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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Immunol Lett. Author manuscript; available in PMC 2016 July 1.
Published in final edited form as:
PMCID: PMC4476929
NIHMSID: NIHMS692211

Surgical cytoreduction restores the antitumor efficacy of a Listeria monocytogenes vaccine in malignant pleural mesothelioma

Abstract

Recent studies suggest that immunotherapy may offer a promising treatment strategy for early-stage malignant pleural mesothelioma (MPM), but advanced tumor burden may limit the efficacy of immunotherapy. Therefore, we hypothesized that surgical cytoreduction could restore the efficacy of vaccine-based immunotherapy for MPM. We developed a murine model of MPM through transduction of a mesothelioma cell line with mesothelin. We used this model to evaluate the efficacy of a Listeria monocytogenes vaccine expressing mesothelin. Tumor growth was significantly inhibited at four weeks in animals vaccinated two weeks prior to tumor cell inoculation as compared to those given an empty vector control (1371 ± 420 mm3 versus 405 ± 139 mm3; p<0.01). Mice vaccinated one week prior to tumor challenge also displayed significant reduction in tumor volume (1227 ± 406 mm3 versus 309 ± 173 mm3; p<0.01). The vaccine had no effect when administered concurrently with tumor challenge, or after tumors were established. Flow cytometry showed reduced mesothelin expression in large tumors, as well as tumor-associated immunosuppression due to increased myeloid derived suppressor cells (MDSCs). These factors may have limited vaccine efficacy for advanced disease. Surgical cytoreduction of established tumors restored the antitumor potency of the therapeutic vaccine, with significantly reduced tumor burden at post-operative day 18 (397 ± 103 mm3 versus 1047 ± 258 mm3; p<0.01). We found that surgery reduced MDSCs to levels comparable to those in tumor-naïve mice. This study demonstrates that cytoreduction surgery restores the efficacy of cancer vaccines for MPM by reducing tumor-related immunosuppression that impairs immunotherapy.

Keywords: malignant mesothelioma, vaccine immunotherapy, surgical cytoreduction

1. Introduction

Mesothelioma is an insidious neoplasm arising in the pleura, pericardium, peritoneum, or tunica vaginalis, with approximately 80% of cases originating in the thorax. Asbestos exposure is the predominant cause of malignant pleural mesothelioma (MPM), which has a latency period of approximately 30 years. 1,2 Though modest improvements in the treatment of MPM have been made in the past decade, the disease remains a therapeutic challenge and the median survival of MPM patients is between 12 and 18 months. 3,4

A growing body of preclinical research supports the development of immunotherapeutic approaches to the treatment of MPM. 1,5-9 The genotype and phenotype of MPM has been well characterized, demonstrating surface expression of the 40 kDa glycoprotein mesothelin, a potential tumor antigen amenable to immunotherapeutic targeting. 10-15 Cancer vaccines have been shown to be effective in other neoplasms, and provide an encouraging method of generating an antigen-specific, cell-mediated immune response to mesothelin. 16-20

Though cancer vaccines show promise, a number of tumor escape mechanisms have posed persistent challenges to the development of effective vaccines. It has been shown that cancer cells may lose or reduce expression of molecules targeted by effector T-cells, such as tumor-associated antigens (TAA), MHC molecules, and molecules associated with antigen processing. 21,22 This loss of antigenicity renders vaccines ineffective against the mutated population of neoplastic cells. 23 Furthermore, vaccines may lose their potency as tumor burden increases, due to complex alterations in the tumor microenvironment and systemic immunosuppression that accompanies advanced tumors. 24-27

Listeria monocytogenes, a Gram-positive intracellular facultative bacteria, is a promising vector for cancer vaccines that may overcome tumor escape via loss of antigenicity. Listeria can infect phagocytic and antigen-presenting cells (APCs), where it is able to move from the phagosome into the cytoplasm of the cell, unlike most other intracellular bacteria. 28,29 This unusual intracellular life cycle allows antigens secreted by Listeria to be processed and presented by both MHC class I and II molecules, resulting in strong CD8+ and CD4+ T-cell-medicated immune responses. 30 Furthermore, it has been shown that Listeria-based vaccines can induce epitope spreading, a phenomenon that may counter tumor cell loss of antigenicity. 31-33 In epitope spreading, APC processing of tumor cells activates secondary tumor-specific T-cell responses, thereby broadening the host immune response beyond the antigen targeted by the vaccine. 34 This process seems critical to the establishment of durable antitumor immune responses, and recombinant strains of Listeria expressing TAA have been shown to generate robust in vivo antitumor responses in melanoma, breast cancer, and HPV-associated neoplasms. 33,35-39

Surgery may also play a role in bolstering the efficacy of cancer vaccines by overcoming the immunosuppressive effects that accompany advanced tumors. Recent work in our laboratory indicates that cytoreduction surgery potentiates the effects of immunotherapy in large tumors by reducing systemic myeloid suppressor cell populations. 25,40,41 Thus, we hypothesized that a Listeria-based vaccine coupled with surgical cytoreduction may overcome some of the challenges that have traditionally hampered the development of cancer vaccines, and offer a novel approach to immunotherapy for the treatment of MPM.

