Identification and Characterization of Tumour Initiating Cells From Gynaecological Malignancies

In the first part of the study, I developed a model to study the CSC/TIC population of the A431 cell line based on the capability of these cells to form spheres in suspension. Putative A431 CSC/TIC were characterized for ‘stemness’ properties such as self-renewal and clone forming capability, presence of a SP and ALDH enzymatic activity, putative stem cell marker expression and in vivo tumorigenicity. The results indicate that the growth of A431 cells, as spheres, was not sufficient by itself to define a stem like population, but it was essential for the emergence of a small population of tumour cells with CSC properties. Then, I investigated CSC/TIC in Epithelial Ovarian Cancer (EOC), with a focus on CD133 and CXCR4 molecules. In a wide range of primary EOC and ascites the expression of CD133 and CXCR4 was found restricted within two separate subpopulations of tumour cells. Enrichment in mRNA levels of OCT4, NANOG and NESTIN was observed with good reproducibility in CXCR4 positive cells, whereas it was more variable in CD133 positive cells. Real Time analyses of ABC transporter expression showed there was a slight increase in CXCR4 positive cells while in CD133 positive cells this increase was much more evident, revealing a clear difference between CXCR4 and CD133 subpopulations in term of chemoresistance potential. Furthermore positive cells isolated from xenograft-derived ascites and sorted for CXCR4 and CD133 were not characterized by higher tumorigenic capacity. In summary, no correlation between CD133 positivity and the ovarian cancer sternness phenotype was found. However detection of CD133 positive cells may be useful to predict the efficacy of specific cytotoxic therapy. On the contrary, CXCR4 positive cells may identify those tumour cells which maintain a partially un-differentiated state (high levels of stem cell marker expression) and possibly responsible for tumour invasion and metastasis

Thymic Epithelium Abnormalities in DiGeorge and Down Syndrome Patients Contribute to Dysregulation in T Cell Development

The thymus plays a fundamental role in establishing and maintaining central and peripheral tolerance and defects in thymic architecture or AIRE expression result in the development of autoreactive lymphocytes. Patients with partial DiGeorge Syndrome (pDGS) and Down Syndrome (DS) present alterations in size and architecture of the thymus and higher risk to develop autoimmunity. We sought to evaluate thymic architecture and thymocyte development in DGS and DS patients and to determine the extent to which thymic defects result in immune dysregulation and T cell homeostasis perturbation in these patients. Thymi from pediatric patients and age-matched controls were obtained to evaluate cortex and medullary compartments, AIRE expression and thymocyte development. In the same patients we also characterized immunophenotype of peripheral T cells. Phenotypic and functional characterization of thymic and peripheral regulatory T (Treg) cells was finally assessed. Histologic analysis revealed peculiar alterations in thymic medulla size and maturation in DGS and DS patients. Perturbed distribution of thymocytes and altered thymic output was also observed. DGS patients showed lower mature CD4+ and CD8+ T cell frequency, associated with reduced proportion and function of Tregs both in thymus and peripheral blood. DS patients showed increased frequency of single positive (SP) thymocytes and thymic Treg cells. However, Tregs isolated both from thymus and peripheral blood of DS patients showed reduced suppressive ability. Our results provide novel insights on thymic defects associated with DGS and DS and their impact on peripheral immune dysregulation. Indeed, thymic abnormalities and defect in thymocyte development, in particular in Treg cell number and function could contribute in the pathogenesis of the immunodysregulation present in pDGS and in DS patients

NKp46-expressing human gut-resident intraepithelial V\u3b41 T cell subpopulation exhibits high anti-tumor activity against colorectal cancer

