RENCA Tumor Cells Research Articles

Development of a New Focal Mouse Model of Bone Metastasis in Renal Cell Carcinoma.

Developing animal models of bone metastasis in renal cell carcinoma (RCC) is challenging as immunodeficient mice are required. The aim of this study was to develop a simple immune model of RCC bone metastasis. RENCA tumor cells were injected into the right femurs of BALB/c mice. Sixty mice were grouped into each twenty-mouse group according to the tumor cell concentration, and the presence or absence and extent of bone metastasis in the total length of the femur were compared using hematoxylin and eosin staining of the excised tissues. Bone metastasis was significantly higher in the high concentration group than in the other groups (p<0.05), with 10 mice developing bone metastasis at two weeks and nine mice developing bone metastasis at three weeks. The extent of bone metastasis was significantly greater in the high concentration group than in the other groups (p<0.05). Multiple logistic regression analysis was performed to examine the factors influencing bone metastasis, and only the high concentration was a significant factor (p<0.05). We developed a normal immunity mouse model of local bone metastasis from RCC. This model could prove valuable for research into the treatment of bone metastases in RCC.

Abstract 6206: Discerning vasculature destruction caused by novel vascular disrupting agents examined by optical and multispectral optoacoustic imaging

Abstract Tumor growth requires a functioning vascular network and offers an attractive therapeutic target. The invasive proliferating neovasculature of tumors lacks pericyte support and exhibits increased permeability thereby offering a unique, potentially selective target for anticancer therapy. Most vascular disrupting agents (VDAs) inhibit microtubule formation, causing rapid morphological changes in endothelia cells resulting in dramatically increased vessel permeability, cellular detachment, vessel occlusion, and vessel wall damage [doi: 10.3390/molecules26092551]. Highly vascular kidney tumors are expected to be particularly sensitive to VDAs. We are evaluating OXi8007, a water soluble indole phosphate pro-drug derivative of the first generation VDA combretastatin. The prodrug OXi8007 is stable in saline but releases active OXi8006 is the presence of alkaline phosphate. Preliminary pharmacokinetic data benchmarked using 13C-labeled OXi8006 and OXi8007 show rapid vascular clearance and conversion to the glucuronide conjugate in vivo. Doses up to 700 mg/kg were tolerated by mice. Luciferase-expressing RENCA tumor cells were implanted orthotopically in the right kidney capsule of syngeneic BALB/c mice. Bioluminescence Imaging (BLI) was applied weekly to assess tumor growth and acute response to OXi8007. Following addition of luciferin substrate subcutaneously BLI signal from the tumor reached a maximum within 10 minutes. When BLI was repeated 4 hrs after 250 or 350 mg/kg OXi8007 IP, the generated signal was &amp;lt;1% of baseline signal indicating acute vascular disruption. Multispectral Optoacoustic Tomography (MSOT) revealed matching acute changes in tumor vascular oxygenation and additionally showed distinct heterogeneity. An oxygen gas breathing challenge indicated lower oxygen saturation in the tumor following treatment. Histology showed massive hemorrhage following both doses. BLI signal recovered after 1 to 2 days and repeated administration of OXi8007 again caused acute vascular shutdown. These results match previous observations in breast tumor models [doi: 10.1016/j.canlet.2015.08.021] indicating opportunities for therapeutic application. Citation Format: Hashini I. Wanniarachchi, Regan Schuetze, Lorena Arango, Khagendra Hamal, Graham J. Carlson, Cyprian Pavlich, Yuling Deng, Mary L. Trawick, Kevin G. Pinney, Li Liu, Ralph P. Mason. Discerning vasculature destruction caused by novel vascular disrupting agents examined by optical and multispectral optoacoustic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6206.

Abstract A012: Mammary fat pad injections: An alternative implantation method for syngeneic tumor models

