Identification of Cells initiating Human Melanoma17-January-08 Nature-06489

Kartheek Dokka

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

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 DC

green

orangeviolet blue pink

yellow

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.

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….

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

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

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

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.

Final Experiment with “nude mouse”

• Flow Cytometric Assessment of ADCC:Anti-ABCB5 mAb: shows higher cytotoxicity [stained more in 3rd(upper right) quadrant]Isotype mAb: lower cytotoxicityNo Ab[untreated] : No toxicity.• Tumor volume calculated comparing with 1st day and on 21st day: The tumor is seen controlled in Anti-ABCB5 mAb treated.

The tumor is higher in Isotype control mAbThe tumor is high in untreated.

Welcome to K-Lab!

My Experiment 1 : July-13th-2021Time: 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.

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.

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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 cellsNegative: Melanoma cells of primary human patients.

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 cellsNegative: cells from last stage of metastases super expressing ABCB5

15th July 2021-3rd August 2001

9:00Am 10:35pm

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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