Enhancement of electroporation facilitated immunogene therapy via T-reg depletion.

Regulatory T cells (T-regs) can negatively impact tumor antigen-specific immune responses after infiltration into tumor tissue. However, depletion of T-regs can facilitate enhanced anti-tumor responses, thus augmenting the potential for immunotherapies. Here we focus on treating a highly aggressive form of cancer using a murine melanoma model with a poor prognosis. We utilize a combination of T-reg depletion and immunotherapy plasmid DNA delivered into the B16F10 melanoma tumor model via electroporation. Plasmids encoding murine granulocyte macrophage colony-stimulating factor and human B71 were transfected with electroporation into the tumor and transient elimination of T-regs was achieved with CD25-depleting antibodies (PC61). The combinational treatment effectively depleted T-regs compared to the untreated tumor and significantly reduced lung metastases. The combination treatment was not effective in increasing the survival, but only effective in suppression of metastases. These results indicate the potential for combining T-reg depletion with immunotherapy-based gene electrotransfer to decrease systemic metastasis and potentially enhance survival.

The BH3 mimetic HA14-1 enhances 5-fluorouracil-induced autophagy and type II cell death in oesophageal cancer cells.

BACKGROUND: Resistance to chemotherapeutic agents has been associated with a failure of cancer cells to induce apoptosis. Strategies to restore apoptosis have led to the development of BH3 mimetics, which inhibit anti-apoptotic Bcl-2 family members. We examined the sensitivity of three oesophageal cancer cell lines to 5-fluorouracil (5-FU) alone and in combination with the BH3 mimetic HA14-1. METHODS: Clonogenic assays, morphology, markers of autophagy and apoptosis were used to assess the involved death mechanisms. RESULTS: In response to 5-FU treatment, OE21 cells induce apoptosis, KYSE450 and KYSE70 cells are more resistant and induce autophagy accompanied by type II cell death. Autophagy induction results in ineffective treatment as substantial numbers of cells survive and re-populate. HA14-1 did not improve 5-FU treatment or reduce colony re-growth in the apoptosis deficient KYSE70 cells. However, the sensitivity of OE21 (apoptotic) and KYSE450 cells (apoptosis deficient/type II cell death) was significantly improved. In OE21 cells, treatment with 5-FU and HA14-1 resulted in augmentation of apoptosis. In KYSE450 cells, the reduction in recovering colonies following combination treatment was due to the enhancement of type II cell death. CONCLUSION: The efficacy of HA14-1 is cell line dependent and is not reliant on apoptosis induction.

The emerging role of viruses in the treatment of solid tumours.

There is increasing optimism for the use of non-pathogenic viruses in the treatment of many cancers. Initial interest in oncolytic virotherapy was based on the observation of an occasional clinical resolution of a lymphoma after a systemic viral infection. In many cancers, by comparison with normal tissues, the competency of the cellular anti-viral mechanism is impaired, thus creating an exploitable difference between the tumour and normal cells, as an unimpeded viral proliferation in cancer cells is eventually cytocidal. In addition to their oncolytic capability, these particular viruses may be engineered to facilitate gene delivery to tumour cells to produce therapeutic effects such as cytokine secretion and anti -tumour immune responses prior to the eventual cytolysis. There is now promising clinical experience with these viral strategies, particularly as part of multimodal studies, and already several clinical trials are in progress. The limitations of standard cancer chemotherapies, including their lack of specificity with consequent collateral toxicity and the development of cross-resistance, do not appear to apply to viral-based therapies. Furthermore, virotherapy frequently restores chemoradiosensitivity to resistant tumours and has also demonstrated efficacy against cancers that historically have a dismal prognosis. While there is cause for optimism, through continued improvements in the efficiency and safety of systemic delivery, through the emergence of alternative viral agents and through favourable clinical experiences, clinical trials as part of multimodal protocols will be necessary to define clinical utility. Significant progress has been made and this is now a major research area with an increasing annual bibliography.

Effective immunotherapy of weakly immunogenic solid tumours using a combined immunogene therapy and regulatory T-cell inactivation.

