Survival-Associated Cellular Response Maintained in Pancreatic Ductal Adenocarcinoma (PDAC) Switched Between Soft and Stiff 3D Microgel Culture

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 prior to 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 toward understanding how invading PDAC cells "remember" the mechanical properties of their diseased ("stiff", elastic modulus, E ≈ 10 kPa) microenvironment even while 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 the 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 downregulation of tumor suppressors (LATS1, BCAR3, CDKN2C), as well as the upregulation 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

The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals - Fig 8

<p>A) Comparative analysis of total protein contents within subcellular structures of MII-oocyte. ^a-c: values with different superscripts are significantly different at p<0.05. B) Protein patterns of oocyte substructures revealed by SDS-PAGE electrophoresis. C) Western blot analysis of NANOG and DNMT3A between NS+S and FS oocyte fragments.</p

Comparative analysis of total mRNA contents between subcellular structures of MII-oocyte.

<p>^a-c: values with different superscripts are significantly different at p<0.05.</p

Investigation of transcript assymetry within oocyte and early embryo.

<p>A) Unfertilized ovine MII-oocytes were micro surgically trisected to generate cortical materials containing MII-spindle material (S) and oocyte hemispheres that were either near to (NS) or far from (FS) the MII-spindle. The pools of S, NS, and FS cytoplasmic fragments were prepared and used for comparative RT-qPCR using 21 developmentally important genes. Obtained profiles revealed four patterns of transcript abundances within the three fragments of the oocyte. B) To understand whether ovine early embryos inherit differential transcripts localization observed within MII-oocyte, the transcriptional distribution of transcripts between early embryonic sister blastomeres was investigated using RT-qPCR. For this purpose, one blastomere of ovine early embryo was labeled with Dil and the embryos were followed during the first and second embryonic divisions to generate pools of leading and lagging blastomeres. The leading and lagging pool of blastomeres were used for quantitative analysis of those transcripts that were found to be differentially located with unfertilized MII oocytes.</p

Developmental competence of corresponding sister blastomeres derived from ovine 2-cell embryos.

<p>The proportion of sister blastomeres that developed to certain developmental stages (y-axis: 2-cell block (2CB), two further cleavage (4-cell block: 4CB), three further cleavage (8-cell block: 8CB), four further cleavage (morula block: MB), and those blastomeres that reached the blastocyst stage (BLS)). The size of circles correspond their relative proportions (numbers within the circles).</p

Topological assessment of sperm entry point (SEP) in ovine eggs.

<p>Fertilzied oocytes were rotated under constant UV-light until MII-spindle was positioned to 3 O’clock. Then, the spatial relationship between SEP and MII-spindle was measured in 4 imaginary oocyte zones. In this scheme, zones I and IV are the closest and the furthest from the MII-spindle, respectively. Table represents the results of topological assessment of SEP.</p

Schematic illustration of the procedure used for microsurgical trisection of ovine unfertilized MII-oocytes using a manual method of oocyte trisection.

<p>A, B) In vitro matured oocytes were denuded of cumulus cells and oocytes with evident first polar body (1Pb) were selected for zona removal. C) Ovine oocytes partially extrude the MII-spindle and associated chromosomes (S) as a cytoplasmic protrusion which is particularly evident following zona removal. D) Using this cytoplasmic protrusion as a reference point, oocytes were first bisected to oocyte hemispheres that were either near-to (NS) or far-from (FS) the MII-spindle (S). E) The obtained NS oocyte hemispheres were then used for preparation of cortical materials containing MII-chromosomes (S) and the enucleated NS oocyte hemispheres.</p

Specific primers used for qRT-PCR.

<p>Specific primers used for qRT-PCR.</p

Contributions of leading and lagging blastomeres to the total cell count of the developed blastocysts.

<p>In majority (75%) of blastocysts, leading blastomeres contributed to significantly higher cells of the blastocyst compared to the lagging counterpart. In the remaining 25%, the contributions of leading and lagging blastomeres to total cell count of the blastocysts were comparable. *: significantly different at P<0.05.</p

Lineage tracing of the leading and lagging blastomeres.

<p>A cohort group of blastocysts derived from leading-labeled two-cell embryos that are observed using appropriate Hoechst-33342 (A) and Rhodamine (B) filters. C) Criteria for sorting of Dil-labeled embryos. Scoring the orientation of the Em-Ab axis in ovine blastocysts relative to the first cleavage plane of the 2-cell embryo. After drawing imaginary lines of the Em-Ab axis and equatorial plane, the blastocysts were classified to three groups depending on the angular departure of the boundary line of the fluorescent/nonfluorescent cells from the equatorial plane: i) specified: when the angular departure was almost 90°, ii) semi-specified: when the angular departure was ≥45°, iii) un-specified: when the angular departure was <45°. D1-F4) Representative images of specified (D1-D4), semi-specified (E1-E4) and un-specified (F1-F4) observed under bright light (D1, E1, & F1), and H33342 (D2, E2, & F2) and Rhodamine (D3, E3, & F3) filters. D4, E4, & F4 images show merged images of H33342 and Rhodamine. Bar represents 100 μm.</p