Akoya Biosciences, Inc., 68 Elm Street, Hopkinton, MA USA (855) 896-8401 www.akoyabio.com

D a y 2 D a y 1 0

0

21 0 1 0

41 0 1 0

61 0 1 0

A S 7 5 0 tu m o r s ig n a l

Ra

dia

nt

Eff

icie

nc

y

C o n tro l

S u lin d a c

Tumor Blood Vessel

LeakinessBar: s.e.m.

n = 8

P < 0.0001

n = 5

P = 0.0007

A Fully-Automated Multiplex Fluorescence IHC Assay with Whole Slide Multispectral Imaging on Mouse Tissue: Phenoptics™ Quantitative Pathology Solutions Translational Workflow

Yi Zheng, Carla Coltharp, Rachel Schaefer, Linying Liu, Ryan Dilworth, Victoria Duckworth, William Kennedy, Darryn Unfricht, Jen-Chieh Tseng, Jeffrey D. Peterson, Milind Rajopadhye, Peter Miller

Background1

Methods2

ConclusionsDeveloping biomarker research strategies for clinically relevant therapies in immuno-oncology (IO) is predicated on the ability to execute fully translational research studies. Here, we demonstrated whole slide analysis utilizing the novel Opal Polaris 7-Color assay plus Opal Polymer anti-Rabbit HRP. The results showed unique tumor vascular signatures and immune context heterogeneity between tumors as well as within tumors. This new application has expanded Phenoptics into whole slide IO tissue research including animal studies, creating a truly translational platform.

4

Results:3

Mouse Breast Cancer Model

BOND RX is for Research Use Only. Not For Use In Diagnostic Procedures.LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH.BOND is a trademark of Leica Biosystems Melbourne Pty Ltd.

Fig 2. Whole Slide Scan of Opal Polaris 7-Color Fluorescent IHC Assay on Mouse Breast Cancer FFPE Section. A) Whole slide MSI of mouse breast cancer section from control group (top) and 10-day Sulindac treatment group (bottom). Functional classes represented in the multi-marker panel include: Proliferative cells (Ki67), Tumor infiltration lymphocytes (CD4 & CD8), Tumor associated macrophage (F4/80), Angiogenesis (CD31) & Anti-apoptotic cells (Bcl-2). Component planes for each individual marker from the whole slide scan from control group (B) and Sulindac treated group (C).

Phenoptics™ quantitative pathology solutions

(QPS) is a comprehensive, end-to-end solution consisting of multiplex fluorescence immunohistochemistry staining along with multispectral imaging and tissue analysis.

Here we describe a robust, fully-automated 7-color Opal staining procedure on Formalin-fixed paraffin-embedded (FFPE) section of mouse breast cancer followed by a multispectral whole slide scan and image analysis.

BALB/c @6 weeks

4T1 mouse BrCacell line injected on

Day -8

Day 0

10-day treatment of Sulindac

Control

• In-vivo Imaging

• Opal™ Multiplex IHC

Day 10

Fig 1. Mouse Breast Cancer Model. BALB/c mice (6 weeks old from Charles River lab) were injected subcutaneously with 1 × 106 4T1 tumor cells in the

right flank. Eight days after inoculation, tumor-bearing mice were treated intraperitoneally with or without with 60 mg/kg Sulindac once daily for

consecutive 10 days.

Mouse in-vivo Imaging

Opal Polaris 7-Color Assay &

Multispectral imaging on Vectra Polaris

Ten days after Sulindac treatment, tumors from both untreated and treated group were collected then fixed with Formalin for processing to formalin fixed paraffin embedded (FFPE) blocks. Tissue sections from mouse breast cancer FFPE blocks were immunostained using Opal™ Polaris 7-Color Automation IHC kit and Opal Polymer Anti-Rabbit HRP Kit on Leica BOND RX ™ automated stainer. Multispectral fluorescence imagery was acquired on a Vectra Polaris® automated imaging system and analyzed with inForm®, Phenochart®, Image J and R software.

