OncoImmunology

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PKHB1, a thrombospondin-1 peptide mimic,

induces anti-tumor effect through immunogenic
cell death induction in breast cancer cells

Kenny Misael Calvillo-Rodríguez, Rodolfo Mendoza-Reveles, Luis Gómez-

Morales, Ashanti Concepción Uscanga-Palomeque, Philippe Karoyan, Ana
Carolina Martínez-Torres & Cristina Rodríguez-Padilla

To cite this article: Kenny Misael Calvillo-Rodríguez, Rodolfo Mendoza-Reveles, Luis Gómez-
Morales, Ashanti Concepción Uscanga-Palomeque, Philippe Karoyan, Ana Carolina Martínez-
Torres & Cristina Rodríguez-Padilla (2022) PKHB1, a thrombospondin-1 peptide mimic,
induces anti-tumor effect through immunogenic cell death induction in breast cancer cells,
OncoImmunology, 11:1, 2054305, DOI: 10.1080/2162402X.2022.2054305

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ONCOIMMUNOLOGY
2022, VOL. 11, NO. 1, e2054305 (15 pages)
https://doi.org/10.1080/2162402X.2022.2054305

ORIGINAL RESEARCH

PKHB1, a thrombospondin-1 peptide mimic, induces anti-tumor effect through

immunogenic cell death induction in breast cancer cells
Kenny Misael Calvillo-Rodríguez a,b,c, Rodolfo Mendoza-Revelesa, Luis Gómez-Moralesa,b,c,d,
Ashanti Concepción Uscanga-Palomequea, Philippe Karoyan b,c,d,e,f*, Ana Carolina Martínez-Torres a
*,
and Cristina Rodríguez-Padillaa,g*
a
Facultad de Ciencias Biologicas, Laboratorio de Inmunología y Virología, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, Mexico;
b
Sorbonne Université, Ecole Normale Supérieure, PSL University, CNRS, Laboratoire des Biomolécules, LBM, 75005 Paris, France; cSorbonne Université,
Ecole Normale Supérieure, PSL University, CNRS, Laboratoire des Biomolécules, DRUG Lab, Site OncoDesign, 25-27 Avenue du Québec, 91140 Les Ulis,
France; dKaybiotix, GmbH, Zugerstrasse 32, 6340 Baar, Switzerland; eKayvisa, AG, Industriestrasse, 44, 6300 Zug, Switzerland; fχ-Pharma, 25 Avenue du
Québec, 91140 Les Ulis, France; gLONGEVEDEN SA de CV, Monterrey, Mexico

ABSTRACT ARTICLE HISTORY

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women Received 16 September 2021
worldwide. Recent advances in the field of immuno-oncology demonstrate the beneficial immunosti­ Revised 3 March 2022
mulatory effects of the induction of immunogenic cell death (ICD). ICD increases tumor infiltration by Accepted 14 March 2022
T cells and is associated with improved prognosis in patients affected by triple negative breast cancer KEYWORDS
(TNBC) with residual disease. The aim of this study was to evaluate the antitumoral effect of PKHB1, Breast cancer; immunogenic
a thrombospondin-1 peptide mimic, against breast cancer cells, and the immunogenicity of the cell cell death; thrombospondin
death induced by PKHB1 in vitro, ex vivo, and in vivo. Our results showed that PKHB1 induces 1; PKHB1; tumor cell lysate;
mitochondrial alterations, ROS production, intracellular Ca2+ accumulation, as well calcium- anticancer vaccine
dependent cell death in breast cancer cells, including triple negative subtypes. PKHB1 has antitumor
effect in vivo leading to a reduction of tumor volume and weight and promotes intratumoral CD8 + T
cell infiltration. Furthermore, in vitro, PKHB1 induces calreticulin (CALR), HSP70, and HSP90 exposure
and release of ATP and HMGB1. Additionally, the killed cells obtained after treatment with PKHB1
(PKHB1-KC) induced dendritic cell maturation, and T cell antitumor responses, ex vivo. Moreover,
PKHB1-KC in vivo were able to induce an antitumor response against breast cancer cells in
a prophylactic application, whereas in a therapeutic setting, PKHB1-KC induced tumor regression;
both applications induced a long-term antitumor response. Altogether our data shows that PKHB1,
a thrombospondin-1 peptide mimic, has in vivo antitumor effect and induce immune system activation
through immunogenic cell death induction in breast cancer cells.

Background
immune response against dead cancer cell antigens.6,7
Breast cancer is the most frequent type of cancer among Additionally, inducing ICD increases tumor tissue infiltration
women; its innate and acquired treatment resistance to current by T cells, which plays an essential role in mediating a positive
therapies is the principal problem to treat it, causing the great­ response to chemotherapy and is associated with improving
est number of cancer-related deaths.1 While systemic therapies clinical outcomes in all subtypes of breast cancer.5,8
have increased the survival rates of breast cancer patients, the We recently designed PKHB1 through a structure–activ­
dramatic variations in response rates of patients with distinct ity relationship studies around the C-terminal Binding
clinicopathologic parameters,2 as well as innate or acquired Domain (CBD) of thrombospondin-1 (TSP-1). PKHB1 is
resistance to current therapies,3 make relevant the search for a peptide mimic stable in the serum of mice and human
new effective treatments for the different molecular subtypes of and able to induce a cell death involving CD47 activation
breast cancer, in particular those associated with poor in different cancer cells, especially in hematological
prognosis. malignancies.9–12 The ability of PKHB1 to induce cell
Recently, several clinical studies have demonstrated the death was also observed in leukemic cells from patients
beneficial immunostimulatory effects of inducing immuno­ with aggressive and chemo-resistant phenotypes, without
genic cell death (ICD),4,5 recognized as a critical determinant affecting non-tumoral cells from humans or mice.9,11,13
for the efficiency of cancer therapies. Indeed, this peculiar type Additionally, PKHB1 induces ICD in T cell acute lympho­
of cell death is capable of stimulating a long-term antitumor blastic leukemia.11,14 If this peptide is currently under

CONTACT Ana Carolina Martínez-Torres [email protected] Facultad de Ciencias Biologicas, Laboratorio de Inmunología y Virología Facultad de
Ciencias Biologicas, Laboratorio de Inmunología y Virología, Universidad Autónoma de Nuevo León, Mexico; Philippe Karoyan philippe.karoyan@sorbonne-
universite.fr Laboratoire des Biomolécules, LBM, Sorbonne Université, Ecole Normale Supérieure PSL University, CNRS, Paris, France
*Co-senior authors
Supplemental data for this article can be accessed on the publisher’s website.
© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
e2054305-2 K. M. CALVILLO-RODRÍGUEZ ET AL.

