Abstracts
1999 Digestive Disease Week

# 2147 HYPOXIA INCREASES THE MALIGNANT POTENTIAL OF PANCREATIC CANCER
H G Hotz, O J Hines, P Buchler, N Ryder, T Yu, B Hotz, Howard A Reber, UCLA Sch of Medicine, Los Angeles, CA

Tissue hypoxia affects the behavior of cancer cells: hypoxic tumors are resistant to radiation and chemotherapy, subpopulations of hypoxic cells show a higher metastatic potential, and hypoxia stimulates neoangiogenesis via production of factors like vascular endothelial growth factor (VEGF). Pancreatic cancer is typically a scirrhous tumor surrounded by chronic pancreatitis, conditions known to be associated with hypoxia. Thus the aim of the present study was to evaluate the effect of hypoxia on human pancreatic cancer in-vitro and in-vivo. Methods: The two human pancreatic cancer cell lines, MIAPaCa-2 (MP; undifferentiated) and Capan-1 (C1; moderately differentiated), were either exposed to 48 h of hypoxia (0% 02,5% CO2) in a modular chamber or grown under normoxic conditions for 48 h. Proliferation was assessed by counting the cell numbers in a standard hemocytometer after treatment. Cell viability was assayed by the MTT-assay. Cell cycle phase distribution and apoptosis were analyzed by a FACScan flow cytometer. The concentration of VEGF in the cell culture media was assessed by an ELISA. To assess the in-vivo effects of hypoxia, 107 cells of either cell line were subcutaneously (SC) or orthotopically (OT) injected into nude mice (n=8, each) after pretreatment with hypoxia or normoxia. The animals were sacrificed after 14 weeks or when clinical signs of massive tumor burden (e.g. cachexia, ascites) occurred. The volume of the primary tumor was calculated and size and site of local infiltration and systemic spread were measured. Results: Cell cycle analysis revealed a significant increase of the S-phase after hypoxic treatment (Normoxia: 22%; Hypoxia: 41%) in the C1 cells whereas the G0/G1-phase was significantly decreased (Normoxia: 73%; Hypoxia: 55%). The cell cycle phase distribution was not significantly altered in MP cells. Hypoxia increased apoptosis significantly in C1 cells (Normoxia: 2.7%, Hypoxia: 9.6%), and slightly in MP cells (Normoxia: 4.2%, Hypoxia: 5.5%). Conclusions: Despite a reduction of in-vitro proliferation and viability of pancreatic cancer cells, hypoxic treatment selects cells that are more aggressive in-vivo than cells under normoxic conditions. Changes in cell cycle phase distribution, apoptosis, or VEGF-production may be underlying mechanisms of this in-vivo effect. These experimental data support the hypothesis that hypoxic conditions in pancreatic cancer tissue are an important factor for the aggressiveness of this disease.

In-vitro	Proliferation (%; Normoxia=100)		Viabilit (%; Normoxia=100)		VEGF-expression (%, Normoxia=100)
(*p<0.05)	MP	C1	MP	C1	MP	C1
Hypoxia	72.7*	55.7*	51.0*	70.3*	244*	435*
In-vivo (*p<0.05)	Tumor Growth (mm3)         Metastatic score
	C1 SC	MP SC	C1 OT	MP OT	C1 OT	MP OT
Normoxia	40±5	261±68	37±31	2899±435	0.8±0.3	4.8±1.4
Hypoxia	64±9*	476±71*	195±86	4911±887	3.4±0.9*	3.1±1.7

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