Enhanced cytolytic activity of tumor infiltrating lymphocytes (TILs) derived from an ICAM-1 transfected tumor in a murine model.

BACKGROUND: Tumors transfected with intercellular adhesion molecule-1 (ICAM-1), a known activator of resting T-cells, show an increased response to adoptive immunotherapy in vivo. This salutary effect may be due to increased sensitivity of the transfected tumor cells to cell-mediated cytotoxicity or an increased activity of the effector cells in the presence of increased amounts of ICAM-1. MATERIALS AND METHODS: MCA105 fibrosarcoma cells were transfected with the gene for ICAM-1, and a clone (Cl149) demonstrating significantly increased expression of ICAM-1 by fluorescent-activated cell sorter (FACS) and enzyme-linked immunosorbent assay (ELISA) compared to parental tumor was selected and cultured. Tumor infiltrating lymphocytes (TILs) were cultured in vitro from MCA105 and Cl149 tumors. K562 tumor cells were used as controls. RESULTS: TILs derived from MCA105 tumors lysed MCA105 (32% at 40:1) and Cl149 (52% at 40:1) target cells but not K562 (3%) demonstrating TIL specificity. TILs derived from Cl149 showed increased lysis of both target cells tested: MCA105 (62% at 40:1, P < 0.05) and Cl149 (98%) compared to lysis of the same target cells by MCA105 TILs as well as being specific (K562, 1%). CONCLUSIONS: These studies demonstrate that increased expression of ICAM-1 by a tumor cell results in increased lysis by TILs derived from either a tumor with enhanced ICAM-1 expression or a parental tumor, compared to the lysis of parental tumor target cells. In addition, TILs derived from a tumor with enhanced expression of ICAM-1 have significantly increased antitumor efficacy compared to TILs from the parental tumor, suggesting a possible mechanism for previously observed in vivo antitumor effects. These results suggest a new strategy for improving the efficacy of adoptive immunotherapy, by using lymphocytes derived from genetically altered tumors. The study of lymphocytes from genetically modified tumor cells may enable the elucidation of properties of various molecules believed important in cellular cytotoxicity.

Expression of ICAM-1 enhances in vivo lymphocyte adhesion in a murine fibrosarcoma.

BACKGROUND AND OBJECTIVES: ICAM-1 is essential for lymphocyte-endothelial cell interactions. We have demonstrated that increased expression of ICAM-1 in tumors results in an enhanced response to adoptive immunotherapy. We undertook this study to determine whether increased expression of ICAM-1 results in increased lymphocyte adhesion in vivo. METHODS: Parental MCA-105 tumor cells were cotransfected with ICAM-1 and the NeoR plasmid. A neomycin resistant clone (Cl149) was selected and increased expression of ICAM-1 confirmed by FACS analysis. Tumor fragments (MCA-105 or Cl149) were placed in a dorsal skin-fold chamber on day 0 in C57BL/6 mice. Lymphocytes were fluorescently labeled using 0.5% acridine orange and activity recorded on videotape at 700x magnification. Lymphocyte activity was quantitated over 30 second intervals in postcapillary venules as either passing or rolling/sticking (R/S). The % R/S was calculated for each category and evaluated using chi 2 analysis. RESULTS: Whereas 38% of lymphocytes were classified as R/S in normal tissue, 32% were classified as R/S (P > .05) in the MCA-105 tumor. However, in the ICAM-1 transfected CL149, there was significantly greater R/S at 53% (P < .05). CONCLUSIONS: These data demonstrate increased lymphocyte adhesion in tumors with enhanced expression of ICAM-1 by direct in vivo observations and may partially explain the salutary effect of increased ICAM-1 expression on adoptive immunotherapy. This suggests the possible application of adhesion molecule expression in the cellular therapy of cancer.

ICAM-1 increases in vitro adhesion and cytotoxicity in a murine fibrosarcoma.