Abstract Syngeneic tumor models used for discovery of immune therapeutics should have several features such as a long study duration, responsiveness to checkpoint inhibitors, high immune cell infiltration and a high homogeneity in tumor growth. Moreover, models should consider the ethical rules (3R reduce, refine, replace). At present, the standard implantation method for syngeneic tumor models is subcutaneous tumor cell inoculation. We have developed an alternative implantation method for syngeneic tumor models: inoculation into the mammary fat pad. Both implantation sides are heterotopic related to the original tumor entity except for syngeneic breast tumor cells. In addition, both tumor inoculation methods can easily be applied and monitored by calipering reducing the costs. We compared the two implantation methods with models MC38-CEA, Ct26wt, Hepa1-6, RENCA, LL-2, AB12, CloneM3, B16.F10, 4T1 and EMT-6 tumor cells in respect to growth characteristics and immune response. Intra-mammary tumor growth showed more homogeneity with higher final tumor volumes compared to the subcutaneous tumor growth. Moreover, in all tested syngeneic models, tumor ulceration was prevented by almost 100% when injecting the tumor cells into the mammary fat pad. In contrast, animals of the subcutaneous tumors were mainly euthanized due to tumor ulceration. Both findings favor the mammary fat pad injection with regard to the 3R rules by strongly reducing tumor ulceration (refinement) and animal number due to a more homogenous growth (reduction). In addition, the immune checkpoint inhibitor treatment was tested and found comparable between the intra-mammary and subcutaneous models. The presence of immune cell populations was investigated in the Ct26wt colon tumor model time-dependently by flow cytometry using a 17 marker-staining panel . The number of isolated cells per gram tumor mass was more than doubled in the intra-mammary tumors. In conclusion, tumor models of the heterotopic intra-mammary implantation side are found to be superior compared to the traditional subcutaneous tumor models: a higher tumor homogeneity with no tumor ulceration, combined with a high number of immune cells and connective stroma tissue, making the mammary fat pad implantation of syngeneic tumor cells the implantation side of choice. Citation Format: Cynthia Obodozie, Susanne Ruf, Gojko Bijelic, Sandra Moor, Bianca Giesen, Ulrike Leisegang, Sebastian Dempe, Holger Weber. Mammary fat pad injections: An alternative implantation method for syngeneic tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A012. doi:10.1158/1535-7163.TARG-19-A012

In vitro and in vivo evaluation of drug-eluting microspheres designed for transarterial chemoembolization therapy

Poly(d,l-lactic acid) biodegradable microspheres, loaded with the drugs cisplatin and/or sorafenib tosylate, were prepared, characterized and studied. Degradation of the microspheres, and release of cisplatin and/or sorafenib tosylate from them, were investigated in detail. Incubation of the drug-carrying microspheres in phosphate buffered saline (pH=7.4) revealed slow degradation. Nevertheless, significant release of cisplatin and sorafenib tosylate from microspheres loaded with both drugs was apparent in vitro; this can be attributed to their porous structure. Supernatants from microspheres loaded with both drugs showed strong toxic effects on cells (i.e. endothelial cells, fibroblast cells and Renca tumor cells) and potent anti-angiogenic effect in the matrigel endothelial tube assay. In vivo anti-tumor effects of the microspheres were also observed, in a Renca tumor mouse model. The poly(d,l-lactic acid) microspheres containing both cisplatin and sorafenib tosylate revealed highest therapeutic efficacy, probably demonstrating that combined local administration of cisplatin and sorafenib tosylate synergistically inhibits tumor growth in situ. In conclusion, this study demonstrates the applicability of biodegradable poly(d,l-lactic acid) microspheres loaded with cisplatin and sorafenib tosylate for local drug delivery as well as the potential of these microspheres for future use in transarterial chemoembolization.

Phase I/II trial of intraperitoneal implantation of agarose-agarose macrobeads (MB) containing mouse renal adenocarcinoma cells (RENCA) in patients (pts) with advanced colorectal cancer (CRC).