development in pre-clinical studies addressing CLL (χ- Intracellular Ca2+ levels assay
Pharma), little is known about the cell death capacity,
5 × 104 cells/well in 24 wells dishes (Life Science) were left
mechanism, immunogenicity, and the antitumor effect of
untreated or pre-incubated with 2.5 mM BAPTA, and then
PKHB1 in solid cancers with different molecular character­
treated for 2 h with PKHB1 (CC50) or left untreated in med­
istics and poor prognosis such as breast cancer (including
ium. Then, cells were detached, washed with RINGER buffer
the triple negative subtype).
without Ca2+, and resuspended in 200 μL of the same RINGER
Therefore, the aim of this study was to evaluate the
buffer with 0.001 μg/mL of Fluo-4 AM (Life Technologies) and
antitumor potential of PKHB1 in breast cancer cells
0.001 μg/mL of Pluronic F-127 (Life Technologies), incubated
(in vitro and in vivo) including triple negative subtypes
37°C for 30 min. Next, cells were washed with RINGER buffer
and to determine whether it induces antitumor immune
w/o Ca2+ and assessed by BD Accuri C6 flow cytometer (BD
system activation through ICD induction (ex vivo and
Biosciences, Franklin Lakes, NJ, USA) (total population 10,000
in vivo).
cells), and data was analyzed using FlowJo software (LLC,
Ashland, OR, USA).
Methods
Peptide synthesis In vivo model
PKHB1 and 4NGG peptides were synthesized manually, using This study was approved by The Animal Research and Welfare
Fmoc-protected amino acids and standard solid phase peptide Ethics Committee (CEIBA), of the College of Biological
synthesis (SPPS) (supplemental material and methods), as Sciences, number: CEIBA-2018-003. All experiments were
described previously.6 conducted according to Mexican regulation NOM-062-ZOO
-1999. Female BALB/c mice (6- to 8-week-old; 22 ± 2 g weight)
were maintained in controlled environmental conditions (25°C
Cell culture and 12 h light/dark cycle) and supplied with rodent food
MCF-7, MDA-MB-231, and 4T1 cell lines were obtained from (LabDiet, St. Louis, MO, USA) and water ad libitum, and
the ATCC. MCF-7 and MDA-MB-231 cell lines were main­ they were monitored daily for health status. Mice were ran­
tained in DMEM-F12 medium (GIBCO by Life Technologies, domly assigned to different groups for all studies. All experi­
Grand Island, NY, USA), while 4T1 cell line was maintained in ments were designed in accordance with the ARRIVE
RPMI-1640 medium, both were supplemented with 10% of guidelines for animal care and protection (supplemental
fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin- material).15
streptomycin (GIBCO by Life Technologies, Grand Island, NY,
USA), and incubated at 37°C in a controlled humidified atmo­
Tumor establishment
sphere with 5% CO2. Cell count was performed following the
ATCC’s standard protocols. 5 × 105 live 4T1 cells in 100 μL of PBS were injected subcuta­
neously in the left hind. Tumor volume and mice weight were
measured three times per week using a caliper (Digimatic
Cell death induction and inhibition analysis Caliper Mitutoyo Corporation, Japan) and a digital scale
5 x 10 4 cells were plated in 24 wells dishes and left (American Weigh Scale-600-BLK, USA), respectively. Tumor
untreated or treated for 2 h with 100 μM, 200 μM, volume was determined with the formula: tumor volume
300 μM, or 400 μM of PKHB1 (KRFYVVMWKK), or (mm3) = (Length × width2)/2. When tumor reached 70–
300 μM of 4NGG (KRFYGGMWKK). Annexin- 120 mm3, 3 days after inoculation with tumor cells, mice
V-allophycocyanin (Ann-V-APC 0.1 μg/ml; BD were treated daily with 400 μg of PKHB1 in 200 μL of sterile
Pharmingen, San Jose CA, USA), and propidium iodide water by intraperitoneal injection, control mice were treated
(PI, 0.5 μg/ml Sigma-Aldrich) were used to assess phos­ with 200 μL of sterile water. Sixteen days after inoculation with
phatidylserine exposure, cell death, and cell viability quan­ tumor cells, mice were anesthetized with ketamine (i.p. 80 mg/
tification, respectively, in a BD AccuryC6 flow cytometer kg body weight) and xylazine (i.p. 10 mg/kg body weight) and
(BD Biosciences, Franklin Lakes, NJ, USA) (total popula­ were euthanized by cervical dislocation. Tumors from Control
tion 10,000 cells). Data was analyzed using FlowJo soft­ or PKHB1-treated mice, were obtained and fixed in 3.7%
ware (LLC, Ashland, OR, USA). neutral formalin, embedded in paraffin, sectioned (5 μm thick­
The calcium chelator BAPTA (5 mM), the pan-caspase inhi­ ness) and stained with H&E (MERCK). Histopathological ana­
bitor Z-VAD-FMK (Z-VAD, 50 μM), the antioxidant N-Acetyl lyses were done by an external veterinarian pathologist
Cysteine (NAC, 5 mM), the necroptotic inhibitor Necrostatin-1 (National professional certificate 2,593,012).
(Nec-1, 50 μM), the phospholipase C (PLC) inhibitor U73122
(1.25 μM) and the ER receptor inhibitors dantrolene (50 μM)
T cells evaluation
and 2-aminoethoxydiphenyl borate (2-APB, 40 μM) were incu­
bated 30 minutes with the indicated agent, before treatment Sixteen days after tumor inoculation, mice treated (n = 6) or
with PKHB1 (CC50), epirubicin (42.5 μM for MCF-7 and MDA- untreated (n = 6) were anesthetized and sacrificed as described
MB-231 and 5 μM for 4T1 for 24 h), or H2O2 (25 μM for all cell above. Blood was obtained by cardiac puncture and isolation of
lines for 24 h) when indicated. the peripheral blood mononuclear cells (PBMCs) was
 ONCOIMMUNOLOGY e2054305-3

performed by density gradient centrifugation using Ficoll- ATP and High-mobility group box 1 release assay
Hypaque-1119 (Sigma-Aldrich, St Louis, MO, USA). The
2.5 × 105 cells/well in 6-well dishes were left untreated
spleen, lymph node, and tumor were harvested and filtered
(Control) or treated with PKHB1 (CC50) for 2 h.
through a cell strainer (70 μM) with PBS (PBMCs were obtained
Supernatants were recovered, centrifuged at 1600 rpm/10 min­
from the spleen as described above), then 1 × 106 cells/mL were
utes and used to assess extracellular ATP by a luciferase assay
plated and the percent of CD3+, CD4+ and CD8 + T cells was
(ENLITEN kit, Promega, Madison, WI, USA), or HMGB1
observed by flow cytometry with the Mouse T lymphocyte sub­
using the HMGB1 ELISA kit for MDA-MB-231, MCF-7 and
set antibody cocktail CD3 (clone 145–2C11), CD4 (clone RM4-
4T1 cells (BioAssay ELISA kit human or mouse, respectively;
5), and CD8 (clone 53–6.7) (from BD Bioscience) following the
US Biological Life Science Salem, MA, USA) following the
manufacturer’s instructions.
manufacturer’s instructions. Bioluminescence was assessed in
a microplate reader (Synergy HT, Software Gen5; BioTek,
Myeloid-derived suppressor cells (MDSCs) and Tregs Winooski, VT, USA) at 560 nm, and absorbance was assessed
evaluation at 450 nm.

