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Wu hypoxia

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Hypoxia and ctc

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Wu hypoxia

  1. 1. Hypoxia and Circulating Tumor Cells in Pancreatic Cancer Douglas Wu Lambowitz lab
  2. 2. Pancreatic cancer http://i.usatoday.net/yourlife/_photos/2011/10/05/Jobs-battle-with-pancreatic-cancer-RRESHAC-x-large.jpg - Low survival rate - Highly metastatic 0 5 10 15 20 15−49 50−59 60−69 70−79 80−99 Age group 5−yearsurvivalrate(%) Men Women
  3. 3. Figure 1 Clinically end prod events, w metastas sion, tum (local inv temically distant o survive a ments of tion, met depicted tive inv tumor c program and int invasion stress-fi Rho/RO sion’’ p Metastasis Valastyan and Weignburg, Cell. 2013
  4. 4. Circulating tumor cells
  5. 5. http://www.verastem.com/research/ http://www.eurostemcell.org/de/node/16637 Cancer stem cells
  6. 6. Epithelial-mesenchymal transition promotes invasion Peinado, et al. Nat Rev. 2007 http://blogs.scientificamerican.com/guest-blog/ 2013/10/30/the-hallmarks-of-cancer-6-tissue- invasion-and-metastasis/
  7. 7. Hypoxic environment promotes EMT Peinado & Cano. Nature Cell Biology. 2008 http://cascadeprodrug.com/main/img_1271875515_14862_1283460123_mod_618_316.jpg
  8. 8. Peinado & Cano. Nature Cell Biology. 2008 Hypoxic environment promotes EMT Normoxia HIF1-a Hydroxyprolination Ubiquitination by VHL Proteasome Hypoxia HIF1-a Hydroxyprolination inhibited No ubiquitination by VHL Transcription
  9. 9. Hypoxic environment promotes CTC production, intravasation and tumorigenicity Hypothesis
  10. 10. Hypothesis: Hypoxic environment promotes CTC production, intravasation and tumorigenicity Challenge: No clear definition of CTCs Single-Cell RNA Sequencing Identifie Matrix Gene Expression by Pancreat Cells Graphical Abstract Highlights Pancreatic CTCs can be enriched with antigen-agnostic micro- fluidic technology Single-cell RNA sequencing of pancreatic CTCs reveals three distinct CTC populations Extracellular matrix genes are highly expressed in mouse and human CTCs The extracellular matrix protein SPARC contributes to pancre- Au Da ma Co ma (S. ha In Cir ric bu de flu se fyi ge ca ex ric to Ac GS GS GS Ting et al.. Cell Reports. 2014 Figure 1. CTC Single-Cell Isolation (A) Schematic of the CTC-iChip-negative inertial focusing device system. (B) Mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion is shown in log10. (C) CTC enumeration by immunofluorescent staining (CK+/CD45-/DAPI+) from normal and cancer mice. Bar represents mean. (D) Representative image of CK-positive CTCs. DAPI (blue), CK (red), and CD45 (green). Scale bar, 20 mm. Bright-field image highlighting lack of im- munomagnetic anti-CD45 beads on CK+ CTCs (white circle). ECM genes by CTCs may contribute to the dissemination of cancer to distal organs. RESULTS CTC-iChip
  11. 11. 0 25 50 75 100 Count Subtype CTC EMT+ CTC platelet CTC epithelial Ting et al.. Cell Reports. 2014 CTC heterogeneity Hypothesis: Hypoxic environment promotes CTC production, intravasation and tumorigenicity Article Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells Graphical Abstract Highlights Pancreatic CTCs can be enriched with antigen-agnostic micro- Authors David T. Ting, Ben S. Wittner, ..., Shya- mala Maheswaran, Daniel A. Haber Correspondence maheswaran@helix.mgh.harvard.edu (S.M.), haber@helix.mgh.harvard.edu (D.A.H.) In Brief Circulating tumor cells (CTCs) are en- riched for the precursors of metastasis, but their composition has not been fully defined. Ting et al. have utilized a micro- fluidic device to perform single-cell RNA sequencing of pancreatic CTCs, identi- fying three distinct populations that sug- gest multiple paths in the metastatic cascade. Extracellular matrix gene expression in particular was highly en- riched in CTCs, pointing to a contribution to distal spread of cancer. Accession Numbers GSE51372
  12. 12. p−value = 0.000382 Hif1a 0 30 60 90 normalizedmeancount annotation circulating tumor cells (n=109) primary tumor (TuGMP3) (n=38) HIF1-a mRNA is more abundant in CTCs Yasuda et al. BBRC. 2014 • HIF1-a can be post-transcriptionally regulated through its 3’ UTR Thanks to Pubmed’s GEO Datasets!
