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Phenoptics™ research solutions to assess the tumor microenvironment in primary and secondary brain tumors

Jadranka Macas1, 5*, Björn Wendik2*, Virginie Goubert2, Yvonne Reiss1,3,4,5§, Karl H. Plate1,3,4,5§

1Institute of Neurology (Edinger Institute), University Hospital, Goethe University, Frankfurt, Germany. 2Akoya Biosciences, Marlborough, MA, USA. 3German Cancer

Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt, Germany.4German Cancer Research Center (DKFZ), Heidelberg, Germany. 5Frankfurt Cancer Institute, Frankfurt, Germany. *§Equal contribution

Introduction

The brain tumor microenvironment (TME) is emerging as a critical regulator of cancer progression in brain malignancies. Brain tumors represent a heterogeneous

group of central nervous system neoplasms that are classified into primary or secondary brain tumors according to their origin1. The most frequent primary brain

tumor in adults is glioblastoma multiforme (GBM) which is characterized by a high heterogenicity within and across patients, a highly aggressive infiltrative

growth and resistance to therapy2. Brain tumor progression is angiogenesis-dependent and associated with a dysfunctional blood-brain barrier3 (BBB). Secondary

malignant brain tumors (brain metastases) disseminate most often from mamma carcinoma, lung adenocarcinoma and melanoma into the brain by passing the

blood-brain-barrier and adjusting to brain niche1. Despite advances in treatment of GBM and brain metastases which includes a combination of surgery,

radiotherapy and chemotherapy, these tumors remain lethal within 6-24 months1,4.

Malignant brain tumors are further characterized by an immunosuppressive microenvironment that impedes dendritic cell maturation and T cell cytotoxicity5. The

abundance of tumor-associated macrophages has been linked to poor clinical outcome5. A series of secreted and molecular factors within the TME allow brain

tumors to evade the host immunosurveillance5. We have recently shown that the combination of immune checkpoint and anti-angiogenic therapy shows efficacy in

glioblastoma, which is considered to be “macrophage-rich” and “non-T cell-inflamed” 6,7. Our findings highlight how immune therapy efficacy can be improved by

also targeting angiogenic factors in this type of cancer6,7.

We employ Phenoptics™ research solutions (Akoya Biosciences, Inc.) to assess immunosuppressive macrophages and cytotoxic T lymphocytes in the context of

the tumor vasculature in glioblastoma and brain metastases.

Methods

Sequentially applied specific antibodies

Human GBM Human Brain Metastasis

CD3 Opal 690 CD3 Opal 690

CD8a Opal 520 CD8a Opal 520

Von Willebrandt Factor Opal 480 Von Willebrandt Factor Opal 570

CD163 Opal 620 CD163 Opal 620

Iba-1 Opal 780 Iba-1 Opal 780

CD47 Opal 570 HER-2 Opal 480

Nuclei DAPI Nuclei DAPI

(A) FFPE sections of brain tumor patients were stained using Opal Polaris 7 colour kit (NEL861001KT, Akoya Biosciences, Inc.) based on thyramide signal

amplification immunostaining technique. To illustrate, the 7plex staining panels targeting anti-human CD3, CD8a, vonWillebrandt Factor, CD163, Iba-1, CD47 or

HER-2 were performed on LabSat™ Research automated staining instrument (Lunaphore Technologies SA) with a turnaround time of 4h 35min. (B) Multiplex

stainings were acquired on Vectra Polaris (Akoya Biosciences, Inc.) using MOTiF™ technology which provided unmixed whole slide scans in a streamlined

workflow within 6-15 min. at 0,5 µm/pixel. Whole slide multispectral image analysis was performed using Phenochart®, version 1.0.12, InForm® Image Analysis

Software (Akoya Biosciences, Inc.), and HALO™ software (Indica Labs). PhenoptrReports package supports InForm data analysis and visualisation.

