Citation Garg AD, Dudek AM, Ferreira GB, Verfaillie T, Vandenabeele P, Krysko DV, Mathieu C, Agostinis P, 2013 ROS-induced autophagy in cancer cells assists in evasion from determinants of immunogenic cell death Autophagy. 2013 Sep;9 Archived version Author manuscript: the content is identical to the content of the published paper, but without the final typesetting by the publisher Published version https://www.landesbioscience.com/journals/autophagy/2012AUTO0449R3.pdf Journal homepage https://www.landesbioscience.com/journals/autophagy. Author contact your email [email protected]your phone number + 32 (0)16 37 75 36 IR https://lirias.kuleuven.be/handle/123456789/415137 (article begins on next page)
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References:
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Figure Legends:
Figure 1. GPX4 overexpression and L-histidine suppress Hyp-PDT induced ecto-CALR and secreted ATP
in cancer cells. (A, B) T24 cells were preincubated with 25 mM of L-histidine for 30 min followed by
treatment with indicated doses of Hyp-PDT (medium – 1.35 J/cm2, high – 2.16 J/cm2) or left untreated
(CNTR) and recovered 1 h post-PDT. On one hand, the surface proteins were biotinylated and
immunoblotted (A) while on the other, the resulting conditioned media was analyzed for the presence of
ATP (B). In (B), data are presented as relative light unit (RLU) values (3 independent experimental
determinations with duplicate determinations in each; mean ± s.e.m.; *P<0.05 as indicated by bars). (C, D)
HeLa cells stably expressing either empty vector (Neo) or overexpressing GPX4 were treated with the
indicated doses of Hyp-PDT (or with medium dose wherever not mentioned) or left untreated (CNTR). The
cells were recovered 1 h post-PDT and in one case the surface proteins were biotinylated followed by
immunoblotting (C) while on the other, the resulting conditioned media was analyzed for the presence of
ATP (D). In D, data are presented as relative light unit (RLU) values (3 independent experimental
determinations with duplicate determinations in each; mean ± s.e.m.; *P<0.05 as indicated by the bar). (E,
F) L929 cells stably expressing either empty vector (Neo) or overexpressing GPX4 were treated with
medium Hyp-PDT dose or left untreated (CNTR). The cells were recovered 1 h post-PDT and in one case
the surface proteins were biotinylated followed by immunoblotting (E) while on the other, the resulting
conditioned media was analyzed for the presence of ATP (F). In F, data are presented as relative light unit
(RLU) values (4 experimental determinations; mean ± s.e.m.; *P<0.05 as indicated by the bar). Here,
‘+BIO’ indicates controls exposed to buffer with biotin and ‘-BIO’ indicates controls exposed to buffer
without biotin.
Figure 2. ATG5 knockdown or knockout does not affect Hyp-PDT induced ATP secretion. (A, B) T24
cells were transfected with scrambled (SCR) siRNA or an ATG5-specific siRNA (ATG5KD) (A) and
A375m cells expressing control (CO = vector expressing scrambled shRNA) or an ATG5-specific shRNA
(ATG5KD) (B) were lysed followed by immunblotting. ATG5 and actin protein bands were quantified for
the integrated band density via Image J and their ratios calculated (T24 – A; A375m - B). Data are
presented as ATG5/actin ratio values normalized to those of SCR/CO samples multiplied by 100 (3
independent experimental determinations; mean ± s.e.m.). (C) T24 cells or A375m cells were treated with
Hyp-PDT doses of 2.16 J/cm2 or 2.43 J/cm2, respectively. They were recovered 1 h post-PDT and the
resulting conditioned media was analyzed for the presence of ATP. Data are presented as relative light unit
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(RLU) values (3 independent experimental determinations with duplicate determinations in each; mean ±
s.e.m.; statistical analysis was done using the Student’s t-test; *P<0.05 vs. CNTR, #P<0.05 as indicated by
the bars). (D-F) T24 cells (D) or A375m cells (E) transfected with/expressing respective siRNAs/shRNAs
(causing ATGKD as applicable) and wild-type MEF cells (Atg5+/+) or MEF cells lacking atg5 (atg5-/-) (F)
were treated with indicated Hyp-PDT doses (medium - 1.35 J/cm2 and high – 2.16 J/cm2) or the medium
dose (1.35 J/cm2) in case of MEFs; or left untreated (CNTR). They were recovered 1 h post-PDT and the
resulting conditioned media was analyzed for the presence of ATP. Data are presented as relative light unit
(RLU) values (3 independent experimental determinations with duplicate determinations in each; mean ±
s.d.; *P<0.05 vs. respective CNTRs, N.S. = not significant, as indicated by the bars).
