Cancer Primary Cultures Research Articles

1,2,3-Triazole tethered 1,2,4-trioxanes: Studies on their synthesis and effect on osteopontin expression in MDA-MB-435 breast cancer cells

Artemisinin and its analogs have shown potent anticancer activity in primary cancer cultures and cell lines by inhibiting cancer proliferation, metastasis, and angiogenesis. Despite its apparent compatibility to normal cells and low IC50 values in comparison to the commonly used anticancer drugs, the underlying mechanisms behind their cytotoxic effects are not yet fully understood. Surprisingly, the efficacy of synthetic 1,2,4-trioxanes against cancer has not been explored yet. Given the high antitumor activity of artemisinin dimers in comparison to their monomers, we report here the synthesis of simple 1,2,3-triazole conjugated 1,2,4-trioxanes and their potential antitumor activity by studying their inhibitory effect on osteopontin (OPN) expression in MDA-MB-435 breast cancer cells. It may be noted that despite being a strong marker to identify human tumor metastasis, no study on effect of artemisinin and its synthetic and semisynthetic derivatives on OPN expression has ever been studied. Although our initial studies did not notice any straight-line relationship between the number of trioxane units in a molecule to the extent of inhibition of OPN protein expression, we could observe better results in some cases in comparison to artemisinin. We have observed that artemisinin did not show appreciable OPN downregulation in MDA-MB-435 cancer cells, but dihydroartemisinin (DHA) and some synthetic 1,2,4-trioxane monomers and dimers showed downregulation of OPN expression. Therefore, these compounds may act as an anti-metastatic agent in controlling breast cancer cells metastasis.

Abstract 191: Extracellular signaling linked to PI3K/AKT triggers the formation of vasculogenic mimcry in ovarian cancer

Abstract Introduction: The presence of an endothelial cell-free perfusion pathway, known as Vasculogenic Mimicry (VM), strongly correlates with reduced patient survival, however the mechanisms by which a tumor can create a self-generated irrigation system remain unknown. Currently there is no mechanism of VM formation reported in fluid conducting tubular structures. Herein, using a standardized in vitro model, we sought to understand the pathways required for VM formation. Methods: We utilized confocal microscopy and Imaris reconstruction to demonstrate that cancer cells can create an internal tubular network in cultured cancer cell lines, cancer spheres and primary cancer cultures of ovarian cancer. Matrigel and 3D matrices containing specific protein components allowed the deciphering of the extracellular matrix proteins involved in VM formation. Intercellular pathways were resolved using chemical inhibitors. Results: VM only occurs when cells are grown in 3D culture and only 30% of primary cancer cultures undergo this process. The presence of laminin 1 is sufficient to trigger the formation of tubular structures and this may involve the alpha2beta1 integrin and metalloproteases. Downstream FAK and PI3K/AKT intercellular pathways are required to form functional tubular networks. Microarray data elucidated the presence of the mRNA transcripts that were increased in cells that undergo VM. Discussion: We demonstrate that Laminin 1, a component of the extracellular matrix (ECM), can promote the process of VM, which further involves the downstream signaling of the potentially druggable targets of the PI3K pathway. As VM is associated with reduced patient survival, elucidation of the mechanisms of formation may deliver new cancer therapies. Funding: FONDECYT 1180241, CONICYT FONDAP 15130011, IMII P09/016F Citation Format: Andres Valdivia, Gabriel Mingo, Varina Aldana, Alejandra Sandoval, Alejandro Corvalan, Francisco Nualart, Gareth I. Owen. Extracellular signaling linked to PI3K/AKT triggers the formation of vasculogenic mimcry in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 191.

A study aimed at the establishment and clinical application of a novel digestive system cancer primary culture method.

A study aimed at the establishment and clinical application of a novel digestive system cancer primary culture method.