Here, we report the development of an in vivo model of MPM using a murine mesothelioma cell line transduced with the mesothelin gene. Using this model, we investigated a recombinant Listeria-based vaccine for mesothelin, which exhibited robust antitumor effects when delivered prior to tumor cell inoculation. Finally, we demonstrate that cytoreduction surgery in large tumors restores the antitumor efficacy of the Listeria vaccine.

2. Materials and Methods

2.1. Animals

Female C57BL/6 (B6, Thy1.2) mice were purchased from Charles River Laboratories (Wilmington, MA). All mice were maintained in pathogen-free conditions and used for experiments at ages 8 week or older. The Animal Use Committees of the Children's Hospital of Philadelphia, The Wistar Institute and the University of Pennsylvania approved all protocols in compliance with the Guide for the Care and Use of Laboratory Animals.

2.2. Cell Lines

Three murine mesothelioma cell lines (AE17, AB12, AB1) that grow in syngeneic mouse strains were transduced with a lentivirus expressing human mesothelin. Flow cytometric sorting was used to purify cells that were successfully transduced with the mesothelin gene. The transduced AE17 cell line was maintained in RPMI media supplemented with 10% heat-inactivated fetal bovine serum, 1% glutamine, and 1% penicillin and streptomycin (P/S). The transduced AB12 and AB1 cell lines were maintained in Dulbecco's Modified Eagle Media supplemented with 10% heat-inactivated fetal bovine serum, 1% glutamine, and 1% penicillin and streptomycin (P/S). Cells were cultured at 37°C in a humidified incubator containing 5% CO2.

2.3. Viral Transduction

Using a third-generation self-inactivating lentiviral expression vector encoding human mesothelin driven by the EF-1α promoter (a generous gift from Dr. Carl June), high-titer repilication-defective lentiviral vectors were produced and concentrated as previously described.42 250,000 to 500,000 cells were seeded in 2 ml of their respective serum supplemented growth medium (described above) per well in a 6 well plate. The following day LV-mesothelin was added to the tumor cells at an MOI of 5:1. Transduced cells expressing high levels of mestohelin were collected using a BD FACSAria cell sorter and cultured as described above.

2.4. Immunostaining

Mice were euthanized at which time tumors were harvested and frozen in Tissue-Tek OCT compound (Sakura Finetek USA Inc., Torrance, CA) to be stored at 80 °C, and 5 μm sections were cut. The frozen tumor tissue sections were fixed with 4% paraformaldehyde, washed with PBS and incubated with 10% goat serum in PBS + 0.1% Tween-20 for 60 min, followed by labeling with anti-human mesothelin antibody (K1 clone, Cat # SIG-3623, Covance) overnight at 4 °C. After incubation, sections were washed and further incubated with PE conjugated sheep anti-mouse secondary antibody (Cat # P8547, Sigma) for 1 hr at room temperature. Sections were counterstained with DAPI (invitogen) and then mounted with slow fade mounting medium (Invitrogen). Sections were visualized under a Nikon E600 microscope.

2.5. Listeria Vaccine

A modified Listeria monocytogenes vector expressing human mesothelin (CRS-207) was provided by Anza Therapeutics, Inc. (Concord, MA). Mice were vaccinated via intraperitoneal injection with CRS-207 at a dose of 1 × 107 colony forming units (cfu) per mouse two weeks prior to tumor challenge. An empty Listeria control vector (ANZ-100) was also provided by Anza Therapeutics. Control mice were vaccinated with ANZ-100 in a similar fashion.

2.6. Animal flank tumor models

C57BL/6 mice were inoculated by subcutaneous injection on the right flank with 1×106 AE17M cells. When the tumors reached 250 mm3 the mice were randomized and grouped into cohorts. Tumor dimensions were measured with calipers and tumor size was determined by the equation: π/6 × length × width × height. All experiments had at least five mice per group and were replicated at least once.

2.7. Surgery

Surgery was performed on mice bearing flank tumors using an established partial resection model. 43 Partial resection was performed on C57BL/6 mice inoculated subcutaneously with 1 ×106 AE17M cells. Surgery was performed when tumors reached ~300 mm3. Mice were anesthetized with intramuscular ketamine (80 mg/kg) and xylazine (10 mg/kg) and shaved with hair clippers prior to surgery. A 1 to 2 cm incision was made adjacent to the tumor and 90% of the tumor was removed using standard blunt dissection technique. The incision was closed using sterile silk 4-0 sutures. Buprenorphine (0.2 mg/kg) was administered at the time of surgery and 6 hours afterward as postoperative analgesia. Preoperative treatment was unknown to the investigator performing surgery and making tumor measurements.

