Human U251MG Glioma Cells Expressing the Membrane

Human U251MG glioma cells expressing the membrane form of macrophage colony-stimulating factor (mM-CSF) are killed by human monocytes in vitro and are rejected within immunodeficient mice via paraptosis that is associated with increased expression of three different heat shock proteins Martin R Jadus,1,2 Yijun Chen,1,2 Mehrdokht Tarbiyat Boldaji,1,2 Christina Delgado,1,3 Ramon Sanchez,1,3 Thomas Douglass,3 Usama Al-Atar,1,2 William Schulz,4 Cheri Lloyd,1,2 and H Terry Wepsic1,2 1Department of Diagnostic and Molecular Medicine, Box 151 Veterans Affairs Medical Center, 5901 E. 7th Street, Long Beach, California 90822, USA; 2Pathology Department, University of California, Irvine, California 92697, USA; 3Biological Sciences Department, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, California 90840, USA; and 4Immunology Department, University of Southern California, Norris Comprehensive Cancer Center, Los Angeles, California 90033, USA.

Human U251MG glioma cells retrovirally transduced with the human gene for the membrane form of macrophage colony- stimulating factor (mM-CSF) were investigated. The clones, MG-2F11 and MG-2C4, that expressed the most mM-CSF, but not the viral vector or the parental U251MG cells, were killed by both murine and human monocyte/macrophages in cytotoxicity assays. MG-2F11 cells failed to form subcutaneous tumors in either nude or NIH-bg-nu-xidBR mice, while mice inoculated with the U251MG viral vector (MG-VV) cells developed tumors. Electron microscopy studies showed that 4 hours after subcutaneous injection, the mM-CSF-transduced cells began dying of a process that resembled paraptosis. The dying tumor cells were swollen and had extensive vacuolization of their mitochondria and endoplasm reticulum. This killing process was complete within 24 hours. Macrophage-like cells were immediately adjacent to the killed MG-2F11 cells. Immunohistological staining for the heat shock proteins HSP60, HSP70 and GRP94 (gp96) showed that 18 hours after inoculation into nude mice, the MG-2F11 injection site was two to four times more intensely stained than the MG-VV cells. This study shows that human gliomas transduced with mM- CSF have the potential to be used as a safe live tumor cell vaccine. Cancer Gene Therapy (2003) 10, 411–420. doi:10.1038/sj.cgt.7700583 Keywords: macrophage colony-stimulating factor; macrophages; human glioma; heat shock proteins; paraptosis

