Multiple Myeloma Cell Drug Responses Differ in Thermoplastic vs PDMS Microfluidic Devices

Poly­(dimethylsiloxane) (PDMS) is a commonly used elastomer for fabricating microfluidic devices, but it has previously been shown to absorb hydrophobic molecules. Although this has been demonstrated for molecules such as estrogen and Nile Red, the absorption of small hydrophobic molecules in PDMS specifically used to treat cancer and its subsequent impact on cytotoxicity measurements and assays have not been investigated. This is critical for the development of microfluidic chemosensitivity and resistance assay (CSRA) platforms that have shown potential to help guide clinical therapy selection and which rely on the accuracy of the readout involving interactions between patient-derived cells and cancer drugs. It is thus important to address the issue of drug absorption into device material. We investigated drug absorption into microfluidic devices by treating multiple myeloma (MM) tumor cells with two MM drugs (bortezomib (BTZ) and carfilzomib (CFZ)) in devices fabricated using three different materials (polystyrene (PS), cyclo-olefin polymer (COP), and PDMS). Half-maximal inhibitory concentrations (IC₅₀) were obtained for each drug–material combination, and an increase in IC₅₀ of ∼4.3× was observed in PDMS devices compared to both thermoplastic devices. Additionally, each MM drug was exposed to polymer samples, and samples were analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS) to characterize adsorption and absorption of the drugs into each material. ToF-SIMS data showed the bias observed in IC₅₀ values found in PDMS devices was directly related to the absorption of drug during dose–response experiments. Specifically, BTZ and CFZ absorption in both PS and COP were all in the range of ∼100–300 nm, whereas BTZ and CFZ absorption in PDMS was ∼5.0 and ∼3.5 μm, respectively. These results highlight the biases that exist in PDMS devices and the importance of material selection in microfluidic device design, especially in applications involving drug cytotoxicity and hydrophobic molecules

Phylogenetic analysis of YTHDF proteins in plants.

(A) Schematic representation of Viridiplantae evolution and relative position of the species used in this study. The architecture of the diagram and the age (million years, My) indicated on some nodes are a simplified version of the trees from Su et al. [59] and Bowman [62]. The length of the branches has been adjusted for illustrative purposes. (B) Phylogenetic tree of YTHDF proteins in plants (S1–S4 Datasets), with color-coding and abbreviations of species names as in A. Three YTHDF proteins from taxa outside but evolutionarily close to Viridiplantae are included as outgroup. Arabidopsis thaliana (Ath) YTHDF proteins are named after the original nomenclature for proteins containing an Evolutionarily Conserved C-Terminal Region (ECT) established by Ok et al. [40]. Proteins from other plant species adhere to the nomenclature established by Scutenaire et al. [26], with small variations reflecting additional clades (DF-E, -F) and the split of the original DF-C clade into -C and -D. Statistical support is calculated as approximate likelihood-ratio test (aLRT), and indicated by grayscale-coded spheres and values on the most relevant nodes. For simplicity, only a subset of proteins is labelled–those from one representative species of the main taxa highlighted in bold in A–, but a comprehensive representation of the same tree with all protein names and aLRT values is available in S1 Fig. The length of the branches represents the evolutionary distance in number of amino acid substitutions per site relative to the scale bar. Double lines crossing the longest branches indicate that the branch has been collapsed due to space problems, but a schematic representation of their relative lengths is shown on the lower-left corner, and not-collapsed branches can be found in S1 Fig.</p

Analysis of the IDRs of YTHDF proteins from representative species of the main taxa of land plants.

(A) Representation of charges (top panels) and slab simulations (bottom panels) of the IDR sequences of YTHDF proteins from the main taxa of land plants. The proteins are sorted according to land plant evolution (vertical axis) and plant DF clades (horizontal axis). Isoetes taiwanensis (Ita) DF-CD (Fig 1B) is excluded from the analysis because its N-terminus is very short (S1 Dataset) and does not behave like an IDR. (B) Relationship between excess transfer free energy from dilute to dense phase (ΔGtrans = RT ln [cdilute / cdense]) and average stickiness (λ) of the IDR residues of the proteins in A. The proteins are color-coded according to plant DF clades. Homo sapiens (Hs) YTHDF2 is included as a reference. Same as for the Arabidopsis thaliana (Ath) ECT set in S19B Fig, the content of sticky residues defined by the CALVADOS model [78] mainly governs the different phase separation propensity among the IDRs. A clade-dependent separation of the proteins according to these properties is apparent. (PDF)</p

Analysis of the IDRs of the YTHDF proteins assayed in this study (extended data).

(A) Time-averaged density profiles of the slab simulations of the ECT proteins shown in Fig 5B. (B) Relationship between excess transfer free energy from dilute to dense phase (ΔGtrans = RT ln [cdilute / cdense]) and average stickiness (λ) of the IDR residues. (C-D) Comparative amino acid composition (C) and representation of charges (D) along the IDR sequences of the different YTHDF proteins assayed in this study. In D, arrows highlight the overall trends in charge change along the IDRs. Notice that the length in amino acids (aa) is not at scale to simplify the representation. (PDF)</p

ECT1 simulation.

Coarse-grained molecular dynamics simulation of 100 ECT1 IDRs using the CALVADOS 2 forcefield (7.5 microseconds simulation time) [126]. The proteins are colored differently to distinguish individual chains. (MP4)</p

Analysis of ECT1/9/11 functional regions.

