therapeutic efficacy of cancer stem cell vaccines in the...

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Therapeutic efficacy of cancer stem cell vaccines in the adjuvant setting

Yangyang Hu1,2,*, Lin Lu1,3,*, Yang Xia1,4,*, Xin Chen1,5, Alfred E. Chang1, Robert E

Hollingsworth6, Elaine Hurt6, John Owen1, Jeffrey S. Moyer1, Mark E.P. Prince1, Fu Dai4, Yangyi

Bao4, Yi Wang4, Joel Whitfield1, Jian-chuan Xia3, Shiang Huang2, Max S. Wicha1, Qiao Li1

1University of Michigan Comprehensive Cancer Center, Ann Arbor, MI; 2Center for Stem Cell

Research and Application, Institute of Hematology, Union Hospital, Tongji Medical College,

Huazhong University of Science and Technology, Wuhan, China; 3State Key Laboratory of

Oncology in Southern China and Department of Experimental Research, Sun Yat-sen University

Cancer Center, Guangzhou 510060, China; 4The Third Affiliated Hospital of Anhui Medical

University, Hefei, China; 5Department of Oncology, Wuhan University, Renmin Hospital, Wuhan,

China; 6MedImmune Inc. Gaithersburg, MD

*Yangyang Hu, Lin Lu and Yang Xia contributed equally to this work.

Running title: Therapeutic efficacy of CSC vaccine in the adjuvant setting

Keywords: cancer stem cell, dendritic cell, vaccine, immunotherapy, adjuvant

Research. on October 12, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 20, 2016; DOI: 10.1158/0008-5472.CAN-15-2664

http://cancerres.aacrjournals.org/

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Financial support: This work was supported by the Elsa U. Pardee Foundation, partially

supported by the Gillson Longenbaugh Foundation and the University of Michigan MICHR

Grant UL1TR000433, as well as the National Science Fund of China (81072170 and 81202093)

and NCI research grant 1R-35CA 197585 (MW).

Correspondence to: Qiao Li or Max S. Wicha, University of Michigan Comprehensive Cancer

Center, 3520B MSRB-1, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-5652, Phone:

(734)615-1977, Fax: (734)998-2440 Email: [email protected] or 1500 E. Med. Ctr. Dr.,

6302CC, Ann Arbor, MI 48109-5942, Phone: (734)936-1831, Fax: (734)647-9271 Email:

[email protected] or Shiang Huang, Center for Stem Cell Research and Application,

Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of

Science and Technology, Wuhan, China. Tel.: +86-27-85726114, Fax: +86-27-857736343

Email: [email protected]

Conflicts of interest: All authors have declared there are no conflicts of interest in regards to

this work.




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Abstract

Dendritic cell (DC)-based vaccine strategies aimed at targeting cancer stem-like cells (CSC) may

be most efficacious if deployed in the adjuvant setting. In this study, we offer preclinical

evidence this is the case for a CSC-DC vaccine as tested in murine models of SCC7 squamous

cell cancer and D5 melanoma. Vaccination of mice with an ALDHhigh SCC7 CSC-DC vaccine

after surgical excision of established SCC7 tumors reduced local tumor relapse and prolonged

host survival. This effect was augmented significantly by simultaneous administration of

anti-PD-L1, an immune checkpoint inhibitor. In the minimal disease setting of D5 melanoma,

treatment of mice with ALDHhigh CSC-DC vaccination inhibited primary tumor growth, reduced

spontaneous lung metastases and increased host survival. In this setting, CCR10 and its ligands

were downregulated on ALDHhigh D5 CSCs and in lung tissues respectively after vaccination

with ALDHhigh D5 CSC-DC. RNAi-mediated attenuation of CCR10 blocked tumor cell

migration in vitro and metastasis in vivo. T cells harvested from mice vaccinated with ALDHhigh

D5 CSC-DC selectively killed ALDHhigh D5 CSCs, with additional evidence of humoral

immunological engagement and a reduction in ALDHhigh cells in residual tumors. Overall, our

results offered a preclinical proof of concept for the use of ALDHhigh CSC-DC vaccines in the

adjuvant setting to more effectively limit local tumor recurrence and spontaneous pulmonary

metastasis, as compared with traditional DC vaccines, with increased host survival further

accentuated by simultaneous PD-L1 blockade.




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Introduction

Although surgical resection has been a standard treatment for solid malignancies, therapeutic

efficacy is limited by both local and distant recurrence (1-3). There are many factors associated

with tumor recurrence (4, 5). Several reports have described strategies to eliminate residual

tumor cells after surgery (3, 6). However, effectively preventing local tumor recurrence remains

a significant challenge. The existence of micro metastasis at the time of tumor resection

represents an even greater therapeutic challenge, since 90% of tumor deaths are due to tumor

metastasis. There is increasing evidence that many cancers are driven and maintained by a

subpopulation of cells that display stem cell properties. Cancer stem cells (CSCs) can self-renew,

mediate tumor growth and contribute to tumor recurrence and metastasis (7-9). Targeting CSCs

may thus increase the therapeutic efficacy of current cancer treatment.

ALDEFLUOR/ALDH (aldehyde dehydrogenase) activity has been successfully used as a marker

to enrich CSC populations in a variety of cancers (10-17). We previously demonstrated that

ALDHhigh murine squamous carcinoma SCC7 and D5 melanoma cells were highly enriched for

tumor initiating capacity (15). Their protective immunogenicity was evaluated by administering

CSC-based dendritic cell (DC) vaccines in syngeneic immunocompetent hosts (15). In a recent

study (17), we demonstrated significant therapeutic efficacy conferred by an ALDHhigh CSC-DC

vaccine in the treatment of established tumors following localized radiation therapy (RT).