\u3b3\u3b4 T cells account for a large fraction of human intestinal intraepithelial lymphocytes (IELs) endowed with potent anti-tumor activities. However, little is known about their origin, phenotype and clinical relevance in colorectal cancer (CRC). To determine \u3b3\u3b4 IEL gut-specificity, homing and functions, \u3b3\u3b4 T cells were purified from human healthy blood, lymph nodes, liver, skin, intestine either disease-free or affected by CRC or generated from thymic precursors. The constitutive expression of NKp46 specifically identifies a new subset of cytotoxic V\u3b41 T cells representing the largest fraction of gut-resident IELs. The ontogeny and gut-tropism of NKp46pos/V\u3b41 IELs depends both on distinctive features of V\u3b41 thymic precursors and gut-environmental factors. Either the constitutive presence of NKp46 on tissue-resident V\u3b41 intestinal IELs or its induced-expression on IL-2/IL-15 activated V\u3b41 thymocytes are associated with anti-tumor functions. Higher frequencies of NKp46pos/V\u3b41 IELs in tumor-free specimens from CRC patients correlate with a lower risk of developing metastatic III/IV disease stages. Additionally, our in vitro settings reproducing CRC tumor-microenvironment inhibited the expansion of NKp46pos/V\u3b41 cells from activated thymic precursors. These results parallel the very low frequencies of NKp46pos/V\u3b41 IELs able to infiltrate CRC, thus providing new insights to either follow-up cancer progression or develop novel adoptive cellular therapies

HER2/neu gene amplification determines the sensitivity of uterine serous carcinoma cell lines to AZD8055, a novel dual mTORC1/2 inhibitor

To evaluate c-erbB2 gene amplification in a series of primary uterine serous carcinoma (USC) cell lines. To assess the efficacy of AZD8055, a novel dual mTORC1/2 inhibitor against primary HER2/neu amplified vs HER2/neu not amplified USC cell lines. Twenty-two primary USC cell lines were evaluated for c-erbB2 oncogene amplification by FISH assays. In vitro sensitivity to AZD8055 was evaluated by flow-cytometry-based viability and proliferation assays. Cell cycle profile and downstream cellular responses to AZD8055 were assessed by measuring the DNA content of cells and by phosphorylation of the S6 protein by flow-cytometry. Nine of 22 (40.9%) USC cell lines demonstrated c-erbB2 gene amplification by FISH. AZD8055 caused a strong differential growth inhibition in USC cell lines, with high HER-2/neu-expressors demonstrating significantly higher sensitivity when compared to low HER-2/neu-expressors (AZD-8055 IC50 mean±SEM=0.27±0.05μM in c-erbB2 amplified versus 1.67±0.68μM in c-erbB2 not amplified tumors, P=0.03). AZD8055 growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells blocked in the G0/G1 cell cycle phase and a dose-dependent decline in pS6 levels in both c-erbB2 amplified vs c-erbB2 not amplified USC cell lines. AZD8055 may represent a novel targeted therapeutic agent in patients harboring advanced/recurrent/refractory USC. c-erbB2 gene amplification may represent a biomarker to identify USC patients who may benefit most from the use of AZD8055. •AZD8055 may represent a novel targeted therapeutic agent for advanced/recurrent/refractory USC.•c-erbB2 gene amplification may identify USC patients who may benefit most from the use of AZD8055

Inhibition of Phosphatidylcholine-Specific Phospholipase C Interferes with Proliferation and Survival of Tumor Initiating Cells in Squamous Cell Carcinoma

<div><p>Purpose</p><p>The role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them.</p><p>Results</p><p>Compared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 μg/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).</p><p>Conclusions</p><p>These data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential.</p></div

Abstract 4498: Afatinib, an irreversible ErbB family inhibitor, demonstrates activity against HER2 mutated cervical cancer in vitro

