Preferential killing of multidrug-resistant KB cells by inhibitors of glucosylceramide synthase

This study has compared the preferential killing of three multidrug-resistant (MDR) KB cell lines, KB-C1, KB-A1 and KB-V1 by two inhibitors of glucosylceramide synthase, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP), to the killing produced by these compounds in the drug-sensitive cell line, KB-3-1. Both of the inhibitors caused much greater induction of apoptosis in each of the three MDR cell lines than in the drug-sensitive cell line, as judged by morphological assay and confirmed by poly-(ADP-ribose)-polymerase cleavage. The highest level of apoptosis was produced following 24-h exposure to 5 μM PPPP. This treatment produced 75.8 (± 7.1)%, 73.6 (± 9.8)% and 75.3 (± 6.4)% apoptotic cells in the three MDR cell lines respectively, compared to 19.0 (± 9.8)% in the drug-sensitive cell line. A reduction in glucosylceramide level following inhibitor treatment occurred in KB-3-1 cells as well as in the MDR cell lines, suggesting that the increased apoptotic response in the MDR cells reflected a different downstream response to changes in the levels of this lipid in these cells compared to that in the drug-sensitive cells. These results suggest that the manipulation of glucosylceramide levels may be a fruitful way of causing the preferential killing of MDR cells in vitro and possibly in vivo. © 1999 Cancer Research Campaig

Abstract P3-02-02: Influence of Body Mass Index on Presence of Disseminated Tumor Cells in Clinical Stage I-III Breast Cancer Patients

Abstract Background: Disseminated tumor cells (DTCs) are found in approximately one third of clinical stage I-III breast cancer (BC) patients, and published reports show that presence of DTCs is an independent predictor of outcome. While higher body mass index (BMI) is associated with increased risk of breast cancer recurrence and lower survival rates in BC patients, women with lower BMIs may have lower bone density and higher bone turnover. We hypothesized that increases in bone turnover may result in the release of bone growth and “homing” factors that facilitate BC metastasis to bone and provide a “pre-metastatic niche” for BC cells. The purpose of this study was to determine if a correlation existed between DTCs and BMI in early stage BC patients. Methods: We obtained informed consent and collected bone marrow samples from 262 clinical stage I-III BC patients who were participants in an IRB-approved clinical study from 2/2005- 2/2010. All marrow samples were collected at the time of surgery for the primary tumor. DTCs were assessed using anti-pancytokeratin (CK) antibody cocktail (AE1/AE3, CAM5.2, MNF116, CK8 and 18) following cytospin. The presence of one or more CK positive cells meeting morphologic criteria for malignancy was considered a positive result for DTC. Patients with a BMI of (18.5 — 24.9) kg/m2 were considered “normal weight”, those with a BMI of (25 - 29.9) kg/m2 “overweight” and a BMI greater than 30 kg/m2 was used to designate them as “obese”. Information on clinicopathological factors including BMI (measured on initial presentation) was obtained from a prospective database. Statistical analyses used Chi-square and non-parametric tests for trend. Results: Median follow-up was 19 months and mean age was 53 (25-80) years. Eighty-four patients (32%) were normal weight, 85 (32%) were overweight and 91 (35%) were obese. Seventy-eight (30%) patients had DTCs present at the time of assessment. Obese patients were significantly less likely to show presence of DTCs as compared to those who had a BMI &amp;lt; 30 kg/m2 (20/78; 26% vs. 71/184; 39%) {O.R. = 0.55, 95% C.I. = 0.29- 0.96, P = 0.03}. DTCs were also less likely to be found in patients with BMI ≥25 kg/m2 as compared to those with BMI &amp;lt; 25 kg/m2 (40/78; 51% vs. 136/184; 74%); {O.R. = 0.42, 95% C.I. = 0.04- 1.02, P = 0.03}. No statistically significant correlation was observed between primary tumor characteristics (ER, PR, HER2, lymph node status, tumor grade) and presence of DTCs. Finally, a non-parametric analysis demonstrated a trend in occurrence of DTCs across the ordered levels of patients’ BMI values (P= 0.013). Conclusions: DTCs were much more common in patients with lower BMI. Further studies are needed to determine if patients with low BMI have unique microenvironmental factors within the bone that predisposes them to tumor cell dissemination. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-02.</jats:p

Abstract P3-02-01: Is Ethnicity a Predictor of Micrometastatic Disease in Early Stage Breast Cancer Patients?