  13. 13. 1. Investigate up-regulation of molecules in the hypoxia signaling pathway in CTCs. 2. Identify the effects of hypoxia on CTC biogenesis. 3. Investigate hypoxia-mediated enhanced tumorigenicity of CTCs. Specific aims Hypothesis: Hypoxic environment promotes CTC production, intravasation and tumorigenicity
  14. 14. Hypoxia signaling is up-regulated in CTCs Hypothesis Aim 1: Investigate up-regulation of molecules in the hypoxia signaling pathway.
  15. 15. Aim 1: Investigate up-regulation of molecules in the hypoxia signaling pathway. • KPC mice A R T I C Hingorani , et al. CANCER CELL. 2005 Tumor model
  16. 16. Ting et al.. Cell Reports. 2014 http://www.cancerresearchuk.org/prod_consump/groups/ cr_common/@cah/@gen/documents/image/ To achieve deep RNA-sequencing profiles of CTCs at the single-cell level, we applied an inertial focusing-enhanced micro- fluidic device, the CTC-iChip, which allows high-efficiency nega- tive depletion of normal blood cells, leaving CTCs in solution where they can be individually selected and analyzed as single cells (Ozkumur et al., 2013). This antigen-agnostic isolation of CTCs enables the characterization of CTCs with both epithelial and mesenchymal characteristics. Further, the high quality of Figure 1. CTC Single-C (A) Schematic of the CTC focusing device system. (B) Mouse WBC depletion normal and cancer mouse is shown in log10. (C) CTC enumeration b staining (CK+/CD45-/DAP cancer mice. Bar represen (D) Representative image DAPI (blue), CK (red), and C 20 mm. Bright-field image munomagnetic anti-CD45 (white circle). ECM genes by CTCs the dissemination of organs. RESULTS Isolation of Mouse P The CTC-iChip combi namic size-based sep ated cells (leukocytes away from red bloo and plasma, with s focusing of the nuclea gle streamline to achi in-line magnetic sor epitopes are highly v surface markers are applying magnetic-co to this very high-throu cell-separation device the vast majority of small number of untagged CTCs (Figure 1A). ing using 100 anti-CD45 beads per WBC ac tion in normal mice, mice bearing orthotop KPC mice (Figure 1B). We first tested the efficacy of the CTC-i tagged mouse PDAC cell line (NB508). CTC the CTC-iChip was measured to be 95% (m ing GFP-tagged NB508 cells spiked into w 1. Immunofluorescent staining 2. Hypoxia microarray Aim 1: Approach
  17. 17. a he er or as o he re e. Hypoxia-Induced EMT in Pancreatic Cancer Aim 1: Immunofluorescence stain Salnikov, et al. PLoS One. 2013
