DOX produces significant shifts in the P-gp expression levels of H460 cells only

The distribution of P-gp in the different cell populations was assessed during four consecutive days to characterise their dynamics. Five initial proportions of sensitive (NCI-H460) and resistant (NCI-H460R) cells (S:R ratios equal to 1:0, 0:1, 1:1, 3:1, 7:1) were employed to analyse the changes in the P-gp expression both in the absence and presence of DOX (50 nM). Figure 2 shows how P-gp expression levels were modified in each cell population under various culture conditions and during a period of 72 h. For H460 cells, only in the presence of DOX there was a statistically significant shift towards higher P-gp expression levels (Fig. 2, left panel). For H460/R cells a slight shift towards lower P-gp expression levels appeared, although it was not statistically significant (Fig. 2, middle panel). For an initial 1:1 mixture of H460 and H460/R cells the kinetics was dramatically different in the absence/presence of DOX. Under DOX there was a statistically significant shift towards higher P-gp expression levels (Fig. 2, right panel). The corresponding p-values for all these experiments are displayed in Table S1 (Supplementary Information).

Figure 2 Experimental results for H460 (left), H460/R (middle) and mix 1:1 (right) cells. Arrows account for the processes that are responsible for the changes observed in P-gp expression. Notice that the areas under each curve, which represent the number of cells, are equal in all cases (all cell samples were of the same size \(\simeq 2\times {10}^{4}\) in the flow cytometry analyses). Full size image

Transport model captured the P-gp expression kinetics of all measured H460 and H460/R cell populations

Our mathematical model captured the experimentally observed cell growth kinetics of the different cell populations, both in the absence and in the presence of the drug DOX, and with various initial cell ratios (S:R ratios equal to 1:0, 0:1, 1:1, 3:1, 7:1). When assessing cell proliferation in real-time, a number of doses of DOX (0, 10, 50 and 100 nM) were used to quantify the effect over the total number of cells on an initial population of 4000 sensitive NCI-H460 cells via the xCELLigence Real Time Cell analyser. Our experimental results show that the higher the administered DOX doses were the slower was the cell growth (see Figs S4 and S5 in the Supplementary Information). This was most prominent for doses above 50 nM. These results allowed us to estimate the parameters entering into our model equations and specifically in the therapy function (see Methods and Supplementary Information), which accounts for the response to the administered chemotherapeutic agent with respect to the P-pg expression level. To minimize possible artefacts caused by nutrient depletion and release of metabolic products that eventually may become toxic, particularly when the cells reach confluence (which first occurred in the absence of DOX after 100 h, see Fig. S5), and may affect the antiproliferative activity of the drug58,59, all comparisons were made during the first 96 hours. Figure 3(a) evidences that our model was able to reproduce the experimental results without the drug, when the transfer of MVs among sensitive and resistant cells is the only process that could lead to alterations in the P-gp expression levels, but also in the presence of the drug (50 nM of DOX), where the processes of selection and induction become relevant, as depicted in Fig. 3(b). Supplementary Figs S6 to S13 further support the good agreement obtained between the model and the experiments for isolated sensitive and resistant cells as well as with different initial mixtures of these, both in the absence and in the presence of DOX. In the absence of the drug, there were no statistically significant changes in the P-gp expression levels whereas, under stress conditions, sensitive cells grown isolated or in mixtures showed highly significant changes in the observed P-gp level profiles (see Table S1 in the SI where p-values between initial and final conditions are collected). In contrast, resistant cells did not display any significant changes in their P-gp levels either in the absence or presence of the drug.

Figure 3 Evolution of H460 and H460/R cell mix 1:1 (a) without and (b) with DOX. Cells were seeded at t = 0 h and measured at subsequent intervals of 24 h. Dashed and solid lines represent the experimental results and the numerical simulations of our model, respectively. Full size image