2.8 In Vivo Depletion of CD8 T-cells

To deplete specific T-cell populations in our model, mice were injected i.p. with monoclonal antibodies purified from the anti-CD8 hybridoma 53-6.7 (American Type Tissue Culture Collection, Manassas, VA). Mice were given 300 μg of purified antibody i.p. dissolved in 200 μL of PBS for CD8+ antibodies. Antibodies against CD8 were administered twice weekly for 3 weeks after tumor resection. Depletions were confirmed by flow cytometry of splenic suspension.

2.9 Detection of Tumor Neutralizing CD8 T-Cells (Winn Assay)

Splenocytes (3 mice/group) or splenic CD8 T cells from tumor bearing mice at various time points or control mice were used. CD8 T-cells were isolated using MACS isolation system (CD8a [Ly-2] mouse MicroBeads -- Miltenyi Biotec, Auburn, Calif). Isolated cells consisted of greater than 90% CD8 T-cells. Fresh AE17 cells were mixed with CD8 T-cells in a ratio of 3 CD8 T-cells to 1 AE17 tumor cell. The resulting mixture (1.5×106 CD8 T-cells to 0.5×106 AE17 tumor cells) was injected subcutaneously into the flanks of 5 naïve C57Bl/6 mice. A control group of 5 mice were injected with AE17 cells alone (0.5×106 cells) in an analogous fashion. Tumor size was assessed over the next 10 days.

2.10. Flow cytometric analysis

For flow cytometric analysis, tumors were removed from euthanized mice and minced into 1–2 mm fine pieces in digestion buffer containing 0.1 mg/mL DNase I and 2.0 mg/mL collagenase type IV (Sigma, St. Louis, MO). Samples were incubated in digestion buffer at 37°C for 30 minutes, filtered through a 70-μm filter, and washed twice with DMEM supplemented with 10% FBS (vol/vol). Fc receptors were blocked with anti-mouse CD16/CD32 antibodies (BD Biosciences PharMingen, San Diego, CA). After 1 wash with staining buffer (PBS supplemented with 2% FBS (vol/vol)), cells were incubated for 30 minutes at 4°C with appropriate antibodies obtained from Abcam (Boston, MA) and BD Biosciences PharMingen (San Diego, CA) and used at the indicated dilutions for flow cytometry. Samples were then washed and re-suspended in staining buffer for immediate flow cytometric analysis. Flow cytometry was completed using a Becton Dickinson FACS Calibur flow cytometer (BD Biosciences), and analyzed using FlowJo software (Ashland, OR).

2.11. Statistical analyses

For flow cytometry, immunohistochemistry, and flank tumor volume studies comparing differences between two groups, we used unpaired Student's t-tests. For studies comparing more than two groups, ANOVA with appropriate post hoc testing was implemented. Differences were considered significant when p < 0.05. Data are presented as mean (standard error), unless otherwise noted.

3. Results

3.1. Generation of murine mesothelioma cell lines expressing human mesothelin

Three murine mesothelioma cell lines (AE17, AB12, AB1) which grow in syngeneic mouse strains were transduced with a lentivirus expressing human mesothelin. Flow cytometric sorting was used to purify cells that were successfully transduced with the mesothelin gene. [Figure 1A]. Once a purified line was achieved by transduction and flow cytometric sorting, syngeneic mice were inoculated subcutaneously with tumor cells in order to develop a functioning heterotopic murine model. Transduced AE17 mesothelin (AE17M) cell lines that retained expression of the mesothelin antigen grew reproducibly in mice [Figure 1B]. Immunofluorescence staining was performed using harvested AE17M mouse tumors and confirmed expression of the mesothelin antigen [Figure 1C].

Figure 1
Establishment of a murine model of malignant mesothelioma

3.2. Vaccination with mesothelin-expressing Listeria vaccine (CRS-207) slows tumorigenesis

An initial pilot study was performed to determine if pre-vaccination with a Listeria vaccine expressing the mesothelin antigen (CRS-207) would protect immunocompetent mice from developing malignant mesothelioma. It was hypothesized that this exposure would lead to development of an immune response against the mesothelin antigen. CRS-207 was tested on the heterotopic murine model of malignant mesothelioma. Five mice were vaccinated intraperitoneally with CRS-207 (1 × 107 cfu per mouse) two weeks prior to tumor challenge. Five control mice received the same dose of empty Listeria control vector (ANZ-100). All of the mice were boosted with an additional dose of the appropriate therapy one week later. The following week, 1 × 106 AE17M tumor cells were injected subcutaneously in the flank. The animals were re-vaccinated at one week and two weeks post tumor cell inoculation. Tumor volume, toxicity, and survival were monitored over this time period. Tumor growth was significantly inhibited at four weeks in animals that had been exposed to the therapeutic vaccine (1371 ± 420 mm3 versus 405 ± 139 mm3; P=0.001) [Figure 2A]. This study indicates that CRS-207 is effective in prevention of growth of malignant mesothelioma in mice.

