Alicia I. Bravo

Targeted inhibition of galectin-1 gene expression in tumor cells results in heightened T cell-mediated rejection: A potential mechanism of tumor-immune privilege

Despite the existence of tumor-specific immune cells, most tumors have devised strategies to avoid immune attack. We demonstrate here that galectin-1 (Gal-1), a negative regulator of T cell activation and survival, plays a pivotal role in promoting escape from T cell-dependent immunity, thus conferring immune privilege to tumor cells. Blockade of immunosuppressive Gal-1 in vivo promotes tumor rejection and stimulates the generation of a tumor-specific T cell-mediated response in syngeneic mice, which are then able to resist subsequent challenge with wild-type Gal-1-sufficient tumors. Our data indicate that Gal-1 signaling in activated T cells constitutes an important mechanism of tumor-immune escape and that blockade of this inhibitory signal can allow for and potentiate effective immune responses against tumor cells, with profound implications for cancer immunotherapy.

Dendritic Cells Charged with Apoptotic Tumor Cells Induce Long-Lived Protective CD4+ and CD8+ T Cell Immunity against B16 Melanoma

Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4+ and CD8+ T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to ∼0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4+ and CD8+ T cells were efficiently primed in vaccinated animals, as evidenced by IFN-γ secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8+ T cells in vitro, and cytolytic activity against tyrosinase-related protein 2180–188-pulsed target cells was observed in vivo. When either CD4+ or CD8+ T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.

Secreted protein acidic and rich in cysteine produced by human melanoma cells modulates polymorphonuclear leukocyte recruitment and antitumor cytotoxic capacity.

The expression of secreted protein acidic and rich in cysteine (SPARC) has been associated with the malignant progression of different types of human cancer. SPARC was associated with tumor cell capacity to migrate and invade, although its precise role in tumor progression is still elusive. In the present study, we show that SPARC produced by melanoma cells modulates the antitumor activity of polymorphonuclear leukocytes (PMN). Administration to nude mice of human melanoma cells in which SPARC expression was transiently or stably knocked down by antisense RNA (SPARC-sup cells) promoted PMN recruitment and obliterated tumor growth even when SPARC-sup cells accounted for only 10% of injected malignant cells. In addition, SPARC-sup cells stimulated the in vitro migration and triggered the antimelanoma cytotoxic capacity of human PMN, an effect that was reverted in the presence of SPARC purified from melanoma cells or by reexpressing SPARC in SPARC-sup cells. Leukotrienes, interleukin 8, and growth-related oncogene, in combination with Fas ligand and interleukin 1, mediated SPARC effects. These data indicate that SPARC plays an essential role in tumor evasion from immune surveillance through the inhibition of the antitumor PMN activity.

A phase I study of an allogeneic cell vaccine (VACCIMEL) with GM-CSF in melanoma patients.

We investigated whether recombinant human granulocyte-monocyte-colony-stimulating factor (rhGM-CSF) increased the immunogenicity of VACCIMEL, a vaccine consisting of 3 irradiated allogeneic melanoma cell lines. A phase I clinical trial was performed on 20 melanoma patients in stages IIB (n=2), III (n=10), and IV (n=8), who were disease free after surgery (n=16) or had minimal disease (n=4). Cohorts of 4 patients were vaccinated 4 times with VACCIMEL and bacillus Calmette Guerin (BCG) as adjuvant. Besides, the patients received placebo (group 1) or GM-CSF: 150 μg (group 2), 300 μg (group 3), 400 μg (group 4), and 600 μg (group 5) per vaccine. The combination of VACCIMEL and GM-CSF had low toxicity. Only in group 5, grade 2 thoracic pain (3/4 patients) and abdominal cramps (2/4 patients) were observed. Delayed-type hypersensitivity increased after vaccination and it was highest in group 4. Phytohemagglutinin stimulation of peripheral blood lymphocytes was analyzed in 9 patients: 4/9 had normal stimulation; 3/9 had low basal stimulation, which recovered after vaccination; and 2/9 were not stimulated. Antimelanoma antibodies preexisted in 9/19 patients; in 3/19 patients, antibodies anti-33 kd, 90 kd, and 100 kd antigens were induced by vaccination. IgG2 but not IgG1 antibodies were detected. Anti-BCG antibodies, mostly IgG2, reached the highest post/prevaccination ratio in group 4. Median serum interleukin-12 was lower in progressing patients (61.6 pg/mL) than in those without evident disease (89 pg/mL). Thus, its low toxicity and the induction of a predominantly cellular immune response suggest that the addition of 300 to 400 μg GM-CSF to VACCIMEL is useful in increasing the immune response.

