Kartheek Dokka=Chap23


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

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Kartheek Dokka=Chap23

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