very year approximately 17,000 new patients are further research into methods to generate better immune Ediagnosed within the United States with glioblastoma responses so that more glioma patients may be treated multiforme.1 These high-grade gliomas recur after surgi- using immuno-based methods. cal resection with standard chemo- and radiation thera- Various proinflammatory cytokines (interleukin 2, 4, 6, pies. The prognosis of these patients is still extremely 7, 12, IFN-g, granulocyte–macrophage colony-stimulat- poor.2,3 Several immunotherapy approaches have in- ing factor (GM-CSF)) have proved useful in prophylactic cluded the following: administering immune adjuvants, vaccination against rodent gliomas.11–18 Both CD4+ antiglioma and growth factor receptor-specific antibodies, and CD8+T-cell responses were generated and produced cytokine transduced glioma cell vaccines and ex vivo immunity against the rechallenge of parental tumors. stimulated tumor infiltrating lymphocytes (TIL) and Parney et al19 transduced the human D54MG glioblasto- lymphokine-activated killer cells (LAK).4–10 These im- ma cell line in vitro with genes encoding GM-CSF and the munotherapeutic modalities occasionally cured a few T-cell costimulatory molecule B7-2. When SCID/beige patients. These positive responses have encouraged mice were reconstituted with human PBL, these ‘‘huma- nized’’ mice then inhibited the growth of these dual Received July 22, 2002. transduced tumors. Although these tumors still grew at a Address correspondence and reprint requests to: Dr Martin R much slower rate, it suggested that the human effector Jadus, Ph.D. Box 113, Diagnostic and Molecular Medicine Service, cells were actively mounting an immune response against Veterans Affairs Medical Center, 5901 E 7thStreet, Long Beach,CA, the tumor cells in vivo. The best vaccination occurred USA. E-mail: [email protected] when the tumor cells were transduced with both the Immunodeficient mice reject human gliomas making mM-CSF MR Jadus et al 412 GM-CSF and B7-2 genes. These studies indicate that it is The cells were propagated in Dulbecco’s modified Eagle’s possible to induce a human antiglioma immune response medium supplemented with 10% fetal bovine serum (FBS, using gene-modified human glioma cells in a complex in Gemini BioProducts, Calabassas, CA), with antibiotics in vivo setting without risking patient safety. a humidified atmosphere of 5% CO2 at 371C. All culture Previously, we have shown that malignant rat T9 supplies were screened and selected on the basis of being glioma tumor cells transduced with mM-CSF, but not the endotoxin-free (under the limits of detection by limulus secreted form of M-CSF, are directly killed by macro- amebeocyte lysate assay). Cells were determined to be phages in vitro through an mM-CSF dose-dependent mycoplasma free, based on Strategene’s mycoplasma PCR pathway that relied upon phagocytosis of the transduced analysis kit (Strategene Inc., San Diego, CA). tumor cells.20,21 Not only were these mM-CSF expressing T9 tumor cells rejected, but concurrently a strong T-cell- Transfection of mM-CSF gene and viral vector into mediated immunity was induced that was capable of tumor cells rejecting an intracranial T9 glioma.22,23 Our studies show The U251MG cells were infected in six-well cluster dishes that immune responses induced by mM-CSF-transduced (Corning, Corning, NY). A total of 100,000 exponentially cells is quite similar to tumor cells cotransduced with both growing cells were incubated either in the presence or the GM-CSF and B7-2 genes. Besides being able to absence of the supernates of the mM-CSF- or viral vector- vaccinate rats against this tumor, rats bearing small 1–3 transducing retroviruses overnight. The cells were refed day established intracranial T9 glioma could be success- with fresh media containing 1 mg/mL G418. After 2 fully treated 93–83% of the time by a peripheral weeks of G418 selection, the cells were cloned by limiting vaccination with mM-CSF expressing tumor cells.24 dilution in 96-well microtiter plates. For mM-CSF Immunization with living mM-CSF T9 tumor cells detection, cells were tested by flow cytometric analysis stimulated cross-protective immunity that resisted differ- as described below. ent syngeneicRT2 and F98 gliomas. 21 This type of cross- protective resistance has not been reported with other cytokine transfection tumor models. Antibodies and flow cytometry Our animal data using the mM-CSF-transduced rat Tumor cells were incubated with the anti-M-CSF anti- gliomas is promising, but it does not guarantee that the body or normal goat serum on ice for 1 hour. Goat anti- mM-CSF expressing human glioma cells will provoke human M-CSF antibody was purchased from R&D identical types of immune responses. This study shows Systems (Minneapolis, MN). Human monocytes were that mM-CSF-transduced human glioblastoma cells are identified by incubating the cells on ice with the anti- killed by both murine and human monocytes/macro- human CD64 antibody (mouse IgG) purchased from phages in vitro. In vivo studies using immunodeficient Pharmingen (San Diego, CA). The cells were washed once mice showed that these transfected tumor cells are with ice-cold PBS in a refrigerated centrifuge and then infiltrated by monocytes/macrophages within 4 hours. incubated in a 1:10 dilution of a fluorescein isothiocya- At this time the tumor cells start to be killed via a nate (FITC)-labeled rabbit antiprimary antibody (Vector process that resembles paraptosis that is independent of Laboratories, Burlingame, CA) for an additional hour on phagocytosis. As a result of this killing, tumors will not ice. The cells were washed three times in cold PBS. A total form in either nude or NIH-bg-nu-xid mice. After 18 of 10,000 cells were analyzed on the flow cytometer. Data hours of the implantation of the mM-CSF-transfected were analyzed by Multi2D (Phoenix Flow Systems, San cells, these tumor cells expressed three different forms of Diego, CA). the heat shock proteins, HSP60, HSP70, GRP94(gp96). These induced proteins have been associated with the Bone marrow-derived macrophage cultures induction of tumor immunity in several tumor models and Mouse bone marrow cells were cultured in RPMI-1640 could help explain why mM-CSF-transfected tumor cells media (Life Technologies, Grand Island, NY) containing function as immunogens. We have demonstrated that 5% FBS and M-CSF supernate derived from mM-CSF- mM-CSF-transfected tumor cells also produce immuno- transfected T9-C2 cells.20 After 1 week of culture at 371C protection in a weakly immunogenic MADB106 breast in a humidified, 5% CO atmosphere, the media were cancer model25 and a nonimmunogenicHepal-6 liver 2 replaced with fresh 33% conditioned media every 3–4 cancer model.26 This study suggests that mM-CSF- days. Macrophages were removed by washing off the transfected human glioma cells can be used as a safe tissue culture media and then incubating the cells in tumor vaccine and may have immunizing properties, phosphate-buffered saline (PBS) for at least 30 minutes to which are not seen when prekilled tumor cells or dendritic 1 hour at 41C. The cells were dislodged using a cell cells pulsed with tumor-derived antigenic peptides are scraper. This procedure results in 495% viability of the used as the vaccine. macrophages, which were phenotyped being 490% positive for Mac1, Mac2, Mac3 and F4/80. Materials and methods Cell lines and cell culture Human effector cells The human U251MG glioma cell line was obtained from After signing informed consent, human donors’ periph- Dr Dennis Deen (University of California, San Francisco). eral blood mononuclear cells from cancer patients

