Survival-associated cellular response maintained in pancreatic ductal adenocarcinoma (PDAC) switched between soft and stiff 3D microgel culture
Data files
Apr 26, 2024 version files 6.52 GB
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Figure2_3wt__average_modulus.csv
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Figure2_5wt__average_modulus.csv
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Figure2_representative_3wt.__indentation_curve.csv
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Figure2_representative_5wt.__indentation_curve.csv
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Figure3a_data.csv
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Figure3b_data.csv
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Figure5_soft_6h_1.txt
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Figure5_soft_6h_2.txt
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Figure5_stiff_6h_1.txt
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Figure5_stiff_6h_2.txt
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Figure7a_soft_to_soft.tif
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Figure7a_soft_to_stiff.tif
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Figure7a_stiff_to_soft.tif
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Figure7a_stiff_to_stiff.tif
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Figure7b_calculations.csv
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Figure7b_raw_image_data.zip
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Figure7c_soft_to_soft.tif
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Figure7c_soft_to_stiff.tif
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Figure7c_stiff_to_soft.tif
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Figure7c_stiff_to_stiff.tif
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Figure7d_calculations.csv
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Figure7d_raw_image_data.zip
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FigureS10_3wt.__radial_tubulin_intensity.csv
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FigureS10_5wt.__radial_tubulin_intensity.csv
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FigureS2_3wt._diameter.csv
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FigureS2_5wt._diameter.csv
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FigureS3_color_heatmap_raw_data.nd2
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FigureS4_3wt.__Live_dead_stain_celltrackergreen488_propidiumiodidered.nd2
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FigureS4_5wt.__Live_dead_stain_celltrackergreen488_propidiumiodidered_01.nd2
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FigureS4_5wt.__Live_dead_stain_celltrackergreen488_propidiumiodidered_02.nd2
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FigureS7_3wt._.nd2
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FigureS7_5wt._.nd2
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FigureS8_2D_1.txt
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FigureS8_2D_2.txt
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FigureS8_2D_3.txt
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README.md
Abstract
Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of all pancreatic cancer cases. Five-year survival rates have remained below 12% since the 1970s, in part due to the difficulty in detection before metastasis (migration and invasion into neighboring organs and glands). Mechanical memory is a concept that has emerged over the past decade that may provide a path towards understanding how invading PDAC cells “remember” the mechanical properties of their diseased (“stiff,” elastic modulus, E ≈ 10 kPa) microenvironment even whilst invading a healthy (“soft,” E ≈ 1 kPa) microenvironment. Here, we investigated the role of mechanical priming by culturing a dilute suspension of PDAC (FG) cells within a 3D, rheologically tunable microgel platform from hydrogels with tunable mechanical properties. We conducted a suite of acute (short-term) priming studies where we cultured PDAC cells in either a soft (E ≈ 1 kPa) or stiff (E ≈ 10 kPa) environment for 6 h, then removed and placed them into a new soft or stiff 3D environment for another 18 h. Following these steps, we conducted RNA-seq analyses to quantify gene expression. Initial priming in 3D culture showed persistent gene expression for the duration of the study, regardless of the subsequent environments (stiff or soft). Stiff 3D culture was associated with the down-regulation of tumor suppressors (LATS1, BCAR3, CDKN2C ), as well as the up-regulation of cancer-associated genes (RAC3). Immunofluorescence staining (BCAR3, RAC3) further supported the persistence of this cellular response, with BCAR3 upregulated in soft culture, and RAC3 upregulated in stiff-primed culture. Stiff-primed genes were stratified against patient data found in The Cancer Genome Atlas (TCGA). Upregulated genes in stiff-primed 3D culture were associated with decreased survival in patient data, suggesting a link between patient survival and mechanical priming.
