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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Clin Cancer Res. Author manuscript; available in PMC Oct 1, 2011.
Published in final edited form as:
PMCID: PMC3016438
NIHMSID: NIHMS251455

Superior Efficacy of Tumor Cell Vaccines Grown In Physiologic Oxygen

Abstract

Purpose

Atmospheric oxygen (~20% O2) has been the universal condition employed to culture tumor cells used as vaccine antigen. We tested the hypothesis that reducing oxygen tension would increase the efficacy of tumor cell lysate vaccines.

Experimental Design

GL261 glioma cells and EMT6 breast carcinoma cells were grown in 5% or 20% O2. Syngeneic tumor-bearing mice were vaccinated with these tumor cell lysates mixed with CpG oligodeoxynucleotides as an adjuvant. Tumor infiltrating T cells and apoptotic GL261 cells were quantified by immunohistochemistry. Tumor-reactive immunoglobulin was detected by western blot. Ovalbumin and gp100-derived peptides were mixed with GL261 lysates as marker antigens to detect changes in presentation of exogenous antigen on major histocompatibility complex (MHC) class I in vitro, and in vivo following adoptive transfer of gp100-specific CD8+ T cells.

Results

Mice bearing orthotopic glioma and breast carcinoma survived significantly longer when vaccinated with 5% O2 lysates. Antigen-specific cytotoxic T lymphocyte (CTL) activation was significantly enhanced following stimulation with lysates derived from GL261 cells grown in 5% O2 versus 20% O2 through a mechanism that involved enhanced cross presentation of exogenous antigen on MHC I. Vaccination with 5% O2 GL261cell lysates caused a significant increase in CTL proliferation, tumoricidal function, and trafficking into brain tumor sites, whereas 20% O2 lysate vaccines predominantly evoked an antibody response.

Conclusions

Tissue culture oxygen functions as an “immunologic switch” by dictating the cellular and humoral immune responses elicited by tumor cell lysates. These results have profound implications for cancer vaccines that utilize tumor cells as the source of antigen.

Keywords: Hypoxia, Toll-like receptors, Vaccine, Glioma, Immunotherapy

Introduction

Therapeutic cancer vaccines have been administered in over 100 clinical trials in fifteen countries (13). Immunologic and clinical responses have been documented in patients with numerous cancers including glioma, colon and renal cell carcinoma (1, 35). However, clinical responses typically occur in a small percentage of patients and the majority of randomized clinical trials have failed to demonstrate a survival advantage in patients receiving cancer vaccines (1, 3). Tumor cells are frequently used as the source of vaccine antigen, functioning as a personalized immunotherapy, targeting multiple, patient-specific tumor antigens. Vaccines that utilize tumor cells as the source of antigen include tumor lysate-pulsed dendritic cells (DCs), DC/tumor cell fusions, tumor-derived heat shock proteins (HSPs), cytokine-secreting tumor cells, and direct administration of tumor cell lysate. In spite of this, tumor cell extracts are typically poorly immunogenic. There is evidence that tumor cell lysates suppress DC maturation and restrain the priming of T cells (6, 7). Although various strategies have been attempted to increase tumor cell immunogenicity (e.g., heat shock, irradiation, genetic engineering; 1, 7, 8), the limited efficacy of cancer vaccines in randomized clinical trials demonstrates the need for novel approaches.

