Isolation Of Cancer Cells Research Articles

Circulating Tumor Cell Isolation and Analysis.

Isolation and analysis of cancer cells from body fluids have significant implications in diagnosis and therapeutic treatment of cancers. Circulating tumor cells (CTCs) are cancer cells circulating in the peripheral blood or spreading iatrogenically into blood vessels, which is an early step in the cascade of events leading to cancer metastasis. Therefore, CTCs can be used for diagnosing for therapeutic treatment, prognosing a given anticancer intervention, and estimating the risk of metastatic relapse. However, isolation of CTCs is a significant technological challenge due to their rarity and low recovery rate using traditional purification techniques. Recently microfluidic devices represent a promising platform for isolating cancer cells with high efficiency in processing complex cellular fluids, with simplicity, sensitivity, and throughput. This review summarizes recent methods of CTC isolation and analysis, as well as their applications in clinical studies.

Scaffold-integrated microchips for end-to-end in vitro tumor cell attachment and xenograft formation.

Microfluidic technologies have substantially advanced cancer research by enabling the isolation of rare circulating tumor cells (CTCs) for diagnostic and prognostic purposes. The characterization of isolated CTCs has been limited due to the difficulty in recovering and growing isolated cells with high fidelity. Here, we present a strategy that uses a 3D scaffold, integrated into a microfludic device, as a transferable substrate that can be readily isolated after device operation for serial use in vivo as a transplanted tissue bed. Hydrogel scaffolds were incorporated into a PDMS fluidic chamber prior to bonding and were rehydrated in the chamber after fluid contact. The hydrogel matrix completely filled the fluid chamber, significantly increasing the surface area to volume ratio, and could be directly visualized under a microscope. Computational modeling defined different flow and pressure regimes that guided the conditions used to operate the chip. As a proof of concept using a model cell line, we confirmed human prostate tumor cell attachment in the microfluidic scaffold chip, retrieval of the scaffold en masse, and serial implantation of the scaffold to a mouse model with preserved xenograft development. With further improvement in capture efficiency, this approach can offer an end-to-end platform for the continuous study of isolated cancer cells from a biological fluid to a xenograft in mice.

The potential complementary role of targeted alpha therapy in the management of metastatic melanoma.

Standard treatments for metastatic melanoma have recently extended survival although many patients still succumb. Targeted alpha therapy (TAT) is a new therapeutic approach in which a cancer-targeting vector is labeled with an alpha-emitting radioisotope. Alpha-particles have the shortest range and highest energy transfer, and produce localized, high-density and lethal ionization damage to DNA. Thus, the targeted radiation can kill isolated cancer cells circulating in blood and lymphatic vessels, regress metastatic cancer cell clusters, and disrupt the vasculature of solid tumors. Preclinical and clinical studies of TAT for metastatic melanoma demonstrate its safety and anti-tumor activity. We recommend ways in which TAT can be used to treat small-volume disease sometimes in conjunction with cytoreductive anti-melanoma therapies.

Abstract 1520: Plasticity of CD44+ colorectal cancer stem cells depends on TGF-beta-induced epithelial mesenchymal transition (EMT): evidences from ex vivo culture system

