Assessment of the number of local cytotoxic T lymphocytes required for degradation of micrometer-size tumor spheroids.
ABSTRACT Adoptive immunotherapy with human cytotoxic T lymphocytes (CTL) is a promising cancer treatment. Previously we showed that human CTLs against various types of tumors can be efficiently produced by coculturing peripheral blood cells with target cells. The aims of this study were to simulate the interaction of CTLs and micrometer-size tumor tissues in vitro and to assess the required number of CTLs at local tumor sites for degradation of a tumor. Allogeneic CTLs against a human transitional cell carcinoma cell line and autologous CTLs against a renal cell carcinoma cell derived from a surgical specimen were generated. The cytotoxic activities of CTLs against tumor cells in monolayer culture and tumor spheroids formed in U-bottom 96-well culture plates were assessed. Both allogeneic and autologous CTLs showed greater destructive activity than lymphokine activated killer (LAK) cells against target tumor spheroids. CTLs inoculated at E/T ratios of 0.1 to 1 coexisted with the tumor spheroid for 5 to 6 days and then increased in number with apparently lethal activity against the tumor spheroid. In contrast to CTLs, the increase in LAK cell numbers was scarcely observed, and the proliferated LAK cells did not show cytotoxicity against the tumor spheroid. These observations suggest that, when a small number of CTLs reach a local tumor site, they can destroy micrometer-size tumors after considerable local proliferation.
- SourceAvailable from: Wolfgang Mueller-Klieser[Show abstract] [Hide abstract]
ABSTRACT: The present article highlights the rationale, potential and flexibility of tumor spheroid mono- and cocultures for implementation into state of the art anti-cancer therapy test platforms. Unlike classical monolayer-based models, spheroids strikingly mirror the 3D cellular context and therapeutically relevant pathophysiological gradients of in vivo tumors. Some concepts for standardization and automation of spheroid culturing, monitoring and analysis are discussed, and the challenges to define the most convenient analytical endpoints for therapy testing are outlined. The potential of spheroids to contribute to either the elimination of poor drug candidates at the pre-animal and pre-clinical state or the identification of promising drugs that would fail in classical 2D cell assays is emphasised. Microtechnologies, in the form of micropatterning and microfluidics, are also discussed and offer the exciting prospect of standardized spheroid mass production to tackle high-throughput screening applications within the context of traditional laboratory settings. The extension towards more sophisticated spheroid coculture models which more closely reflect heterologous tumor tissues composed of tumor and various stromal cell types is also covered. Examples are given with particular emphasis on tumor-immune cell cocultures and their usefulness for testing novel immunotherapeutic treatment strategies. Finally, tumor cell heterogeneity and the extraordinary possibilities of putative cancer stem/tumor-initiating cell populations that can be maintained and expanded in sphere-forming assays are introduced. The relevance of the cancer stem cell hypothesis for cancer cure is highlighted, with the respective sphere cultures being envisioned as an integral tool for next generation drug development offensives.Journal of Biotechnology 07/2010; 148(1):3-15. · 3.18 Impact Factor
Cytotechnology 37: 31–40, 2001.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
Assessment of the number of local cytotoxic T lymphocytes required for
degradation of micrometer-size tumor spheroids
Koji Kawai1,2, Hitoshi Hayashi1,2, Yoshinobu Ozaki1, Kaoru Saijo1, Shu Qin Liu1,
Hideyuki Akaza2& Tadao Ohno1∗
1RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), Koyadai, Tsukuba Science City,
Ibaraki 305, Japan;2Department of Urology, Institute of Clinical Medicine, University of Tsukuba, Tennodai,
Tsukuba Science City, Ibaraki 305, Japan (∗Author for correspondence; E-mail: email@example.com; Fax:
+81 0298 36 9049)
Received 16 January 2001; accepted 7 June 2001
Key words: adoptive immunotherapy,cytotoxic T lymphocyte, spheroid, three-dimensional culture, tumor
Adoptive immunotherapywith human cytotoxic T lymphocytes(CTL) is a promising cancer treatment. Previously
we showed that human CTLs against various types of tumors can be efficiently produced by coculturing peripheral
blood cells with target cells. The aims of this study were to simulate the interaction of CTLs and micrometer-size
tumor tissues in vitro and to assess the required number of CTLs at local tumor sites for degradation of a tumor.