Obstacles to effective immunotherapeutic anti-cancer approaches include poor immunogenicity of the tumour cells and the presence of tolerogenic mechanisms in the tumour microenvironment. We report an effective immune-based treatment of weakly immunogenic, growing solid tumours using a locally delivered immunogene therapy to promote development of immune effector responses in the tumour microenvironment and a systemic based T regulatory cell (Treg) inactivation strategy to potentiate these responses by elimination of tolerogenic or immune suppressor influences. As the JBS fibrosarcoma is weakly immunogenic and accumulates Treg in its microenvironment with progressive growth, we used this tumour model to test our combined immunotherapies. Plasmids encoding GM-CSF and B7-1 were electrically delivered into 100 mm(3) tumours; Treg inactivation was accomplished by systemic administration of anti-CD25 antibody (Ab). Using this approach, we found that complete elimination of tumours was achieved at a level of 60% by immunogene therapy, 25% for Treg inactivation and 90% for combined therapies. Moreover, we found that these responses were immune transferable, systemic, tumour specific and durable. Combined gene-based immune effector therapy and Treg inactivation represents an effective treatment for weakly antigenic solid growing tumours and that could be considered for clinical development.

Curcumin induces apoptosis-independent death in oesophageal cancer cells.

BACKGROUND: Oesophageal cancer incidence is increasing and survival rates remain extremely poor. Natural agents with potential for chemoprevention include the phytochemical curcumin (diferuloylmethane). We have examined the effects of curcumin on a panel of oesophageal cancer cell lines. METHODS: MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assays and propidium iodide staining were used to assess viability and DNA content, respectively. Mitotic catastrophe (MC), apoptosis and autophagy were defined by both morphological criteria and markers such as MPM-2, caspase 3 cleavage and monodansylcadaverine (MDC) staining. Cyclin B and poly-ubiquitinated proteins were assessed by western blotting. RESULTS: Curcumin treatment reduces viability of all cell lines within 24 h of treatment in a 5-50 muM range. Cytotoxicity is associated with accumulation in G2/M cell-cycle phases and distinct chromatin morphology, consistent with MC. Caspase-3 activation was detected in two out of four cell lines, but was a minor event. The addition of a caspase inhibitor zVAD had a marginal or no effect on cell viability, indicating predominance of a non-apoptotic form of cell death. In two cell lines, features of both MC and autophagy were apparent. Curcumin-responsive cells were found to accumulate poly-ubiquitinated proteins and cyclin B, consistent with a disturbance of the ubiquitin-proteasome system. This effect on a key cell-cycle checkpoint regulator may be responsible for the mitotic disturbances and consequent cytotoxicity of this drug. CONCLUSION: Curcumin can induce cell death by a mechanism that is not reliant on apoptosis induction, and thus represents a promising anticancer agent for prevention and treatment of oesophageal cancer.

Immune gene therapy as a neoadjuvant to surgical excision to control metastatic cancers.

We investigated if the range of efficacy of a gene-based immunotherapy of solid tumours against systemic disease could be extended when used as a neoadjuvant to surgery. Hundred percent mice whose subcutaneous tumours were surgically removed 4 days post-electroporation with GM-CSF and B7-1 plasmid were systemically resistance to tumour rechallenge. In mice bearing both subcutaneous and hepatic tumours, neoadjuvant treatment of the primary tumour resulted in significant reduction in, or elimination of, hepatic tumour growth, with a significant increase in survival, indicating that control of the primary tumour by immunotherapy was not a prerequisite for containment of systemic disease.

Laparoscopic peritoneal lavage for generalized peritonitis due to perforated diverticulitis.

BACKGROUND: The standard approach to generalized peritonitis due to perforated diverticulitis involves open surgery and diversion of faecal content. This study assessed the feasibility of laparoscopic peritoneal lavage. METHODS: A prospective multi-institutional study of 100 patients was undertaken. All consenting patients with perforated diverticulitis causing generalized peritonitis underwent attempted laparoscopic peritoneal lavage. The degree of peritonitis, according to the Hinchey grading system, was recorded. Primary endpoints were operative success and resolution of symptoms. RESULTS: Patients had a median age of 62.5 (range 39-94) years, a male : female ratio of 2 : 1 and a median American Society of Anesthesiologists grade of III (range II-V). Eight patients with grade 4 diverticulitis had conversion to an open Hartmann's procedure. The remaining 92 patients were managed by laparoscopic lavage, with morbidity and mortality rates of 4 and 3 per cent respectively. Two patients required postoperative intervention for a pelvic abscess. Only two patients re-presented with diverticulitis at a median follow up of 36 (range 12-84) months. CONCLUSION: Laparoscopic management of perforated diverticulitis with generalized peritonitis is feasible, with a low recurrence risk in the short term.