We hypothesized that tissue culture oxygen would modify the immunogenicity of tumor cells because oxygen is a regulator of many processes including cell metabolism and gene transcription (810). As available oxygen decreases, hypoxia inducible factors (HIFs) accumulate, changing expression of genes such as those encoding angiogenic cytokines, transcription factors, and regulators of stem cell self-renewal (8, 9, 11, 12). Recent studies demonstrated profound changes in glioma cell gene expression when comparing cells grown in conventional atmospheric oxygen (~20% O2) to more physiologically relevant oxygen tensions that likely exist in the tumor bed, ranging from 1–7% O2 (1113). Of particular importance to immunotherapy is work demonstrating that hypoxia-associated injury is sufficient to induce inflammation. Ischemia-induced hypoxia is associated with release of alarmins such as heat shock proteins and high-mobility group box 1 (HMGB1)(14, 15). Some HSPs and HMGB1 are recognized as danger signals by cells expressing toll like receptors (TLRs), recently understood to serve a dual function as sensors of infection or physiologic stress (16). The TLRs play a pivotal role in shaping innate and adaptive immune responses. For instance, unmethylated CpG Oligodeoxynucleotides (ODN) signal through TLR 9 on DCs to increase expression of inflammatory cytokines and co-stimulatory molecules that promote naïve T cell activation and differentiation (17). Thus, TLR ligands are often used as vaccine adjuvants in experimental studies and clinical trials (18).

The purpose of this study was to compare the immunogenicity of tumor lysates derived from cells cultured in conventional atmospheric oxygen (~20% O2) to a more tumor physiologic oxygen (5% O2). We extensively characterized the differential immune response in a murine model of glioma, which led to the identification of oxygen as an “immunologic switch” that affects both cell-mediated and humoral immune responses elicited by tumor cell lysates. Our data suggest that the efficacy of tumor cell vaccines could be markedly improved by reducing tissue culture oxygen.

Materials and Methods

Animal Models

Tumors were implanted into female mice (6–8 weeks old) that were purchased from Jackson Laboratory and maintained in a specific pathogen-free facility according to the guidelines of the University of Minnesota Animal Care and Use Committee. The GL261 model was established in C57BL/6 (BL6) mice by inoculation with 15,000 GL261-Luc cells in 1 µl of phosphate-buffered saline (PBS). Tumors were implanted stereotactically into the right striatum; coordinates were 2.5-mm lateral, and 0.5-mm anterior of bregma, and 3-mm deep from the cortical surface of the brain (19). The EMT6 model was established in BALB/c mice by injection of 1×106 EMT6 cells in 50 ml of PBS into the left superior mammary fat pad as described (20). Tumor cells used to establish both models were cultured in atmospheric oxygen.

Cell Culture

GL261 cells were cultured in media consisting of DMEM/F12 (1:1) with L-glutamine, sodium bicarbonate, penicillin/streptomycin (100 U/ml), B27 and N2 supplements, and 0.1-mg/ml normocin. EMT6 cells, splenocytes and T cells were cultured in RPMI 1640. GL261-Luc cells were cultured in DMEM; both DMEM and RPMI media contained 10% FBS, penicillin/streptomycin (100 U/ml), and 0.1 mg/ml normocin. Unless otherwise stated, all cultures were maintained exclusively in atmospheric oxygen. Cultures referred to as “5% O2” were briefly exposed to atmospheric oxygen 1–2 times per week for media changes and for cytokine feeding. Cultures were maintained in the indicated oxygen tension uninterrupted for at least 48 hrs prior to use in all experiments. A Thermo Scientific Forma Series II multi-gas incubator set to 5% O2, or an incubator perfused with atmospheric oxygen were used; both incubators were maintained at 5% CO2.

Immunization Protocol

Tumor cells were dissociated with non-enzymatic cell dissociation buffer (Sigma), washed thrice with PBS, resuspended in 500 µl of PBS, and frozen initially by placing in −80°C overnight. Cells were further lysed by five cycles of freezing in liquid nitrogen and thawing in a 56°C water bath. Cell debris was pelleted by centrifugation at 14,000 RCF, and the protein concentration of the supernatant was determined using a Bradford assay. Pellets were resuspended, and lysates were stored at −80°C until use. Each vaccine was prepared at the time of vaccination and consisted of 65 µg of protein lysate mixed with 50 µg of phosphorothioated type-B CpG ODN 685 (5'-tcgtcgacgtcgttcgttctc-3'; SBI Biotech, Japan) in a final volume of 100 µl injected intradermally (50 µl in the lower neck and 50 µl in the hind flank on the right thigh). Vaccination was administered weekly starting three days after tumor implantation for a total of six doses.

