Abstract
Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by extensive local invasion and systemic spread. In this study, we employed a three-dimensional organoid model of human pancreatic cancer to characterize the molecular alterations critical for invasion. Time-lapse microscopy was used to observe invasion in organoids from 25 surgically resected human PDAC samples in collagen I. Subsequent lentiviral modification and small-molecule inhibitors were used to investigate the molecular programs underlying invasion in PDAC organoids. When cultured in collagen I, PDAC organoids exhibited two distinct, morphologically defined invasive phenotypes, mesenchymal and collective. Each individual PDAC gave rise to organoids with a predominant phenotype, and PDAC that generated organoids with predominantly mesenchymal invasion showed a worse prognosis. Collective invasion predominated in organoids from cancers with somatic mutations in the driver gene SMAD4 (or its signaling partner TGFBR2). Reexpression of SMAD4 abrogated the collective invasion phenotype in SMAD4-mutant PDAC organoids, indicating that SMAD4 loss is required for collective invasion in PDAC organoids. Surprisingly, invasion in passaged SMAD4-mutant PDAC organoids required exogenous TGFβ, suggesting that invasion in SMAD4-mutant organoids is mediated through noncanonical TGFβ signaling. The Rho-like GTPases RAC1 and CDC42 acted as potential mediators of TGFβ-stimulated invasion in SMAD4-mutant PDAC organoids, as inhibition of these GTPases suppressed collective invasion in our model. These data suggest that PDAC utilizes different invasion programs depending on SMAD4 status, with collective invasion uniquely present in PDAC with SMAD4 loss. Significance: Organoid models of PDAC highlight the importance of SMAD4 loss in invasion, demonstrating that invasion programs in SMAD4-mutant and SMAD4 wild-type tumors are different in both morphology and molecular mechanism.
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Organoids were isolated from 48 primary human PDAC samples acquired from surgical resection specimens at The Johns Hopkins Hospital – these included 25 PDAC samples (PCO1-PCO25) for immediate invasion analysis and 23 PDAC samples (PCO26-PCO48) for additional analyses, including immunofluorescence, lentiviral transduction, and pharmacological manipulation. This study was approved by the Institutional Review Board of The Johns Hopkins Hospital, and written informed consent was obtained from patients prior to sample acquisition. Organoids from 25 patients were isolated from fresh primary tumor samples as previously described ( 14 ). Briefly, PDAC tissue was digested and filtered; organoids were isolated by differential centrifugation. PDAC organoids were embedded into collagen I gels for time lapse imaging using a Nikon Eclipse Ti inverted microscope, with images collected at 30 minute intervals for 3–7 days. Immunofluorescence assays were used to assess expression of CK (1:100, #4545, Cell signaling Technologies), VIM (1:100, #5741, Cell signaling Technologies), SOX9 (1:100, #D8G8H, Cell signaling Technologies), Nkx6.1 (1:100, BDB563022, Fisher scientific), and GATA4 (1:100 #560327, BD Biosciences) in a subset of these organoids using established protocols with secondary antibodies coupled to Alexa Flour 488, 568 or 647 (1:1000, (A11001, A10042, A21235, Invitrogen). Primary PDAC tissue from these cases was analyzed by immunohistochemistry for SMAD4 (clone RBT-SMAD4, predilute; Bio SB), ECAD (clone EP700Y, predilute; Cell Marque), VIM (clone V9, predilute; Roche), P40 (clone BC28, 1:100; Biocare), and GATA6 (R&D Cat. Number AF1700,1:1000) using established protocols. We also isolated genomic DNA from the primary PDACs in our cohort for targeted sequencing of 11 PDAC driver genes using Ion AmpliSeq library preparation with a previously described primer panel ( 19 ), followed by sequencing on an Ion Torrent PGM. For re-expression of SMAD4, the SMAD4 coding sequence was subcloned into pCW57.GFP-P2A-MCS for doxycycline-inducible expression, and lentivirus was produced from HEK-293T cells using the pLKO.1 lentiviral vector protocol. For lentiviral