Stefan Löb

Inhibitors of indoleamine-2,3-dioxygenase for cancer therapy: can we see the wood for the trees?

Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme capable of inhibiting a destructive maternal T cell response against allogeneic fetuses. Expression of IDO is evident in tumours and is thought to enable escape from immunologically mediated rejection. Consequently, clinical trials using an inhibitor of IDO, 1-methyltryptophan (1MT), have been initiated. However, a review of the current literature indicates that we are far from understanding the biological relevance of IDO expression during tumorigenesis. A better understanding of IDO biology is needed to comprehend the effect of IDO inhibitors and to provide a rationale for their therapeutic application in cancer.

IDO1 and IDO2 are expressed in human tumors: levo- but not dextro-1-methyl tryptophan inhibits tryptophan catabolism

ObjectivesIndoleamine-2,3-Dioxygenase (IDO) is an immunosuppressive molecule inducible in various cells. In addition to classic IDO (IDO1), a new variant, IDO2, has recently been described. When expressed in dendritic cells (DCs) or cancer cells, IDO was thought to suppress the immune response to tumors. A novel therapeutic approach in cancer envisages inhibition of IDO with 1-methyl-tryptophan (1MT). The levo-isoform (l-1MT) blocks IDO1, whereas dextro-1MT (d-1MT), which is used in clinical trials, inhibits IDO2. Here we analyze IDO2 expression in human cancer cells and the impact of both 1-MT isoforms on IDO activity.MethodsSurgically extirpated human primary tumors as well as human cancer cell lines were tested for IDO1 and IDO2 expression by RT-PCR. IDO1 activity of Hela cells was blocked by transfection with IDO1-specific siRNA and analysed for tryptophan degradation by RP-HPLC. The impact of d-1MT and l-1MT on IDO activity of Hela cells and protein isolates of human colon cancer were studied.ResultsHuman primary gastric, colon and renal cell carcinomas constitutively expressed both, IDO1 and IDO2 mRNA, whereas cancer cells lines had to be induced to by Interferon-gamma (IFN-γ). Treatment of Hela cells with IDO1-specific siRNA resulted in complete abrogation of tryptophan degradation. Only l-1MT, and not d-1MT, was able to block IDO activity in IFN-γ-treated Hela cells as well as in protein isolates of primary human colon cancer.ConclusionsAlthough IDO2 is expressed in human tumors, tryptophan degradation is entirely provided by IDO1. Importantly, d-1MT does not inhibit the IDO activity of malignant cells. If ongoing clinical studies show a therapeutic effect of d-1MT, this cannot be attributed to inhibition of IDO in tumor cells.

Are Indoleamine-2,3-dioxygenase Producing Human Dendritic Cells a Tool for Suppression of Allogeneic T-cell Responses?

Background. Suppressive dendritic cells (DCs) are a promising tool for tolerance induction in transplantation. A human DCs subpopulation, which constitutively expresses indoleamine-2,3-dioxygenase (IDO), a molecule shown to prevent the rejection of fetus during pregnancy, has recently been described. This subset, characterized by nonadherence and CD123/CCR6 expression, exhibited sustained IDO production if exposed to interleukin (IL)-10. In the present work, we generated human nonadherent, CD123+/CCR6+ DCs secreting IL-10. Methods. Monocytes were separated by plastic adherence and differentiated to DCs in the presecence of IL-3 and IL-4. Expression of IDO was determined by reverse-transcriptase polymerase chain reaction and enzyme activity by reverse-phased high-performance liquid chromatography. Mixed lymphocyte cultures were performed with allogeneic nylon wool-purified T-cells. Results. Contradicting previous findings, CD123+/CCR6+ DCs did not express IDO. Maturation of these cells with inducer-cytokines up-regulated IDO, but the allogeneic T-cell stimulatory capacity of these DCs was even stronger than that of immature IDO− DCs, and chemical abrogation of IDO activity did not increase T-cell proliferation. In parallel, we generated mature IDO− DCs, but these cells also did not induce stronger T-cell stimulation than their IDO+ counterpart. Conclusions. In conclusion, CD123+/CCR6+ DCs do not constitutively express IDO and “induced” IDO+ DCs, even coexpressing anti-inflammatory IL-10, do not suppress allogeneic T-cell responses.

Role of IDO in organ transplantation: promises and difficulties.

Induction of donor-antigen-specific immunological tolerance still remains the “holy grail” in organ transplantation. Recently, Indoleamine-2,3 Dioxygenase (IDO)—a tryptophan degrading enzyme—has been shown to be implicated in one of natures most impressive examples of tolerance, which is maternal acceptance of the semi-allogeneic foetus. Although many experimental findings propose IDO as a key player in induction and maintenance of peripheral tolerance, scepticism exists as to whether IDO represents a promising therapeutic target with clinical relevance. In this review article we will discuss the role of IDO in transplantation and take a critical look at IDO-based therapeutic strategies.

