Cells with sphere forming capacity, spheroid cells, are present in the malignant ascites of patients with epithelial ovarian cancer (EOC) and represent a significant impediment to efficacious treatment due to their putative role in progression, metastasis and chemotherapy resistance. The exact mechanisms that underlie EOC metastasis and drug resistance are not clear. Understanding the biology of sphere forming cells may contribute to the identification of novel therapeutic opportunities for metastatic EOC. Here we generated spheroid cells from human ovarian cancer cell lines and primary ovarian cancer. Xenoengraftment of as few as 2000 dissociated spheroid cells into immune-deficient mice allowed full recapitulation of the original tumor, whereas >105 parent tumor cells remained non-tumorigenic. The spheroid cells were found to be enriched for cells with cancer stem cell-like characteristics such as upregulation of stem cell genes, self-renewal, high proliferative and differentiation potential, and high aldehyde dehydrogenase (ALDH) activity. Furthermore, spheroid cells were more aggressive in growth, migration, invasion, scratch recovery, clonogenic survival, anchorage-independent growth, and more resistant to chemotherapy in vitro. 13C-glucose metabolic studies revealed that spheroid cells route glucose predominantly to anaerobic glycolysis and pentose cycle to the detriment of re-routing glucose for anabolic purposes. These metabolic properties of sphere forming cells appear to confer increased resistance to apoptosis and contribute to more aggressive tumor growth. Collectively, we demonstrated that spheroid cells with cancer stem cell-like characteristics contributed to tumor generation, progression and chemotherapy resistance. This study provides insight into the relationship between tumor dissemination and metabolic attributes of human cancer stem cells and has clinical implications for cancer therapy.

It has been reported that levo-1-methyl tryptophan (L-1MT) can block indoleamine-2,3-dioxygenase (IDO) expressed by human dendritic cells (DC), whereas dextro-1-methyl tryptophan (D-1MT) is inefficient. However, whether L-1MT or D-1MT can efficiently reverse IDO-induced arrest of human T-cell proliferation has not been clarified. Here, we show a marked immunosuppressive effect of IDO derived from INDOtransfected 293 cell, IDO + ovarian cancer cells, and monocytederived DCs on CD4 + Th1 cells, CD8 + T cells, and natural killer cells derived from peripheral blood, ascites, and tumors of ovarian cancer patients. We found that, whereas L-1MT and D/L-1MT can restore proliferation of tumor-derived and peripheral blood T-cell subsets, D-1MT does not effectively restore IDO-induced arrest of T-cell proliferation. Although D-1MT inhibited kynurenine production at high concentrations, L-1MT was more effective in abrogating kynurenine generation and tryptophan depletion, whereas tryptophan was completely depleted by IDO even in the presence of high amounts of D-1MT. Together, the results indicate that, whereas the generation of tryptophan metabolites (kynurenines) by IDO is important in mediating suppression of T-cell proliferation, the degree to which tryptophan depletion is restored by 1MT is also critical in overcoming IDO-induced arrest of T-cell proliferation. [Cancer Res 2009;69(13):5498-504]

BackgroundPancreatic cancer is a deadly disease with a very low 5-year patient survival rate of 6–8%. The major challenges of eliminating pancreatic cancer are treatment resistance and stromal barriers to optimal drug access within the tumor. Therefore, effective molecular targeting drugs with high intra-tumor access and retention are urgently needed for managing this devastating disease in the clinic.MethodsThis study has used the following in vitro and in vivo techniques for the investigation of exceptional anticancer drug FL118’s efficacy in treatment of resistant pancreatic cancer: cell culture; immunoblotting analysis to test protein expression; DNA sub-G1 flow cytometry analyses to test cell death; MTT assay to test cell viability; pancreatic cancer stem cell assays (fluorescence microscopy tracing; matrigel assay; CD44-positive cell colony formation assay); human luciferase-labeled pancreatic tumor orthotopic animal model in vivo imaging; pancreatic cancer patient-derived xenograft (PDX) animal models; and toxicology studies with immune-competent BALB/cj mice and beagle dogs.ResultsOur studies found that FL118 alone preferentially killed cisplatin-resistant cancer cells, while a combination of FL118 with cisplatin synergistically killed resistant pancreatic cancer cells and reduced spheroid formation of treatment-resistant pancreatic cancer stem-like cells. Furthermore, using in vivo-imaging, we found that FL118 in combination with cisplatin strongly inhibited both drug-resistant pancreatic xenograft tumor growth and metastasis. In PDX model, we demonstrated that FL118 alone effectively eliminated PDX tumors, while FL118 in combination with gemcitabine eliminated PDX tumors that showed relative resistance (less sensitivity) to treatment with FL118. These FL118 efficacy results are consistent with our molecular-targeting data showing that FL118 inhibited the expression of multiple antiapoptotic proteins (survivin, Mcl-1, XIAP, cIAP2) and ERCC6, a critical regulator of DNA repair, in treatment-resistant pancreatic stem-like cancer cells. Furthermore, FL118 toxicity studies in BALB/cj mice and beagle dogs indicated that FL118 exhibits favorable hematopoietic and biochemical toxicities.ConclusionTogether, our studies suggest that FL118 is a promising anticancer drug for further clinical development to effectively treat drug-resistant pancreatic cancer alone or in combination with other pancreatic cancer chemotherapeutic drugs.

IDO2 is a newly discovered enzyme with 43 % similarity to classical IDO (IDO1) protein and shares the same critical catalytic residues. IDO1 catalyzes the initial and rate-limiting step in the degradation of tryptophan and is a key enzyme in mediating tumor immune tolerance via arrest of T cell proliferation. The role of IDO2 in human T cell immunity remains controversial. Here, we demonstrate that similar to IDO1, IDO2 also degrades tryptophan into kynurenine and is inhibited more efficiently by Levo-1-methyl tryptophan (L-1MT), an IDO1 competitive inhibitor, than by dextro-methyl tryptophan (D-1MT). Although IDO2 enzyme activity is weaker than IDO1, it is less sensitive to 1-MT inhibition than IDO1. Moreover, our results indicate that human CD4(+) and CD8(+) T cell proliferation was inhibited by IDO2, but both L-1MT and D-1MT could not reverse IDO2-mediated arrest of cell proliferation, even at high concentrations. These data indicate that IDO2 is an inhibitory mechanism in human T cell proliferation and support efforts to develop more effective IDO1 and IDO2 inhibitors in order to overcome IDO-mediated immune tolerance.