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Eliminating microscopic residual disease in the tumor bed is important in preventing local

disease recurrence. Administration of CSC-based vaccines after surgical excision of tumor,

where local recurrence is high, may reduce local tumor relapse and distant metastasis and

possibly improve survival. Furthermore, since CSCs mediate tumor metastasis, targeting this cell

population in the adjuvant setting may eliminate micro metastasis prolonging survival. In this

study, we evaluated the potential therapeutic efficacy of this approach in the adjuvant setting

using CSC-DC vaccination following surgical resection of the tumor, or by treatment of minimal

disease.

We developed a vaccination strategy utilizing cell lysates from ALDHhigh SCC7 or D5 CSCs to

pulse dendritic cells (CSC-DC). DCs pulsed with ALDHlow SCC7 or D5 non-CSC lysate

(ALDHlow-DC), or with heterogeneous, unsorted cell lysate (H-DC) served as controls.

Vaccination with ALDHhigh CSC-DC in immunocompetent mice significantly inhibited SCC7

local tumor recurrence after surgery, and inhibited minimal D5 tumor growth with prolonged

survival significantly more than either ALDHlow-DC or H-DC vaccination. Furthermore, this

effect was accentuated by simultaneous PD-L1 immune checkpoint blockade.




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Materials and Methods

Mice. Female C3H/HeNCr MTV (C3H) mice and C57BL/6 (B6) mice were purchased from

Jackson lab and Charles River Laboratories (15). The University of Michigan Laboratory of

Animal Medicine approved all animal protocols.

Culture of Tumor cells. The squamous carcinoma cell line, SCC7, produces a poorly

immunogenic tumor and is syngeneic to C3H mice. D5 is a clone of the melanoma cell line B16,

which is syngeneic to B6 mice, and was originally established by our laboratory. The cell lines

were grown in complete medium consisting of RMPI 1640 and supplements (15).

ALDEFLUOR assay. The ALDEFLUOR™ Kit (StemCell Technologies, British

Columbia, Canada) was used to isolate ALDEFLUOR+/ALDHhigh CSCs from the SCC7 and D5

cells (15).

Preparation of Dendritic Cell vaccine. Tumor cell lysates of unsorted,

ALDEFLUOR+/ALDHhigh or ALDEFLUOR-/ALDHlow SCC7 and D5 cells were prepared as

previously described (15). Bone marrow-derived murine cells were cultured in 10 ml complete

medium (CM) supplemented with 20 ng/mL GM-CSF at a concentration of 0.2 - 0.4 x 106

cells/ml in non-tissue culture petri dishes (Corning, Tewksbury , MA) on day 0. Fresh medium




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supplemented with 20 ng/mL GM-CSF was added On days 3, 10 ml. On day 6 and day 8, 10 ml

of cultured cell suspension was taken from each dish respectively, centrifuged, and the pellet

re-suspended in 10 ml of fresh CM with 20 ng/ml of GM-CSF, and added back to each dish. On

day 10, DCs were harvested by dispenser and enriched by Opti-Prep density gradient medium.

Lysate of unsorted, ALDHlow or ALDHhigh cells was added to DCs at a 1:3 cell equivalent ratio.

The DCs were then incubated at 37°C for 24 h with 5% CO2. After incubation, the unsorted

tumor cell lysate-pulsed DCs (H-DC), ALDHlow lysate-pulsed DCs (ALDHlow-DC) or ALDHhigh

lysate-pulsed DCs (ALDHhigh-DC, e.g. CSC-DC) will be used as vaccine as specified in the

subsequent experiments.

Tumor model and treatment protocols. C3H mice were inoculated subcutaneously (s.c.) with 0.5

million SCC7 cells on day 0. On day 21, the mice were subjected to surgical tumor resection

except for one group serving as control. The animals with the s.c. tumor removed were then

divided into 4 groups (n=5), and administrated with PBS, H-DC, ALDHlow SCC7-DC and

ALDHhigh SCC7 CSC-DC vaccine respectively 24 hours after tumor resection. The vaccination

was repeated on day 29 and day 36 respectively. Each mouse was inoculated s.c with 2 million

DCs per vaccine. In additional experiments when SCC7 CSC-DC is used in combination with

an anti-PD-L1 antibody (MedImmune Inc., Gaithersburg, MD), the vaccination was only

repeated once on day 29 with anti-PD-L1 administration. In the minimal tumor model, B6

mice were inoculated s.c. with 5,000 D5 cells. The 1st vaccine was administered s.c. 24 hours




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after tumor inoculation, followed by a 2nd vaccine on day 8. Each vaccine comprised 2 million

DCs. The long and short diameters of tumor masses were measured, as well as the tumor

volumes measured three times per week. The volumes were calculated as: tumor volume =

(width2 * length)/2. Survival was monitored and recorded as the percentage of survivors after

tumor inoculation.

Hematoxylin and eosin (H&E) staining for histologic analysis. At the conclusion of the

experiments, the lungs were harvested and stained with H&E to discern the histopathological

response.

Measurement of chemokine receptor and PD-L1 expression on tumor cells. Freshly harvested

s.c. D5 tumors were disaggregated into single cell suspensions (18). Unsorted, ALDHhigh and

ALDHlow D5 cells were then respectively incubated with PE-anti-CCR10 for flow cytometry

analysis with a BD LSR-cytometer. To evaluate the PD-L1 levels in the CSC and non-CSC

populations post-treatment, D5 tumors were harvested at the end of therapy to prepare tumor cell

suspensions. These tumor cells were then incubated with PE-anti-PD-L1 (BioLegend, San

Diego, CA), followed by staining with ALDEFLUOR (FITC) for ALDHhigh and ALDHlow

population isolation as described in ALDEFLUOR assay. The ALDHhigh (CSC) and ALDHlow

(non-CSC) D5 tumor cells were then examined by flow cytometry for PD-L1 expression.