Flow Cytometry

A Becton Dickinson Canto three-laser flow cytometer was used for data acquisition. The following antibodies were used: CD11c-alexafluor700, CD80-PE, CD86-FITC, CD8-alexafluor700, iCOSL-PE, GITRL-PE, Vβ13-FITC, anti-SIINFEKL/H-2Kb-PE (all from eBioscience), and H-2Db-hgp100 tetramer-APC (kind gift from Dr. Robert Prins, UCLA). Antibody staining was conducted by staining 4–5 × 105 cells resuspended in 100 µl of PBS with specific antibodies according to manufacturer’s protocol, incubating for 20 min at 4°C, washing three times, and analyzing. Appropriate isotype controls were used.

For co-stimulatory molecule analysis splenocytes were harvested from BL6 mice and exposed to 156 mM ammonium chloride to eliminate erythrocytes. Remaining splenocytes were then washed thrice and cultured in complete RPMI media with 2 µg of GL261 lysate from cells grown in 5% or 20% O2. Following a 48-hrs of incubation, splenocytes were analyzed by flow cytometry.

CTL-mediated Interferon Gamma Secretion and Cross Presentation

For measurement of IFNγ BL6 mouse splenocytes were plated at a concentration of 4 × 105 cells / well in a 96 well plate in complete RPMI 1640 media. Splenocytes were pulsed with 10 µg of the indicated tumor lysate with or without 10 µg of human (h) gp10025–33 peptide (KVPRNQDWL) and incubated for 24 hrs. CD8+ T lymphocytes were purified from Pmel mouse (21) splenocytes using negative immunomagnetic selection according to the manufacturer’s protocol (Miltenyi Biotec). Purity was confirmed to be greater than 95% by flow cytometry; 2 × 105 purified Pmel CTLs were co-cultured with splenocytes. Forty-eight hrs after co-culture, IFNγ was quantified in the tissue culture supernatant using a flow cytometric bead array according to the manufacturer’s protocol (BD Biosciences).

For measurement of presented chicken ovalbumin (OVA) peptide, BL6 mouse splenocytes were plated at a concentration of 4 × 105 cells / well in a 96 well plate in complete RPMI 1640 media. Splenocytes were pulsed with 10 µg of the indicated tumor lysate with or without 10 µg of a peptide containing the core OVA-derived SIINFEKL/H-2Kb epitope (EVSQLEQLESIINFEKLTEEWTSSNVM) and incubated for 24 hrs. Cells were harvested, stained and analyzed by flow cytometry as described above.

Immunohistochemistry

Mice were perfused with 30 ml of PBS followed by 30 ml of 10% formalin. Standard processing, embedding and rehydration methods were employed to generate tissue sections (5 µm thick) for staining. Following rehydration, sections are put into ReVeal, 6.0 pH citrate buffer (Biocare Medical) and placed in a steamer for 30 mins followed by a 20 min cool-down. Slides are rinsed well in tap water and place into a PBS bath for 5 minutes. Sections were incubated with Sniper blocking reagent (Biocare Medical) for 30 min followed by incubation at 4°C overnight with the first primary antibody: rabbit monoclonal CD3 (Lab Vision); 1:400. The following day a biotinylated rabbit link (Covance) was applied for 30 min at RT. Samples were rinsed with PBS then placed into 3% H2O2 in PBS for 10 min, followed by a tap water rinse and incubation in a PBS bath for 5 min. An SA-HRP label (Covance) was applied for 30 min; samples were rinsed in PBS followed by a 2–5 min incubation in DAB. Slides were rinsed in tap water, then incubated for 5 min with the denaturing solution (Biocare Medical) before applying the second primary antibody against cleaved Caspase 3 (Biocare Medical); 1:400 dilution overnight at 4°C. The following day a biotinylated rabbit link (Covance) was applied for 30 min at RT followed by a tap water rinse and a 5 min PBS bath. This was followed with a 4+ Streptavidin AP label (Biocare Medical) for 30 min at RT and rinsing with PBS. Vulcan Fast Red (Biocare Medical) was applied for 5–15 min at RT. The samples were counterstained with Harris hematoxylin for 1 min, decolorized, rinsed in tap water for 5 min followed by routine dehydration and cover slipped using permount. Cleaved caspase 3+ and CD3+ cell number was quantified manually by a blinded review of histological sections. All tumors were harvested from mice that were sacrificed when moribund (five sections per animal, three animals per treatment group).