Abstract Background: Ethnicity plays a role in breast cancer (BC) outcome, highlighted by the fact that African-American women have a higher BC mortality rate than do Caucasian women. Microscopic disease, including disseminated tumor cells (DTCs) in bone marrow and circulating tumor cells (CTCs) in peripheral blood, has been shown to predict worse outcomes as well. We sought to determine whether ethnicity was a significant predictor for the presence of DTCs and/or CTCs in stage I-III BC patients. Methods: Patients provided informed consent to participate in an IRB-approved study involving collection of blood and bone marrow at the time of surgery for their primary BC. CTCs (per 7.5 ml blood) were detected using the Cell SearchTM system (Veridex) and were defined as nucleated cells lacking CD45 but expressing cytokeratins (CK) 8, 18, or 19; for this study we considered one or more positive cells meeting these criteria a positive result. DTCs were assessed using an anti-CK antibody cocktail (AE1/AE3, CAM5.2, MNF116, CK8 and 18) following cytospin. A positive result for DTCs was defined by presence of one or more CK positive cells meeting morphologic criteria for malignancy. Information on clinicopathological factors including ethnicity was obtained from a prospective database. Statistical analyses used Chi-square test on STATA IC11. Results: We prospectively evaluated 224 patients undergoing surgery for stage I-III BC. Median follow-up was 22 months and mean age was 53 years. One hundred sixty seven patients (75%) were Caucasians, 22 (10%) were African-American (AA), 30 (14%) were Hispanic and 3 (1%) belonged to other ethnicities. CTCs were found in 25% (57/224) and DTCs in 30% (67/224) of patients. Patients of AA ethnicity were significantly more likely to have CTCs (50%, (11/22)) compared to the other ethnic groups (22%, (43/194)); {O.R. = 2.5, 95% C.I. = 1.35- 7.80, P = 0.002}, and had significantly higher numbers of CTCs (≥2 CTCs or ≥3 CTCs/7.5mL blood) than other ethnic groups (P = 0.001 and P &amp;lt; 0.001, respectively). No statistically significant correlation was observed between other ethnic groups and CTCs. Patients of Hispanic origin were more likely to have DTCs (60%, (18/30)) as compared to other ethnic groups (25%, (49/194)); {O.R. = 4.4, 95% C.I. = 1.85- 10.80, P &amp;lt; 0.0001}, while DTCs were less likely to be found in Caucasians (26%, (44/167)) as compared to the other ethnicities (40%, (23/57)); {O.R. = 0.52, 95% C.I. = 0.27 — 1.05, P = 0.046}. No significant association was found between occurrence of DTCs and AA ethnicity. In a multivariate analysis considering lymph node status, tumor size and tumor markers, ethnic origin was an independent predictor of microscopic disease. Conclusions: Nearly one-third of primary BC patients have CTCs and/or DTCs. African-American women were much more likely to have CTCs and Hispanic patients had significantly more DTCs than did patients of other ethnicities. Ethnicity was an independent predictor of microscopic disease. These findings may shed some light on the higher BC mortality rates found in certain ethnic groups. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-01.</jats:p

P4-06-04: Detection of HER2 Gene Amplification in Circulating Tumor Cells and Disseminated Tumor Cells by Fluorescence In Situ Hybridization Using OncoCEE™.