e14517 Background: RENCA tumor cells encapsulated in two concentric agarose layers which release signals to treat advanced cancers is a novel concept that has been substantiated in in vitro and in vivo models as reported in Cancer Research 71(3), 716-735, 2011.We report results of a phase I/II study in advanced CRC pts. Methods: Previously-treated advanced CRC pts (ECOG PS 0-2) were enrolled with informed consent obtained. Pts had intraperitoneal implantation up to 4 times via laparoscopy with 8 (37/40 pts- established dose) or 16 (3/40 pts) RENCA MB/kg. Serial exams, lab profiles, and imaging were done pre- and 3 months (mo.) post-implant to assess efficacy. Endpoints of safety, tumor marker decrease, response, and overall survival (OS) were evaluated. Results: 40 pts were implanted with RENCA MB (12 pts phase I; 28 pts phase II); 17M: 23F. Mean age 58.3. 14/40 pts had &gt;1 implant (≥2: 14 pts; ≥3: 2 pts; =4: 2 pts). Response to MB is marked by a prominent initial rise in CRP, ESR, and IL-6, indicating a systemic inflammatory response (SIR) (100% of pts) and a parallel decrease in CEA and/or CA 19-9 in approximately 70%. SIR including its accompanying fatigue and anorexia lasts days to 3 wks. Overall, there was a statistically significant difference (p=0.009) in OS between the 70% of pts showing a decrease in tumor markers by at least 20% during the first 30 days post-implant (OS; 9.7 mo., C.I. 6.5-13.5) and those who did not (OS; 4.4 mo., C.I. 1.1-7.6). On PET-CT imaging, a striking feature of response, most often seen in pts with the longest OS, is tumor necrosis. MB were well-tolerated and no Grade ≥3 adverse events were treatment-related. Conclusions: For advanced, treatment-resistant CRC pts, early response to MB implantation characterized by tumor marker decrease in association with SIR correlates with a statistically significant increase in OS. RENCA MB represent a possible new therapeutic option for late-stage CRC. Clinical trial information: NCT01053013.

Loss of Inducible Nitric Oxide Synthase Expression in the Mouse Renal Cell Carcinoma Cell Line RENCA Is Mediated by MicroRNA miR-146a

Tumor-associated macrophages can potentially kill tumor cells via the high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS); however, tumor-associated macrophages actually support tumor growth, as they are skewed toward M2 activation, which is characterized by low amounts of NO production and is proangiogenic. We show that the mouse renal cell carcinoma cell line, RENCA, which, on stimulation, expresses high levels of iNOS mRNA, loses its ability to express the iNOS protein. This effect is mediated by the microRNA miR-146a, as inhibition of RENCA cells with anti-miR- 146a restores iNOS expression and NO production (4.8 ± 0.4 versus 0.3 ± 0.1 μmol/L in uninhibited cells, P < 0.001). In vivo, RENCA tumor cells do not stain for iNOS, while infiltrating tumor-associated macrophages showed intense staining, and both cell types expressed iNOS mRNA. Restoring iNOS protein expression in RENCA cells using anti-miR-146a increases macrophage-induced death of RENCA cells by 73% (P < 0.01) in vitro and prevents tumor growth in vivo. These results suggest that, in addition to NO production by macrophages, tumor cells must produce NO to induce their own deaths, and some tumor cells may use miR-146a to reduce or abolish endogenous NO production to escape macrophage-mediated cell death. Thus, inhibiting miR-146a may render these tumor cells susceptible to therapeutic strategies, such as adoptive transfer of M1-activated macrophages.

Suppression of tumor growth in renal cancer treatment with tumor vaccination after haploidentical bone marrow cell reconstitution

To investigate whether whole tumor cell vaccination strategies in combination with bone marrow transplantation (BMT) can stimulate graft-versus-tumor effect (GVT). Twenty-six BALB/c mice were randomly divided into 3 groups: BMT group (group A, n = 10), BMT + vaccination group (group B, n = 10), control group (group C, n = 6). (BALB/c × C57BL/6) F1 mice [CB6F1, H-2K(b/d)] were used as donors. BALB/c mice of group C were only inoculated with Renca cell (2.6 × 10(6)). Mice of group A and B were conditioned with 8 Gy irradiation, followed by infusion by bone marrow cell of CB6F1 mice on day 1, then inoculated with Renca cell (2.6 × 10(6)) on day 8. All mice of group B were immunized subcutaneous on the back with 5 × 10(5) irradiated Renca tumor cells on day 9 and day 16. All mice of group C were inoculated with Renca cell (2.6 × 10(6)) on day 8. In group A and B, all mice were analyzed by fluorescence activated cell sorter (FACS) on day 14, and 28 day after BMT. Mice were killed on day 32 after inoculation with tumor cell and collected blood sample. All tumors were taken out to be weighed and then fixed in 10% buffered formalin, embedded in paraffin, and cut into 5 µm slices. The slices were stained with HE and examined by TdT mediated-dUTP nick end labeling (TUNEL). Liver, skin, intestine, and spleen were biopsied for histopathological examination. The results of chimera showed that engraftments of group A, B were full donor chimerism, and the chimerism of those remained above 90% and preserved even after 28 days. The tumor weight, tumor volume increment in the group B was lower than group A and C (P < 0.05). The tumor suppressing rates of the group A and B were 54%, 60% respectively. The area ratio of tumor necrosis and apoptosis index (AI) of the tumor in the group B were higher than group A and C (P < 0.05). Graft-versus-host disease was not observed in each group. The mechanism of GVT after haploidentical allogeneic bone marrow transplantation with tumor vaccination may be the promotion of tumor necrosis and apoptosis.