Allogeneic CTLs against a human transitional cell carcinoma cell line and autologous CTLs against a renal cell
carcinoma cell derived from a surgical specimen were generated. The cytotoxic activities of CTLs against tumor
cells in monolayer culture and tumor spheroids formed in U-bottom 96-well culture plates were assessed. Both
allogeneic and autologous CTLs showed greater destructive activity than lymphokine activated killer (LAK) cells
against target tumor spheroids. CTLs inoculated at E/T ratios of 0.1 to 1 coexisted with the tumor spheroid for 5 to
6 days andthen increasedin numberwith apparentlylethal activityagainst the tumorspheroid. Incontrast to CTLs,
the increase in LAK cell numbers was scarcely observed, and the proliferated LAK cells did not show cytotoxicity
against the tumor spheroid. These observations suggest that, when a small number of CTLs reach a local tumor
site, they can destroy micrometer-size tumors after considerable local proliferation.
Abbreviations: CTL, cytotoxic T lymphocyte; LAK cell, lymphokine activated killer cell; TIL, tumor-infiltrating
lymphocyte; E/T ratio, effector/target ratio; PBMC, peripheral blood mononuclear cell; CV assay, crystal violet
Human cytotoxic T lymphocytes (CTL) can be effi-
ciently generated in vitro by coculturing peripheral
blood mononuclear cells (PBMC) with cultured tar-
get tumor cells or with formalin-fixed and paraffin-
embedded tumor sections using a medium containing
interleukin (IL)-1, 2, 4 and 6 (Liu et al., 1995, 1996,
1998). The CTLs killed over 80% of monolayer-
cultured target tumor cells at a low effector/target
(E/T) ratio within 24 hr (Liu et al., 1995, 1996, 1998).
These results suggest that adoptive immunotherapy
with in vitro generated CTLs is promising for can-
cer patients. Indeed, preliminary clinical studies using
in vitro activated T cells showed therapeutic benefit
against several cancers such as renal cell carcinoma,
glioma and malignant melanoma (Plautz et al., 1998;
Chang et al., 1997). However, these reports also sug-
gest that the present adoptive immunotherapy with
CTLs is not powerful enough to completely degrade
tumor mass. Several reasons for the limited efficacy
were proposed: heterogeneity of the population of in-
effector cells to tumor sites, and sub-optimal activity
of the effector cells at the local tumor sites (Yee et al.,
Many investigators consider that the CTL activ-
ity of tumor-infiltrating lymphocytes (TIL), which
are the most widely used effector cells in adoptive
immunotherapy, plays a major role in tumor regres-
sion (Rosenberg et al., 1988; Topalian et al., 1989;
Bukowski et al., 1991). However, the median percent
111In injectate per gram of tumor was reported to be
only 0.0021in the study of111In-labeledTIL infusion
for melanoma patients (Pockaj et al., 1994). These
observations suggest that the efficacy of adoptive im-
munotherapy in vivo depends primarily on the activity
of a small fraction of the effector cells. Thus, more
precise knowledge of effector cell activities at local
tumor sites and a more efficient method of delivery
of effector cells are necessary. Two points need to be
determined. First, whether the high level of cytotoxic
activity of CTLs observed in monolayer culture truly
carries over to tumor tissue composed of packed mul-
tilayers tumor cells. Second, how many CTLs must
be delivered to the local tumor site to exert sufficient
In order to address these questions, we conduc-
ted experiments with multi-cellular tumor spheroids
as a modelofmicrometer-sizetumortissues. Although
lackingappropriatestromaandbloodvessels, the mul-
ticellular tumor spheroid model is thought to provide
a useful experimental model to analyze the interaction
between lymphokine activated killer (LAK) cells and
solid tumor cells at the local tumor sites (Jaaskelainen
et al., 1989; Iwasaki et al., 1990). In the present study,
we used U-bottom 96-well culture plates (Koide et al.,
1990) to evaluate the interaction between human al-
logeneic and autologous CTLs and solid uniform-size
tumor spheroids. The results suggest that CTLs can
efficiently degrade the spheroid after their numbers
increased at the local site.
