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. 2022 Mar 15;12(3):1337-1371.
eCollection 2022.

Time-extended exposure of gastric epithelial cells to secretome of Helicobacter pylori-activated fibroblasts induces reprogramming of gastric epithelium towards pre-cancerogenic and pro-invasive phenotype

Gracjana Krzysiek-Maczka  1 , Aneta Targosz  1 , Tomasz Wrobel  2 , Milena Paw  2 , Urszula Szczyrk  1 , Janusz Opila  3 , Malgorzata Strzalka  1 , Mateusz Wierdak  4 , Piotr Major  4 , Tomasz Brzozowski  1 , Jarosław Czyz  2 , Agata Ptak-Belowska  1
Affiliations

Time-extended exposure of gastric epithelial cells to secretome of Helicobacter pylori-activated fibroblasts induces reprogramming of gastric epithelium towards pre-cancerogenic and pro-invasive phenotype

Gracjana Krzysiek-Maczka et al. Am J Cancer Res. .

Abstract

Despite of the improvement in gastric cancer (GC) therapies patients still suffer from cancer recurrence and metastasis. Recently, the high ratio of these events combined with increased chemoresistance has been related to the asymptomatic Helicobacter pylori (Hp) infections. The limited efficiency of GC treatment strategies is also increasingly attributed to the activity of tumor stroma with the key role of cancer-associated fibroblasts (CAFs). In order to investigate the influence of Hp infection within stromal gastric tissue on cancer initiation and progression, we have exposed normal gastric epithelial cells to long-term influence of Hp-activated gastric fibroblast secretome. We have referred obtained results to this secretome influence on cancer cell lines. The invasive properties of cells were checked by time-lapse video microscopy and basement membrane assays. The expression of invasion-related factors was checked by RT-PCR, Western Blot, immunofluorescence and Elisa. Hp-activated gastric fibroblast secretome induced EMT type 3-related shifts of RGM1 cell phenotype; in particular it augmented their motility, cytoskeletal plasticity and invasiveness. These effects were accompanied by Snail1/Twist activation, the up-regulation of cytokeratin19/FAP/TNC/Integrin-β1 and MMPs, and by the induction of cMethigh/pEGFRhigh phenotype. Mechanistic studies suggest that this microevolution next to TGFβ relies also on c-Met/EGFR signaling interplay and engages HGF-Integrin-Ras-dependent Twist activation leading to MMP and TNC upregulation with subsequent positive auto- and paracrine feedback loops intensifying this process. Similar shifts were detected in cancer cells exposed to this secretome. Collectively, we show that the secretome of Hp-infected fibroblasts induces reprogramming/microevolution of epithelial and cancer cells towards type 3 EMT-related invasive phenotype in a manner reciprocally reliant next to TGFβ on cMet/Integrin-β1/p-EGFR-dependent axis. Apparently, the phenotypical plasticity of Hp-activated fibroblast reprogrammed gastric epithelial cells determines their susceptibility to the pro-invasive signaling, which results in re-organization of gastric niches and provides the cues for GC promotion/progression.

Keywords: Helicobacter pylori-activated fibroblasts; cancer associated fibroblasts; epithelial-mesenchymal transition; epithelial-myofibroblast transition; gastric cancer; mesenchymal-epithelial transition; metastasis.

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Conflict of interest statement

None.