Cellular adhesion and specific cytotoxicity are two essential components for the successful cellular therapy of cancer. Intercellular adhesion molecule-1 (ICAM-1) is an essential participant in lymphocyte-endothelial cell adhesion and may also play a role in lymphocyte-mediated cytotoxicity. To study the effect of ICAM-1 on adhesion and cytotoxicity in vitro, MCA-105 tumor cells were cotransfected with ICAM-1 and the gene for neomycin resistance (NeoR). Two clones (Clones 81 and 149) with confirmed enhancement of ICAM-1 expression were selected. Studies were performed examining adhesion of lymphocytes to HUVECs, MCA-105, Clone 81 or Clone 149 alone, or combinations of the three tumor cell lines with HUVECs. Peripheral blood lymphocytes labeled with 51Cr were used and adhesion was determined by counting in a gamma-counter after rinsing away nonadherent cells. Cytotoxicity was performed using 51Cr-labeled MCA-105, NeoR, Clone 81, and Clone 149 target cells. LAK cells cultured from splenocytes of normal mice were used as the effector cells and a chromium release assay was performed. Adhesion data showed significant increases in adhesion (P < 0.05) for Clones 81 and 149 compared to MCA-105. However, the combination of HUVECs and tumor cells to mimic the in vivo condition had a variable effect on adhesion compared to tumor cells alone. Cytotoxicity experiments demonstrated that Clone 149 was significantly (P < 0.05) more susceptible to lysis by normal LAK cells compared to MCA-105, NeoR, and Clone 81. These results suggest that increased ICAM-1 expression enhances the susceptibility of tumor cells to lysis by LAK cells.

Tumor necrosis factor alpha mediates the antitumor effect of combined interleukin-2 and whole body hyperthermia.

Combined whole body hyperthermia (WBHT) and interleukin-2 (IL-2) significantly reduces the growth rate of subcutaneous 10 day MCA-105 tumors in C57BL/6 mice, but not in 3-day tumors. Others have shown that macroscopic tumors show reduced growth with tumor necrosis factor alpha (TNF alpha) therapy compared with microscopic tumors. We sought to determine if the antitumor effect of combined WBHT+IL-2 is mediated by TNF alpha. After inducing MCA-105 sarcoma in the right hind limb on Day 0, C57BL/6 mice were randomized to treatment groups (six each) on Day 10: control, WBHT alone, IL-2 alone, and WBHT+IL-2. Pooled serum was assessed by ELISA for TNF alpha level: control (350 pg/ml), WBHT (250 pg/ml), IL-2 alone (>2450 pg/ml), and WBHT+IL-2 (>2450 pg/ml). Using the same tumor model, animals were treated in the following groups: control, WBHT+IL-2, anti-TNF alpha Ab alone, and WBHT+IL-2+Ab. Mice in the control, Ab alone, and WBHT+IL-2+Ab groups had similar tumor growth rates (P > 0.05). However, the growth rate for WBHT+IL-2 was significantly lower (P < 0.05) compared to the other three groups. These data demonstrate significantly increased TNF alpha levels in mice treated with combined therapy and abrogation of the antitumor effect of WBHT+IL-2 therapy by the addition of anti-TNF alpha Ab with a tumor growth rate similar to that observed in untreated mice, suggesting that the antitumor effect of WBHT+IL-2 is mediated at least in part by TNF alpha.

Transfection of a murine fibrosarcoma with intercellular adhesion molecule-1 enhances the response to adoptive immunotherapy.

BACKGROUND: Increasing the ability of antitumor effector cells to leave the vasculature and gain access to tumor cells may improve therapeutic efficacy. We undertook this study to determine whether increased expression of intercellular adhesion of molecular-1 (ICAM-1) by gene transfection would result in an improved response to adoptive immunotherapy in vivo. METHODS: C57BL/6 mice received 1 x 10(6) tumor cells on day 0. Tumor cells examined were MCA-105 (parental), NeoR (MCA-105 transfected with the neomycin resistance gene), or Clones 81 or 149 (MCA-105 cotransfected with NeoR and the gene for ICAM-1 and highly express ICAM-1). Animals were treated by use of no treatment, interleukin-2 alone (days 10 through 14), hyperthermia alone (days 10 and 13), or interleukin-2 + hyperthermia, and tumor growth was reported as a ratio to size on day 10. In vitro cytotoxicity was assayed by using murine lymphokine-activated killer cells. RESULTS: Tumors transfected with ICAM-1 and treated with hyperthermia + immunotherapy grew significantly (p < 0.05) slower (mean, 0.78 +/- 0.16 on day 19) than parental tumor (size, 1.35 +/- 0.22) or tumor cells transfected with NeoR alone (1.21 +/- 0.19). Tumors containing both MCA-105 and Clone 81 treated with hyperthermia + immunotherapy grew significantly slower (1.58 +/- 0.49 on day 19, p < 0.05) than untreated Clone 81 (2.38 +/- 0.46) or treated MCA-105 (2.49 +- 0.29) but more rapidly than treated Clone 81 (1.18 +/- 0.08), suggesting a paracrine efect for ICAM-1. CONCLUSIONS: These findings show that increased expression of ICAM-1 by tumor cells results in a significant increase in antitumor efficacy of combined interleukin-2 and hyperthermia in a murine model. Although the mechanism has yet to be elucidated, modulation of cellular adhesion may play a role in the therapeutic efficacy of cellular immunotherapy.