Figure 2
Vaccination with CRS-207 inhibits tumorigenesis

To determine the optimal time interval for pre-vaccination, an experiment was performed in which five control and five experimental group mice received a single vaccine dose one week prior to tumor challenge. Additional doses were given at the time of tumor cell inoculation, and then one week, and two weeks after tumor cell inoculation. The details of the remainder of the experiment were identical to the first experiment. Again, CRS-207 demonstrated efficacy in slowing the growth of the tumor. At four weeks, average tumor volume was 1227 ± 406 mm3 in the control group compared to 309 ± 173 mm3 in the treated group (p=0.001) [Figure 2B].

To determine whether the vaccine was effective when administered concurrently with tumor challenge, we inoculated five control and five experimental group mice subcutaneously with 1 × 106 AE17M cells in the flank and then vaccinated with ANZ-100 or CRS-207 intraperitoneally one hour prior to tumor cell inoculation. Booster vaccines were given at one and two weeks. Although statistical significance was not achieved, there was some protection provided by concurrent vaccination. Average tumor volume at the conclusion of the experiment was 1370 ± 409 mm3 in the untreated group compared to 967 ± 154 mm3 in the treated group (p=0.07) [Figure 2C]. These data provide proof of principle that CRS-207 can generate an immune response to slow tumor growth. Vaccination prior to or concurrent with tumor cell inoculation inhibited tumor cell growth and slowed disease progression.

3.3. CRS-207 does not inhibit growth of established primary tumor

CRS-207 was tested for its ability to treat mice with established malignant mesothelioma tumors. Five mice were inoculated subcutaneously with 1 × 106 AE17M cells in the flank. Once tumors (average volume 200 mm3) had been established, mice received CRS-207 vaccine intraperitoneally. The animals were boosted with additional doses at one week and two weeks. There was no measurable benefit from CRS-207 therapy [Figure 3A].

Figure 3
CRS-207 is not effective against established tumors

3.4. Loss of antigenicity may partially explain lack of CRS-207 efficacy in advanced tumor

It has been well established that cancer cells can downregulate tumor-associated antigens to reduce their immunogenicity. We hypothesized that this loss of antigenicity may have played a role in the lack of response of established tumors to CRS-207. To evaluate this theory, we analyzed mesothelin expression levels in small (~100 mm3) and advanced (~500 mm3) AE17M tumors using flow cytometry, finding reductions in mesothelin expression levels in advanced tumors [Figure 3B].

3.5 Adaptive immune responses are responsible for vaccine efficacy

Due to the inability of CRS-207 to effectively treat established tumors, we investigated whether cytoreduction surgery would enhance the antitumor efficacy of the vaccine in established tumors. This notion was based upon past work in our laboratory indicating that advanced tumor burden is characterized by an immunosuppressive environment, and that surgical cytoreduction may lessen tumor-associated immunosuppression.

In order to test this hypothesis, mice were injected subcutaneously with 1 × 106 AE17M and at the same time point were given either CRS-207 or ANZ-100. Mice were boosted with appropriate vaccines after one week. When tumor reached ~300 mm3 in mice, 90% of the tumor was resected and mice were given an additional dose of CRS-207 or ANZ-100. Recurrent tumor growth was monitored after surgery and it was observed that CRS-207 significantly inhibited tumor growth (397 ± 103 mm3) compared to ANZ-100 at post-operative day 18 (1047 ± 258 mm3) (p = 0.002) [Figure 4].

Figure 4
Cytoreduction surgery restores antitumor efficacy of CRS-207

Previous reports have described that immune responses are a primary mechanism of the effects of adaptive immune response. To more precisely define this role in our model, mice with recurrent AE17 flank tumors after surgery were randomized to four groups: (1) ANZ-100 (control), (2) ANZ-100 with CD8 T-cell depletion, (3) CRS-207, or (4) CRS-207 with CD8 T-cell depletion. Depletion of the T-cell population was confirmed weekly by analysis of spleen cells by flow cytometry (data not shown). Although mice receiving CRS-207 had smaller nodules after 14 days of tumor growth, effects were negated in the presence of CD8 T-cell depletion (Fig. 5A). These results confirm the role of CD8 T-cells as an essential element in anti-tumor effects of Listeria vaccination protocols in our recurrent tumor model.

Figure 5
Listeria vaccination increases CD8 T-cell lymphocyte infiltration and systemic populations more effectively than control vector

To further confirm that CD8 T-cells were responsible for this antitumor effect, we isolated CD8 T-cells from mice 3 to 5 days following the first dose of Listeria vaccination. These CD8 T-cells were injected along with fresh AE17 tumor cells (in a ratio of 3 CD8 T-cells: 1 AE17 cell) into the flank of tumor naïve C57bl/6 mice. The growth of these nodules was then observed for 7 days. We found that CD8 T-cells obtained from mice receiving CRS-207 were better able to neutralize AE17 cells as compared to mice receiving ANZ-100 (p=0.02) (Fig. 5B). These results suggest that CRS-207 decreases tumor burden primarily by augmentation of anti-tumor CD8 T-cell function.

