Cervical cancers, a malignancy associated with oncogenic papilloma viruses, remain a major disease burden in the absence of effective implementation of preventive strategies. CD66+ cells have previously been identified as a tumor-propagating subset in cervical cancers. We investigated the existence, differentiation state, and neoplastic potential of CD66+ cells in a precancer cell line harboring HPV31b episomes. The gene expression profile of CD66high cells overlaps with differentiated keratinocytes, neoplastic mesenchymal transition, cells of the squamocolumnar junction, and cervical cancer cell line–derived spheroids. There is elevated expression of DNMT1, Notch1, and the viral gene product E1⁁E4 in CD66high cells. Thus, CD66high cells, in the absence of differentiating signals, express higher levels of key regulators of keratinocytes stemness, differentiation, and the viral life cycle, respectively. We also find a striking association of neoplastic traits, including migration, invasion, and colony formation, in soft agar with CD66high cells. These properties and a distinct G2–M–enriched cell-cycle profile are conserved in cells from cervical cancers. Principally, using a precancerous cell line, we propose that CD66high cells have an intermediate differentiation state, with a cellular milieu connected with both viral replication and neoplastic potential, and validate some key features in precancer lesions. Such pathophysiologically relevant systems for defining cellular changes in the early phases of the disease process provide both mechanistic insight and potential therapeutic strategies. Collectively, our data provide a rationale for exploring novel therapeutic targets in CD66+ subsets during cancer progression. Cancer Res; 74(22); 1–11. ©2014 AACR.

Cervical cancers, a malignancy associated with oncogenic papillomaviruses, remain a major disease burden in the absence of effective implementation of preventive strategies. CD66+ cells have previously been identified as a tumor propagating subset in cervical cancers. We investigated the existence, differentiation state and neoplastic potential of CD66+ cells in a pre-cancer cell line harboring HPV31b episomes. The gene expression profile of CD66High cells overlaps with differentiated keratinocytes, neoplastic mesenchymal transition, cells of the squamo-columnar junction and cervical cancer cell line derived spheroids. There is elevated expression of DNMT1, Notch1 and the viral gene product E1^E4, in CD66High cells. Thus CD66High cells, in the absence of differentiating signals, express higher levels of key regulators of keratinocytes stemness, differentiation and the viral life cycle respectively. We also find a striking association of neoplastic traits including migration, invasion and colony formation in soft agar with CD66High cells. These properties and a distinct G2M enriched cell cycle profile are conserved in cells from cervical cancers. Principally, using a precancerous cell line, we propose that CD66High cells have an intermediate differentiation state with a cellular milieu connected with both viral replication and neoplastic potential and validate some key features in pre-cancer lesions. Such pathophysiologically relevant systems for defining cellular changes in the early phases of the disease process provide both mechanistic insight and potential therapeutic strategies. Collectively, our data provides a rationale for exploring novel therapeutic targets in CD66+ sub-sets during cancer progression.

Stage III/IV melanoma remains incurable in most cases due to chemotherapeutic resistance. Thus, predicting and monitoring chemotherapeutic responses in this setting offer great interest. To overcome limitations of existing assays in evaluating the chemosensitivity of dissociated tumor cells, we developed a label-free monitoring system to directly analyze the chemosensitivity of undissociated tumor tissue. Using a preparation of tumor micro-fragments (TMF) established from melanoma biopsies, we characterized the tissue organization and biomarker expression by immunocytochemistry. Robust generation of TMF was established successfully and demonstrated on a broad range of primary melanoma tumors and tumor metastases. Organization and biomarker expression within the TMF were highly comparable with tumor tissue, in contrast to dissociated, cultivated tumor cells. Using isolated TMF, sensitivity to six clinically relevant chemotherapeutic drugs (dacarbazine, doxorubicin, paclitaxel, cisplatin, gemcitabine, and treosulfan) was determined by impedance spectroscopy in combination with a unique microcavity array technology we developed. In parallel, comparative analyses were performed on monolayer tumor cell cultures. Lastly, we determined the efficacy of chemotherapeutic agents on TMF by impedance spectroscopy to obtain individual chemosensitivity patterns. Our results demonstrated nonpredictable differences in the reaction of tumor cells to chemotherapy in TMF by comparison with dissociated, cultivated tumor cells. Our direct impedimetric analysis of melanoma biopsies offers a direct ex vivo system to more reliably predict patient-specific chemosensitivity patterns and to monitor antitumor efficacy. Cancer Res; 74(22); 1–11. ©2014 AACR.

Stage III/IV melanoma remains incurable in most cases due to chemotherapeutic resistance. Thus, predicting and monitoring chemotherapeutic responses in this setting offer great interest. To overcome limitations of existing assays in evaluating the chemosensitivity of dissociated tumor cells, we developed a label-free monitoring system to directly analyze the chemosensitivity of undissociated tumor tissue. Using a preparation of tumor micro-fragments (TMF) established from melanoma biopsies, we characterized the tissue organization and biomarker expression by immunocytochemistry. Robust generation of TMF was established successfully and demonstrated on a broad range of primary melanoma tumors and tumor metastases. Organization and biomarker expression within the TMF was highly comparable to tumor tissue, in contrast to dissociated, cultivated tumor cells. Using isolated TMF, sensitivity to six clinically relevant chemotherapeutic drugs (dacarbazine, doxorubicin, paclitaxel, cisplatin, gemcitabine, treosulfan) were determined by impedance spectroscopy in combination with a unique microcavity array technology we developed. In parallel, comparative analyses were performed on monolayer tumor cell cultures. Lastly, we determined the efficacy of chemotherapeutic agents on TMF by impedance spectroscopy to obtain individual chemosensitivity patterns. Our results demonstrated non-predictable differences in the reaction of tumor cells to chemotherapy in TMF by comparison to dissociated, cultivated tumor cells. Our direct impedimetric analysis of melanoma biopsies offers a direct ex vivo system to more reliably predict patient-specific chemosensitivity patterns and to monitor antitumor efficacy.

Metabolic rearrangements subsequent to malignant transformation are not well characterized in endometrial cancer. Identification of altered metabolites could facilitate imaging-guided diagnosis, treatment surveillance, and help to identify new therapeutic options. Here, we used high-resolution magic angle spinning magnetic resonance mass spectroscopy on endometrial cancer surgical specimens and normal endometrial tissue to investigate the key modulators that might explain metabolic changes, incorporating additional investigations using qRT-PCR, Western blotting, tissue microarrays (TMA), and uptake assays of [3H]-labeled choline. Lipid metabolism was severely dysregulated in endometrial cancer with various amino acids, inositols, nucleobases, and glutathione also altered. Among the most important lipid-related alterations were increased phosphocholine levels (increased 70% in endometrial cancer). Mechanistic investigations revealed that changes were not due to altered choline transporter expression, but rather due to increased expression of choline kinase α (CHKA) and an activated deacylation pathway, as indicated by upregulated expression of the catabolic enzymes LYPLA1, LYPLA2, and GPCPD1. We confirmed the significance of CHKA overexpression on a TMA, including a large series of endometrial hyperplasia, atypical hyperplasia, and adenocarcinoma tissues, supporting a role for CHKA in malignant transformation. Finally, we documented several-fold increases in the uptake of [3H]choline in endometrial cancer cell lines compared with normal endometrial stromal cells. Our results validate deregulated choline biochemistry as an important source of noninvasive imaging biomarkers for endometrial cancer. Cancer Res; 74(23); 1–11. ©2014 AACR.