Phenoptics™ research solutions to assess the tumor microenvironment in primary and secondary brain tumors Jadranka Macas 1, 5 *, Bj örn Wendik 2 *, Virginie Goubert 2 , Yvonne Reiss 1,3,4,5§ , Karl H. Plate 1,3,4,5§ 1 Institute of Neurology (Edinger Institute), University Hospital, Goethe University, Frankfurt, Germany. 2 Akoya Biosciences, Marlborough, MA, USA. 3 German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt, Germany. 4 German Cancer Research Center (DKFZ), Heidelberg, Germany. 5 Frankfurt 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 origin 1 . 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 therapy 2 . Brain tumor progression is angiogenesis-dependent and associated with a dysfunctional blood-brain barrier 3 (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 niche 1 . 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 months 1,4 . Malignant brain tumors are further characterized by an immunosuppressive microenvironment that impedes dendritic cell maturation and T cell cytotoxicity 5 . The abundance of tumor-associated macrophages has been linked to poor clinical outcome 5 . A series of secreted and molecular factors within the TME allow brain tumors to evade the host immunosurveillance 5 . 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 cancer 6,7 . 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 1 Louis 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 Cancer 2 Ohgaki H, Kleihues P (2005) Epidemiology and Etiology of Gliomas. Acta Neuropathologica, 109: 93–108 3 Liebner 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 4 Stupp 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. 5 Quail, DF. & Joyce JA (2017), The Microenvironmental Landscape of Brain Tumors. Caner Cell, Volume 31, Issue 3, 13 March 2017, Pages 326-341. 6 Scholz 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. 7 Di 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 annotations Tissue segmentation WSS view for batch analysis Fused 7plex spectrally unmixed image 7plex spectrally unmixed ROI A B C D CD3 CD8 CD47 CD163 Iba-1 vWF DAPI CD3 CD8 CD47 CD163 Iba-1 vWF DAPI DAPI CD3 CD8 HER-2 CD163 vWF Iba-1 2 mm Fused image Fused image Experimental GL261 Mouse Glioma Model CD3 CD8a CD31 Iba-1 DAPI
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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
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