Enhanced expression of ICAM-1 in a murine fibrosarcoma reduces tumor growth rate.

Intercellular adhesion molecule-1 (ICAM-1) plays an essential role in lymphocyte adhesion to endothelium and migration across endothelial cell barriers. We undertook this study to determine the growth of a murine fibrosarcoma transfected with the ICAM-1 gene. MCA-105 tumor cells were cotransfected with ICAM-1 and the plasmid for neomycin resistance (NeoR). Selected G418-resistant clones were expanded and cell surface ICAM-1 expression was verified using a fluorescence-activated cell sorter. Integration of the ICAM-1 gene and ICAM-1 mRNA expression were verified by Southern and Northern blot hybridization analysis, respectively. C57BL/6 mice were divided into five groups (six animals/group): Control, NeoR only, ICAM-1 (low expressing, Clone 25), ICAM-1 (high expressing, Clone 81), and a 1:1 mixture of NeoR:Clone 81; animals received 1 x 10(6) cells on Day 0 and tumor measurements began on Day 7 and were measured in mm2. At 19 days, tumors from cell lines expressing ICAM-1 were significantly (P < .05) smaller than both the parental cell line and tumor-containing NeoR only (364 mm2 vs 466 mm2 and 527 mm2, respectively). This decrease in tumor growth may be a result of increased lymphocyte migration or increased anti-tumor cytotoxicity by infiltrating lymphocytes. The results from the mixed tumor experiment suggest a possible paracrine effect by cells expressing ICAM-1. Studies are currently under way to investigate the effect of immunotherapy on tumors derived from ICAM-1-cloned transfectants.

Combined local hyperthermia and immunotherapy treatment of an experimental subcutaneous murine melanoma.

Immunotherapy (IT) has become an accepted therapeutic modality for a limited number of tumor types. One of the limiting factors in the use of interleukin-2 (IL-2) has been dose-related toxicity. We undertook these studies to study the effects of combined therapy on a murine melanoma. The B16 melanoma was implanted in the right hindlimb of C57BL/6 mice and therapy begun on day 3 (microscopic tumor model) or day 10 (macroscopic tumor model). Animals were divided into four groups: No therapy, local hyperthermia (HT) alone (45 degrees C x 15 minutes on days 3 and 6 or days 10 and 13), HT+IL-2 at 300,000 IU ip tid, and HT+IL-2 at 600,000 IU ip tid. We have shown in multiple previous experiments that IL-2 alone at these doses has no effect on tumor growth; these groups were omitted. In the microscopic model, tumors in the no treatment group were an average of 400 mm2. Animals treated with HT alone had a mean tumor size of 300 mm2. However, tumors in animals receiving both therapeutic modalities measured a mean of 100 mm2 (300,000 IU IL-2 ip tid) and 80 mm2 (600,000 IU IL-2 ip tid). In the macroscopic tumor model, tumors in animals receiving no treatment were an average of 7.5 times larger than on day 10, in animals receiving HT alone were an average of 5 times larger, animals receiving IL-2 were 2.95 times larger (both dose levels). These results show that combined IT+HT therapy resulted in significantly (P < .05) reduced growth with both microscopic and macroscopic tumors compared to HT alone or no therapy in a murine subcutaneous melanoma model using doses significantly lower than those usually needed to observe a therapeutic response with IL-2 used alone. This study further supports the use of this combined modality approach in patients with advanced malignancies.

Hyperthermia increases intercellular adhesion molecule-1 expression and lymphocyte adhesion to endothelial cells.