Cancer Gene Therapy Immunodeficient mice reject human gliomas making mM-CSF MR Jadus et al 413 undergoing various therapies at the University of South- assays were analyzed using Student’s t-test. Values were ern California Norris Comprehensive Cancer Center (Los considered significantly different at the Po.05 levels. Angeles, CA) were collected by leukapheresis and ficolled. These cells were determined to be HIV and hepatitis B Tumor growth in animals and C free as determined by PCR. The HLA-A2+tissue- typed human peripheral blood mononuclear cells Animal use was carried out according to the Animal (PBMC) were kindly provided by Dr Jeffrey Weber Studies Committee approved protocols. Nude (Crl:nu/ (USC). The cells were preserved in standard 10% DMSO nu-nuBR) mice and NIH-bg-nu-xidBR (SCID/biege/ freezing media and were stored in liquid nitrogen at a xid) mice were obtained from Charles River (Wilmington, concentration of 5 Â 107/ml. Before starting the cytotoxi- MA). The animals were housed in our AAALAC city study, the viability of the thawed PBMC was accredited facility. The immunodeficient mice were examined by trypan blue exclusion and was 490%. housed in sterile microisolators. Manually restrained mice were injected subcutaneously on the ventral side PlasticPetri dishes were overlayed with 2 ml FBS and 5 6 incubated for 15 minutes at 371C. The FBS was aspirated with 3 Â 10 –10 cells in a volume of 50 ml. After 5–7 and the dishes were used immediately. The cryopreserved days, when the tumors were palpable they were measured human PBL cells were thawed out and resuspended at 3– with metric calipers according to the length, width 5 Â 106 cells/ml in RPMI-1640 media supplemented with and height of the tumor three times a week. The tumor 10% FBS. The cell suspension was added to the FBS- volume was then calculated by the equation: volume ¼ pretreated dishes (5 ml/dish) and incubated at 371C for 1 length Â width Â height Â p/6. The data are expressed as 7 hour. The dishes were gently washed three times with the mean tumor volume SEM. Data at each time point PBS. The adherent cells were detached using 5 ml of were then compared using a Student’s t-test. A P-value versene buffer (Life Technologies). The remaining ad- o.05 was considered significant. herent cells were dislodged using a cell scraper after 1 ml of FCS was added to the versene. Electron microscopic studies The mice were injected with 5 Â 106 cells resuspended in HLA tissue typing of U251MG glioma cell line 50 ml PBS on the ventral side. Tissues taken at 4 hours The U251MG cells were tissue typed for MHC class I by postinjection were fixed for 1–2 hours with 2.5% PCR techniques by Dr Robertson Parkman, Children’s glutaraldehyde at pH 7.3 in 0.1 M Sorensen’s phosphate Hospital of Los Angeles, Department of Research buffer. The tissues were then rinsed in Sorensen’s buffer, Immunology and Bone Marrow Transplantation (Los postfixed for 1 hour in 1% OsO4, and rinsed again in Angeles, CA). These cells were identified as HLA-A2+, Sorensen’s buffer. Following dehydration with a cold B18+, Bw6+ and C5+. graded ethanol series and subsequent transfer to propy- lene oxide, the tissues were infiltrated with Spurr’s resin Cytotoxicity studies over a period of 36 hours. Sections taken with a diamond knife were stained with uranyl acetate and lead citrate and Human and mouse macrophage-medicated cytotoxicity then observed using a JEOL-1200EX II transmission studies were performed according to our previously used electron microscope (Peabody, MA). methods.20,21 Exponentially growing tumor cells in 10 ml of complete media were internally labeled with 10 mCi of 3H-thymidine (3H-TdR) (ICN, Costa Mesa, CA) over- Immunohistology night. The tissue culture media were replaced with fresh At 4–18 hours after subcutaneous inoculation in nude media and allowed to incubate a further 1–3 hours to mice, the MG-2F11 and U251MG-VV tumors were reduce spontaneous release by the tumor cells. A total of removed and sectioned in a cryostat. The rabbit antiheat 10,000 target cells were incubated in 200 ml of media with shock protein-70 antibody was obtained from Chemicon graded doses of monocytes/macrophages ranging from Intl (Temecula, CA). Rabbit antiheat shock protein-60 20:1 to 2.5:1 at 371C in a humidified 5% CO2 incubator and anti-GRP94 (also known as gp96) antibodies were for 24–48 hours. Afterward, 100 ml of supernatant was purchased from Stress Gen (Victoria, BC, Canada). The removed and placed in 2 ml of scintillation fluid. antibodies were diluted 1:100 in PBS. The tissue was Maximum release is calculated by taking 104 target cells incubated 2 hours with the primary antibody in a and rapidly freeze–thawing them three times in liquid humidified chamber at 41C. The slides were washed three nitrogen. Specific release is calculated using the standard times in PBS and the 1:100 diluted biotinylated goat equation for cytotoxicity reactions: antiprimary antibody (Pharmingen Inc., San Diego, CA) CPM experimental À CPM spontaneous release was incubated at room temperature for 30 minutes. The % killing ¼ CPM maximum release À CPM spontaneous release slides were washed three times in PBS and incubated for 30 minutes in the streptavidin conjugated peroxidase Â 100 (DAKO Inc., Carpinteria, CA). The tissue was rinsed Cytotoxicity data from triplicate cultures at each macro- three times in PBS and then the DAB substrate (DAKO) phage : tumor cells ratios are presented as the mean 7SD. was added. The tissue was briefly counterstained in Cytotoxicity is not considered relevant if values are less freshly filtered Mayer’s hematoxylin (DAKO) for 15 than 10% specific killing. Data from the cytotoxicity seconds and then mounted with a cover slide using

