multispectral imaging reveals unique macrophage profiles … · 2018-11-15 · multispectral...

1
Introduction Spectral imaging microscopy is a powerful technique that enables quantification and identification of distinct intrahepatic macrophage phenotypes in situ in human FFPE liver tissue The multiplex staining panel and imaging analysis software algorithms that we have developed allows these elusive cells to be studied in the context of intact hepatic architecture. The unique macrophage profiles that we have identified will be correlated with patient outcomes. Table 1. Antibodies used to identify intrahepatic macrophages using this platform Macrophage: Parent cell markers Tissue resident Kupffer cells CD68+ Tissue remodeling / Pro-fibrotic CD163+ Systemic monocytes Mac387+ Markers analyzed by level of expression on parent cells Classical (pro-inflammatory) macrophages CD14++/CD16- Intermediate macrophages CD14++/CD16+ Non-classical (anti-inflammatory) macrophages CD14+/CD16++ Acknowledgments Results Intrahepatic macrophages greatly impact the composition of the hepatic microenvironment, host immune response and development of fibrosis. Studies of human intrahepatic macrophages can be challenging for several reasons: (i) they are difficult to isolate from human liver tissue (ii) they become activated and change their phenotype when isolated or manipulated (iii) in vitro and mouse model systems of HCV infection or fatty liver disease do not closely mimic the long-term, chronic infections that are observed in humans We are using spectral imaging microscopy with advanced imaging analysis software programs to analyze intrahepatic macrophages in situ in human liver biopsies. This platform is optimized for multiplex immunofluorescence staining of formalin- fixed paraffin-embedded tissues and does not compromise the hepatic architecture. This approach will allow us to gain an in-depth understanding of how variations in human hepatic macrophage profiles affect the host immune response and development of hepatic fibrosis. Methods Conclusions Figure 1. Multiplex images obtained from staining FFPE liver biopsies from control patients compared to patients with three common chronic liver diseases (NASH, AIH and HCV). Biopsies were stained with the macrophage multiplex panel (CD68, CD163, Mac387, CD14, CD16, and DAPI) and representative images (20X) were acquired. Panel A: Fluorescent multispectral image after staining with the multiplex panel. Panel B: The multiplex images were then analyzed with Visiopharm phenotyping applications and each color represents a unique cellular phenotype. Panel C: The t-SNE plots highlight the unique profiles of macrophages that were identified in the livers of patients with chronic liver diseases versus controls. Panel D: Shows differences in the numbers of Mac387+ macrophages in the portal tracts and lobules in patients with either NASH, AIH or HCV, compared to control patients. Results shown are representative of three different patients with similar hepatitis activity scores (i.e., MHAI) and fibrosis stages (using the Ishak criteria). NASH: Nonalcoholic steatohepatitis, AIH: Autoimmune hepatitis; HCV: hepatitis C virus, P tract: Portal tract, tSNE: t-distributed Stochastic Neighbor Embedding (tSNE) algorithm. Results OPAL- 6 Color Multiplex IHC CD68 MAC87 CD16 CD14 CD163 Contact Information Moody Endowment Research Award UT Systems Rising STARs Award Perkin Elmer: Kevin Mottershead Visiopharm: Dr. Ben Freiberg and Dr. Alex Villa University of Michigan: Dr. Arvind Rao Rice University: Santhoshi Krishnan Multispectral Imaging Reveals Unique Macrophage Profiles Associated with Type of Liver Disease and Fibrosis Stage Omar A. Saldarriaga 1 , Adam L. Booth 1 , Jared K. Burks, Netanya S. Utay 2 , MinKyung Yi 3 , Monique Ferguson 4 , Laura Beretta 5 and Heather L. Stevenson 1 . 1 Department of Pathology, University of Texas Medical Branch, Galveston, TX; 2 University of Texas Health Science Center at Houston, Houston, TX; 3 University of Texas Medical Branch, Galveston, TX; 4 University of Texas MD Anderson Cancer Center, Houston, TX Heather Stevenson-Lerner, M.D., PhD Assistant Professor, UTMB, Dept. of Pathology [email protected] ; Office: 409-772-8554 Omar A. Saldarriaga, DVM., PhD Research Scientist, UTMB, Dept. of Pathology [email protected] ;Office: 409-747-0275 Adam L. Booth, MD Chief Resident, PGY3, UTMB, Dept. of Pathology [email protected] , Office: 903-918-1790 Figure 2. Differences in intrahepatic macrophage profiles in HCV+ patients (genotype 1a+) with minimal fibrosis or cirrhosis versus controls. We obtained low power images of two different patient’s liver biopsies after staining with Masson’s trichrome (blue = fibrosis). Both patients are males of similar age that report being infected with HCV approximately 20 years ago. Why did these patients respond so differently to chronic hepatitis C? The first patient's biopsy (A) shows minimal fibrosis (Ishak stage: 1/6) and the second patient (B) has cirrhosis with large areas of parenchymal extinction. We used the multiplex macrophage panel to determine differences in the intrahepatic macrophage profiles in the HCV+ patients when compared to controls. (C) t-SNE unsupervised analysis of macrophage markers highlights the unique populations identified in these patients. Panel A Panel B Panel C Panel D Fig. 1 Control 0 2 4 6 8 10 12 14 P Tract Lobule MAC387 + CELLS (%) Non-alcoholic Steatohepatitis (NASH) 0 2 4 6 8 10 12 14 P Tract Lobule MAC387+ CELLS (%) Autoimmune Hepatitis (AIH) 0 2 4 6 8 10 12 14 P Tract Lobule MAC387+ CELLS (%) Chronic Viral Hepatitis (HCV) 0 2 4 6 8 10 12 14 P Tract Lobule MAC387+ CELLS (%) t-SNE t-SNE 1 t-SNE t-SNE t-SNE 1 t-SNE 1 A. t-SNE 1 t-SNE CD68 - Opal 520 CD163 - Opal 650 Mac387- Opal 690 CD16 - Opal 620 CD14 - Opal 540 Macrophage phenotypes in NASH t-SNE 1 t-SNE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 CD68 (520) Mac387 (690) CD16 (620) CD14 (540) CD163 (650) -1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00 B. Figure 3. Unsupervised analysis shows multiple macrophage phenotypes in FFPE liver biopsies obtained from patients with NASH after staining with the macrophage panel. (A) t- SNE plots use dimensional reduction to facilitate visualization of macrophage marker expression. Cells with similar properties appear close together in a two-dimensional map and red (or “hot”) markers show cells with relatively more expression of that specific marker when compared to blue (or “cold”) markers, which indicate absent or minimal expression. (B) The phenotypic matrix algorithm identified 16 distinct macrophage phenotypes (#: 1-16) in one multiplex image obtained from a patient with NASH. Other cell populations, which likely include hepatocytes, lymphocytes and epithelial cells, appear blue in the phenotype matrix map (#: 17-25). Control HCV+ Minimal fibrosis HCV+ Cirrhosis Fig. 2. HCV+ minimal fibrosis HCV+ Cirrhosis t-SNE Plot A. B. C. Spectral Imaging Library Tyramide signal amplification A. @DrHSLovesLiver @ALBoothMD @UTMB_Pathology

