Our Phenotypic drug screening panel's latest addition with Patient derived Gliomas / Astrocytomas / Oligodendromas

1. Onco - NeuroPrime
TM
All Rights Reserved | Saarum Sciences Pvt. Ltd., 1st Floor, AIMSR Building, Apollo Health City, Jubilee Hills, Hyderabad 500096, India | http://www.saarum.com
SAARUM
SCIENCES
Phenotypic Platforms for Drug & Biomarker Discovery
Figure 3. High grade glioma cells were cultured in serum free media on non-adherent
plates for 10 to 15 days to form three dimensional growth known as Neurospheres.
These 3D Neurospheres are true in vitro representation of human brain tumors and
can be used for drug screening and imaging based studies. For comparison,
Neurospheres generated from U87MG cell line is shown in the right panel.
Bendamustine Concentration
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Absorbanceat450nm
Bendamustine
cells + DMSO 0.8 um 4 uM 20 uM 100 uM 500 uM
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mn054taecnabrosbA
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OncoPrime
TM
Use a primary cell-based platform that truly represents the clinical diversity of brain cancer - Switch to
“
Patient cells characterized across several passages
“ Why use 2D cell lines when you can take advantage of
3D primary cell cultures“
OncoPrime
TM
platform is ideally suited for drug screening & lead optimization
“ U87MG - IC25/IC75 Titration
Cells + 0.225% DMSO IC 25 IC 75
1000 cells 2500 cells 5000 cells
P1, Day 4 P1, Day 12
P2, Day 4 P2, Day 12
U87MG cell line, Day 10
U87MG cell line, Day 15
High grade glioma, Day 10
High grade glioma, Day 15
10X
10X
10X
10X
Figure 4. Effect of reference compounds. Cytotoxicity assay on neurospheres treated with Bendamustine at doses IC 25 and IC 75
Figure 2. Cells derived from patient tumours proliferate well in primary cultures
and maintain their morphology through successive generations. Representative
phase contrast images of High grade glioma. Images acquired at 100X mag.
Figure 1b. Diversity of samples available in our biobank. With steady inflow of patient
samples, we have banked a generous diversity of Breast cancer samples. The chart
represents high level categories only. Brain cancer subtypes as defined by WHO criteria.
Figure 1a. Glioma classification based on lineage. Schematic representation of
the differentiation process of neural stem cells into different cell lineages of the
CNS and putative cells of origin of gliomas. WHO grading is shown in table.
ASTROCYTOMA OF BRAIN
ADENOCARCINOMA, METASTATIC,
NOS
LOW-GRADE FIBROMYXOID
SARCOMA
GLIOBLASTOMA MULTIFORME OF
BRAIN
NON-HODGKIN'S LYMPHOMA
GLIOBLASTOMA NOS
PITUITARY ADENOMA
MENINGIOMA NOS
NEOPLASM OF BRAIN
GANGLIOGLIOMA
GEMISTOCYTIC ASTROCYTOMA
CEREBRAL ARTERIOVENOUS
MALFORMATION
TRIGEMINAL SCHWANNOMATUBERCULOSIS
PRIMITIVE NEUROECTODERMAL
TUMOR
TRICHILEMMAL CYSTEPIDERMOID CYST
OLIGODENDROGLIOMA,
ANAPLASTIC
MENINGOTHELIAL MENINGIOMA
CRANIOPHARYNGIOMA
TRANSITIONAL MENINGIOMA
CARCINOMA, METASTATIC, NOS
DIFFUSE NON-HODGKIN'S
LYMPHOMA, LARGE CELL
PILOCYTIC ASTROCYTOMA
ANAPLASTIC ASTROCYTOMA OF
BRAIN
EPENDYMOMA NOS
ANGIOMATOUS MENINGIOMA
INFLAMMATORY DISORDER
HEMATOMA
Our Biobank Inventory
Grade
Grade I tumor (juvenile
pilocytic astrocytoma)
Grade II tumor
(astrocytoma)
Grade III tumor
(anaplastic astrocytoma)
Grade IV tumor
(glioblastoma)
Comments
Benign, slow-growing tumor; usually associated
with long-term survival; least likely to recur
Increased hypercellularity; no mitosis;
no vascular proliferation; no necrosis;
can recur as a higher-grade tumor
High rate of hypercellularity; high rate of
mitosis; no vascular proliferation; no necrosis;