BACKGROUND: We observed that the synergistic combination of immunotherapy and whole-body hyperthermia is active against large well-vascularized tumors but not microscopic tumors, and we therefore hypothesized that hyperthermia may act on lymphocyte-endothelial cell interactions. We undertook these studies to evaluate the effect of hyperthermia on lymphocyte-endothelial cell adhesion. METHODS: Cultured human umbilical vein endothelial cells (HUVEC) and normal peripheral blood lymphocytes were used. HUVEC were cultured to confluence. Treatment groups included control, hyperthermia alone (41 degrees C for 2 hours), interferon-gamma (IFN-gamma) alone, or hyperthermia + interferon-gamma. 51Cr-labeled peripheral blood lymphocytes were allowed to adhere to treated HUVEC, and nonadhering cells were washed away. Adherent cells were lysed and counted in a gamma-counter, calculating an adhesion index compared to controls. The experiment was then conducted with the addition of anti-intercellular adhesion molecule (ICAM) antibody. Cell surface ICAM expression was determined with double monoclonal antibody staining and fluorescence-activated cell sorter analysis, and soluble ICAM secretion was determined with enzyme-linked immunosorbent assay in each group. RESULTS: In a representative experiment, interferon-gamma increased adhesion by a factor of 1.81 (p < 0.05) compared with control and hyperthermia by 1.38 (p < 0.05) and combined treatment by a factor of 2.43 (p < 0.05). Anti-ICAM antibody abrogated the increased adhesion caused by hyperthermia but did not abrogate the effect of interferon-gamma. Although only 26% of control cells expressed ICAM-1 on the cell surface, interferon-gamma increased expression to 53% (p < 0.05), hyperthermia increased expression to 38% (p < 0.05), and combined treatment increased expression to 61% (p < 0.05). Soluble ICAM-1 was not increased 12 hours after treatment, but by 24 hours significant (p < 0.05) differences (control 0.262 ng/ml, IFN alone 1.50, hyperthermia alone 1.57, and combined 2.71) were noted. CONCLUSIONS: These results suggest that hyperthermia has a significant effect on lymphocyte adhesion to endothelial cells, at least in part mediated by ICAM-1. Cell surface ICAM-1 is increased at 12 hours, and soluble ICAM-1 is increased at 24 hours. These data suggest that hyperthermia may function by increasing lymphocyte adhesion, providing another locus of action to improve clinical results with immunotherapy.

Local hyperthermia abrogates the anti-immunotherapeutic effect of interleukin-8.

The therapeutic efficacy of cellular immunotherapy depends not only on the anti-tumor activity of the administered effector cells but also on their ability to gain access to the tumor by extravasation. Although interleukin-8 (IL-8) has been shown to prevent the vascular leak associated with IL-2, it also abrogates the anti-tumor effect of IL-2. We undertook these studies to determine if LHT could abrogate the anti-immunotherapeutic effect of IL-8, since IL-8 inhibits leukocyte adhesion. C57BL/6 mice were divided into four groups of six animals each after induction of MCA-105 fibrosarcoma inoculated into the right hindlimb on day 0 and were treated beginning on day 3 as follows: no therapy, IL-2 alone (1.02 x 10(6) IU ip tid on days 3-7), IL-2 + IL-8 (9.6 ng ip tid on days 3-7), and IL-2 + IL-8 + LHT (45C x 15 min on days 3, 5, and 7). Tumor growth was measured on days 10-21 and analyzed by Wilcoxon rank-sum analysis. IL-2 reduced tumor growth significantly (P < .05) compared to no therapy, and IL-8 abrogated the anti-tumor effect of IL-2, resulting in tumor growth in animals receiving IL-2 + IL-8, similar to the no therapy group (P > .05). However, addition of LHT to IL-2 + IL-8 resulted in significantly (P < .05) less tumor growth than no therapy or IL-2 + IL-8. Activity of the mice was scored as an indicator of systemic toxicity. We found that IL-8 was able to increase the activity (P = .07) of the mice when administered with IL-2. These results suggest that IL-8 may protect the tumor-bearing animal from the systemic toxicity of IL-2, while LHT abrogates the anti-immunotherapeutic effect of IL-8.

Target cell membrane modification and susceptibility to lymphokine-activated killer cell-mediated lysis. II. Sodium periodate has a differential effect on the lysis of human and murine tumor cells.

Little is known about the mechanism by which lymphokine-activated killer (LAK) cells and natural killer (NK) cells recognize and lyse target cells. We undertook this study to investigate the effects of target cell membrane modification by sodium periodate (PI) on the lysis of tumor cells by human and murine LAK cells (HLAK, MLAK) and NK cells (HNK, MNK). LAK and NK cells were generated and used as effector cells in 4-hr 51Cr release assays against a variety of PI-treated and untreated target cells. PI significantly increased the MLAK cell-mediated lysis of YAC and MH134 tumor cells, but had no effect on the MNK lysis of YAC or MH134 tumor cells. PI had no effect on the HLAK or HNK lysis of YAC or MH134 tumor cells. PI had no effect on the MLAK or MNK lysis of K562 or U937 human tumor cells. Similarly, PI had no effect on the HLAK lysis of K562, but did significantly increase the HNK lysis of K562. PI increased both the HLAK and HNK lysis of U937. Cold target inhibition studies revealed that PI-treated YAC cells inhibited the MLAK cell-mediated lysis of untreated YAC cells. The variable effects of PI on LAK cell- and NK cell-mediated lysis support the hypothesis that LAK cells are a distinct population of cells rather than a subpopulation of NK cells on the basis of mechanism of target cell recognition. The results of the cold target inhibition study suggest that PI-treated tumor cells are identified by effector cells in a manner similar to that of untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined hyperthermia and immunotherapy treatment of multiple pulmonary metastases in mice.

