Advanced Development and Validation of 3D Spheroid Culture of...

Advanced Development and Validation of 3D Spheroid Culture of Primary Cancer Cells using Nano3D Technology

This work is supported by the NCI IMAT Award R33 CA206949

Assist. Prof. Timothy Spicer- Discovery Biology and [email protected] Tel: (561) 228-2150

http://hts.florida.scripps.edu/

December 7th 2017

Poster#8

TSRI © 2017. All rights reserved.

• Screening in 2D formats has left us wanting for more relevant and physiologically appropriate assays-Hanging drop and other low throughput assays exist but aren’t effective to rapidly translate cost effective strategies to HTS

• Pancreatic cancer remains a leading cause of cancer deaths, with 5-year survival rate less than 5%.

• Genetic alterations are associated with pancreatic cancer, including oncogene (KRAS), tumor suppressors (TP53, CDKN2A, SMAD4, ARID1A and MLL3 ).

• We received the following cell lines:

Name Tissue Mutations

hM1-CAF a resected metastatic lung lesion, same as hM1 Kraswt (fibroblast, SV40 immortalized)

hM1 a resected metastatic lung lesion KrasG12D, p53R175H (epithelial-like)

hT1-CAF resected primary tumors, same as hT1 Kraswt (fibroblast, SV40 immortalized)

hT1 resected primary tumors KrasG12V, P53loss, SMAD4loss, CDKN2Ahom del((epithelial-like)

hM1-CAF/hM1, hT1-CAF/hT1 are CAF/cancer pairs derived from the same tissue.


Figure 1: Greiner cell repellent plates readily form spheroids using cells combined with Nanoshuttle. This can be done on thebottom side of the plates using a magnetic driver. Note that the shape of the bottom of the wells is compatible with automatedconfocal microscopy.

Incubation hours

Detach cells

384 well format 1536 well format


Condition Outcome

No cells No ATP produced – no signal

Cells-Vehicle More ATP production – High signal

Cells-Cytotoxiccompound Less ATP production – Low signal

The assay determines the number of viable cells based on the amount of ATP present. The amount of ATP is detected via CellTiter-Glo reagent involving enzymatic reactions

which generate a stable luminescent signal. The amount of ATP is proportional to the number of viable cells in culture.

A

B


3D: hT1-2D

-12 -11 -10 -9 -8 -7 -6 -5 -4 -30

150

300

450

600

750 OXADOXGEM5-FUSN-38

LogIC50HillSlope

IC50

OXA~ -2.275-0.9961~ 0.005303

DOX-5.947-1.2851.131e-006

GEM 5-FU~ -0.8444-0.7499~ 0.1431

SN-38~ 1.022-0.5791~ 10.53

Log[Compound], M

RLU

Z stack Confocal Image20X objective

2D: hT1-2D

-12 -11 -10 -9 -8 -7 -6 -5 -4 -30

500

1000

1500

2000

2500 OXADOXGEM5-FUSN-38

LogIC50HillSlope

IC50

OXA~ 1.367-0.3842~ 23.26

DOX-6.484-1.4713.282e-007

GEM-7.488-0.93163.249e-008

5-FU~ 8.392~ -0.2352~ 2.464e+008

SN-38-7.434-0.46573.685e-008

Log[Compound], M

RLU

4X objective

Cells in 2D monolayer and in 3D spheroid/organoid format demonstrated different responses to therapeutic compounds. Drug resistance was observed in 3D model. Complete optimization of all 4 cell lines in 3D format followed by drug testing and HTS

2D HTS 3D HTS


1250 cells per well

Condition: 1250 hT1 cells per well were seed in 1536 well plate (5uL), with 4hr printing time and read at 4 dayspost seeding

These conditions are robust- passed Z’. CRC of 5 controls in 1536 wpf is consistent with that in 384 wpf.

EC100: Medium + DMSO

EC100: cells + DMSO

%CV S/N Z Z'8.60 210.23 0.740 0.731

%CV S/N Z Z'25.26 244.22 0.238 0.639

n3D/1536: hT1 CRC

-12 -10 -8 -6 -40

500

1000

1500

2000

2500OXA

GEMDOX

5-FUSN-38

LogIC50HillSlope

IC50

OXA~ -6.702~ -13.71~ 1.986e-007

DOX-6.013-1.6069.705e-007

GEM~ 3.238-0.2045~ 1730

5-FU~ 0.7373-0.5561~ 5.461

SN-38-6.112-3.3367.732e-007

Log[Compound], M

RLU


Run Statistics (n = 6 plates)• 3290 compounds tested at 2.6 µM nominal concentration in duplicate• Z’ = 0.72±0.05• S:B = 184.94±13.41• Hit Cutoff (Interval cut-off) = 38.91%• Hits = 21 (0.64%)

High control is Medium only + DMSO; Low control is Cells + DMSO

This assay showed excellent Z’ values. Doxorubicin IC50 matched expected. Applying interval-based cutoff, 21 hits were identified in

this screen.

