CXCL12-loaded-hydrogel (CLG): A new device for metastatic circulating tumor cells (CTCs) capturing and characterization

Abstract

Background: Circulating Tumor Cells (CTCs) represent a small, heterogeneous population that comprise the minority of cells able to develop metastasis. To trap and characterize CTCs with metastatic attitude, a CXCL12-loaded hyaluronic-gel (CLG) was developed. CXCR4+cells with invasive capability would infiltrate CLG.

Methods: Human colon, renal, lung and ovarian cancer cells (HT29, A498, H460 and OVCAR8 respectively) were seeded on 150 μl Empty Gels (EG) or 300 ng/ml CXCL12 loaded gel (CLG) and allowed to infiltrate for 16 h. Gels were then digested and fixed with 2 % FA-HAse for human cancer cell enumeration or digested with HAse and cancer cells recovered. CLG-recovered cells migrated toward CXCL12 and were tested for colonies/spheres formation. Moreover, CXCR4, E-Cadherin and Vimentin expression was assessed through flow cytometry and RT-PCR. The clinical trial "TRAP4MET" recruited 48 metastatic/advanced cancer patients (8 OC, 8 LC, 8 GBM, 8 EC, 8 RCC and 8 EC). 10 cc whole blood were devoted to PBMCs extraction (7 cc) and ScreenCell™ filters (3 cc) CTCs evaluation. Ficoll-isolated patient's PBMCs were seeded over CLG and allowed to infiltrate for 16 h; gels were digested and fixed with 2 % FA-HAse, cells stained and DAPI+/CD45-/pan-CK + cells enumerated as CTCs.

Results: Human cancer cells infiltrate CLG more efficiently than EG (CLG/EG ratio 1.25 for HT29/1.58 for A498/1.71 for H460 and 2.83 for OVCAR8). CLG-recovered HT29 cells display hybrid-mesenchymal features [low E-cadherin (40 %) and high vimentin (235 %) as compared to HT29], CXCR4 two-fold higher than HT29, efficiently migrate toward CXCL12 (two-fold higher than HT29) and developed higher number of colonies (171 ± 21 for HT29-CLG vs 131 ± 8 colonies for HT29)/larger spheres (spheroid area: 26561 ± 6142 μm² for HT29-CLG vs 20297 ± 7238 for HT29). In TRAP4MET clinical trial, CLG-CTCs were isolated in 8/8 patients with OC, 6/8 with LC, 6/8 with CRC, 8/8 with EC, 8/8 with RCC cancer and 5/8 with GBM. Interestingly, in OC, LC and GBM, CLG isolated higher number of CTCs as compared to the conventional ScreenCell™ (CLG/SC ratio = 1.88 for OC, 2.47 for LC and 11.89 for GBM). Bland and Altman blot analysis and Passing and Bablok regression analysis showed concordance between the methodological approaches but indicate that SC and CLG are not superimposable suggesting that the two systems select cells with different features.

Conclusion: CLG might represent a new and easy tool to isolate invasive CTCs in multiple cancers such as OC, LC and GBM at today orphan of reliable methods to consistently detect CTCs.

Keywords: Artificial niche; CXCL12/CXCR4; Cancer trap; Metastatic CTCs.

Fig. 1

Human cancer cells efficiently infiltrate CLG. (A) Schematic representation of Gel infiltration assay. (B) Human colon, renal, lung, and ovarian cells (HT29, A498, H460 and OVCAR8) were seeded on empty (EG) or CXCL12-loaded Hydrogel (CLG) on 8-well chamber slide in Serum Free (SF) media and allowed to infiltrate 16 h. Cells were fixed with 2%FA + Hyaluronidase (HAase) for 6 h and stained with DAPI for enumeration at fluorescent microscope. B 100, 500 or 1000 A498, HT29, H460 and OVCAR8 were allowed to infiltrate EG or CLG. Data in bar graphs represent mean ± SEM of at least two independent experiments. A two-tailed t-test was used to assess significance. *P < 0·05, **P < 0·01 ***P < 0·001.

Fig. 2

HT29-CLG cells express high CXCR4, migrate efficiently toward CXCL12 and show mesenchymal features. (A) HT29-EG- (HT29-EG) and CLG- (HT29-CLG) HT29-derived immortalized cells CXCR4 expression; (B) CXCL12-induced migration; (C) colony assay; (D) spheroid formation capability and (E) E-cadherin and Vimentin RNA expression. Data in bar graphs represent mean ± SEM of at least two independent experiments. A two-tailed t-test was used to assess significance. *P < 0·05, **P < 0·01 ***P < 0·001.

Fig. 3

CLG efficiently recovered CTCs from cancer patient's blood (TRAP4MET clinical trial). (A) Schematic representation of CTCs isolation using CLG. (B) CTCs isolated from patients from the TRAP4MET clinical trial using CLG. 7 cc patient's blood were Ficoll-paqued and isolated cells suspended in Serum Free media and placed on CLG in 8-well chamber slide. Cells were allowed to migrate 16h and then fixed in 2 % FA-HAase and stained with DAPI, Alexa488-anti human CD45 and Alexa594-anti-human panCK. As negative control, HD blood samples were searched for DAPI+/pan-CK+/CD45-cells (DAPI+/pan-CK+/CD45-mean count/cc of 0,33 ± 0,32).

Fig. 4

CLG isolated CTCs with higher efficiency than Screen Cell in OC, LC and GBM patients. Ten milliliters of blood were collected, 7 cc used for CLG-CTCs and 3 cc for Screen Cell™ CTCs isolation. (A) Total CTCs isolated with CLG vs SC; (B) Correlation analysis (Passing-Bablok regression and Bland-Altman plot); (C) Mann-Whitney test for independent samples analysis; (D) Wilcoxon test for paired samples analysis.