Materials and methods
Target tumor cells and formation of uniform spheroids
Human bladder transitional carcinoma cell line T24
obtained from RIKEN Cell Bank was maintained in
modified Eagle’s medium (MEM) containing 10%
fetal bovine serum (FBS). Renal cell carcinoma
TUHR10TKB cells were derived from the surgically
resected specimen of a 69-year-old man and were
maintained in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% FBS. For spheroid forma-
tion and maintenance, the medium RHAMα contain-
ing 5% heat-inactivated FBS was used. RHAMα is a
mixture of RPMI 1640, Ham’s F12, and MEMα at the
ratio of 3:1:1 by volume that has been developed for
serum-free culture of LAK cells (Kawai et al., 1992).
Spheroids were formed in U-bottom 96-well culture
plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan).
The tumor cells, 7.5×103in 100 µl of medium, were
seeded in each well of the plates. After one hour in-
cubation at 37◦C in a humidified CO2 incubator, the
and the culture was continued. A uniform-size spher-
oid was formed in each well within 24 hr and was
maintained with a half medium change every other
Generation of CTLs and LAK cells
Generation of CTLs was carried out as described pre-
viously (Liu et al., 1995, 1998). Briefly, PBMCs
from heparinized peripheral blood were separated by
suspended at a concentration of 1×106cell ml−1
in RHAMα medium supplemented with autologous
plasma(5%)or heat-inactivatedFBS (5%), IL-1(Gen-
zyme Co., 167 U ml−1), IL-2 (Sionogi Co., Ltd.,
67 U ml−1), IL-4 (Genzyme Co., 67 U ml−1), and
IL-6 (Genzyme Co., 134 U ml−1). The PBMCs were
cocultured in 6-well culture plates which contained
a confluent monolayer of the target cancer cells pre-
viously irradiated with 30 Gy. Half of the culture
medium was changed every other day. After com-
plete lysis of irradiated target cells (usually 2 weeks),
with live target cells at an E/T ratio of 10.
LAK cells were prepared by culturing PBMCs
(1×106cell ml−1) in RHAMα medium supplemen-
ted with autologous plasma (5%) or heat-inactivated
FBS (5%) and IL-2 (500 U ml−1) for 2–4 weeks. The
mouse anti-human CD3 monoclonalantibody (OKT3,
Nichirei Co.) at a final concentration of 0.1 µg ml−1
was added weekly for the first 4 weeks. Allogeneic
CTL and LAK cells against T24 (T24-CTL and T24-
LAK, respectively) were induced from PBMCs of a
healthy volunteer, and autologous CTL and LAK cells
(TU-CTL and TU-LAK, respectively) against the tar-
get TUHR10TKB cells were induced from PBMCs of
the renal cell carcinoma patient TUHR10.
Figure 1. Phase-contrast photomicrographs and histology of spheroids. (a) A typical T-24 spheroid 1 hr after inoculation of T24-CTL at E/T
ratio of 0.1; (b) and (c) T24-spheroids after 7 days coculture with LAK cells (b) or with CTL (c) at the initial E/T ratio of 0.1. Lymphocytes
proliferated around the spheroids. Note that the surface has become indistinct and lost integrity in the spheroid treated with CTL (c).
Figure 1d-e. (d) and (e) Hematoxylin-eosin stained cross-sections (×200, the length of one grid is 500 µm) of control T24-spheroid (d) and
TU-spheroid (e) at 7 days after the initiation of spheroidal culure. Note that viable cancer cells arranged in multilayers lack central necrosis.
Cytotoxicity of effector cells against monolayer target
The cytotoxic activity of CTLs against target cells in
monolayer culture was assayed by crystal violet (CV)
staining as described previously (Liu et al., 1995).