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Detection of chemokine expression in lung tissues. The mRNA expression levels of

chemokines CCL27 and CCL28 in lung tissues were analyzed using real time quantitative PCR

(qRT-PCR) (17). The preparations of the total RNA and cDNA were previously described (19).

The data was expressed as the relative fold changed.

CCR10 gene silencing. Equal doses of CCR10 siRNA and negative siRNA (QIAGEN Sciences,

Germantown, MD) were used according to the manufacture’s instructions to transfer unsorted,

ALDHhigh, and ALDHlow D5 cells for 48 hours to inhibit CCR10 expression. 106 cells were then

resuspended and RNA extracted using RNeasy Mini Kit (QIAGEN Sciences). Five mg of total

RNA was reverse transcribed (M-MLV, Invitrogen, Carlsbad, CA) to generate cDNA for

subsequent RT-PCR. Platinum Sybr Supermix (Invitrogen) was used to amplify sequences for

CCR10 (forward: CAGTCTTCGTGTGGCTGTTGTC and reverse

TCACAGTCTGCGTGAGGCTTTC) and GAPDH (forward TGAAGCAGGCATCTGAGGG

and reverse CGAAGGTGGAAGAGTGGGAG ) using a standard 3-step protocol (35 cycles of:

30 seconds each of 95°C , 58°C , 72°C). Melting point analysis verified the presence of single

products.

Chemotaxis assay. 500 μl RMPI 1640 containing 1×106 D5 cells or CCR10 siRNA transferred

D5 cells were added to the upper chamber of a transwell (insert pore size, 8μm; Corning, New

York, NY). Chemokines CCL27 and CCL28 (R&D Systems, Minneapolis, MN) were added to




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the lower chamber in a volume of 750μl RMPI 1640 which contained 20% FBS. After 37°C

incubation for 27 hours, the cells that migrated to the lower surface of membrane were stained

with Diff-QuikTM set (Siemens Healthcare Diagnostics Inc, Newark, DE). Cells were

photographed under the microscope at 200×magnifications, and counted in 5 fields of triplicate

membranes.

Purification and culture of host B cells and T cells. Spleens were harvested from animals

subjected to various treatments at the end of the experiments. Splenic B cells were purified and

activated in CM supplemented with lipopolysaccharide (LPS, SIGMA, St. Louis, MO),

anti-CD40 (AdipoGen, San Diego, CA) and IL-2 (Prometheus Laboratories Inc., San Diego, CA)

(15). The culture supernatants were collected and stored at -20°C for future experiments.

Splenic T cells were purified and activated to generate CTLs that were analyzed in LDH

cytotoxicity assays (15).

CSC binding by immune supernatant and antibody/complement mediated cytotoxicity. Sorted

ALDHihgh or ALDHlow D5 cells were incubated with the immune supernatants collected from the

cultured B cells with equal quantities of IgG followed by incubation with the second

FITC-conjugated anti-mouse IgG. The binding of supernatant antibody to ALDHhigh vs. ALDHlow

D5 cells was assessed using flow cytometry (15). Antibody and complement-mediated

cytotoxicity against CSCs was measured as previously described (15).




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Statistics. Survival analysis was determined by the log-rank test. Analysis for the presence of

lung metastasis was performed using the Fisher exact test. Other data were evaluated by unpaired

Student’s t-test (2 cohorts) or one-way analysis of variance (ANOVA) (> 2 cohorts).




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Results

1. An ALDHhigh CSC-DC vaccine significantly inhibited tumor recurrence and prolonged

animal survival after surgical resection of head and neck SCC7 tumors

We previously demonstrated that administration of ALDHhigh SCC7 CSC-DC vaccines in

immunocompetent mice induces protection against subsequent SCC7 challenge (15). In this

study, we examined the therapeutic potential of CSC-DC vaccination to prevent local tumor

recurrence, reduce metastasis, and prolong survival when deployed in the adjuvant /early disease

settings. The first model employed surgical excision of SCC7 s.c head and neck squamous

carcinomas, a tumor in which local recurrence contributes to patient mortality and morbidity (20,

21). C3H mice were inoculated s.c. with 0.5 x 106 SCC7 tumor cells. Resulting tumors were

surgically excised 21 days after inoculation, followed by vaccination with DCs pulsed with

lysates of heterogeneous unsorted SCC7 cells (H-DC), ALDHlow SCC7 cells (ALDHlow-DC) or

ALDHhigh SCC7 cells (ALDHhigh –DC). Vaccines were administrated once per week for 3 weeks

starting on the second day post-surgery. Mice were subsequently monitored for local tumor

recurrence and survival.

As shown in Figure 1, there was 100% mortality in tumor bearing mice without tumor resection

by day 40 due to progressive tumor growth. In PBS control mice, tumor recurrence was noted

beginning on day 30 and all mice ultimately died by day 55 due to tumor growth. The H-DC




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and ALDHlow-DC vaccination delayed tumor recurrence, resulting in prolonged animal survival

compared with control mice. More importantly, the ALDHhigh-DC (CSC-DC) vaccine

significantly reduced tumor recurrence compared with the PBS control (p<0.0001), H-DC

(p=0.0221) and ALDHlow-DC (p=0.0495) vaccination, respectively (Figure1A). As a result, the

ALDHhigh-DC treatment significantly increased animal survival compared to the other treatments

or control mice (Figure1B). While only a 50% of the mice in H-DC and ALDHlow-DC treated

groups survived to day 65 all of the mice treated with the ALDHhigh-DC vaccine survived to that

time point. These results demonstrate the ability of the ALDHhigh-DC vaccine to reduce local

recurrence and prolong survival in this model of SCC.