transduction, organoids from 3 patients were passaged in Matrigel and then digested into single cells, transduced, and re-established in Matrigel, followed by puromycin selection three days after transduction. To characterize the invasion of modified organoids, the organoids were transferred from Matrigel to collagen I and treated with doxycycline (5mg/L) for 3 days to induce the expression of SMAD4 followed by TGFβ1 treatment (5ng/mL) for an additional 3 days. Then, 30 organoids from each group were observed for 3–5 days by time-lapse microscopy, and the number of invasive organoids was quantified. Invasive organoids were divided into two groups based on the extent of their invasive protrusions into the surrounding collagen I gel: moderately invasive organoids (invasive protrusions 20–50 um) and extensively invasive organoids (invasive protrusions >50 um). Organoids with no protrusions or protrusions less than 20um were considered non-invasive. Protein expression in the treated organoids was assessed by immunofluorescence (CK, VIM – see details above) and Western blot (SMAD4 [1:50, #sc-7966, Santa Cruz], ECAD [1:1000, #3195, Cell Signaling Technologies], VIM [1:1000, #D21H3, Cell Signaling Technologies], total SMAD2 [1:500, #5339S, Cell Signaling Technologies], p-SMAD2 [1:500, #3108S, Cell signaling Technologies]) using established protocols. Secondary antibodies for Western blot included anti-Mouse IgG-HRP (1:10000, #NA931V, GE Healthcare) and anti-Rabbit IgG-HRP (1:10000, #NA934V, GE Healthcare). Proliferation and apoptosis of modified organoids were assessed by CellTiter-Glo® 3D Cell Viability Assay (Promega) and CellTiter-Glo® caspase 3/7 Assay (Promega) according to manufacturer’s instructions. We also treated passaged unmodified organoids of both SMAD4 genotypes with TGFβ1 (5ng/mL) and inhibitors of RAC1 (20uM, #553508, Calbiochem), CDC42 (50uM, #500503, Calbiochem), and ROCK1 (which inhibits signaling downstream of RHOA) (25uM, #688002, Calbiochem). After transfer from Matrigel to collagen I and treatment with TGFβ1 +/− inhibitors, 30 organoids of each group were observed by time-lapse microscopy for approximately 3 days to assess invasion. We analyzed activity of RAC1 and CDC42 by GTPase pull-down assay (Thermo Scientific) following TGFβ1 and inhibitor treatment. Additional details are provided in the Supplementary Methods .
Show full methods section
Organoids were isolated from 48 primary human PDAC samples acquired from surgical resection specimens at The Johns Hopkins Hospital – these included 25 PDAC samples (PCO1-PCO25) for immediate invasion analysis and 23 PDAC samples (PCO26-PCO48) for additional analyses, including immunofluorescence, lentiviral transduction, and pharmacological manipulation. This study was approved by the Institutional Review Board of The Johns Hopkins Hospital, and written informed consent was obtained from patients prior to sample acquisition. Organoids from 25 patients were isolated from fresh primary tumor samples as previously described ( 14 ). Briefly, PDAC tissue was digested and filtered; organoids were isolated by differential centrifugation. PDAC organoids were embedded into collagen I gels for time lapse imaging using a Nikon Eclipse Ti inverted microscope, with images collected at 30 minute intervals for 3–7 days. Immunofluorescence assays were used to assess expression of CK (1:100, #4545, Cell signaling Technologies), VIM (1:100, #5741, Cell signaling Technologies), SOX9 (1:100, #D8G8H, Cell signaling Technologies), Nkx6.1 (1:100, BDB563022, Fisher scientific), and GATA4 (1:100 #560327, BD Biosciences) in a subset of these organoids using established protocols with secondary antibodies coupled to Alexa Flour 488, 568 or 647 (1:1000, (A11001, A10042, A21235, Invitrogen). Primary PDAC tissue from these cases was analyzed by immunohistochemistry for SMAD4 (clone RBT-SMAD4, predilute; Bio SB), ECAD (clone EP700Y, predilute; Cell Marque), VIM (clone V9, predilute; Roche), P40 (clone BC28, 1:100; Biocare), and GATA6 (R&D Cat. Number AF1700,1:1000) using established protocols. We also isolated genomic DNA from the primary PDACs in our cohort for targeted