Abstract 789: Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells

Abstract Introduction: A key step in cancer progression is tumor irrigation. The process of angiogenesis can be complemented in sub-sets of highly aggressive cancers by the process of vasculogenic mimicry, which is the formation of tubular structures by tumor cells. Herein, we sought to establish an in vitro assay of vasculogenic mimicry and determine the percentage of ovarian and gastrointestinal primary cultures capable of undergoing this process. Materials & Methods: Gastric cancer cell lines, AGS and Hs746T, and the ovarian cancer cell lines SKOV-3 and HEY were used in conjunction with gastrointestinal and ovarian primary cultured cancer cells extracted from peritoneal fluid. Cells were seeded on matrigel and monitored for a week. Hollow channels formation was evaluated by fluorescent dye microinyection, periodic acid Schiff staining, confocal microscopy and X-Ray microtomography (Micro-CT). Results: SKOV-3, HEY and AGS cell lines underwent the process of vasculogenic mimicry. Both confocal microscopy and Micro-CT reconstruction were able to show the presence of a lumen of the structures in 3D. In 22 primary cancer cultures (Ovarian, Colon and Gastric) only 38% could underwent vasculogenic mimicry. Discussion: We have standardized an in vitro assay to assay and quantify vasculogenic mimicry. Regardless of origin, only a low percentage of cultures have the ability to undergo vasculogenic mimicry. An understanding of this process could shed light on new therapies for this sub-set of highly aggressive cancers. Funding: FONDECYT 1120292, 3150028 & 1140970. CORFO L2 13IDL2-18608, BMRC 13CTI 21526-P6, IMII P09/016-F, CONICYT-FONDAP #1513001 Citation Format: Andres Valdivia, Dusan Racordon, Raul Aravena, Gabriel Mingo, Alejandra Sandoval, Maria Loreto Bravo, Mauricio A. Cuello, Sumie Kato, Rafaela Erices, Carolina Ramirez, Pamela Gonzalez, Beatriz Sanchez, Alejandro H. Corvalan, Gareth I. Owen. Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2017-789

VEGFA links self-renewal and metastasis by inducing Sox2 to repress miR-452, driving Slug

Cancer stem cells (CSC) appear to have increased metastatic potential, but mechanisms underlying this are poorly defined. Here we show that VEGFA induction of Sox2 promotes EMT and tumor metastasis. In breast lines and primary cancer culture, VEGFA rapidly upregulates SOX2 expression, leading to SNAI2 induction, EMT, increased invasion and metastasis. We show Sox2 downregulates miR-452, which acts as a novel metastasis suppressor to directly target the SNAI2 3′-untranslated region (3′-UTR). VEGFA stimulates Sox2- and Slug-dependent cell invasion. VEGFA increases lung metastasis in vivo, and this is abrogated by miR-452 overexpression. Furthermore, SNAI2 transduction rescues metastasis suppression by miR-452. Thus, in addition to its angiogenic action, VEGFA upregulates Sox2 to drive stem cell expansion, together with miR-452 loss and Slug upregulation, providing a novel mechanism whereby cancer stem cells acquire metastatic potential. Prior work showed EMT transcription factor overexpression upregulates CSC. Present work indicates that stemness and metastasis are a two-way street: Sox2, a major mediator of CSC self-renewal, also governs the metastatic process.

A 22q11.2 amplification in the region encoding microRNA-650 correlates with the epithelial to mesenchymal transition in breast cancer primary cultures of Mexican patients.

Breast cancer ranks first in incidence and mortality in working age women. Cancer initiation and progression relies on accumulation of genetic and epigenetic aberrations that alter cellular processes, among them, epithelial to mesenchymal transition (EMT) denotes particularly aggressive neoplasias given its capacity to invade and metastasize. Several microRNAs (miRNA) have been found able to regulate gene expression at the core of EMT. In this study, the Affymetrix CytoScan HD array was used to analyze three different primary tumor cell isolates from Mexican breast cancer patients. We found an amplification in band 22q11.2 shared by the three samples, in the region that encodes miRNA-650. Overexpression of this miRNA has been associated with downregulation of tumor suppressors ING4 and NDRG2, which have been implicated in cancer progression. Using the Pathway Linker platform the ING4 and NDRG2 interaction networks showed a significant association with signaling pathways commonly deregulated in cancer. Also, several studies support their participation in the EMT. Supporting the latter, we found that the three primary isolates were E-cadherin negative, vimentin positive, presented a cancer stem cell-like phenotype CD44+CD24−/low and were invasive in Transwell invasion assays. This evidence suggests that the gain of region 22q11.2 contributes to trigger EMT. This is the first evidence linking miR-650 and breast cancer.