IL-12 and IL-10 Expression Synergize to Induce the Immune-Mediated Eradication of Established Colon and Mammary Tumors and Lung Metastasis

Preclinical studies demonstrated that certain cytokines are potentially useful for the induction of antitumor immune responses. However, their administration in clinical settings was only marginally useful and evoked serious toxicity. In this study, we demonstrate that the combination of autologous inactivated tumor cells expressing IL-12 and IL-10 induced tumor remission in 50–70% of mice harboring large established colon or mammary tumors and spontaneous lung metastases, with the consequent establishment of an antitumor immune memory. Mice treatment with tumor cells expressing IL-12 was only marginally effective, while expression of IL-10 was not effective at all. Administration of the combined immunotherapy stimulated the recruitment of a strong inflammatory infiltrate that correlated with local, increased expression levels of the chemokines MIP-2, MCP-1, IFN-γ-inducible protein-10, and TCA-3 and the overexpression of IFN-γ, but not IL-4. The combined immunotherapy was also therapeutically effective on established lung metastases from both colon and mammary tumors. The antitumor effect of the combined immunotherapy was mainly dependent on CD8+ cells although CD4+ T cells also played a role. The production of IFN-γ and IL-4 by spleen cells and the development of tumor-specific IgG1 and IgG2a Abs indicate that each cytokine stimulated its own Th pathway and that both arms were actively engaged in the antitumor effect. This study provides the first evidence of a synergistic antitumor effect of IL-12 and IL-10 suggesting that a Th1 and a Th2 cytokine can be effectively combined as a novel rational approach for cancer immunotherapy.

Tumor Associated Stromal Cells Play a Critical Role on the Outcome of the Oncolytic Efficacy of Conditionally Replicative Adenoviruses

The clinical efficacy of conditionally replicative oncolytic adenoviruses (CRAd) is still limited by the inefficient infection of the tumor mass. Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter-based CRAds since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC. Interestingly, both CRAds displayed cytotoxic activity on SPARC positive-transformed human microendothelial HMEC-1 cells and WI-38 fetal fibroblasts. Both CRAds were therapeutically effective on SPARC positive-human melanoma tumors growing in nude mice but exhibited restricted efficacy in the presence of co-administered HMEC-1 or WI-38 cells. Conversely, co-administration of HMEC-1 cells enhanced the oncolytic efficacy of Ad(I)-F512-TK on SPARC-negative MIA PaCa-2 pancreatic cancer cells in vivo. Moreover, conditioned media produced by stromal cells pre-infected with the CRAds enhanced the in vitro viral oncolytic activity on pancreatic cancer cells, but not on melanoma cells. The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.

Metallothionein expression in colorectal cancer: Relevance of different isoforms for tumor progression and patient survival

Metallothioneins are a family of small, cysteine-rich proteins with many functions. Immunohistochemical evaluation of all metallothionein 1 + 2 isoforms in colorectal tumors has demonstrated an important down-regulation compared with normal tissue, although its prognostic significance is unclear. Moreover, the contribution of individual isoforms to overall metallothionein down-regulation is not known. To address these important issues, we analyzed the messenger RNA expression levels of all functional metallothionein 1 + 2 isoforms by quantitative reverse transcription polymerase chain reaction in 22 pairs of normal and tumor-microdissected epithelia and correlated these to the overall immunohistochemical protein expression. Our results showed that 5 isoforms (MT1G, 1E, 1F, 1H, and 1M) were lost during the transition from normal mucosa to tumor, whereas MT1X and MT2A were less down-regulated, and their expression was correlated with overall protein positivity. Second, we showed that MT1G hypermethylation occurred in cell lines and in 29% of tumor samples, whereas histone deacetylase inhibitors are able to induce most isoforms. Furthermore, we analyzed by immunohistochemistry 107 normal mucosae, 25 adenomas, 81 carcinomas, and 19 lymph node metastases to evaluate metallothionein expression during different stages of cancer development and to assess its relationship to patient survival. A lower immunohistochemical expression was associated with poorer survival, although it was not an independent predictor. Overall, this study identifies for the first time the relevant metallothionein isoforms for colorectal cancer progression, supports the concept that their loss is associated with worse prognosis, and suggests 2 mechanisms for epigenetic repression of metallothionein expression in colorectal tumors.

