Fig. 2: Monitoring protein misfolding in myeloma cells during synergic and individual lenalidomide and bortezomib treatment.

a, Schematic diagram of LEN and BTZ treatment in myeloma cells. b, Amide I map of multiple myeloma cancer cells (MM1.S) pretreated with 10 μM of LEN for 72 h and subsequently treated with 100 nM of BTZ for 24 h, then imaged at 1,650 cm⁻¹ (Mt4). The white crosses indicate the myeloma cells selected for spectral analysis. c, Differential spectra of the LEN/BTZ-treated myeloma cells shown in b (red line) and the untreated myeloma cells shown in d (blue line) at 96 h. The spectrum in red shows a band at 1,620 cm⁻¹, assigned to an intermolecular β-sheet of misfolded proteins, which was not found in untreated cells. Spectra are presented as mean ± s.d. d, Amide I map of untreated myeloma cells imaged at 1,650 cm⁻¹ after being in culture for 96 h. The white crosses indicate the myeloma cells selected for spectral analysis. e, Differential spectra of LEN/BTZ-treated MM1.S cells at different time points (green, 92 h; red, 96 h). The differential spectrum of untreated cells is shown in blue. MiROM can detect the effect of proteasome inhibition after 92 h of LEN and BTZ combined treatment (n = 5 independent experiments). f, Differential spectra of MM1.S cells treated with 25 μM of LEN for 144 h compared to untreated cells maintained in culture for 144 h. LEN-treated cells show formation of the intermolecular β-sheet structure, which is not present in the untreated cells (n = 3 independent experiments). Spectra are presented as mean ± s.d. g, Differential spectra of MM1.S cells treated with 100 nM of BTZ for 18 h compared to untreated cells maintained in culture for 18 h. BTZ-treated cells show formation of the intermolecular β-sheet structure, which is not present in the untreated cells (n = 3 independent experiments). Spectra are presented as mean ± s.d. h, Differential spectra of MM1.S cells treated with 1 μM of doxorubicin (DOX) for 2 h compared to untreated cells maintained in culture for 2 h. The intermolecular β-sheet structure is not visible in either differential spectra (untreated and DOX-treated cells, n = 3 independent experiments). Spectra are presented as mean ± s.d. i, NMF components extracted from spectral data (n = 700 spectra). j, Component 2 represents intermolecular β-sheet secondary structures (1,682–1,618 cm⁻¹). k, Violin plots from kernel density estimate the time evolution coefficients of NMF component 2 obtained from 700 spectra from 5 independent experiments acquired in LEN/BTZ-treated cells and untreated cells. Component 2 increased by 174% during the treatment. Boxplot minima and maxima inside the violin plots indicate the interquartile range, the white circles indicate the mean values, and the whiskers indicate the standard deviation (coefficient = 1). l, A t-SNE map representing the distribution of the 5 components identified in LEN/BTZ-treated myeloma cells. Spectra acquired from the cell medium (dashed orange circle) and spectra acquired from treated cells after 96 h of treatment (dashed magenta circle) are clustered together.