Figures

Figure 11
Figure 11
Flowchart of the experimental procedures.
Figure 1
Figure 1
Motility of RGM1, l.t.EMT-RGM1 and l.t.EMT+RGM1 cells in Hp-AGF supernatant, GF supernatant and DMEM+10% FBS. Total length of cell trajectory (µm), velocity of cell movement (speed; total length of cell trajectory/time of recording; µm/min) and total length of cell displacement (i.e. the distance from the starting point directly to the cell’s final position; µm) were quantified with the Hiro program. Column charts show migration parameters at the population level (registered for 8 h; N=50): distance (A) and displacement (B). Cell trajectories are presented as circular diagrams (axis scale in µm) drawn with the initial point of each trajectory placed at the origin of the plot. Circular diagrams represent migration parameters at the single-cell level of all three cell types in: Hp-AGF supernatant (C), in GF supernatant (D) and in DMEM+10% FBS (E). Distance over displacement ratio for each cell type in respective media (F). For constant directionality the parameter equals 1, for random movement the parameter tends to zero. The movement strategies of l.t.EMT-RGM1 cells (G) and l.t.EMT+RGM1 cells (H, I).
Figure 2
Figure 2
Hp-AGF induces phenotypical diversity and plasticity of actin cytoskeleton organization in l.t.EMT+RGM1 cells associated with nuclear actin localization. The composition of Nomarski contrast and immunofluorescence micrographs of actin cytoskeleton in RGM1 cells characterized by the thin bundles of actin filaments in the perinuclear zone and thick cortical bundles of actin filaments at cellular peripheries, which formed a tangential system at the periphery of epithelial islets (A); in l.t.EMT-RGM1 cells showing their reprogramming towards fibroblast-like phenotype: blue arrows point stress fibers (B), white arrow points lamellipodium (C) and in l.t.EMT+RGM1 cells characterized by enhanced formation of filopodia and broad diversity of actin cytoskeleton organization (D). Nuclear localization of actin in l.t.EMT+RGM1 cells (respectively: actin, chromatin and composite); white arrows indicate filopodia, yellow arrows indicate nuclear actin (E). Red: F-actin and blue: Hoechst.
Figure 3
Figure 3
Hp-AGF secretome prompts invasive properties of long-term RGM1 cells. Geltrex invasion assay of RGM1, l.t.EMT-RGM1 and l.t.EMT+RGM1 cells towards Hp-AGF supernatant and towards DMEM+10% FBS showing enhanced invasiveness of l.t.EMT+RGM1 cells (A). RT-PCR analysis of the expression of 18S mRNA and of FAP and MMP 2, 3 and 9 mRNA expression in l.t.EMT-RGM1, l.t.EMT+RGM and original RGM1 cells and the ratio of selected genes over 18S mRNA showing their strong upregulation in l.t.EMT+RGM1 cells (B). Phase contrast microscopy of l.t.EMT-RGM1 and l.t.EMT+RGM1 Geltrex metalloproteinase activity assay (1:1) showing the ability of l.t.EMT+RGM1 to degrade basement membrane components (C). Results are mean ± SEM of four to six independent experimental repeats. Asterisk (*) indicates a significant change (P<0.05) as compared to the control RGM1 value. Hash (#) indicates a significant change (P<0.05) as compared to l.t.EMT-RGM1 value.
Figure 4
Figure 4
Dynamics of cell groups/aggregates in 3D Geltrex BM. Nomarski contrast of initial and final distribution of cell groups in 3D Geltrex BM flooded with appropriate culture medium: RGM1 cells in DMEM+10% FBS (A), l.t.EMT-RGM1 cells in GF supernatant (B) and l.t.EMT+RGM1 in Hp-AGF supernatant (C). Upper and lower pictures show the initial and final distribution of cell groups (8 hrs after seeding and 16 hrs after seeding) with visible computed ellipses (magenta) coinciding with error ellipse (white). Cell group dynamics was registered with Leica DMI6000B time-lapse system for 8 hrs. The computed parameters of relevant aggregates e.g. standard deviations (σX, σY), centroid positions (µX, µY) and correlation coefficient r for given cell type are shown in the tables: RGM1 cells in DMEM+10% FBS (D), l.t.EMT-RGM1 in GF supernatant (E) and l.t.EMT+RGM1 cells in Hp-AGF secretome (F). The changing values of correlation coefficient reflect the aggregate shape changes and standard deviations changes reflect the area changes. Decreasing value of correlation coefficient points to striving round shape adaptation (evolution of linearly elongated aggregates into more circularly shaped ones) and decreasing values of standard deviations point to the overall area decrease. Nomarski contrast of l.t.EMT+RGM1 (G) and l.t.EMT-RGM1 (H) depicting aggregated cells in 3D Geltrex basement membrane 24 hrs after seeding and 48 hrs after seeding and showing invasive properties of l.t.EMT+RGM1 cells. Computed ellipses (magenta) and computed parameters of relevant aggregates: standard deviations (σX, σY), centroid positions (µX, µY) and correlation coefficient r. The aggregate area was calculated according to the formula: S=9πσXσY. Cell group dynamics was registered with Leica DMI6000B.