Is IDO a key enzyme bridging the gap between tumor escape and tolerance induction

BackgroundShaping immune responses to prevent tumor-induced tolerance or transplant rejection after solid organ transplantation is a permanently expanding field of research. Immunological tolerance, in this case, is a double-edged sword. Tumors escape immune surveillance by creating an abnormal state of tolerance towards their own antigens, whereas transplantation medicine is challenged to develop new strategies to induce allograft-specific immunological tolerance. One mechanism possibly capable of achieving immunoregulation is based on indoleamine-2,3-dioxygenase (IDO).ObjectiveThis overview article focuses on IDO-mediated tryptophan catabolism with special regard to its role in cancer and transplantation immunology.ResultsThe historical view about IDO as a host’s antimicrobial defence mechanism has been extended by the observation that its expression is essential for successful allogeneic pregnancy. Subsequent studies analysing IDO as an immune-regulatory enzyme describe its implications in cancer immune escape, as chemical abrogation of enzyme activity with 1-methyl-tryptophan (1-MT), results in enhanced antitumor responses in animal models. Therefore, a clinical trial treating cancer patients with 1-MT has been started. IDO also seems to play an essential role in the control of allo- and autoreactive T cell responses. CTLA4-Ig is able to induce IDO expression in dendritic cells (DCs) and consequently renders them tolerogenic, which might provide one explanation for the observed therapeutic effects of abatacept and belatacept.ConclusionThere is evidence that IDO achieves immune modulation in several animal models. However, in humans, this remains controversially discussed.

Increased pretransplantation plasma kynurenine levels do not protect from but predict acute kidney allograft rejection.

Indoleamine 2,3-dioxygenase (IDO), an enzyme expressed in many cell types, catalyses degradation of tryptophan (Trp) to kynurenine (Kyn) and may exert immunosuppressive functions, mediated mainly by kynurenines. Therefore, increased Kyn concentrations would be expected to protect allografts from rejection. We conducted this study to examine whether Kyn has predictive value for kidney graft outcome. End-stage renal disease patients (n = 210) demonstrated an increased Kyn/Trp ratio compared with healthy controls (n = 30). Both Kyn and Trp levels were significantly higher in patients who subsequently developed acute rejection than in patients who did not (p < 0.001 and p < 0.001, respectively). Furthermore, pretransplantation Kyn and Trp plasma concentrations were significantly different in patients who went on to develop acute rejection (high values) or acute tubular necrosis (low values) (p = 0.007 and p = 0.021, respectively). After transplantation Kyn levels decreased. Approximately 3 days before biopsy-confirmed rejection, Kyn was significantly increased in patients with rejection compared with those without rejection (p < 0.001). Contrary to expectation, high Kyn plasma levels before transplantation were not predictive of low rejection risk. Although informative in overall terms, at the present stage, Kyn levels do not allow the concise risk differentiation of individual patients.

Rapamycin-Induced Impaired Wound Healing Is Associated with Compromised Tissue Lactate Accumulation and Extracellular Matrix Remodeling

Introduction: Extracellular matrix (ECM) remodeling involving matrix metalloproteinases (MMPs) and wound lactate accumulation are essential elements of tissue repair. The aim of this study was to investigate whether rapamycin-induced impaired healing is associated with compromised wound fluid lactate accumulation and altered ECM remodeling. Methods: Polyvinyl alcohol sponges were subcutaneously implanted in male C57/BL6 mice. Animals were randomized to daily intraperitoneal treatment with either vehicle or 1.5 mg/kg rapamycin. After 7 or 14 days, sponges were harvested to collect wound fluid for subsequent analyses. Wounds were excised for assessment of tensile strength. Results: After 7 days, wound hydroxyproline content was significantly decreased due to rapamycin therapy, whereas the observed difference in tensile strength marginally failed to show statistical significance. In addition, rapamycin reduced wound lactate accumulation and enhanced MMP-2 protein expression, and both MMP-2 and MMP-9 activity. At day 14, wound tensile strength and hydroxyproline content were significantly lower along with an increase in MMP-2 and MMP-9 activity in rapamycin-treated mice. Similarly, wound fluid lactate concentration and MMP-2 protein expression were found to be persistently decreased and increased, respectively. Conclusions: Rapamycin affects tissue repair by interfering with fundamental mechanisms involved in healing, namely lactate accumulation and ECM remodeling.