A B Image Analysis Platform

Phenochart® 1.0.12

HALO™ 2.1 InForm® 2.4.6

phenoptrReports

Results

1Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Ellison DW, Figarella-Branger D, Perry A, Reifenberger G, Deimling von A (2016) WHO

Classification of Tumours of the Central Nervous System, 4 ed International Agency for Research on Cancer2Ohgaki H, Kleihues P (2005) Epidemiology and Etiology of Gliomas. Acta Neuropathologica, 109: 93–1083Liebner S, et al., (2018) Functional Morphology of the Blood-Brain Barrier in Health and Disease. Acta Neuropathologica, 311–336. doi:

10.1007/s00401-018-1815-1

4Stupp R, Hegi ME, Mason WP, et al. Effects of Radiotherapy with Concomitant and Adjuvant Temozolomide versus Radiotherapy Alone on

Survival in Glioblastoma in a Randomised Phase III study: 5-year Analysis of the EORTC-NCIC Trial. Lancet

Oncology. 2009;10(5):459–466.5Quail, DF. & Joyce JA (2017), The Microenvironmental Landscape of Brain Tumors. Caner Cell, Volume 31, Issue 3, 13 March 2017,

Pages 326-341.6Scholz A., et al. (2016), Endothelial Cell-derived Angiopoietin-2 is a Therapeutic Target in Treatment-naive and Bevacizumab-resistant

Glioblastoma. EMBO Mol. Med. 8, 39–57.7Di Tacchio M., Macas J., Weissenberger J., et al., (2019) Tumor Vessel Normalization, Immunostimulatory Reprogramming, and Improved

Survival in Glioblastoma with Combined Inhibition of PD-1, Angiopoietin-2, and VEGF. Cancer Immunology Research. doi:

10.1158/2326-6066.CIR-18-0865.

Literature

(A) The Opal Polaris 7plex fluorescent

IHC assay for mamma carcinoma brain

metastasis includes the membrane

bound tumor marker HER-2, T-cell

markers CD3 and CD8, myeloid cell

markers Iba-1 and CD163 and

endothelial cell marker von Willebrandt

factor. The staining was reviewed and

annotated in Phenochart® 1.0.12 whole

slide viewer and spectrally unmixed in

InForm®.. Scale bar 100µm.

For initial screening of primary (A) and recurrent (B) glioblastoma mircroenvironment we performed

the Opal Polaris fluorescent IHC assay for CD3+ and CD8+ T cells, Iba-1+ and CD163+ myeloid

cells, vWF+ endothelial cells and CD47+ cells, a myeloid checkpoint frequently highly expressed on

glioblastoma cells. The first results confirm the heterogeneity of CD8+, CD3+ and CD163+

expression between and across patients (C). Additional biomarkers i.e. FoxP3, PD-1, PD-L1, CD49d,

CD206, Ki67, Caspase 3 are included into different panel combinations in our lab for further analysis.

Opal Polaris fluorescent IHC assay is currently being established also on experimental mouse brain

tumor models (D). Scale bar 100µm (ROIs in A, B, D). Scale bar 2mm (fused image in A, B).

Multispectral analysis of dynamic cell interactions within the malignant brain TME is

essential for understanding biology of tumor progression and angiogenesis and has become

indispensable to assess the effect of multimodal therapies.

Phenoptics™ Research Solutions provide tools for staining, scanning and analysis of 6

biomarkers on whole FFPE sections without a selection bias, an interference of spectral

overlap or autofluorescence and with high flexibility through combination of several image

analysis software packages and fully customizable image analysis options.

Conclusions

Figure 1: Whole Slide Scan and Multispectral Image Analysis Workflow on Mamma Carcinoma Brain Metastasis

(B) Large numbers of spectrally unmixed images can be batch processed in InForm®, exported and fused in a

whole slide scan qptiff image for multiple analysis in HALO™, a modular designed image analysis platform.

Displayed are field of views for a better illustration. The Highplex FL and the Tissue Classifier Add-On enable

simultaneous analysis of unlimited fluorescent signals and identification of specific cell phenotypes within

specific tissue segments across a whole FFPE section. Tissue Classifier additionally provides options to turn tissue

segments into annotations and therefore identify specific regions to include (tumor in red, stroma in green) or

exclude (vessel lumen in grey) from analysis. Spatial analysis module enables proximity and nearest neighbour

analysis of specific cell phenotypes or cell infiltration analysis.

A B

Figure 2: Multispectral Image Analysis of Glioma Patient Biopsy Samples

Cell phenotyping Proximity analysis of all CD8+

to HER-2+

Tissue annotationsTissue segmentationWSS view for batch

analysis

Fused 7plex spectrally

unmixed image7plex spectrally unmixed ROI

A B C DCD3

CD8

CD47

CD163Iba-1 vWF DAPI

CD3

CD8

CD47

CD163Iba-1 vWF DAPI

DAPI

CD3

CD8

HER-2

CD163 vWFIba-1

2 mm

Fused image Fused image

Experimental GL261 Mouse Glioma Model

CD3

CD8a

CD31

Iba-1

DAPI