Encapsulation and adhesion of nanoparticles as a potential biomarker for TNBC cells metastatic propensity

Abstract

Metastasis is the main cause of cancer-related mortality; therefore, the ability to predict its propensity can remarkably affect survival rate. Metastasis development is predicted nowadays by lymph-node status, tumor size, histopathology, and genetic testing. However, all these methods may have inaccuracies, and some require weeks to complete. Identifying novel prognostic markers will open an essential source for risk prediction, possibly guiding to elevated patient treatment by personalized strategies. Cancer cell invasion is a critical step in metastasis. The cytoskeletal mechanisms used by metastatic cells for the invasion process are very similar to the utilization of actin cytoskeleton in the endocytosis process. In the current study, the adhesion and encapsulation efficiency of low-cost carboxylate-modified fluorescent nanoparticles by breast cancer cells with high (HM) and low metastatic potential (LM) have been evaluated; benign cells were used as control. Using high-content fluorescence imaging and analysis, we have revealed (within a short time of 1 h), that efficiency of nanoparticles adherence and encapsulation is sufficiently higher in HM cells compared to LM cells, while benign cells are not encapsulating or adhering the particles during experiment time at all. We have utilized custom-made automatic image analysis algorithms to find quantitative co-localization (Pearson's coefficients) of the nanoparticles with the imaged cells. The method proposed here is straightforward; it does not require especial equipment or expensive materials nor complicated cell manipulations, it may be potentially applicable for various cells, including patient-derived cells. Effortless and quantitative determination of the metastatic likelihood has the potential to be performed using patient-specific biopsy/surgery sample, which will directly influence the choice of protocols for cancer patient's treatment and, as a result, increase their life expectancy.

Figure 1

A typical images (selected cells × 5 magnification from Supplementary 1) of internalized and internalized and adhered carboxylate modified 100 nm and 200 nm nanoparticles by benign cells, cells with low (LM) and high (HM) metastatic potential. HM and LM cells were extensively washed with Trypsin or PBS after 1 h incubation with fluorescent nanoparticles, resulting in the presence of internalized or internalized and adhered beads, respectively. The scale bar is 10 µm.

Figure 2

3D reconstruction images of adhered and internalized (A) and internalized only (B) carboxylate modified 200 nm nanoparticles by high MP breast cancer cells. The scale bar is 10 µm.

Figure 3

Scattergrams of Pearson coefficients for (A) 200 nm and (B) 100 nm diameter nanoparticles colocalization with breast cancer cells with high and low MP (MDA-MB-231 and MCF7, respectively) and benign cells (MCF10-A). Mean value is marked by + , median by − . Statistical significance for (A) and (B) is shown in Tables 1 and 2 respectively.

Figure 4

(A) Pearson coefficients obtained with 100 (light gray) and 200 (dark gray) nm particles for all tested cell types; n > 110. (B) Normalized to control background values of Pearson coefficients obtained with 100 (light gray) and 200 (dark gray) nm particles for high and low MP breast cancer cells. Error bars are standard errors; * − P < 0.0001.

Figure 5

Results of flow cytometry analysis of HM (black) and LM (gray) cells with internalized (dotted line) and internalized and adhered (solid line) 200 nm fluorescent beads.