Capillary constrictions prime cancer cell tumorigenicity through PIEZO1

Abstract

Metastasis is a hallmark of cancer and is responsible for the majority of cancer-related deaths. Evidence suggests that even a single cancer cell can spread and seed a secondary tumour. However, not all circulating tumour cells have this ability, which implies that dissemination and distal growth require adaptative mechanisms during circulation. Here we report that constriction during microcapillary transit will trigger reprogramming of melanoma cells to a tumorigenic cancer stem cell-like state. Using a microfluidic device mimicking physiological flow rates and gradual capillary narrowing, we showed that compression through narrow channels lead to cell and nuclear deformation, rapid changes in chromatin state and increased calcium handling through the mechanosensor PIEZO1. Within minutes of microcapillary transit, cells show increased regulation of transcripts associated with metabolic reprogramming and metastatic processes, which culminates in the adoption of cancer stem cell-like properties. Squeezed cells displayed elevated melanoma stem cell markers, increased propensity for trans-endothelium invasion, and characteristics of enhanced tumorigenicity in vitro and in vivo. Pharmacological disruption of channel activity inhibited the stem cell-like state, while the selective PIEZO1 activator Yoda1 primed this state irrespective of constriction. Finally, deletion of PIEZO1 led to complete abrogation of the constriction-induced stem cell-like state. Together, this work demonstrates that compressive forces during circulation can reprogram circulating cancer cells to tumorigenic stem cell-like states that are primed for extravasation and metastatic colonization.

Fig. 1. Compressive forces induce melanoma cell state changes in a model microcapillary device.

(A) Simplified schematic illustrating the design and the different areas in the microcapillary-like microfluidic device. The device consists of a series of parallel constrictive channels of 30 μm (i), 20 μm (ii), 10 μm (iii), 5 μm (iv) in diameter. Red areas represent the constrictive channel (CC), while the green areas represent the relaxation chamber (RC). (B) Images of melanoma cells passing through the microfluidic device via squeezing through constrictions (top) and within the relaxation chambers (bottom). (C) Quantification of viable cells in control group (CTRL) compared to squeezed (SQZD) group. Cells were stained with Trypan Blue and counted using a hemocytometer. The results are expressed as the mean from three independent experiments. (D) Box and Whisker Plot representing the deformation index (DI) of melanoma cells transiting the microfluidic device. The DI value reflects how much deformation has occurred relative to the original size of the cells. A higher DI indicates greater deformation. Red boxes are indicative of DI into the constrictive channels, while green box indicate the DI measure for cells after passing through the constrictive channel. (E) Inverse relationship between microchannel diameter (black, left Y-axis) and corresponding median deformation (% Median Deformation, red, right Y-axis) for melanoma cells transiting sequential microfluidic constrictions. (F) Bar graph representing the % of Median deformation in 10μm and 5μm constrictive channels (red) and the corresponding adjacent relaxation chamber (green). (G) Representative fluorescence images of Hoechst for cells before (CTRL) and 4 hours after been squeezed (SQZD) into the microfluidic device. (H) Bar graph displaying the nuclei area for CTRL and SQZD cells. (I) Bar graph displaying the fluorescence intensity of Hoechst signal for CTRL and SQZD cells. (L) Representative staining of H3K9ac in CTRL and SQZD melanoma cells. (M) Bar graph displaying the H3K9ac levels in CTRL and SQZD cells. (N) Representative staining of H3K9me3 in CTRL and SQZD melanoma cells. (O) Bar graph displaying the H3K9me3 levels in CTRL and SQZD cells.

Fig. 2. Transcriptomics and immunofluorescence after constriction indicate increased invasiveness and evidence for stem cell-like states.