Cancer Gene Therapy Immunodeficient mice reject human gliomas making mM-CSF MR Jadus et al 414 Vector’s Laboratories Mounting Media (Burlingame, remained stable over the last 3 years. The parental CA). The staining of the cells was then scored by at least U251MG cells transduced with an empty viral vector two people using a multiheaded microscope. Scoring was control retrovirus (MG-viral vectors) and were negative assessed by staining intensity and the total number of cells for mM-CSF expression (data not shown). positively stained above the negative controls. Mouse macrophages kill the mM-CSF-transfected human glioma cells Initial cytotoxicity experiments were performed with Results mouse bone marrow-derived macrophages since these mM-CSF transduction of the U251MG cells cells were readily available at the time these mM-CSF- The human U251MG cells were transduced using retro- transduced clones were being characterized. The mouse viral vectors as previously described.20 Once the cells were macrophages killed the MG-2F11 and MG-2C4 cells, but positively selected with G418, they were cloned by the not MG parental cells or MG-viral vector cells in two limiting dilution method. Three dozen clones were separate cytotoxicity assays (Fig 2). At macrophage to analyzed by flow cytometry for the expression of mM- tumor ratios from 2.5:1 to 10:1, the macrophages killed CSF. The two best expressing clones, MG-2F11 and MG- the MG-2F11 glioma cells in a dose-dependent manner. 2C4, were selected for further use (Fig 1). The MG-2F11 The difference in killing between the two mM-CSF- cells are roughly four times as fluorescent as the MG-2C4 transduced cell lines was significantly different (Po.05) cells. The expression of mM-CSF on these cells has from the parental U251MG cells and MG-viral vector cells. These data show that the mM-CSF-transduced clones are killed by mouse macrophages. Since the MG- 2F11 cells had more mM-CSF than the MG-2C4 cells and were killed by the macrophages, we chose to use this clone for all further work.