Ex Vivo Proliferation and CTL Analyses

These assays were conducted as previously described (22, 23). Briefly, for proliferation experiments, two million carboxyfluorescein succinimidyl ester (CFSE)-labeled Pmel splenocytes were adoptively transferred by i.v. injection. Glioma-bearing mice were vaccinated with a mixture of CpG (50 µg), lysate (65 µg), hgp10025–33 (10 µg) by intradermal injection above the shoulder and flank. Seventy-two hours following the first vaccination, draining inguinal and cervical lymph nodes were harvested, dissociated, and analyzed by flow cytometry. For the CTL assay, 72 hrs following the second vaccination, draining inguinal and cervical lymph nodes were harvested, dissociated, and incubated with CFSE labeled GL261 cells for 4 hrs, and analyzed for cytotoxicity according the manufacturer’s protocol (Immunochemistry, LLC). Briefly, following incubation, the percentage of CFSE labeled target cells that incorporated 7-AAD was determined by flow cytometry and plotted as the percent lysis.

Western Blot

GL261 tumor cells cultured in 5% O2 were washed, pelleted, and lysed in RIPA buffer containing protease and phosphatase inhibitors (Pierce). Protein concentration was determined using BCA colorimetric method (Pierce). GL261 lysates were diluted in reducing sample buffer and forty micrograms were loaded per lane on a 4–12% SDS-PAGE Gel and run at 160 volts. Gels were then transferred to nitrocellulose at 5 volts overnight (BioRad), blocked using 5% NFDM/TBS/0.1% Tween-20 for 1 hr, incubated in 1:1000 serum in blocking buffer for 1 hr and washed six times over 1 hr in TTBS. Blots were then incubated in 1:50,000 anti-mouse IgG HRP (Jackson ImmunoResearch) in blocking buffer for 1 hr and washed six times over 1 hr in TTBS. Nitrocellulose was then incubated in ECL Plus chemiluminescent substrate (GE) for 5 min, drained and exposed to HyBlot CL Autoradiography film (Denville Scientific) for 30 sec.

Statistical Analysis

Statistical comparisons were made by ANOVA, followed by post hoc comparisons using a 2-tailed t-test. Differences in animal survival were evaluated by log-rank test. All tests were performed with Prism 4 software (Graph Pad Software, Inc). P values <0.05 were considered significant.

Results

Superior efficacy of 5% O2 lysate / CpG ODN vaccine at clinically relevant dose

We previously demonstrated that vaccination of glioma-bearing mice with 20% O2 lysates mixed with CpG ODN doubled median survival by a mechanism that required CTLs and the combination of lysate mixed with CpG ODN (24). Consistent with our previous study, glioma-bearing mice treated with lysate or CpG ODN alone did not survive significantly longer than saline controls (Supplementary Fig. 1). However, our previous study employed a clinically unachievable dose of protein lysate; we dosed mice at 50 mg/kg (1 mg protein from 4×106 cells), which would require over a billion tumor cells to generate enough lysate for a single vaccination in a human. We therefore compared the potency of tumor lysate vaccination using 65 µg of protein lysate (3-mg/kg), which would require 107–108 tumor cells per vaccine in a human (routinely achieved in clinical trials; reviewed in (1)). Mice bearing intracranial GL261 glioma were treated by vaccination with GL261 cell lysates from 5% or 20% O2 cultures mixed with CpG ODN as an adjuvant. The group treated by 5% O2 lysate exhibited a 41% increase in median survival relative to saline controls (55 vs. 39 days, respectively). Twenty-seven percent of the 5% O2 lysate–treated mice survived beyond 100 days (Fig. 1A). In contrast, mice treated by 20% O2 lysate did not survive significantly longer than saline controls. Thus, tumor lysate vaccination was efficacious at a clinically relevant antigen dose only when the cells were cultured in 5% O2.