(A) MDS of the two groups control (X1, X2, X3 CTRL) and squeezed (X1, X2, X3 SQZD) analysed in triplicates, where X and Y axes represent dimensions that capture the largest differences between samples based on their gene expression profiles. (B) corresponding Venn diagram showing unique and overlapping genes between the two groups. (C) Volcano plot indicating significantly differentially expressed mRNAs between SQZD and CTRL. The grey horizontal line shows P-value cut-off (*p<0.01) and the vertical dashed lines indicate up/down regulated genes (<−1.5 and >1.5 fold change). (D) Heatmap of the differentially expressed genes with. (E) Bar graph displaying number of genes involved in the GO biological processes and KEGG pathways associated with metabolic reprogramming (orange), tumorigenicity and metastasis (red), cell-matrix interactions (green) and cell cycle (blue). Ticks in the gene number x axis are every 5 genes. (F) Representative fluorescence images of melanoma cancer cells, seeded on glass before (CTRL) and 4 hours after been squeezed (SQZD) into the microfluidic device, for biomarkers relative to stemness characteristic, e.g., CD44, CD271, ABCB5, PRDM14. (G) Bar graph showing the fold change of fluorescence signal of stemness-related biomarkers for SQZD cells compared to CTRL cells. The results are expressed as the mean from five independent experiments.

Fig. 3. Microcapillary-like constrictions enhance melanoma cell tumorigenicity.

(A) Images of control (CTRL) and squeezed (SQZD) melanoma cells forming tumorsphere in serum-free media after 7 days of culture. (B) Graph representative of the mean tumorsphere area increasing over time when cultured in serum-free media, for CTRL (blue) and SQZD (red) cells. (C) Bar graph displaying the area of tumorsphere for CTRL and SQZD cells at day 7. The result is expressed as the mean from four independent experiments. (D) IVIS Imaging of lung metastases that developed after 30 days from injecting CTRL and SQZD luciferase melanoma cells into the lateral tail vein of 7-week-old female Balb/c Nude mice. [Note the change in scale between the groups]. (E, F) Representative H&E-stained lung sections from mice injected with CTRL cells (E) and SQZD cells (F) harvested 30 days post tail vein injection. (G) Whole body luciferase signal of mice injected intra-cardially at week 4, the final timepoint at which all mice in both groups were accounted for. (H-L) Photon flux from individual metastatic sites revealed increased median signals in the (H) lung and significantly higher signals in (I) bone and (L) brain in the SQZ group compared to controls. Each dot represents an individual animal. (M) Metastatic burden and Kaplan–Meier survival curves following IC injection with control (black) and squeezed (red) cells. Data represent pooled results from each group (CTR, n = 8; SQZ, n = 10).

Fig. 4. PIEOZ1 activity directs stem cell-like state and tumorigenicity after microconstriction.

(A) Representative fluorescence images of PIEZO1expression for cells before (CTRL) and 4 hours after been squeezed (SQZD) into the microfluidic device. (B) Bar graph displaying the increase of PIEZO1 fluorescence signal for squeezed cells compared to control. (C) Representative fluorescence images showing melanoma cells as they squeeze through the constrictive channels. The images highlight the deformation of cells due to confinement in the narrower channels. Bar graph relative to the fold change increased of intracellular calcium concentration, detected with calcium sensitive fluorescent dye as melanoma cells squeeze into the constrictive channels. (D) Representative fluorescence images of melanoma cancer cells adhered to glass for 4 hours, showing stemness biomarkers such as CD44, CD271, ABCB5, and PRDM14, under control conditions (CTRL) and after treatment with 20 μM Yoda1 and 30 μM Ruthenium Red (RR). (E) Bar graph showing the fold change of fluorescence signal of stemness-related biomarkers for cells treated with Yoda1 and RR compared to CTRL cells. The result is expressed as the mean from three independent experiments. (F) Images of CTRL and treated melanoma cells, with Yoda1 and RR, forming tumorsphere in serum-free media after 7 days of culture. (G) Graph representative of the mean tumorsphere area increasing over time when cultured in serum-free media, for CTRL (red) and treated with Yoda1 (pink) or RR (grey). (H) Bar graph displaying the area of tumorsphere for CTRL, Yoda1 and RR cells at day 7. The result is expressed as the mean from four independent experiments.

Fig. 5. Loss of PIEZO1 affects the adaptive changes of stem-like phenotype during microcapillary-induced deformation.

