Onco -Breast CancerPrimeTM

OncoPrimeTM

OncoPrimeTM

Use a primary cell-based platform that truly represents the clinical diversity of breast cancer - Switch to “

All Rights Reserved | Saarum Sciences Pvt. Ltd., IICT, Tarnaka, Hyderabad 500007, India. http://www.saarum.com

Figure 2. Diversity of samples available in the biobank. With steady inflow of patient samples, we have banked a generous diversity of Breast cancer samples. The chartsrepresents high level categories only. Breast cancer staging as per WHO guidelines.

Triple -ve HER2+ Luminal B Luminal AMolecularSubtypes

% 15-20% 10-15% 20% 40%ReceptorExpression

Histologicgrade

Prognosis

Responseto therapy

Our Biobank Diversity

HER2 ER+ / PR+

High (grade III) Low (grade I)

Poor Good

Chemotherapy

TrastuzumabHormone Rx

Figure 1. Disease Diversity. Based on biomarker expression breast cancer can be categorized into four major subtypes. Triple negative (TNBC) tumors respond best to chemotherapy similar to other aggressive cancers. Luminal A tumors respond best to endocrine therapy e.g. antiestrogen or aromatase inhibitor.

Patient cells characterized across several passages“ Why use 2D cell lines when you can take advantage of 3D primary cell cultures“

Figure 3. Cells derived from patient tumours proliferate well in primary cultures and maintain their morphology through successive generations. Representative phase contrast images of SB-Br-523 (Infiltrating Ductal Carcinoma Grade III, ER +ve, PR +ve, and HER2 +ve). Images were acquired with a 10X objective.

P1 P2

P3 P4

Figure 4. Human mammary epithelial cells isolated from normal breast tissue by collagenase digestion were cultured as monolayers (Top panel) phase contrast images shown. Bottom panel shows Mammospheres (arrows) grown in 2% agarose, 48h after passage. These 3D cell cultures are ideal for drug testing and other assays.

P1 P2

P2

100X

100X

ER/PR -ve / Her2 +ve

ER/PR +ve / Her2 +ve

ER/PR +ve / Her2 -ve ER+ve/PR +ve /

Her2 +ve

ER-ve /PR -ve / Her2 -ve

Breast cancer - ER/PR/HER2 STATUS

platform is ideally suited for drug screening & lead optimization“

Figure 5. Effect of reference compounds. Cell viability assay demonstrates effect of standard of care (SOC) compunds on three main categories of patient derived breast cancer cells

Er/Pr Negative, Her2 PositiveLog Concentration

DoxorubicinPaclitaxel5-FU

Cel

l via

bilit

y

Er/Pr Positive, Her2 Negative

Doxorubicin5-FUGemcitabine

Log concentration

Cel

l via

bilit

y

Triple negativeLog Concentration

Cel

l via

bilit

y

Doxorubicin5-FU

27% 12% 28% 33%

Breast Cancer Stages

Our Breast Cancer Sample Collection

I A

II A

II B

III A

III B III C

OncoPrimeTM

OncoPrimeTM

Onco -Breast CancerPrimeTM

A comprehensive array of phenotypic characterizations...“

Some early evidence for Epithelial to Mesenchymal Transition (EMT) Model“

Applications of the platform“ Advantages of the platform“

All Rights Reserved | Saarum Sciences Pvt. Ltd., IICT, Tarnaka, Hyderabad 500007, India. http://www.saarum.com All Rights Reserved | Saarum Sciences Pvt. Ltd., IICT, Tarnaka, Hyderabad 500007, India. http://www.saarum.com

Figure 6. Cells of SB-Br-6170 in primary culture have a diploid amount of genomic DNA. A high percentage of cells are arrested at the G0/G1 phase at the time of sampling.

4.84% 79.16% 4.76% 12.10%

62.85% 62.62%

48.54% 6.93% 0.06%

Figure 8. Representative images of immunofluoresence staining of SB-Br-6045 cells in culture (ER and PR negative, positive for HER2). These cells show differential staining for epithelial and mesenchymal markers. Images were captured by confocal microscope with a 60X objective.

DAPI EpCAM Vimentin Merged

SB-Br-6045

Figure 7b. Human mammary epithelial cells isolated from normal breast were culturedand stained with antibodies against EpCAM and CK 8/18. Similarly the human mammary fibroblasts were stained with antibodies against Vimentin and alpha-SMA. Images were captured by confocal microscope with a 40X objective.

CK 8/18EpCAM alpha-SMAVimentin

60X60X40X40XNormal Breast

• 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

MorphologyMorphological evaluation at each passageCell surface markers – common and specificIncluding sterility and Mycoplasma testing

Mutation and Epigenetic Profiling - Breast cancerBRCA1/2, EGFR, B-RAF, K-Ras, HER-2/Neu, TP53, C-Met, C-myc, PI-3K, MEK, FLT-3, PDGFR, TERT1/2

Functional assays - Reference drug dataCell migration and invasivenessColony formation and sphere formationStandard of care (SOC) data for drug screening

Regulated model of Epithelial Mesenchymal TransitionTarget discovery or validation,ID & screening of new drugs / combinationsID novel EMT / Metastasis biomarkersTherapeutics targeting EMT will target the crucial step of - metastasis and the formation of secondary sites of cancer.

Day 0 Day 7 Day 11

Day 12 Day 14 Day 15

35 mm 35 mm 35 mm

48 well 48 well 24 well

Figure 7a. Top Panel: Epithelial to mesenchymal transition after transfection of primary normal breast epithelial cells with our provisional patented constructs.Bottom Panel: At Passage 1, induced cells show positive staining (+++) for mesenchymal markers and minimal staining (+) for epithelial marker which is reversed by Passage 2. DAPI - blue, CK 8/18 & EpCAM - green, SMA - Red

DAPI, EpCAM & Vimentin

DAPI, CK 8/18 & SMA