Hit Cutoff

n3D/1536: hT1 CRC

-12 -10 -8 -6 -40

300

600

900

1200

1500OXA

GEMDOX

5-FUSN-38

LogIC50HillSlope

IC50

DOX-6.065-1.9658.615e-007

GEM-5.582-1.3552.617e-006

5-FU~ -4.133~ -10.59~ 7.366e-005

SN-38-6.431-2.2633.708e-007

Log[Compound], M

RLU


The pancreatic cancer cells and CAFs are compatible with N3D technologies. CRC of control drugs are reproducible overtime and show differential response in 3D vs 2D HTS of ~3200 approved drugs in both 2D and 3D demonstrates marked difference in sensitivity

-10 -9 -8 -7 -6 -5-25

0

25

50

75

100

125

hT1-3DhT1-CAF-3DhM1-3DhM1-CAF-3DhT1-2DhT1-CAF-2DhM1-2DhM1-CAF-2D

Log[Disulfiram]

% In

hibi

tion

D

Drug3D assay 2D assay Resistance Factor

hT1 hT1-CAF hM1 hM1-CAF hT1 hT1-CAF hM1 hM1-CAF hT1 hT1-CAF hM1 hM1-CAFBortezomib 41.1E-9 59.3E-9 128.1E-9 38.5E-9 10.3E-9 10.E-9 16.2E-9 8.7E-9 4.0 5.9 7.9 4.4Carfilzomib 55.1E-9 83.4E-9 63.1E-9 74.3E-9 16.6E-9 10.3E-9 19.5E-9 10.2E-9 3.3 8.1 3.2 7.3Romidepsin 19.E-9 1.8E-9 46.E-9 3.5E-9 7.91E-10 5.38E-10 1.6E-9 2.4E-9 24.0 3.3 28.8 1.5

Homoharringtonine 3.34E-7 1.75E-7 1.56E-7 1.41E-7 1.82E-7 19.2E-9 16.7E-9 10.3E-9 1.8 9.1 9.3 13.7Trametinib 4.7E-9 1.4E-6 16.5E-9 2.8E-6 >5.0E-6 >5.0E-6 2.7E-9 >5.0E-6 <0.0009 <0.3 6.1 <0.6Disufliram 2.1E-7 >6.58E-6 9.9E-8 >6.58E-6 >6.58E-6 >6.58E-6 3.1E-7 2.0E-7 <0.03 / 0.3 >32.9

E

Approved Drugs vs. hT1

-100 -50 0 50 100-100

-50

0

50

100

% Response (2D)

% R

espo

nse

(3D

)

I

II

III

Approved Drugs vs. hT1-CAF

-100 -50 0 50 100-100

-50

0

50

100

% Response (2D)

% R

espo

nse

(3D

)

I

II

III

Approved Drugs vs. hM1

-50 0 50 100-50

0

50

100

% Response (2D)

% R

espo

nse

(3D

)

I

II

III

Approved Drugs vs. hM1-CAF

-100 -50 0 50 100-100

-50

0

50

100

%Response (2D)

%R

espo

nse

(3D

)

I

II

III

F


Mechanical and Automation testing • Plate stability with Carousel rotation (384w and 1536w) • Robotic gripper plate handing• Testing of automated plate placement and removal

Robotic Gripper can flawlessly place plates onto driver• Magnet alignment appears correct• No positioning errors induced with

carousel rotation.• Metal lid prevents lateral movement of

plate.


Using hT1-CAF cells, 1536 drive V3.0 achieved similar CRC of controls as the offline test. Spinning of plates (1250rpmX5min) after dispense CTG suffices in 1536w test.

n3D/1536: hT1-CAF CRC (offline)

-12 -10 -8 -6 -40

200

400

600

800

1000OXA

GEMDOX

5-FUSN-38

LogIC50HillSlope

IC50

OXA-0.4305-1.0980.3711

DOX-6.668-1.4662.147e-007

GEM-7.241-0.90085.740e-008

5-FU12.70-0.28574.960e+012

SN-38-8.124-0.74087.522e-009

Log[Compound], M

RLU

n3D/1536: hT1-CAF CRC (Online V3.0)

-12 -10 -8 -6 -40

400

800

1200

1600OXA

GEMDOX

5-FUSN-38

LogIC50HillSlope

IC50

OXA-7.766-1.2911.712e-008

DOX-6.708-1.6611.957e-007

GEM-7.601-0.80932.505e-008

5-FU~ -0.2451~ -1.069~ 0.5688

SN-38-8.476-0.72813.338e-009

Log[Compound], M

RLU

S/N Z Z'255.1 -0.37 0.71

S/N Z Z'238.2 -0.44 0.56

Condition Z’ Conclusion

Before spin 0.608+0.08 Shaking may be necessaryfor 384w test, but not in 1536w;

Spin is necessary to remove bubbles, and homogenize the signal.

After spin 0.627+0.08

After shaking 0.622+0.07

384w

1536w

before spin

after spin after shaking


This work is supported by the NCI IMAT Award R33 CA206949

1. Madoux F, Tanner A, Vessels M, Willetts L, Hou S, Scampavia L, Spicer T. A 1536-Well 3D Viability Assay to Assess the Cytotoxic Effect of Drugs on Spheroids. SLAS Discovery. 2017 Jan. PMID:28346088

2. Singhera F, Cooper E, Scampavia L, Spicer T. Using bead injection to model dispensing of 3-D multicellular spheroids into microtiter plates. Talanta. 2017 Sep.

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