One of the major recent advances in tumor immunotherapy has been the development of

strategies to block the immunosuppressive components of the tumor microenvironment (22, 23).

We next performed experiments where SCC7 s.c tumors were surgically excised as in Figure 1A,

and animals were treated as indicated in Figure 1C with or without anti-PD-L1 administration.

SCC7 ALDHhigh-DC (CSC-DC) vaccination plus anti-PD-L1 administration significantly

inhibited tumor relapse (Figure 1C) and prolonged animal survival (Figure 1D) compared to

either treatment alone. These experiments clearly demonstrate that immunologically targeting

CSCs, while simultaneously blocking PD-1/PD-L1-mediated immune suppression, has the

potential to significantly enhance the efficacy of cancer immunotherapies.




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2. CSC-DC vaccination inhibited tumor growth and prevented spontaneous lung metastasis in

D5 melanoma

To evaluate the therapeutic efficacy of the CSC-DC vaccine in the setting of micrometastatic

disease, we utilized the highly metastatic D5 mouse melanoma model. In order to test the

efficacy of the CSC-DC vaccine in treating micro-metastatic disease, it was administered 24

hours following inoculation of tumor cells. Syngeneic B6 mice were inoculated with 5,000 D5

melanoma cells s.c. followed by vaccination 24 hours later (day 1) with DCs pulsed with the

lysate of ALDHhigh D5 CSCs (CSC-DC), ALDHlow D5 cell lysate (ALDHlow-DC), heterogeneous

unsorted D5 cell lysate (H-DC), or with PBS, respectively. The treatment was repeated on day 8.

As shown in Figure 2A, no significant difference in primary tumor growth was observed among

PBS, H-DC or ALDHlow-DC treated mice. However, administration of the CSC-DC vaccine

treatment resulted in significant inhibition of tumor growth compared to controls (p<0.02 vs. all

other groups). The CSC-DC treated mice also survived longer than controls (Figure 2B). These

data indicate that treatment of s.c tumor-bearing mice in the setting of minimal tumor with

CSC-DC vaccination generated significant antitumor immunity, resulting in inhibited s.c. tumor

growth and prolonged survival of the tumor-bearing hosts.

To investigate the effect of these treatments on the development of lung metastases, we harvested

the lungs at the end of the experiments and accessed lung metastases. Representative histology of




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the lungs is shown in Figure 2C. Mice subjected to PBS treatment, H-DC or ALDHlow-DC

vaccine all displayed numerous large lung metastases. In contrast, there were significantly

reduced lung metastases detected in the lungs harvested from ALDHhigh CSC-DC vaccinated

hosts (Figure 2C). The ALDHhigh-DC vaccine significantly inhibited tumor metastasis to the

lung compared with PBS, H-DC and ALDHlow-DC vaccine treatments (p<0.05, Figure 2D).

Only 2 of 11 total mice developed lung metastasis after ALDHhigh-DC vaccination, while 9 of 11

mice treated with PBS or ALDHlow-DC; and 8 of 11 mice treated with H-DC developed lung

metastases (Figure 2D). Together, these results indicated that CSC-DC vaccine significantly

inhibited tumor growth and lung metastases resulting in increased animal survival. In addition,

as shown in Figure 2E, after ALDHhigh-DC vaccination, PD-L1 expression on ALDHhigh cells

(CSC) was decreased to 5.3% compared to PBS (17.2%), H-DC (8.3%) or ALDHlow-DC

(11.4%) treatment. Similarly, PD-L1 expression on ALDHlow cells (non-CSC) was decreased

after ALDHhigh-DC vaccination to 4.5% (Figure 2F) compared to PBS (8.2%), H-DC (6.2%)

or ALDHlow-DC (7.6%) treatment. Statistically, when the mean+/-SE of the three experiments’

data were compared (Figure 2G), ALDHhigh-DC vaccination significantly (p<0.05) reduced the

PD-L1 expression on both ALDHhigh cells (CSC) and ALDHlow (non-CSC) cells.

3. CSC-DC vaccination significantly down-regulated CCR10 expression on ALDHhigh CSCs

Chemokines play a significant role in tumor metastasis (24-26). We examined the expression of




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CCR10 on tumor cells from mice treated in the minimal disease setting, and compared its

expression in ALDHhigh CSCs vs. ALDHlow non-CSCs. CCR10 expression was accessed by flow

cytometry in D5 tumors harvested from animals subjected to various vaccines (Figure 3A and

3B). CSC-DC vaccination significantly decreased expression of CCR10 in unsorted bulk tumor

cells (p< 0.01 vs. all other groups). With CSC-DC vaccination, the expression of CCR10 on D5

tumor cells was significantly decreased to approx. 3% compared with PBS treatment (>20%), or

with H-DC and ALDHlow-DC vaccination (both around 15%) (Figure3A, 3B). We then sorted

ALDHhigh and ALDHlow cells from freshly harvested D5 tumors subjected to vaccine therapy, and

assessed their CCR10 expression. We found that the expression of CCR10 was significantly

(p<0.0001) higher on D5 ALDHhigh CSCs (>60%) than on ALDHlow non-CSCs (<20%) (Figure

3C, 3D, PBS groups). ALDHhigh CSC-DC vaccination significantly decreased the expression of

CCR10 on D5 ALDHhigh as well as on ALDHlow cells (Figure 3C, 3D). Finally, using qRT-PCR,

we found that mRNA for the corresponding chemokine ligands for CCR10 in the lung tissues,

CCL27 and CCL28, were both significantly decreased after ALDHhigh CSC-DC vaccine

treatment (p<0.01 vs. all other groups) (Figure 3E). Collectively, these data suggest that

CSC-DC vaccination may inhibit pulmonary metastasis of the local tumor by significantly

down-regulating the expression of CCR10 on primary tumor cells, particularly on the ALDHhigh

CSCs in the primary tumor, as well as reducing the production of CCR10 ligands, CCL27 and

CCL28 in the lung tissues.