Sulindac Reduces Tumor Neovascularization

7-Color Whole Slide Multispectral Imaging

Co

ntr

ol

Su

lin

da

c

Whole slide Tissue Segmentation and Cell Phenotyping

Ki67-Opal Polaris 780 CD31-Opal 690

Bcl-2 –Opal 520 CD8a-Opal Polaris

480

F4/80-Opal 620 CD4-Opal 570

C Phenotype Densities (cells/mm2) in both Live & Dead

Zones

A

Ctrl-Live Zone Sulin-Live Zone

Sulin-Dead ZoneCtrl-Dead Zone

B

Fig 4. Mouse Breast Cancer Tumor Angiogenesis. A) Tumor Inflammation and Angiogenesis/Leakiness were measured by the intensity of PS 750 and AS750 respectively using in-vivo imaging. Data is represented as an average of tumor Radiant Efficiency, a measure of total fluorescence in the tumors. B) Representative CD31 staining component planes from Fig. 2

In-vivo imaging results give an overall information that Sulindac effectively reduces tumor angiogenesis. Additionally, CD31 staining from multiplex IHC shows the heterogeneous nature of breast cancer samples and indicates the mechanism of reduced angiogenesis in Sulindac treated group due to reduced micro vessels in the Live Zone of tumor.

A

B

Fig 3. Immune Profiling on Whole Slide View Based on Opal Polaris 7-Color Fluorescent IHC. A) Representative image from whole slide tissue segmentation of mouse breast cancer section based on Ki67 staining (Ki67+: Live Zone & Ki67-: Dead Zone). B) Zoomed in views of composite images (top) and tissue segmentation with cell phenotypes(i&ii) or CD31 object segmentation (iii) (bottom). C) Cell densities of each phenotype marker within Live Zone and Dead Zone of each tissue section.

A Whole Slide Tissue Segmentation

Tumor Proliferation

Fig 5. Proliferation of Mouse Breast Cancer Tumors. Percentages of Live Zone and Dead Zone of each tumor were calculated by using areas from tissue segmentation based on Ki67 staining.

Bcl-2

Ki67

Bcl-2+Ki67+

CD8a

CD4

F4/80

CD31

B Cell Phenotyping on Whole Slide MSI in both Live Zone & Dead

Zone

i iiiii

Ki67-Opal Polaris 780 CD31-Opal 690

Bcl-2 –Opal 520 CD8a-Opal Polaris

480

F4/80-Opal 620 CD4-Opal 570

C

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Ctrl Sulin

F4/80+

0

1000

2000

3000

4000

5000

6000

7000

8000

Ctrl Sulin

Bcl2+

0

300

600

900

1200

1500

1800

2100

Ctrl Sulin

Bcl-2+ F4/80+

0

10

20

30

40

50

60

70

Ctrl Sulin

CD4+

Dead Zone

Live Zone

Live Zone

Dead Zone

Control Sulindac

Control Sulindac

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Live Zone

Dead Zone

Control Sulindac1 2

3

1 2

3

Non-invasive assessment of Sulindac effects on tumors were assessed on day 2 and 10 post-treatment by fluorescent imaging on the IVIS® SpectrumCT. Treated and untreated 4T1 tumor-bearing mice were anesthetized and injected with either AngioSense 750 EX (AS750, NIR fluorescent vascular probe) or ProSense 750EX (PS750, NIR fluorescent cathepsin activatableprobe). Epifluorescence images were captured 24h later, and flank tumor regions were quantified by careful analysis using Living Image® 4.5.

D a y 2 D a y 1 0

0

51 0 9

11 0 1 0

1 .51 0 1 0

21 0 1 0

P S 7 5 0 tu m o r s ig n a l

Ra

dia

nt

Eff

icie

nc

y

C o n tro l

S u lin d a c

Tumor Inflammation

n = 8

P = 0.0661

n = 5

P = 0.8821