Abstract BACKGROUND: The status of HER2 gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might provide useful information for monitoring response to trastuzumab therapy, and may provide a basis for consideration of trastuzumab in patients with HER2 negative primary tumors who have HER2 positive CTCs and/or DTCs. The majority of techniques utilized for detection of minimal residual disease are limited in their ability to allow detailed phenotypic and genotypic evaluation of the cells. We report the utility of a microfluidic platform (OncoCEE™,Biocept, San Diego) for detecting HER2 gene amplification in CTCs and DTCs in patients with non-metastatic breast cancer. METHODS: Peripheral blood (10ml) and bone marrow (BM) (1-2ml) were collected from patients with clinical stage I-III breast cancer in acid citrate dextrose solution (BD, Franklin Lakes, NJ) and anti-clumping reagent (OncoCEE-Sure™). Mononuclear cells were recovered using a Percoll density gradient method, incubated with a mixture of 10 primary capture antibodies (Abs), introduced into CEE™ microchannels, stained with fluorescent anti cytokeratin (CK) and CD45 abs and finally processed for fluorescence in situ hybridization (FISH) using probes specific to centromere 17 (spectrum green) and HER2 (spectrum arrange). The ratio of HER2:CEP17 &amp;gt;2.2 in any CK+/CD45- and CK-/CD45- cell was regarded as positive for HER2 gene amplification. RESULTS: Peripheral blood and/or BM from 78 patients (65 BM; 70 blood; 57 matched blood and BM) with T1NO (39), T1N1 (8), T2N0 (12), T2N1 (2), T2N2 (1), T2N3 (3), T3N0 (2), T3N1 (2), T3N2 (1), T4N0(5), T4N1 (3), with HER2+ (n=12) and HER2− (n=58) primary invasive breast tumors were studied. The 12 patients with HER2+ primary tumors had HER2+ DTCs in 3/12 (25%) and HER2+ CTCs in 1/9 (11%) cases respectively. HER2+ DTCs and HER2+ CTCs occurred in 12/55 (24%) and in 4/63 (6%) of the patients with HER2− primary breast tumors. HER2+ CTCs and DTCs occurred simultaneously in only 2 patients and in either blood (3) or BM (13) in the remaining patients. CONCLUSION: 1. The cell enrichment and extraction microfluidic technology (OncoCEE™) provides a sensitive platform for evaluation of HER2 gene amplification of CTCs and DTCs. 2. HER2+ primary tumors were associated with either HER2+ CTCs or DTCs in 25% of the patients. 3. HER2+ CTCs or DTCs occurred in 28% of patients with HER2−primary tumor. 4. Discordant HER2 status was contributed mainly by HER2+ DTCs occurring in HER2 - primary tumors. 5. The clinical significance of evaluating the status of HER2 gene amplification in CTCs and DTCs in the management of patients with breast cancer needs to be evaluated prospectively in larger clinical trials. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-06-04.</jats:p

Novel inflammatory breast cancer cell line, MDA-IBC-1 expresses and secretes WISP3, a putative tumor suppressor in inflammatory breast cancer.

Abstract Abstract #1051 Wnt-1-induced secreted protein 3 (WISP3) is a member of the CCN family of proteins, which also includes connective tissue growth factor (CTGF), Cy61, Nov, WISP1, and WISP2. WISP3, as well as the other CCN family members, plays important roles in cellular processes such as proliferation, migration, and survival. WISP3 was originally isolated as Lost in Inflammatory Breast Cancer (LIBC) based on its expression in only 20% of human IBC tumors. Lack of WISP3 expression has also been reported in the IBC-derived cell line SUM-149. In SUM149 cells WISP3 has been reported to function as a tumor suppressor capable of decreasing proliferation and maintaining an epithelial phenotype. Here we examine the expression of WISP3 in a novel aggressive IBC cell line with epithelial-mesenchymal transition (EMT) morphology and abundant WISP3 expression. MDA-IBC-1 cells were isolated from the pleural fluid of a patient with ER+, PR-, HER2/neu- IBC. In 2-dimensional (2D) adherent culture, MDA-IBC-1 cells exhibit fibroblast-like EMT morphology and express EMT-associated proteins such as N-cadherin, vimentin, and fibronectin. Correspondingly, E-cadherin protein expression is absent in 2D cultures. MDA-IBC-1 cells derived from 2D cultures form mammospheres when cultured in 3-dimensional (3D) progenitor promoting conditions. Interestingly, the expression of EMT molecular markers is absent in 3D progenitor promoting mammosphere culture. MDA-IBC-1 cells exhibit robust WISP3 protein expression in both 2D and 3D culture (1.8-fold higher in 3D). In addition, MDA-IBC-1 cells secrete WISP3 as indicated by the presence of WISP3 protein in MDA-IBC-1 derived conditioned media. WISP3 binding is reported to decrease insulin-like growth factor-1 (IGF-1) activation of the IGF1R signaling cascade in SUM-149 cells. IGFR1 protein is present in 2D and 3D cultured MDA-IBC-1 cells. IGF-1 protein overlay assays using conditioned media demonstrate a WISP3:IGF-1 interaction, suggesting functional folding. Serum-free MDA-IBC-1 conditioned media did not increase basal SUM149 proliferation and it inhibited IGF-1 stimulated proliferation to serum-free (basal) levels (p≤0.05). In addition, culturing SUM-149 cells in conditioned media obtained from IBC-1 cells significantly alters SUM-149 to a completely fibroblastic morphology. These data demonstrate that MDA-IBC-1 cells represent an IBC cell line with novel expression and regulation of WISP3. MDA-IBC-1 cells may better represent the cohort of IBC patients whose tumors express WISP3. This data suggest WISP3 may not function as a tumor suppressor in all IBC patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1051.</jats:p

Populations of cells exhibiting epithelial-mesenchymal transition (EMT) or stem cell characteristics can be propagated from a single inflammatory breast cancer (IBC) cell line based on culture conditions.