Figure 1
Tumor cells grown in 5% O2 have superior therapeutic benefit as vaccine antigen

Vaccination with 5% O2 lysates increased the number of tumor infiltrating T cells and apoptotic tumor cells

To determine the potential mechanisms responsible for the improved survival after GL261 challenge, T cell infiltration and tumor cell apoptosis were quantified at the brain tumor site. Immunohistochemistry (IHC) for CD3 revealed significantly more T cells in the tumors of mice treated by 5% O2 lysate relative to other groups (Fig. 1B). Accordingly, IHC for activated caspase 3 demonstrated greater numbers of apoptotic tumor cells in the 5% O2 lysate group (Fig. 1C). Closer examination by double labeling revealed T cells forming putative immunological synapses with tumor cells undergoing various stages of apoptosis in the 5% O2 lysate-treated mice (Fig. 1D). There were more putative immunological synapses between T cells and apoptotic tumor cells in mice treated by 5% O2 lysate, although this trend did not reach statistical significance (P=0.16; data not shown). Flow cytometry on brain infiltrating lymphocytes was performed to further characterize T cell subsets at the tumor site. Glioma-bearing mice were adoptively transferred with Pmel CD8+ T cells that express a T cell receptor which reacts with both human and murine gp10025–33 epitopes (21), the later of which is expressed in GL261 cells (25). There was a greater than four-fold increase in the number of hgp10025–33/H2-Db tetramer+CD8+ T cells in the brains of mice that were vaccinated with 5% O2 lysate relative to other groups (Supplementary Fig. 2).

5% O2 lysates enhanced antigen-specific CD8+ T cell activation

To directly measure the effect of cell lysates on CD8+ T cell activation, we developed a cell culture assay whereby GL261 cell lysates were or were not mixed with hgp10025–33. Lysate / hgp10025–33 combinations were pulsed onto BL6 mouse splenocytes and purified Pmel CD8+ T cells were co-cultured with the splenocytes for 48 hrs. CD8+ T cell activation was quantified by interferon gamma (IFNγ) release in the tissue culture supernatant. There was no appreciable IFNγ measured when splenocytes were not pulsed with hgp10025–33 (Fig. 2A). Lysates alone did not result in significant IFNγ production above background, revealing minimal reactivity to endogenous mouse gp10025–33 in the lysate (Fig. 2A). When pulsed in conjunction with hgp10025–33, 5% O2 lysates more than doubled IFNγ secretion compared to hgp10025–33 or lysate alone. In contrast, the 20% O2 cell lysates significantly suppressed antigen-specific IFNγ secretion (Fig. 2A). Thus, 5% O2 lysates significantly increased antigen-specific CD8+ T cell activation but had no intrinsic ability to induce IFNγ secretion from the cultured antigen presenting cells.

Figure 2
5% O2 lysates increase CD8+ T cell activation via cross presentation

The adjuvant property of 5% O2 lysates was associated with an increase in cross presentation of exogenous antigen on MHC I

We investigated if the adjuvant properties of the 5% O2 lysates could be attributed to increasing expression of co-stimulatory molecules involved in T cell activation. Splenocytes from BL6 mice were pulsed with GL261 cell lysates from 5% or 20% O2 cultures. Forty-eight hrs later CD11c+ cells (dendritic cell marker) were analyzed by flow cytometry for co-expression of CD80, CD86, iCOSL, and GITRL. Relative to unpulsed controls, the only significant change was an increase in CD86 expression in cells pulsed with 20% O2 cell lysates (Fig. 2B). Therefore, the adjuvant properties of the 5% O2 lysates were not attributable to changes in expression of these co-stimulatory molecules, implying other mechanisms.