Abstract Background EpCAMhigh CD44+ colorectal cancer (CRC) cells are thought to be cancer stem cells. Recently CD44- CRC cells are also suggested to acquire a character of cancer stem cells, but the molecular mechanisms have not been clarified. Epithelial-mesenchymal transition (EMT) is a possible process of cancer cells to acquire stemness. However, it is unclear whether EMT can be induced in primary human CRC. Patients and Methods We obtained surgical specimens from 51 CRC patients, and cultured isolated cancer cells on matrigel-coated dish with medium containing several growth factors. For induction of EMT, TGF-beta was added into the culture medium. Immunocytochemistry staining, flow cytometric analysis of the spheroid cells and single cell PCR analysis using 48-gene set containing and Embryonic stem cell (ES)-related genes EMT-related genes were performed. Results Cancer cells proliferated and formed sphere (>50 μm in diameter) in 15 out of 51 samples after one week culture. The sphere-forming ability was related with clinical stage (Stage1 and 2:16.7% Stage 3 and 4:41.3%). Sorted single CD44+ cell had higher sphere-forming ability, compared to CD44- cell. The higher expression of ES- and EMT-related genes was observed in sphere-forming cells than in non-sphere forming cells. To confirm the heterogeneity of sphere forming cells derived from sorted single cell, we performed single cell PCR using C1 single cell auto prep system. Cells consisted of a sphere were classified into 2 different population on the basis of primary component analysis. In addition, flow cytometric analysis revealed that sphere forming CD44+ cells gave rise to CD44- cells. These results suggest that CD44+ cells have an ability to reconstruct the heterogeneous population. Next we isolated sphere-forming cells and cultured on the same condition. Isolated sphere-forming CD44+ cells could form sphere with high efficiency. This result suggests that CD44+ cell have a self-renewal ability. Previously, it has been reported that EMT was induced after TGF-beta treatment in cancer cell lines and these cells acquired cancer stem cell properties. In our culture system, we could detect very few CD44+ cells after 8-days culture of CD44- cells, and TGF-beta treatment also increased the number of CD44+ cells. These CD44 expressing cells expressed N-cadherin. Furthermore N-cadherin expressing cells expressed ES-and EMT-related genes. At last, TGF-beta enhanced sphere-forming ability in CD44- cells. Conclusions To our knowledge, this is the first report that CD44- cells from primary CRC samples undergo EMT and induced CD44+ cells by TGF-beta treatment. Investigation whether these induced CD44+ cells have cancer stem cell like properties such as tumorigenic potential in immunosuppressive mouse is underway. Also we are going to identify cancer stem cell specific gene expression in our culture system using DNA microarray. Citation Format: Michitaka Nakano, Hioshi Ariyama, Shingo Tamura, Taichi Isobe, Kohta Miyawaki, Yuta Okumura, Hitoshi Kusaba, Takashi Ueki, Eishi Baba, Koichi Akashi. Plasticity of CD44+ colorectal cancer stem cells depends on TGF-beta-induced epithelial mesenchymal transition (EMT): evidences from ex vivo culture system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1520. doi:10.1158/1538-7445.AM2015-1520

Biodegradable nano-films for capture and non-invasive release of circulating tumor cells

Selective isolation and purification of circulating tumor cells (CTCs) from whole blood is an important capability for both clinical medicine and biological research. Current techniques to perform this task place the isolated cells under excessive stresses that reduce cell viability, and potentially induce phenotype change, therefore losing valuable information about the isolated cells. We present a biodegradable nano-film coating on the surface of a microfluidic chip, which can be used to effectively capture as well as non-invasively release cancer cell lines such as PC-3, LNCaP, DU 145, H1650 and H1975. We have applied layer-by-layer (LbL) assembly to create a library of ultrathin coatings using a broad range of materials through complementary interactions. By developing an LbL nano-film coating with an affinity-based cell-capture surface that is capable of selectively isolating cancer cells from whole blood, and that can be rapidly degraded on command, we are able to gently isolate cancer cells and recover them without compromising cell viability or proliferative potential. Our approach has the capability to overcome practical hurdles and provide viable cancer cells for downstream analyses, such as live cell imaging, single cell genomics, and in vitro cell culture of recovered cells. Furthermore, CTCs from cancer patients were also captured, identified, and successfully released using the LbL-modified microchips.

Lactate promotes PGE2 synthesis and gluconeogenesis in monocytes to benefit the growth of inflammation-associated colorectal tumor.

Reprogramming energy metabolism, such as enhanced glycolysis, is an Achilles' heel in cancer treatment. Most studies have been performed on isolated cancer cells. Here, we studied the energy-transfer mechanism in inflammatory tumor microenvironment. We found that human THP-1 monocytes took up lactate secreted from tumor cells through monocarboxylate transporter 1. In THP-1 monocytes, the oxidation product of lactate, pyruvate competed with the substrate of proline hydroxylase and inhibited its activity, resulting in the stabilization of HIF-1α under normoxia. Mechanistically, activated hypoxia-inducible factor 1-α in THP-1 monocytes promoted the transcriptions of prostaglandin-endoperoxide synthase 2 and phosphoenolpyruvate carboxykinase, which were the key enzyme of prostaglandin E2 synthesis and gluconeogenesis, respectively, and promote the growth of human colon cancer HCT116 cells. Interestingly, lactate could not accelerate the growth of colon cancer directly in vivo. Instead, the human monocytic cells affected by lactate would play critical roles to 'feed' the colon cancer cells. Thus, recycling of lactate for glucose regeneration was reported in cancer metabolism. The anabolic metabolism of monocytes in inflammatory tumor microenvironment may be a critical event during tumor development, allowing accelerated tumor growth.