Figure 5
Figure 5
L.t.EMT+RGM1 cell pro-invasive and pro-metastatic abilities are related to Twist protein expression. RT-PCR analysis of the expression of 18S mRNA and of Snail and Twist mRNAs expression in l.t.EMT-RGM1 cells, l.t.EMT+RGM1 cells and the original RGM1 cells and the ratio of selected genes over 18S mRNA (A). Western Blot analysis of Snail Twist and Cytokeratin19 expression in total cellular proteins isolated from l.t.EMT-RGM1 cells, l.t.EMT+RGM1 cells and original RGM1 cells and the semi-quantitative densitometry analysis of the ratio of selected proteins over GAPDH showing the upregulation of Snail, Twist and Cytokeratin19 in l.t.EMT+RGM1 cells. 10 µg of total cellular proteins were loaded per each lane for Snail and Cytokeratin 19 and 30 µg of Twist (B). The composite of Nomarski contrast and immunofluorescence of chromatin and Twist protein in RGM1 cells, l.t.EMT-RGM1 and l.t.EMT-RGM1 cells showing the appearance of Twist and additionally its translocation into the nucleus (sequentially: composite and Twist). Green: Twist and blue: chromatine (Hoechst 33258) (C). Results are mean ± SEM of four to six independent experimental repeats. Asterisk (*) indicates a significant change (P<0.05) as compared to the control RGM1 value. Hash (#) indicates a significant change (P<0.05) as compared to l.t.EMT-RGM1 value.
Figure 6
Figure 6
Pro-migratory and pro-metastatic potential of l.t.EMT+RGM1 cells is associated with the activation of HGF/TNC/Integrin-β1 pathway. RT-PCR analysis of the adhesion and motility related mRNA expression (18S, cMet, HGF, EGFR and TNC) in l.t.EMT-RGM1, l.t.EMT+RGM1 and original RGM1 cells and the ratio of selected genes over 18S mRNA (A). Elisa analysis of HGF content in the secretome. The cells were transferred to DMEM+10% FBS and allowed for 48 hrs secretion. The analysis showed that only l.t.EMT+RGM1 cells were able to secrete HGF (B). Western Blot analysis of c-Met, p-EGFR, integrin-β1 and TNC protein expression in total cellular proteins isolated from l.t.EMT-RGM1, l.t.EMT+RGM1 and the original RGM1 cells and the semi-quantitative densitometry analysis of the ratio of selected proteins over GAPDH showing the upregulation of c-Met, p-EGFR, Integrin-β1 and TNC proteins in l.t.EMT+RGM1 cells. 10 µg and in the case of EGFR and TNC 25 µg of total cellular proteins were loaded per each lane (C). Results are mean ± SEM of four to six independent experimental repeats. Asterisk (*) indicates a significant change (P<0.05) as compared to the control RGM1 value. Hash (#) indicates a significant change (P<0.05) as compared to l.t.EMT-RGM1 value.
Figure 7
Figure 7
Hp-hAGFs elicit EMT in short-term cultures of HaCaT cells. Nomarski contrast of the phenotype of HaCaT cells placed in DMEM+10% FBS (control), hGF supernatant and Hp-hAGF supernatant (A). The trajectories of cell movement in respective supernatants and control medium presented as circular diagrams (axis scale in µm) drawn with the initial point of each trajectory placed at the origin of the plot (B). Corresponding motility parameters: total length of cell trajectory (µm), velocity of cell movement (speed; total length of cell trajectory/time of recording; µm/min) and total length of cell displacement (i.e. the distance from the starting point directly to the cell’s final position; µm) were quantified with the Hiro program. Column charts show migration parameters at the population level (registered for 8 h; N=50): distance and displacement (C). RT-PCR analysis of the expression of mRNA for 18S, Snail, Twist and EMT markers in HaCaT cells cultured in DMEM+10% FBS (control), hGF supernatant and Hp-hAGF supernatant and the ratio of selected genes over 18S mRNA. Results are mean ± SEM of four independent experimental repeats. Asterisk (*) indicates a significant change (P<0.05) as compared to the control value. Hash (#) indicates a significant change (P<0.05) as compared to the value for cells cultured in hGF secretome (D).