3.6. Surgical cytoreduction restores antitumor efficacy of CRS-207 by systemic reduction in myeloid derived suppressor cells

Past work in our laboratory has shown that surgery reduces systemic myeloid suppressor cell (MDSC) populations to restore the efficacy of T-cell mediated immunotherapy in a mouse model of non-small cell lung cancer. 25 To determine whether a similar phenomenon was responsible for the restoration of the antitumor effects of CRS-207 in our model of mesothelioma, we evaluated the number of MDSCs (CD11b+Gr1+) present in mice without tumors, mice bearing established tumors (~300 mm3), and at postoperative day 3. Mice were sacrificed and spleens were evaluated for MDSC populations using flow cytometry. We found a significant increase in MDSCs as a fraction of CD45 splenocytes in mice bearing tumors compared to tumor-naïve mice (3.2% to 57.5%; p < 0.01). Three days after cytoreduction surgery, the percentage of MDSCs decreased to levels comparable to those in tumor-naïve mice (4.7%; p<0.01) [Figure 6]. These data indicate that surgery has the ability to restore the efficacy of cancer vaccines by reducing the tumor-related immunosuppression networks that impair CD8 T-cells.

Figure 6
Surgical cytoreduction reduces tumor-associated immunosuppression

4. Discussion

Cancer vaccines represent a promising immunotherapy strategy that has produced some striking responses in animal models for a variety of malignant diseases. 18 However, these results have translated poorly to human trials, and a number of challenges have hampered the development of clinically effective vaccines, especially for advanced tumors. 44 It has been demonstrated that cancer cells are able to lose or reduce their immunogenicity through a number of mechanisms, including downregulation of TAA and MHC molecules. 21,22 Here, we show a significant reduction in expression of mesothelin in large tumors, further demonstrating the loss of antigenicity that may significantly limit the efficacy of vaccines for advanced disease. Further work needs to be completed in order to determine if this phenomenon is truly due to selection pressure against mesothelin in tumor cells compared with simply loss of mesothelin expression over time.

Advances in molecular immunology, coupled with increased knowledge of pathogen physiology, have facilitated the use of attenuated bacteria as vaccine vectors, a strategy that may counter loss of antigenicity through epitope spreading. Epitope spreading is a process by which the T-cell response is expanded beyond the antigen targeting the vaccine, thereby broadening and enhancing vaccine efficacy. 45 Listeria monocytogenes seems to be especially promising in this regard, and several studies have demonstrated its propensity to induce epitope spreading in a number of malignant neoplasms. 31-33

In this study, we add to the relatively abundant literature demonstrating the potency of Listeria-based vaccines targeting model tumor antigens. We report the establishment of a mouse model of MPM by the successful transduction of a murine mesothelioma cell line with mesothelin, a surface protein overexpressed in MPM. Using this model, we show robust antitumor activity elicited in a response to a recombinant Listeria vector expressing mesothelin. Tumorigenesis was significantly inhibited when mice were pre-vaccinated at two time points over a two-week period prior to tumor cell inoculation. Inhibition of tumor growth was also observed in mice vaccinated with a single dose either one week prior to or concurrently with tumor cell inoculation.

Despite its effectiveness in slowing tumorigenesis when administered prior to or concurrently with cancer cell inoculation, the mesothelin-expressing Listeria vaccine did not exhibit antitumor activity against established primary tumors, a result that is consistent with previous studies of other cancer vaccines, and one that points to another persistent challenge in the realization of the potential of vaccine-based immunotherapy. 40 Past work in our laboratory and elsewhere has demonstrated that local and systemic host immunosuppression accompanies advanced tumor burden and may limit vaccine effectiveness. This immunosuppression appears to be mediated by a complex array of factors, including extensive systemic suppressive cell populations, poor T-cell trafficking, and increased levels of immunosuppressive cytokines. 24,25,27

Recently, a number of studies have suggested that surgery may play a role in restoring immunotherapy for advanced tumors. Surgery may enhance immunotherapy not only through simple mechanical cytoreduction, but also by reducing systemic tumor-related immunosuppression. 25,41,46 Here, we demonstrate that surgery reduces systemic myeloid derived suppressor populations, which are known to inhibit CD8 T-cell responses. This approach is not without limitations, as surgery has been shown to generate a transiently immunosuppressive tumor microenvironment that allows increased tumor growth. 47,48 However, the results of this study provide support for pairing cytoreductive surgery with neoadjuvant immunotherapy in advanced disease to re-establish the effectiveness of immunotherapy. We demonstrate that surgical debulking of established tumors restores the antitumor efficacy of the mesothelin-expressing Listeria vaccine.

5. Conclusions

In summary, the results of this study add to the growing body of literature demonstrating the effectiveness of Listeria-based cancer vaccines, and suggest a role for a mesothelin-targeted vaccine in the treatment of MPM, a cancer that remains a significant therapeutic challenge. A recent clinical trial of a similar Listeria vaccine expressing mesothelin found that the immunotherapy was well-tolerated and induced immune activation in patients with various advanced cancers. 49 This result underscores the potential clinical application of our findings. Finally, we expand upon the concept of a synergy between surgery and immunotherapy, demonstrating that debulking of tumor restores the antitumor efficacy of vaccine-based immunotherapy for MPM by reducing systemic MDSC populations. Ultimately, the combination of a mesothelin-expressing Listeria vaccine and surgical cytoreduction warrants further study and may one day offer an attractive therapeutic strategy for patients with advanced MPM refractory to conventional therapy.