Cyclooxygenase-2 inhibitors demonstrate anti-proliferative effects in oesophageal cancer cells by prostaglandin E(2)-independent mechanisms.

The incidence of oesophageal cancer (OC) has risen in recent decades, with survival rates remaining poor despite surgical treatment and adjuvant chemotherapy. Studies have reported cyclooxygenase-2 (COX-2) overexpression in OC and current evidence suggests NSAIDs have major potential for chemoprevention through COX-2 inhibition. However, several reports have questioned the specificity of these inhibitors, suggesting they may act through mechanisms other than COX-2. We evaluated the effects of specific COX-2 inhibitors, NS-398 and nimesulide, on cell lines of both histological types of OC. COX-2 protein expression varied in the cell lines and corresponded with levels of prostaglandin E(2) (PGE(2)) production. Following treatment with low concentrations of NS-398 (0.1 microM), PGE(2) production was reduced dramatically, indicating inhibition of COX-2 activity. Examination of cellular morphology, caspase-3 activity and mitochondrial membrane integrity found no major induction of apoptotic cell death at concentrations below 100 microM. Tumour cell proliferation was significantly reduced at high concentrations (50-100 microM) of both inhibitors over 6 days. Cellular responses were more evident in NS-398-treated adenocarcinoma cells. However, concentrations required to inhibit proliferation were up to 1000-fold higher than those needed to inhibit enzyme activity. Addition of exogenous PGE(2) to NS-398-treated adenocarcinoma cells failed to reverse the inhibitory effects, indicating PG and COX-2 independence. It remains possible that in vivo COX-2 is the primary target, as enzyme inhibition can be achieved at low concentrations, however, inhibition of proliferation is not the primary mechanism of their anti-tumour activity.

Beta-catenin transcriptional activity is inhibited downstream of nuclear localisation and is not influenced by IGF signalling in oesophageal cancer cells.

Aberrant expression/localisation of beta-catenin has been implicated in the progression of oesophageal cancer. As a member of the Wnt-signalling pathway, activated beta-catenin translocates into the nucleus and drives gene transcription. Insulin-like growth factors (IGFs) have been implicated in modulation of beta-catenin localisation and transcriptional activity. We have demonstrated that beta-catenin is abundantly expressed by oesophageal cancer cells, and is both cytoplasmic and nuclear in location. beta-catenin was transcriptionally inactive in 4 of 5 cell lines. All cells expressed the IGF-1 receptor. Addition of exogenous IGFs activated the PI-3 kinase pathway but did not enhance beta-catenin/T-cell factor- (TCF) mediated transcription. Activation of Wnt signalling by lithium induced beta-catenin stabilisation in 2 cell lines but this did not increase transcriptional activity. In contrast 2 cell lines without lithium-enhanced stabilisation or re-distribution of beta-catenin did exhibit beta-catenin/TCF-mediated transcriptional activity. This study shows that beta-catenin accumulation and nuclear localisation is not indicative of transcriptional activity, and therefore is not supportive of a major role in these oesophageal cancer cells. It also questions the value of immunohistochemical studies that examine only expression. Co-operative signalling from other growth factors or adhesive molecules is likely to be required to relieve nuclear inhibition of transcriptional activity, and the nature of this is currently unknown.

Local involvement of the urinary bladder in primary colorectal cancer: outcome with en bloc resection.

BACKGROUND: Colorectal cancers that adhere to the urinary bladder require en bloc partial or total cystectomy to achieve negative tumor margins. METHODS: This prospective study evaluated the outcome of combined bladder resection for carcinoma of the colon or rectum at a unit specializing in gastrointestinal cancer. RESULTS: Patients (n = 63) with colorectal tumors adherent to the bladder at operation and without distal metastases were followed. Fifty-eight patients (92%) had tumors of the sigmoid colon or upper rectum. Operative morbidity and mortality rates were 18% and 1.5%, respectively. Histological staging demonstrated bladder adherence in 46% (29/63) and invasion in 54% (34/63). Overall disease-specific survival was 54%, with a mean follow-up of 7.6 (range 5-12) years. Five-year survival for margin negative patients was 72% (26/36) and 27% (4/15) for node negative and positive tumors, respectively. The bladder was closed primarily in 48 patients and reconstructed by enterocystoplasty in five, with ten patients requiring urinary diversion. CONCLUSIONS: En bloc bladder resection for adherent or invading tumors of the colon and rectum achieves good local control, but an infiltrative extravesical margin denotes poor prognosis. The potential for cure in completely excised node negative tumors is good. Bladder reconstruction is achievable in most patients.

