P1-03-02: The Norton-Simon Hypothesis and Cancer Stem Cells: How Cancer Stem Cells May Explain the Effectiveness of Dose-Dense Chemotherapy.
Authors: Landis MD , Dobrolecki LE , Wong H , Lai Q , Vahdat LT , Chang JC 
Published: June 2012

Abstract
Background: Systemic therapies are effective initially in controlling and reversing tumor growth; however, residual cancers will invariably re-grow despite this initial response. We have published data from paired human breast cancer samples that standard therapy every three weeks kills dividing daughter cells but not tumor-initiating cells (TICs), so that samples obtained after therapy are enriched for CD44+/CD24−/low putative “tumor-initiating” or “cancer stem” cells, indicating that standard treatment regimens are missing the critical targets, TICs. Interestingly, we have recent data in human breast tumors that indicate that TICs may in fact be chemosensitive initially, with a decrease in TICs observed within two days of chemotherapy, but shortly thereafter, TICS are actually induced by chemotherapy.
Materials and Methods: To evaluate TICs response to chemotherapy, mice with human breast tumor xenograft lines BCM-2665a and BCM-2147 were treated with vehicle, 10-, or 33-mg/kg docetaxel, and then tumors were collected for TIC assays and molecular analysis at both 48 and 72h after treatment. Using Affymetrix gene expression microarrays and reverse phase protein array (RPPA) analysis with 119 different validated antibodies, we identified pathways involved in regulation of TICs. Results: According to flow cytometric analysis for TIC markers and mammosphere (MS) formation efficiency, BCM-2665 TICs were reduced by docetaxel treatment compared to vehicle-treated at 48h post 10 mg/kg docetaxel (4-fold decrease) and 48 and 72h post 33 mg/kg docetaxel compared to control (14- and 2-fold decrease, respectively). Although the BCM-2147 TICs did not significantly decrease at any time points or doses tested, they were clearly induced at 72h post-treatment compared to control. Additionally, BCM-2665 TICs were increased within 72h post 10 mg/kg docetaxel, indicating that these time points are ideal for defining the mechanisms responsible for induction of TICs. Ingenuity Pathway Analysis of Affymetrix microarray data for both BCM-2665 and BCM-2147 revealed induction of inflammatory pathways, suggesting leukocyte infiltration associated with induction of TICs. Furthermore, RPPA analysis confirmed gene expression changes from the microarray data, and implicated apoptosis and inflammatory pathways. Sixteen of 28 proteins significantly changed with activation of CSC are involved in development of leukocytes. Discussion: These findings are consistent with the Norton-Simon Hypothesis in that chemotherapy regimens given more frequently may in fact eliminate TICs, thereby explaining the proven increased effectiveness of dose-dense chemotherapy. Based on when TICs became chemoresistant, we are comparing dose-dense treatment (4 mg/kg docetaxel every 3 days) to a traditional single dose of 32 mg/kg, in an effort to eliminate the tumor cells that cause tumor recurrence. Furthermore, our analysis of gene expression at both the RNA and protein level implicated the immune cells as TICs inducers. Since our immunocompromised mice lack T- and B- cells but have active macrophages, macrophages are indicated as inducers of TICs. We are focusing our current efforts at identifying how immune cells activate TICs and thus enhance tumorigenesis.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-02.