Figure 3. ATG5 knockdown in cancer cells increases Hyp-PDT induced, ecto-CALR and accumulation of
oxidatively damaged proteins. (A, B) T24 cells transfected with scrambled (SCR) siRNA or an ATG5-
specific siRNA (ATG5KD) (A) and A375m cells expressing the control (CO = vector expressing scrambled
shRNA) or an ATG5-specific shRNA (ATG5KD) (B) were treated with the indicated Hyp-PDT doses
(medium – 1.35 J/cm2, high – 2.16 J/cm2, higher – 2.43 J/cm2) or left untreated (CNTR). The cells were
recovered 1 h post-PDT and the surface proteins were biotinylated followed by immunoblotting (A). Here
in A and B, ‘+BIO’ indicates controls exposed to buffer with biotin and ‘-BIO’ indicates controls exposed
to buffer without biotin. Ecto-CALR and FAS protein bands were quantified for the integrated band density
via Image J and their ratios calculated (T24 – A; A375m – B; 3 independent experimental determinations,
mean ± s.e.m.; *P<0.05 as indicated by the bars). (C, D) T24 cells (C) and A375m cells (D) transfected
with/expressing respective siRNA/shRNA constructs (as detailed in A, B) were treated with high Hyp-PDT
dose (2.16 J/cm2) or left untreated. They were recovered 1 h post-PDT and the levels of carbonylated
proteins were estimated. In (C and D), the carbonyl content was calculated as, nmols of carbonylated
proteins per mg of total proteins. Data are presented as carbonyl content values normalized to the
respective untreated controls (3 independent experimental determinations - mean ± s.e.m.; *P<0.05 as
indicated by the bars).
Figure 4. Hyp-PDT-induced autophagy in cancer cells suppresses maturation of the interacting dendritic
cells (DCs) in an ecto-CALR-independent manner. (A) A375m cancer cells expressing control CO-shRNA
(indicated by ‘-‘) (CO = vector expressing scrambled shRNA) or an ATG5-specific shRNA (ATG5KD;
indicated by ‘+’) were treated with Hyp-PDT (2.43 J/cm2) or left untreated (CNTR). Thereafter the
respective cells were recovered 24 h post-PDT and cocultured with human-immature DCs (hu-iDCs) for 24
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h. As a positive control, hu-iDCs were stimulated with LPS for 24 h. After coculturing, the cells were
immunostained for CD86/HLA-DR positivity and scored by FACS analysis. Data are presented as
percentage of CD86-positive and HLA-DR-positive cells (3 independent experimental determinations with
duplicate determinations in each; mean ± s.e.m.; *P<0.05, vs. respective CNTR; as indicated by bars). (B-
D) The A375m-hu-iDC coincubation conditioned media obtained during the experiments detailed in (A)
were collected followed by analysis for concentrations of IL10 (B), IL1B (C) and IL6 (D). Absolute
concentrations are depiction of one representative (four replicate determinations; mean ± s.e.m.; *P<0.05 or
N.S. = non-significant, as indicated by the bars) of 3 independent experiments. Data for hu-iDCs only is
not depicted since they produced no recordable levels of these three cytokines. (E) Respective A375m
cancer cells were treated as in (A), and recovered 24 h post-PDT, incubated with either chicken anti-CALR
or isotype IgY antibodies (Ab) and cocultured with hu-iDCs for 24 h. After coculturing, the cells were
immunostained for CD86/HLA-DR positivity and scored by FACS analysis. Data are presented as
percentage of CD86-positive and HLA-DR-positive cells (3 experimental determinations; mean ± s.e.m.;
*P<0.05 as indicated by the bars).
Figure 5. Hyp-PDT-induced autophagy in cancer cells reduces proliferation of CD4+ or CD8+ T cells. (A,
B) A375m cancer cells expressing control shRNA (indicated by ‘+‘ in CO-shRNA A375m line) (CO =
vector expressing scrambled shRNA) or an ATG5-specific shRNA (indicated by ‘+‘ in ATG5-shRNA
A375m line) were treated with Hyp-PDT (2.43 J/cm2) or left untreated (CNTR). Thereafter the respective
cells were recovered 24 h post-PDT and cocultured with human-immature DCs (hu-iDCs) for 24 h. As a
positive control, hu-iDCs were stimulated with LPS for 24 h. After that, the cells were further cocultured
with naïve human T cells for 5 days followed by immunostaining for CD3/CD4 positivity (A) or CD3/CD8
positivity (B); and scoring by FACS analysis. ‘T cells’ represents negative control for T cells proliferation.
Data are presented as percentage of respective proliferating T cells (4 experimental determinations, across
T cells from two separate healthy human donors; mean ± s.e.m.; statistical analysis was done using the
Student’s t-test; *P<0.05 as indicated by bars). (C) The A375m-hu-iDC-T cell coincubation conditioned
media obtained during the experiments detailed in (A, B) were collected followed by analysis for
concentration of IFNG. Hu-iDCs stimulated with LPS for 24 h represent positive controls whereas “T
cells” and “T cells + DCs” are the negative controls for IFNG production. Data are presented as fold
change in absolute concentrations (3 experimental determinations; mean ± s.e.m.; *P<0.05 or N.S. = non-
significant as determined by one-way ANOVA comparison between data sets as indicated by bars). (D)
Schematic overview of cancer cell-associated autophagy’s role in Hyp-PDT induced immunogenic cell
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death (ICD). Hyp-PDT treatment causes photo-oxidative (phox)-ER stress in the treated cancer cells which
is associated with increased surface expose of calreticulin (ecto-CALR), ATP secretion, DC maturation
characterized by increased CD86/HLA-DR positivity and production of IL6 as well as increased
proliferation of CD4+/CD8+ T cells. Here, we observed that autophagy induced following Hyp-PDT
treatment of these cancer cells suppresses various crucial determinants of ICD i.e. ecto-CALR induction
(without affecting ATP secretion), maturation of interacting DCs and IL6 production and proliferation of