Isolating cancer cells with acoustic waves

Isolating cancer cells with acoustic waves

Proper genomic profiling of (BRCA1-mutated) basal-like breast carcinomas requires prior removal of tumor infiltrating lymphocytes

IntroductionBRCA1-mutated breast carcinomas may have distinct biological features, suggesting the involvement of specific oncogenic pathways in tumor development. The identification of genomic aberrations characteristic for BRCA1-mutated breast carcinomas could lead to a better understanding of BRCA1-associated oncogenic events and could prove valuable in clinical testing for BRCA1-involvement in patients. MethodsFor this purpose, genomic and gene expression profiles of basal-like BRCA1-mutated breast tumors (n = 27) were compared with basal-like familial BRCAX (non-BRCA1/2/CHEK2*1100delC) tumors (n = 14) in a familial cohort of 120 breast carcinomas. ResultsGenome wide copy number profiles of the BRCA1-mutated breast carcinomas in our data appeared heterogeneous. Gene expression analyses identified varying amounts of tumor infiltrating lymphocytes (TILs) as a major cause for this heterogeneity. Indeed, selecting tumors with relative low amounts of TILs, resulted in the identification of three known but also five previously unrecognized BRCA1-associated copy number aberrations. Moreover, these aberrations occurred with high frequencies in the BRCA1-mutated tumor samples. Using these regions it was possible to discriminate BRCA1-mutated from BRCAX breast carcinomas, and they were validated in two independent cohorts. To further substantiate our findings, we used flow cytometry to isolate cancer cells from formalin-fixed, paraffin-embedded, BRCA1-mutated triple negative breast carcinomas with estimated TIL percentages of 40% and higher. Genomic profiles of sorted and unsorted fractions were compared by shallow whole genome sequencing and confirm our findings. ConclusionThis study shows that genomic profiling of in particular basal-like, and thus BRCA1-mutated, breast carcinomas is severely affected by the presence of high numbers of TILs. Previous reports on genomic profiling of BRCA1-mutated breast carcinomas have largely neglected this. Therefore, our findings have direct consequences on the interpretation of published genomic data. Also, these findings could prove valuable in light of currently used genomic tools for assessing BRCA1-involvement in breast cancer patients and pathogenicity assessment of BRCA1 variants of unknown significance. The BRCA1-associated genomic aberrations identified in this study provide possible leads to a better understanding of BRCA1-associated oncogenesis.

Enhancing sensitivity and specificity in rare cell capture microdevices with dielectrophoresis.

The capture and subsequent analysis of rare cells, such as circulating tumor cells from a peripheral blood sample, has the potential to advance our understanding and treatment of a wide range of diseases. There is a particular need for high purity (i.e., high specificity) techniques to isolate these cells, reducing the time and cost required for single-cell genetic analyses by decreasing the number of contaminating cells analyzed. Previous work has shown that antibody-based immunocapture can be combined with dielectrophoresis (DEP) to differentially isolate cancer cells from leukocytes in a characterization device. Here, we build on that work by developing numerical simulations that identify microfluidic obstacle array geometries where DEP-immunocapture can be used to maximize the capture of target rare cells, while minimizing the capture of contaminating cells. We consider geometries with electrodes offset from the array and parallel to the fluid flow, maximizing the magnitude of the resulting electric field at the obstacles' leading and trailing edges, and minimizing it at the obstacles' shoulders. This configuration attracts cells with a positive DEP (pDEP) response to the leading edge, where the shear stress is low and residence time is long, resulting in a high capture probability; although these cells are also repelled from the shoulder region, the high local fluid velocity at the shoulder minimizes the impact on the overall transport and capture. Likewise, cells undergoing negative DEP (nDEP) are repelled from regions of high capture probability and attracted to regions where capture is unlikely. These simulations predict that DEP can be used to reduce the probability of capturing contaminating peripheral blood mononuclear cells (using nDEP) from 0.16 to 0.01 while simultaneously increasing the capture of several pancreatic cancer cell lines from 0.03-0.10 to 0.14-0.55, laying the groundwork for the experimental study of hybrid DEP-immunocapture obstacle array microdevices.