Highlights

  • A murine model of malignant pleural mesothelioma was developed.
  • A Listeria vaccine expressing mesothelin was tested against an empty vector control.
  • Mice vaccinated prior to tumor inoculation had significant reduction in tumor volume.
  • There was no effect on established tumors due to tumor-related immunosuppression.
  • Surgery restored vaccine efficacy by reducing tumor-related immunosuppression.

Acknowledgements

This work was partly funded by an National Institutes of Health P01 CA087971 (SS).

6. Vitae

Gregory T. Kennedy is a medical student at the Perelman School of Medicine at the University of Pennsylvania. He graduated summa cum laude from Princeton University, where he was elected to Phi Beta Kappa, and received a graduate degree from the University of Cambridge, where he was a Rotary Ambassadorial Scholar. He plans to pursue a career in academic surgery.

Brendan F. Judy is a medical student at Jefferson Medical College. He graduated from the University of Richmond in 2010 with a degree in political science. He subsequently worked in the Thoracic Surgery Research Laboratory before beginning medical school in 2013. He is interested in a career in surgery.

Pratik Bhojnagarwala graduated from the University of Illinois at Urbana-Champaign with his B.S. in Molecular Biology. He also received his MS in Biotechnology from the University of Pennsylvania. Before joining Dr. Singhal's lab, Pratik worked in the Department of Pharmacology at UPenn. Currently, his primary focus involves developing a better understanding of the function of myeloid cells in lung cancer. This will hopefully lead to design of better immunotherapies in the future. Future plans involve getting a Ph.D. in immunology and eventually working to bring new and novel therapies to the market.

Edmund K. Moon is a junior faculty member in Pulmonary, Allergy, and Critical Care at the University of Pennsylvania. Two and half years of his training and two years of his initial faculty career have been spent doing basic science research under the mentorship of Dr. Steven Albelda in the Thoracic Oncology Research Group. Dr. Moon's research interest is in adoptive T cell immunotherapy for thoracic and pleural malignancies with the overarching goal of becoming established as a successful physician-scientist in academic medicine.

Zvi G. Fridlender is a senior physician in Pulmonary Medicine and Internal Medicine at the Hadassah Medical Center in Jerusalem, Israel. He received his medical degree and M.Sc. in Developmental Biology from Hebrew University in Jerusalem. He received residency and fellowship training at Hadassah Medical Center, and was a postdoctoral researcher in the Thoracic Oncology Research Laboratory from 2007-2009.

Steven M. Albelda is the Director of the Thoracic Oncology Research Laboratory and Professor of Medicine at the University of Pennsylvania School of Medicine. He received his medical degree, residency, and fellowship training from the University of Pennsylvania. His laboratory focuses on translational research, with a special emphasis on gene therapy of thoracic malignancies.

Sunil Singhal is the Director of the Thoracic Surgery Research Laboratory and Assistant Professor of Surgery at the University of Pennsylvania School of Medicine. He obtained his Doctor of Medicine degree from the University of Pennsylvania and received post-graduate training at Johns Hopkins and the University of Pennsylvania. His research interests include nanotechnology, intraoperative immune suppression, muscle physiology, patient outcomes, myeloid derived suppressor cells, and gene-expression and profiling.