We hypothesized that the 5% O2 lysates could enhance cross presentation of exogenous antigen on MHC I. In order to test this directly, splenocytes were pulsed with a 26 amino acid peptide derived from OVA (OVA248–274) that must be cleaved in order for the SIINFEKL epitope to be presented on H-2Kb; the H-2Kb/SIINFEKL complex is then detectable by a specific antibody using flow cytometry (26). Splenocytes were pulsed with OVA248–274 alone or in combination with GL261 cell lysates from 5% or 20% O2 cultures. Cells were analyzed by flow cytometry 24 hrs later. CD11c+ splenocytes that were pulsed with OVA248–274 mixed 5% O2 lysates exhibited an appreciable increase in cell surface levels of the H-2Kb/SIINFEKL complex (Fig. 2C). The mean fluorescent intensity quantifying the amount of the H-2Kb/SIINFEKL complex on CD11c+ cells was significantly increased in the 5% O2 lysate group relative to all other groups (Fig. 2D). These data revealed that one mechanism by which 5% O2 lysates enhance antigen-specific CD8+ T cell activation was by increasing cross presentation of exogenous antigens on MHC I.

5% O2 lysate vaccination enhanced CTL proliferation and tumoricidal function

We next investigated the effects of 5% O2 lysate on in vivo proliferation and ex vivo tumoricidal function of CTLs. Glioma-bearing mice were adoptively transferred with CFSE-labeled Pmel splenocytes. Mice were vaccinated with 5% or 20% O2 GL261 lysates mixed with hgp10025–33 and CpG ODN one day after adoptive transfer. Lymphocytes were isolated from the draining lymph nodes (DLN) 72 hrs following vaccination and analyzed for proliferation by flow cytometry. Pmel CTLs exhibited significantly increased proliferation in 5% O2 lysate-treated mice compared to other treatment groups (Fig. 3A–B). To measure tumoricidal function, vaccinations were repeated and lymphocytes from the DLN were harvested 72 hrs following the second vaccination and used as effector cells in a CTL assay. Significantly greater GL261 cell lysis was documented in the 5% O2 lysate group (Fig. 4A). In contrast, western blot analysis revealed a tumor-reactive antibody response of greater breadth and magnitude in the serum from mice treated with 20% O2 lysate relative to 5% O2 lysate (Fig. 4B).

Figure 3
Glioma cells grown in 5% O2 induce superior CD8+ T cell proliferation in vivo
Figure 4
Vaccination with 5% O2 induced increased tumoricidal function of CTL and diminished tumor-reactive antibody responses

These data demonstrate that 5% O2 lysates significantly enhanced CTL responses whereas 20% O2 lysates predominately evoked tumor-reactive antibody responses.

Efficacy of 5% O2 lysate vaccination was not dependent on a specific cell line or mouse strain

The broader reproducibility of the survival data obtained using the GL261 glioma model was tested in another tumor model. BALB/c mice were inoculated with syngeneic EMT6 breast carcinoma in the mammary fat pad. Tumor-bearing mice were vaccinated with EMT6 cell lysates from 5% or 20% O2 cultures mixed with CpG ODN as an adjuvant. Vaccination with 5% O2 lysates was even more efficacious than the GL261 model, rendering 60% of the mice long-term survivors with no apparent evidence of residual tumor (Fig. 5 and data not shown). Similarly to the GL261 studies, mice treated by 20% O2 EMT6 lysates did not survive significantly longer than saline controls. These data suggest that the effect of oxygen on the therapeutic efficacy of tumor lysate vaccination is not restricted to a specific cell line, tumor type, or genetic background.