Adoptively-Infused NK Cells Maintain Their Antitumor Effects in Vivo in the Presence of CyclosporineA (CSA)

Animal models show infusions of donor NK cells given after allogeneic HCT can prevent GVHD while simultaneously mediating a graft-vs-tumor effect. However, it is unclear whether adoptively infused NK cells can mediate these beneficial effects in the presence of CSA, which is commonly given after HCT to prevent GVHD. In this study, we analyzed the in vitro effects of pharmacological concentrations of CSA on NK cell phenotype, cell proliferation, and tumor cytotoxicity. We also evaluated in vivo whether CSA administration would reduce the anti-tumor effects of adoptively infused NK cells in tumor bearing mice. PBMCs collected from healthy donors were labeled with CFSE then were stimulated in vitro with IL-2 for 7 days in the presence or absence of CSA (1000ng/ml). CFSE proliferation assays on fresh PBMC showed CSA inhibited IL-2 stimulated CD3+ T-cell proliferation more than CD3−/CD56+ NK cell proliferation (mean percentage inhibition of proliferation 49.4% vs. 22.2% for T cells and NK cells respectively; p<0.05). CD3−/CD56+ NK cells were then isolated from PBMCs of healthy donors and expanded in vitro with irradiated EBV-LCL and IL-2 for 10 days. In contrast to T-cells, CSA only minimally inhibited IL-2 induced proliferation of expanded NK cells (mean 9.5% inhibition of proliferation by CFSE staining). T cells and NK cells were next isolated from PBMCs and stimulated with either OKT3, PMA-Ionomycin (PI), or IL-2. A [3H] TdR uptake assay showed T cell proliferation was inhibited at a substantially higher level by CSA (mean stimulation index for OKT3: 0.03, for PI: 0.35, for IL-2: 0.55) compared to that of expanded NK cells (mean stimulation index for IL-2: 0.82, p<0.05). Furthermore, an ELISA assay showed CSA treatment reduced IL-2 induced secretion of INF-g by T cells more than expanded NK cells (mean reduction in INF-g secretion in T cells of 94.7 % vs. 36.5 % in NK cells, p<0.05). Compared to controls, culturing in vitro expanded NK cells in CSA did not alter surface expression of the activating receptors NKp30, NKp42, and NKG2D but did reduce surface expression of NKp44 and TRAIL (mean reduction in surface expression 36% and 36.3% respectively). Cytotoxic granule release assessed by CD107a staining was inhibited by CSA in CD8+ melanoma specific T cells co-cultured with melanoma cells (mean 12.2 % inhibition) in contrast to NK cells co-cultured with K562 cells where CSA increased CD107a expression a mean 29.7% (p<0.05). Furthermore, at a 20:1 E:T ratio, 51Cr cytotoxity assays showed CSA did not reduce the cytotoxicity of in vitro expanded NK cells against renal cell carcinoma (RCC) cells (58% mean lysis) compared to NK cells cultured in control media (55% mean; p=n.s.). In contrast, melanoma specific T-cell killing of tumor targets was significantly lower in CTL cultures containing CSA compared to control media (38.0% vs. 50.2% respectively P<0.05). Next, we assessed the impact of CSA administration on the anti-tumor effects of adoptive NK cell infusions in tumor bearing animals where syngeneic NK cell infusions following bortezomib treatment have been shown to delay tumor progression and prolong survival. BALB/c mice injected with 100,000 luciferase transfected RENCA tumor cells i.v. received 3 weekly treatments with the combination of bortezomib (5ug/mouse i.v.) and 2×106 syngeneic NK cells i.v. with or without daily administration of CSA (15mg/kg sc). Bioluminescence imaging in controls that did not receive CSA showed tumor growth was slower and survival was prolonged in mice receiving adoptive NK cell infusions (median survival 53 days) compared to mice that did not receive NK cells (median survival 30.2 days; p<0.05). CSA administration did not impair the anti-tumor effects of adoptive NK cell infusions; mice receiving CSA and adoptive NK cell infusions had similar tumor growth and survival as recipients of NK cells without CSA (median survival 47 vs. 53 days; p=n.s.). These results show that CSA is significantly more immunosuppressive to T cells compared to NK cells and provide evidence that the anti-tumor effects of adoptively infused in vitro expanded NK cells are maintained even in the presence CSA.