Abstract Uterine cervical cancer (UCC) is the second leading cause of cancer deaths among women worldwide and results in 275,000 deaths annually. Functional characterization of cancer-associated genetic alterations may lead to new therapeutic approaches using molecularly targeted therapies which have the potential to improve patient outcomes. Consistent with this view, whole exome sequencing studies in a variety of human tumors including cervical cancer have recently identified somatic mutations in genes that encode for the ErbB family receptors. Importantly, some of these mutations have been shown to be “drivers” and to correlate with tumor sensitivity to the exposure to ErbB tyrosine kinase inhibitors in vitro as well as in vivo. In this study we have evaluated whether genetic alterations in the c-erbB2 gene determine the sensitivity of UCC primary cell lines to Afatinib, an EGFR, HER2, and HER4 irreversible tyrosine kinase inhibitor. Ten primary UCC cell lines, (two harboring c-erbB2 gene mutations in the extracellular HER2/neu domain [i.e., the S280F and E375D mutations-ref genome hg18] and eight harboring wild type c-erbB2), established as long term cultures in vitro, were analyzed in our study. The effect of Afatinib on cell growth, cell-cycle distribution and signaling were assessed using flow cytometry and western blot analysis following incubation of primary UCC cell lines with scalar concentrations of Afatinib for 72 hours in vitro. We found that despite similar ErbB2 mRNA expression levels by qRT-PCR in the mutated versus the wild type c-erbB2 groups, IC50 values in response to Afatinib were significantly lower in the group of mutated cell lines than in the non-mutated control UCC (MEAN±SEM = 0.55±0.11 vs. 1.64±0.09 μM, P<0.05). Furthermore, Afatinib growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells blocked in the G1 cell cycle phase as well as a dose-dependent dephosphorylation of HER2, S6, AKT and ERK in both c-erbB2 mutated and wild type UCC cell lines. Our data suggests that Afatinib, a recently FDA approved drug, is highly effective against c-erbB2 mutated UCC in vitro and could represent a valid therapeutic option for patients harboring c-erbB2 mutated advanced/recurrent cervical cancers unresponsive to radiation and/or chemotherapy. Citation Format: Salvatore Lopez, Emiliano Cocco, Bellone Stefania, Ileana Bortolomai, Elena Bonazzoli, Roberta Nicoletti, Carlton Schwab, Diana P. English, Corrado Terranova, Roberto Angioli, Alessandro D. Santin. Afatinib, an irreversible ErbB family inhibitor, demonstrates activity against HER2 mutated cervical cancer in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4498. doi:10.1158/1538-7445.AM2014-449

Effects of PC-PLC inhibition on EGFR, ERK and AKT phosphorylation in the HaCat and A431-AD cells.

<p>Representative Western blot analyses of total cell lysates from HaCaT (left panels) and A431-AD (right panels) cells cultured in the presence or absence of 50 μg/ml of D609. Cell lysates were immunoblotted with the following antibodies: pEGFR (Tyr1068), EGFR, pERK1/2 (Thr202/Tyr204), ERK 1/2, pAKT (Ser473), AKT and β-actin. β-actin was used as a quantitative loading control. Histograms below each panel represent the relative optical densities of phospho-protein levels normalized to the total protein level (mean values ± SD of three independent experiments) and are presented relative to the untreated sample at 24h. Statistical analyses were performed between treated samples (24h and 48 h) and untreated control sample at 24h, using the t-test.</p

Effects of the PC-PLC inhibitor D609 on PC-PLC activity, PC-PLC protein expression and cell proliferation in HaCaT keratinocytes and in the squamous carcinoma cell line A431-AD.

<p><b>A</b>) Proliferation assays performed on cells seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell count (mean ± SD, n = 3) of live cells was obtained by Trypan blue exclusion assays and by automated cell counter, as described in the Materials and Methods section. <b>B</b>) Cell counting (mean percentage ± SD, n = 3) of either live (white columns) or dead cells (black columns) measured by Trypan blue exclusion test in the cultures used for the proliferation assays shown in panel A. <b>C</b>) Top panel: PC-PLC activity (mean ± SD, n = 3) measured by Amplex Red assay in total lysates of control (untreated = black columns) or 50 μg/ml D609-treated cells (grey columns). Statistical analyses were performed using t-test; HaCaT, P = 0.006 at 24h and P = 0.002 at 48h; A431-AD, P<0.0001 at 24h and at 48h. Bottom panel: Representative Western blot analyses of PC-PLC protein expression performed in total lysates of cells cultured in the presence or absence of 50 μg/ml D609 (n = 3 independent experiments); t0 = untreated cells at 72 hours after seeding; β actin was used as quantitative loading control.</p

PC-PLC protein expression and activity in the non-tumoral keratinocyte HaCaT and in the A431-AD squamous carcinoma cell lines.