README: Survival-associated cellular response maintained in pancreatic ductal adenocarcinoma (PDAC) switched between soft and stiff 3D microgel culture
https://doi.org/10.5061/dryad.z34tmpgn8
Co-corresponding authors: Siddharth Dey, sdey@ucsb.edu; Angela Pitenis, apitenis@ucsb.edu
File List
A) Figure2_representative_3wt.%_indentation_curve.csv
B) Figure2_representative_5wt.%_indentation_curve.csv
C) Figure2_3wt%_average_modulus.csv
D) Figure2_5wt%_average_modulus.csv
E) Figure3a_data.csv
F) Figure3b_data.csv
G) Figure5_soft_6h_1.txt
H) Figure5_soft_6h_2.txt
I) Figure5_stiff_6h_1.txt
J) Figure5_stiff_6h_2.txt
K) Figure6_soft_to_soft_1.txt
L) Figure6_soft_to_soft_2.txt
M) Figure6_soft_to_soft_3.txt
N) Figure6_soft_to_soft_4.txt
O) Figure6_soft_to_stiff_1.txt
P) Figure6_soft_to_stiff_2.txt
Q) Figure6_soft_to_stiff_3.txt
R) Figure6_stiff_to_soft_1.txt
S) Figure6_stiff_to_soft_2.txt
T) Figure6_stiff_to_soft_3.txt
U) Figure6_stiff_to_soft_4.txt
V) Figure6_stiff_to_stiff_1.txt
W) Figure6_stiff_to_stiff_2.txt
X) Figure6_stiff_to_stiff_3.txt
Y) Figure6_stiff_to_stiff_4.txt
Z) Figure7a_soft_to_soft.tif
AA) Figure7a_soft_to_stiff.tif
AB) Figure7a_stiff_to_soft.tif
AC) Figure7a_stiff_to_stiff.tif
AD) Figure7b_calculations.csv
AE) Figure7c_soft_to_soft.tif
AF) Figure7c_soft_to_stiff.tif
AG) Figure7c_stiff_to_soft.tif
AH) Figure7c_stiff_to_stiff.tif
AI) Figure7d_calculations.csv
AJ) FigureS2_3wt.%diameter.csv
AK) FigureS2_5wt.%diameter.csv
AL) FigureS3_color_heatmap_raw_data.nd2
AM) FigureS4_3wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered.nd2
AN) FigureS4_5wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered_01.nd2
AO) FigureS4_5wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered_02.nd2
AP) FigureS7_3wt.%.nd2
AQ) FigureS7_5wt.%.nd2
AR) FigureS8_2D_1.txt
AS) FigureS8_2D_2.txt
AT) FigureS8_2D_3.txt
AU) FigureS10_3wt.%_radial_tubulin_intensity.csv
AV) FigureS10_5wt.%_radial_tubulin_intensity.csv
AW) Figure7b_raw_image_data.zip
AX) Figure7d_raw_image_data.zip
FIGURE 2: microtribometer
A) Figure2_representative_3wt.%_indentation_curve.csv: representative indentation curve of 3 wt.% bulk polyacrylamide hydrogel
• row 1: labels for column data with units
• column 1: time, in seconds, of indentation
• column 2: normal force, Fn, in micro-Newtons, or the y-axis in the indentation curve
• column 3: standard deviation in the normal force measurement, in micro-Newtons
• column 4: z-stage displacement in micrometers, or the x-axis in the indentation curve
• rows 2 and beyond: data for each of these columns
B) Figure2_representative_5wt.%_indentation_curve.csv: representative indentation curve of 5 wt.% bulk polyacrylamide hydrogel
• row 1: labels for column data with units
• column 1: time, in seconds, of indentation
• column 2: normal force, Fn, in micro-Newtons, or the y-axis in the indentation curve
• column 3: standard deviation in the normal force measurement, in micro-Newtons
• column 4: z-stage displacement in micrometers, or the x-axis in the indentation curve
• rows 2 and beyond: data for each of these columns
C) Figure2_3wt%_average_modulus.csv: average moduli values calculated from indentations on the micro tribometer for the 3 wt.% bulk polyacrylamide hydrogel
• row 1: column labels
• column 1: position number on a gel for a set of measurements
• column 2: cycle number per position on a gel
• column 3: maximum force obtained during measurement, in micro-Newtons
• column 4: pressure, in kPa
• column 5: contact radii, in micrometers
• column 6: measured E* elastic modulus using a Hertz fit, in kPa
D) Figure2_5wt%_average_modulus.csv: average moduli values calculated from indentations on the microtribometer for the 5 wt.% bulk polyacrylamide hydrogel
• row 1: column labels
• column 1: position number on a gel for a set of measurements
• column 2: cycle number per position on a gel
• column 3: maximum force obtained during measurement, in micro-Newtons
• column 4: pressure, in kPa
• column 5: contact radii, in micrometers
• column 6: measured E* elastic modulus using a Hertz fit, in kPa
FIGURE 3: rheology
E) Figure3a_data.csv: data for angular frequency sweep reporting storage moduli for both the 3 wt.% and 5 wt.% microgel particles
• row 1: column labels for angular frequency, storage modulus, and loss modulus
• row 2: units corresponding to each column
• row 3: replicate number for each sample
• row 4 and beyond: data for the frequency, storage, and loss moduli