Local involvement of the urinary bladder in primary colorectal cancer: outcome with en-bloc resection.

BACKGROUND: Colorectal cancers that adhere to the urinary bladder require en-bloc partial or total cystectomy to achieve negative tumor margins. METHODS: This prospective study evaluated the outcome of combined bladder resection for carcinoma of the colon or rectum at a unit specializing in gastrointestinal cancer. RESULTS: Patients (n = 63) with colorectal tumors adherent to the bladder at operation and without distal metastases were followed. Fifty-eight patients (92%) had tumors of the sigmoid colon or upper rectum. Operative morbidity and mortality rates were 18% and 1.5%, respectively. Histological staging demonstrated bladder adherence in 46% (29/63) and invasion in 54% (34/63). Overall disease-specific survival was 54% with a mean follow-up of 7.6 years (range 5-12). Five-year survival for margin-negative patients was 72% (26/36) and 27% (4/15) for node-negative and -positive tumors, respectively. The bladder was closed primarily in 48 patients and reconstructed by enterocystoplasty in 5, with 10 patients requiring urinary diversion. CONCLUSIONS: En-bloc bladder resection for adherent or invading tumors of the colon and rectum achieves good local control, but an infiltrative extravesical margin denotes poor prognosis. The potential for cure in completely excised node-negative tumors is good. Bladder reconstruction is achievable in most patients.

Evaluation of cellular uptake and gene transfer efficiency of pegylated poly-L-lysine compacted DNA: implications for cancer gene therapy.

Recent success in phase I/II clinical trials (Konstan, M. W.; Davis, P. B.; Wagener, J. S.; Hilliard, K. A.; Stern, R. C.; Milgram, L. J.; Kowalczyk, T. H.; Hyatt, S. L.; Fink, T. L.; Gedeon, C. R.; Oette, S. M.; Payne, J. M.; Muhammad, O.; Ziady, A. G.; Moen, R. C.; Cooper, M. J. Hum. Gene Ther. 2004, 15 (12), 1255-69) has highlighted pegylated poly-L-lysine (C1K30-PEG) as a nonviral gene delivery agent capable of achieving clinically significant gene transfer levels in vivo. This study investigates the potential of a C1K30-PEG gene delivery system for cancer gene therapy and evaluates its mode of cellular entry with the purpose of developing an optimally formulated prototype for tumor cell transfection. C1K30-PEG complexes have a neutral charge and form rod-like and toroid-like nanoparticles. Comparison of the transfection efficiency achieved by C1K30-PEG with other cationic lipid and polymeric vectors demonstrates that C1K30-PEG transfects cells more efficiently than unpegylated poly-L-lysine and compares well to commercially available vectors. In vivo gene delivery by C1K30-PEG nanoparticles to a growing subcutaneous murine tumor was also demonstrated. To determine potential barriers to C1K30-PEG gene delivery, the entry mechanism and intracellular fate of rhodamine labeled complexes were investigated. Using cellular markers to delineate the pathway taken by the complexes upon cellular entry, only minor colocalization was observed with EEA-1, a marker of early endosomes. No colocalization was observed between the complexes and the transferrin receptor, which is a marker for clathrin-coated pits. In addition, complexes were not observed to enter late endosomes/lysosomes. Cellular entry of the complexes was completely inhibited by the macropinocytosis inhibitor, amiloride, indicating that the complexes enter cells via macropinosomes. Such mechanistic studies are an essential step to support future rational design of pegylated poly-L-lysine vectors to improve the efficiency of gene delivery.

Local gene therapy of solid tumors with GM-CSF and B7-1 eradicates both treated and distal tumors.

Gene therapy-induced expression of immunostimulatory molecules at tumor cell level may evoke antitumor immune mechanisms by recruiting and enhancing viability of antigen-processing cells and specific tumoricidal lymphocytes. The antitumor efficacy of a plasmid, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and the B7-1 costimulatory immune molecule, delivered into growing solid tumors by electroporation was investigated. Murine fibrosarcomas (JBS) growing in Balb/C mice (

Non-viral in vivo immune gene therapy of cancer: combined strategies for treatment of systemic disease.