<p>Subcellular localization of PC-PLC (grey) on the plasma membrane of unfixed cells (<b>A</b>) and in different cellular compartments of fixed and permeabilized cells (<b>B)</b> detected by CLSM analyses. Single central optical sections are shown. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. <b>C</b>) Western Blot analysis of the relative PC-PLC protein expression in HaCaT and A431-AD total cell lysates (left) and in their cytoplasmic and nuclear fractions (right). Nucleoporin and β-actin were used to ensure the quality of fractions’ separation and protein quantitative loading, respectively. <b>D)</b> Absolute PC-PLC activity (pmoles/μg protein x min; mean ± SD, n = 6) measured by Amplex Red assay in total cell lysates. P = 0.0001.</p

Abstract 732: Iron oxide nanoparticles functionalized with the Clostridium Perfringens Enterotoxin carboxi-terminal fragment peptide: A novel diagnostic and therapeutic approach to specifically target ovarian cancer

Abstract Ovarian carcinoma remains the cancer with the highest mortality among gynecological tumors. Development of novel strategies for the diagnosis and treatment of chemotherapy resistant ovarian disease remains a high priority. Magnetic iron oxide nanoparticles (NP) consist of an iron oxide core and hydrophilic coating. NP can establish magnetic field gradients that alter proton relaxation properties, thus providing an excellent source of contrast for magnetic resonance imaging (MRI). Moreover, since they can be heated in alternating magnetic fields (AMF), they may also provide an effective form of hyperthermia for cancer therapy. Unfortunately, currently available iron oxide NP, although potentially able to allow MRI detection of single cells in vivo, are not tumor specific. Our group has recently analyzed the genetic fingerprints of ovarian cancer identifying extremely high expression of the genes encoding the proteins claudin-3 and -4, the epithelial receptors for Clostridium perfringens enterotoxin (CPE). Since the C-terminus domain (C-CPE) of the toxin is sufficient for CPE binding and it is not toxic to cells, we used iron oxide NP complexed to C-CPE peptide to target chemotherapy resistant ovarian cancer cells overexpressing claudin-3 and -4. We report that NP functionalized by the fluorescent C-CPE peptide effectively bind and rapidly internalize into ovarian cancer cells using flow cytometry,confocal microscopy and Prussian blue staining. A 5-fold increase in iron cellular uptake after incubation of tumor cells with C-CPE NP was consistently detected when compared to tumor cells exposed to non specific control NP [i.e., iron concentration of (mean ± STDV) 20.3 ± 0.5 vs 4.1 ± 1.3 nmoles/5*10 cells respectively; p<0.01]. Importantly, only tumor cells exposed to C-CPE NP were detectable in MRI in vitro experiments using a Bruker 4.7Tmagnet. Finally, to assess the potential of C-CPE NP to be used for hyperthermia therapy against chemotherapy resistant ovarian cancer, we characterized the magnetic heating capacity of C-CPE NP using an induction heating system (Ambrell, Easy Heat). Our results show a temperature increase of 7.85 ± 0.6 °C (mean ± STDV) when C-CPE NP were subjected to an AMF of 343 kHz of frequency and 2 KW of power for 10 minutes. Our data clearly suggest that C-CPE NP may represent a novel theranostic tool to specifically target chemotherapy resistant ovarian cancer. Citation Format: Emiliano Cocco, Ileana Bortolomai, Stefania Bellone, Sara Gasparrini, Diana English, Erik Shapiro, Sergio Pecorelli, Dan-Arin Silasi, Masoud Azodi, Elena Ratner, Thomas Rutherford, Peter Schwartz, Alessandro Santin. Iron oxide nanoparticles functionalized with the Clostridium Perfringens Enterotoxin carboxi-terminal fragment peptide: A novel diagnostic and therapeutic approach to specifically target ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 732. doi:10.1158/1538-7445.AM2013-73