Abstract 1562: miRNAs in exosomes from prostate cancer cells modify normal fibroblasts and osteoblasts favoring tumor spread and metastasis

Abstract Introduction: Exosomes from bulk and cancer stem cells (CSCs) from prostate cancer primary cultures present a differential pattern of miRNAs. The most abundant miRNA in all exosomes is miR-100, while miR-21 and miR-139 were the most abundant differentially expressed miRNAs in bulk and CSCs exosomes, respectively. Bioinformatics analysis suggested that these miRNAs were related with tumor progression. The functional effect of these miRNAs on targets related with changes at primary and pre-metastatic microenvironment was evaluated in prostatic stromal cells and osteoblasts. Methods: Normal human undifferentiated osteoblasts (hFOB1.19) and prostate fibroblasts (WPMY-1) cell lines were transfected, by separated, with 25 nM of the following miRNas: miR-100-5p, miR-21-5p, miR-139-5p. let7c was included as transfection control. After 48 hours, changes in the expression of metalloproteinases (MMP)-2, -9 and-13, RANKL, OPG and IL-8 at mRNAs and protein levels were evaluated by qPCR and western blot, respectively. As transfection control, expression of specific targets previously described for each miRNA was evaluated. Besides, the effect of miRNAs in fibroblast migration was evaluated by transwell assay. Results: In fibroblasts, transfection with miR-21, -100 and -139 increased significantly expression of RANKL, IL8 and all MMPs, with a higher effect in MMP-9. Besides, miR-21 increased fibroblast migration. In osteoblasts, transfection with miRNAs changed the balance that triggers vicious circle at the bone, increasing RANKL and diminishing OPG expression. miRNAs also increased MMPs expression. The greater effect was observed with miR-21 transfection. Conclusions: Through exosomes, prostate cancer cells recruit normal cells to favor their growth and spread. miRNAs in exosomes increase expression of proteins (in particular, MMPs and RANKL) and cell migration of fibroblasts that create a favorable microenvironment for tumor progression at the primary niche. Besides, through bloodstream, tumoral exosomes can reach the bone, preparing the premetastatic niche. miRNAs from these exosomes change the balance RANKL/OPG that triggers osteoclast recruitment facilitating metastases by activation of the bone vicious circle. miR-21, overexpressed in bulk cells, the main cellular component of tumor, has the higher effect in modify microenvironment, being a potential therapeutic target. (FONDECYT 11121525 and 1140417). Citation Format: Eliana Andahur, Enrique A. Castellon, Chistian Ramos, Juan A. Fulla, Catherine A. Sanchez. miRNAs in exosomes from prostate cancer cells modify normal fibroblasts and osteoblasts favoring tumor spread and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1562.

Abstract 4385: Colorectal cancer lung metastasis treatment with lung-selective PI3K pathway inhibition

