Abstract 2063: Fate of hypoxic cancer cells in microscopic and fully developed macroscopic tumors
Authors: Li Xiao-Feng , Ma Yuanyuan , O'Donoghue Joseph 
Published: July 2012

Abstract
We previously reported, in mouse cancer models of human colorectal cancer HT29 cells, microscopic tumors (&lt;1 mm) were severe hypoxia and poor perfusion, in contrast, larger ones (1-4 mm) were well-perfused and not significantly hypoxic, as tumors increased in size beyond this level, the typical pattern of macroscopic tumor hypoxia reappeared (Cancer Res, 2007; 67: 7646). The fate of hypoxic cells at each stage of cancer development is largely unknown. This issue is critically important for planning anticancer therapy; hypoxia induces cancer therapy resistance.
Methods: We injected HT29 cells into peritoneal cavity, ascites tumors (floating in ascites, in dormant status and &lt;1mm in diameter) were evident 4-5 wk later, ascites tumors were harvested 1-7 d followed exogenous hypoxia marker pimonidazole (PIMO) administration, the spatial localization of PIMO adducts visualized by histochemical staining and their relating to viable cells or cell debris in necrosis was used to estimate the lifespan of hypoxic cancer cells, fate of hypoxic cells was also observed in fully developed HT29 subcutaneous xenografts (∼10 mm in diameter). Changes in hypoxia during early stage of tumor development were investigated in an intradermal tumor model using dual hypoxia markers staining technique: various days after cancer cells inoculation (i.e. various size of tumors), we injected PIMO to hosts 1h before their sacrifice to label “current” hypoxic cells, this compared with hypoxia induced protein carbonic anhydrase 9 (CA9) expression, CA9 is able to mark “historic” hypoxia because it has 3 days half-life even after reoxygenation. Stroma formation and angiogenesis were also observed.
Results: PIMO adducts closely associated with viable cancer cells in dormant ascites tumors 7 d followed PIMO injection, there was little necrotic tissue in ascites tumors. However, Major PIMO adducts were in necrotic zones 2-3 d later in fully developed macroscopic xenografts. Changes in hypoxia during early stage of tumor development were investigated: 3 to 5 d after tumor initiation, a sole cluster of pure cancer cells (&lt;1 mm) was found, majority of cancer cells had high PIMO binding and high CA9 expression. Seven days after cell inoculation when tumors reached to 2-3 mm in diameter, cancer cells had little PIMO binding but still strong CA9 expression, abundant stroma along with functional blood vessels were evident in the tumors, Which indicated previous hypoxic cells had been recently oxygenated due to stroma formation/angiogenesis switch.
Conclusion: Hypoxic cancer cells in submillimeter dormant microscopic tumors have very long lifespan, which maybe oxygenate after angiogenesis and tumor growth. Hypoxic cell in fully developed tumors generally go to necrosis. Hypoxia is critical important in microscopic tumors, hypoxic cells may resist cancer therapies and microscopic tumors develop to macroscopic tumors in a future time.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2063. doi:10.1158/1538-7445.AM2011-2063