(A) Box and Whisker Plot representing the deformation index (DI) of knockout (KO) melanoma cells transiting the microfluidic device. Red boxes are indicative of DI into the constrictive channels, while green box indicate the DI measure for cells after passing through the constrictive channel. (B) Quantification of viable KO melanoma cells in control group (CTRL) compared to squeezed (SQZD) group. Cells were stained with Trypan Blue and counted using a hemocytometer. The results are expressed as the mean from three independent experiments. (C) Representative fluorescence images showing Wild type (WT) and KO melanoma cells as they squeeze through the 10μm and 5μm constrictive channels. (D) Bar graph relative to the fold change decreased of intracellular calcium concentration, detected with calcium sensitive fluorescent dye (Calbryte 520 AM), as KO melanoma cells squeeze into the constrictive channels, compared to squeezed WT cells. (E) Representative fluorescence images of Hoechst for KO cells before (CTRL) and 4 hours after been squeezed (SQZD) into the microfluidic device. (F) Bar graph displaying the nuclei area for CTRL and SQZD-KO cells. (G) Representative staining of H3K27ac in CTRL and SQZ-KO melanoma cells. (H) Bar graph displaying the H3K9ac Euchromatin level in CTRL and SQZD-KO cells. (I) Bar graph displaying the H3K9me3 Heterochromatin level in CTRL and SQZD-KO cells. (L) Representative fluorescence images of WT and OK melanoma cancer cells, seeded on glass 4 hours after been squeezed into the microfluidic device, for biomarkers relative to stemness characteristic, e.g., CD44, CD271, ABCB5, PRDM14. (M) Bar graph showing the fold change of fluorescence signal of stemness-related biomarkers for squeezed WT and KO cells compared to their relative CTRL cells. The results are expressed as the mean from five independent experiments. (N) Images of control WT and KO melanoma cells, under CTRL and SQZD experimental condition, forming tumorsphere in serum-free media after 7 days of culture. (O) Graph representative of the mean tumorsphere area increasing over time when cultured in serum-free media, for WT CTRL cell (blue), WT SQZD cell (red), WT SQZD cell with the addition of Ruthenium Red during perfusion (orange), KO CTRL cell (green), KO SQZD cell (light green). (P) Bar graph displaying the area of tumorsphere for all conditions at day 7. The result is expressed as the mean from three independent experiments.

Fig. 6. Micro constrictions facilitate increased engagement of melanoma stem cell-like cells with endothelial cells and model blood vessels.

(A) Representative image showing Crystal Violet dye signal from WT and KO melanoma cells migrating through the transwell membrane before and after been squeezed into the microfluidic chip. The image highlights the cells that have successfully migrated through the membrane pores and adhered to the bottom surface of the transwell insert. (B) Bar graph showing the fold change of WT and KO cell coverage area, compared to their relative controls. (C) A simplified schematic illustrating the co-culture of melanoma cancer cells with a functional monolayer of endothelial cells. (D) Representative fluorescence images of vascular endothelial proteins, e.g. Vascular endothelial cadherin junction (VE-Cadherin) and Vascular Cell Adhesion Protein (VCAM), when the endothelial monolayer is co-culture is WT and KO cells under control (CTRL) and squeezed (SQZD) experimental conditions. Asterisks are reported in monolayer’s area, where VE-Cadherin remodel and leave gap in between cells (E) Bar graph displaying the fluorescence expression of VCAM protein from endothelial cells cultured under CTRL condition and with the presence of WT and KO cells, before or after been squeezed into the microfluidic device. (F) Schematic illustrating the microfluidic chip used for melanoma extravasation assay and composed of two independents vascular channels and a central tissue chamber, communicating with a membrane of pores. After a dynamic culture protocol of 3 days to obtain the functional biological barrier into the vascular channel, melanoma cells are injected and allowed to extravasate into the tissue chamber during overnight. (G) Representative fluorescence images of the mature blood vessel. Inset showing the expression of VE-Cadherin at cell-cell border, indicating a function biological barrier. (H) Representative fluorescence images of WT melanoma cells (red) after 24 hours of residence into the blood vessel under dynamic condition. These images illustrate the heightened invasiveness of SQZD WT cells compared to CTRL cells, with most SQZD WT cells having penetrated through the membrane pores into the tissue chamber. (I) Bar graph showing permeability coefficient comparison for CTRL (cell-free device), Vessel, WT-CTRL (Vessel + WT melanoma cells non treated), WT-SQZD (Vessel + WT squeezed melanoma cells), KO-SQZD (Vessel + KO squeezed melanoma cells).