Liposome-tethered supported lipid bilayer platform for capture and release of heterogeneous populations of circulating tumor cells.

Because of scarcity, vulnerability, and heterogeneity in the population of circulating tumor cells (CTCs), the CTC isolation system relying on immunoaffinity interaction exhibits inconsistent efficiencies for all types of cancers and even CTCs with different phenotypes in individuals. Moreover, releasing viable CTCs from an isolation system is of importance for molecular analysis and drug screening in precision medicine, which remains a challenge for current systems. In this work, a new CTC isolation microfluidic platform was developed and contains a coating of the antibody-conjugated liposome-tethered-supported lipid bilayer in a developed chaotic-mixing microfluidic system, referred to as the "LIPO-SLB" platform. The biocompatible, soft, laterally fluidic, and antifouling properties of the LIPO-SLB platform offer high CTC capture efficiency, viability, and selectivity. We successfully demonstrated the capability of the LIPO-SLB platform to recapitulate different cancer cell lines with different antigen expression levels. In addition, the captured CTCs in the LIPO-SLB platform can be detached by air foam to destabilize the physically assembled bilayer structures due to a large water/air interfacial area and strong surface tension. More importantly, the LIPO-SLB platform was constructed and used for the verification of clinical samples from 161 patients with different primary cancer types. The mean values of both single CTCs and CTC clusters correlated well with the cancer stages. Moreover, a considerable number of CTCs were isolated from patients' blood samples in the early/localized stages. The clinical validation demonstrated the enormous potential of the universal LIPO-SLB platform as a tool for prognostic and predictive purposes in precision medicine.

Proliferative ability of circulating tumor cells is a prognostic factor in Early-Stage lung adenocarcinoma.

INTRODUCTION: Circulating tumor cells (CTCs) and their proliferative ability in lung adenocarcinoma (LUAD) were not well-investigated. We developed a protocol combining an efficient viable CTC isolation and in-vitro cultivation for the CTC enumeration and proliferation to evaluate their clinical significance. METHOD: The peripheral blood of 124 treatment-naïve LUAD patients were processed by a CTC isolation microfluidics, DS platform, followed by in-vitro cultivation. LUAD-specific CTCs were defined by immunostaining of DAPI+/CD45-/(TTF1/CK7)+ and were enumerated upon isolation and after 7-day cultivation. The CTC proliferative ability was evaluated by both the cultured number and the culture index, a ratio of cultured CTC number to the initial CTC number in 2 mL of blood. RESULT: All but two LUAD patients (98.4%) were detected with at least one CTC per 2 mL of blood. Initial CTC numbers did not correlate with metastasis (75 ± 126 for non-metastatic, 87 ± 113 for metastatic groups; P = 0.203). In contrast, both the cultured CTC number (mean: 28, 104, and 185 in stage 0/I, II/III, and IV; P < 0.001), and the culture index (mean: 1.1, 1.7 and 9.3 in stage 0/I, II/III, and IV; P = 0.043) were significantly correlated with the stages. Overall survival analysis within the non-metastatic group (N = 53) showed poor prognosis for patients with elevated cultured counts (cutoff ≥ 30; P = 0.027). CONCLUSION: We implemented a CTC assay in clinical LUAD patients with a high detection rate and cultivation capability. Cultured CTC count and proliferative ability, rather than the crude CTC numbers, highly associated with cancer prognosis.

Uncovering synthetic lethal interactions for therapeutic targets and predictive markers in lung adenocarcinoma.

Two genes are called synthetic lethal (SL) if their simultaneous mutation leads to cell death, but mutation of either individual does not. Targeting SL partners of mutated cancer genes can selectively kill cancer cells, but leave normal cells intact. We present an integrated approach to uncover SL gene pairs as novel therapeutic targets of lung adenocarcinoma (LADC). Of 24 predicted SL pairs, PARP1-TP53 was validated by RNAi knockdown to have synergistic toxicity in H1975 and invasive CL1-5 LADC cells; additionally FEN1-RAD54B, BRCA1-TP53, BRCA2-TP53 and RB1-TP53 were consistent with the literature. While metastasis remains a bottleneck in cancer treatment and inhibitors of PARP1 have been developed, this result may have therapeutic potential for LADC, in which TP53 is commonly mutated. We also demonstrated that silencing PARP1 enhanced the cell death induced by the platinum-based chemotherapy drug carboplatin in lung cancer cells (CL1-5 and H1975). IHC of RAD54B↑, BRCA1↓-RAD54B↑, FEN1(N)↑-RAD54B↑ and PARP1↑-RAD54B↑ were shown to be prognostic markers for 131 Asian LADC patients, and all markers except BRCA1↓-RAD54B↑ were further confirmed by three independent gene expression data sets (a total of 426 patients) including The Cancer Genome Atlas (TCGA) cohort of LADC. Importantly, we identified POLB-TP53 and POLB as predictive markers for the TCGA cohort (230 subjects), independent of age and stage. Thus, POLB and POLB-TP53 may be used to stratify future non-Asian LADC patients for therapeutic strategies.