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4. The role played by CCR10 in the metastasis of tumor cells was significantly blocked by

CCR10 siRNA inhibition

To substantiate the role for CCR10 and its ligands in tumor metastasis, we used CCR10 siRNA

to inhibit CCR10 gene expression as described in Materials and Methods. To test the effect of

CCR10 siRNA on the inhibition of D5 cell migration in vitro, we carried out a chemotaxis assay.

As shown in Figure 4A, CCR10 siRNA treated D5 cells demonstrated significantly (p<0.001)

less migration ability than non-treated D5 cells towards the CCL27 and CCL28 added to the

bottom of the transwell at the concentrations as indicated. To test the effect of CCR10 siRNA on

the inhibition of ALDHlow and ALDHhigh D5 cell migration in vivo, we compared the metastasis

of non-treated ALDHlow and ALDHhigh D5 cells with that of CCR10 siRNA transferred ALDHlow

and ALDHhigh D5 cells when 1x106 cells of each group were i.v. injected into the normal B6

mice. As expected, ALDHhigh D5 cells generated significantly (p=0.03) more metastasis than

ALDHlow D5 cells (Figure 4B). Importantly, CCR10 siRNA-treated ALDHlow and ALDHhigh D5

cells generated significantly less metastasis than non-treated ALDHlow (p=0.0002) or ALDHhigh

(p=0.0003) D5 cells respectively. These experiments strongly suggest that CCR10 plays an

important role in the migration and therefore the metastasis of D5 tumor cells.

To confirm the efficacy of CCR10 siRNA in CCR10 gene silencing, equal doses of CCR10

siRNA and control siRNA were used to transfer unsorted D5 cells for various time periods, e.g.




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24, 48, and 72 hours. Figure 4C shows that transfer of unsorted D5 cells for 48 hours begin to

demonstrate significantly (p=0.0005) silenced CCR10 gene. We therefore transferred unsorted

D5 cells for 48 hours in Figure 4A as well as for ALDHhigh and ALDHlow D5 cells in Figure 4B.

In addition, Figure 4D revealed that CCR10 gene expression in ALDHhigh D5 cells is higher

(p<0.0001) than that in ALDHlow D5 cells. Importantly, CCR10 siRNA treated ALDHlow and

ALDHhigh D5 cells showed significantly down-regulated CCR10 gene expression compared

with non-treated ALDHlow (p<0.05) and ALDHhigh (p<0.05) D5 cells, respectively.

5. CSC-DC vaccination conferred host CSC-specific antibody responses

To provide experimental evidence that CSC-DC vaccination induces specific anti-CSC

immunity, we collected the spleens following the full treatment course in the minimal D5 tumor

model, purified splenic B cells, and activated them in vitro with LPS and anti-CD40. We then

accessed the specificity of CSC-DC vaccine-primed antibody by binding assays of the B cell

culture supernatants to ALDHhigh D5 CSCs vs. ALDHlow D5 non-CSCs respectively. Immune

supernatants produced by B cells from mice that received ALDHhigh-DC treatment bound to

ALDHhigh D5 CSCs (60.8%) (Figure 5A) much more effectively than the binding of immune

supernatants collected from PBS-treated (12.3%), H-DC vaccinated (29.8%), or ALDHlow-DC

treated (15.7%) mice. In contrast, the immune supernatants produced by B cells harvested from

H-DC or ALDHlow-DC vaccinated mice bound to the ALDHlow non-CSCs (45.8% and 50.2%




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respectively) significantly more than the binding of immune supernatants produced by B cells

harvested from the CSC-DC vaccinated mice (6.8%) or from PBS-treated controls (18.8%).

Figure 5B shows the results of multiple binding assays, indicating that the immune supernatants

produced by CSC-DC vaccine-primed B cells bound to the ALDHhigh D5 CSCs much more

effectively (p<0.01 vs. all other groups). In contrast, the ALDHlow-DC vaccine-primed immune

supernatants bound to the ALDHlow non-CSCs similar to the binding by H-DC vaccine-primed

immune supernatants, but significantly more than the binding of PBS or CSC-DC

vaccine-primed immune supernatants (Figure 5C); demonstrating CSC-DC vaccine induced

CSC-specific humoral immunity.

To examine the functional consequence of CSC-specific antibody induced by CSC-DC

vaccination, we performed antibody and complement-dependent cytotoxicity (CDC) assays

(Figure 5D). ALDHhigh CSC-DC vaccine-primed immune supernatant killed ALDHhigh D5

CSCs significantly more than the immune supernatants collected from other groups (p<0.001 vs.

all other groups). In contrast, the immune supernatant harvested from H-DC or ALDHlow

non-CSC vaccinate-treated host resulted in significant ALDHlow D5 cell lysis, while the immune

supernatant from the ALDHhigh CSC-DC vaccinated hosts produced minimal lysis of the

ALDHlow targets. Together these data support the conclusion that ALDHhigh D5 CSC-DC

vaccine confers significant host anti-CSC humoral immunity by producing D5 CSC-specific

antibodies that specifically bind and kill D5 CSCs.




