Kartheek Dokka=Chap23
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Molecular Biology of The Cell : Chapter 23 and a Science PAper related to the Chapter.

Molecular Biology of The Cell : Chapter 23 and a Science PAper related to the Chapter.

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  • 1. Identification of Cells initiating Human Melanoma 17-January-08 Nature-06489 Kartheek Dokka
  • 2. The Target!
    • Tumor initiating cells from:
      • Human hematological malignancies
      • Solid Cancer
    • Human Malignant Melanoma Initiating Cells [MMIC]
      • Defined by expression of Chemo-resistance Mediator “ABCB5”
      • ATP Binding Cassette subfamily B [ABC Transporter]
      • Trans-membrane Protein
      • Molecular Marker
    • Specific Targeting of MMIC inhibits Tumor Growth
    MMIC ABCB5
  • 3. Relation of ABCB5 with malignant melanoma
    • ImmunoHistoChemical staining of an established Melanoma Progression Tissue MicroArray :
      • A : Benign Melanocytic Nevi [Birth marks/moles]
      • B : Primary Cutaneous Melanoma
      • C : Metastases to Lymph Nodes
      • D : Metastases to Viscera
    • ABCB5 closely associated with CD166
    • [marker of advanced disease]
    • Expression of ABCB5:
    • B,C,D > A
    • Thick B > Thin B
    • C > D
    A B D C green orange violet blue pink yellow
  • 4. Expression of other markers/identifiers
    • 7 Specimen [melanoma patients] were taken and when assayed in their single-cell suspension , ABCB5 was consistently expressed in all the 7.
    • CD20 - 4/7
    • NESTIN - 7/7
    • TIE 1 - 7/7
    • CD144 - 5/7
    • BMPRIA - 7/7
    • CD31 - 6/7
    • Comparing expression levels with ABCB5+
    • and ABCB5 ¯[4-7 patients]
    • Controls:
    • CD20 and CD31 are not Expressed hence are “ negative controls”
    • Positive Control : Marker expressing in last stage of metastases can be used.
  • 5. Subset defined by ABCB5
    • They showed that ABCB5 was expressed in all stages of the melanoma and also affected the expression of other markers.
    • To compare the abilities of ABCB5+ versus ABCB5- melanoma cells to initiate tumor formation in-vivo….
  • 6. ABCB5+ vs ABCB5 ¯ 1 ° patient tumor cells NOD/SCID mice XenoTransplants ABCB5+ ABCB5- Unsegregated Repurification Secondary Tumor Formation Total: US -7/23, + :14/23, - :1/23 + :10/18 - :0/18 Immuno-Magnetic Separation
  • 7. ImmunoHistoChemistry usage for Progression of ABCB5
    • Parent tumor: Primary Patient cells [human]
    • First Passage: Primary Xenograft with SCID mouse.
    • Second Passage: Secondary Xenograft shows the re-establishment of parent tumor heterogeneity
  • 8. Flow Cytometry readings for ABCB5 progression
    • ABCB5+ : DsRED[red fluoroscent protein]
    • ABCB5- : EYFP[Enhanced yellow-green fluoroscent protein]
    • Cell Inoculum: Initial contents of ABCB5 in the Xenotransplant cells
    • In-Vivo Tumor: After 6 weeks of XT: Dense growth of DsRED and reduced growth of EYFP.
    • Controls: Just the cells without any stains
    • Cells stained with only DsRED
    • Cells stained with EYFP
  • 9. Final Experiment with “nude mouse”
    • Administered mAb directed at ABCB5 in a human-nude mouse Xenograft.
    • Nude mice capable of ADCC[antibody dependent Cell mediated cytotoxicity] unlike SCID/NOD.
    • They administered non-specific mAb and also studied untreated mice.
    • Controls:
    • Positive: healthy nude mice cells.
    • Negative : Over Expressed ABCB5 mice cells
    • Graph:
    • Tumor formation low in cells treated with anti-ABCB5 mAb
    • Tumor in Isotype mAb almost same as in untreated.
  • 10. Final Experiment with “nude mouse”
    • Flow Cytometric Assessment of ADCC:
    • Anti-ABCB5 mAb: shows higher cytotoxicity [stained more in 3 rd (upper right) quadrant]
    • Isotype mAb: lower cytotoxicity
    • No Ab[untreated] : No toxicity.
    • Tumor volume calculated comparing with 1 st day and on 21 st day:
    • The tumor is seen controlled in Anti-ABCB5 mAb treated.
    • The tumor is higher in Isotype control mAb
    • The tumor is high in untreated.
  • 11. Welcome to K-Lab!
    • My Experiment 1 : July-13 th -2021
    • Time: 6:00 AM
    • Took 3 humans with melanoma. 2 healthy Humans. 10 NUDE mice.
    • Made some Primary sample of melanoma cells.
    • Using RNAi technique, I silenced the gene expressing ABCB5.
    • XenoTransplanted these cells with NUDE mice cells.
    • Using flowCytometry, I checked for Tumor formation in the xenotransplants.
  • 12. My results! July 13-2021 Time: 11:30pm
    • Xenotransplants without the RNAi
    • -Since expression not suppressed, gradual
    • growth of ABCB5 as Time moves on forming
    • metastases.
    Tumor Formation Time Tumor Formation Time Xenotransplants with RNAi - As the gene was silenced, tumor formation was suppressed for 3 hours and as the ABCB5 was re-made, tumor was induced. Controls: Positive: Healthy Human cells Negative: Melanoma cells of primary human patients.
  • 13. Experiment 2: Chemical Carcinogen Patch
    • Took 35 NOD/SCID mice:
    • Group A : Knocked out the gene expressing ABCB5 in 10.
    • Group B: Transfected 10 mice with genes over-expressing ABCB5.
    • Group C : 10 mice got lucky and got one patch only. [Used as a control]
    • Applied a Chemical Carcinogen Patch [Dimethylanthracene] on the bellies of Mice Groups A and B. Did it for 5 days forming layers of patch.
    • Made a biopsy of the 3 groups everyday for 24 days and observed the cell-growth.
    • Controls:
    • Positive: 5 Untreated SCID mice cells
    • Negative: cells from last stage of metastases super expressing ABCB5
    15 th July 2021-3 rd August 2001 9:00Am 10:35pm Tumor Formation Tumor Formation Tumor Formation Time [days] Time [days] Time [days]
  • 14. References
    • http://melanoma.blogsome.com/wp-admin/images/fig9a.jpg
    • http://www.melanoma.ca/images/melanocyte4.gif
    • http://infotrek.er.usgs.gov/mercury/images/lab.jpeg
    • http://www.icq.com/img/friendship/usercreated/static/card_408_r.jpg
    • http://www.nature.com/nature/journal/v451/n7176/extref/nature06489-s1.pdf