Antitumor effect of SCV-07 in murine lung and renal cancer models

14012 Background: SCV-07 (γ-Glu-Trp) is an immunomodulatory peptide with demonstrated antitumor effects in the murine B16 melanoma model. Herein, the antitumor effects of SCV-07 were evaluated in mice bearing Lewis lung carcinoma (LLC) or renal carcinoma (RenCa) cells. The effect on growth of cultured LLC and RenCa cells was also evaluated to address whether the antitumor effect of SCV-07 is mediated by cytotoxicity. Methods: LLC and RenCa cells were implanted subcutaneously (sc) into the right axillary area of C57BL/6 and Balb/C mice, respectively. SCV-07 was sc administered (at a site different from tumor implantation) once daily at doses of 10 or 20 mg/kg (for LLC) and 5 or 10 mg/kg (for RenCa) for 14 consecutive days. Cisplatin (CDDP, given at 6 mg/kg on days 1, 6, and 12) and cyclophosphamide (CTX, given at 40 mg/kg every other day) were positive controls for LLC and RenCa tumors, respectively. The animals were euthanized on the termination day (day 16 in LLC model, day 17 in RenCa model) and tumors excised and weighed. For the evaluation of cytotoxic effect, LLC or RenCa cells were cultured in 96-well plates with SCV-07 at various concentrations, ranging up to 100 μg/mL, representing the plasma concentrations after effective doses in the mouse models. After 48- (LLC) or 72- (RenCa) hour incubation, inhibitory effects on cell proliferation were determined by the MTT method. Results: In both LLC and RenCa cancer models, SCV-07 treatment led to significant inhibition of tumor growth compared to vehicle control. On the day of study termination, decreases of 16.84% (p<0.05) and 37.45% (p<0.01) in LLC tumor weight were seen after SCV-07 treatment at 10 and 20 mg/kg, respectively, and decreases of 30.02% (p<0.01) and 28.33% (p<0.01) in RenCa tumor weight seen at 5 and 10 mg/kg, respectively. Growth inhibition in the controls were 77.35% (p<0.01) for LLC and 90.12% (p<0.01) for RenCa. In the cultured LLC or RenCa cells, SCV-07 did not inhibit cell proliferation at any tested concentration. Conclusions: Administration of SCV-07 once daily for 14 consecutive days via the sc route is effective against growth of LLC and RenCa tumors. SCV-07 does not induce in vitro cytotoxic effects in cultured LLC and RenCa tumor cells, indicating the antitumor effect of SCV-07 is related to its immunomodulatory actions, rather than cytotoxicity. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration SciClone Pharmaceuticals, Inc. SciClone Pharmaceuticals, Inc SciClone Pharmaceuticals, Inc.

Treatment of Transforming Growth Factor-Beta-Insensitive Mouse Renca Tumor by Transforming Growth Factor-Beta Elimination

The mouse renal cell carcinoma line, Renca, is insensitive to transforming growth factor-beta (TGF-beta) in vitro. The present study was conducted to determine whether removal of TGF-beta from these tumor cells would inhibit tumor progression in vivo. TGF-beta elimination was accomplished either by administration of neutralizing TGF-beta antibody into mice receiving intravenous injection of Renca tumor cells or infection of TGF-beta antisense expression vector into these tumor cells before subcutaneous injection into recipient mice. Although a low dose of TGF-beta antibody (5 mg/kg every 3 days) was without any effect, a high dose of TGF-beta antibody (50 mg/kg every 3 days), administered to recipient mice, resulted in a significant reduction in lung metastasis and was accompanied by increased apoptosis in the tumor cells. When the tumor cells were transfected with a TGF-beta1 antisense expressing vector, a significant reduction occurred in the tumor incidence, as well as the tumor burden. However, in nude mice, cells with reduced TGF-beta1 production grew almost as well as did the unmodified Renca cells, suggesting that the host's immune system might play an antitumor role. These results indicate that progression of Renca tumor can be inhibited by eliminating TGF-beta from the tumor cells. Our results also suggest that, although insensitive to TGF-beta under in vitro conditions, Renca tumors could be inhibited by TGF-beta removal through the systemic host environment.