Human PBMC and monocyte/macrophages can kill the mM-CSF-transfected human glioma cells Human PBMC from several HLA-A2-matched individuals were tested for their cytotoxicity towards the

Figure 2 The mouse macrophages kill MG-2F11 and MG-2C4 cells but not MG parental or viral vector cells. The clones from Figure 1 were radiolabeled overnight with 3H-Tdr and used the next day as Figure 1 Flow cytometric expression of mM-CSF on MG-2F11 and target cells for mouse bone marrow-derived macrophages in a MG-2C4 cells. U251MG cells were transfected with mM-CSF- cytotoxicity assay. The macrophages were serially diluted (from 10:1 transducing retroviruses and subsequently cloned. Two subclones, to 2.5:1) and cultured with the tumor cells (104 cells/well). Four cell MG-2F11 and MG-2C4, were characterized. An isotypic control types were studied in parental U251MG cells, U251 Viral Vector antibody is included as a control. A total of 10,000 cells were (MG-VV)-transduced cells, and two clones expressing the mM-CSF. analyzed on the EPICS flow cytometer. The isotypic control was The results from the two mM-CSF-transduced target cells were subtracted from the signal derived from the mM-CSF-stained cells significantly different (Po.05) at all macrophage:target cell ratios and is represented by the shaded area. from the parental and viral vector results as noted by the asterisks.

Cancer Gene Therapy Immunodeficient mice reject human gliomas making mM-CSF MR Jadus et al 415 MG-2F11 and U251MG parental cells. Four out of the five donors’ PBMC significantly (Po.05) killed the MG- 2F11 cells at the 100 : 1 PBMC : tumor cell ratio, while none of the PBMC lysed the U251MG parental cells. The data from a representative experiment are shown in Figure 3a. To show that the human monocytes/macrophages were killing the mM-CSF-transduced cells, we enriched the monocytes by adherence to plastic culture dishes. The adherent cells proved to be positive (495%) for the monocyte/macrophage marker CD64. These adherent monocytes were tested in cytotoxicity assays towards MG-2F11 and MG parental cells. A representative experiment performed with two separate donors in two independent experiments showed that the MG-2F11 cells were killed in a dose-dependent manner. Aliquots of these same monocytes failed to kill the MG parental cells when tested in simultaneous parallel studies (Fig 3b). mM-CSF-transfected U251MG cells did not grow in subcutaneous sites in immunodeficient mice Nude and NIH-bg-nu-xidBR mice were implanted subcutaneously with 3 Â 105 MG-VV or MG-2F11 cells on day 0. Mice implanted with the MG-VV control cells formed tumors within 3 weeks in seven out of eight with both nude (Fig 4a) and NIH-bg-nu-xidBR (Fig 4b) mice. These tumors grew consistently until the animals were euthanized. All of the eight nude mice that received an equivalent dose of MG-2F11 cells failed to form tumors. All eight NIH-bg-nu-xidBR mice failed to form MG-2F11 Figure 4 The mM-CSF-transduced MG-2F11 cells do not form tumors during the first 50 days after inoculation (Fig 4b). tumors in immunodeficient mice. Mice (panel A: nude mice; panels B One NIH-bg-nu-xidBR mouse eventually formed the and C: NIH-bg-nu-xidBR) were injected subcutaneously with 3 Â 105 MG-VV or MG-2F11 cells on day 0. Panel C: half of the NIH-bg-nu- xidBR were treated with anti-asialo GM1 five times before being injected with 1 Â 106 MG-2F11 cells on day 0. On a Monday– Wednesday–Friday basis from Days 3 to 50, the tumors on the mice were measured with metric calipers. The size of each tumor from the groups was pooled. The average and SE of the mean were plotted. The asterisks indicate that the Student’s t-test P-values are significantly different (Po.05) from the control animals. In panel A, eight nude mice were used in each group. In panel B, eight NIH-bg- nu-xid mice were used in each group. In Panel C, five NIH-bg-nu-xid mice were used in each group.

