Fig 1.
In vitro cancer recurrence model based on platinum-resistant colon cancer cells HCT116.
(A) Schematic presentation of the model. (B) Representative images of cisplatin-resistant cells HCT116 (HCT116 cspl-R) at 1-33 days after exposure to cisplatin. Arrows on the microscopic images indicate areas of vacuolization (black) and multinucleation (red). All images are shown at the same scale; scale bars represent 100 μm. Uncropped images are available in Supplementary S2 Fig.
Fig 2.
Cell cycle dynamics and proliferation gene expression in FUCCI-expressing oxaliplatin-resistant HCT116 cells following 150 µM oxaliplatin treatment for 6 hours.
(A) Representative fluorescent microscopy images showing FUCCI signals over time. Red (mKO2-Cdt1) marks G1 phase; green (Clover-Geminin) marks S/G2/M phases. Scale bars: 100 µm. (B) Schematic representation of the experimental timeline and cellular redistribution following oxaliplatin treatment. The diagram illustrates the transition of cells from a proliferating state to a maximally enriched population of resting/dormant cells by day 6 post-oxaliplatin treatment. The sequential fluorescence of FUCCI markers is depicted, showing cells in G1 phase (red fluorescence); сells in S/G2/M phases (green fluorescence); сells transitioning between phases (yellow/orange (red + green overlap)). The dynamic shift in fluorescence reflects cell cycle arrest and enrichment of quiescent/dormant cells following chemotherapy-induced stress. (C) Quantification of cell cycle phase distribution over time. Bars represent the mean percentage of cells in G0/G1, S, or G2/M phases based on FUCCI signal classification. (D) Normalized expression of proliferation-related genes MKI67, AURKA, cyclin A, cyclin B after oxaliplatin exposure (days 0-10), relative to day 0, measured by qPCR, with GAPDH as endogenous control. Bars represent mean relative expression (± SD) from triplicate samples at days 0, 3, 6, and 10 post-treatment.
Fig 3.
Dynamics of cell cycle regulators in cancer reсurrence model based on cisplatin-resistant colon cancer cells HCT116 cspl-R.
(A,B) qPCR analysis of proliferation markers and CDK inhibitors following cisplatin exposure (days 0-33). Expression normalized to day 0, with GAPDH as endogenous control. Bars represent 0, 1, 4, 6, 12, 18, 28, and 33 days after cisplatin exposure. (C) Immunoblot analysis of p27/Kip1 and Cyclin A protein levels (α-Tubulin loading control). Numbers indicate normalized densitometry values relative to day 0. (D) Immunoblots with p-p38 and p-ERK antibodies. (E) Quantitative p-ERK:p-p38 ratio (%) based on the densitometry data of normalized p-p38 and p-ERK expression (stacked barplot) and (F) schematic representation of signaling balance during dormancy-recurrence transition. All data represent biological triplicates, mean ± SEM.
Fig 4.
Relative viability (MTT assay) of proliferating (day 0) and quiescent (day 6 after 6-hour exposure to 150 µM oxaliplatin) platinum-resistant colon cancer cells HCT116 oxpl-R after 48-hour exposure to chemotherapeutic drugs: 25 µM cisplatin (CSPL), 10 µM oxaliplatin (OXPL), 30 µM etoposide (ETOP), 50 µM irinotecan (IRI), 20 µM 5-fluorouracil (5-FU), 0.5 µg/ml doxorubicin (DXR).
Data represent triplicate samples and are displayed as mean ± SD.
Fig 5.
Autophagy in in vitro cancer reсurrence model based on platinum-resistant colon cancer cells.
(A,B) LysoTracker-positive acidic lysosomal compartments in quiescent cells. Oxaliplatin-resistant HCT116 oxpl-R cells (A) and cisplatin-resistant HCT116 cspl-R cells (B) before (day 0) and after (day 8) platinum treatment (oxaliplatin or cisplatin, respectively), stained with LysoTracker Green™ and MitoTracker Orange™. Scale bars represent 50 µm. (C,D) Dynamics of autophagy marker expression in the in vitro cancer reсurrence model based on cisplatin-resistant colon cancer cells HCT116 cspl-R, analyzed by immunoblotting (C) and qPCR (D). (C) Immunoblots probed with LC3, Beclin and Survivin antibodies. α-Tubulin was used as a loading control. Quantification values shown beneath each lane represent band intensity normalized to loading control and relative to day 0 control (set as 1.0). (D) Normalized expression of autophagy-related genes (lc3, beclin) after cisplatin exposure (days 0-33), relative to day 0. Expression of the GAPDH gene served as the endogenous control. Data represent biological triplicate experiments and are displayed as mean ± SEM.
Fig 6.
Markers of EMT and stemness in cancer reсurrence model in vitro based on cisplatin-resistant colon cancer cells HCT116 cspl-R, qPCR data.
(A) Normalized expression of genes related to EMT after cisplatin exposure (days 0-33), relative to day 0. (B,C) Dynamics of pluripotency markers OCT4, SOX2, NANOG (B) and cancer stem cell markers clusterin and CD44 (C) expression, qPCR data. Normalized expression of stemness-related genes after cisplatin exposure (days 0-33), relative to day 0. (D,E) Peak activation level of stem cell markers in resistant cells at day 6 after oxaliplatin treatment compared to their expression in human induced pluripotent stem cells (iPSCs) AD3. Expression of the GAPDH gene served as the endogenous control. Data represent biological triplicate experiments and are displayed as mean ± SEM.
Fig 7.
Differences in ROS levels between proliferative (d0) and quiescent/dormant (d6) HCT116 oxpl-R and HCT116 cspl-R cells.
Representative fluorescent images of DCF-stained HCT116 oxpl-R (A) and HCT116 cspl-R (B) cells. Histograms (C,D) show relative DCF fluorescence levels, demonstrating the distribution of proliferating (green) and dormant (grey) cells according to flow cytometry data. Diagrams (E,F) present mean DCF intensity as relative ROS levels (fold change) based on flow cytometry. Data represent biological triplicate experiments and are displayed as mean ± SEM.
Fig 8.
Testing alternative parameters for cancer reсurrence modeling.
Relative viability (MTT assay) of platinum-resistant colon cancer cells: oxaliplatin-resistant HCT116 oxpl-R (A) and cisplatin-resistant HCT116 cspl-R (B) in the cancer reсurrence model in vitro after different regimens of platinum exposure (50 or 150 µM oxaliplatin/ 25 or 75 µM cisplatin for 6 or 24h). Time points of full repopulation are marked with a star. Data are representative of 6 samples from two independent experiments and are displayed as mean ± SD.