We used our model to analyse the role of initial conditions for the seeded cell populations on their subsequent P-gp expression dynamics. In Fig. 4 (see also Figs S6–S13 in the SI) a comparison of the P-gp expression with different ratios of sensitive and resistant cells is plotted together with the corresponding cell numbers during 72 h after seeding; the distinct colours encode the initial proportions (blue for sensitive only and light red for resistant only). Figure 4(a) shows that, in the absence of drug (0 nM of DOX), a small tail of cells having higher P-gp levels developed when the initial fraction of resistant cells was larger. Also, the cell number grows faster for sensitive cells and progressively decreases with the ratio of resistant cells (see inset of Fig. 4(a)). However, in the presence of drug (50 nM of DOX), see Fig. 4(b), for the very same initial proportions of Fig. 4(a), not only significant shifts in the P-gp expression profiles after 72 h were observed for initially sensitive cells and mixtures 1:1, 3:1 and 7:1 (see also Table S1 in the SI), but the corresponding cell numbers displayed noticeable differences. Except for resistant cells only, all mixtures evidenced a transient reduction in the cell numbers and, after a varying amount of hours depending on the initial ratios (see inset of Fig. 4(b)), the surviving tumor cells increased in all cases evidencing the emergence of subpopulations tolerant to DOX.

Figure 4 P-gp distribution changes after 72 h under (a) 0 nM and (b) 50 nM of DOX for initial sensitive, resistant and mixed cells (sensitive:resistant fractions 1:1, 3:1 and 7:1). Insets: Cell number versus time for initial populations of sensitive, resistant and mixed cells with 0 and 50 nM of DOX. Full size image

The presence of DOX influences the rate of P-gp transfer to H460 cells

Among all the processes implicated in the development of resistances, the one involving P-gp transfer via MVs was analysed independently. To this end, the P-gp expression of sensitive cells was measured both in standard and in conditioned media, the latter obtained from a culture of resistant cells. In previous works it has been demonstrated that MVs’ shedding constantly occurs among cancer cells28 and that MVs released by resistant cells carry P-gp molecules31. The upper row in Fig. 5 collects the results of both experimental conditions (with and without DOX) showing practically no difference in the absence of DOX. These differences were, however, statistically significant in the presence of the drug (at the significance level α = 0.05, see Table S2 in the SI), with an additional shift towards higher P-gp values when both DOX and the conditioned media from the resistant cells were combined. To test for reproducibility, three additional independent replicas of all these experiments were carried out. The results displayed very similar behaviour in all cases and identical conclusions were obtained in the statistical analyses (for details, see Fig. S14 in the SI).

Figure 5 Detection of P-gp transfer during 48 h in different culture media with sensitive cells (\(\simeq 2\times {10}^{4}\)). Upper/lower rows represent the experimental results and the model predictions, with the corresponding calculated MV transfer function (inset). The culture media comprised only the H460 cells (blue solid curves), H460 cells grown in a conditioned medium (CM) exchanged from the H460/R cells to the H460 cells medium (red dashed curves), H460 cells grown in the presence of 50 nM of DOX (yellow dotted curves) and H460 cells grown both in the presence of 50 nM of DOX and CM (violet dashed-dotted curves). Asterisks in the upper row denote the p-values for pairwise comparisons: (**)p < 0.005 and (***)p < 0.0005. Full size image

The lower plots in Fig. 5 show that our model was also capable of reproducing the experiments of medium exchange in the presence and in the absence of DOX. One remarkable effect predicted by our model was the different MVs uptake rates displayed by sensitive cells depending on whether DOX was administered or not. The presence of MVs in the microenvironment appears to have a higher impact on the P-gp level of sensitive cells under the action of DOX. In the inset of Fig. 5 the transfer functions used in the simulations to fit the experimental data are plotted. Notice that the transfer function exhibits a ten fold change in the presence of the drug with respect to its absence. To support this finding, similar statistical analyses applied to the previous data were performed on the calculated distribution curves. Without DOX, the P-gp expression curves showed no significant difference between normal and conditioned medium. However, in the presence of DOX, our analysis displayed a difference at the level of α = 0.05 (see Table S2 in the SI) which was statistically significant.

Increases in the P-gp expression levels of H460 cells were reversible

To answer the question of whether the observed changes in the P-gp expression levels of the initially sensitive cells, due to transfer, were permanent (within the considered time duration of our experiments) or else reversible, we performed an additional experiment outlined below. This is an important aspect from a clinical perspective as it has a direct impact when designing personalized dosing strategies. Indeed, if previously sensitive cells to a certain drug acquire a transfer-mediated-resistance phenotype which is transient, this implies that the initial drug may be employed repeatedly on those same cells once their characteristic return period to that drug has been identified. This is also at the heart of combining several drugs; the tumor cells exhibit distinct time-varying responses to each one of them but, in principle, while the different chemo agents are administered there will be no need to permanently discard subsets of these and introduce new ones. If, in contrast, previously sensitive cells to a certain drug, after acquiring a resistant phenotype, do not return to a sensitive state once the drugs are removed, then subsets of the employed chemo agents will be compromised and new therapeutical approaches will have to be developed for that specific tumor16.