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6. CSC-DC vaccination conferred host CSC-specific CTL function

We next examined the ability of CSC-DC vaccination to generate host CSC-specific CTL activity.

As in Fig. 5, we collected the spleens at the end of the treatment in the minimal D5 tumor model;

purified splenic T cells, and activated them in vitro with anti-CD3/anti-CD28 followed by

expansion in IL-2. This activation procedure generates cytotoxic T cells (>95 CD3 cells) (15).

We then measured the CTL activity of these T cells on ALDHhigh D5 CSCs vs. ALDHlow D5

non-CSCs respectively. D5 ALDHhigh-DC-primed CTLs mediated significantly greater

cytotoxicity in D5 ALDHhigh CSCs at all E:T ratios compared with the CTLs generated from PBS,

H-DC or ALDHlow DC-primed CTLs (p<0.05 Figure 6A). In contrast, CTLs generated from

splenocytes of mice subjected to ALDH low DC and H-DC vaccination selectively killed ALDHlow

D5 cells (Figure 6B). These experiments indicate that CSC-DC vaccination conferred host CTL

reactivity as well as humoral responses against CSCs in the treatment of minimal tumor disease.

7. CSC-DC vaccination significantly reduced the population of ALDHhigh CSCs in vivo

As described above, ALDHhigh CSC-DC vaccination induced significant host cellular and

humoral immune responses against CSCs. To confirm that CSCs are effectively targeted by

CSC-induced immunity, we determined the effect of CSC-DC vaccination on the proportion of

ALDHhigh CSC in vivo. Assessment of the ALDHhigh population was performed by flow




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cytometry using the Aldefluor assays as previously described (15). We mixed the tumor cells

from mice of each experimental group, and generated representative flow cytometric graphs to

demonstrate the ALDHhigh populations in each group (Figure 7A). Subcutaneous tumors

harvested from CSC-DC treated mice contained only 1.7% ALDHhigh cells, which was

significantly less than that present in the s.c. tumors subjected to PBS (13.4%), H-DC (7.5%) or

ALDHlow-DC (8.3%) treatments. As shown in Figure 7B, CSC-DC vaccination significantly

reduced the percentage of ALDHhigh populations compared with PBS, H-DC, or ALDHlow-DC

treatments (p=0.0002, 0.0002 and 0.0029, respectively) in this tumor model. Together, these

studies demonstrate that in the D5 minimal disease model, CSC-DC vaccine elicits both humoral

and cellular immune responses reducing the proportion of CSC, resulting in decreased tumor

growth, lung metastases and prolonged survival.




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Discussion

Utilizing two tumor models, we demonstrate the efficacy of a CSC-DC vaccine when used to

treat minimal disease in the adjuvant setting. Several reports have described the generation of

CSC-specific CD8 T effector cells in vitro (27-30); the killing of CSCs via non-specific

immune effector cells (31-34) as well as by oncolytic viruses (35) and antibodies (36). We

previously reported therapeutic efficacy of CSC-DC vaccination in the treatment of established

tumors following localized radiation therapy (RT) (17). However, since CSC may be responsible

for local tumor recurrence following resection (37) as well as mediating tumor metastasis

(38-41), CSC targeted therapeutics may have their greatest utility when they are utilized in the

adjuvant-minimal disease setting. We examined this utilizing two different mouse tumor

models.

The SCC7 squamous carcinoma model was designed to determine the efficacy of CSC-DC

vaccination following surgical removal of the primary tumor. This model is clinically relevant

since in squamous carcinoma of the head and neck, resection of bulky SCC primary tumors has

been associated with a high rate of local tumor recurrence associated with significant morbidity

and mortality (20, 21). Using the murine SCC7 tumor model, we found that the SCC7 ALDHhigh

CSC-DC vaccine significantly inhibited tumor recurrence and prolonged animal survival

following surgical resection compared with SCC7 H-DC or SCC7 ALDHlow-DC vaccinations.

Simultaneous administration of an anti-PD-L1 mAb significantly enhanced the therapeutic




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efficacy of SCC7 CSC-DC vaccine in the adjuvant setting.

The second model involved treatment of D5 murine melanoma in an early disease setting 24

hours after tumor inoculation. While the H-DC vaccination and the ALDHlow-DC had minimal

effects on the local tumor growth and only modestly prolonged survival, the ALDHhigh CSC-DC

vaccine was significantly more effective in inhibiting tumor growth, resulting in prolonged

survival.

To date, the mechanisms that are involved in CSC-DC vaccine-mediated therapeutic efficacy

have not been fully defined, and limited experimental evidence was provided for direct targeting

of CSCs by CSC-DC vaccine-induced anti-CSC immunity. CSCs are responsible for tumor

metastasis and progression (38-41). In this study we found that the therapeutic efficacy of

CSC-DC vaccine was associated with significantly inhibited metastasis of the s.c. tumor to the

lung. A number of studies have suggested that tumor cell metastasis is determined by the

expression level of chemokine receptors on the malignant tumor cells and the expression of

corresponding chemokine ligands in the target organs (24-26, 42-46).

We demonstrated high levels of CCR10 (>20%) in tumor cells isolated from control mice.