Upload: others

Post on 09-Apr-2020

6 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Multispectral Imaging Reveals Unique Macrophage Profiles … · 2018-11-15 · Multispectral Imaging Reveals Unique Macrophage Profiles Associated with Type of Liver Disease and Fibrosis

Introduction

• Spectral imaging microscopy is a powerful technique that enables quantification

and identification of distinct intrahepatic macrophage phenotypes in situ in human

FFPE liver tissue

• The multiplex staining panel and imaging analysis software algorithms that we have

developed allows these elusive cells to be studied in the context of intact hepatic

architecture.

• The unique macrophage profiles that we have identified will be correlated with

patient outcomes.

Table 1. Antibodies used to identify intrahepatic macrophages using this platform

Macrophage: Parent cell markers

Tissue resident Kupffer cells CD68+

Tissue remodeling / Pro-fibrotic CD163+

Systemic monocytes Mac387+

Markers analyzed by level of expression on parent cells

Classical (pro-inflammatory) macrophages CD14++/CD16-

Intermediate macrophages CD14++/CD16+

Non-classical (anti-inflammatory) macrophages CD14+/CD16++

Acknowledgments

Results

Intrahepatic macrophages greatly impact the composition of the hepatic

microenvironment, host immune response and development of fibrosis.

Studies of human intrahepatic macrophages can be challenging for several

reasons:

(i) they are difficult to isolate from human liver tissue

(ii) they become activated and change their phenotype when isolated or

manipulated

(iii) in vitro and mouse model systems of HCV infection or fatty liver disease do

not closely mimic the long-term, chronic infections that are observed in humans

We are using spectral imaging microscopy with advanced imaging analysis

software programs to analyze intrahepatic macrophages in situ in human liver

biopsies.

This platform is optimized for multiplex immunofluorescence staining of formalin-

fixed paraffin-embedded tissues and does not compromise the hepatic architecture.

This approach will allow us to gain an in-depth understanding of how variations in

human hepatic macrophage profiles affect the host immune response and

development of hepatic fibrosis.

Methods

ConclusionsFigure 1. Multiplex images obtained from staining FFPE liver biopsies from control patients compared to

patients with three common chronic liver diseases (NASH, AIH and HCV). Biopsies were stained with the

macrophage multiplex panel (CD68, CD163, Mac387, CD14, CD16, and DAPI) and representative images (20X)

were acquired. Panel A: Fluorescent multispectral image after staining with the multiplex panel. Panel B: The

multiplex images were then analyzed with Visiopharm phenotyping applications and each color represents a

unique cellular phenotype. Panel C: The t-SNE plots highlight the unique profiles of macrophages that were

identified in the livers of patients with chronic liver diseases versus controls. Panel D: Shows differences in the

numbers of Mac387+ macrophages in the portal tracts and lobules in patients with either NASH, AIH or HCV,

compared to control patients. Results shown are representative of three different patients with similar hepatitis

activity scores (i.e., MHAI) and fibrosis stages (using the Ishak criteria). NASH: Nonalcoholic steatohepatitis, AIH:

Autoimmune hepatitis; HCV: hepatitis C virus, P tract: Portal tract, tSNE: t-distributed Stochastic Neighbor

Embedding (tSNE) algorithm.