Self-propelled carbon nanotube based microrockets for rapid capture and isolation of circulating tumor cells.

Here, we report a non-invasive strategy for isolating cancer cells by autonomously propelled carbon nanotube (CNT) microrockets. H2O2-driven oxygen (O2) bubble-propelled microrockets were synthesized using CNT and Fe3O4 nanoparticles in the inner surface and covalently conjugating transferrin on the outer surface. Results show that self-propellant microrockets can specifically capture cancer cells.

Dielectrophoretic discrimination of cancer cells on a microchip

The analysis of single cell type typically requires expensive equipments in combination with labeling techniques. As a label-free alternative, in this letter, the characteristic dielectric properties of various cancer cell lines (MCF-7, SKOV-3, MDA-MB-231, and LnCap) and healthy peripheral blood mononuclear cells were examined and compared using the dielectrophoretic (DEP) crossover frequency technique. We found that each type of the cancer cells shows a distinct DEP crossover frequency with an order of SKOV-3, MDA-MB-231, MCF-7, and LnCap from low to high frequency, from which the specific cell membrane capacitance and membrane conductance could be derived. Cell fixation and antibody coupling were found to have minimal or no effects on the cell dielectric properties while cell permeabilization significantly changed the DEP crossover frequency. These findings suggested that the DEP crossover frequency is promising to be used as a “dielectric finger print” to discriminate different cell types and may even enable the specific manipulation of certain cell types, for example, to isolate cancer cells from blood.

Abstract 1228: Clinical relevance of FGFR2 amplification in diffuse gastric cancer

Abstract Diffuse gastric cancer (DGC) is a poorly differentiated adenocarcinoma characterized by infiltration of signet ring cells into the stomach wall and a lack of tumor mass formation. DGC has rapid disease progression with a dismally poor prognosis and is resistant to chemo- and radiotherapy. There is no available targeted therapy, and preclinical models that reliably predict clinical activity of novel compounds are also lacking. In addition, molecular analyses of DGC have been hampered by difficulty in obtaining primary tumors of sufficient tumor cellularity without contaminating normal fibroblasts. To overcome the scarcity of cancer cells and the abundance of surrounding stromal tissue, we isolated cancer cells from malignant ascites of DGC patients. Red blood cells were removed by Ficoll separation and CD45+ hematopoietic cells were depleted using antibody-conjugated magnetic beads. Exome sequencing was performed on cancer cells and peripheral blood cells from the same patient to identify somatic alterations in DGC. We also generated patient-derived xenografts (PDX) using isolated cancer cells to establish preclinical cancer models for DGC. Exome sequencing revealed FGFR2 and KRAS amplifications as well as p53 mutations. Cancer cells isolated from malignant ascites formed subcutaneous tumors that recapitulated the histology of primary DGC tumors with characteristic signet ring cells and intracellular mucin. FGFR2 amplification in PDX tumors was confirmed by fluorescence in situ hybridization (FISH), with correspondingly high levels of FGFR2 transcripts and protein expression. We performed drug treatments on PDX tumors using FGFR2 inhibitor AZD4547 and found that FGFR2 amplification status alone was sufficient to predict the sensitivity of PDX tumors to AZD4547. Tumor volume of FGFR2-amplified tumors decreased or remained the same following AZD4547 treatment compared to vehicle-treated tumors which showed increased tumor volume. In contrast, AZD4547 treatment had little or no effect on tumor growth of non-FGFR2 amplified tumors. Furthermore, these PDX tumors regardless of FGFR2 amplification status responded poorly to cisplatin, a standard drug currently used for treatment of DGC. In conclusion, we report a high prevalence of FGFR2 gene amplification in DGC and the establishment of reliable PDX models that can predict tumor response to targeted therapy. Citation Format: Wen Min Lau, Zhijiang Zang, Eileen Teng, Kakoli Das, Wei Peng Yong, Ming Teh, Tania Chia, Jin Wei Tan, Amy Tay, Asim Shabbir, Koji Kono, Jimmy So, Patrick Tan, Shing Leng Chan. Clinical relevance of FGFR2 amplification in diffuse gastric cancer. [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 1228. doi:10.1158/1538-7445.AM2014-1228