DNA Synthesis in Estrogen Receptor-Positive Human Breast Cancer Takes Place Preferentially in Estrogen Receptor-Negative Cells

The primary breast tumors of 27 patients were analyzed for the expression of estrogen receptors (ER) and DNA synthesis. Seventeen tumors were ER‐positive, and the simultaneous expression of ER and DNA synthesis could be analyzed in 14 ER‐positive tumors. DNA synthesis was measured through the thymidine labeling index (TLI). ER expression was detected by immunohistochemistry with monoclonal antibodies. In these tumors, 38.6% ± 13.1% of the cells were ER‐positive (average TLI = 0.60% ± 0.70%), as opposed to the presence of 61.4% ± 13.1% of ER‐negative cells (average TLI = 0.65% ± 0.53%). In 12 of 14 tumors, both ER‐positive and ER‐negative cells were found to be engaged in DNA synthesis, whereas in two tumors only ER‐negative cells were synthesizing DNA. On the basis of the TLI and the proportion of ER‐negative and ER‐positive cells in the total population, it is suggested that the ER‐positive and ER‐negative compartments are interrelated in most tumors. In five tumors, the ER‐negative compartment would be a precursor of the ER‐positive segment, whereas in six tumors the ER‐positive segment appears to be a precursor of the ER‐negative one. In three tumors, no evidence of an interrelationship between both segments could be found. In the 14 tumors analyzed, it also was found that 69.1% ± 21.3% of the DNA‐synthesizing cells were ER‐negative; this probably accounts for the temporary remissions observed after hormonal treatment in breast cancer.

IL-2- or IL-15-activated NK cells enhance Cetuximab-mediated activity against triple-negative breast cancer in xenografts and in breast cancer patients.

Triple-negative breast cancer (TNBC) patients do not benefit from target-specific treatments and is associated with a high relapse rate. Epidermal growth factor receptor is frequently expressed in TNBC and is a candidate for new therapies. In this work, we studied Cetuximab-mediated immune activity by NK cells. Thirteen activating/inhibitory receptors were examined on peripheral blood and tumor infiltrating NK cells. NK-cell functionality was evaluated using as effectors tumor-modulated NK cells and NK cells from patients. We evaluated the treatment with Cetuximab plus IL-2 or IL-15 in vivo in TNBC xenografts. Tumor NK-cells receptor profile showed upregulation of inhibitory receptors and downregulation of activating ones. Tumor-modulated NK cells were less cytotoxic. They could perform antibody-dependent cellular cytotoxicity (ADCC) triggered by Cetuximab, although impaired, it could still be restored by stimulation with IL-2 or IL-15. Patients with advanced disease displayed diminished levels of ADCC compared to healthy volunteers. ADCC was restored and potentiated with both cytokines, which were also effective in enhancing the therapeutic activity of Cetuximab in vivo. The combination of Cetuximab with IL-15 and IL-2 may be considered an attractive therapeutic approach to enhance the clinical efficacy of Cetuximab in TNBC.

Protein expression changes during human triple negative breast cancer cell line progression to lymph node metastasis in a xenografted model in nude mice

Triple negative breast cancers (TNBC) lacking hormone receptors and HER-2 amplification are very aggressive tumors. Since relevant differences between primary tumors and metastases could arise during tumor progression as evidenced by phenotypic discordances reported for hormonal receptors or HER-2 expression, in this analysis we studied changes that occurred in our TNBC model IIB-BR-G throughout the development of IIB-BR-G-MTS6 metastasis to the lymph nodes (LN) in nude mice, using an antibody-based protein array to characterize their expression profile. We also analyzed their growth kinetics, migration, invasiveness and cytoskeleton structure in vitro and in vivo. In vitro IIB-BR-G-MTS6 cells grew slower but showed higher anchorage independent growth. In vivo IIB-BR-G-MTS6 tumors grew significantly faster and showed a 100% incidence of LN metastasis after s.c. inoculation, although no metastasis was observed for IIB-BR-G. CCL3, IL1β, CXCL1, CSF2, CSF3, IGFBP1, IL1α, IL6, IL8, CCL20, PLAUR, PlGF and VEGF were strongly upregulated in IIB-BR-G-MTS6 while CCL4, ICAM3, CXCL12, TNFRSF18, FIGF were the most downregulated proteins in the metastatic cell line. IIB-BR-G-MTS6 protein expression profile could reflect a higher NFκB activation in these cells. In vitro, IIB-BR-G displayed higher migration but IIB-BR-G-MTS6 had more elevated matrigel invasion ability. In agreement with that observation, IIB-BR-G-MTS6 had an upregulated expression of MMP1, MMP9, MMP13, PLAUR and HGF. IIB-BR-G-MTS6 tumors presented also higher local lymphatic invasion than IIB-BR-G but similar lymphatic vessel densities. VEGFC and VEGFA/B expression were higher both in vitro and in vivo for IIB-BR-G-MTS6. IIB-BR-G-MTS6 expressed more vimentin than IB-BR-G cells, which was mainly localized in the cellular extremities and both cell lines are E-cadherin negative. Our results suggest that IIB-BR-G-MTS6 cells have acquired a pronounced epithelial-to-mesenchymal transition phenotype. Protein expression changes observed between primary tumor-derived IIB-BR-G and metastatic IIB-BR-G-MTS6 TNBC cells suggest potential targets involved in the control of metastasis.