Figure 5
Superior efficacy of 5% O2 lysate vaccination against orthotopic breast carcinoma

Discussion

The results of this study identify oxygen as a master “immunologic switch” that can be manipulated to augment the efficacy of cancer vaccines. There are several possible mechanisms by which tissue culture oxygen changes tumor cell lysate immunogenicity. Such mechanisms could include altering levels of immunomodulatory adenosine (27), expression of target antigens, endogenous alarmins (e.g., TLR ligands, HSPs), or proteins that modulate antigen presentation by novel means. We were unable to find evidence that 5% O2 lysates activated CD11c+ dendritic cells as assessed by upregulation of co-stimulatory molecules (Fig. 2B), or increasing secretion of IFNγ, IL-4, IL-6, IL-10, or IL-12 (Fig. 2A and data not shown). Although target antigen expression could change as a function of oxygen tension, the differences measured in Pmel CD8+ T cell activation were not attributable to changes in endogenous mouse gp100 expression (Supplementary Fig 3). Nevertheless, our data demonstrate that 5% O2 lysates significantly increased T cell activation as shown by antigen-dependent IFNγ secretion from highly purified CD8+ Pmel T cells. Pulsation of splenocytes with OVA248–274 confirmed that 5% O2 lysates increased presentation of exogenous protein antigens on MHC class I relative to 20% O2 lysates. It was therefore not surprising that 5% O2 lysates enhanced T cell priming. This is the first study to demonstrate that tumor lysates from low O2 environments enhance antigen-specific CD8+ T cell proliferation, trafficking to tumor sites, and tumor-specific cytolytic responses, resulting in increased survival in two mouse tumor models. Identification and purification of the molecule(s) that are responsible for the enhancement in cross presentation could yield a valuable new reagent to increase the efficacy of cancer vaccines. Although our study focused on CTL responses, the effect of 5% O2 lysates on the presentation of MHC II-restricted peptides and effector CD4+ T cell responses should be addressed in futures studies.

Western blot analysis demonstrated that vaccination with CpG/lysate primed a polyclonal tumor-reactive antibody response in both the 5% O2 and 20% O2 groups; each band revealed an antibody reactive to an antigen in the GL261 cell lysate (Fig. 4B). Relative to the 20% O2 group, the antibody response in the 5% O2 group was diminished as illustrated by a reduction in the number and intensity of bands. This could reflect an intrinsic humoral immune response bias to vaccination with 20% O2 lysates or recognition of different antigens present in each lysate. Oxygen concentration exhibits inter- and intratumoral heterogeneity in the glioma microenvironment, ranging from 0.1 to 10% and trending towards lower oxygen as tumor grade increases (28). Thus, lowering oxygen in cell culture may select for expression of antigens that are in greater abundance on the tumor in situ. If true, vaccination with lysates from a more tumor physiologic oxygen tension should increase the “signal to noise” ratio by focusing the adaptive immune response against antigens relevant at the tumor site. Further studies will be required to understand if tumor physiologic oxygen tension selects for antigens that are more abundantly expressed in the tumor.

Although it was beyond the scope of this study to conduct immune monitoring in the breast carcinoma model, the survival data in this model illustrates that the translational significance of our findings are not limited to gliomas. An additional consideration with translational significance is the dose of tumor cell lysate used in vaccination. Many investigators, including our group, have shown effective immunotherapy using an excess of one million tumor cells grown in 20% O2 as vaccine antigen in the mouse (24). However, our analysis of the protein content in GL261 cells suggests that this corresponds to a clinically unfeasible cell dose (50 mg/kg dose of protein lysate requiring over a billion tumor cells in a human). When reduced to a protein lysate dose of 3 mg/kg, requiring ~107–108 cells in human, tumor cells grown in 20% O2 were not effective as vaccine antigen. This observation illustrates the importance of the dose of tumor antigen relative to the size of the animal. We speculate that one factor contributing to the poor predictability of murine tumor models is the discordant doses of vaccine antigen used on mg/kg basis.