• columns A-F: data for 5 wt.% polyacrylamide microgel
• columns G-L: data for 3 wt.% polyacrylamide microgel
F) Figure3b_data.csv: data for oscillatory strain sweep
• row 1: columns labels for oscillatory strain, storage, and loss moduli
• row 2: corresponding units for each column
• row 3: replicate number for each column
• row 4 and beyond: data for the oscillatory strain, storage, and loss moduli
• columns A-F: data for 5 wt.% polyacrylamide microgel
• columns G-L: data for 3 wt.% polyacrylamide microgel
FIGURE 5: cells cultured for 6 h in microgel; data analyzed with bulk-mRNA-seq R script
G) Figure5_soft_6h_1.txt: gene count data for replicate 1 of soft-microgel-cultured PDAC cells after 6 h
• row 1: list of column numbers
• column 1: list of gene names
• column 10: list of gene counts
• other columns: NA, 0-filled matrices
H) Figure5_soft_6h_2.txt: gene count data for replicate 2 of soft-microgel-cultured PDAC cells after 6 h
• row 1: list of column numbers
• column 1: list of gene names
• column 10: list of gene counts
• other columns: NA, 0-filled matrices
I) Figure5_stiff_6h_1.txt: gene count data for replicate 1 of stiff-microgel-cultured PDAC cells after 6 h
• row 1: list of column numbers
• column 1: list of gene names
• column 10: list of gene counts
• other columns: NA, 0-filled matrices
J) Figure5_stiff_6h_2.txt: gene count data for replicate 2 of stiff-microgel-cultured PDAC cells after 6 h
• row 1: list of column numbers
• column 1: list of gene names
• column 10: list of gene counts
• other columns: NA, 0-filled
FIGURE 6, S5, and S9: mechanical switch RNA-seq data, analyzed with bulk-mRNA-seq R script
K) Figure6_soft_to_soft_1.txt: gene count file for replicate 1 of the soft-to-soft condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
L) Figure6_soft_to_soft_2.txt: gene count file for replicate 2 of the soft-to-soft condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
M) Figure6_soft_to_soft_3.txt: gene count file for replicate 3 of the soft-to-soft condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
N) Figure6_soft_to_soft_4.txt: gene count file for replicate 4 of the soft-to-soft condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
O) Figure6_soft_to_stiff_1.txt: gene count file for replicate 1 of the soft-to-stiff condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
P) Figure6_soft_to_stiff_2.txt: gene count file for replicate 2 of the soft-to-stiff condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
Q) Figure6_soft_to_stiff_3.txt: gene count file for replicate 3 of the soft-to-stiff condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
R) Figure6_stiff_to_soft_1.txt: gene count file for replicate 1 of the stiff-to-soft condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
S) Figure6_stiff_to_soft_2.txt: gene count file for replicate 2 of the stiff-to-soft condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
T) Figure6_stiff_to_soft_3.txt: gene count file for replicate 3 of the stiff-to-soft condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
U) Figure6_stiff_to_soft_4.txt: gene count file for replicate 4 of the stiff-to-soft condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
V) Figure6_stiff_to_stiff_1.txt: gene count file for replicate 1 of the stiff-to-stiff condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
W) Figure6_stiff_to_stiff_2.txt: gene count file for replicate 2 of the stiff-to-stiff condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
X) Figure6_stiff_to_stiff_3.txt: gene count file for replicate 3 of the stiff-to-stiff condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
Y) Figure6_stiff_to_stiff_4.txt: gene count file for replicate 4 of the stiff-to-stiff condition shown in Figure 6b
• row 1: labels of each column
• column 1: list of gene names
• column 5: list of gene counts
• other columns: NA, 0-filled
FIGURE 7: immunofluorescence
Z) Figure7a_soft_to_soft.tif: raw image of soft-to-soft condition appearing in Figure 7a
• processed using FIJI into a SUM intensity projection over 4.65 micrometers (slices 9-14)
• green channel shows BCAR3 staining, and blue channel shows nuclear staining
AA) Figure7a_soft_to_stiff.tif: raw image of soft-to-stiff condition appearing in Figure 7a
• processed using FIJI into a SUM intensity projection over 4.65 micrometers (slices 9-14)
• green channel shows BCAR3 staining, and blue channel shows nuclear staining