CSC-DC vaccination significantly reduced the expression of CCR10 to 3%. More importantly,

we found that the expression of CCR10 was significantly higher on D5 ALDHhigh CSCs (>60%)




24

than on ALDHlow non-CSCs (<20%), and ALDHhigh CSC-DC vaccine significantly decreased the

expression of CCR10 on D5 ALDHhigh cells to <15%. In a group of experiments, we found that

CCR10 siRNA treatment to inhibit CCR10 gene expression significantly blocked tumor cell

migration in vitro and metastasis in vivo. In addition, ligands for CCR10, including CCL27 and

CCL28 were significantly decreased in the lung tissues harvested from the animals treated with

CSC-DC vaccination. These data suggest that decreased CCR10, CCL27 and CCL28 may play

an important role in CSC-DC vaccination-induced inhibition of tumor metastasis. Chemokine

receptors can activate downstream effectors, such as mitogen-activated protein kinases, by

complex mechanisms (47). The molecular and biochemical signaling pathways by which

CSC-DC vaccination induces down-regulation of CCR10, CCL27 and CCL28 remain to be

identified.

We examined the ability of CSC-DC vaccines to elicit CSC specific humoral and cellular

immune responses. Using splenocytes collected from the treated mice, we generated CTLs. D5

ALDHhigh-DC-primed CTLs significantly killed the D5 ALDHhigh CSCs compared with the CTLs

generated from PBS, H-DC or ALDHlow DC-primed CTLs. In contrast, CTLs generated from

splenocytes of mice subjected to ALDH low DC and H-DC vaccination selectively killed ALDHlow

D5 cells. These experiments indicate that CSC-DC vaccination confers host CTL activity that

specifically target CSCs. In addition, the ALDHhigh CSC-DC vaccine-primed host B cells

produced antibody specifically bound to ALDHhigh CSCs (>60%), which was significantly higher




25

than the binding by antibodies produced PBS, H-DC or ALDHlow-DC-primed B cells (10-30%).

In contrast, H-DC or ALDHlow-DC vaccine-primed B cells produced antibody preferentially

bound to ALDHlow D5 cells (45-50%), which was significantly higher than the binding by

antibodies produced by PBS or ALDHhigh CSC-DC vaccine-primed B cells (18% and 6%

respectively). The immunological consequence of antibody binding of the CSCs was the lysis

of the CSCs in the presence of complement, demonstrating that CSC-DC vaccines elicit

significant humoral immune responses against CSCs as well as CSC specific cellular immune

responses. While we demonstrated that antibodies and CTLs were induced against cancer stem

cells, the identity of any recognized antigens has yet to be elucidated. Identification of CSC

antigen(s) represents an active research focus in our lab and warrants further investigation.

The induction of cytotoxic T cells to CSCs has been observed in 2 different animal histologies

using the CSC lysate vaccine both in our previous protection study (15) and in this therapeutic

study. To date, we have not observed immune tolerance in our model system of CSC-DC

vaccination. However, an immune adjuvant may enhance the induction of a tumor lysate-DC

vaccine. We previously reported that the use of a second signal agent such as anti-4-1BB mAb

augmented the anti-tumor efficacy of DC-based vaccines (48). We did not use this approach in

this report in order to focus on the use of CSC-DC vaccines by themselves. Nevertheless, the

use of adjuvant agents may enhance T, B cell activation as a method to improve CSC-DC

vaccine-induced anti-CSC immunity.




26

Our experiments provide direct evidence that CSC-DC vaccine can induce anti-CSC immunity

by targeting CSCs. As a result, ALDHhigh CSC populations in the residual tumor of the mice

subjected to CSC-DC vaccine were significantly decreased to <2% compared with the

PBS-treated control (~15%), and was significantly lower than those of the animals subjected to

H-DC or ALDHlow-DC treatment (7-9%). In our previous publications we have demonstrated

that a reduction in ALDH expression is strongly associated with reduction in tumor initiating

capacity as accessed by extreme limiting dilution analysis (49, 50). The values of reduction of

ALDH associated with treatments shown in this study are highly statistically significant. Future

studies accessing the ability of CSC-DC vaccines to reduce tumor initiating capacity of treated

tumor cells transplanted into secondary animals are warranted. We propose that the significant

reduction of the residual CSCs after CSC-DC immunotherapy is due to CSC-DC

vaccine-induced cellular and humoral targeting of CSCs. Together these studies suggest the

potential clinical efficacy of utilizing CSC-DC vaccines in the adjuvant/early tumor setting, a

strategy that may be augmented by PD-l/PD-L1 immune checkpoint blockade.

.




27

Acknowledgement

We thank Jill Granger for valuable assistance in editing the manuscript.




28

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Figure legends

Fig. 1: (A) DCs pulsed with ALDHhigh SCC7 CSCs significantly inhibited tumor recurrence.

Twenty-one days after inoculation of SCC7 cells, s.c, tumors were surgically removed and

animals were treated with different vaccines as indicated on day 22, day 29 and day 36 except for

the “no tumor resection” group as a control. Tumor volume (mean±SEM) is shown. (B) DCs

pulsed with ALDHhigh SCC7 CSCs significantly prolonged the animal survival after surgical

resection of the s.c. SCC7 tumors. Data are representative of three experiments independently

performed. (C) Administration of anti-PD-L1 significantly inhibited tumor recurrence in animals

treated with suboptima1 doses (2 vs. 3 in A) ALDHhigh SCC7 CSC-DC vaccinations after

surgical resection of the s.c. SCC7 tumors. SCC7 s.c tumors were surgically excised 21 days

after inoculation as in (A). Animals were then treated with different vaccines as indicated on day

22 and day 29 except for the “no tumor resection” group as a control. In addition, anti-PD-L1

(0.05mg/mouse) was intraperitoneally injected on days 22 and 25, days 29 and 32, either alone or

with the ALDHhigh-DC vaccine. (D) Administration of anti-PD-L1 significantly prolonged the

survival of animals treated in (C). Two experiments were independently performed.

Fig. 2: In the minimal D5 tumor model, the ALDHhigh CSC–DC vaccine significantly inhibited

tumor growth and prolonged the survival which was associated with prevented lung metastasis.