Radiation-induced apoptosis along with local and systemic cytokine elaboration is associated with DC plus radiotherapy-mediated renal cell tumor regression

Utilizing melanoma and sarcoma tumor models syngeneic to C57BL/6 mice, we previously reported the antitumor effects of intratumoral (i.t.) administration of dendritic cells (DC) combined with localized radiotherapy (RT). However, the mechanisms underlying the augmented therapeutic effects have yet to be fully defined. Using the BALB/c host, we explored in this study the capacity of RT to augment the therapeutic efficacy of DC in the syngeneic renal cell cancer, Renca. I.t. DC administration combined with RT inhibited tumor growth in a synergistic manner. This extends our previous findings using a different host strain and two histologically distinct tumor models. More importantly, we provide evidence in this report that RT induced significant apoptosis and necrosis in Renca tumor cells, which involved down-regulated expression of Bcl-2 and a concurrent up-regulated expression of Bax. We also found significantly elevated expression of TNFα in RT plus DC-treated Renca tumors. Furthermore, splenocytes isolated from DC plus RT-treated mice elaborated higher levels of IL-2, IL-4, IFNγ and IgG, IgM in response to tumor cells compared with splenocytes from monotherapy-treated hosts. These data support the conclusion that radiotherapy enhanced DC vaccination by inducing tumor cell apoptosis in BABL/c host, and the significantly augmented therapeutic efficacy by RT + DC treatment was associated with an increased local production of TNFα as well as an amplified systemic antitumor responses conferred by the combined therapy. I.t. DC administration in concert with localized RT may represent a promising novel regimen for human cancer therapy.

Adoptive Infusion of Alloreactive Donor NK Cells Reduces GVHD, Mediates Anti-Tumor Effects and Prolongs Survival in Recipients of MHC-Matched Hematopoietic Cell Transplantation.

Alloreactive natural killer (NK) cells with killer immunoglobulin-like receptor (KIR) ligand incompatibility have been implicated to reduce graft-vs-host disease (GVHD) and leukemia relapse in humans undergoing MHC-mismatched T-cell depleted allogeneic hematopoietic cell transplantation (HCT). Analogous to human NK KIR, murine NK cells express subgroups of MHC-specific receptors of the C-type lectin superfamily, termed Ly49, that regulate their function. In MHC-mismatched HCT, the infusion of alloreactive Ly49-mismatched NK cells decreases the incidence of GVHD. We investigated if an infusion of alloreactive NK cells would reduce GVHD and further mediate anti-tumor effects in mice undergoing MHC-matched allogeneic HCT. Balb/c mice were injected intravenously with 105 RENCA tumor cells. Ten days following tumor injection, mice were irradiated (950cGy) and transplanted with 15×106 and 8×106 splenocytes and bone-marrow cells respectively from MHC-matched B10.d2 donors. Five days following transplantation, recipients were given a single infusion of either 0.5–1×106 Ly49 ligand-matched (Ly49G2; non-alloreactive to Balb/c) or Ly49 ligand-mismatched (Ly49C; alloreactive to Balb/c) donor NK cells. Recipients of Ly49 ligand-mismatched NK cells had a significant reduction in the incidence of GVHD (p<0.01-figure), and prolonged survival (median 84 vs 39 days; p<0.01-figure) compared to HCT recipients not receiving NK cells. Recipients of Ly49 ligand-matched NK cells had the same incidence of GVHD and similar survival compared to controls not receiving NK cells. Pulmonary tumor burden was significantly (p<0.01) lower in recipients of Ly49-mismatched or Ly49-matched NK cells compared to mice not receiving NK cells. These data provide in vivo evidence that a single infusion of alloreactive donor NK cells can prolong survival by both reducing GVHD and mediating anti-tumor effects following MHC-matched allogeneic HCT. [Display omitted]

