DOI: 10.5061/dryad.z34tmpgn8
title: Survival-associated cellular response maintained in pancreatic ductal adenocarcinoma (PDAC) switched between soft and stiff 3D microgel culture

description: 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.
description: # 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](https://doi.org/10.5061/dryad.z34tmpgn8) Co-corresponding authors: Siddharth Dey, [sdey@ucsb.edu](mailto:sdey@ucsb.edu); Angela Pitenis, [apitenis@ucsb.edu](mailto: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](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](https://doi.org/10.1038/s41598-021-84787-5)