It is possible that our results with CpG/lysate vaccination are relevant to other approaches that utilize tumor cells as the source of antigen (e.g., lysate-pulsed dendritic cells, irradiated tumor cells). This is an ongoing area of investigation in our laboratory. Further study of the effect of oxygen on tumor cell immunogenicity using clinically scalable lysate doses is warranted. It will be important to determine if reducing oxygen below the 5% threshold used in these studies can further enhance CTL responses against tumors. We propose that exploitation of oxygen as a master immunologic switch could improve the efficacy of cancer vaccines in the foreseeable future.

Translational Relevance

Thousands of cancer patients have been treated with vaccines that utilize tumor cells as the source of antigen. Tumor cell vaccines have demonstrated modest, albeit encouraging results in clinical trials. Atmospheric oxygen (~20% O2) has been the universal condition employed to culture tumor cells used as vaccine antigen. Here we report that reducing the oxygen tension used in tissue culture to 5% O2 markedly enhances the efficacy of tumor lysate vaccines. The enhancement in survival was reproducible in mouse models of glioma and breast carcinoma. These data suggest the clinical efficacy of vaccines that utilize tumor cells as the source of antigen could be increased by reducing tissue culture oxygen tension.

Supplementary Material

Supp Fig 1

Supplementary Figure 1. Monotherapy vaccine is ineffective against GL261 glioma. BL6 mice bearing GL261 gliomas where vaccinated with 65 µg of tumor lysate derived from 5% or 20% O2 GL261 cultures or 50 µg CpG ODN or saline. Vaccinations were given weekly, starting three days after tumor implantation, for a maximum of six doses. There was no significant difference in survival between any group (N=10/group; P<0.05).

Supp Fig 2

Supplementary Figure 2. 5% O2 lysates increase CD8+ T cell infiltration into the brain. Glioma-bearing mice were adoptively transferred with two million CFSE-labeled Pmel splenocytes. One day and seven days later mice were vaccinated with a mixture of CpG (50 µg), lysate (65 µg), and hgp10025–33 (10 µg) by intradermal injection. Seventy-two hours following the second vaccination, brain-infiltrating lymphocytes (BILs) were isolated from the CNS, pooled (3 mice/group), counted, and stained for CD8 and hgp10025–33/H-2Db tetramer. The absolute number of CD8+ tetramer+ cells was calculated by multiplying the percentage of positive cells by the total number of BIL. A greater than four-fold increase in the number of CD8+ hgp10025–33/H-2Db tetramer cells was observed from the brains of mice that were vaccinated with 5% O2 lysate relative to other groups. Note, error bars and statistics not shown because data represent pooled samples.

Supp Fig 3

Supplementary Figure 3. Oxygen did not change endogenous mgp100 expression in GL261 glioma cells. GL261 cells were cultured in 5% or 20% O2, washed, pelleted and lysed in RIPA buffer containing protease and phosphatase inhibitors (Pierce). Protein concentrations were determined using the BCA colorimetric method (Pierce). Lysates were put in SDS PAGE reducing sample buffer, resolved by 4–12% SDS-PAGE (Invitrogen), transferred to nitrocellulose (BioRad), blotted against goat anti-mouse GP100 (Santa Cruz) and detected with ECL Plus (GE).

Acknowledgments

This work was supported by grants from the National Institutes of Health/National Institute of Neurological Disorders & Stroke (NIH/NINDS) (NIH IR21-NS055738 (JRO), American Cancer Society RSG-09-189-01-LIB (JRO), Randy Shaver Cancer Research and Community Fund (JRO), Children’s Cancer Research fund (JRO) and NIH R01 CA72669 (BRB).

Footnotes

Author Contributions: M.R.O., B.M.A, F.P., W.C., B.R.B., and J.R.O designed research; M.R.O., B.M.A, D.M.Z, P.T.G, Z.X., C.F., C.S. and K.S. performed research and analyzed data; M.R.O, B.M.A, D.M.Z, B.R.B., and J.R.O edited the manuscript.

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