Abstract 1947: Plasticity of CD44+ colorectal cancer stem cells depends on TGF-beta-induced epithelial mesenchymal transition(EMT): Evidences from an ex vivo culture

Abstract Background EpCAMhigh CD44+ colorectal cancer (CRC) cells are thought to be cancer stem cells. Recently CD44- CRC cells are also suggested to gain a character of cancer stem cells, but the molecular mechanisms have not been clarified. Epithelial-mesenchymal transition (EMT), which is observed in several caner cell lines, could be a possible process of cancer cells to obtain stemness. However, whether primary human CRC cells underwent EMT has not been proven. Patients and Methods We obtained surgical specimens of 30 cases of advanced CRC, and cultured isolated primary cancer cells in matrigel-based medium containing several growth factors. For induction of EMT, TGF-beta was added into the culture of CD44+ and CD44- cells. Immunocytochemistry staining and flow cytometry of the spheroid cells and single cell PCR analysis of the cultured cells using 48-gene set that included EMT-related genes (BioMark HD System:Fluidigm Corporation) were performed. Results Cancer cells grew spherically (50 μm> diameter) in 20 out of 30 samples after a week culture of whole isolated cancer cells. The sphere mostly consisted of CD44+ cells, and non-sphere forming single cells were CD44-negative. Isolated CD44+ cells from primary samples gave rise to CD44+ and CD44- cells in a week culture. Interestingly, 10% of CD44- cell from primary samples also expressed CD44. Similar to the previous findings of cell lines that TGF-beta-induced EMT could convert CD44- cells into CD44+ cancer initiating cells, increased number of CD44+ cells appeared from primary CD44- cells cultured with TGF-beta. In this culture, N-cadherin+ cells increased time-dependent manner and most of this N-cadherin expressing cells co-expressed CD44. In single cell PCR analysis, increased expression of EMT-related genes, vimentin and TWIST1, were preferentially detected in CD44+N-Cadherin+ cells. Conclusions To our knowledge, this is the first report that CD44- cells from primary samples undergo EMT and convert into CD44+ cells by TGF-β treatment. Investigation whether these CD44+ converted cells have cancer stem cell properties such as tumorigenic potential in immunodeficient mouse, self renewing ability and drug resistance is underway. Citation Format: Michitaka Nakano, Hiroshi Ariyama, Shingo Tamura, Taichi Isobe, Kohta Miyawaki, Yuta Okumura, Hitoshi Kusaba, Eishi Baba, Koichi Akashi. Plasticity of CD44+ colorectal cancer stem cells depends on TGF-beta-induced epithelial mesenchymal transition(EMT): Evidences from an ex vivo culture. [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 1947. doi:10.1158/1538-7445.AM2014-1947

SU‐E‐J‐03: A Comprehensive Comparison Between Alpha and Beta Emitters for Cancer Radioimmunotherapy

Purpose:The purpose of this study is to perform a comprehensive comparison of the therapeutic efficacy and cytotoxicity of alpha and beta emitters for Radioimmunotherapy (RIT). For each stage of cancer development, specific models were built for the separate objectives of RIT to be addressed:a) kill isolated cancer cells in transit in the lymphatic and vascular circulation,b) regress avascular cell clusters,c) regress tumor vasculature and tumors.Methods:Because of the nature of short range, high LET alpha and long energy beta radiation and heterogeneous antigen expression among cancer cells, the microdosimetric approach is essential for the RIT assessment. Geant4 based microdosimetric models are developed for the three different stages of cancer progression: cancer cells, cell clusters and tumors. The energy deposition, specific energy resulted from different source distribution in the three models was calculated separately for 4 alpha emitting radioisotopes (211At, 213Bi, 223Ra and 225Ac) and 6 beta emitters (32P, 33P, 67Cu, 90Y, 131I and 177Lu). The cell survival, therapeutic efficacy and cytotoxicity are determined and compared between alpha and beta emitters.Results:We show that internal targeted alpha radiation has advantages over beta radiation for killing isolated cancer cells, regressing small cell clusters and also solid tumors. Alpha particles have much higher dose specificity and potency than beta particles. They can deposit 3 logs more dose than beta emitters to single cells and solid tumor. Tumor control probability relies on deep penetration of radioisotopes to cancer cell clusters and solid tumors.Conclusion:The results of this study provide a quantitative understanding of the efficacy and cytotoxicity of RIT for each stage of cancer development.

