Human glioblastoma cells exposed to long-term hypoxia and nutrient starvation stimulated induction of secondary T-cell leukemia in mice

Secondary leukemia occurs long after remission of the primary tumor with a history of chemo and radiation therapy; however, the mechanism of secondary leukemogenesis is not fully understood. In this study, we demonstrate cases of secondary T-cell leukemia in mice after complete regression of xenograft primary glioblastoma independent of chemo and radiotherapy. Recently, antiangiogenic therapy, often in combination with conventional chemotherapy, has been clinically validated for solid tumors. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are major regulators of angiogenesis; indeed, a monoclonal anti-VEGF antibody (bevacizumab) and small molecular weight inhibitors of VEGFRs (sorafenib and sunitinib) have improved the therapeutic indexes of advanced malignancies, including the increase in disease-free survival in breast cancer, colorectal cancer, non-small cell lung carcinoma, renal cell carcinoma, hepatocellular carcinoma and glioblastoma. However, antitumor effect of anti-VEGF antibody and VEGF receptor inhibitors concomitant with increased invasiveness, and distant metastasis have been reported using preclinical cancer models. In addition, high-grade glioma patients following treatments with anti-VEGF antibody in combination with irinotecan demonstrated an effective response by the primary tumor, but also a high rate of distant tumor progression including tumor infiltration and vascular co-option. We previously reported that long-term hypoxia and nutrient starvation double-deprivation cycles increased cellular migration, invasion and distant metastasis in a murine cancer model, suggesting that this extreme tumor microenvironment could be a mechanism of the tumor aggressiveness following antiangiogenic therapy. Glioblastoma is the most common brain tumor in adults and is often rapidly fatal with median survival of less than a year from diagnosis. In contrast, brain tumors in fetal to young individuals may achieve complete regression of the primary tumor with surgical resection along with standard chemotherapy and/or radiotherapy. A major clinical problem of brain tumors in fetal and young individuals can be recurrence of the primary tumor or development of secondary leukemia years after remission of the primary tumor. We hypothesized that non-tumorigenic human glioblastoma (T98G) cells exposed to hypoxia and nutrient starvation may become aggressive and also affect host cells, causing leukemia. On this basis, we developed a cell culture system to maintain cells under hypoxia and nutrition starvation double deprivationstress (DDS) cycles, which at least partially reflected in vitro the hypoxic and nutrient-starved tumor microenvironment under antiangiogenesis therapy. Using this system, we generated T98G-DDS10 cells exposed to 10 or more cycles of hypoxia and nutrient deprivation stress cycles (Figure 1a), because most cancer cells including T98G cells cannot be maintained under the prolonged hypoxia and nutrient starvation DDS for longer than 72 h in vitro (Figure 1b). To examine whether hypoxia and nutrient starvation facilitate the induction of tumorigenicity in non-tumorigenic T98G cells, 1 10 of T98G-DDS10 cells were subcutaneously inoculated into BALB/c nu/nu nude mice (n1⁄4 5) along with the original T98G cell xenograft in control mice (n1⁄4 5). T98G-DDS10 tumor xenograft mice demonstrated initial formation of tumors (3/5), but not original T98G tumor xenograft mice (0/5) (Figure 1c). The T98G-DDS10 primary tumor showed complete regression within 2 weeks. We examined whether T98G-DDS10 xenograft cells induced recurrence of the tumor or affected host cells to induce leukemia after remission of the primary tumor. T98G-DDS10 xenograft mice were disease free for 1 year, but suddenly, around day 365–375 after inoculation of T98GDDS10, these three mice almost simultaneously lapsed into a cachexic condition with an enlarged spleen, liver, lymph node and invasion of immature lymphocytes in hemorrhagic ascites of the abdominal cavity (Figure 1d, Supplementary Figure 1). Quantitative real-time PCR was performed using human-specific primers against hb-actin and human glial fibrillary acidic protein (hGFAP) for the detection of T98G glioblastoma cells. In addition, mouse-specific primers against T-cell pan-marker mCD3 and mouse B-cell pan-marker mCD19 were used for the detection of mouse-derived secondary leukemia in the spleen and liver tissue specimens of T98G-DDS10 xenograft mice (Supplementary Table 1). As shown in Figure 2a, significant upregulation of mCD3 mRNA levels was observed, but hbactin and hGFAP mRNA was not detected in the spleen or liver tissues of mice with T98G-DDS10 xenograft. The mRNA of mCD19, a B-cell marker, was found at similar levels in the spleens of T98G and T98G-DDS10 xenograft mice, and was markedly low in the livers of these mice compared with normal spleens (positive controls), suggesting that mCD19 expression in leukemic cells is negative at the mRNA level (Figure 2a). Histological examination by hematoxylin-eosin (HE) staining showed massive invasion of leukemic cells associated with scattered mitotic cells into the liver and spleen (Figure 2b). Immunohistochemical staining using anti-mCD3 and hGFAP antibodies was performed in 4% paraformaldehyde-fixed spleen and liver specimens of T98G-DDS10 xenograft mice containing a tumor compared with the spleen and liver of the original T98G xenograft mice. Immunohistochemical staining revealed that the leukemic cells observed in the spleen and liver of T98G-DDS10 xenograft mice were CD3-positive, but GFAPnegative (Figure 2b), suggesting that the tumor had not relapsed with human glioblastoma cells. We suggest that non-tumorigenic T98G human glioblastoma cells become aggressive and stimulate the induction of T-cell leukemia following long-term hypoxia and nutrient starvation DDS. Amplification or insertion of a human oncogene can result in the development of secondary leukemia. To examine whether oncogenes derived from T98G-DDS10 cells were integrated into the leukemic cells, PCR amplification of the human Alu-repetitive sequence, which is thought to be co-integrated with ascertain human genes, was conducted using a spleen with leukemic cells of T98G-DDS10 xenograft cachexia mice and Citation: Blood Cancer Journal (2011) 1, e6; doi:10.1038/bcj.2011.5 & 2011 Macmillan Publishers Limited All rights reserved 2044-5385/11