Abstract Colorectal cancer (CRC) is the second leading cause of cancer death in the US, and its frequency is increasing worldwide. Lung metastases are seen in 10-20% of CRC patients with overall 25-40% 5-year survival after metastasectomy despite advances in imaging and surgical technique. Novel approaches are needed for the treatment of CRC lung metastasis. Selective lung delivery and release of potent PI3K inhibitors may offer a new approach to improve outcome of patients with CRC lung metastasis. The purpose of this study was to evaluate PI3K/Akt pathway activation and expression in CRC lung metastasis patient samples and to develop a nanocarrier for lung selective PI3K drug delivery. METHODS. Metastatic human (HT29, KM20) and mouse (CT26) CRC cell lines were used in our study. Primary colon cancer cultures were isolated from patient samples. Cell viability and IC50 concentrations were measured using a sulforhodamine B colorimetric (SRB) cytotoxicity assay. Expression of PARP, cyclin D1 and pAkt (p473) was assessed by western blot. Akt1, Akt2 and pAkt (S473) expression was analyzed in normal lung, primary and metastatic CRC patient samples by immunohistochemistry. Fluorescent nanoparticle accumulation patterns in vivo were analyzed with IVIS Spectrum imaging and confocal microscopy of fixed frozen tissue sections. Polymeric nanoparticles were loaded with hydrophobic PI3K inhibitors - Wortmannin, BEZ235, PIK-90, and PI-103. RESULTS. We found strong Akt2 and pAkt (S473) expression in all matched primary CRC and CRC lung metastasis patient samples. PI3K drug loaded nanoparticles effectively induced cell cycle arrest and apoptosis in metastatic cancer cell lines and primary cancer cultures. IVIS Spectrum imaging and confocal microscope imaging of fixed frozen tissue sections confirmed absence of fluorescent nanoparticle accumulation in liver, spleen and kidney after intravenous administration in vivo. Lung selective PI3K pathway inhibition in vivo was confirmed by western blot analysis of protein extracts from lung, liver, spleen and kidney. CT26 lung metastasis treatment with lung selective wortmannin-loaded nanoparticles demonstrated a marked suppression of lung metastasis growth and absence of side effects normally associated with PI3K drug treatment. CONCLUSIONS. The introduction of PI3K/Akt pathway inhibitors as anticancer agents against metastatic CRC is still in an early stage. We developed a safe and efficient lung selective drug nanocarrier and demonstrate that lung selective PI3K inhibition is a viable treatment strategy. Therefore, lung selective PI3K inhibition have a considerable promise to maximize the potential of PI3K/AKT inhibitors towards effective CRC lung metastasis treatment. Citation Format: Piotr G. Rychahou, Younsoo Bae, Yekaterina Zaytseva, Eun Y. Lee, Heidi L. Weiss, B. Mark Evers. Colorectal cancer lung metastasis treatment with lung-selective PI3K pathway inhibition. [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 4385. doi:10.1158/1538-7445.AM2015-4385

Metformin, at Concentrations Corresponding to the Treatment of Diabetes, Potentiates the Cytotoxic Effects of Carboplatin in Cultures of Ovarian Cancer Cells

The use of the type 2 diabetics drug metformin has been correlated with enhanced progression-free survival in ovarian cancer. The literature has speculated that this enhancement is due to the high concentration of metformin directly causing cancer cell death. However, this explanation does not fit with clinical data reporting that the women exposed to constant micromolar concentrations of metformin, as present in the treatment of diabetes, respond better to chemotherapy. Herein, our aim was to examine whether micromolar concentrations of metformin alone could bring about cancer cell death and whether micromolar metformin could increase the cytotoxic effect of commonly used chemotherapies in A2780 and SKOV3 cell lines and primary cultured cancer cells isolated from the peritoneal fluid of patients with advanced ovarian cancer. Our results in cell lines demonstrate that no significant loss of viability or change in cell cycle was observed with micromolar metformin alone; however, we observed cytotoxicity with micromolar metformin in combination with chemotherapy at concentrations where the chemotherapy alone produced no loss in viability. We demonstrate that previous exposure and maintenance of metformin in conjunction with carboplatin produces a synergistic enhancement in cytotoxicity of A2780 and SKOV3 cells (55% and 43%, respectively). Furthermore, in 5 (44%) of the 11 ovarian cancer primary cultures, micromolar metformin improved the cytotoxic response to carboplatin but not paclitaxel or doxorubicin. In conclusion, we present data that support the need for a clinical study to evaluate the adjuvant maintenance or prescription of currently approved doses of metformin during the chemotherapeutic treatment of ovarian cancer.

Abstract IA14: Exploiting the translational and functional opportunities for homologous recombination deficiency