For MDSCs assessment, PBMCs were obtained from the

blood of mice as described above. Cells were labeled with T cell isolation
a cocktail of CD11b-PE (clone M1/70), Gr-1-APC (clone Mice were anesthetized and sacrificed as described above, and
RB6/8C5), and Ly-6 G-FITC (clone 1A8) using the Mouse blood was obtained by cardiac puncture. PBMCs isolation was
MDSC Flow kit (from Biolegend) following the manufac­ performed as described above. Murine CD3+ cells were iso­
turer’s instructions. lated from total PBMCs by positive selection using magnetic-
For Tregs evaluation, PBMCs were obtained from the blood activated cell sorting (MACS) microbead technology with anti-
of mice as described above. Cells were labeled using a True CD3ε-biotin and anti-biotin microbeads (Miltenyi Biotech;
Nuclear One Step Staining Mouse Treg Flow kit (FOXP3- >98% purity and >98% viability), as stated by manufacturer’s
AlexaFluor488, CD25-PE, CD4-PerCP; Biolegend) following instructions.
the manufacturer’s instructions.
Cells were assessed in a BD Accuri C6 flow cytometer (BD
Biosciences, Franklin Lakes, NJ, USA), and data was analyzed Differentiation of bone marrow-derived dendritic cells
using FlowJo software (LLC, Ashland, OR, USA). (BMDCs)
After sacrifice of anesthetized mice (n = 6), bone marrow was
removed from the femur and tibia by flushing into RPMI-1640.
Calreticulin, HSP70, and HSP90 exposure
Eluted cells were cultured for 5 days with 20 ng/mL of IL-4 and
5 × 104 cells/well were plated in 24-well plates and treated GM-CSF (R&D Systems, Minneapolis, MN, USA) until
with PKHB1 (CC50) for 2 h or epirubicin (42.5 μM for approximately 70% of the cells were CD11c+.
MCF-7 and MDA-MB-231 and 5 μM for 4T1 for 24 h).
Then, cells were detached, washed, and incubated for 1 h at
room temperature (RT) with 2 μg/mL of anti-Calreticulin Evaluation of DCs maturation
(FMC-75, Enzo Life Science), 0.8 μg/mL anti-HSP70 (F-3, CD11c, MHC-II, CD80, and CD86 were evaluated by flow
Santa Cruz Biotechnology), and 0.8 μg/mL anti-HSP90 cytometry with the fluorescent label-conjugated antibodies,
(F-8, Santa Cruz Biotechnology) in FACS buffer; cells antiCD11c-Alexa-fluor 488 (N418, R&D Systems), anti-MHC
were washed and incubated for 30 min in darkness at RT Class II-PE (REA813, Miltenyi Biotec), anti-CD80-FITC (16–
with goat anti-mouse IgG (Alexa Fluor 488) (H + L, Life 10A1, R&D Systems), and antiCD86-APC (GL1) from BD
Technologies) (1:1500) in FACS buffer; cells were then Biosciences (San Jose, CA, USA). In brief, 1 × 106 DCs /mL
washed and incubated in the dark for 10 min at RT with were stained in 100 μL of FACS buffer with the indicated
7-AAD (Life Technologies) (1:1000) in FACS buffer. The antibodies at RT for 30 minutes and then were washed twice
surface exposure of CALR, HSP70 and HSP90 was deter­ with PBS, centrifugated at 1600 rpm/10 min, resuspended in
mined by flow cytometry in non-permeabilized (7-AAD- 100 μl of FACS buffer and assessed by Flow Cytometer as
negative) cells. described previously. For MHC-II and CD80 evaluation by
flow cytometry, CD11c was added with MHC-II or CD86, we
then gated CD11c+ cells and we next assessed MHC-II or
Immunofluorescence microscopy CD86 MFI.
2.5 × 105 cells/well in 6-well dishes were left untreated
(Control) or treated with PKHB1 (CC50) and incubated for
PKHB1-KC and EPI-KC preparation
2 h. Then, cells were washed with PBS and stained with
Calreticulin-PE antibody (FMC-75, 2 μg/ml) and Hoechst 4T1 cells (1.5 × 106 cells/mL per mice) were plated and, after
33,342 (0.5 μg/ml) (Thermo Scientific Pierce, Rockford, IL, adherence, cells were then treated with 400 μM of PKHB1 for
USA), incubated for 1 h in FACS buffer at RT, washed twice, 2 h or 10 μM of EPI for 24 h, to obtain 80–90% of killed cells
maintained in PBS, and assessed by confocal microscopy (KC). After treatments, cells were obtained, centrifuged at
(Olympus X70; Olympus, Tokyo, Japan). 1600 rpm/10 min and resuspended in 100 μL of serum-free
e2054305-4 K. M. CALVILLO-RODRÍGUEZ ET AL.

medium/mice. Cell death was confirmed using Trypan blue positive or negative 4T1 cells were assessed in a BD
staining and flow cytometry. Finally, the PKHB1-KC or EPI- AccuryC6 flow cytometer (BD Biosciences) (total popula­
KC were inoculated by subcutaneous injection in the right tion 10,000 cells). Data was then analyzed using FlowJo
flank. software.

Freeze and thaw-killed cells preparation Prophylactic vaccination

4T1 cells (3 × 106 cells/mL per mice) were first frozen at −80°C for Vaccination was carried out as follows: PKHB1-KC
15 min, then thawed 10 min at 37°C in a water bath. The freeze– (n = 10) or EPI-KC (n = 10) were obtained as previously
thaw (F-T) cycles were repeated three times in rapid succession. described, and then inoculated s.c. in 100 μL of serum-free
After the final thaw, killed cells were resuspended in PBS. medium into the right hind leg (day −7), 7 days later, viable
(5 × 105) 4T1 cells were inoculated into the left hind leg
DCs’ co-culture with PKHB1-KC, EPI-KC, or FT-KC (day 0). Tumor volume and weight were measured as
described above.
DCs were resuspended in fresh medium (1 × 106 cells/mL), left
untreated (control) or incubated with 3 × 106 4T1 killed cells/
mL obtained after treatment with PKHB1, EPI, or FT, to give PKHB1-KC and EPI-KC treatment
a range of 1:3 (DCs to killed cells); co-culture was left for 24 h.
Then the supernatant was obtained, and the well was washed Tumor was established by subcutaneous injection of 5 × 105
twice with PBS before the next co-culture. 4T1 cells in 100 μL of PBS, in the left hind. Tumor volume and
mice weight were measured as described above. When tumor
reached 70–120 mm3 the first treatment of PKHB1-KC (n = 10)
DCs + T lymphocytes co-culture or EPI-KC (n = 10) was applied. Killed cells were inoculated
Control DCs or DCs previously co-cultured with PKHB1-KC, subcutaneously in 100 µl of serum-free medium, in the right
EPI-KC, or FT-KC were maintained in fresh medium at hind, twice a week for a total of four applications in a 2-week
1 × 106 cells/mL. Then, allogeneic BALB/c mCD3+ cells period. Control mice were treated with 100 µl of serum-free
were added at 3 × 106 cells/mL to give a range of 1:3 (DCs medium.
to CD3+ cells), co-culture was left for 96 h. Then, lympho­
cytes were collected (in the supernatant), washed with PBS,
and resuspended in fresh medium at 5 × 106 cells/mL for their Long-term antitumor effect evaluation
use in the next co-culture. Mice in complete remission after prophylactic (n = 9) or
therapeutic (n = 9) 4T1-PKHB1-KC application were re-
T-Lymphocytes + 4T1 cells co-culture challenged with 5 × 105 4T1 viable cells in 100 μL of PBS in
the left hind and tumor volume was measured as described
1 × 105 cells/mL viable 4T1 cells were plated. Then, allogeneic above.
BALB/c mCD3+ cells were added to each well at 5 × 105 cells/
mL, unprimed (previously co-cultured with control DCs) or
primed (previously co-cultured with DCs-PKHB1-KC, EPI- Long-term splenocytes-cytotoxicity
KC, or FT-KC) to give a range of 1:5 (tumor to effector). Co-
culture was left for 24 h. Mice in complete remission after prophylactic (n = 4) or
therapeutic (n = 4) PKHB1-KC application were re-
challenged with 5 × 105 4T1 viable cells in 100 μL of PBS in
Cytokine release assay the left hind. Three days after tumor inoculation mice were
Supernatants from the indicated co-cultures were obtained for sacrificed, spleens were harvested, filtered through a cell strai­
the assessment of IL-2, IL-4, IL5, and TNFα (BD CBA Mouse ner (70 μM) with PBS, and PBMCs were obtained as described
Th1/Th2 Cytokine Kit, San Jose, CA, USA) by flow cytometry above. Splenocytes were recovered and co-cultured with 4T1
following manufacturer’s instructions. IFNγ was assessed using cells (previously stained with calcein-AM) at 44:1 ratio (respec­
an ELISA kit (Sigma-Aldrich) and the Synergy HTTM (BioTek tively). Finally, calcein positive or negative cells were assessed
Instruments, Inc., Winooski, VT, USA) plate reader at 570 nm as described above.
wavelength, following manufacturer’s instructions.
Statistical Analysis
Calcein assay
Mice were randomly assigned to different groups for all in vivo
4T1 cells (1 × 106 cells/mL) were stained with (0.1 mL/ studies. At least three independent experiments were repeated
mL) Calcein-AM from BD Biosciences (San Jose, CA) in three independent times. Mann-Whitney tests and two-tailed
FACS buffer at 37°C and 5% CO2 for 30 min, washed unpaired Student’s t-tests were performed using GraphPad
twice with PBS. Thus, primed or unprimed T cells were Prism Software (San Diego CA, USA) and presented as mean
added in a 1:5 (tumor to effector) ratio. Co-culture was values ± SD. The p values were considered significant as fol­
incubated at 37°C and 5% CO2 for 24 h. Finally, calcein lows: p< 0.05.
 ONCOIMMUNOLOGY e2054305-5