AB) Figure7a_stiff_to_soft.tif: raw image of stiff-to-soft condition appearing in Figure 7a
• processed using FIJI into a SUM intensity projection over 4.65 micrometers (slices 8-13)
• green channel shows BCAR3 staining, and blue channel shows nuclear staining
AC) Figure7a_stiff_to_stiff.tif: raw image of stiff-to-stiff condition appearing in Figure 7a
• processed using FIJI into a SUM intensity projection over 4.65 micrometers (slices 10-15)
• green channel shows BCAR3 staining, and blue channel shows nuclear staining
AD) Figure7b_calculations.csv: intensity measurements for Figure 7b
• row 1: column labels
• column A: sample number
• column B: minimum intensity
• column C: maximum intensity
• column D: integrated fluorescent density
• column E: area measurement, in micrometers squared
• column F: average fluorescence intensity (integrated density/unit area; column D / column E), in fluorescence unit/micrometers squared
• row 2 and beyond: data
AE) Figure7c_soft_to_soft.tif: raw image of soft-to-soft condition appearing in Figure 7c
• processed using FIJI to create a SUM intensity z-projection across the entire stack
• green channel shows RAC3 staining, and blue channel shows nuclear staining
AF) Figure7c_soft_to_stiff.tif: raw image of soft-to-stiff condition appearing in Figure 7c
• processed using FIJI to create a SUM intensity z-projection across the entire stack
• green channel shows RAC3 staining, and blue channel shows nuclear staining
AG) Figure7c_stiff_to_soft.tif: raw image of stiff-to-soft condition appearing in Figure 7c
• processed using FIJI to create a SUM intensity z-projection across the entire stack
• green channel shows RAC3 staining, and blue channel shows nuclear staining
AH) Figure7c_stiff_to_stiff.tif: raw image of stiff-to-stiff condition appearing in Figure 7c
• processed using FIJI to create a SUM intensity z-projection across the entire stack
• green channel shows RAC3 staining, and blue channel shows nuclear staining
AI) Figure7d_calculations.csv:
• row 1: column labels
• column A: sample number
• column B: minimum intensity
• column C: maximum intensity
• column D: integrated fluorescent density
• column E: area measurement, in micrometers squared
• column F: average fluorescence intensity (integrated density/unit area; column D / column E), in fluorescence unit/micrometers squared
• row 2 and beyond: data
AW) Figure7b_raw_image_data.zip
• this compressed file has four folders for the four different conditions of our experiment: soft-to-soft, soft-to-stiff, stiff-to-soft, and stiff-to-stiff
• files in each of these subfolders are TIFF images of BCAR3 immunofluorescent staining
• channel 1 shows a nuclear stain
• channel 2 shows the BCAR3 stain
AX) Figure7d_raw_image_data.zip
• this compressed file has four folders for the four different conditions of our experiment: soft-to-soft, soft-to-stiff, stiff-to-soft, and stiff-to-stiff
• files in each of these subfolders are TIFF images of RAC3 immunofluorescent staining
• channel 1 shows a nuclear stain
• channel 2 shows the RAC3 stain
• channel 3 shows actin staining that was only used to identify the cell body
FIGURE S2: microgel size distribution
AJ) FigureS2_3wt.%diameter.csv: microgel size information for 3 wt.% polyacrylamide microgel particles
• row 1: column labels
• column 1: number of measurements from 1 - 250 for n = 250 samples counted
• column 2: radii measurements for each sample, in micrometers
• column 3: diameter measurements for each sample, in micrometers
AK) FigureS2_5wt.%diameter.csv: microgel size information for 5 wt.% polyacrylamide microgel particles
• row 1: column labels
• column 1: number of measurements from 1 - 250 for n = 250 samples counted
• column 2: radii measurements for each sample, in micrometers
• column 3: diameter measurements for each sample, in micrometers
FIGURE S3: color depth heatmap
AL) FigureS3_color_heatmap_raw_data.nd2: raw image processed in FIJI
• maximum intensity projection was created after using the Z-stack Depth Colorcode 0.0.2 plugin
FIGURE S4: cell viability
AM) FigureS4_3wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered.nd2:
• propidium iodide staining for cells cultured in 3 wt.% microgels for 24 h
• cell tracker green is in channel one for the identification of cells
• dead stain in the second channel identifies dead cells
AN) FigureS4_5wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered_01.nd2
• propidium iodide staining for cells cultured in 5 wt.% microgel for 24 h