Abstract Cancer is a disease of DNA, and the processes by which cells repair DNA damage are critical, not only in causing the disease but also in determining how the cancer cell will respond to treatment. Of the six major pathways by which DNA damage is repaired, homologous recombination (HR) is perhaps the best studied in ovarian cancer. This pathway is responsible for repairing double strand breaks and uses at least forty proteins, including both BRCA1 and BRCA2. We have shown that 50% of high grade serous cancers are defective in homologous recombination and this group are more likely to be sensitive to platinum chemotherapy and have a better prognosis than tumours which are competent for the pathway. As may be expected, this group also includes all the tumours occurring in women with germline mutations of BRCA1 and BRCA2. HR status has also been suggested to be predictive of response to PARP inhibitors and it is proposed that this is an example of synthetic lethality in which inactivation of one pathway is not harmful to a cell but inhibition of two (one by the PARP inhibitor and the other the underlying HR defectiveness) will render the cell non viable. We have confirmed in a large panel of ovarian cancer primary cultures and cell lines that HR defectiveness is indeed a very strong predictor of ex vivo drug sensitivity with positive and negative predictive values close to 100%. These data provide the basis for developing biomarker directed therapy for PARP inhibitors and provide a rationale for increasing the indication for these drugs to tumours other than those caused by germline mutations. Challenges still exist however and current work is seeking to investigate the interplay of the different DNA damage repair pathways and how these may be used to further refine the biomarkers currently available. Citation Format: Richard John Edmondson. Exploiting the translational and functional opportunities for homologous recombination deficiency. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA14.

Involvement of Heregulin/HER3 in the Primary Culture of Human Urothelial Cancer

We previously established a novel method of human colorectal cancer primary culture. This method, termed the cancer tissue originated spheroid method, involves the preparation of multicellular spheroids of primary cancer cells that are cultured so that cell-cell contact is maintained. We applied this method to human urothelial cancer. Cancer tissue originated spheroids were prepared from xenografts or primary human bladder urothelial cancer tumors following the same protocol used for human colorectal cancer. Cancer tissue originated spheroids were characterized using immunohistochemistry, Western blot and polymerase chain reaction. We established a xenograft from a primary bladder urothelial cancer, and isolated and cultured cancer tissue originated spheroids from the xenograft tumor. Cancer tissue originated spheroids retained the characteristics of the original tumor and those of the xenograft. Heregulin promoted cancer tissue originated spheroid growth, and inhibitors of PI3K and mTOR inhibited heregulin induced growth, as did lapatinib but not erlotinib. We also prepared cancer tissue originated spheroids from primary bladder urothelial cancer. The success rate of establishing primary cancer tissue originated spheroids from nonmuscle invasive urothelial cancer was 90.7% and that from muscle invasive cancer was 68.2%. The overall success rate was 84.2%. Heregulin promoted the growth of primary cancer tissue originated spheroids from 4 of 7 patients. We report a method of establishing primary cultures of human urothelial cancer cells. Growth stimulation by heregulin in cancer tissue originated spheroids from xenografts and primary tumors suggests the possibility of molecular targeting therapy against HER3 signaling for human urothelial cancer. The cancer tissue originated spheroid method might be useful for selecting patients for molecular targeting drugs such as lapatinib.

Abstract P1-05-12: Metastatic xenograft models of human estrogen receptor negative breast cancer primary cultures are driven by the recruitment of myeloid-derived suppressor cells

Abstract The study of early stage and metastatic breast cancer relies heavily on the use of established cell lines often derived from metastatic lesions rather than from primary tumors, limiting our understanding of primary tumor development. Study of the mechanisms governing the metastatic process in its entirety is not always feasible with these types of cellular and xenograft models, as often the primary tumor needs to be excised, or breast cancer cells are introduced into circulation of host mice. While this approach has allowed a better understanding of metastasis, it may unintentionally enhance the colonization efficiency and pass over important events occurring in the tumor microenvironment. In order to provide a cellular model that more closely recapitulates breast cancer development and progression, we generated breast cancer cellular cultures by dissociating specimens of human estrogen receptor-negative primary breast tumors and placing them in culture. Within our group of dissociated tumor (DT) cell cultures, we had shown that five cultures are formed by breast cancer cells and these were classified by PAM50 predictor analysis as belonging to the ER−/PR−/Her2+ and triple negative subtypes. DT cells are tumorigenic in NOD/Scid and NOD/Scid gamma (NSG) mice. Our first aim was to assess the metastatic potential of the DT cells in two mouse models. We show that 3 of the 5 cultures are metastatic to lymph nodes, liver and or lungs; common sites of metastasis in breast cancer patients. Spontaneous metastases were observed without the requirement of primary tumor excision. The metastatic frequency ranged from 25–90% in NOD/Scid mice, and from 80–100% in NSG mice. Moreover, expression of key proteins such as epidermal growth factor receptor (EGFR) detected in primary tumors was also found in the metastatic lesions. Because the DT cells have been isolated from primary breast tumors they represent a clinically relevant and valuable model to study breast cancer and metastasis. A critical factor influencing tumor progression and metastasis is the infiltration of immune cells including myeloid-derived suppressor cells (MDSCs), which have been shown to be increased in a tumor-dependent manner in xenograft models of breast cancer. Previous work from our lab has shown that stroma of tumor-bearing mice exhibited gene expression changes, including increased expression of calcium binding proteins S100A8 and S100A9, that are consistent with myeloid immune cell infiltration. Our second aim was thus to analyze the recruitment of S100A8+ MDSCs by xenografted DT tumors. We show that the recruitment of S100A8+ MDSCs is detected in DT tumors and sites of metastasis, and more importantly, their presence was significantly greater in the tumors that metastasized compared to those that did not. These data strongly suggest that the metastatic potential of breast cancer cells may be driven at least in part by the recruitment of S100A8+ MDSCs. Taken together, our observations and our closer-to-primary xenograft models may lead to a better understanding of metastatic disease, as well as to the development and pre-clinical screening of targeted breast cancer therapeutics, particularly in the metastatic setting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-12.