Results sup. 5). The decrease in tumor volume was correlated with the
decrease in tumor weight, going from 1.5 grams in the controls
PKHB1 induces breast cancer cell death
to 0.40 grams in PKHB1-treated mice (Figure 2(b)). The
Although the potency of PKHB1was previously demonstrated decrease of tumor volume in mice treated with PKHB1, led us
in hematopoietic malignancies,9,11 its effectiveness was not yet to evaluate the involvement of T cells in the observed effect. First,
evaluated for solid tumors in vivo. Thus, after peptide synthesis we analyzed histological sections of tumors from control
and characterization (Supplemental material and methods, (Figure 2(c)) or PKHB1-treated mice (Figure 2(d)); results
table sup.1 and figure sup.1), we evaluated here its effect in revealed that tumors from control mice showed tumor cells
two types of human breast cancer cell lines, I) MCF-7 (luminal (black arrows) with moderate mitotic activity (blue arrows),
subtype) and II) MDA-MB-231 (triple negative subtype), as whereas the PKHB1-treated mice showed sporadic mitotic activ­
well as on the murine 4T1 cell line (mimics triple negative ity, extensive necrosis with abundant accumulation of cellular
subtype).(16) We observed that PKHB1 induces cell death in debris (green arrows), and abundant inflammatory exudate,
a concentration-dependent way in MCF-7 (Figure 1(a)), MDA- composed of polymorphonuclear elements, eosinophils, and
MB-231 (Figure 1(b)), and 4T1 (Figure 1(c)) cells, as they lymphoplasmacytic cells (red arrows).
showed an increase in the percentage of double-positive Ann- Additionally, we evaluated if the cell number and distribu­
V-APC/PI staining (figure sup. 2). We determined that the tion of T lymphocytes in peripheral blood, spleen, lymph
cytotoxic concentration that induces approximately 50% of nodes, and tumor site, changed after PKHB1 treatment. We
cell death (CC50) in MDA-MB-231 and MCF-7 is 200 μM observed (Figure 2(e)) that the percentage of CD3+ cells
whereas in 4T1 is 300 μM. increased in blood, lymph nodes, and tumors of PKHB1-
Next, we evaluated mitochondrial damage and cytosolic Ca2+ treated mice, while it was maintained in spleen. When we
augmentation, and observed that PKHB1 (CC50) induced loss of assessed CD4+ cells, the percentage of cells significantly aug­
mitochondrial membrane potential (Figure 1(d)), ROS produc­ mented in lymph nodes, whereas it was significantly dimin­
tion (Figure 1(e)) and increase of intracellular Ca2+ (Figure 1(f)) ished in the tumor site (Figure 2(f)). Furthermore, CD8 + T
in all cell lines. Afterwards, we searched to determine the cell cells significantly increased in peripheral blood and specially in
death effectors and evaluated ROS-dependence, also, we used tumor site (gating strategy in figure sup. 6), while they signifi­
inhibitors of caspases (Z-VAD), necroptosis (Nec-1), and the cantly diminished in lymph nodes (Figure 2(g)). To extend this
Ca2+-chelator (BAPTA) and assessed cell death. We used epir­ analysis, we evaluated the proportion of myeloid-derived sup­
ubicin (EPI) and H2O2 as controls for inhibition. We found that pressor cells (MDSCs) and Tregs in peripheral blood of control
Z-VAD inhibited the cell death induced by EPI but not PKHB1- and PKHB1-treated mice. We found a significative decrease in
induced cell death (Figure 1(g)), while NAC and NEC-1 inhib­ the percent of MDSCs (mean from 48% to 23%) and Tregs
ited the cell dead induced by H2O2 but not PKHB1-mediated (mean from 12% to 3%) in mice treated with PKHB1, when
cell death (Figure 1(h,i)). Finally, we observed that the Ca2+ compared with control mice (Figure 2(h,i)). Additionally, we
chelator BAPTA inhibited the Ca2+ augmentation (figure determine a significative increase in the ratio of CD8/Tregs
sup. 3) and cell death (Figure 1(j)), induced by PKHB1. This (mean 2% to 14%) in mice treated with PKHB1, when com­
Ca2+ dependence was previously observed for leukemic cells,9,11 pared with control mice (Figure 2(j)). Finally, we determined
suggesting a similar cell death mechanism and common signal­ whether splenocytes from PKHB1-treated mice could induce
ing pathway among solid and liquid cancers. an antitumor cell cytotoxicity. For this purpose, we evaluated
To assess this hypothesis, we used a phospholipase C (PLC) the calcein negative 4T1 cells after co-culture with splenocytes
inhibitor (U73122) and ER receptor inhibitors (dantrolene, for obtained from control or PKHB1-treated mice. In Figure 2(k),
ryanodine receptors, and 2-APB, for IP3 receptors). We deter­ results show that splenocytes from PKHB1-treated mice
mined that PKHB1-cell death is significatively inhibited when induced a significant increase in calcein negative 4T1 cells
blocking the ER-Ca2+-channels with U73122, dantrolene and (75%) in comparison with control mice (40%). These results
2-APB (Figure 1(j,k) and Figure 1(l)) in breast cancer cells, improve the knowledge of the immune system-involvement in
confirming a similar cell death pathway induced by PKHB1 in the antitumor effect mediated by PKHB1 treatment.
breast cancer cells, as previously found in leukemic cells.
PKHB1 induces DAMPs exposure and release in breast
PKHB1 has antitumor effects in breast cancer and cancer cell lines
promotes intratumorally CD8 + T cell infiltration
As we observed that PKHB1 induced cell death in breast cancer
To evaluate in vivo the potential antitumor effect of PKHB1, 4T1 cell lines and CD8 + T lymphocyte-recruitment in tumor site and
breast cancer cells were grafted into BALB/c mice. Daily treat­ the decrease of immunosuppressive cells, we wondered if cell
ments were initiated when tumor volume reached approximately death induced by PKHB1 was able to induce DAMPs’ exposure/
100 mm3, and 16 days after the cell transplant, tumor volume of release in breast cancer cells. The first step was to evaluate the
the control mice had reached 1500 mm3, requiring the sacrifice exposure of CALR (one of the principal DAMPs related with
of the animals, while the tumor volume of the PKHB1-treated ICD).17 Our results show that PKHB1-treatment and EPI-
mice reached a maximum volume of 890 mm3 (at day 8) which treatment were able to induce a significative increase of CALR
started to decrease, reaching a volume of 570 mm3 at day 16 positive cells in MCF-7 (Figure 3(a)), MDA-MB-231 (Figure 3
(Figure 2(a)) (individual growth curves in figure sup. 4). Also, (b)), and 4T1 (Figure 3(c)) cells. The CALR exposure induced by
daily treatment with PKHB1 did not affect mice weight (figure PKHB1 was confirmed by immunofluorescence microscopy,
e2054305-6 K. M. CALVILLO-RODRÍGUEZ ET AL.