MG-2F11 tumor three months after inoculation. The other seven mice remained tumor free. The growth difference between the MG-VV and MG-2F11 tumors in nude and NIH-bg-nu-xidBR mice was statistically significant (Po.01) beginning on day 33 for the nude mice and day 38 for the NIH-bg-nu-xid mice. The nude mice that rejected the MG-2F11 cells were then rechallenged with the MG-VV cells to determine whether immunity was induced. All of the eight mice that rejected the MG-2F11 clone failed to resist the growth Figure 3 Human PBMC and enriched monocytes kill the mM-CSF- of the U251MG parental cells (data not shown), demon- transfected human glioma cells. U251MG and MG-2F11 cells were strating that there was no inherent immunological used as target cells by human PBMC and enriched monocytes in a memory stimulated in these immunocompromised mice 48-hour cytotoxicity assay. The asterisks represent significantly against the U251MG tumor cells. different data from that generated with the control nontransduced To show that the MG-2F11 cells could form tumors tumor cells (Po.05). in mice, we pretreated NIH-bg-nu-xidBR mice with

Cancer Gene Therapy Immunodeficient mice reject human gliomas making mM-CSF MR Jadus et al 416 anti-asialo GM1 antibody. Previously, it was shown that this antibody could deplete mice of their NK cells, activated T cells and activated macrophages.27,28 Since the NIH-bg-nu-xidBR mice lack NK and T cells, this antibody will deplete the activated macrophages. Even when 3.3 times more MG-2F11 cells (106 cells) were injected into these immunodeficient mice, none of the control mice developed a tumor, while four out of five mice pretreated with the anti-asialo GM1 antibody formed the MG-2F11 tumor (Fig 4c). The mice pretreated with the anti-asialo GM1 antibody allowed the MG-VV tumors to grow as well (data not shown).

Electron microscopy indicates that the MG-2F11 cells were killed by a process that resembles paraptosis Electron microscopy was used to study the mechanism through which the tumor cells were being eliminated. Studies showed that after implantation of the MG-2F11 cells, there was no evidence that the tumor cells were growing; that is mitoticfigures and celldivision. Figure 5a demonstrates that the MG-2F11 cells displayed a swollen phenotype 4 hours after being injected subcutaneously. The type of cell death that best describes these dying tumor cells is called paraptosis.29 Swelling of the cells was evident as a loss of electron-dense material. Macrophage- like cells were found to be adjacent to the dying tumor cells. Most of the MG-2F11 cells were swollen, and vacuolated with rare membrane blebbing (zeiosis), while the viral vector control cells never appeared this way. The MG-2F11 cells had some areas of condensed nuclear chromatin, but these dying tumor cells rarely formed smaller apoptoticbodies. One notable feature was that the mM-CSF-transfected tumor cells were not killed by direct phagocytosis. During the course of these studies the macrophages did show signs of activity that resulted in morphological changes. Normal monocytes/macrophages were more electron dense than the tumor cells. As the macrophages became activated, they appeared to become more heavily electron dense. Some activated macrophages showed signs of becoming apoptotic by membrane blebbing and cellular break-up (Fig 5b).

Immunohistochemical staining of the MG-2F11 injection site reveals enhanced heat shock protein staining The results of the electron microscopy suggested that nonphagocytic cytotoxic functions of the macrophages were being induced by the mM-CSF-transfected U251MG cells. Cells dying of necrosis have been demonstrated to display increased amounts of the various 30 Figure 5 Electron microscopic analysis of the MG-2F11 cells heat shock proteins (HSP). Since little is known of cells 4 hours after subcutaneous inoculation. Panel A shows two dying of paraptosis, we analyzed these dying tumor cells monocytic/macrophage-like cells attacking the MG-2F11 cells. The for the production of the heat shock proteins. Tumor tumor cell has become a swollen heavily vacuolated cell. The sections were then stained for three different members of magnification is Â 3000. Panel B shows an apoptotic macrophage in this diverse family of HSPs (HSP60, HSP70 and GRP94). the process of undergoing zeiosis and cellular break-up. The When the tissue was examined after 4 hours, neither the magnification of panel B is Â 6000. MG-2F11 tumor nor the MG-viral vector (MG-VV) tumor displayed any significant amount of HSP (data not

Cancer Gene Therapy Immunodeficient mice reject human gliomas making mM-CSF MR Jadus et al 417

