Dissociated Tumor Cells - Whitepaper
Discovery Life Sciences - Aug. 17, 2017

Dissociated Tumor Cells

A Viable Alternative to Fresh Tumor Tissue
Shawn Fahl, PhD
Justin Sleeman

INTRODUCTION
Primary tumor tissue is an integral part of therapeutic anddiagnostic research, but the logistics and budgetary challenges ofobtaining fresh tissues can often lead to significant delays forresearch projects. The purpose of Dissociated Tumor Cells (DTCs)
is to serve as a viable alternative to acquiring fresh tumor tissue.
DTCs can be acquired quickly from inventory, from a variety ofdifferent solid tumor indications and unique patient donors, andare more cost effective than acquiring fresh tissue and working
through dissociation protocols in house. These viable single cellsuspensions are useful for many of the same downstream
applications as fresh tissue (Box 1A). DTCs contain all of thevarious cell populations present in tumor microenvironment:
tumor, immune and support cells (Box 1B).

A.

Potential Applications:- Cell isolation studies- Flow Cytometry- Cell Culturing- Patient Derived Xenografts(PDX Models)- Drug response and targetdiscovery- 3D Organoids- Sequencing

B.

Ovarian Cancer

EpCAM

Dr. Shawn Fahl has led a thorough examination of Discovery Life
Science’s DTC samples, conducted on 26 post-thaw samples
across five major oncology indications. The body of thiswhitepaper presents the data and analysis from this study. Some
important highlights from the findings are listed below:

Box 1.

Key Points:- DTCs contain high percentages of viable cells bothpre-freeze (81.3%) and post-thaw (69.7%)- DTCs consist of the full cellular composition of the tumor
microenvironment (tumor, immune and support cells)

CD45- All lymphoid subsets can be found in dissociatedtumor cells (T cells, B cells and NK cells)- Myeloid cells can be found in DTCs

2018
DiscoveryLife
LifeSciences©©
2021
DiscoverySciences

www.dls.com/biopharma/biospecimens/tissue-dissociations

Dissociated Tumor Cells - Whitepaper
Discovery Life Sciences - Aug. 17, 2017

Dissociated Tumor Cells, or DTCs, are single cell suspensions
generated from fresh primary tumors using both enzymatic andmechanical dissociation. Our tumor dissociation protocol has
been validated for 5 cancer indications: colorectal cancer, kidneycancer, lung cancer, melanoma, and ovarian cancer. This gentle
procedure results in an average viability of 81.3% across allindications (Figure 1). Following isolation, single cell suspensions
are cryopreserved and stored in liquid nitrogen. Cells are thawed
at 37°C, and following cryopreservation, an average viability of
69.7% was observed across all validated indications (Figure 1).
Our dissociation protocol, therefore, provides excellent viability of
DTCs and greater than 1 million viable cells per vial.

Figure 1.
Viability of DTCs in Validated Indications
Prior to and Following Cryopreservation.

% Viable Cells

Dissociated Tumor Cells (DTCs) Are Viable Following
Dissociation and Cryopreservation

DTCs Recapitulate the Cellular Composition of theTumor Microenvironment
The tumor microenvironment is composed of numerous cell
populations in addition to tumor cells, including immune cells andtumor support cells such as fibroblasts and endothelial cells1. To
determine if our dissociated tumor cells accurately represented
the cellular components of the tumor microenvironment, we
performed flow cytometric analysis of each of the 5 validated
indications for the presence of tumor and immune cells. Immune
cells, identified using the pan-hematopoietic cell marker CD45,
were present in all indications tested (Figure 2 A-B). Unlike
immune cells, which ubiquitously express CD45, tumor cells fromdifferent indications have differing cell surface expression.
Epithelial cell adhesion molecule, or EpCAM, is expressed on manydifferent tumors2, and we were able to readily identify EpCAM+
tumor cells in colorectal, lung, and ovarian cancer DTCs (Figure 2A).
Melanoma tumor cells, on the other hand, do not express EpCAM3,
but do often express melanoma cell adhesion molecule, orCD1464. Indeed, melanoma tumor cells, as identified as CD146+,
were observed in Melanoma DTCs (Figure 2B). Across allindications, we observed patient-specific variability in thepercentage of tumor cells (Figure 2C) and immune cells (Figure 2D),
highlighting the inherent differences between different patientsand indications.

2018
DiscoveryLife
LifeSciences
Sciences©©
2021
DiscoveryThe viability of DTCs was measureddirectly following dissociation prior tocryopreservation and immediately
following thawing. Viability wasmeasured on a Nexcelom Cellometerby dual-fluorescence using acridine
orange and propidium iodide. Data iscompiled from 91 unique DTC samples
across the 5 validated indications.

www.dls.com/biopharma/biospecimens/tissue-dissociations

Dissociated Tumor Cells - Whitepaper
Discovery Life Sciences - Aug. 17, 2017

Figure 2.

Ovarian Cancer

B.

CD45

CD45D.