Sphere Formation Assay is not an Effective Method for Cancer Stem Cell Derivation and Characterization from the Caco-2 Colorectal Cell Line

Although the existence of cancer stem cells (CSCs) has been demonstrated in colorectal cancer, further investigation is hindered by controversies over their surface markers. The sphere formation assay is widely used as in vitro method for derivation and characterization of CSCs based on the intrinsic self-renewal property of these cells. Isolated cancer cells that form tumorspheres are generally recognized as CSCs with self-renewal and tumorigenic capacities. In this study, colon spheres grown from Caco-2 cells in the sphere formation assay were separated from other differentiated cells and characterized. Compared with Caco-2 cells, the derived colon spheres lost several CSC properties. The colon spheres contained decreased levels of specific colorectal CSC surface markers as well as low levels of ATP-binding cassette (ABC) transporters typically overexpressed in CSCs, resulting in the near loss of their chemoresistance ability. Furthermore, cells that developed as colon spheres with strong self-renewal ability in vitro lost their tumorigenic capacity in vivo compared with Caco-2 cells, which could establish tumors in non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice. The results indicated that the Caco-2 cell derived colon spheres did not consist of colorectal CSCs. Thus, the well-accepted sphere formation assay may not be an effective method for CSC isolation and characterization from the Caco-2 colorectal cancer cell line.

The Angiogenic Activity of Ascites in the Course of Ovarian Cancer as a Marker of Disease Progression

Ovarian cancer cells are able to create invasive implants in the peritoneum and their growth is directly associated with the angiogenetic potential. This effect is probably stimulated by vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), which are both found in ascites. The aim of this study was to assess the influence of ascites produced by ovarian cancer on the angiogenesis. Peritoneal fluid was collected from patients with advanced ovarian cancer; cancer cells were separated from CD45+ leukocytes. Angiogenesis was assessed in mice, after intradermal injection of full cellular suspension together with supernatant or phosphate buffered saline, purified cancer cells suspension, or CD45+ leukocytes suspension. The angiogenesis index (AI) was assessed after 72 hours. VEGF and Il-8 were measured in the supernatant and cellular suspension. AI was the highest in the isolated cancer cells suspensions as well in the group stimulated with supernatant. Both VEGF and IL-8 were high in supernatants from ascites rich in cancer cells (>45%). A significant correlation was revealed between IL-8 concentration and AI. We conclude that ascites in patients with advanced ovarian cancer stimulates angiogenesis and this mechanism is dependent mostly on cancer cells activity and enhanced by cooperation with infiltrating leukocytes.

Prognostic significance of subtype and pathologic response in operable breast cancer; a pooled analysis of prospective neoadjuvant studies of JBCRG.

In the past decade, JBCRG has conducted three studies of neoadjuvant chemotherapy which have examined sequential combination of fluorouracil, epirubicin and cyclophosphamide, and docetaxel. The present study is a pooled analysis of these studies performed to determine the prognostic significance of pathologic complete response (pCR) and predictive variables for pCR. A total of 353 patients were included. pCR was defined as the absence of invasive cancer or only a few remaining isolated cancer cells in the breast (quasi-pCR, QpCR). Disease-free survival (DFS) and overall survival (OS) were not significantly different among studies, and patients who achieved a QpCR had significantly better prognosis (DFS, p<0.001; OS, p=0.002). Patients with triple-negative (TN) tumors had worse prognosis than patients with the other subtypes (DFS, p=0.03; OS, p=0.10). A Cox proportional hazards model showed node-positive, TN, and QpCR were the significant predictors for DFS and OS among study, age, tumor size, nuclear grade, nodal status, subtype, clinical response, and pathologic response (DFS; node-positive, HR=2.29, p=0.001; TN, HR=3.39, p<0.001; QpCR, HR=0.27, p<0.001: OS; node-positive, HR=3.05, p=0.003; TN, HR=4.92, p<0.001; QpCR, HR=0.12, p<0.001). In a logistic regression analysis, subtype and clinical response before surgery were the significant predictive variables for QpCR (luminal/Her2-positive, odds ratio (OR)=4.15, p=0.002; Her2-positive, OR=6.24, p<0.001; TN, OR=4.24, p<0.001; clinical response before surgery, OR=2.41, p=0.019). This study confirmed the prognostic significance of QpCR and nodal status and the predictive and prognostic significance of subtype in neoadjuvant chemotherapy.