Results

OPAL- 6 Color Multiplex IHC

CD68 MAC87 CD16

CD14 CD163

Contact Information

Moody Endowment Research Award

UT Systems Rising STARs Award

Perkin Elmer: Kevin Mottershead

Visiopharm: Dr. Ben Freiberg and Dr. Alex Villa

University of Michigan: Dr. Arvind Rao

Rice University: Santhoshi Krishnan

Multispectral Imaging Reveals Unique Macrophage Profiles

Associated with Type of Liver Disease and Fibrosis StageOmar A. Saldarriaga1, Adam L. Booth1, Jared K. Burks, Netanya S. Utay2, MinKyung

Yi3, Monique Ferguson4, Laura Beretta5 and Heather L. Stevenson1. 1Department of Pathology, University of Texas Medical Branch, Galveston, TX; 2University of Texas Health Science Center at Houston,

Houston, TX; 3University of Texas Medical Branch, Galveston, TX; 4University of Texas MD Anderson Cancer Center, Houston, TX

Heather Stevenson-Lerner, M.D., PhD

Assistant Professor, UTMB, Dept. of Pathology

[email protected]; Office: 409-772-8554

Omar A. Saldarriaga, DVM., PhD

Research Scientist, UTMB, Dept. of Pathology

[email protected];Office: 409-747-0275

Adam L. Booth, MD

Chief Resident, PGY3, UTMB, Dept. of Pathology

[email protected], Office: 903-918-1790

Figure 2. Differences in intrahepatic macrophage profiles in HCV+ patients (genotype 1a+)

with minimal fibrosis or cirrhosis versus controls. We obtained low power images of two different

patient’s liver biopsies after staining with Masson’s trichrome (blue = fibrosis). Both patients are

males of similar age that report being infected with HCV approximately 20 years ago. Why did these

patients respond so differently to chronic hepatitis C? The first patient's biopsy (A) shows minimal

fibrosis (Ishak stage: 1/6) and the second patient (B) has cirrhosis with large areas of parenchymal

extinction. We used the multiplex macrophage panel to determine differences in the intrahepatic

macrophage profiles in the HCV+ patients when compared to controls. (C) t-SNE unsupervised

analysis of macrophage markers highlights the unique populations identified in these patients.

Panel A Panel B Panel C Panel D

Fig. 1 Control

0

2

4

6

8

10

12

14

P Tract Lobule

MA

C3

87

+

CE

LL

S (%

)

Non-alcoholic Steatohepatitis (NASH)

0

2

4

6

8

10

12

14

P Tract Lobule

MA

C3

87

+ C

EL

LS

(%

)

Autoimmune Hepatitis (AIH)

0

2

4

6

8

10

12

14

P Tract Lobule

MA

C3

87

+ C

EL

LS

(%

)

Chronic Viral Hepatitis (HCV)

0

2

4

6

8

10

12

14

P Tract Lobule

MA

C3

87

+ C

EL

LS

(%

)

B.

t-S

NE

t-SNE 1

t-S

NE

t-S

NE

t-SNE 1 t-SNE 1

A.

t-SNE 1

t-S

NE

CD68 - Opal 520 CD163 - Opal 650 Mac387- Opal 690 CD16 - Opal 620

CD14 - Opal 540 Macrophage phenotypes in NASH

t-SNE 1

t-S

NE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

CD68 (520)

Mac387 (690)

CD16 (620)

CD14 (540)

CD163 (650)

-1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00

B.

Figure 3. Unsupervised analysis shows multiple macrophage phenotypes in FFPE liver

biopsies obtained from patients with NASH after staining with the macrophage panel. (A) t-

SNE plots use dimensional reduction to facilitate visualization of macrophage marker expression.

Cells with similar properties appear close together in a two-dimensional map and red (or “hot”)

markers show cells with relatively more expression of that specific marker when compared to blue

(or “cold”) markers, which indicate absent or minimal expression. (B) The phenotypic matrix

algorithm identified 16 distinct macrophage phenotypes (#: 1-16) in one multiplex image obtained

from a patient with NASH. Other cell populations, which likely include hepatocytes, lymphocytes

and epithelial cells, appear blue in the phenotype matrix map (#: 17-25).

Control

HCV+ Minimal

fibrosis

HCV+ Cirrhosis

Fig. 2. HCV+ minimal fibrosis HCV+ Cirrhosis t-SNE Plot

A. B. C.Spectral Imaging Library

Tyramide signal amplification

A.

@DrHSLovesLiver

@ALBoothMD

@UTMB_Pathology