Figure 3. Secretion of inflammatory cytokines from CAFs activates STAT3 in PDA organoids. (A) Quantification of secretome dot blots of conditioned media from mouse tumor organoid monocultures, PSC monocultures, co-cultures, or Matrigel-only controls (MG). Results show normalized mean ± SD of three biological replicates. (B) Schematic illustration of the trans-well culture platform. (C and D) qPCR analysis of GP130 signaling ligands and receptors in mouse PSCs (C) or tumor organoids (D) cultured in monoculture or trans-well culture. Results show mean ± SD of four biological replicates. n.d., not detected. (E) ELISA of IL-6, IL-11, and LIF from conditioned media of PSC monocultures, organoid monocultures, or co-cultures. Results show mean ± SD of three biological replicates. (F) Quantification of secretome dot blots of conditioned media from human primary CAF monocultures, patient-matched tumor organoid monocultures, or co-cultures (n = 2). Results show mean ± SD of two technical replicates for each condition. (G) Representative IF image of KPC mouse tumor stained for phosphorylated STAT3 (Tyr705; green, pSTAT3) and the epithelial marker Cytokeratin 19 (Krt19, red; n = 2). Counterstain, DAPI (blue). Bar, 75 µm. (H) Western blot analysis of pSTAT3 in organoids treated with either 10 ng/ml recombinant IL-6, 10 ng/ml recombinant IL-11, or 50 ng/ml recombinant LIF, in the presence or absence of neutralizing antibodies or isotype controls (n = 2). Loading control, Actin. Molecular weights in kilodaltons. (I) Western blot analysis of pSTAT3 in organoids treated with co-culture conditioned media in the presence or absence of neutralizing antibodies against IL-6, IL-11, or LIF (n = 3). Loading control, Actin. Molecular weights in kilodaltons. (J) Passaging of organoids in reduced media conditions in monoculture or co-culture with WT (PSC WT) or IL-6 KO PSCs (PSC IL-6 KO). Red dot indicates the passage number when all organoids were found dead. Green dot indicates the passage number of surviving organoids when the experiment was terminated. Each dot represents one biological replicate. Bars indicate the average number of passages for each condition. (K) qPCR analysis of Il6, Il11, Lif, and Acta2 transcript levels in PSCs cultured with control media (Matrigel-only conditioned media) or tumor organoid conditioned media. Results show mean ± SD of five biological replicates for Il6, Lif, and Acta2, and three biological replicates for Il11. (L) Western blot analysis of PSCs cultured with control media or tumor organoid conditioned media (n = 3). Loading control, Hsp90α. Molecular weights in kilodaltons. (M and N) qPCR analysis of Il6 and Acta2 in three primary PSC lines (M) and two KPC mouse CAFs (N) cultured with control media or tumor organoid conditioned media. Results show mean ± SD of two technical replicates for each line. (O) qPCR analysis for IL6 and ACTA2 transcript levels in human primary CAFs cultured with control media or conditioned media from the corresponding patient-matched tumor organoids. Results show mean ± SD of 2 technical replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired Student’s t test.
Figure 3.

Secretion of inflammatory cytokines from CAFs activates STAT3 in PDA organoids. (A) Quantification of secretome dot blots of conditioned media from mouse tumor organoid monocultures, PSC monocultures, co-cultures, or Matrigel-only controls (MG). Results show normalized mean ± SD of three biological replicates. (B) Schematic illustration of the trans-well culture platform. (C and D) qPCR analysis of GP130 signaling ligands and receptors in mouse PSCs (C) or tumor organoids (D) cultured in monoculture or trans-well culture. Results show mean ± SD of four biological replicates. n.d., not detected. (E) ELISA of IL-6, IL-11, and LIF from conditioned media of PSC monocultures, organoid monocultures, or co-cultures. Results show mean ± SD of three biological replicates. (F) Quantification of secretome dot blots of conditioned media from human primary CAF monocultures, patient-matched tumor organoid monocultures, or co-cultures (n = 2). Results show mean ± SD of two technical replicates for each condition. (G) Representative IF image of KPC mouse tumor stained for phosphorylated STAT3 (Tyr705; green, pSTAT3) and the epithelial marker Cytokeratin 19 (Krt19, red; n = 2). Counterstain, DAPI (blue). Bar, 75 µm. (H) Western blot analysis of pSTAT3 in organoids treated with either 10 ng/ml recombinant IL-6, 10 ng/ml recombinant IL-11, or 50 ng/ml recombinant LIF, in the presence or absence of neutralizing antibodies or isotype controls (n = 2). Loading control, Actin. Molecular weights in kilodaltons. (I) Western blot analysis of pSTAT3 in organoids treated with co-culture conditioned media in the presence or absence of neutralizing antibodies against IL-6, IL-11, or LIF (n = 3). Loading control, Actin. Molecular weights in kilodaltons. (J) Passaging of organoids in reduced media conditions in monoculture or co-culture with WT (PSC WT) or IL-6 KO PSCs (PSC IL-6 KO). Red dot indicates the passage number when all organoids were found dead. Green dot indicates the passage number of surviving organoids when the experiment was terminated. Each dot represents one biological replicate. Bars indicate the average number of passages for each condition. (K) qPCR analysis of Il6, Il11, Lif, and Acta2 transcript levels in PSCs cultured with control media (Matrigel-only conditioned media) or tumor organoid conditioned media. Results show mean ± SD of five biological replicates for Il6, Lif, and Acta2, and three biological replicates for Il11. (L) Western blot analysis of PSCs cultured with control media or tumor organoid conditioned media (n = 3). Loading control, Hsp90α. Molecular weights in kilodaltons. (M and N) qPCR analysis of Il6 and Acta2 in three primary PSC lines (M) and two KPC mouse CAFs (N) cultured with control media or tumor organoid conditioned media. Results show mean ± SD of two technical replicates for each line. (O) qPCR analysis for IL6 and ACTA2 transcript levels in human primary CAFs cultured with control media or conditioned media from the corresponding patient-matched tumor organoids. Results show mean ± SD of 2 technical replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired Student’s t test.

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