Tumor and Immune Cells are Both
Present in DTCS.
A. Ovarian Cancer DTCs were analyzed
for the presence of tumor cells (EpCAM+),immune cells (CD45+), and tumor support
cells (EpCAM- CD45-). Live cells wereidentified using propidium iodide and red
blood cells were excluded by GlyA surface
expression.

Immune Cells

B. Melanoma DTCs were analyzed for thepresence of tumor cells (CD146+),
immune cells (CD45+) and tumor support
cells (CD146- CD45-) after removal of red
blood cells by GlyA and dead cells bypropidium iodide.
C. Compilation of the percentage oftumor cells (defined as either EpCAM+ orCD146+ depending on indication) from 26
unique DTC samples across the 5validated indications.

% CD45+ Cells

Tumor Cells% Tumor Cells

C.

MelanomaCD146

EpCAM

A.

D. Compilation of the percentage ofimmune cells (defined as CD45+) from 26
unique DTC samples across the 5validated indications.

All Major Immune Cell Subsets are Present in DTCs
The immune cell filtration into the tumor microenvironment is a heterogeneous mixture of cells including
lymphoid cells, such as T cells and B cells5, as well as various myeloid cell populations6. As the percentage ofCD45+ immune cells was maintained following dissociation and cryopreservation, we next evaluated what
immune cell subsets constituted the CD45+ cells present in our DTC samples by flow cytometry. We werereadily able to detect CD4 T cells, CD8 T Cells, B cells, and NK cells from the dissociated tumor samples(Figure 3A). Furthermore, there was patient-specific variability in the percentage of each lymphocyte subset
across the 5 validated indications (Figure 3B). The major lymphoid subsets, therefore, are easily identified
within our DTC samples.
Tumor-infiltrating myeloid cells have recently become a very active area of research, as they play a potentiallyvital role during tumor pathogenesis6. Examination of the CD45+ immune cells present within our dissociatedtumor cells revealed that myeloid cells were maintained during the dissociation and cryopreservation process.
Indeed, we were able to detect CD11b+ myeloid cells, as well as CD14+ monocytic and CD15+
polymorphonuclear cells, within our DTC samples (Figure 4A). Similar to the lymphocyte subsets, the myeloidsubsets demonstrated patient-specific variability across our 5 validated indications (Figure 4B). Collectively,
the immune cell profiling of our DTC samples demonstrated that the major lymphoid and myeloid cells aremaintained and viable following dissociation and cryopreservation.

© 2018 Discovery Life Sciences© 2021 Discovery Life Sciences

www.dls.com/biopharma/biospecimens/tissue-dissociations

Dissociated Tumor Cells - Whitepaper
Discovery Life Sciences - Aug. 17, 2017

Figure 3.

CD8 T Cells

Lymphocyte Subsets are Present in DTCs.
A. Lung cancer DTCs were analyzed forthe presence of CD4 T cells (CD3+ CD4+),
CD8 T cells (CD3+ CD8+), and B cells(CD19+). Ovarian cancer DTCs wereanalyzed for the presence of NK cells(CD3- CD56+). Live cells were identified
by propidium iodide and immune cells
were identified by CD45.

CD8CD4 T Cells

CD4A.

CD3

B Cells

NK Cells

SSC

CD3CD3B. The percentage of each lymphocyte
population of CD45+ cells from a
representative DTC sample for eachvalidated indication of 5-6 unique DTC
samples per indication analyzed.

CD19CD56B.

© 2018 Discovery Life Sciences© 2021 Discovery Life Sciences

www.dls.com/biopharma/biospecimens/tissue-dissociations

Dissociated Tumor Cells - Whitepaper
Discovery Life Sciences - Aug. 17, 2017

Figure 4.

A.

Myeloid Cells are Present in DTCs.

SSC

CD14

A. Ovarian DTCs were analyzed for thepresence of myeloid cells (CD11b+),
monocytes (CD14+), andpolymorphonuclear cells (CD15+). Livecells were identified by propidium iodide
and immune cells were identified by CD45.

CD11b

CD15

B. The percentage of each myeloid
population of CD45+ cells from a
representative DTC sample for eachvalidated indication of 5-6 unique DTC
samples per indication analyzed.

B.

2018
DiscoveryLifeLifeSciences©©
2021
DiscoverySciences

www.dls.com/biopharma/biospecimens/tissue-dissociations

Dissociated Tumor Cells - Whitepaper
Discovery Life Sciences - Aug. 17, 2017

References:
1. Chen, F. et al. (2015) BMC Med 13:45
2. Spizzo, G. et al. (2011) J Clin Pathol 64:415-20
3. Odashiro, D. et al. (2006) Cancer Cell Int 6:26
4. Lei, X. et al. (2015) Cancer Cell Int 15:3
5. Jochems, C. and Schlom, J. (2011) Exp Biol Med 236:567-79
6. Elliott, L. et al (2017) Front Immunol 8:86

2018DiscoveryLife
LifeSciences
Sciences©©
2021
Discovery

www.dls.com/biopharma/biospecimens/tissue-dissociations