(A) Topology of the YTH domain of Homo sapiens (Hs) YTHDF1 with secondary structure elements drawn in pink [61]. The two grey helices represent the only structural elements outside the YTH domain as defined by Stoilov et al. [8]: α0 immediately upstream, and α4 at the C-terminus of the protein. Superimposed on the diagrams are the non-conservative amino acid substitutions found in Ath ECT1, ECT9 and ECT11 compared to the majority of Ath ECTs (see alignment in S16 Fig). The localization of these amino acids in the predicted 3D structures, predominantly on the surface, and the spatial integrity of the aromatic cage in the homology models of ECT1/9/11, can be found in S17 Fig. (B) Slab simulations of the IDRs of the indicated proteins showing their tendency to remain in condensed phase or disperse in solution. Source videos of ECT2, ECT1 and ECT11 simulations are provided as S1–S3 Movies (C) Time-averaged density profiles for a subset of the proteins in B. The analyses of the remaining Ath ECTs are shown in S19A Fig. (D) Calculated free energy change for the transition between aqueous solution and condensed phase (ΔGtrans = RT ln [cdilute / cdense]) for the IDRs of the indicated proteins. (E) Cumulative charge distribution of the IDRs of the indicated proteins. The length in amino acids (aa) is at scale. The same analysis for all Ath ECTs can be found in S19D Fig.</p

Expression levels of ECT paralogs in tissues of <i>Arabidopsis thaliana</i>.

Expression levels of ECT1-ECT11 across different tissues according to public mRNA-Seq data [73]. TPM, transcripts per million. (PDF)</p

Isolation of <i>ECT1-TFP</i> transgenic lines and <i>ect1</i> T-DNA insertion alleles.

(A) Western blot using antibodies against GFP (that recognize TFP) in different ECT1p:gECT1-TFP-ECT1t independent lines. Ponceau (Ponc.) staining of the membrane is used as loading control. (B) Northern blot using the probe (P) specified in Fig 3G to detect ECT1 mRNA. Although the probe recognizes specifically ECT1 in the ECT1p:gECT1-TFP-ECT1t Line #21 (marked with an asterisk in A), the endogenous expression levels of ECT1 in Col-0 wild type are below detection limit by northern blot. (C) Schematic representation of the Arabidopsis thaliana ECT1 locus (At3g03950). Exons are represented as boxes and introns as lines. Untranslated regions (UTRs) are coloured grey, the sequence encoding the YTH domain is purple, and the rest of the ECT1-coding sequence is black. The IDs and positions of the T-DNA insertions assigned to ect1-1, ect1-2 and ect1-3 alleles are marked, and so is the location of primers used for their genotyping. (D-F) 1% agarose gels showing EtBr-stained PCR fragments corresponding to the genotyping of ect1-1 (D), ect1-2 (E) and ect1-3 (F) in plants germinated from seeds provided by the Nottingham Arabidopsis Stock Center (NASC) as indicated. The primer set for each PCR (‘T-DNA’ detects the insertion, and ‘WT’ detects the wild type allele) and the length of the resulting amplicons are indicated to the left of each gel. The sequence of all primers can be found in S2 Table. The progeny of plants homozygous for each T-DNA insertion, highlighted in shades of blue, was selected for crosses and further characterization. (PDF)</p

Knockout of <i>ECT1</i> does not affect arabidopsis plant development.

(A) Abbreviations for combinations of double (d), triple (t), or quadruple (q) ect (e) mutants following the nomenclature proposed by Arribas-Hernández et al. [17]. The prefix ‘G’ identifies allele combinations having only T-DNA insertions belonging to the GABI-KAT collection [129]. (B) Morphological appearance of seedlings with or without ECT1 in the different backgrounds indicated, at 9 or 17 days after germination (DAG). (C) Analyses of the percentage of trichomes with 3, 4, 5 or 6 spikes in plants with or without ECT1 in the different backgrounds indicated. n, number of trichomes assessed for each genotype. NS, non-significant differences according to statistical analysis performed as in [16]. (D) Analysis of root growth rate and directionality upon mutation of ECT1 in the ect2-3 and ect2-3/ect3-2 (Gde23) knockout backgrounds. Upper panels represent the overlayed silhouettes of actual roots as they grow on vertically disposed MS-agar plates. n, number of plants assessed for each genotype. VGI, vertical growth index; HGI, horizontal growth index. Root phenotypic analyses were conducted as in [17]. (PDF)</p

Dissection of ECT1/9/11 functional regions.

(A) Schematic representation of the strategy followed to express chimeras with N-terminal IDR and YTH domains of different ECT proteins. (B) Weighed averages of the complementation rates observed for each chimeric construct measured as in Fig 2B and 2C. The raw complementation rates and transformation efficiencies of each independent transformation (I.T.) can be found in S7B and S9 Figs respectively, and photographs of the scored 10-day-old T1 seedlings with mCherry fluorescence are shown in S15 Fig. (C) 10-day-old primary transformants of rdr6-12/te234 with the indicated transgenes. Dashed outlines are magnified below to show mCherry fluorescence. (D) Same genotypes as in C at 26 days after germination. Additional independent transgenic lines, developmental stages and controls for C and D can be found in S10 Fig. All transgenes are expressed from the US7Y promoter and contain mCherry fused to the C-terminus.</p