This low-cost CV assay is as sensitive for assessment
of the killing activity of lymphocytes against adherent
target cells as the standard51Cr-release cytotoxicity
assay (Liu et al., 1995) in the range of E/T ratios of
10 or below. Above this E/T ratio, underestimation,
though slight, of the killing activity of effector cells
Briefly, the target cells, 1×104cells/well in
200 µl culture medium, were seeded in each well of
96-well plates and were precultured overnight. After
the culture plate was washed with PBS, the cultured
lymphocytessuspended in 200 µl of LAK cell or CTL
culture medium were added as effector cells to each
well at the indicated E/T ratio. The cells were cocul-
turedfor12hr, andthen werewashedoncegentlywith
PBS. Adherent target cells were fixed for 1 hr with
10% (v/v) formalin (200 µl/well), then stained with
crystal violet solution (0.4% in water, 100 µl/well) for
30 min at room temperature. The plate was washed
with tap water and dried at room temperature. To each
well, 200 µl of 80% methanol was added and the OD
at 570 nm of each well was determined.
Figure 2. Numberof cells per spheroid and percentage of viable cells inthe spheroidal culture. Each data point represents the mean oftriplicated
determinations with SD bars.
The mean of triplicated samples was determined,
and the percentage of surviving target cells was ex-
pressed as follows:
Surviving target cells (%) = (B–C)/A×100
where A is the absorbance of control target cells pre-
cultured in a separate plate just before the addition of
the effector cells, B is that of remaining target cells
to which effector cell were added, and C is that of
effector cell only. Each value in the figures represents
the mean of triplicates accompaniedby a SD errorbar.
Note that some results show more than 100% survival
because of rapidgrowthof target cells duringthe 12 hr
incubation. Experiments for the determination of the
cytotoxicity were repeated twice or more.
Determination of spheroid volume and cell numbers
in a spheroid
In the spheroid culture, all tests were performed after
confirming the formation of a single spheroid in each
U-bottomed well of the 96-well culture plate. After
incubation with or without effector cells, the tumor
spheroid was gently aspirated with micropipette tip
fixed with 50 µl of 10% formalin. The spheroid size
was measured under a microscope. The volume (V)
of the spheroid was calculated from the 2 orthological
diameters (a and b) using V = 3.14 aXb2/6 where b
is the longer axis. Morphological analysis was car-
ried out after hematoxylin-eosin (H–E) -stained sec-
tions were cut from formalin-fixed paraffin-embedded
To count cells, the spheroids were dissociated with
0.2% trypsin, and the total number of viable cells
was counted on a hemocytometer by the conventional
trypan-blue staining method. Each experiment was
repeated at least twice.
For cytometric analyses, 2×105effector cells were
stained with FITC-conjugated monoclonal antibody
formed with a FACScan (Becton-Dickinson). FITC-
conjugated monoclonal antibodies used were anti-
CD3, anti-CD4 and anti-CD8 (Nichirei Co., Tokyo,
Japan, OKT-3, OKT-4 and OKT-8, respectively) and
anti-CD45 (Dako Japan Inc.).
◦C for 30 min. Flow cytometry was per-
Figure 3. Cytotoxicity of CTL and LAK cells against monolayer-cultured target tumor cells. (a) and (b) Cytotoxicty of CTL and LAK cells
against monolayer-cultured T24 cells (a) and TUHR10TKB cells (b) determined by the 12-hr killing assay. (c) Specific cytotoxicty of allogeneic
T24-CTL determined by the 12- hr killing assay. Each data point represents the mean of triplicated determinations with SD bars.
Formation and viability of the micrometer-size tumor
Both T24 and TUHR10TKB cells formed a spheroid
in each well without satellite aggregate formation or
fragmentation (Figures 1a, d and e). Diameters of
T24-shperoids and TU-spheroids were 382±35 µm
(mean±SD, n = 9) and 589±95 µm, respectively,
just after the spheroid formation. The spheroids re-
tained their morphology for at least 2 weeks in the
culture. T24 cells continued proliferation in the spher-
oidal culture for two weeks and, consequently, the
cell number in the spheroid doubled. In contrast,
TUHR10TKB cells did not exhibit obvious prolifer-
ation in the spheroid (Figure 2). Trypan blue vital
staining showed that over 90% of cells forming the
spheroids were viable (Figure 2, dotted lines). H-E
staining of the spheroids after 7 days of culture re-
vealed no central necrosis in the spheroids (Figures 1d
Cytotoxic activities of CTLs against monolayer target
Both allogeneic T24-CTL and autologous TU-CTL
were generated against monolayer cultured target T24
cells and TUHR10TKB cells, respectively. They con-
sisted of over 95% CD3+, over 95% CD8+and 1–5%
Figure 4. Cytotoxicity of allogeneic CTL and LAK cells against the tumor spheroids at the E/T ratio of 4. (a) Cytotoxicity of CTL and LAK
cells against T24-spheroids determined by sphere volume. (b) Total number of cells and CD45-negative cells per well in which T24-spheroids
were treated with LAK cells or CTL for 24 hrs. Each point represents the mean of triplicated determinations with SD bars.