Figure 6 Immunohistology of the subcutaneous MG-2F11 tumor injection site in nude mouse. Panel A: negative control; panel B: anti-HSP60; panel C: anti-HSP70; panel D: anti-GRP94(gp96). All magnifications are Â 200. shown). However, when we assayed the tumor sites after subcutaneous tumor vaccine to treat 3-day-old established 18 hours, the MG-2F11 tissue proved to be 2–4 times intracranial 9L gliomas. We have seen that the immunity more strongly positive for the HSP60 (Fig 6b), HSP70 induced by vaccinating with these mM-CSF-transfected (Fig 6c) and GRP94(gp96) (Fig 6d) than the MG-VV T9-C2 cells could also immunize rats against other tumor. The immunohistology showed that HSP70 and syngeneicRT2 and F98 gliomas. These unique findings GRP94(gp96) staining was four times more intense in the rat glioma model encouraged us to investigate than that seen with the MG-VV. HSP60 staining was whether human mM-CSF-transduced gliomas could only twice the intensity seen with the MG-viral vector behave in a similar manner in the hope of developing cells. The kinetics of this reaction suggests that this these cells into an allogeneic human tumor vaccine. paraptotickilling of the mM-CSF expressing MG-2F11 In the present study, we report that the human cells does appear to produce a multiple heat shock protein U251MG glioma upon transfection with mM-CSF was response. killed in vitro by mouse macrophages. HLA-A2-matched human PBL and the enriched human monocytes/macrophages also killed these tumor cells in vitro. We observed Discussion that the best killing by PBL occurred at a ratio of 100:1, when the highest effector : target ratio was used. We Our previous experience using the rat T9 glioma cells, also speculate that this ratio had the optimal number of known as 9L, transduced with mM-CSF showed that monocytes present to kill the mM-CSF-transduced MG- tumor rejection and subsequent immunity against the 2F11 tumor cells, since the PBMC only contain 3–5% parental tumor occurs in vivo. The immunity that was monocytes. When we enriched for monocytes, the optimal produced by the therapeutic vaccination could even killing of the MG-2F11 cells was at a ratio of 20:1 and protect rats with small 1–4-day-old established intracra- 10:1. No killing was observed against the MG parental nial tumors.24 These last results were comparable or even cells at these same monocyte : tumor ratios. This is the better than those reported by Okada, et al17 when they first time that our lab has shown that human monocytes/ ran identical studies using IL-4 transfected 9L cells as the macrophages can kill mM-CSF-transduced human tumor

