors. Blocking antibodies against a3b1, but not
stained with anti-human CD45 in order to distinguish leukocytes from tumor cells. CD
antigen expression data was acquired on a BD LSR II flow cytometer equipped with a
a6b4, completely reversed the effect of Ln-5. Transfected HCC cell line a3b1, but not
96-well plate reader and analyzed using FloJo software. Heatmap analysis was performed
the wild type or the scramble control, evaded Sorafenib’s effect in the presence of Ln-
with Multiexperiment Viewer (MeV) software.
5. We found that NF-kB, p-Akt, Akt, ERK1/2, c-Myc, P-53, survivin and p-p38 were not
involved in this mechanism, whereas FAK was phosphorylated by Ln-5 and HSC-CM
Results: We observed significant intertumoral heterogeneity with regards to the fractions
of CD45-negative HCC cells expressing different CD antigens, validating the heterogeneity and de-phosphorylated by Sorafenib. In the presence of Sorafenib, a3b1 positive but not
observed in clinical, histopathological, and genetic studies. However, we were able to negative HCC cells showed FAK phosphorylation if exposed to Ln-5. a3b1 transfected
identify 20 antigens that were consistently expressed in more than 35% of HCC cells HCC cells, but not the wild or scramble type, showed FAK phosphorylation in the presence
in all samples analyzed. We also identified 50 CD antigens expressed consistently in of Sorafenib if Ln-5 was also present
a small fraction of cells (0.5-5%) in all samples analyzed. The vast majority of these
consistently expressed antigens are not currently recognized to play a defined role in Conclusions: To our knowledge this is the first study showing a mechanism of resistance
HCC pathobiology. to Sorafenib, whereby Ln-5 phosphorylates FAK via a3b1, offsetting Sorafenib’s
effectiveness.
Conclusions: High throughput flow cytometry screening is a viable platform for biological
discovery in the context of human HCC. Our further studies are aimed at i) exploring
whether the commonly expressed antigens can be utilized as novel cell surface markers of
bulk HCC cells with potential clinical applicability as novel HCC biomarkers; ii) determining
whether the infrequently expressed markers may represent novel populations of HCC
TICs; and iii) investigating whether defined cell surface marker profiles correlate with
clinical outcomes in the patients from whom the HCC specimens were obtained.