CD4+cells. T24-CTL showed higher cytotoxicity
than T24-LAK cells derived from the PBMCs of the
same subject. Higher cytotoxicity was also observed
in TU-CTL than in TU-LAK cells generated from the
autologous PBMCs of the patient TUHR10. These
two CTLs killed over 90% of the correspondingtarget
tumor cells within 12 hr at E/T ratios of 4 and 10, re-
spectively(Figures3a andb). ThecytotoxicityofT24-
CTL was specific to T24 cells so far examined. T24-
CTL did not kill two uterine cancer cell lines, OMC-1
laboratory (Figure 3c). TU-CTL were also specific to
the autologous renal carcinoma, TUHR10TKB cells,
and did not kill renal carcinoma cell lines Hpt.10,
Hpt.15, TUHR3TKB or TUHR4TKB, which were es-
tablished in our laboratory as previously reported (Liu
et al., 1998).
Cytotoxicity of CTLs against tumor cell spheroids
T24-CTL inoculated at an E/T ratio of 4 (3×104
cells/well) significantly reduced the volume of T24-
spheroids within 24 hr, and the T24-spheroids had
totally disintegrated by 48 hr (Figure 4a). In contrast,
T24-spheroids treated with T24-LAK cells showed
Figure 5. Cytotoxicity of CTL and LAK cells against the tumor spheroids at the E/T ratio of 0.1 and 1. (a) Effector cell proliferation in the
coculture. Allogeneic CTL or LAK cells were inoculated at 0.75×103cells/well into each well containing a T24 spheroid (E/T ratio = 0.1 at
day 0). Autologous CTL or LAK cells were inoculated at 7.5×103cell /well to each well containing a TU-spheroid (E/T ratio = 1 at day 0).
(b) and (c) Cytotoxicity of CTL and LAK cells against T24-spheroid (b) and TU-spheroid (c) determined by the sphere volume.
a transient decrease in volume at 24 hr, but their
volume increased again with further incubation up to
48 hr (Figure 4a). When cocultured with T24-CTL,
the surface of the T24-spheroids became indistinct
and lacked integrity under microscopic examination.
These morphologicalchangeswere hardlyobservedin
the T24-spheroids treated with T24-LAK cells.
To determine the number of tumor cells in each
well, spheroids with or without effector cells were
dissociated by trypsinization to form single-cell sus-
pensions after 24 hr coculture, and the number of
whole cells in the well was counted with a hemocyto-
meter. The number of the tumor cells was calculated
from the ratio of CD45-negative cells in the cell
suspension after staining with anti-CD45 monoclonal
antibody and the flow cytometry. As shown in Fig-
ure 4b, the number of tumor cells was significantly
decreased when T24-spheroids were cocultured with
T24-CTL. In contrast, there was no significant de-
crease in the numberof tumorcells in the wells treated
with T24-LAK cells.
When T24-CTLand TU-CTL were inoculatedinto
spheroid cultures at the E/T ratios of 0.1 and 1, re-
spectively, they did not instantly reduce the volume of
thespheroids.At theseE/T ratios, bothoftheT24-and
TU-spheroids were morphologically stable for at least
5 days. During this period, the number of T24-CTLs
and TU-CTLs increased constantly in the coculture
(Figure 5a). However, both T24-LAK cells and TU-
LAK cells maintained very slow growth for 8 and 12
days in coculture, respectively.
In contrast to the CTL growth, the increase in
the volume of the CTL-treated T24-spheroids was
suppressed for 5 days and began to decrease rapidly
after 5 day in coculture. Complete degeneration of the
T24-spheroids was observed after 7 days in coculture,
although an apparent volume increase was observed
in the control T24-spheroids and the LAK cell-treated
T24-spheroids (Figures 1b and c, Figure 5b). In the
TU-spheroids, as well, rapid destruction was observed
after 5 days of coculture. T24-LAK cells and TU-
LAK cells did not show destructive activity against
corresponding tumor spheroids (Figures 5b and c).