• cell tracker green is in channel one for the identification of cells
• dead stain in the second channel identifies dead cells
AO) FigureS4_5wt.%_Live_dead_stain_celltrackergreen488_propidiumiodidered_02.nd2
• propidium iodide staining for cells cultured in 5 wt.% microgel for 24 h, second image
• cell tracker green is in channel one for the identification of cells
• dead stain in the second channel identifies dead cells
FIGURE S6 and S7: microgel packing
AP) FigureS7_3wt.%.nd2:
• image was processed in FIJI using the local thickness native analysis
• image was thresholded, and packing density was calculated as the percentage of fluorescence per slice
• local thickness values were extracted from FIJI, in micrometers
AQ) FigureS7_5wt.%.nd2:
• image was processed in FIJI using the local thickness native analysis
• image was thresholded, and packing density was calculated as the percentage of fluorescence per slice
• local thickness values were extracted from FIJI, in micrometers
FIGURE S8: 2D and 3D comparison, analyzed with bulk-mRNA-seq R script
AR) FigureS8_2D_1.txt: Gene counts of 2D tissue culture plastic-culture PDAC cells after 6 h
• gene count data is given in column 5
• row 1 shows column labels
• column 1 shows gene names
• data in all other columns are NA (shown as 0s)
• comparisons were made to files K-Y
AS) FigureS8_2D_2.txt: gene counts of 2D tissue culture plastic-culture PDAC cells after 6 h, replicate 2
• gene count data is given in column 5
• row 1 shows column labels
• column 1 shows gene names
• data in all other columns are NA (shown as 0s)
• comparisons were made to files K-Y
AT) FigureS8_2D_3.txt: gene counts of 2D tissue culture plastic-culture PDAC cells after 6 h, replicate 3
• gene count data is given in column 5
• row 1 shows column labels
• column 1 shows gene names
• data in all other columns are NA (shown as 0s)
• comparisons were made to files K-Y
FIGURE S10: radial tubulin expression
AU) FigureS10_3wt.%_radial_tubulin_intensity.csv: data on tubulin expression across a cell diameter for PDAC cells cultured in soft microgel for 24 h
• row 1: column labels for distance in microns (A, E, I, M), intensity (called gray value, B, F, J, N), normalized distance (from 0 to 1, C, G, K, O), and normalized intensity (gray value normalized, D, H, L, P) for each sample
• row 2: sample number information
• row 3 and below: data
• column Q maps distance values from 0 to 1 for the x-axis of the plots in Figure S10
• column R shows average intensity values calculated as averages of columns D, H, L, and P
• column S shows standard deviations in intensity values from columns D, H, L, and P
• sample 1: columns A-D
• sample 2: column E-H
• sample 3: columns I-L
• sample 4: columns M-P
AV) FigureS10_5wt.%_radial_tubulin_intensity.csv: data on tubulin expression across a cell diameter for PDAC cells cultured in stiff microgel for 24 h
• row 1: column labels for distance in microns (A, E, I, M), intensity (called gray value, B, F, J, N), normalized distance (from 0 to 1, C, G, K, O), and normalized intensity (gray value normalized, D, H, L, P) for each sample
• row 2: sample number information
• row 3 and below: data row 4 and below: data
• column Q maps distance values from 0 to 1 for the x-axis of the plots in Figure S10
• column R shows average intensity values calculated as averages of columns D, H, L, and P
• column S shows standard deviations in intensity values from columns D, H, L, and P
• sample 1: columns A-D
• sample 2: column E-H
• sample 3: columns I-L
• sample 4: columns M-P
R script provided: RNA-seq analysis R script
Functions utilized:
A) BiocManager version 3.14
B) DESeq2
C) plyr
D) apeglm
E) ggplot2
F) pheatmap
G) vsn
H) RColorBrewer
I) EnhancedVolcano
J) magrittr
K) ashr
L) pasilla
M) tidyverse
N) hrbrthemes
O) viridis
P) plotly
Q) heatmaply
R) ComplexHeatmap
S) psych
T) reshape2
ACCESSED DATA
Data for generating survival curves, based on mRNA data from the pancreas, appearing in Figures 5d and 6b used The Cancer Genome Atlas (TCGA) Data, Resources, and Materials originally published by the National Cancer Institute. Data was accessed through kmplot.com. The data obtained from TCGA can be published under CC0. The results published here are in part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga
• Nagy A, Munkacsy G, Gyorffy B: Pancancer survival analysis of cancer hallmark genes, Sci Rep., 2021 Mar 15;11(1):6047, https://doi.org/10.1038/s41598-021-84787-5