THE EFFECT OF PNEUMOPERITONEUM ON DISSEMINATION AND SCAR IMPLANTATION OF INTRA-ABDOMINAL TUMOR CELLS

The role of laparoscopy for the treatment of cancer remains controversial, and a particular concern is port site metastases after laparoscopic surgery. Since laparoscopy is being performed with increasing frequency, the question arises as to whether it is a safe oncological procedure. After intraperitoneal inoculation of renal cell carcinoma cells in a mouse model, we compare abdominal wall scar implantation following laparoscopic trocar insertion and pneumoperitoneum with standard laparotomy, and examine the effects on tumor dissemination in the peritoneal cavity. Following intra-abdominal RENCA cell inoculation, Balb/c mice were randomized into group 1-20 mice that underwent carbon dioxide pneumoperitoneum and telescope trocar insertion, group 2-20 subjected to laparotomy and group 3-10 anesthetized only. All animals were sacrificed 2 weeks after inoculation, and abdominal wall metastases and intraperitoneal tumor distribution were evaluated. Overall, intra-abdominal implantation of inoculated RENCA tumor cells was detected in 15 of 20 animals (75%) in group 1, 14 of 20 (70%) in group 2 and 10 of 10 (100%) in group 3. Wound metastases developed in 46.7% of the mice in group 1 and 50% in group 2. There was no difference among the groups in the pattern of intraperitoneal tumor implants and scar seeding incidence. Pneumoperitoneum does not facilitate port site metastases.

Cytokine depot formulations as adjuvants for tumor vaccines. I. Liposome-encapsulated IL-2 as a depot formulation.

In an attempt to mimic cytokine gene-transfected tumor cells and to develop an alternative approach to cancer immunotherapy, the authors vaccinated mice with mixtures of inactivated tumor cells and cytokine-containing depots. The RenCa mouse renal carcinoma and the B16 mouse melanoma were used as animal tumor models, with interleukin-2 (IL-2) as a cytokine and liposomes as a depot form. The results obtained show that vaccines consisting of mixtures of irradiated tumor cells and cytokine-containing liposomes can be used as highly effective tumor vaccines. These vaccines are very easy to prepare and, in contrast to vaccines consisting of cytokine gene transfected tumor cells, their composition (cell dosage, cytokine dosage) can be easily varied. Vaccination efficiency depended on (a) on the immunogenicity of the tumor cells: RenCa tumor cells are more immunogenic than B16 melanoma cells; (b) vaccination frequency: a single vaccination with irradiated tumor cells and 10 micrograms of IL-2 in liposome-encapsulated form was sufficient to induce lasting protective immunity against the RenCa tumor, whereas several (four to six) vaccinations in weekly intervals were needed to obtain a similar degree of protective immunity to the B16 melanoma; and (c) the dose of the cytokine encapsulated in the admixed liposome depots: immunity to the tumors could be induced only within a narrow cytokine-dose range ("IL-2-dose window"). The results obtained indicate that, because of the easiness of preparation and handling, vaccine formulations consisting of irradiated tumor cells and IL-2 in depot formulations are candidates for tumor vaccines for the treatment of tumor patients.

Molecular Mechanisms of Immune-Mediated Lysis of Murine Renal Cancer: Differential Contributions of Perforin-Dependent Versus Fas-Mediated Pathways in Lysis by NK and T Cells

Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-gamma or TNF-alpha, and the combination of IFN-gamma + TNF-alpha synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-gamma and TNF-alpha are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-gamma and TNF-alpha.

Interferon-gamma-inducing factor elicits antitumor immunity in association with interferon-gamma production.

Interferon-gamma-inducing factor (IGIF) is a novel cytokine that stimulates T-cell proliferation, augments natural killer (NK) cell lytic activity, and induces interferon-gamma (IFN-gamma) production in established type 1 T-helper (Th1) cells in the presence of anti-CD3 antibody. The in vitro induction of IFN-gamma by recombinant murine IGIF in these cells was more potent than that induced by murine interleukin-12 (IL-12) and occurred apparently independent of murine IL-12. Here we report that subcutaneous injection into mice of tumor cells transfected with murine IGIF complementary DNA (cDNA) resulted in > or = 10-fold increase of mitogen-stimulated IFN-gamma production in cultured splenocytes. In addition, IGIF-transfected Renca and K1735 tumor cells can be rejected in vivo. The IGIF antitumor effect was abrogated in mice that were sublethally irradiated or depleted of both CD4+ and CD8+ T cells but not in mice depleted of either subpopulation alone. The antitumor effect mediated by IGIF appears to be dependent on IFN-gamma production, because in vivo neutralization of IFN-gamma was accompanied by growth of IGIF-transfected tumors in 100% of the animals. Taken together, our results show that murine IGIF can elicit T-cell-dependent antitumor immunity associated with IFN-gamma induction.