Phenotypic analyses of the increased number of
CTLs cocultured with tumor spheroids revealed that
T24-CTL consisted of over 99% CD3+, 1.4% CD4+
and 95% CD8+, and TU-CTL consisted of over 99%
CD3+, 21% CD4+and 86% CD8+.
In the present study, we cultured tumor-cell spheroids
as an experimental model of micrometer-size tumor
tissues in order to evaluate activities at the local tumor
sites. First, in the spheroid model, the CTL showed
higher destructive activity than LAK cells against the
allogeneic and autologous tumor spheroids, as has
been observed against the monolayer culture of the
target tumor cells. Second, CTLs inoculated at E/T
ratios of 0.1 and 1 initially coexisted with the tumor
spheroids for 5–6 days, and then rapid reduction of
the volume of the tumor spheroids was observed (Fig-
ure 5). Third, the proliferated lymphocyte population
was CD8+ dominant, similar to the population at the
start of the coculture. In contrast to CTLs, prolifer-
ation was not observed in the allogeneic T24-LAK
cells (Figure 5a). Autologous TU-LAK cells showed
moderate proliferation, but the proliferated LAK cells
tumor spheroids (Figure 5c). At the E/T ratios of 0.1
and 1, the number of CTLs may have not been suffi-
cientto degradethetumorspheroidintheinitial5 days
of coculture (Figures 5b and c). After 5 days of co-
culture with the tumor spheroid, T24-CTLs numbered
3×104per well. Thus, the estimated local E/T ratio at
day 5 was above 5. Similarly, the estimated local E/T
ratio in the coculture of TU-CTL and the TU-spheroid
exceeded 6 at day 5. These observations suggest that
some minimum E/T ratio in which the effectors cells
are definitively superior to the target cells is essential
at the local tumor site to degrade the tumor tissues.
While CTLs showed promising activity against
solid-tumor spheroids in the present in vitro study, it
may be difficult to directly deliver sufficient numbers
of effectorcells to tumortissue to achievehighenough
E/T ratio in vivo because of non-specific entrapping
of infused lymphocytes (Lotze et al., 1980). Although
there has been some controversy about tracking pat-
gene marking studies did not support tumor-homing
ability of bulk TILs (Economou et al., 1996; Mer-
rouche et al., 1995). To overcome this limitation, two
methods to achieve definitively superior E/T ratio in
vivo are possible: One is to increase the homing ca-
pacity of effector cells to localize preferentially into
the tumor tissues, and another is to support the pro-
liferation of effector cells at the local tumor site. Sev-
eral animal studies suggested that the in situ-activated
CTLs but not bulk TILs may have significant capa-
city to localize to tumor tissues (Matsumura et al.,
1994; Park et al., 1995). In addition, recent ana-
lysis of the cell-surface phenotypes of TILs revealed
that the TIL which expresses high levels of several
adhesion molecules including cutaneous lymphocyte
antigen, showed enhanced integrin-mediatedadhesion
to dermal micro-vascularendotheliumin vitro (Adams
et al., 1997). These observations suggest that modi-
fication of the cell-surface adhesion molecules may
increase a phenotype with selective homing capacity
in vivo. The present multicellular tumor spheroid is
thoughtto bea usefulexperimentalmodelforinvestig-
ating the adhesion molecules that mediate interaction
between CTL and solid tumor cells. The expansion
of CTLs in situ would be the other possible strategy
to achieve the high local E/T ratio. The present study
demonstrated that the CTL is a promising effector cell
having superior ability to multiply at the local tumor
site while retaining cytotoxicity compared to LAK
cells. Althoughthe results were obtainedunderthe op-
timal environment for CTL growth, the present model
might be useful to investigate the optimal microenvir-
onment that supports CTL proliferation and activity at
local tumor sites. These findings will be quite help-
CTL adoptive immunotherapy.
The present study was supported in part by the Spe-
cial CoordinationFundof the Science andTechnology
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