  18. 18. Salnikov, et al. PLoS One. 2013 Aim 1: Immunofluorescence stain
  19. 19. Expected results in CTCs Vimentin HIF1-a DAPI Aim 1: Immunofluorescence stain
  20. 20. 1. Immunofluorescence staining shows HIF1-a in CTCs 2. Downstream targets also up-regulated Aim 1: Investigate up-regulation of molecules in the hypoxia signaling pathway. Predictions
  21. 21. HIF1-a is a master regulator in hypoxia signaling HIF1-a Survival - bcl2 - Vegfa Proliferation - Igf2 Immmunosuppress - Mif - Il10 Metabolism - Ldha - Pdk1 Motility - Met - Mmp Angiogenesis - Vegfa EMT - Snai2 - Twist1 ECM modulation - Mmp
  22. 22. Single cell RNA-seq of CTCs had active hypoxia signaling Downstream targets of HIF1-a are also up-regulated p−value = 0.0436 p−value = 0.00225 p−value = 0.256 p−value = 0.000142 p−value = 0.00254 p−value = 0.00145 p−value = 1.72e−05 p−value = 0.0195 p−value = 0.0315 p−value = 0.192 p−value = 0.00205 p−value = 1.03e−08 Bcl2 Igf2 Il10 Ldha Met Mif Mmp14 Pdk1 Snai2 Twist1 Vegfa Vim 0 100 200 300 0 25 50 75 100 0 1 2 3 4 5 0 1000 2000 3000 0 100 200 300 400 0 200 400 600 0 500 1000 1500 0 25 50 75 0 2 4 0 1 2 0 1 2 3 0 2500 5000 7500 10000 normalizedmeancount annotation circulating tumor cells (n=109) primary tumor (TuGMP3) (n=38)
  23. 23. MP2.1 MP2.11MP2.17 MP2.18MP2.2 MP2.20 MP2.21MP2.24 MP2.26MP2.30 MP2.32 MP2.36 MP2.4MP3.15MP3.17 MP3.2 MP3.21MP3.3MP3.5MP3.8MP3.9MP4.1MP4.13MP4.14MP4.17MP4.20MP4.22 MP4.24 MP4.28MP4.29MP4.3MP4.31MP4.32MP4.4MP4.6MP4.7MP4.8MP6.10 MP6.11 MP6.15 MP6.16 MP6.17 MP6.18 MP6.19MP6.2MP6.20MP6.21MP6.3MP6.4MP6.5 MP6.6 MP6.7 MP6.9 MP7.1 MP7.12 MP7.13 MP7.16 MP7.18 MP7.20MP7.21 MP7.25 MP7.29 MP7.3 MP7.30MP7.31 MP7.33MP7.34MP7.37MP7.4 MP7.40 MP7.41MP7.42 MP7.7 MP7.8MP7.9 TuGMP3.1.051413TuGMP3.14.051413TuGMP3.15.051413TuGMP3.16.051413TuGMP3.17.051413TuGMP3.19.051413TuGMP3.2.051413TuGMP3.20.051413TuGMP3.22.051413TuGMP3.24.051413TuGMP3.25.051413TuGMP3.26.051413TuGMP3.31.051413 TuGMP3.34.051413 TuGMP3.35.051413 TuGMP3.4.051413 TuGMP3.5.051413TuGMP3.6.051413TuGMP3.7.051413TuGMP3.8.051413 Bcl2 Igf2 Il10 Ldha Met Mif Mmp14 Pdk1 Snai2 Twist1 Vegfa Vim −4 0 4 0.0 2.5 5.0 7.5 10.0 PC1 PC2 Cell type a a circulating tumor cells primary tumor (TuGMP3) Single cell RNA-seq of CTCs had active hypoxia signaling Biplot: HIF1-a downstream targets introduce variations between CTCs and primary tumor cells
  24. 24. Summary • CTCs show EMT markers with hypoxia phenotype • Downstream targets of HIF1-a up- regulated in CTCs Aim 1: Investigate up-regulation of molecules in the hypoxia signaling pathway.