Figure 1. PKHB1 induces cell death in breast cancer cell lines. Cell death analysis in (A) MCF-7, (B) MDA-MB-231 and (C) 4T1 cells, without treatment (Control),
treated with the control peptide 4NGG (300 μM) or PKHB1 (100, 200, 300, or 400 μM) for 2 h. (D) Representative graphs and quantification of the loss of ΔΨm measured
through TMRE, (E) ROS levels measured through Hydroethidine staining and (F) intracellular Ca2+ by Fluo-4 staining, by flow cytometry in cells left alone or treated with
the CC50 of PKHB1 (200 μM for MCF-7 and MDA-MB-231 and 300 μM for 4T1) for 2 h. (G-I) Cell death induced by PKHB1 (CC50), Epirubicin (42.5 μM for MCF-7 and MDA-
MB-231 and 5 μM for 4T1) and H2O2 (25 μM for all cell lines) was assessed in cells left without pre-treatment (-) or pre-treated (30 minutes) with Z-VAD-FMK (Z-VAD),
N-Acetyl Cysteine (NAC) and Necrostatin-1 (NEC-1). (J) Cell death induced by PKHB1 (CC50) was assessed in cells left without pre-treatment (-) or pre-treated (30 minutes)
with BAPTA. Cell death induced by PKHB1 (CC50) was assessed as in J in cells left without pre-treatment (-) or pre-treated (30 minutes) with dantrolene, 2-APB or U73122.
Graph represents the means (±SD) of triplicates of three independent experiments. NS = Not significant.

where we observed that PKHB1 induced CALR exposure in all the exposure (Figure 3(g)), 2.7 ± 0.6, 2 ± 0.6, and 7 ± 1.85-fold of
cases Figure 3(d,e) and Figure 3(f). Additionally, PKHB1 treat­ HSP90 exposure (Figure 3(h)) in MCF-7, MDA-MB-231 and 4T1
ment induced 24 ± 3, 3.2 ± 1.3 and 4.23 ± 2-fold of HSP70 cells, respectively, when compared with untreated cells.
 ONCOIMMUNOLOGY e2054305-7

a b

c d

e f g

h i j k

Figure 2. PKHB1 treatment induces tumor reduction and T cells distribution. Mice were inoculated s.c. with 5 × 105 4T1 viable cells and when tumor reached 70–
120mm3 were treated with sterile water (Control, n = 6) or PKHB1 (400 μg of PKHB1 daily, n = 6), tumor volume was measured three times per week. (A) Graphs indicate
the mean of the tumor volume in PBS-treated group (Control, n = 6) or PKHB1-treated group (PKHB1, n = 6). (B) Graphs indicate the tumor weight of control and treated
mice at day 16. (C,D) Histology from tumors of control or PKHB1-treated mice stained with H&E. Tumor cells (black arrows), mitotic cells (blue arrows), cellular debris
(green arrows) and immune system cells infiltration (red arrows). (E, F, and G) Graphs show the percent of CD3+, CD4+, and CD8+ cells in blood, spleen, lymph node,
and tumor of control or PKHB1-treated mice at day 16. (H-I) Graphs show the percent of MDSCs and Tregs in blood of control or PKHB1-treated mice at day 16. (J) Graph
show the ratio of CD8/Tregs cells of control or PKHB1-treated mice at day 16. (K) Graphs show the percent of calcein negative 4T1 cells after the co-culture with
splenocytes of control (n = 4), or PKHB1-treated mice (n = 4). NS = Not significant.

Finally, we wondered if PKHB1 induced the release of evaluated as we show in the schema of Figure 4(a). Thus,
HMGB1 and ATP, two important DAMPs related with bone marrow-derived murine DCs were left untreated
ICD.7 Therefore, the presence of HMGB1 and ATP was (Control) or pulsed for 24 h with the PKHB1-KC, EPI-
assessed in the supernatants of treated and untreated breast KC, FT-KC, or LPS (1 μg/mL). After co-culture, DCs
cancer cells. In Figure 3, results showed a significant release pulsed with the different stimulus (killed cells or LPS)
of HMGB1 (Figure 3(i)) and ATP (Figure 3(j)) in the maintained the expression of the DCs marker CD11c
supernatants of PKHB1-treated cells, when compared with (Figure 4(b)), while only LPS induced a significative
untreated cells. increase of the MHC-II in cell surface (Figure 4(c)).
However, the PKHB1-KC, EPI-KC, and LPS induced
a significant increase of the co-stimulatory molecule CD86
PKHB1-KC induce maturation of bone marrow-derived while no difference was observed in the DCs stimulated
DCs and antitumor T cell responses with the FT-KC (Figure 4(d)). Additionally, DCs pulsed
To assess the immunogenicity of the dead cells obtained with PKHB1-KC show a significant increase in CD80 cell
upon treatment with PKHB1, 4T1 cells were treated with surface expression and TNFα release in comparison with
400 μM of PKHB1, epirubicin (EPI) or freeze and thaw unstimulated DCs (figure sup. 7A-C).
cycles (FT) as positive or negative controls (respectively) of Once we determined DCs markers after co-culture with
ICD. The PKHB1-killed cells (PKHB1-KC), EPI-KC, or FT- killed cells, we assessed if the DCs pulsed with the different
KC were then prepared as described in the methods sec­ killed cells (PKHB1-KC, EPI-KC or FT-KC) were able to prime
tion, and its ability to induce DCs maturation was T cells. First, primary T lymphocytes (CD3+ cells) were co-
e2054305-8 K. M. CALVILLO-RODRÍGUEZ ET AL.