Mitochondrial translocation of EGFR regulates mitochondria dynamics and promotes metastasis in NSCLC.

Dysfunction of the mitochondria is well-known for being associated with cancer progression. In the present study, we analyzed the mitochondria proteomics of lung cancer cell lines with different invasion abilities and found that EGFR is highly expressed in the mitochondria of highly invasive non-small-cell lung cancer (NSCLC) cells. EGF induces the mitochondrial translocation of EGFR; further, it leads to mitochondrial fission and redistribution in the lamellipodia, upregulates cellular ATP production, and enhances motility in vitro and in vivo. Moreover, EGFR can regulate mitochondrial dynamics by interacting with Mfn1 and disturbing Mfn1 polymerization. Overexpression of Mfn1 reverses the phenotypes resulting from EGFR mitochondrial translocation. We show that the mitochondrial EGFR expressions are higher in paired samples of the metastatic lymph node as compared with primary lung tumor and are inversely correlated with the overall survival in NSCLC patients. Therefore, our results demonstrate that besides the canonical role of EGFR as a receptor tyrosine, the mitochondrial translocation of EGFR may enhance cancer invasion and metastasis through regulating mitochondria dynamics.

CDK inhibitor p57 (Kip2) is downregulated by Akt during HER2-mediated tumorigenicity.

HER2/neu oncogene is frequently deregulated in cancers, and the (PI3K)-Akt signaling is one of the major pathways in mediating HER2/neu oncogenic signal. p57 (Kip2) , an inhibitor of cyclin-depependent kinases, is pivotal in regulating cell cycle progression, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to p57 (Kip2) regulation, and that Akt is a negative regulator of p57 (Kip2) . Ectopic expression of Akt can decrease the expression of p57 (Kip2) , while Akt inhibition leads to p57 (Kip2) stabilization. Mechanistic studies show that Akt interacts with p57 (Kip2) and causes cytoplasmic localization of p57 (Kip2) . Akt phosphorylates p57 on Ser 282 or Thr310. Akt activity results in destabilization of p57 by accelerating turnover rate of p57 and enhancing p57 ubiquitination. Importantly, the negative impact of HER2/Akt on p57 stability contributes to HER2-mediated cell proliferation, transformational activity and tumorigenicity. p57 restoration can attenuate these defects caused by HER2. Significantly, Kaplan-Meier analysis of tumor samples demonstrate that in tumors where HER2 expression was observed, high expression levels of p57 (Kip2) were associated with better overall survival. These data suggest that HER2/Akt is an important negative regulator of p57 (Kip2) , and that p57 restoration in HER2-overexpressing cells can reduce breast tumor growth. Our findings indicate the applicability of employing p57 regulation as a therapeutic intervention in HER2-overexpressing cancers.

The ability of LCRMP-1 to promote cancer invasion by enhancing filopodia formation is antagonized by CRMP-1.

Metastasis is a predominant cause of death in patients with cancer. It is a complex multistep process that needs to be better understood if we are to develop new approaches to managing tumor metastasis. Tumor cell invasion of the local stroma is suppressed by collapsin response mediator protein-1 (CRMP-1). Recently, we identified a long isoform of CRMP-1 (LCRMP-1), expression of which correlates with cancer cell invasiveness and poor clinical outcome in patients with non-small-cell lung cancer (NSCLC). Here, we report that LCRMP-1 overexpression in noninvasive human cell lines enhanced filopodia formation, cancer cell migration, and invasion via stabilization of actin. This effect required a highly conserved N-terminal region of LCRMP-1 as well as the WASP family verprolin-homologous protein-1/actin nucleation pathway (WAVE-1/actin nucleation pathway). Furthermore, LCRMP-1 appeared to act downstream of Cdc42, a Rho family protein known to be involved in actin rearrangement. In addition, LCRMP-1 associated with CRMP-1, which downregulated cancer cell metastasis by interrupting the association of LCRMP-1 and WAVE-1. Finally, we found that high-level expression of LCRMP-1 and low-level expression of CRMP-1 were associated with lymph node metastasis and poor survival in patients with NSCLC. In sum, we show that LCRMP-1 and CRMP-1 have opposing functions in regulating cancer cell invasion and metastasis and propose that this pathway may serve as a potential anticancer target.