  25. 25. 1. Investigate up-regulation of molecules in the hypoxia signaling pathway in CTC. 2. Identify the effects of hypoxia on CTC biogenesis. 3. Investigate hypoxia-mediated enhanced tumorigenicity of CTCs. Specific aims Hypothesis: Hypoxic environment promotes CTC production, intravasation and tumorigenicity
  26. 26. Loss of HIF1-a reduces CTCs production Hypothesis Aim 2: Identify the effects of hypoxia on CTC biogenesis
  27. 27. HIF1a knockdown MIA PaCa-2 cells ! Xenograft Figure 1. CTC Single-Cell Isolation (A) Schematic of the CTC-iChip-negative inertial focusing device system. (B) Mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion is shown in log10. (C) CTC enumeration by immunofluorescent staining (CK+/CD45-/DAPI+) from normal and cancer mice. Bar represents mean. (D) Representative image of CK-positive CTCs. DAPI (blue), CK (red), and CD45 (green). Scale bar, 20 mm. Bright-field image highlighting lack of im- munomagnetic anti-CD45 beads on CK+ CTCs (white circle). 1. Single cell RNA-seq 2. CTC count 3. Tumor Size Schwab et al. Breast Cancer Research 2012 Yang and Kang. Yonsei Med J. 2008 MIA PaCa-2 cells Aim 2: Approach
  28. 28. Schwab et al. BCR. 2012 • Tumor size • correlate to previous study Percenttumors<500 mm3 log-rank p<0.0001 median WT, 64 days KO, 127 days WT KO Tumorvolume,mm3 KO WT A Tumorvolume,mm3 Tumorweight,g p=0.0091 * Tumorburden,%BW p=0.0343 * WT WT WTKO KO KO p=0.0068 B C ab et al. Breast Cancer Research 2012, 14:R6 /breast-cancer-research.com/content/14/1/R6 Page 10 of Aim 2: Predictions HIF1-a knockdown cells tumor xenograft shows slower growth rate
  29. 29. Published Expected 0 25 50 75 100 Count Subtype CTC EMT+ CTC platelet CTC epithelial • CTC count • Decrease in HIF1-a knockout • Change in CTC subtypes • Decrease in CTC EMT+ subtype Aim 2: Predictions Mice implanted with HIF1-a knockdown cells have fewer CTCs
  30. 30. • Tumor size decreases • Fewer CTCs • Change in CTC count is due to loss of EMT+ subtype Summary Aim 2: Identify the effects of hypoxia on CTC biogenesis
  31. 31. 1. Investigate up-regulation of molecules in the hypoxia signaling pathway in CTC. 2. Identify the effects of hypoxia on CTC biogenesis. 3. Investigate hypoxia-mediated enhanced tumorigenicity of CTCs. Specific aims Hypothesis: Hypoxic environment promotes CTC production, intravasation and tumorigenicity
  32. 32. Aim 3: Investigate hypoxia-mediated enhanced tumorigenicity of CTCs. Loss of HIF1-a reduces CTC tumorigenicity Hypothesis
  33. 33. ! Xenograft Figure 1. CTC Single-Cell Isolation (A) Schematic of the CTC-iChip-negative inertial focusing device system. (B) Mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion is shown in log10. (C) CTC enumeration by immunofluorescent staining (CK+/CD45-/DAPI+) from normal and cancer mice. Bar represents mean. (D) Representative image of CK-positive CTCs. DAPI (blue), CK (red), and CD45 (green). Scale bar, 20 mm. Bright-field image highlighting lack of im- munomagnetic anti-CD45 beads on CK+ CTCs (white circle). ! Xenograft Serial dilution Aim 3: Approcah HIF1a knockdown MIA PaCa-2 cells MIA PaCa-2 cells
  34. 34. ! Xenograft Figure 1. CTC Single-Cell Isolation (A) Schematic of the CTC-iChip-negative inertial focusing device system. (B) Mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion is shown in log10. (C) CTC enumeration by immunofluorescent staining (CK+/CD45-/DAPI+) from normal and cancer mice. Bar represents mean. (D) Representative image of CK-positive CTCs. DAPI (blue), CK (red), and CD45 (green). Scale bar, 20 mm. Bright-field image highlighting lack of im- munomagnetic anti-CD45 beads on CK+ CTCs (white circle). Serial dilution Aim 3: Approcah HIF1a knockdown MIA PaCa-2 cells MIA PaCa-2 cells 3D soft fibrin matrix assay / invasive assay
  35. 35. C GAPDH TRC 1 day 3 days 5 days 7 days * * Control 800 700 600 500 Colonysize(×103µm3) 400 300 200 100 0 1 2 Culture time on plastic (day) 0 0 0.05 Cellstiffness(kPa) 0.1 0.15 0.2 0.25 3 4 5 Rel 6 4 2 0 ition of Sox2 expression and self-renewal of TRCs on 2D rigid subs Aim 3: Investigate hypoxia-mediated enhanced extravasation of CTCs. Alternative: 3D soft fibrin matrix assay Tan, et al. Nature Comm. 2014
  36. 36. • Decrease in tumorigenicity from CTCs collected from mice implanted with HIF1- a knockdown MIA PaCa-2 cells Aim 3: Expected result
  37. 37. Hypoxia HIF1-a EMT Macroscopic Metastasis Summary Modified Peinado & Cano. Nature Cell Biology. 2008

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