sequencing of 11 PDAC driver genes using Ion AmpliSeq library preparation with a previously described primer panel ( 19 ), followed by sequencing on an Ion Torrent PGM. For re-expression of SMAD4, the SMAD4 coding sequence was subcloned into pCW57.GFP-P2A-MCS for doxycycline-inducible expression, and lentivirus was produced from HEK-293T cells using the pLKO.1 lentiviral vector protocol. For lentiviral transduction, organoids from 3 patients were passaged in Matrigel and then digested into single cells, transduced, and re-established in Matrigel, followed by puromycin selection three days after transduction. To characterize the invasion of modified organoids, the organoids were transferred from Matrigel to collagen I and treated with doxycycline (5mg/L) for 3 days to induce the expression of SMAD4 followed by TGFβ1 treatment (5ng/mL) for an additional 3 days. Then, 30 organoids from each group were observed for 3–5 days by time-lapse microscopy, and the number of invasive organoids was quantified. Invasive organoids were divided into two groups based on the extent of their invasive protrusions into the surrounding collagen I gel: moderately invasive organoids (invasive protrusions 20–50 um) and extensively invasive organoids (invasive protrusions >50 um). Organoids with no protrusions or protrusions less than 20um were considered non-invasive. Protein expression in the treated organoids was assessed by immunofluorescence (CK, VIM – see details above) and Western blot (SMAD4 [1:50, #sc-7966, Santa Cruz], ECAD [1:1000, #3195, Cell Signaling Technologies], VIM [1:1000, #D21H3, Cell Signaling Technologies], total SMAD2 [1:500, #5339S, Cell Signaling Technologies], p-SMAD2 [1:500, #3108S, Cell signaling Technologies]) using established protocols. Secondary antibodies for Western blot included anti-Mouse IgG-HRP (1:10000, #NA931V, GE Healthcare) and anti-Rabbit IgG-HRP (1:10000, #NA934V, GE Healthcare). Proliferation and apoptosis of modified organoids were assessed by CellTiter-Glo® 3D Cell Viability Assay (Promega) and CellTiter-Glo® caspase 3/7 Assay (Promega) according to manufacturer’s instructions. We also treated passaged unmodified organoids of both SMAD4 genotypes with TGFβ1 (5ng/mL) and inhibitors of RAC1 (20uM, #553508, Calbiochem), CDC42 (50uM, #500503, Calbiochem), and ROCK1 (which inhibits signaling downstream of RHOA) (25uM, #688002, Calbiochem). After transfer from Matrigel to collagen I and treatment with TGFβ1 +/− inhibitors, 30 organoids of each group were observed by time-lapse microscopy for approximately 3 days to assess invasion. We analyzed activity of RAC1 and CDC42 by GTPase pull-down assay (Thermo Scientific) following TGFβ1 and inhibitor treatment. Additional details are provided in the Supplementary Methods .
Supplementary Material 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
📊 Figures
Figure 1.
Expression of cytokeratin, vimentin, and SOX9 in human PDAC organoids and pancreatic tissue.
(A) Immunofluorescence co-staining of PDAC organoids for cytokeratin (green) and SOX9 (red) reveals that even cytokeratin-negative organoids express SOX9. Scale bars, 25 um. Images show two representa...
Figure 2.
Pattern of invasion is associated with clinical outcome and SMAD4 mutation.
(A) There was no difference in pattern of invasion or VIM expression between patients who did or did not receive neoadjuvant chemotherapy prior to surgical resection. (B) Representative images of prim...
Figure 3.
Re-expression of SMAD4 leads to mesenchymal invasion in presence of TGFu03b2.
(A-B) Representative images showing morphology of unmodified (A) and lentivirally transduced (B) SMAD4- mutant PDAC organoids cultured in Matrigel. (C) Representative images of modified SMAD4 -mutant ...
Figure 4.
RAC1 and CDC42 activation is required for TGFu03b2-induced collective invasion in SMAD4 -mutant PDAC organoids.
(A) Representative images of organoids from a SMAD4- mutant PDAC cultured in collagen I and treated with TGFu03b2 and/or RAC1, ROCK1, or CDC42 inhibitors. (B-C) Relative invasion of organoids from a S...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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