Claudin-1 is a metastasis suppressor and correlates with clinical outcome in lung adenocarcinoma.

RATIONALE: Claudin (CLDN)-1, a key component of tight junction complexes, was down-regulated in human lung adenocarcinomas. OBJECTIVES: To investigate the clinical significance of CLDN1 expression in patients with lung adenocarcinoma and its role in cancer invasion and metastasis. METHODS: We examined the CLDN1 mRNA expression in tumor specimens from 64 patients with lung adenocarcinoma and protein expression by immunohistochemistry in an independent cohort of 67 patients with lung adenocarcinoma. CLDN1 functions in cancer cell migration, invasion, and metastatic colonization were studied by overexpression and knockdown of CLDN1. Affymetrix microarrays were performed to identify gene expression changes associated with CLDN1 overexpression. MEASUREMENTS AND MAIN RESULTS: We found that low-CLDN1 mRNA expression had shorter overall survival (P = 0.027, log-rank test) in 64 patients with lung adenocarcinoma, and we confirmed by immunohistochemistry that low CLDN1 expression had shorter overall survival (P = 0.024, log-rank test) in an independent cohort of 67 patients with lung adenocarcinoma. Overexpression of CLDN1 inhibited cancer cell dissociation in time-lapse imaging of wound healing, and suppressed cancer cell migration, invasion, and metastasis. Knockdown CLDN1 expression increased cancer cell invasive and metastatic abilities. Affymetrix microarrays identified a panel of genes altered by CLDN1 overexpression. CLDN1 increased expressions of cancer invasion/metastasis suppressors (e.g., connective tissue growth factor [CTGF], thrombospondin 1 [THBS1], deleted in liver cancer 1 [DLC1], occludin [OCLN], zona occludens 1 [ZO-1]) and suppressed expressions of invasion/metastasis enhancers (e.g., secreted phosphoprotein 1 [SPP1], cut-like homeobox 1 [CUTL1], transforming growth factor alpha [TGF-alpha], solute carrier family 2 [faciliated glucose transporter] member 3 [SLC2A3], placental growth factor [PGF]), supporting a role for CLDN1 as an invasion and metastasis suppressor. CONCLUSIONS: CLDN1 is a cancer invasion/metastasis suppressor. CLDN1 is also a useful prognostic predictor and potential drug treatment target for patients with lung adenocarcinoma.

Connective tissue growth factor inhibits metastasis and acts as an independent prognostic marker in colorectal cancer.

BACKGROUND & AIMS: Connective tissue growth factor (CTGF) has been shown to be implicated in tumor development and progression. The aim of this study was to investigate the role of CTGF in progression of colorectal cancer (CRC). METHODS: Immunohistochemical staining of specimens from 119 patients with CRC was performed. Liposome-mediated transfection was used to introduce a CTGF expression vector into CRC cell lines. Transfectants were tested in invasive ability and experimental hepatic metastasis in BALB/c mice. Furthermore, a FOPflash/TOPflash reporter assay was performed to investigate CTGF on the beta-catenin/T-cell factor signaling pathway. RESULTS: Patients with stage II and stage III CRC whose tumors displayed high CTGF expression had a significantly higher overall survival and a disease-free advantage over patients with CRC with low CTGF expression. Alterations in the CTGF level in CRC cell lines modulated their invasive ability with an inverse correlation. In addition, a reduction in the CTGF level of CT26 cells after stable transfection with antisense CTGF resulted in increased liver metastasis in BALB/c mice. The activity of the beta-catenin/T-cell factor signaling pathway and its downstream effector gene matrix metalloproteinase 7 in these CTGF-transfected cells was strongly attenuated. Blockage of matrix metalloproteinase 7 with its neutralizing antibodies inhibited increased invasiveness in antisense CTGF-transfected CT26 cells. CONCLUSIONS: Our results implicate CTGF as a key regulator of CRC invasion and metastasis, and it appears to be a useful and better prognosis factor for patients with stage II and stage III CRC.