Characterizing the malignancy and drug resistance of cancer cells from their membrane resealing response

In this report, we showed that two tumor cell characteristics, namely the malignancy and drug-resistance status can be evaluated by their membrane resealing response. Specifically, membrane pores in a number of pairs of cancer and normal cell lines originated from nasopharynx, lung and intestine were introduced by nano-mechanical puncturing. Interestingly, such nanometer-sized holes in tumor cells can reseal ~2–3 times faster than those in the corresponding normal cells. Furthermore, the membrane resealing time in cancer cell lines exhibiting resistance to several leading chemotherapeutic drugs was also found to be substantially shorter than that in their drug-sensitive counterparts, demonstrating the potential of using this quantity as a novel marker for future cancer diagnosis and drug resistance detection. Finally, a simple model was proposed to explain the observed resealing dynamics of cells which suggested that the distinct response exhibited by normal, tumor and drug resistant cells is likely due to the different tension levels in their lipid membranes, a conclusion that is also supported by direct cortical tension measurement.

Cells were maintained at 37°C and 5% CO humidified atmosphere. In addition, a total concentration of 2 × 10 cells/mL was incubated in confocal dishes without serum/growth factor for 24 hours to achieve G0 phase synchronization prior to the actual test.

C. Drugs and protocols for developing drug-resistance
Supply of HSP-90 inhibitor drug, AUY922 was obtained from Novartis  Recent studies have shown that the cortical tension of cells can be measured by indentation with a pyramid or spherical indenter 11, S23 . Similar approach was adopted in this investigation. In particular, a cylindrical probe coated with Bovine Serum Albumin (to eliminate possible adhesion with the cell membrane) was used to indent the cell.
Before any contact take place, Laplace law implies that: where is the intracellular pressure, is the external pressure, γ is the membrane tension and with R is the radius of the cell. Denoting the indentation depth and the corresponding contact radius as δ and B respectively ( Supplementary Fig. 3), we proceed by assuming that the cell deformation is small (i.e. the cell still maintains the spherical shape outside the contact region) and hence these two quantities can be approximately related to each other as: In addition, force equilibrium of the contact part requires that: where F is the force applied by the indenter. Combining Eqs. (S1) -(S3), we finally have: Given that both F and are recorded in the indentation test, Eq. (S4) provides a simple way for us to estimate the cortical tension (γ ). In our experiments, this quantity was measured to be in the range of ~50-200 pN/μm (see Fig. 4(A) and 4(B)), depending on the cell types, which is comparable to those reported in the literature S23, 24 .

E. Resealing response of drug-treated A549 cells
To confirm whether the distinct resealing response observed in this study is caused by the different tension levels in the membrane of cells. We treated A549 cells with three drugs, Jasplakinolide (Jas), Cytochalasin D (CytD) and Blebbistain (Blebst), that are known to alter the cortical tension in cells. Specifically, Jas can interfere with actin assembly/disassembly in the cortex and lead to a reduction in the membrane tension by more ∼80% on average S31, 32 ; CytD promotes the depolymerization of F-actin and hence will also result in a decrease in the cortical tension S33, 34 ; Blebbistatin inhibits the contractility of myosins and hence reduces the tension level in cells 25 . Indeed, our measurements show that the tension level in A549 cells treated with Jas (1.5μM for 30 minutes), CytD (0.5 μM for 30 minutes) and Blebst (75 μM for 30 minutes) was significantly reduced (Fig. 4C). Interestingly, as expected, such decrease in the membrane tension was accompanied by a marked increase in the resealing speed, refer to Fig. 4D. All groups were found to be significantly different from one another (two-sampled t-tests, P < 0.05)

F. Resealing response of A549 cells under elevated surrounding osmotic pressure
In addition to drug treatment, variations in the surrounding osmotic pressure (30, 50, and 70mmHg) were also introduced to A549 cells for 2 hours before membrane resealing test was conducted. Specifically, salt solutions were prepared by dissolving sucrose (IBI Scientific) in DMEM medium (Gibco) with 10% (v/v) fetal bovine serum and 1% (v/v) antimycotic as well as antibiotic solution (Invitrogen). The osmotic pressure ∆π can be calculated by the Morse equation where i is the dimensionless Van't Hoff factor, c is the concentration of newly added sucrose in the culture medium, k > is the Boltzmann constant and T is the absolute temperature. Note that the elevated surrounding osmolarity will induce the shrinkage of cells and hence reduce their membrane tensions. As such, it is conceivable that a faster resealing response will be observed here (similar to that shown in Fig. 4).
Indeed, as demonstrated in Supplementary Figure 4, a higher increase in the medium osmolarity will lead to a shorter membrane resealing time.

Supplementary Figure 4
The resealing time of 1-µm membrane pores in A549 cells under elevated surrounding osmotic pressure. Results shown here were based on measurements on 15 cells (in each case) and a statistical confidence level of no less than 95% by t-test has been achieved.

G. Effective elastic moduli of cancer and normal cells
The effective elastic moduli of different cancer and normal cells were measured by rate-jump indentation S35 . Specifically, the synchronized cells were cultured onto the petri dishes for 24 hours. After that, the indenter was first moved into the cell until an indentation depth of 0.2μm is reached, and then was held for 30s, finally followed by a sudden retraction at a speed of 0.1 μm/s for 10s. All atomic force microscopy measurements were obtained by a JPK NanoWizard® II AFM (Veeco, Santa Barbara, CA) that was mounted onto a Zeiss AxioObserver with the fluid-cell-mounted cantilever (Microlevers, Veeco). The piezo platform and photodiode signal were controlled by JPK NanoWizard® II software (JPK Instrumental). A flat-end cylindrical silicon nitride cantilever (Veeco) of 1μm in diameter with a spring constant of 0.035 N/m was used in all experiments. All tests were conducted at 25℃ and within 1 hour after cells were removed from the incubator.
A typical force curve from such test is shown in Supplementary Figure 5A. From which the reduced modulus E A of the cells can be determined as S35, 36 ∆F C = 2E A a∆δ C where ∆F C is the loading rate jump, ∆δ C is the displacement rate jump, and a is the tip radius. Notice that, here where it has been assumed that ν OPQQ ≈ 0.5 (i.e. the cell body is treated as