Footnotes

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References

1. Haas AR, Sterman DH. Malignant pleural mesothelioma: Update on treatment options with a focus on novel therapies. Clin Chest Med. 2013;34(1):99–111. [PMC free article] [PubMed]
2. Remon J, Lianes P, Martinez S, Velasco M, Querol R, Zanui M. Malignant mesothelioma: New insights into a rare disease. Cancer Treat Rev. 2013;39(6):584–591. [PubMed]
3. Krug LM, Pass HI, Rusch VW, et al. Multicenter phase II trial of neoadjuvant pemetrexed plus cisplatin followed by extrapleural pneumonectomy and radiation for malignant pleural mesothelioma. J Clin Oncol. 2009;27(18):3007–3013. [PMC free article] [PubMed]
4. Flores RM, Krug LM, Rosenzweig KE, et al. Induction chemotherapy, extrapleural pneumonectomy, and postoperative high-dose radiotherapy for locally advanced malignant pleural mesothelioma: A phase II trial. J Thorac Oncol. 2006;1(4):289–295. [PubMed]
5. Robinson BW, Lake RA. Advances in malignant mesothelioma. N Engl J Med. 2005;353(15):1591–1603. [PubMed]
6. Hegmans JP, Veltman JD, Lambers ME, et al. Consolidative dendritic cell-based immunotherapy elicits cytotoxicity against malignant mesothelioma. Am J Respir Crit Care Med. 2010;181(12):1383–1390. [PubMed]
7. Nowak AK, Lake RA, Marzo AL, et al. Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation, cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells. J Immunol. 2003;170(10):4905–4913. [PubMed]
8. Powell A, Creaney J, Broomfield S, Van Bruggen I, Robinson B. Recombinant GM-CSF plus autologous tumor cells as a vaccine for patients with mesothelioma. Lung Cancer. 2006;52(2):189–197. [PubMed]
9. Wong RM, Ianculescu I, Sharma S, et al. Immunotherapy for malignant pleural mesothelioma. current status and future prospects. Am J Respir Cell Mol Biol. 2014;50(5):870–875. [PubMed]
10. Hassan R, Bera T, Pastan I. Mesothelin: A new target for immunotherapy. Clin Cancer Res. 2004;10(12 Pt 1):3937–3942. [PubMed]
11. Hassan R, Ho M. Mesothelin targeted cancer immunotherapy. Eur J Cancer. 2008;44(1):46–53. [PMC free article] [PubMed]
12. Robinson BW, Creaney J, Lake R, et al. Mesothelin-family proteins and diagnosis of mesothelioma. Lancet. 2003;362(9396):1612–1616. [PubMed]
13. Pastan I, Hassan R. Discovery of mesothelin and exploiting it as a target for immunotherapy. Cancer Res. 2014;74(11):2907–2912. [PMC free article] [PubMed]
14. Sterman DH, Albelda SM. Advances in the diagnosis, evaluation, and management of malignant pleural mesothelioma. Respirology. 2005;10(3):266–283. [PubMed]
15. Villena-Vargas J, Adusumilli PS. Mesothelin-targeted immunotherapies for malignant pleural mesothelioma. Ann Cardiothorac Surg. 2012;1(4):466–471. [PMC free article] [PubMed]
16. Antoniu SA, Dimofte G, Ungureanu D. Immune therapies for malignant mesothelioma. Expert Rev Anticancer Ther. 2014:1–9. [PubMed]
17. Bagia M, Nowak AK. Novel targeted therapies and vaccination strategies for mesothelioma. Curr Treat Options Oncol. 2011;12(2):149–162. [PubMed]
18. Guo C, Manjili MH, Subjeck JR, Sarkar D, Fisher PB, Wang XY. Therapeutic cancer vaccines: Past, present, and future. Adv Cancer Res. 2013;119:421–475. [PMC free article] [PubMed]
19. Izzi V, Masuelli L, Tresoldi I, Foti C, Modesti A, Bei R. Immunity and malignant mesothelioma: From mesothelial cell damage to tumor development and immune response-based therapies. Cancer Lett. 2012;322(1):18–34. [PubMed]
20. Schlom J. Therapeutic cancer vaccines: Current status and moving forward. J Natl Cancer Inst. 2012;104(8):599–613. [PMC free article] [PubMed]
21. Marincola FM, Jaffee EM, Hicklin DJ, Ferrone S. Escape of human solid tumors from T-cell recognition: Molecular mechanisms and functional significance. Adv Immunol. 2000;74:181–273. [PubMed]
22. Kageshita T, Hirai S, Ono T, Hicklin DJ, Ferrone S. Down-regulation of HLA class I antigen-processing molecules in malignant melanoma: Association with disease progression. Am J Pathol. 1999;154(3):745–754. [PubMed]
23. Parmiani G, Castelli C, Dalerba P, et al. Cancer immunotherapy with peptide-based vaccines: What have we achieved? where are we going? J Natl Cancer Inst. 2002;94(11):805–818. [PubMed]
24. Barnett BG, Ruter J, Kryczek I, et al. Regulatory T cells: A new frontier in cancer immunotherapy. Adv Exp Med Biol. 2008;622:255–260. [PubMed]
25. Predina JD, Kapoor V, Judy BF, et al. Cytoreduction surgery reduces systemic myeloid suppressor cell populations and restores intratumoral immunotherapy effectiveness. J Hematol Oncol. 2012;5:34–8722-5-34. [PMC free article] [PubMed]
26. Serafini P, De Santo C, Marigo I, et al. Derangement of immune responses by myeloid suppressor cells. Cancer Immunol Immunother. 2004;53(2):64–72. [PubMed]
27. Sulitzeanu D. Immunosuppressive factors in human cancer. Adv Cancer Res. 1993;60:247–267. [PubMed]