Abstract Abstract #103 We have previously reported the isolation of a novel IBC cell line (MDA-IBC-1) from human pleural effusion fluid by passaging cells under 3-dimensional (3D) mammosphere (progenitor-promoting) conditions and isolating attached 2-dimensional (2D) cells from ultralow attachment plates. Mammosphere culture is serum-free and growth factor enriched. Serum-starving 2D cells leads to spontaneous formation of floating mammospheres with the same percentage of CD44+24- cells as 3D cultured mammospheres. 2D cells display distinct fibroblastic morphology. Here we report that passage in 2D (standard) or 3D (mammosphere) culture selects for different molecular phenotypes: EMT in 2D and stem/progenitor-like in 3D. 3D cells are generated from 2D cells for all experiments and are examined at second passage. RT-PCR reveals embryonic stem cell pluripotency factors Oct-4, Nanog, Sox-2 and Rex-1 are expressed in 3D but not 2D MDA-IBC-1 cells. MicroRNAs reported to be involved in self-renewal of breast cancer cells including Let-7a,b,c,d,e and f are decreased in 3D cultures (&amp;gt;5 fold decrease) compared to 2D. MiR16, 21, 125b and 200c are also markedly reduced in 3D vs. 2D cells. On the other hand, EMT proteins, N-cadherin, vimentin, and fibronectin are only expressed in 2D culture and higher expression of snail and slug is observed in 2D compared to 3D. Correspondingly, E-cadherin is absent from 2D cells. Moreover, Notch1, a putative stem cell signaling pathway protein that has been shown to promote EMT by snail induction is upregulated in 2D cells compared to 3D, and in response to radiation in both 2D and 3D. It has been reported in some systems that EMT promotion can induce stem cells. The data herein suggest the MDA-IBC-1 maintains pluripotent cells capable of both EMT and stem-like phenotypes. MDA-IBC-1 provides a unique model to examine the interaction between stem cells and EMT and to examine the mechanisms of resistance and proliferation of both cell types. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 103.</jats:p

Predicting treatment Response based on Dual assessment of magnetic resonance Imaging kinetics and Circulating Tumor cells in patients with Head and Neck cancer (PREDICT-HN): matching 'liquid biopsy' and quantitative tumor modeling

BACKGROUND: Magnetic resonance imaging (MRI) has improved capacity to visualize tumor and soft tissue involvement in head and neck cancers. Using advanced MRI, we can interrogate cell density using diffusion weighted imaging, a quantitative imaging that can be used during radiotherapy, when diffuse inflammatory reaction precludes PET imaging, and can assist with target delineation as well. Correlation of circulating tumor cells (CTCs) measurements with 3D quantitative tumor characterization could potentially allow selective, patient-specific response-adapted escalation or de-escalation of local therapy, and improve the therapeutic ratio, curing the greatest number of patients with the least toxicity. METHODS: The proposed study is designed as a prospective observational study and will collect pretreatment CT, MRI and PET/CT images, weekly serial MR imaging during RT and post treatment CT, MRI and PET/CT images. In addition, blood sample will be collected for biomarker analysis at those time intervals. CTC assessments will be performed on the CellSave tube using the FDA-approved CellSearch® Circulating Tumor Cell Kit (Janssen Diagnostics), and plasma from the EDTA blood samples will be collected, labeled with a de-identifying number, and stored at - 80 °C for future analyses. DISCUSSION: The primary objective of the study is to evaluate the prognostic value and correlation of weekly tumor response kinetics (gross tumor volume and MR signal changes) and circulating tumor cells of mucosal head and neck cancers during radiation therapy using MRI in predicting treatment response and clinical outcomes. This study will provide landmark information as to the utility of CTCs ('liquid biopsy) and tumor-specific functional quantitative imaging changes during treatment to guide personalization of treatment for future patients. Combining the biological information from CTCs and the structural information from MRI may provide more information than either modality alone. In addition, this study could potentially allow us to determine the optimal time to obtain MR imaging and/ or CTCs during radiotherapy to assess tumor response and provide guidance for patient selection and stratification for future dose escalation or de-escalation strategies. TRIAL REGISTRATION: Clinicaltrials.gov ( NCT03491176 ). Date of registration: 9th April 2018. (retrospectively registered). Date of enrolment of the first participant: 30th May 2017