Cancer Gene Therapy Immunodeficient mice reject human gliomas making mM-CSF MR Jadus et al 418 cells, suggesting that our previous work in the rodent synthesizes replacement proteins for those damaged ones. models can be directly translatable with human tumors. These newly synthesized proteins would also likely In vivo studies showed significantly reduced tumor- include potential antigenic peptides. HSPs act as chaper- igenicity of the MG-2F11 tumor in immunodeficient mice ones, which can bind the antigenic peptides in a more when compared to viral vector control U251MG cells. permissive manner than peptides binding to MHC In both nude mice (all eight tested, 0% with tumors) molecules.40,41 Once the tumor cell releases these pep- and NIH-bg-nu-xidBR mice (one out of eight mice, tide/HSP complexes, the host’s antigen-presenting cells 13% with tumors) failed to show growth of subcuta- (APC), both dendritic cells and macrophages, can take up neous inoculated MG-2F11 cells, while the viral vector these antigens via the CD91 receptor.42 Peptides com- control cells grew in the immunodeficient mice. When plexed to HSPs stimulate immunity better against the NIH-bg-nu-xidBR mice were pretreated with anti-asialo antigen than when the antigenicpeptide is not complexed GM1 antibody, which depletes the activated murine to the HSP. HSP70 and gp96 have also been reported to macrophages, the MG-2F11 glioma cells formed tumors enhance dendritic cell maturation.30,43 HSP70 also acts as in four out of five mice. This work demonstrates that a cytokine, which stimulates tumor necrosis factor, IL-1 macrophages actively inhibited the growth of the mM- and IL-6 production from CD14+monocytes,44 which CSF-transfected U251MG gliomas. includes the immature dendritic cells. Thus, we speculate The mechanism through which the macrophages killed that one of the mechanisms through which living mM- the tumor cells resembled a newly described process called CSF transfected cells stimulate antitumor immunity may paraptosis.29 Paraptosis is a form of programmed cell be through the production of the HSPs, which activates death that differs from apoptosis. As a result of an insult the local APCs into becoming better antigen-presenting delivered by the macrophages, the tumor cells began a cells. swelling and vacuolization process that is associated with One of the difficulties of immunogene therapy based on disruption of the endoplasmic reticulum and mitochon- developing an autologous vaccine is the difficulty of dria. In contrast, apoptosis is characterized by cell establishing glioma cell lines from each patient.45,46 Early- shrinkage and membrane blebbing that culminates in passage human gliomas grow much slower than the apoptoticbody formation. Immunohistology revealed established human glioma cell lines. It may take months that the tumor site had the increased expression of three to establish the glioma tumor cell lines. Next it is different members of the HSP family of proteins. This necessary to transfect the tumor cells, so that they express suggests that the tumor cells produced the HSP to mM-CSF and to select the most appropriate clones for counteract the attack delivered by the macrophages. immunotherapy. In many cases, the length of the time Relatively little is known of the immunological vaccinat- necessary to do this entire procedure may exceed the ing properties of paraptotictumor cells.Using the rat T9 lifespan of some patients, since the recurrence of the tumor model, we have seen that both PMNs and glioma may occur within a few months of the initial macrophages contributed to the paraptotic killing of the resection. One possible solution to this problem of lack of mM-CSF-expressing T9-C2 cells.31 However in this study, time is to use allogeneictumor cellsinstead of using we have only observed a monocytic/macrophagic infil- autologous tumor cells as the sources of the vaccine. trate within the implanted MG-2F11 cells. Thus, human We found that in the rat T9 glioma model that tumor cells can be killed via paraptosis by only immunization using living mM-CSF-expressing T9-C2 monocytes/macrophages. cells produced cross-protective immunity against the Cells that die of apoptosis usually do not provoke rechallenge of the syngeneic F98 and RT-2 gliomas.24 immunological responses, although apoptotic cells can be This unique cross-protective immunity suggests that we used to stimulate antitumor immunity with the help of in can develop an allogeneic tumor vaccine composed of vitro cultured dendritic cells. Cells that are killed via a either a single glioma cell line or a pool of human glioma necrotic pathway tend to become more immunogenic cells engineered to express high levels of mM-CSF to when directly compared to apoptotic cells.32–37 This key immunize other HLA-matched glioma patients. For feature is perhaps the basis of the ‘‘Danger Hypothesis’’.38 safety precautions, these mM-CSF-transfected gliomas Released HSPs may be a key component, which alerts the would also be genetically engineered with either thymi- immune system of potential dangers elicited by necrotic dine kinase or cytidine deaminase suicide genes. Since the cells. Necrotic cells will produce heat shock proteins.30,36 living mM-CSF-transfected glioma cells that are being HSPs have been described as ‘‘natural adjuvants’’ since killed via paraptosis do express HSPs, it may be possible they provoke immunity.35 In this regard, paraptoticcells to immunize patients with other MHC haplotypes resemble necrotic cells. Three different members of this different from the vaccines’ MHC composition. HSPs diverse family of proteins (HSP60, HSP70 and GRP94) complexed with antigenic peptides can produce immunity were expressed in the paraptoticMG-2F11 cells.In some in individuals with different MHC haplotypes in a process tumors, HSP70 and gp96 have been identified as tumor- called ‘‘cross-presentation’’.47,48 Thus, theoretically an specific antigens.39 In other cases, HSP70, HSP90 and allogeneic mM-CSF tumor vaccine may work in patients, gp96 have increased the immunogenicity of tumors by if HSPs can cross-present tumor antigens. Studies are now increasing the synthesis of tumor antigens that can be ongoing in anti-asialo GM1-treated immunodeficient recognized by the T cells.40,41 The significance of these mice reconstituted with HLA-A2-matched PBMCs to HSPs is that as the tumor cell is being attacked, the cell prove that MG-2F11 cells can elicit a human immune

Cancer Gene Therapy Immunodeficient mice reject human gliomas making mM-CSF MR Jadus et al 419 response against the parental U251MG tumor. Our production of interleukin 2 and interferon-g. Cancer Res. second aim is to determine whether cross-presentation 1995;55:1902–1910. against different human gliomas can be accomplished 16. Visse E, Siesjo P, Widegren B, and Sjogren O., Regression of using different MHC-matched individuals and their intracerebral rat glioma isografts by therapeutic subcuta- gliomas using mM-CSF-transduced MG-2F11 cells. neous immunization with interferon-g, interleukin-7, or B7- 1-transfected tumor cells. Cancer Gene Ther. 1999;6:37–44. 17. Okada H, Giezeman-Smits KM, Tahara H, et al. Effective Acknowledgments cytokine gene therapy against an intracranial glioma using a retrovirally transduced IL-4 plus HSVtk tumor vaccine. This study was funded in part from grants obtained from Gene Ther. 1996;6:219–226. the Veterans Affairs Medical Center (HTW, MRJ) and 18. 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