Unmasking epithelial-mesenchymal transition in a breast cancer primary culture: a study report

BackgroundImmortalized cancer cell lines are now well-established procedures in biomedicine for a more complete understanding of cellular processes in cancer. However, they are more useful in preparation of fresh tumour tissue, in order to obtain cancer cells with highly preserved individual tumour properties. In the present study we report an analytical investigation on a breast cancer primary cell culture isolated from a surgical specimen obtained from a patient with an infiltrating ductal carcinoma. The objective of the research was to reveal unrecognized aspects of neoplastic cells, typical of the tumour from where the cells were derived, but masked in fixed tissue sections, in order to better predict the aggressive potentiality of the tumour.FindingsUsing a combination of mechanical and enzymatic treatment, the tumour tissue was dissociated immediately after surgical removal. The primary cells were isolated by differential cell centrifugation and grown in selective media. Immunocytochemistry and quantitative RT-PCR analysis were performed to detect the presence of specific biomarkers at protein and transcript level.The isolated primary breast cancer cells displayed phenotypic behaviour, characteristic of malignant cells and expression of several mesenchymal markers, revealing a strong signature for the epithelial-to-mesenchymal transition associated to a stellate morphology with a number of cellular protrusions and the attitude to overgrow as multilayered overlapping cellular foci.ConclusionsOur data are a further meaningful indication that primary cell cultures represent a powerful system that could be applied to those cases deserving a deeper investigation at molecular level in order to design individualized anticancer therapies in the future.

Antitumor Activity of Artemisinin and Its Derivatives: From a Well-Known Antimalarial Agent to a Potential Anticancer Drug

Improvement of quality of life and survival of cancer patients will be greatly enhanced by the development of highly effective drugs to selectively kill malignant cells. Artemisinin and its analogs are naturally occurring antimalarials which have shown potent anticancer activity. In primary cancer cultures and cell lines, their antitumor actions were by inhibiting cancer proliferation, metastasis, and angiogenesis. In xenograft models, exposure to artemisinins substantially reduces tumor volume and progression. However, the rationale for the use of artemisinins in anticancer therapy must be addressed by a greater understanding of the underlying mechanisms involved in their cytotoxic effects. The primary targets for artemisinin and the chemical base for its preferential effects on heterologous tumor cells need yet to be elucidated. The aim of this paper is to provide an overview of the recent advances and new development of this class of drugs as potential anticancer agents.

An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models

BackgroundMany factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression.MethodsPrimary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively.ResultsSignificant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumourogenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells.ConclusionsOur results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.