Figure 3. PKHB1 induces exposure and release of DAMPs in breast cancer cells. Representative FACS histograms of CALR exposure (filled histograms) and IgG
isotype antibodies (open histograms) in non-permeabilized (7-AAD negative cells) (A) MCF-7, (B) MDA-MB-231, and (C) 4T1 cells, untreated (Control) or treated with the
CC50 of PKHB1 (in gray) (200 μM for MCF-7 and MDA-MB-231 and 300 μM for 4T1) for 2 h or EPI (in red) (42.5 μM for MCF-7 and MDA-MB-231 and 5 μM for 4T1) for 24 h.
Calreticulin exposure observed by confocal microscopy in (D) MCF-7, (E) MDA-MB-231, and (F) 4T1 cells untreated (Control) or treated with PKHB1 (CC50) using CALR-PE
staining and Hoechst 33,342. Representative graphs of the ratio of HSP70 (G) or HSP90 (H) exposure in non-permeabilized cells (7-AAD negative cells), untreated
(Control) or treated with PKHB1 (CC50) for 2 h. Representative graphs of the (I) HMGB1 or (J) ATP release in the supernatants of control or PKHB1 (CC50) treated cells.
Graphs shown are means (± SD) of triplicates of three independent experiments. CALR-PE = Calreticulin-PhycoErythrin.

cultured for 96 h with pulsed or unpulsed DCs, and we next step was to carry out the Gold Standard of ICD (prophy­
observed the release of TNFα, IFNγ, and IL-2 in the co- lactic vaccination)18,19 to confirm whether PKHB1 induced
culture of CD3+ and DCs-PKHB1-TCL (table sup. 2). Next, ICD. The vaccine was based in the subcutaneous inoculation
primed (co-cultured with pulsed DCs-PKHB1-KC, EPI-KC, or of the 4T1-PKHB1-KC, 7 days before the transplantation of
FT-KC) or unprimed (co-cultured with unstimulated DCs) viable 4T1 cells, while mice were inoculated with 4T1-EPI-KC
T lymphocytes were collected and co-cultured during 24 h used as a positive control, and controls without KC were
with viable 4T1 cells (previously stained with calcein-AM). injected with serum-free medium (Figure 5(a)). Results
To assess antitumor cell cytotoxicity, we evaluated the increase showed that vaccination with PKHB1-KC prevented tumor
in calcein negative 4T1 cells after co-culture with primed or establishment in 80% (8/10) of mice compared to 70% (7/10)
unprimed T lymphocytes. Results showed that only of mice treated with EPI-KC. No survival (0%) was observed
T lymphocytes co-cultured with pulsed DCs-PKHB1-KC and in the Control group inoculated with serum-free medium
DCs-EPI-KC induced a significant increase in calcein negative (Figure 5(b)) (individual growth curves in figure sup. 8
4T1 cells (Figure 4(e)), in comparison with the lymphocytes A-C). Additionally, survival rates of mice in each group
co-cultured with DCs-FT-KC or unprimed T lymphocytes. were consistent with tumor growth, observing, respectively,
Additionally, lymphocytes stimulated with DCs-PKHB1-KC 80% and 70% of survival in mice vaccinated with PKHB1-KC
show a significant increase in IFNγ and IL-2 release in the co- and EPI-KC by day 60, while control mice perished by day 21
culture with 4T1 cells in comparison with lymphocytes stimu­ (Figure 5(c)).
lated with control DCs (figure sup. 7D and E).

Treatment with PKHB1-KC induces tumor regression

Prophylactic vaccination with PKHB1-KC prevented tumor
After ex vivo and in vivo results, we evaluated if the immu­
establishment of 4T1 cells
nogenicity of PKHB1-KC was able to diminish tumor
Considering that PKHB1 treatment induces tumor decline, growth and improve overall survival in syngeneic mice
infiltration of CD8+ cells into the tumor, DAMPs’ exposure bearing 4T1 tumors. First, 4T1 viable cells were inoculated
and release, and the antitumor immune response ex vivo, the in BALB/c mice. When tumor reached 70–120 mm3,
 ONCOIMMUNOLOGY e2054305-9

Figure 4. PKHB1-Killed cells induce antitumor immune responses ex vivo. (A) Schema of the ex vivo experiments. (B) Representative histograms from flow cytometry
analyses of CD11c expression on DCs left with medium (CTR) or pulsed 24 h with a PKHB1-KC, EPI-KC, FT-KC, or LPS, graphs of the means obtained by FACS (right side).
(C) Representative histograms from flow cytometry analyses of MHC-II expression on DCs treated as in A, graphs of the means obtained by FACS (right side). (D)
Representative histograms from flow cytometry analyses of CD86 expression on DCs treated as in A, graphs of the means obtained by FACS (right side). (E) Graphs
shown are means (± SD) of triplicates of three independent experiments from flow cytometry analyses of 4T1 cells stained with calcein-AM and co-cultured with
T lymphocytes (unprimed or primed). NS = Not significant.

a control group was treated with serum-free medium, PKHB1-KC prophylactic and therapeutic vaccinations
a second group was treated with PKHB1-KC and the induce long-term antitumor effect
third group was treated with EPI-KC. All mice were treated
To assess the long-term antitumor response against 4T1 breast
two times per week for a total of four treatments (Figure 5
cancer cells induced by PKHB1-KC in a prophylactic or ther­
(d)). Tumor growth measurements show that PKHB1-KC-
apeutic application, mice in complete remission (tumor free
treated mice had diminished tumor growth after day 10
>60 days) were re-challenged with living 4T1 cells. Tumor
(7 days after the first treatment), which continued to volume analysis showed that, contrary to naïve mice
decrease until no tumor was detected by day 18, in the (Control), which showed a correct 4T1 tumor establishment,
group of EPI-KC the tumor diminished after day 10 (7 days mice in remission after PKHB1-KC prophylactic or therapeutic
after the first treatment) which continued to decrease until application showed a slight increase in tumor volume at day 3,
no tumor was detected by day 16 (Figure 5(e)). Tumor which immediately disappears by day 6 (Figure 6(a)). These
growth diminution was reflected in overall mice survival, results correlate with mice survival, where we observed that
as PKHB1-KC-treated mice presented a 78% (7/9) of survi­ compared to naïve mice, in which a primary 4T1 cells challenge
val, while the EPI-KC-treated mice presented 67% (6/9) of resulted in a 0% (0/9) of survival by day 23, those that were in
survival, and all control mice perished by day 23 (Figure 5 remission after prophylactic or therapeutic application of
(f)) (individual growth curves in figure sup. 8D-F). PKHB1-KC were completely resistant to a re-challenge with
e2054305-10 K. M. CALVILLO-RODRÍGUEZ ET AL.

b c

e f

Figure 5. PKHB1-KC induce tumor elimination in a prophylactic and therapeutic application. (A) Schema of PKHB1-KC or EPI-KC prophylactic application. (B)
Graph indicates the mean of the tumor growth in mice treated with serum-free medium (Control; n = 10) or mice receiving a prophylactic vaccination with PKHB1-KC
(Prophylactic-PKHB1-KC, n = 10) or EPI-KC (Prophylactic-EPI-KC, n = 10). (C) Kaplan–Meier survival graph of mice treated as in B over time. (D) Schema of PKHB1-KC or
EPI-KC therapeutic application. (E) Graph indicates the mean of the tumor growth in mice treated with serum-free medium (Control; n = 9), PKHB1-KC (Therapeutic-
PKHB1-KC, n = 9) or EPI-KC (Therapeutic-EPI-KC, n = 9), arrows indicate days of KC or serum-free medium inoculation. (F) Kaplan–Meier survival graph of mice treated as
in E over time.