Many patients with various types of cancers have already by the time of presentation, micrometastases in their tissues and are left after treatment in a minimal residual disease state [Am J Gastroenterol 95(12), 2000]. To prevent tumour recurrence these patients require a systemic based therapy, but current modalities are limited by toxicity or lack of efficacy. We have previously reported that immune reactivity to the primary tumour is an important regulator of micrometastases and determinant of prognosis. This suggests that recruitment of specific anti-tumour mechanisms within the primary tumour could be used advantageously for tumour control as either primary or neo-adjuvant treatments. Recently, we have focused on methods of stimulating immune eradication of solid tumours and minimal residual disease using gene therapy approaches. Gene therapy is now a realistic prospect and a number of delivery approaches have been explored, including the use of viral and non-viral vectors. Non-viral vectors have received significant attention since, in spite of their relative delivery inefficiency, they may be safer and have greater potential for delivery of larger genetic units. By in vivo electroporation of the primary tumour with plasmid expressing GM-CSF and B7-1, we aim to stimulate immune eradication of the treated tumour and associated metastases. In this symposium report, we describe an effective gene based approach for cancer immunotherapy by inducing cytokine and immune co-stimulatory molecule expression by the growing cells of the primary tumour using a plasmid electroporation gene delivery strategy. We discuss the potential for enhancement of this therapy by its application as a neoadjuvant to surgical excision and by its use in combination with suppressor T cell depletion.

Paraesophageal lymph node metastasis from prostatic adenocarcinoma in a patient with esophageal squamous carcinoma.

SUMMARY. Esophageal squamous carcinomas induce regional immune suppression in the domain of the tumor while the global immune system remains intact. We report a patient with a squamous esophageal carcinoma, who was discovered at esophagectomy to have paraesophageal lymph node metastases from a prostatic adenocarcinoma. No other sites of metastatic disease were identified. This supports the concept that regional immune suppression by esophageal squamous cancers facilitates growth of metastases in the local lymph nodes.

Combined electric field and ultrasound therapy as a novel anti-tumour treatment.

The permeabilising effects of electric pulses on cell membranes and the use of ultrasound energy of various intensities, for both thermal effects and enhancement of drug and gene delivery, have led to extensive research into the potential applications of these systems in the development of novel anti-cancer treatments. In the present study we have demonstrated for the first time that the application of brief electric pulses 'sensitises' tumour cells to the effects of low intensity ultrasound. The studies were conducted in human tumours established in athymic nude mice and in many instances resulted in the reduction of tumour mass. The combined electric field and ultrasound approach (CEFUS) was applied in vivo to a murine colon adenocarcinoma (C26) and a human oesophageal adenocarcinoma (OE19). The experiments performed demonstrated the anti-tumour effects of the combined therapy. Varying the electrosensitisation parameters used (voltage, waveform, electrode type) contributed to optimise the procedure. Exponential electric pulses with a peak of 1000 V/cm were initially used, but square wave pulses (1000 V/cm, 1 ms, x2, 1 Hz) were found to be just as effective. All ultrasound application parameters were kept constant during the study. The growth rate of C26 tumours treated with CEFUS was significantly reduced with respect to untreated controls at day 7 (96% of average initial tumour volume in CEFUS group versus 615% for controls, P < 0.05). Similar reduction was observed in OE19 tumours treated with CEFUS by day 4 (82% versus 232%, P < 0.032). Our preliminary data suggest that this novel technology could potentially be of wide application in clinical practice for the treatment of solid tumours and is worth further investigation.

Probiotics: an emerging therapy.

There is considerable clinical interest in the utility of probiotic therapy--the feeding of (live) non-pathogenic bacteria, originally derived from the alimentary tract, for disease treatment or health promotion. The microflora of the gastrointestinal tract is essential for mucosal protection, for immune education and for metabolism of fecal residue. Physiological disturbances of these processes, when they occur, result from: i) alteration of a microbial ecosystem, originally conserved by evolution; ii) reduced consumption of microorganisms; iii) invasion of pathogens; or iv) modern interventions. Recent data support the use of proven probiotic organisms in prevention and treatment of flora-related gastrointestinal disorders including inflammatory bowel disease, infectious and antibiotic related diarrheas, and post-resection disorders including pouchitis. Therapeutic activity of probiotic bacteria can be due to competition with pathogens for nutrients and mucosal adherence, production of antimicrobial substances, and modulation of mucosal immune functions. Although a promising treatment, controlled clinical trials are necessary to validate the benefit of probiotics.