28. Wood LM, Guirnalda PD, Seavey MM, Paterson Y. Cancer immunotherapy using listeria monocytogenes and listerial virulence factors. Immunol Res. 2008;42(1-3):233–245. [PMC free article] [PubMed]
29. Portnoy DA, Auerbuch V, Glomski IJ. The cell biology of listeria monocytogenes infection: The intersection of bacterial pathogenesis and cell-mediated immunity. J Cell Biol. 2002;158(3):409–414. [PMC free article] [PubMed]
30. Pamer EG. Immune responses to listeria monocytogenes. Nat Rev Immunol. 2004;4(10):812–823. [PubMed]
31. Wood LM, Pan ZK, Guirnalda P, Tsai P, Seavey M, Paterson Y. Targeting tumor vasculature with novel listeria-based vaccines directed against CD105. Cancer Immunol Immunother. 2011;60(7):931–942. [PMC free article] [PubMed]
32. Seavey MM, Maciag PC, Al-Rawi N, Sewell D, Paterson Y. An anti-vascular endothelial growth factor receptor 2/fetal liver kinase-1 listeria monocytogenes anti-angiogenesis cancer vaccine for the treatment of primary and metastatic her-2/neu+ breast tumors in a mouse model. J Immunol. 2009;182(9):5537–5546. [PMC free article] [PubMed]
33. Maciag PC, Seavey MM, Pan ZK, Ferrone S, Paterson Y. Cancer immunotherapy targeting the high molecular weight melanoma-associated antigen protein results in a broad antitumor response and reduction of pericytes in the tumor vasculature. Cancer Res. 2008;68(19):8066–8075. [PMC free article] [PubMed]
34. Gulley JL. Therapeutic vaccines: The ultimate personalized therapy? Hum Vaccin Immunother. 2013;9(1):219–221. [PMC free article] [PubMed]
35. Gunn GR, Zubair A, Peters C, Pan ZK, Wu TC, Paterson Y. Two listeria monocytogenes vaccine vectors that express different molecular forms of human papilloma virus-16 (HPV-16) E7 induce qualitatively different T cell immunity that correlates with their ability to induce regression of established tumors immortalized by HPV-16. J Immunol. 2001;167(11):6471–6479. [PubMed]
36. Paterson Y, Maciag PC. Listeria-based vaccines for cancer treatment. Curr Opin Mol Ther. 2005;7(5):454–460. [PubMed]
37. Pan ZK, Ikonomidis G, Lazenby A, Pardoll D, Paterson Y. A recombinant listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours. Nat Med. 1995;1(5):471–477. [PubMed]
38. Kim SH, Castro F, Gonzalez D, Maciag PC, Paterson Y, Gravekamp C. Mage-b vaccine delivered by recombinant listeria monocytogenes is highly effective against breast cancer metastases. Br J Cancer. 2008;99(5):741–749. [PMC free article] [PubMed]
39. Hussain SF, Paterson Y. What is needed for effective antitumor immunotherapy? lessons learned using listeria monocytogenes as a live vector for HPV-associated tumors. Cancer Immunol Immunother. 2005;54(6):577–586. [PubMed]
40. Judy BF, Singhal S. How can cytoreduction surgery improve the prospects for cancer patients receiving immunotherapy? Immunotherapy. 2012;4(11):1077–1080. [PubMed]
41. Kruklitis RJ, Singhal S, Delong P, et al. Immuno-gene therapy with interferon-beta before surgical debulking delays recurrence and improves survival in a murine model of malignant mesothelioma. J Thorac Cardiovasc Surg. 2004;127(1):123–130. [PubMed]
42. Parry RV, Rumbley CA, Vandenberghe LH, June CH, Riley JL. CD28 and inducible costimulatory protein src homology 2 binding domains show distinct regulation of phosphatidylinositol 3-kinase, bcl-xL, and IL-2 expression in primary human CD4 T lymphocytes. J Immunol. 2003;171(1):166–174. [PubMed]
43. Predina JD, Judy B, Fridlender ZG, et al. A positive-margin resection model recreates the postsurgical tumor microenvironment and is a reliable model for adjuvant therapy evaluation. Cancer Biol Ther. 2012;13(9):745–755. [PMC free article] [PubMed]
44. Tucker ZC, Laguna BA, Moon E, Singhal S. Adjuvant immunotherapy for non-small cell lung cancer. Cancer Treat Rev. 2012;38(6):650–661. [PubMed]
45. Kudo-Saito C, Schlom J, Hodge JW. Induction of an antigen cascade by diversified subcutaneous/intratumoral vaccination is associated with antitumor responses. Clin Cancer Res. 2005;11(6):2416–2426. [PubMed]
46. Predina JD, Judy B, Aliperti LA, et al. Neoadjuvant in situ gene-mediated cytotoxic immunotherapy improves postoperative outcomes in novel syngeneic esophageal carcinoma models. Cancer Gene Ther. 2011;18(12):871–883. [PMC free article] [PubMed]
47. Predina JD, Judy B, Kapoor V, et al. Characterization of surgical models of postoperative tumor recurrence for preclinical adjuvant therapy assessment. Am J Transl Res. 2012;4(2):206–218. [PMC free article] [PubMed]
48. Liu Y, Wang L, Predina J, et al. Inhibition of p300 impairs Foxp3(+) T regulatory cell function and promotes antitumor immunity. Nat Med. 2013;19(9):1173–1177. [PMC free article] [PubMed]
49. Le DT, Brockstedt DG, Nir-Paz R, et al. A live-attenuated listeria vaccine (ANZ-100) and a live-attenuated listeria vaccine expressing mesothelin (CRS-207) for advanced cancers: Phase I studies of safety and immune induction. Clin Cancer Res. 2012;18(3):858–868. [PMC free article] [PubMed]