4T1 cells, resulting in a 100% (9/9) of survival (Figure 6(b)). Discussion

Furthermore, we determined if splenocytes from re-challenged
Here, we assessed for the first time the characteristics of the
mice can induce an antitumor cell cytotoxicity. For this pur­ cell death induced by PKHB1 in breast cancer cells, includ­
pose, we evaluated the increase in calcein negative 4T1 cells ing the triple negative phenotype, which conserves the
after co-culture with splenocytes obtained from naïve or re- principal molecular characteristics of cell death (caspase-
challenged mice. In Figure 6 (d), results showed that spleno­ independent, calcium-dependent, PLC-dependent, and IP3
cytes from re-challenged mice induced a significant increase in R and RYR receptor-dependent cell death with the presence
calcein negative 4T1 cells (60%) in comparison with naïve of ROS, loss of mitochondrial membrane potential and the
mice (30%). intracellular accumulation of Ca2+) reported mainly in
 ONCOIMMUNOLOGY e2054305-11

Figure 6. PKHB1-KC prophylactic and therapeutic application induces long-term antitumor effect, schematic representation of the PKHB1-effect. Mice in
remission (30 days) after PKHB1 prophylactic or therapeutic PKHB1-KC application were re-challenged with 5 × 105 4T1 viable cells. (A) Graph indicates the mean of the
tumor growth in PBS-treated group (Control; n = 9), prophylactic re-challenged group (Prophylactic remission-4T1 re-challenge, n = 9) or therapeutic re-challenged
group (Therapeutic remission-4T1 re-challenge, n = 9). (B) Kaplan–Meier survival graph of mice treated as in A over time. (C) Graphs show the percent of calcein
negative 4T1 cells after co-culture with splenocytes control (n = 4), prophylactic (n = 4) or therapeutic (n = 4) re-challenged mice. (D) PKHB1 induces ROS production,
intracellular Ca2+ accumulation, loss of mitochondrial membrane potential (ΔΨm), leading to DAMPs exposure and release in breast cancer cells. Neoantigens and
DAMPs exposure/release induced by PKHB1 promotes DCs maturation, which triggers T cell activation to induce cancer cytotoxicity. (E) However, PKHB1-treatment
in vivo induces T cell redistribution in lymph nodes, peripheral blood and intratumorally, leading to tumor reduction. (F) PKHB1-KC prophylactic vaccination prevented
tumor establishment. (G) PKHB1-KC therapeutic application induced tumor remission. (H) PKHB1-treatment, PKHB1-KC prophylactic or therapeutic application induced
tumor-specific splenocytes’ cytotoxicity.

leukemic cells.9,11,12,14 We recently reported the overexpres­ Our results also revealed the antitumor effect of PKHB1
sion of PLCγ1 and its importance in the cell death induced against 4T1-breast cancer cells in vivo, as PKHB1-treatment
by PKHB1 in CLL cells,9 but although here we did not diminished tumor volume and weight. Furthermore, we
assess specially this isoform, it has been demonstrated the observed the distribution of T cells in PKHB1-treated
the overexpression of PLCγ1 in breast cancer patients is mice that involves the increase of T cells (in blood, lymph
correlated with poor clinical outcome,20 and the overex­ node, and tumor), trafficking of CD4+ cells to lymph
pression of PLC-β,¹¯²PLC-ε, and PLC-δ has negative nodes, and tumor CD8+ cells infiltration that its associated
outcomes.21 Thus, the involvement of PLC in the mechan­ to an antitumor response.22 Additionally, PKHB1 induced
ism of PKHB1-cell death, might have an advantage in the the decrease of immunosuppressive cells such as MDSCs
cancer cells that overexpress these proteins. and Tregs in blood, also, we observed an increase in the
e2054305-12 K. M. CALVILLO-RODRÍGUEZ ET AL.

ratio of CD8+/Tregs, which has been related with an tumor volume decreased 7 days after the first administration,
enhanced antitumor immune activity.23,24 Overall, our reaching tumor regression on day 18 in approximately 80% of
results are promising, and indicate that PKHB1 promote mice, while in the group of EPI-KC the tumor diminished 7 days
a robust antitumor immune response as it has been after the first treatment which continued to decrease until tumor
reported that extensive tumor infiltration by cytotoxic regression on day 16. Our results highlight the immunogenicity of
CD8 + T cells, the decrease of MDSCs and Tregs, and the the PKHB1-induced cell death, because the therapeutic applica­
increase in the ratio CD8+/Tregs are strongly associated tion of the PKHB1-KC induced tumor remission even in the
with patient’s survival and response to therapy, even in absence of adjuvants. Additionally, we determined the therapeutic
different phenotypes of breast cancer.23–27 Finally, spleno­ potential of the EPI-KC for the first time, as a novel strategy for
cytes from PKHB1-treated mice were more cytotoxic the application of chemotherapy-ICD inductors. These results
against breast cancer cells than splenocytes from control differentiate the PKHB1-KC from other therapeutic strategies
mice, probably due to the immunogenicity of the cell death with tumor lysates against melanoma, prostate and ovarian can­
triggered by PKHB1.22,28 cer, which have been poorly evaluated and were mainly used in
The low immunogenicity of tumor cells is a main obstacle of combination with adjuvants.46,47 The success of the therapeutic
antitumor therapies; therefore, a way to reactivate potent anti­ application of PKHB1-KC in breast cancer was similar to the
tumor immune responses is through the emission of DAMPs 29 T-ALL model,14 despite their intrinsic molecular
and dead cells-derived antigens, which can be achieved in the differences.16,48–50
ICD.5,30 Here, we demonstrated that PKHB1 is capable of The perspectives for cell therapy against cancer are based on
inducing CALR, HSP70, and HSP90 exposure, HMGB1 and the development of T cell responses, resulting in effective
ATP release, which can promote the uptake of dying cells, and rejection of tumors and long-term protection.51,52 From this
the recruitment, maturation and cross-presentation activity of fact, the induction of ICD eventually results in long-lasting
antigen-presenting cells (APCs).28,31 In this sense, we demon­ protective antitumor immunity.53 Our results demonstrate
strated that the cell death induced by PKHB1 and epirubicin that PKHB1-KC induces long-term antitumor effect since
(our positive control of ICD)32–34 are able to promote a mature 100% of mice in remission after PKHB1-KC prophylactic or
phenotype of DCs,35 both induced a significant increase in the therapeutic application survived at the re-challenge with 4T1
co-stimulatory molecule CD86, such as different ICD cells. Although immunotherapy with pulsed DCs, primed
inductors.36 Additionally, we observed that DCs pulsed with T lymphocytes or CAR-T cells is the main approach used to
the PKHB1-KC and EPI-KC promote the antitumor specific stimulate antitumor immune responses, they represent greater
cytotoxicity of T cells, which confirm the phenotypic and technical complexity, higher cost, among other disadvantages
functional maturation of DCs. Additionally, we observed that regarding the use of crude PKHB1-KC.54–56
the freeze and thaw-killed cells do not stimulate the DCs, as Overall, our results demonstrate that PKHB1 is an ICD
other studies have demonstrated that freeze and thaw-killed inductor in breast cancer cells and highlight a new
cells suppress DCs maturation and function.37 approach for TSP-1 peptides mimic, which could induce
However, vaccination assays involving syngeneic models ICD as a conserved mechanism of cell death in different
are the gold standard to formally identify ICD inducers, since types of tumor cells, including solid cancers, additionally,
this demonstrates the tumor rejection capacity of the immu­ our results provide evidence for a novel strategy in the
nized host.29,38 Our results show that the prophylactic appli­
obtention and application of killed cancer cells against
cation of EPI-KC and PKHB1-KC prevented tumor
breast cancer.
establishment and increased survival in 70 and 80% (respec­
tively) of the mice, without using adjuvants and with only one
vaccination, in comparison with other strategies of prophy­
Acknowledgments
lactic KC-vaccines.39–41 Also, we used Epirubicin, a well-
known ICD inductor with major side-effects in human32,42 We thank the SEP-CONACYT-ECOS-ANUIES grant 291297, PAICYT,
as a positive control, highlighting its ability to prevent the the Laboratory of Immunology and Virology of the Faculty of Biological
sciences, UANL, Kaybiotix and the DRUG Lab from Sorbonne University
establishment of breast cancer in 70% of the mice. Our results
for the financial support and the facilities provided to achieve this work.
are in line with the protective potential of established ICD KMCR, LGM, and ACUP thank CONACYT for their scholarship. LGM
inducers including oxaliplatin, doxorubicin, idarubicin, thanks SU/LBM/DRUG Lab/Kaybiotix for scholarship. PK is grateful to
mitoxantrone, and specific forms of radiotherapy (in colon SATT-Lutech and DRI from SU, Kayvisa/Kaybiotix/χ-Pharma for finan­
cancer) which presented between 80% and 90% of tumor-free cial support and Oncodesign for hosting the LBM DRUG Lab. We thank
Alejandra Reyes-Ruiz and Martin Herrick Ramón Kane for technical help,
mice17,43,44 while the antibody 7A7 (anti-EGFR) induced 50%
Moises Armides Franco-Molina for the facilities and for his help, and
of survival (lung cancer),45 and especially with the fact that an Alejandra Arreola-Triana for editing and proofreading.
ICDinductor should display elevated tumor-free survival
(>50%).19
The therapeutic application of the dead cells killed by Authors’ contributions
a potential ICD inducer can be used as a confirmatory trial for
KMCR, ACUP and RMR carried out cell death, TMRE, and ROS assess­
ICD inductors, to evaluate their ability to mediate therapeutic
ment. KMCR and RMR carried out Ca2+ assessment. LGM carried out
effects depending on the immune system against established peptide synthesis. KMCR performed ex vivo and in vivo experiments.
neoplasms.38 In this sense, our results show that when we treated ACMT and PK conceived and supervised the work. KMCR and ACMT
tumor bearing mice with only four applications of PKHB1-KC, prepared the figures and wrote the manuscript. KMCR, LGM, RMR,
 ONCOIMMUNOLOGY e2054305-13