Sensitive cells overexpressing P-gp were extracted and placed in a new fresh medium. Their resistance level was then monitored during 240 h. We observed that sensitive cells returned to their basal values of P-gp expression (see bottom row in Fig. 6). Therefore, the acquired transfer-mediated-resistance phenotype was reversible and did not involve genetic or epigenetic mutations. To quantify the duration of these changes, we fed our model with the same parameters used in previous simulations. The four upper rows in Fig. 6 depict snapshots at various times (t = 0, 60, 120, 240 h) of the expression levels corresponding to sensitive, resistant and sorted cells. At t = 0 h both resistant and sorted sensitive cells overexpressing P-gp show undistinguishable distributions. When placed in a new fresh medium (without any drug or MVs), the sorted sensitive cells overexpressing P-gp gradually recovered their basal distribution, which already at t = 60 h was manifestly shifted, being complete after ten days as in the experiments. Our model was able to mimic the observed changes without the need to modify the basal P-gp values; these were assumed to be time-independent during the entire examined period as any actual genetic mutation, possibly affecting these basal values, would be expected to take place at longer times (after many cell cycles).

Figure 6 Duration of P-gp changes. The upper four rows correspond to the numerical simulations over a time period of 240 h, while the last row shows the experimental results (at t = 240 h). Full size image

Variability in response to different treatment protocols is driven by initial percentage of resistant cells

To assess how the interplay of Lamarckian induction and nonlocal transfer of extracellular MVs both collectively and individually affect the progression of the tumor cell populations when subjected to different treatment schedules, we considered three protocols (Darwinian selection is always present). We used our model to simulate, during a time period of 240 h, how the total cell numbers changed upon DOX administration (see also Fig. S15) and compared the response with the growth in the absence of drug. The protocols that we examined were:

Protocol 1: Drug during 0–120 h (no drug during 120–240 h), which corresponds to a single administration session.

Protocol 2: Drug during 0–60 h and 180–240 h (no drug during 60–180 h), which corresponds to two administration sessions separated by one resting interval.

Protocol 3: Drug during 0–24 h, 48–72 h, 96–120 h, 144–168 h and 192–216 h (no drug during the remaining 24 h intervals), which corresponds to five administration sessions alternated by four resting intervals.

Notice that the total time under drug pressure and drug absence is the same in the three protocols, 120 h and 120 h, respectively. In addition, each of the three protocols were simulated with three DOX concentrations of 10 nM, 50 nM and 100 nM (see Fig. S16 in SI).

For initial sensitive cells subjected to 100 nM of DOX (see Fig. 7(a)), their response to the three protocols exhibits marked differences; protocol 1 results in a growth delay of about 120 h, protocol 2 shows the largest cell number change, whereas protocol 3 is the one giving rise to a slower growth on average with respect to the other two. In this scenario, induction is the most relevant process towards the emergence of resistance (MV transfer is essentially absent). Of the three protocols, number 2 is the one in which induction is smaller (see Fig. S17).

Figure 7 Treatment response of (a) H460, (b) RH460 and (c) Mix 1:1 cell groups to the three different protocols using a 100 nM drug concentration of DOX and a control group (no drug). The initial cell number was 2 × 104 cells in all cases. Full size image

For initial resistant cells subjected to 100 nM of DOX (see Fig. 7(b)), the three protocols show much smaller differences with respect to the previous scenario as this subpopulation is expected to barely respond to treatment. In this case, the processes of induction and MV transfer do not play any relevant role whatsoever. The residual differences observed in the three protocols come from a small fraction of resistant cells having lower expression levels of P-gp which are be partially responsive to the drug.

When both sensitive and resistant cells are initially present and subjected to 100 nM of DOX (see Fig. 7(c)) both induction and MV transfer do contribute. Of the three protocols, number 2 is the one in which induction and MV transfer is larger (see Figs S17 and S18), hence more similar to the response shown by sensitive cells alone, while the responses to protocols 1 and 3 were more similar to that displayed by resistant cells alone.

Furthermore, when varying the doses and the protocols (see Figs S17 and S18), the largest observed differences occurred for sensitive cells alone and the smallest for resistant cells alone. Therefore, when a relatively large fraction of sensitive cells is present in the tumor, the response to therapy is more dependent on the specific protocol and the differences increase with dose concentration.