Local Peritonectomy Highly Attracts Free Floating Intraperitoneal Colorectal Tumour Cells in a Rat Model

Background/Aims: Intraperitoneal free cancer cells are associated with a higher risk of recurrence and a poorer prognosis in colorectal surgery. Tumour recurrence may occur early after surgery. One potential mechanism is the ability of peritoneal lesions to attract tumour cells. Methods: In Wag-Rija rats, the parietal peritoneum was resected on a defined area, a corresponding control area was marked in the same rat and colorectal tumour cells (CC531) were applied into the abdomen after surgery. Tissue was harvested 6 or 9 days after surgery to evaluate intra-abdominal tumour growth. Additionally tumour cells were applied 2 weeks after peritoneal resection to investigate tumour growth in a healed area of peritonectomy. Specimens were evaluated for macroscopic tumour spread, weight of the abdominal wall and maximal tumour thickness. Results: Macroscopic tumour spread, weight of the abdominal wall and maximal tumour thickness were significantly increased within the area of peritonectomy after both 6 and 9 days compared to the control area. However, only macroscopic tumour expansion was significantly increased in the healed area of peritonectomy. Conclusion: Peritoneal defects may play an important role in the pathogenesis of tumour implantation and might have some impact on tumour recurrence. The peritoneal damage should be kept as low as possible.

Surgery of Colorectal Carcinoma in Patients Aged over 80

Background: The incidence of colorectal carcinoma increases rapidly in aged patients. We investigated retrospectively the differences in treatment relative to the patients’ age. Patients and Methods: A total of 394 patients with colorectal carcinoma (group I: ≥80 years, n = 197; group II: 60–79 years, n = 197) were analyzed in an average period of 4 years in relation to surgery, comorbidities, postoperative morbidity, mortality, survival and recurrence. Results: Patients ≥80 years had a significantly higher rate of comorbid conditions (p = 0.04; cardiovascular, p = 0.01; diabetes mellitus, p < 0.05) and more carcinomas in the sigmoid/rectum (72% vs. 67%; p < 0.05). Tumor stage, R0 resection rate, and overall complication rate were not influenced by age. The 30-day mortality rate was significantly higher in group I (12% vs. 3%; p = 0.02). Emergency surgical procedures were required significantly more often in group I (14%) than in group II (5%; p = 0.003). The 5-year survival rate among patients in group I was 30.1% compared to 50.5% among patients in group II (p < 0.0001). Conclusions: Elderly patients have a higher rate of comorbidity and a higher postoperative 30-day mortality rate. Tumor stage, R0 resection rate, and overall postoperative complication rate do not appear to be influenced by age. The higher rate of emergency operations on patients ≥80 years is associated with the higher 30day mortality. Even in patients aged ≥80 years, attention should focus on the long-term oncological results, after appropriate assessment of the preoperative risk.

Pharmacodynamics of Oxaliplatin-Derived Platinum Compounds During Hyperthermic Intraperitoneal Chemotherapy (HIPEC): An Emerging Aspect Supporting the Rational Design of Treatment Protocols

BackgroundHyperthermic intraperitoneal chemotherapy (HIPEC) is used to treat peritoneal surface malignancies with application of cytostatic drugs such as oxaliplatin (OX) after cytoreductive surgery. Despite its increased use, evidence for optimal drug dosage, and notably duration of HIPEC, is scarce.MethodsIn this study, OX distribution was comprehensively assessed in nine patients during HIPEC (300 mg OX/m2 body surface area in Physioneal solution for 30 min). Oxaliplatin and its derivatives were measured in peritoneal perfusates over time by liquid chromatography coupled with mass spectrometry (LC-MS), and the resulting total platinum concentration in tissue was analyzed by atomic absorption spectrometry. Additionally, a novel impedance-based real-time cytotoxicity assay was used to evaluate the bioactivity of perfusates ex vivo.ResultsCompared with amounts of OX expected in peritoneal perfusates by calculation, only 10–15% of the parent drug could be detected by LC-MS during HIPEC. Notably, the study additionally detected platinum compounds consistent with OX transformation, accounting for a further fraction of the applied drug. The cytotoxic properties of perfusates remained unchanged during HIPEC, with only a slight but significant attenuation evidenced after 30 min.ConclusionsThe bioactivity of peritoneal perfusates ex vivo is a useful parameter for evaluating the actual cytotoxic potential of OX and its derivatives used in HIPEC over time, overcoming important limitations and disadvantages associated with respective drug monitoring only. Ex vivo cytotoxicity assays may be a promising tool to aid guiding future standardization and harmonization of HIPEC protocols based on drug-mediated effects.