Abstract 4114: Evaluation of ERCC1 expression and in vitro drug response to cisplatin based on human NSCLC cell lines and primary cultures of human lung cancers

Abstract Introduction: In vitro experimentation has been suggested as a rapid tool for identifying and evaluating biomarkers associated with drug response. Excision repair cross-complementation 1 (ERCC1) enzyme plays a rate-limiting role in the nucleotide excision repair pathway and its up regulation has been associated with resistance to platinum agents. Recent studies have suggested that low expression levels of ERCC1 are related to a better survival benefit from cisplatin-based chemotherapy among patients with advanced non-small cell lung carcinoma (NSCLC). This study evaluates the relationship between ERCC1 expression and in vitro drug response to cisplatin using an in vitro chemoresponse assay, ChemoFx® Drug Response Marker (DRM), in human NSCLC cell lines and primary cultures of human lung cancers. Methods: The ChemoFx® DRM was performed on 13 immortalized human NSCLC cell lines (NCI-H520, HOP_92, HOP-62, A549, Calu-3, HCC827, OK, NCI-H460, NCI-H596, NCI-H1666, EKVX, NCI-H358, and NCI-H1975) and 30 primary cultures established from de-identified lung cancer surgical specimens. Cells were treated with a 10-dose range of cisplatin for 72 hours before DAPI-nuclear staining and counting. Response Index scores (RI scores), which are derived from an area under the dose-response curve (AUC), were calculated on the resulting dose-response curves. Protein expression of ERCC1 was evaluated with In-Cell Western analysis. Pearson correlation coefficient was used to show the association between ERCC1 expression and in vitro drug response. Result: Increased ERCC1 expression was significantly associated with cisplatin resistance in the 13 immortalized NSCLC cell lines (r=−0.72, p=0.004). A similar trend was observed across 30 primary cultures of human lung cancers, but the association did not reach the level of significance (r=−0.22, p=0.24). Conclusion: Clinical observation of a negative correlation between ERCC1 expression level and improved survival benefit from cisplatin-based chemotherapy was mirrored by in vitro ChemoFx® DRM analysis with strong correlation in immortalized human NSCLC cell lines and in primary cultures from human lung cancers with a correlation trend. This result suggests the value of in vitro chemoresponse assay in identification and evaluation of biomarkers and their association with chemotherapeutic response. The less significant correlation in human lung cancer primary cultures indicated that ERCC1 alone may not be sufficient for clinical prediction of response in human lung cancers across different pathological subtypes and stages, and additional biomarkers may be involved. These results suggest the value of in vitro chemo response assay, in helping to determine cisplatin response in human lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4114. doi:10.1158/1538-7445.AM2011-4114

Metabolic responses of A549 lung cells to cisplatin and radiation exposure studied by 1H NMR spectroscopy

This work aims to characterize the dynamic metabolic responses of A549 lung tumor cells exposed to cisplatin (CDDP) and to 6 Gy ionizing radiation over a period of 48h. Control and CDDP/radiation treated cells, in the form of lysed suspensions, were directly analyzed by 1H High Resolution Magic Angle Spinning (HRMAS) NMR spectroscopy (500 MHz) and the changes in their intracellular metabolic profiles assessed by spectral integration and multivariate analysis. In this way, consistent variation patterns could be detected and specific metabolic effects related to drug and/or radiation exposure could be identified. In particular, significant time-dependent alterations were found in lipids and choline-containing compounds, as well as in low molecular weight metabolites such as some amino acids and nitrogenated bases. The results presented show that 1H NMR spectroscopy is a powerful tool for providing detailed biochemical information about the effects induced on cultured cells by external perturbations, such as a chemotherapy drug and ionizing radiation. In the future, this approach will be applied to lung cancer primary cultures subjected to different treatment regimens.

Genomic action of permanently charged tamoxifen derivatives via estrogen receptor-α

