Conversant Bio has developed drug testing services Goals ...

Conversant Bio has developed drug testing services

Goals: 6-Day Proliferation Study (Study #1)

•Determine the viability / proliferation of samples over 6 days to identify an
acceptable time-frame for compound treatment
•Compare the viability / proliferation of samples grown on tissue culture-
treated and Matrigel-coated plates

Goals: Compound Treatment Study (Study #2)

•Assess the effects of staurosporine treatment on the viability of samples
grown on tissue culture-treated and Matrigel-coated plates
•Assess assay window and reproducibility

Study #1 – assessed EpCAM expression by flow

Colorectal Cancer Lung Cancer Ovarian Cancer

• Most patient samples contain ~30-80% EpCAM+ cells

Study #1 – deep characterization of tested samples

• Tested patient samples show a post-thaw viability of 70-100%
• Tested solid tumor samples generally contain:

~60-80% epithelial cells
<~20% hematopoietic cells
<10% endothelial cells
<7% putative cancer stem cells

Study #1 – general protocol

Thaw cryopreserved samples into FBS-containing media

Determine viable cell counts by Trypan blue exclusion

Plate cells onto tissue culture (TC)-treated or Matrigel-coated plates

Measure daily viability using the Cell Titer Glo assay
(Day 0: 3 hrs post cell plating)

Study #1 – example pictures of cells after 4 days

Patient 6 (ovarian) Patient 7 (NSCLC) Patient 8 (NSCLC)

TC-treated

Matrigel-coated

• A variety of cell sizes and shapes were observed between samples
• A larger percentage of cells attached to the plate bottom in Matrigel-coated plates
compared to TC-treated plates (data not shown); the degree of attachment varied
greatly between samples

Study #1 – comparison of absolute luminescence

Signal-to-noise** Signal-to-noise**

• Similar absolute luminescence readings are observed when cells are plated on TC-treated
or Matrigel-coated plates on Day 0 and Day 4
• Plating 30,000 viable cells / well results in a suitable signal-to-noise reading for most
tested patient samples

* Error bars denote standard deviation
** Signal-to-noise: luminescence from wells containing cells / media only wells on the same plate

Study #1 – comparison of cell proliferation over 6 days

• Fairly similar proliferation rates observed between most patient samples grown on TC-
treated or Matrigel-coated plates over 6 days (exceptions mentioned on the next slide)

• Typically observe %CV < 10%
• Patient samples can be divided into 3 groups:

• Proliferative: A549, #2 (ovarian), #4 (ovarian), #6 (ovarian)
• Non-proliferative, but maintain viability: #1 (NSCLC), #7 (NSCLC), #8 (NSCLC)
• Non-proliferative, with decreased viability: #3 (CRC), #5 (CRC)

* Error bars denote %CV

Study #1 – plate coating does affect proliferation

• Select samples (#2, #4, #8) show an increase in proliferation on TC-treated plates and less so on
Matrigel-coated plates; most evident after 4 days in culture

• This pattern is not observed with all patient samples (i.e. #1) or the A549 cell line
• This pattern is hypothesized to be the result of an out growth of fibroblast cells
• Carrying-out studies on Matrigel-coated plates, limiting studies to 4 days and/or plating isolated
epithelial cells will likely mitigate this effect

• Alternatively, interference from fibrotic our-growth can be controlled and tumor
heterogeneity maintained, by using isolated EpCAM+ / EpCAM- cells separated by a
transwell insert

Study #1 – EpCAM comparison

(immediately post-thaw vs 6 days in culture)

• The percentage of EpCAM+ cells decreases slightly in two tested NSCLC samples but remains the
same in 2 tested ovarian cancer samples when the primary cells were cultured for 6 days
• Culturing human primary cells for 6 days on TC-treated or Matrigel-coated plates does not affect the
percentage of EpCAM+ cells

Study #2 – general protocol for in vitro compound testing

Thaw cryopreserved samples into FBS-containing media
Determine viable cell counts by Trypan blue exclusion

Plate cells onto tissue culture (TC)-treated or Matrigel-coated plates (Day 0)
Treat cells with compound (Day 1)

Measure viability using the Cell Titer Glo assay (Day 4)

Study #2 – staurosporine is our tool compound

Staurosporine

• Prototypical ATP-competitive kinase inhibitor
• Binds to serine / threorine and tyrosine kinases
• Non-kinase selective inhibitor
• Potent inhibitor of PKC
• Low [nM] result in cell-cycle arrest (cytostatic)1
• Higher concentrations induce apoptosis2

1McGahren-Murray, Mollianne, et al., Cancer Research, 2006
2Belmokhtar, Chafke Ahmed, et al., Oncogene, 2001

Study #2 – staurosporine-treated patient samples

NSCLC NSCLC

Ovarian Ovarian

• Staurosporine treatment results in different dose-response curves between samples
• Suitable assay windows are observed
• Typically observe %CV ~ 1-20%

* Error bars denote %CV
** Assay Window: luminescence from DMSO control wells / 20uM treated wells on the same plate

Study #2 – tissue culture treated vs.

Matrigel coated plates

Ovarian (metastatic) NSCLC NSCLC

• Cells from patients #6, #7 and #8 treated with staurosporine show almost identical results
when cells are plated on TC-treated or Matrigel-coated plates
• Suitable assay windows observed
• Typically observe %CV ~ 1-20%

* Error bars denote %CV
** Assay Window: luminescence from DMSO control wells / 20uM treated wells on the same plate

Study #2 – this assay is highly reproducible

Blue vs. red dose-response curves show data collected from
2 separate studies, 1 month apart, by 2 different people

10K cells / well plated 30K cells / well plated 30K cells / well plated 30K cells / well plated

• Highly reproducible dose-response curves observed when the same patient sample is
re-tested a month later by a different person

* Error bars denote %CV

We can conduct any number of cell-based assays

Common assays (monocultures or mixed cultures)

• Viability / proliferation / cytotoxicity assays
• Analysis of released soluble factors
• Cell migration assay
• Pathway signaling studies
• Mixed culture assays with transwells

Specialty assays

• Ex vivo tumor drug testing (3D cultures)
• Cancer stem cell (CSC) relevant assays:

• Colony assay
• Tumor sphere assay
• Serial replating assay to assess self-renewal potential
• Expression of cell-surface and intracellular CSC markers