ACUP, PK, ACMT, and CRP designed experiments, analyzed and inter­ OncoImmunology. 2017 Oct 4;6(12):e1386829. doi: 10.1080/
preted data, and read and approved the final manuscript. 2162402X.2017.1386829. PMID: 29209573 Available from:
https://www.tandfonline.com/doi/full/10.1080/2162402X.2017.
1386829
Availability of data and material 6. Galluzzi L, Buqué A, Kepp O, Zitvogel L, Kroemer G.
Immunogenic cell death in cancer and infectious disease. Nat Rev
The data used to support the findings of this study are available from the Immunol. 2017. Feb;17(2):97–111. doi:10.1038/nri.2016.107.
corresponding authors upon request. 7. Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell
death in cancer therapy. Annu Rev Immunol. 2013;31
(1):51–72. doi:10.1146/annurev-immunol-032712-100008.
Ethics approval 8. Stanton SE, Disis ML. Clinical significance of tumor-infiltrating
The Animal Research and Welfare Ethics Committee (CEIBA), of the lymphocytes in breast cancer [Internet]. Journal for
School of Biological Sciences approved this study: CEIBA-2018-003. All ImmunoTherapy of Cancer BioMed Central Ltd. 2016 [cited
experiments were conducted according to Mexican regulation NOM-062- 2020 Jul 15];4(1):59. Available from http://jitc.bmj.com/lookup/
ZOO-1999. doi/10.1186/s40425-016-0165-6
9. Martinez-Torres AC, Quiney C, Attout T, Boullet H, Herbi L,
Vela L, Barbier S, Chateau D, Chapiro E, Nguyen-Khac F, et al.
Disclosure statement CD47 agonist peptides induce programmed cell death in refractory
chronic lymphocytic leukemia B cells via PLCγ1 activation: evi­
The authors declare the following competing financial interest(s): a patent dence from mice and humans. PLoS Med. 2015;12(3). doi:10.1371/
including results from this paper has been filed. The authors declare that journal.pmed.1001796
no other competing interests exist. 10. Denèfle T, Boullet H, Herbi L, Newton C, Martinez-Torres A-C,
Guez A, Pramil E, Quiney C, Pourcelot M, Levasseur MD, et al.
Thrombospondin-1 mimetic agonist peptides induce selective
Funding death in tumor cells: design, Synthesis, and structure–activity rela­
tionship studies. J Med Chem. 2016 Sep 22 Internet]. [cited 2018
This work was supported by SEP-CONACYT-ECOS-ANUIES, Grant/ Dec 21];59(18):8412–8421. Available from http://pubs.acs.org/doi/
Award Number: 291297; the Laboratory of Immunology and Virology of 10.1021/acs.jmedchem.6b00781
the College of Biological Sciences; UANL; Sorbonne-Université, 11. Uscanga-Palomeque AC, Calvillo-Rodríguez KM, Gómez-
Laboratoire des Biomolécules, DRUGLAB, Kaybiotix. KMCR, LGM, and Morales L, Lardé E, Denèfle T, Caballero-Hernández D, Merle-
RMR hold a Consejo Nacional de Ciencia y Tecnología (CONACYT) Béral H, Susin SA, Karoyan P, Martínez-Torres AC, et al. CD 47
scholarship. LGM hold a Kaybiotix grant; SATTLutech; Kayvisa/ agonist peptide PKHB 1 induces immunogenic cell death in T-cell
Kaybiotix/-Pharma. acute lymphoblastic leukemia cells. Cancer Sci. 110:1. 256–268.
Internet]. 2018 Dec 14 [cited 2018 Dec 21];cas.13885. Available
from https://onlinelibrary.wiley.com/doi/abs/10.1111/cas.13885
ORCID 12. Denèfle T, Pramil E, Gómez-Morales L, Levasseur MD, Lardé E,
Newton C, Herry K, Herbi L, Lamotte Y, Odile E, et al.
Kenny Misael Calvillo-Rodríguez http://orcid.org/0000-0001-7289-
Homotrimerization approach in the design of thrombospondin-1
6166
mimetic peptides with improved potency in triggering regulated
Philippe Karoyan http://orcid.org/0000-0003-1525-6474
cell death of cancer cells. J Med Chem. 2019 Sep 12 Internet]. [cited
Ana Carolina Martínez-Torres http://orcid.org/0000-0002-6183-0089
2019 Sep 26];62(17):7656–7668. Available from: https://pubs.acs.
org/doi/10.1021/acs.jmedchem.9b00024
13. Martinez-Torres A-C, Quiney C, Attout T, Boullet H, Herbi L,
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