high rate of tumor recurrence
Very high rate of hypercellularity; very high
rate of mitosis; presence of vascular proliferation;
presence of necrosis
WHO Grading System for Gliomas

2. Onco - NeuroPrime
TM
All Rights Reserved | Saarum Sciences Pvt. Ltd., 1st Floor, AIMSR Building, Apollo Health City, Jubilee Hills, Hyderabad 500096, India | http://www.saarum.com
SAARUM
SCIENCES
Phenotypic Platforms for Drug & Biomarker Discovery
PCR and sequencing:
IDH1, PTEN, TP53, NF1, EGFR, CDK4, INK4A-ARF, MGMT,
TIMP3, PDGFRA, 1p/19q deletion, B-Raf
Amplification by qPCR
EGFR, CDK4 and PDGFRA
Promoter methylation:
EGFR, INK4A-ARF, MGMT, TIMP3 genes
Table 1. These patient samples were used for Glioma characterization
Age Gender Clinical History Diagnosis
38yr Female Right Frontol SOL High Grade Glioma
49yr Male Right Frontol SOL Anaplastic Oligodendroglioma WHO Gr-III
32yr Male Right Frontal Lesion Anaplastic Astrocytoma WHO Gr-III
Figure 7. Immunofluorescence staining for beta III Tubulin (left panel) with DAPI and
GFAP (right panel) with DAPI of primary cells (High Grade Glioma) in culture.
Arrow shows an astrocyte cell surrounded by possibly diferrentiated astrocytes.
Figure 9. Glioma Panel - Cell Viability Assay. U87MG cell line (left) and Primary Glioma
cells in culture (right) were treated with Bendamustine for 72 h at dose range 500 uM,
50 uM, 5 uM and 0.5 uM
Figure 8. Nested PCR for EGFR / EGFR VIII
U87 MG cell line shows EGFR expression at ~1 KB
Figure 6. Glioma Panel –Senescence-ß-gal Assay. Top row - fibroblast cells used as
positive control. Bottom row - oligo dendro glioma and high grade glioma cells
as indicated. Arrows - show less than 5 % positive staining in primary cells as
compared to ~95% positive staining in fibroblast controls
GFAP-Green +DAPI
60X
60X
beta III Tubulin +DAPI
60X
60X
A comprehensive array of phenotypic characterizations...
“• Expression microarrays mRNA and miRNA identification
• Activation states of cellular kinases and other enzymes
• Phenotypic measurements like cellular toxicity
• Real Time PCR to quantify gene expression
• FACS cell sorting to isolate cell populations of interest
• FISH analysis with specific probes
High grade glioma Oligo dendro glioma
OncoPrime
TM
OncoPrime
TM
Applications of the platform
“ Advantages of the platform
“
Senescence assay to monitor neuronal death
“ Identification of nerve cell type specific markers
“
• Biomarker identification and validation
• Target identification and validation
• NME / NBE / NCE screening
• Drug repurposing
• Biosimilar / Biobetter characterization
• Drug mechanism of action elucidation
• Accurately reflect the disease phenotype and diversity
• Better characterization and easy to adopt
• Cost effective and shorten time to end points
• Accelerated Preclinical Development
• Novel disease models such as EMT and MET
• 3D spheroid cultures to better mimic in vivo conditions
78.05 %
98.68 %
2.88 % 89.22 % 3.22 % 5.09 %
86.61 % 5.02 % 0.20 %
82.74 %
98.67 %
87.03 % 4.24 % 0.11 %
4.46 % 89.85 % 2.08 % 3.94 %
Figure 5. High grade glioma (left panel) and oligo dendro glioma (right panel) in primary
culture have a diploid amount of genomic DNA. A high percentage of cells are found to
arrested at the G0/G1 phase at the time of sampling.
100X
100X
Fibroblast Fibroblast Fibroblast
Oligo dendro glioma Oligo dendro glioma High grade glioma
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%Viability
Log Conc
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%Vuability
Log Conc
U87MG Glioma
Lane Sample
1 100 bp ladder
2 1st round PCR product
3 2nd round PCR product
4 GAPDH
5 1 KB ladder
6 No template control
EGFR
1 2 3 4 5 6