The development of novel flexible electrode arrays for the electrochemotherapy of solid tumour tissue. (Potential for endoscopic treatment of inaccessible cancers).

Therapeutic "electroporation" involves application of electric fields to target cells/tissues, thereby rendering their cell membranes transiently porous, thus making feasible the cellular uptake and efficacy of previously impermeant and ineffective therapeutic agents. The objectives of this research are a) the development of flexible electrode arrays for incorporation into microsystem endoscopic devices, and b) the assessment of their efficacy in delivering selected genetic and pharmaceutical anticancer therapies. Gold electrodes were fabricated on flexible polyimide substrates following predictive modeling and simulation of electric fields using FEMLAB software. Subsequent assessment of electroporation efficiency in-vitro involved 1) enumeration of viable tumour cells after delivery of electric pulses and exposure to low concentrations of bleomycin, otherwise known as electrochemotherapy 2) Efficacy of gene delivery by detection of emitted green fluorescence by cells after electroporation with the pEGFP plasmid and 3) In-vivo efficacy of electrochemotherapy in a variety of human solid tumour masses in nude mouse models (xenografts). The flexible electrode system was found to be successful for electrical delivery of plasmids and drugs in-vitro and in-vivo. We found in-vivo complete regression of prostate, colon, oesophageal, and renal cancers with reduced growth rates for fibrosarcoma and breast cell lines. These flexible electrodes are suitable for electrochemotherapy or gene therapy to solid tumours masses and may be fabricated for application to the treatment of some cancers in humans by transcutaneous or endoscopic delivery systems.

Oral immune tolerance to tumor specific antigens may confer growth advantage to esophageal and gastric cancers.

Oral administration of antigens induces antigen-specific systemic immune tolerance (Oral Tolerance). We postulate that the poorer prognosis of foregut cancers might, in part be explained by the systemic immune tolerizing effect of tumor specific antigens shed into and processed by the gut immune system thus conferring a growth advantage specific to individual cancers. Immunocompetent Balb/c mice were fed by gavage, either tumor tissue (JBS/CarB) in phosphate buffered saline (PBS) or PBS alone, daily for 14 days. On day 15 either subcutaneous tumors were induced or animals were immunized with cells in adjuvant. JBS tumors appeared earlier and grew faster in the JBS tumor fed mice than in either the PBS (P = 0.025) or CarB (P = 0.168) fed animals. The delayed type hypersensitivity response in tolerized mice was significantly abrogated (P < 0.01) compared to controls. These experiments demonstrate antigen specific oral immune tolerance for tumors, which is reflected in a faster growth rate and impaired delayed type hypersensitivity response. Similar mechanisms may be operational in human esophagogastric malignancy and may in part explain their poorer outcome.

Fas ligand mediates immune privilege and not inflammation in human colon cancer, irrespective of TGF-beta expression.

Many cancers express Fas ligand (FasL/CD95L) in vivo, and can kill lymphoid cells by Fas-mediated apoptosis in vitro. However, overexpression of recombinant FasL in murine tumour allografts revealed a potential antitumour effect of FasL, via recruitment of neutrophils. Transforming growth factor-beta1 (TGF-beta1) could inhibit these neutrophil-stimulatory effects of FasL. In the present study, we sought to determine directly whether FasL contributes to immune privilege or tumour rejection in human colon cancers in vivo, and whether TGF-beta1 regulates FasL function. Serial tumour sections were immunostained for FasL and TGF-beta1. Neutrophils and tumour infiltrating lymphocytes (TILs) were detected by immunohistochemistry for lactoferrin and CD45, respectively. Apoptotic TIL were identified by dual staining for TUNEL/CD45. FasL expression by nests of tumour cells was associated with a mean four-fold depletion of TILs (range 1.8-33-fold, n=16, P<0.001), together with a two-fold increase in TIL apoptosis (range 1.6-2.5-fold, n=14, P<0.001), relative to FasL-negative nests within the same tumours. The overall level of neutrophils present in all tumours examined was low (mean 0.3%, n=16), with FasL expression by tumour nests associated with a mean two-fold decrease in neutrophils, irrespective of TGF-beta1 expression. Together, our results suggest that tumour-expressed FasL is inhibitory rather than stimulatory towards antitumour immune responses.