Differential Effect of Ischemia/Reperfusion Injury on the Primary and Metastatic Growth of Lewis Lung Carcinoma

Abstract

Background: A growing body of evidence points toward an active role of inflammation and ischemia in tumor biology. Reperfusion injury is an inflammatory process involving natural IgM antibodies and complement activation that occurs following acute ischemia of tissues throughout the body. We hypothesized that ischemia-reperfusion (I/R) injury can affect tumor growth and spread. Methods: The Lewis Lung carcinoma (3LL) cell line was implanted by intramuscular injection into the hindlimbs of wild-type C57/BL6 or Rag2 -/- mice, which are deficient in lymphocytes and antibodies. Subsequently, tumor was subjected to varying times of tourniquet induced hind-limb ischemia, followed by tourniquet removal and reperfusion. Control mice underwent hindlimb I/R to the contralateral, non-tumor bearing hindlimb or were sham-treated. Growth of the primary tumor was followed using external caliper measurement and wet weight at sacrifice. Surface lung metastasis were counted using a stereo microscope. Tumor and surrounding hindlimb muscle were examined for histologic signs of injury. Presence of activated caspase-3 and immunoglobulin M was assessed using immunohistochemistry. Statistical analysis was performed using the Wilcoxon test. Results: I/R injury decreased 3LL lung metastasis compared to control, uninjured mice (mean number: 6.9 (sham) vs. 1.4 (I/R), p < .001). Conversely, primary tumor growth was enhanced in I/R treated animals compared to sham controls (mean weight: 9.7 g (sham) vs. 12.9 g (I/R), p < .0001). Control I/R injury to the non-tumor bearing hindlimb had no significant effect on primary (p<0.13) or metastatic (p<0.59) 3LL growth. Following I/R injury, 3LL tumors showed histologic evidence of necrosis and presence of activated caspase-3 staining, a marker of apoptosis. Deposition of IgM on I/R treated but not sham treated 3LL was seen. In 3LL implanted in Rag2 -/- mice, there was no difference in primary tumor growth in I/R injured and sham groups. However, reduction of 3LL lung metastasis following I/R treatment was preserved in Rag2 -/- mice. Conclusions: This study demonstrates that I/R injury can significantly impact tumor behavior in vivo. I/R injury inhibited 3LL lung metastasis in a lymphocyte-independent manner. In contrast, I/R injury enhanced primary 3LL tumor growth in a lymphocyte-dependent manner. These effects correlated histologically with 3LL tumor necrosis and apoptosis following I/R injury. Moreover, altered tumor behavior required the direct, local effect of I/R injury and not its systemic effects alone. Further studies are required to delineate the pathways underlying the differential impact of I/R injury on primary and metastatic tumor growth.