Abstract B056: p70S6K activity drives local relapse in breast cancer

Abstract For early breast cancer (EBC) patients local relapse represents what mostly influences disease outcome. Notably, 90% of local recurrences occur at or close to the same quadrant of the primary cancer. Surgery itself and the consequent process of wound healing have been proposed to stimulate local recurrences via pathway(s) still to be clarified. Our previous studies implicated p70S6K pathway in breast cancer cell response to post-surgical inflammation, supporting the hypothesis that it may be crucial also for breast cancer recurrence. We designed an in vivo experimental model resembling the course of human BC, in which MDA-MB-231 breast cancer cells were bilaterally injected in nude mice mammary fat pads and, when primary tumors were grown, masses were surgically removed under anesthesia. After recovering, mice were followed up to detect appearance of local relapse. Using this model, we dissected the role of p70S6K during breast cancer growth and recurrence by modulating its activity using both genetic and pharmacologic approaches. Our results showed that p70S6K positively contributed to proliferation and survival programs in breast cancer cell lines. In vivo, the analysis of the impact of p70S6K on primary tumor growth highlighted that a robust p70S6K signaling was required for the initiation of the tumor masses. But, more importantly, interfering with p70S6K activity strongly impaired local relapse. A three-day schedule of peri-operative treatment using specific pharmacological inhibition of p70S6K1 was sufficient to reduce by 83% the rate of local recurrence. We showed that inhibition of p70S6K activity induced apoptosis in cells challenged to survive in a hostile environment, supporting the idea that p70S6K signaling is necessary for the survival of isolated cancer cells in breast microenvironment, following surgery. The significance of our results was confirmed in human EBC specimens, proving that p70S6K activity is robustly induced by surgery, also in human patients. Taken together, our results provide a biological rationale for peri-operative treatment targeting p70S6K pathway, directed to compensating the harmful consequences of surgery and to restraining local recurrence in early breast cancer patients. Citation Format: Ilenia Segatto, Stefania Berton, Maura Sonego, Samuele Massarut, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. p70S6K activity drives local relapse in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B056.

Biological and molecular aspects of lymph node metastasis in gastro-intestinal cancer

Recently, the existence of lymph node micrometastasis, including isolated tumor cells, has been the focus of the development of molecular diagnostic tools for lymph node metastasis in various malignant neoplasms, including those of the GI tract. In this review, we summarize recent molecular biological studies that might provide two reasons to explain the survival of single isolated cancer cells in lymph nodes. One is the specific characteristics of cancer cells, which can exist under severe circumstances, along with recent technological innovations to obtain expression profiles and sequencing from a single cell. The other is microenvironmental factors that support the formation of micrometastasis even in small numbers of cancer cells. The expression profile of whole transcriptome sequencing, genomic sequencing and epigenetic sequencing of a single cancer cell with tumorigenic properties in lymph node metastases should be disclosed in the near future.

Cancer cell separator using size-dependent filtration in microfluidic chip

We have developed a microfluidic chip that can separate gastric cancer cells from peritoneal washes. Gastric cancer cells tend to be agglomerated in the peritoneal wash, and are larger than the other cells and the soft tissue fragments. This paper presents a novel microfluidic chip to enrich low-concentrated cancer cells from the peritoneal wash by size-dependent cell filtration. This microfluidic chip has the advantage that it can separate agglomerated cells in high speed because larger objects are filtered by a gap in the microchannel. Two-step exposure was performed to fabricate a precise uneven channel for cell filtration. The main and shallow channels have heights of 100μm and 8μm, respectively. Furthermore, inserting fins in the microchannel that assist sheath flow enhanced continuous and robust separation against flow fluctuation in the microchannel. The microfluidic channel was designed to have high filtration efficiency and a high throughput while having a compact profile. We used patient's peritoneal washes to isolate cancer cells. As a result, we were able to enrich the cancer cells in peritoneal wash.