Figure 8
Figure 8
Hp-hAGF secretome induces EMT related changes of motility mode in cancer cells. Nomarski contrast of the phenotype of HT29 cells placed in DMEM+10% FBS (control), hGF supernatant and Hp-hAGF supernatant (A) and of AGS cells placed in DMEM/F12HAM+10% FBS, hGF supernatant, Hp-hAGF supernatant and Hp-hAGF supernatant with the addition of EGFR inhibitor tyrphostin A46 (0.1 mM) (B). Cell trajectories of epithelioid AGS cells in DMEM/F12HAM+10% FBS, hGF supernatant, Hp-hAGF supernatant and Hp-hAGF supernatant with the addition of A46 (0.1 mM) (C) and corresponding motility parameters (D). Cell trajectories of fibroblastoid AGS cells in DMEM/F12HAM+10% FBS, hGF supernatant, Hp-hAGF supernatant and Hp-hAGF supernatant with the addition of A46 (0.1 mM) and corresponding motility parameters (E). Cell trajectories are presented as circular diagrams (axis scale in µm) drawn with the initial point of each trajectory placed at the origin of the plot. Total length of cell trajectory (µm), velocity of cell movement (speed; total length of cell trajectory/time of recording; µm/min) and total length of cell displacement (i.e. the distance from the starting point directly to the cell final position; µm) were quantified with the Hiro program. Column charts show migration parameters at the population level (registered for 8 h; N=50): distance and displacement.
Figure 9
Figure 9
Hp-hAGF secretome prompts Twist related EMT type 3 pro-pluripotent changes in cancer cells. RT-PCR analysis of the expression of mRNA for 18S, Snail, Twist and pluripotency-related Yamanaka factors: c-Myc, Oct4 and Sox-2 in HT29 (A) and AGS (B) cells cultured for 96 hrs in control medium, hGF and Hp-hAGF supernatants and the ratio of selected genes over 18S mRNA. Additionally AGS cells were cultured in Hp-hAGF secretome with the addition of A46 (0.1 mM). The analysis shows Snail mRNA expression increase and strong Twist transcriptional upregulation. Results are mean ± SEM of four independent experimental repeats. Asterisk (*) indicates a significant change (P<0.05) as compared to the control value. Hash (#) indicates a significant change (P<0.05) as compared to the value for cells cultured in hGF secretome.
Figure 10
Figure 10
Hp-hAGF secretome induces increased invasiveness of cancer cells. This phenomenon in adenocarcinoma cells is related to cMet/HGF/Integrin-β1/TNC and EGFR signaling. Geltrex invasion assay of HT29 and AGS cells towards culture medium, hGF and Hp-hAGF supernatants and in the case of AGS cells towards Hp-hAGF secretome with the addition of tyrphostin A46 (0.1 mM) showing enhanced invasiveness towards Hp-hAGF supernatant and the decrease after A46 (A). Fluorescent microscopy of transmigrated AGS cells attached to the bottom side of the geltrex/transwell membrane stained with bisbenzimide (Hoechst 33342) (B). The phase contrast microscopy showing HT29 and AGS cells that transmigrated through invasion chambers towards Hp-hAGF supernatant. Three independent experiments were performed for each condition (C). RT-PCR analysis of the expression of 18S, Fap, MMP 2, 3, 9 and mRNA in HT29 (D) and AGS (E) cells cultured in culture medium, hGF, Hp-hAGF supernatants and in the case of AGS cells in Hp-hAGF secretome with addition of tyrphostin A46 (0.1 mM) and the ratio of selected genes over 18S mRNA showing their strong upregulation in Hp-hAGF secretome. Phase contrast microscopy of Geltrex metalloproteinase activity assay (1:1) of HT29 (F) and AGS (G) flooded with culture medium, hGF, Hp-hAGF supernatants and in the case of AGS cells with Hp-hAGF secretome with the addition of A46 (0.1 mM), showing enhanced ability of cells flooded with Hp-hAGF supernatant to cross basement membrane components already after 24 hrs. RT-PCR analysis of the mRNA expression for 18S, cMet, HGF, EGFR and Integrin-β1 in AGS cells cultured in control medium, hGF, Hp-hAGF and in Hp-hAGF secretome with the addition of A46 (0.1 mM) and the ratio of selected genes over 18S mRNA (H). Results are mean ± SEM of four independent experimental repeats. Asterisk (*) indicates a significant change (P<0.05) as compared to the control cell value. Hash (#) indicates a significant change (P<0.05) as compared to the value of cells grown in GF supernatant.
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