Tamoxifen is a selective estrogen receptor modulator widely used in oncology and reproductive endocrinology. In order to decrease its non-desirable effects and elucidate mechanisms of action, permanently charged tamoxifen derivatives (PCTDs) have been reported. Whether PCTDs have genomic effects remains controversial. Since the clinical relevance of tamoxifen, the necessity to have new anticancer drugs, and in order to gain insights into the mechanisms of action of PCTDs, we obtained six quaternary ammonium salts derived from tamoxifen including three new compounds. We characterized them by nuclear magnetic resonance, X-ray diffraction, electron microscopy, and/or high performance liquid chromatography, and detected them in cell lysates by liquid chromatography coupled to mass spectrometry. We evaluated their binding to estrogen receptor-α (ERα, their effect on the transcriptional activity mediated by ERα (gene reporter assays), and the proliferation of cancer cells (MCF-7 and cells from a cervical cancer primary culture). Structural studies demonstrated the expected identity of the molecules. All PCTDs did bind to ERα, one of them induced ERα-mediated transcription while two others inhibited such genomic action. Accordingly, PCTDs were detected in cell lysates. PCTDs inhibited cell proliferation, noteworthy, two of them displayed higher inhibition than tamoxifen. Structure–activity analysis suggests that PCTDs permanent positive charge and the length of the aliphatic chain might be associated to the biological responses studied. We suggest genomic effects as a mechanism of action of PCTDs. The experimental approaches here used could lead to a better design of new therapeutic molecules and help to elucidate molecular mechanisms of new anticancer drugs.

Gene Expression Profiles for Predicting the Efficacy of the Anticancer Drug 5-Fluorouracil in Breast Cancer

Chemotherapy is an important postsurgery adjuvant therapy in the treatment of breast cancer. However, because of the individual genotype differences of patients, the drug efficacy differs from person to person, even when the same chemotherapy drug is administered. The purpose of this research was to probe the gene expression profiles to predict the efficacy of 5-fluorouracil (5-FU), the common drug used in chemotherapy for various type of cancers, in Taiwanese breast cancer patients. Microarray analysis was conducted on the cancer cell line ZR-75-1 with and without 5-FU stimulation to identify the differentially expressed genes. The significant overexpressed gene groups were selected after bioinformatics software analysis to explore the molecular mechanism of 5-FU. Six strains of breast cancer cell line purchased from American Type Culture Collection were used to analyze the expression profiles of the above target gene groups. IL18, CCL28, CXCL2, SOD1, HRAS, FDXR, and CHI3L1 genes were significantly differentially expressed in 5-FU responder and nonresponder cell lines. The selected gene groups were validated with 20 strains of breast cancer primary cultures established previously in our laboratory. The experimental results demonstrated that FAM46A, IL18, CCL28, TNF, CXCL2, PLEKHA8, HRAS, FDXR, and CHI3L1 genes showed statistically significant differential expression between primary breast cancer culture cells that respond and nonrespond to 5-FU. Six genes, IL18, CCL28, CXCL2, HRAS, FDXR, and CHI3L1, showed significant differential expression pattern in both American Type Culture Collection and primary breast cancer cultured cells. The findings of this study may serve as basis for predicting the effectiveness of 5-FU on breast cancer.

Monocyte Chemotactic Protein-1 Secreted by Primary Breast Tumors Stimulates Migration of Mesenchymal Stem Cells

Major barriers to effective adenovirus-based gene therapy include induction of an immune response and tumor-specific targeting of vectors. The use of mesenchymal stem cells (MSC) as systemic delivery vehicles for therapeutic genes has been proposed as a result of their combined ability to home in on the tumor site and evade the host immune response. This study is aimed at investigating factors mediating homing of human MSCs to breast cancer primary cultures and cell lines in vitro and in vivo. Fluorescently labeled MSCs were given to mice bearing breast cancer xenografts, and tumor tissue was harvested to detect MSC engraftment. MSC migration in response to primary breast tumors in vitro was quantified, and chemokines secreted by tumor cells were identified. The role of monocyte chemotactic protein-1 (MCP-1) in cell migration was investigated using antibodies and standards of the chemokine. Serum MCP-1 was measured in 125 breast cancer patients and 86 healthy controls. Engrafted MSCs were detected in metastatic breast tumors in mice after systemic administration. There was a significant increase in MSC migration in response to primary breast tumor cells in vitro (6-fold to 11-fold increase). Tumor explants secreted a variety of chemokines including GROalpha, MCP-1, and stromal cell-derived factor-1alpha. An MCP-1 antibody caused a significant decrease (37-42%) in MSC migration to tumors. Serum MCP-1 levels were significantly higher in postmenopausal breast cancer patients than age-matched controls (P < 0.05). These results highlight a role for tumor-secreted MCP-1 in stimulating MSC migration and support the potential of these cells as tumor-targeted delivery vehicles for therapeutic agents.