(A) The CSC-DC vaccination significantly inhibited subcutaneous tumor growth. 24 hours

after s.c inoculation of D5 cells, animals were treated with different vaccines as indicated, and




33

the treatment was repeated one week later. Tumor volumes (mean ± SEM) are shown. (B)

CSC-DC vaccine significantly prolonged the survival of s.c D5-bearing mice. (C, D) CSC-DC

vaccination significantly prevented the lung metastasis of the s.c injected tumor. (C) Lung

metastasis was verified by hematoxylin and eosin (H&E) staining. Representative graphs show

the histologic alternation of the lung tissues. Lung tissue harvested from a normal B6 mouse

served as control. The red arrows point to the tumor lesions in the lung tissues. (D) p values

comparing lung metastasis (n=11) among groups treated as indicated. Data are representative of

three independent experiments performed. (E, F) Representative flow cytometry data of one of

the three experiments performed to show decreased PD-L1 expression on ALDHhigh cells (CSC,

E) and ALDHlow cells (non-CSC, F) after ALDHhigh-DC vaccination. (G) Statistically,

ALDHhigh-DC vaccination significantly (p<0.05) reduced the PD-L1 expression on both

ALDHhigh cells (CSC) and ALDHlow (non-CSC) cells.

Fig. 3: The expression of CCR10 was significantly down-regulated on ALDHhigh D5 cancer stem

cells in animals subjected to ALDHhigh D5 CSC-DC vaccine treatment. (A) Flow cytometry

graphs of CCR10, which were generated using mixed D5 cells harvested from multiple animals

in each treatment group as indicated. (B) Bar graph shows the p value with SE using the D5 cells

harvested from each experiment group. Data are representative of two independently

performed experiments. (C) Flow cytometry graphs of CCR10 expression on D5

ALDHhigh-CSCs vs. ALDHlow-non CSCs post treatment of the minimal tumor with PBS,




34

ALDHlow-DC, H-DC, and ALDHhigh-DC respectively. (D) p values comparing CCR10

expression on D5 ALDHhigh-CSCs vs. D5 ALDHlow-non CSCs from animals treated as indicated.

(E) PCR analyses showed that the ALDHhigh-DC (CSC-DC) vaccine significantly reduced the

mRNA levels of CCR10 ligands, e.g. CCL27 and CCL28, in lung tissues harvested from

D5-bearing host subjected to treatments in minimal disease.

Fig.4: CCR10 siRNA significantly blocked the role played by CCR10 in tumor metastasis. (A)

The effect of CCR10 siRNA on the inhibition of D5 cell migration in vitro in a Chemotaxis assay.

Data are representative of three chemotaxis assays independently performed. (B) The effect of

CCR10 siRNA on the inhibition of metastasis of ALDHlow and ALDHhigh D5 cells. (C) Equal

doses of CCR10 siRNA and control siRNA were used to transfer unsorted D5 cells for various

periods of time as indicated to inhibit CCR10 expression. The data was expressed as the relative

fold changed. Relative fold changes of CCR10 gene expression as shown in C represent the

averages of 3 replicates for each group analyzed via the 2-∆∆CT method. (D). ALDHlow and

ALDHhigh D5 cells showed significantly down-regulated CCR10 gene expression by CCR10

siRNA treatment. Data as shown represent the averages of 3 replicates for each group analyzed

via the 2-∆∆CT method.

Fig. 5: Antibody produced by D5 CSC-DC vaccine-primed B cells bound and killed D5 CSCs

specifically. (A) Flow cytometry histograms using culture supernatant of mixed B cells from




35

each treatment group. (B) Statistic analysis of the binding to ALDHhigh CSCs by immune

supernatants primed by PBS, H-DC, ALDHlow-DC or ALDHhigh-DC respectively. (C) Statistical

analysis of the binding to ALDHlow D5 cells by immune supernatants primed as indicated.

Binding experiments were repeated three times. (D) CSC-DC vaccine-primed antibody

selectively targeted CSCs via CDC. 105 viable ALDHhigh or ALDHlow D5 cells were incubated

with the culture immune supernatants of purified and activated spleen B cells collected from the

animals subjected to treatments as indicated. The cells were then incubated with rabbit

complement for 1 hour. The trypan blue staining was used to assess the cell lysis，which was

expressed as: % viable cells = the number of viable cells after immune supernatant and

complement incubation/105. Each experiment was repeated once.

Fig. 6: T cells harvested from D5 ALDHhigh DC vaccinated animals selectively and significantly

killed the ALDHhigh D5 cells. CTLs were generated as described in the Materials and Methods

from the spleens harvested from the animals subjected to PBS, H-DC, ALDHlow–DC or

ALDHhigh–DC vaccination respectively. Cytotoxicity mediated by CTLs was measured by

LDH release assay. (A) ALDHhigh–DC (CSC-DC)-primed CTLs selectively and significantly

killed the ALDHhigh D5 CSCs (p<0.05 compared with all other groups). (B) CTLs generated

from the splenocytes of mice vaccinated with ALDHlow–DC or H-DC killed ALDHlow D5 cells

specifically.




36

Fig. 7: The ALDHhigh CSC-DC vaccine treatment significantly decreased the percentage of

ALDHhigh cells in the s.c minimal residual tumors. (A) Representative flow cytometry showing

the percentage of ALDHhigh cells in the residual tumor after different treatment as indicated. (B)

The bar graph shows the mean+/-SE and p values using multiple animals from each group.




Published OnlineFirst June 20, 2016.Cancer Res Yangyang Hu, Lin Lu, Yang Xia, et al. adjuvant settingTherapeutic efficacy of cancer stem cell vaccines in the

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