The combination of immunotherapy and hyperthermia results in a greater reduction in tumour growth compared to either therapy used alone in a murine subcutaneous tumour model. To evaluate this combination further we tested it in a murine pulmonary metastasis model. Mice were given 5 x 10(5) MCA-105 sarcoma cells on day 0 intravenously resulting in the formation of pulmonary metastases. Mice were treated with local hyperthermia to the left hemithorax with a transcutaneous microwave applicator or with whole-body hyperthermia to 41 degrees C for 30 min on days 3 and 6. Immunotherapy included 5 x 10(7) syngeneic LAK cells administered on days 3 and 6 and interleukin-2 given intraperitoneally three times daily on days 3-7. Animals were killed on day 12 and pulmonary nodules enumerated. While the addition of whole-body hyperthermia to immunotherapy had no significant effect on tumour growth, the combination of local hyperthermia and immunotherapy significantly decreased the number of pulmonary nodules by 94% compared to controls in combined experiments. The mechanism of this beneficial effect may be related to increased trafficking of immune active cells to the tumour-bearing site, an increase in the sensitivity of tumour cells to lysis, or perhaps a local release of cytokines induced by hyperthermia. This study established the efficacy of combined immunotherapy and hyperthermia for the treatment of visceral metastases and provides impetus for the initiation of clinical trials.

Immunotherapy and whole-body hyperthermia as combined modality treatment of a subcutaneous murine sarcoma.

Interleukin-2 and hyperthermia have been used individually to treat a variety of tumors in both experimental and human trials. Combined adoptive immunotherapy and hyperthermia is an exciting new line of investigation. Previous work in our laboratory has shown that combined local hyperthermia and rIL-2 therapy can significantly decrease the rate of tumor growth. In this study, we investigated the effect of combined whole-body hyperthermia (WBHT) and rIL-2 on the growth of subcutaneous MCA-105 murine tumors in C57BL/6 mice. Treatment of both microscopic (day 3) and macroscopic (day 10) tumors was evaluated. In the treatment of microscopic tumors, animals received either no treatment; rIL-2 (3 x 10(5) IU ip tid) on days 3-7; plus WBHT (41 degrees C for 30 min) on days 3, 5, and 7; or WBHT only days 3, 5, and 7. In treating macroscopic tumors, animals received either no treatment; rIL-2 on days 10-14; plus WBHT on days 10, 12, and 14; or WBHT only on days 10, 12, and 14. While combined treatment and WBHT alone had no significant effect on the growth of microscopic tumors, combined IL-2 and WBHT significantly reduced the rate of tumor growth of macroscopic tumors. These results suggest that the tumor microenvironment plays a critical role in combined WBHT and rIL-2 therapy, and may be due to effects of WBHT on the tumor vasculature.

Lymphocyte migration to tumors after hyperthermia and immunotherapy.

We have previously shown that the combination of immunotherapy with interleukin 2 (IL-2) and local hyperthermia (LHT) abrogates the growth of murine subcutaneous tumors significantly more than either modality alone. This study was undertaken to investigate whether the beneficial effect of combined modality therapy could be attributed to increased trafficking of effector cells to the tumor. After inducing MCA-105 sarcomas in the hindlimbs of C57BL/6 mice, animals were given no therapy, LHT, IL-2, or IL-2 + LHT followed by an iv injection of 51Cr-labeled syngeneic splenocytes or LAK cells. Select organs and the tumor-bearing extremity were counted in a gamma counter. IL-2 or LHT alone did not affect lymphocyte migration, while IL-2 + LHT significantly decreased trafficking (P less than 0.001) to the tumor. LAK cells showed increased migration to the tumor site compared to splenocytes in all treatment groups (P less than 0.02). IL-2 caused increased migration of LAK cells but not splenocytes to the lung; this was not affected by LHT. LAK cell trafficking to the spleen was decreased by IL-2 and IL-2 + LHT, while splenocyte migration was decreased in the LHT and combined treatment groups. LHT and IL-2 had no effect on trafficking of either effector cell type to liver or kidney. These results show that the beneficial effect of combined modality therapy may not be due to increased trafficking of lymphocytes to the tumor area. In addition, LAK cells traffic more to subcutaneous tumors than splenocytes, and this cannot be explained by the differential trafficking to other organs. The results of this study will be important in the planning of future experiments with combined adoptive immunotherapy and hyperthermia.